key: cord- - q l f authors: lewis, lon d.; phillips, robert w. title: treatment of the calf with diarrhea date: - - journal: vet clin north am large anim pract doi: . /s - ( ) -x sha: doc_id: cord_uid: q l f nan from the colostrum as well as that actively produced by the calf itself; irregular feeding or overfeeding; the degree of confinement and crowding, which determines the pathogenic challenge to which the calf is exposed; and a multitude of stress factors such as inclement weather and difficult birth. calf scours may affect calves up to two months of age, but it is generally the greatest problem in calves less than two weeks of age, and frequently occurs in those less than one week old. in contrast, coccidiosis is more common in older calves and is generally hemorrhagic. clostridial enterotoxemia causes sudden death more commonly than it causes diarrhea. although large numbers of clostridia are present in the small intestine of some calves with diarrhea, they may not be the primary pathogen; therefore, their presence does not confirm clostridial enterotoxemia as the cause of the diarrhea. salmonellosis occurs most often in calves two weeks to four months of age that are debilitated, stressed, and under close confinement. in some areas it may be endemic and therefore may be the most common cause of diarrhea in those calves. however, in most areas, calf scours is by far the most common cause of diarrhea in the calf. frequently, the primary etiologic agent is not identified. the two clinical forms of enteritis in the calf are septicemia endotoxemia and diarrheal dehydration. the septicemic-endotoxemic form is most common in calves that have been deprived of colostrum and occurs most commonly in the first few days oflife. this form of the disease has an acute, fatal course. afflicted calves are often found dead without ever having been observed to be sick, and even before diarrhea has occurred. they account for less than per cent of the cases reported, although in some herds, this form of the disease may be more prevalent. the most common clinical manifestation of enteritis is diarrhea which results in extensive losses of fluids and electrolytes. these losses and their effects have been described in detail elsewhere and are quite similar regardless of the causative agent. therefore, the treatment of the calf with diarrhea is also quite uniform regardless of the etiology of the disease. the most important aspect in the treatment of the diarrheic calfis the administration of fluids to replace the extensive losses of water and electrolytes that occur and which are responsible for the clinical signs and potential death of the calf. these fluids should also supply energy, particularly in patients requiring more than one or two days of treatment, since anorexia often accompanies the disease. optimal administration of fluids requires familiarity with several general principles of fluid therapy. in addition, the selection of the route and rate of administration of fluids and the type of fluid used is based upon the acuteness of the illness and the degree of dehydration. the degree of dehydration is best estimated from the clinical signs described in table . the amount of fluid needed as well as the condition of the animal can be determined from the degree of dehydration. for example, if a kg calf is per cent dehydrated, the amount of fluid needed to correct this dehydration is kg times per cent, or liters. although the hematocrit and plasma protein concentrations increase linearly as diarrheal dehydration in the calf progresses, they are poor indicators of the degree of dehydration. both parameters may vary widely in normal calves; therefore changes from an average value may be meaningless in any individual animal. however, changes in hematocrit and plasma protein concentration as well as in body weight are quite useful in determining whether fluid therapy is correcting the dehydration and compensating for the continuing loss of fluid. weighing the patient periodically is one of the simplest and most reliable modes of assessment; it is also the most frequently overlooked method. all fluids, regardless of the route of administration, should always be administered at body temperature. warming cold blood to body temperature prior to massive blood transfusion decreased the incidence of cardiac arrest from per cent to per cent in one study in humans. ringer's lactate solution given intravenously at °c to five dogs in hemorrhagic shock resulted in three deaths. however, when five dogs with a similar blood loss were given the same amount of fluid warmed to body tempera- ture prior to administration, no deaths occurred. cold fluids given intravenously have a direct effect on the sino-atrial node, decreasing heart rate, cardiac output, blood pressure, and coronary artery flow, and causing death from arrhythmia and diminished contractility. cold fluids given by nonintravenous routes are absorbed more slowly than are warm fluids. oral. oral administration of fluids is always the route of choice in the treatment of the diarrheic calf, unless the condition is so acute that intestinal absorption is not rapid enough to keep up with losses of fluid or unless the animal is too severely dehydrated. if the animal is more than per cent dehydrated or if the disease is fulminating, fluids must be given parenterally (table ). in these cases, however, giving fluids by both routes is beneficial and lessens the amount of fluids that must be given parenterally. oral fluid therapy has several advantages over the parenteral administration of fluids: the ability to give large volumes rapidly; a sustained input as fluids are absorbed; a lesser expense since the fluids need not be sterile and may be given by the owner; a less critical composition of the fluid compared with fluids given parenterally; and the relative safety of this route. for best results, fluids administered orally should be formulated to afford the maximal rate of intestinal absorption. this is accomplished by using fluids which contain proteins or amino acids, glucose, sodium, and bicarbonate. these solutes have a synergistic effect on the rate of intestinal absorption of each other and, therefore, of water. for example, the rate of intestinal absorption of sodium is greatly increased if glucose is present; conversely, the rate of intestinal absorption of glucose is more than doubled if sodi) m is present. of course, through salivary, pancreatic, and hepatic secretion, some sodium is always present in the intestine, but the quantity may not always be sufficient for maximal rates of absorption to occur. absorption of water is passive; as solutes are absorbed, it is absorbed. thus, to obtain the maximal rate of water absorption a fluid designed to attain the maximal rate of solute absorption is required. a number of fluids designed for this purpose are available commercially* or can be formulated as shown in table . oral fluids constitute excellent media for microbial growth and are nonsterile; therefore, after use, the remainder should be refrigerated and, if not used within one to two days, it should be discarded. the energy, amino acids, and proteins derived from oral fluids have a dual purpose. they not only enhance intestinal absorption but they also *life guard oral-norden labs, lincoln, nebraska; resorb-beecham-massengil, bristol, tennessee. assist in the nutritional maintenance of the animal. although the energy and protein that most oral fluids provide are certainly of benefit, they are not sufficient to meet the animal's requirements. therefore, additional energy must be provided if more than two days of therapy are required. although the administration of oral fluids may increase the loss of fecal fluid, this does not negate their benefit. their use will either reduce the net loss of water from the body or will result in a net gain of water. this is demonstrated in the example shown in table , in which, prior to receiving oral fluids, the calf had a net water loss and a decrease in body weight of i kg per day. when oral fluids were given, fecal losses doubled, but the calf then had a net water gain and an increase in body weight of i kg per day. weighing the patient eight hours or so after administration of oral fluids will confirm whether or not the fluids given orally or by any other in preruminant calves and lambs, suckled fluids will bypass the rumen and reticulum and go directly into the omasum and abomasum. nursed fluids, therefore, reach the intestine more rapidly than those given by stomach tube and are absorbed.more quickly. thus, the calf should first be encouraged to nurse as much fluid as possible. the remainder of the fluids needed may be given by stomach tube. most of the fluid administered by stomach tube is deposited in the rumen and takes longer to reach the intestine where absorption occurs. in some cases, this may be of benefit in providing a more prolonged therapeutic delivery. the subcutaneous route of fluid therapy is quite useful in animals other than the horse and pig which lack sufficient subcutaneous space for the administration of significant volumes by this route. fluids given subcutaneously are absorbed in four to six hours following administration. they provide a slow but sustained delivery of fluid during this period, which may be adequate iflosses of fluid from the body are not occurring faster than the rate of absorption. in more severe cases, the subcutaneous administration of fluids in conjunction with intravenous fluids may be quite beneficial and may lessen the amount of fluids that must be given intravenously. if the degree of dehydration is greater than per cent, peripheral circulation is decreased to the extent that fluids given subcutaneously will not be absorbed or will be absorbed more slowly. after correcting dehydration, the continued absorption of additional fluids given subcutaneously can help in replacing continuing losses. fluids given subcutaneously should be sterile and warmed to body temperature. they should be from to y times isosmotic. electrolytes should be y to times isosmolality; they may contain from to y per cent glucose and generally contain at least meq per liter of sodium (table ). if their content varies from this formula, they will draw fluids or electrolytes out of the blood and may worsen the condition, at least initially. addition of <?ther ingredients to the fluid depends on the specific condition being treated. up to ml of fluid per kg of body weight may be given subcutaneously with a maximum of to ml per kg given in one area. the rate of administration is not important as long as excessive pressure does not cause discomfort to the patient. fluids given subcutaneously may be . isosmotic electrolytes + grams glucose per liter or ml or per cent glucose per liter; total osmolality, mosm administered by gravity flow or they may be given more rapidly under pressure. a three-way stopcock* and syringe may be used (one stopcock attachment going to the needle inserted subcutaneously, one to the fluid bottle, and one to a syringe which is used to draw the fluid from the bottle and i~ect it into the animal), or a collapsible fluid administration bag that can be squeezed works nicely. intravenous. the intravenous route of fluid therapy is the route of choice when the animal is more than per cent dehydrated (table ) , when the loss of fluids from the body is so rapid that replacement fluids given by other routes may not be absorbed fast enough to compensate for the losses, when shock is present, or when the animal shows clinical signs of altered concentrations of plasma electrolyte or glucose. intravenous administration of fluids has the advantage that, since absorption is not a factor, the rate at which the fluid enters the body water pool can be controlled. because of this, the rate at which fluids are given intravenously must be controlled according to the needs of the individual patient, the purpose of therapy, and the composition of the fluids given. too rapid a rate of administration of any fluid may result in shivering, apprehension, tachycardia, pulmonary edema, or death. the intravenous administration of more than a small quantity of fluid requires catheterization. a number of commercially available catheters may be used. if a jugular intracath (that is, a catheter on the inside of a needle) is used in a calf, it should not be over cm ( in) in length. a cm ( in) catheter may induce ventricular fibrillation and death. a to cm ( . to in) or gauge extracath (catheter on the outside of the needle) works well in a calf and does not kink, which may occur with intracaths. to insert the extracath, clip and disinfect the skin over thejugular vein, pinch the skin up and away from the vein, and make a single stab incision with a no. bard parker blade. this procedure does not require local anesthesia or suturing after removal of the catheter. next, place a tab on the hub of the catheter using two pieces of adhesive tape, approximately one half inch by inches in size. the sticky sides should be together with the catheter hub between them. attach a syringe to the catheter needle and insert the needle and catheter through the skin incision and into the vein, aspirating slightly as you do so. as soon as blood is drawn into the syringe, slide the catheter off the needle and down the vein. tape the catheter to the calfs neck using adhesive tape. encircle the calfs neck about times with the tape which should be firmly stuck to the tab previously attached to the catheter hub. to prevent pulling on the catheter during the administration of fluid, the tubing from the fluid bottle to the catheter should be taped to the calfs forehead with a piece of tape encircling the calfs head just in front of the ears. intraperitoneal. the intraperitoneal route of fluid administration is *becton-dickinson co. , rutherford, new j ersey. not routinely used. it has no advantage over the subcutaneous route because the absorption of fluids occurs at about the same rate by either route of administration . if fluids are poorly absorbed from the subcutaneous area because of the degree of dehydration, they will also be poorly absorbed from the peritoneal cavity. the intraperitoneal administration of fluids may result in peritonitis, adhesions, or damage by the needle to internal organs. if the diarrheic calf is not over per cent dehydrated (table ) and if the losses are not so rapid that they exceed absorptive capacity, the administration of oral fluids alone is often adequate. give enough to rehydrate the calf and to maintain its body weight. this will generally require to liters of the oral fluids previously described two to three times a day. if the calf is over per cent dehydrated or if the fluid losses exceed the intestinal absorptive capacity, as indicated by continued weight loss, increasing hematocrit, or past experience in the herd, fluids must be given parenterally. however, even in these cases the administration of fluids orally is beneficial and lessens the amount of parenteral fluids required. fluids should be given orally to any calf that is not over per cent dehydrated (table ) . if the calf is to per cent dehydrated (table ), in addition to giving fluids orally, give liters subcutaneously in four or five locations under the loose skin on each side of the neck, shoulders, and back. do not give over ml in anyone area. if the state of hydration is greater than per cent, or iflosses occur more rapidly than oral and subcutaneous fluids can be absorbed, fluids must be given intravenously. in this instance a minimum of three liters of fluid is needed initially by the to kg calf. administer liter intravenously over a period of about one hour, that is, at a rate of to ml.per kg per hr. the additional liters may also be given intravenously at a rate no faster than half of this rate, that is, ml per hr per calf. if if is not possible to give all fluids slowly over this prolonged period of time, give the first liter intravenously and the additional fluid needed may be given more rapidly subcutaneously. a second treatment, administered to hours later, if necessary, again may be either by the intravenous or subcutaneous routes or by a combination of both. in summary, at per cent dehydration or less, administer to liters of fluid orally two to three times a day. if the state of dehydration is to per cent, give fluids orally and liters subcutaneously. at greater than per cent dehydration, liters of fluid should be administered. give liter intravenously over a period of one hour; the remaining liters should be given intravenously over a period of not less than hours, or the remaining liters may be given subcutaneously. in acute cases, it may be necessary to administer fluids intravenously, regardless of the degree of dehydration present when therapy is instituted, in order to compensate fo'r diarrheal fluid losses. even though certain fluid and electrolyte compositions may be more effective than others, the administration of nearly any fluid to the diarrheic calf will be beneficial since the most important ingredien t is water. however, to be of maximal benefit, therapy should be aimed at not only correcting dehydration and vascular imbalances, but at correcting whole body imbalances. the plasma potassium concentration of a severely diarrheic calf is nearly doubled. this fact may be very misleading since diarrhea causes extensive loss of potassium from the body, resulting in a total body deficit of potassium and a decrease in the intracellular concentration of potassium. potassium should be given to correct the total body deficit; it must pass into body cells to correct the extracellular increase and intracellular decrease in concentration of potassium. glucose and bicarbonate are both beneficial in this regard. glucose is needed for the following reasons: it enhances the movement of potassium into cells; hypoglycemia is often present in diarrheic calves; and it serves as a source of energy. bicarbonate is needed to treat metabolic acidosis. acidosis, which is both intracellular and extracellular, causes an increase in the efflux of potassium from cells. correcting acidosis reverses this effect, causing movement of hydrogen ions out of and movement of potassium into cells. ringer's lactate solution is often used to treat acidosis; the lactate must be metabolized to be effective, however. in the diarrheic calf, more lactate is present than can be metabolized as indicated by an increase in plasma lactate levels. in addition, half of the lactate in lactated solutions is the d-isomer. only the l-isomer is normally metabolized in the body. the d-isomer is poorly metabolized even by healthy animals. thus, it is better if a lactate solution is not used for the treatment of the diarrheic calf. based on existing data and clinical results, ringer's bicarbonate is recommended instead. however, if ringer's bicarbonate is not available, ringer's lactate, acetate, or gluconate may be used. the following should be added to any of these fluids used: for the calf that is dehydrated per cent or more (table ) , meq of potassium chloride, meq of sodium bicarbonate, and ml of per cent glucose or dextrose solution should be added to the first liter of fluids given intravenously. to any additional fluids given intravenously, to all fluids given subcutaneously, and for the calf that is less than per cent dehydrated, meq of potassium chloride, meq of sodium bicarbonate, and ml of per cent glucose or dextrose solution should be added to each liter. if ringer's bicarbonate, acetate, gluconate, or lactate are not available, mix equal parts of a per cent glucose solution either with isotonic saline or ringer's . solution. then add the same amount of potassium chloride, meq per liter more sodium bicarbonate than that given in either case mentioned above, and omit the glucose. the easiest way to add the potassium chloride and sodium bicarbonate is to inject the correct number of milliliters of a concentrated solution of these substances into the fluid used. ringer's lactate solutions often contain calcium, and therefore should be administered immediately after adding sodium bicarbonate. if this is not done, the calcium will combine with the bicarbonate to form a precipitate. it takes two to three hours for the precipitate to be seen. in the treatment of severe, acute diarrhea, orally administered antimicrobial drugs are often of questionable value. controlled studies have failed to show benefit or harm from the oral administration of a number of different antimicrobial drugs in the treatment of acute undifferentiated diarrhea in the calf. in some bacterial cases of diarrhea, normal intestinal flora have been shown to be instrumental in clearing enteropathogenic organisms from the intestinal tract. therefore, antibacterial suppression of the normal flora may succeed only in prolonging the disease without affecting the signs. it is also difficult to determine to which antibacterial drug the actual enteropathogenic agent is sensitive. if the diarrhea is vir ally induced, as many are, none of the antibacterial agents is effective, but they may be effective against secondary opportunistic bacteria such ase. coli. in vitro determinations of antibacterial sensitivities to fecal swabs may show little or no correlation to the actual sensitivity in the calf. if the calf is still getting milk, tetracyclines will be bound to calcium, decreasing their effectiveness despite the fact that the causative agent may be sensitive to tetracycline. if given in conjunction with intestinal adsorbents, antimicrobial drugs may be bound to the adsorbent with subsequent decreased effectiveness. however, the clinical impressions of many have indicated that oral antibacterial drugs are of benefit in the treatment of acute, severe diarrhea in the calf. if used at all, they should be restricted to acute, severe cases, and they should be given only at the proper dosage for an interval of two to three days. the antibacterials generally thought to be the most clinically beneficial in these cases in our area are the nitrofurans, gentami- cin, chloramphenicol, and polymyxin b (fig. ) . the dosages used are: nitrofurans (enterfur*), two boluses initially, followed by one twice a day per calf; chloramphenicol, mg per kg initially, followed by mg per kg two to three times a day; gentamicin, to mg per kg twice a day; and polymyxin b, to mg per kg twice a day. these drugs were the only ones found by the kirby-bauer technique to effectively inhibit in vitro the majority of e. coli isolated from diarrheic calves over a two year period. it must be remembered, however, that chloramphenicol is not approved for use in food-producing animals. the nitrofurans are currently being carefully scrutinized by the food and drug administration; they are suspected to be potential carcinogens and may be removed from the market in the near future. in addition, because of manufacturing problems, enterfur is not presently available. gentamicin is approved for use . in food-producing animals, but is extremely expensive. regardless of whether antimicrobial drugs are given orally, for severe cases of diarrhea in the calf they are indicated systemically to prevent or treat respiratory disease. pneumonia commonly occurs in conjunction with or secondary to diarrhea in the calf. i n one study imohing field cases of acute, severe diarrhea in which the calves were given extensive fluid therapy intravenously but were given no antibacterial drugs systemic-*norden labs, lincoln , nebraska. ally, died. all deaths occurred after the diarrhea had resolved and when the calves were well hydrated with normal blood electrolytes, glucose, and acid-base values. based upon evaluation at necropsy, the death of each of these calves was a result of pneumonia. systemic antimicrobial drugs may be given parenterally or, in the case of those such as chloramphenicol which are readily absorbed from the intestinal tract, they may be given orally. nitrofurans and polymyxin b are poorly absorbed following oral administration. systemic administration of nitrofuran is not recommended. the best method of treatment for any disease for which prevention has failed is to correct the primary alteration which has caused the disease. for diarrhea, this means the correction of the intestinal dysfunction. currently, knowledge of the intestinal defects responsible for diarrhea is minimal, which makes rational treatment difficult or impossible. in addition, there have been few controlled trials of therapy to correct intestinal dysfunctions responsible for diarrhea in the calf. this includes the treatments given below. intestinal protectants, such as kaolin and pectin products, are of doubtful value in severe diarrhea. they have not been shown to alter losses of fluids and electrolytes. although they may bind toxins and protect the mucosal wall, they have not been shown to be of any definitive benefit or harm. it is not known whether the drugs that alter intestinal motility are beneficial or detrimental. anticholinergic agents decrease intestinal peristalsis, segmentation, and sphincter tone. a decrease in all of these parameters may allow an increase in bacterial migration or multiplication of bacteria in the more cranial aspects of the small intestine. although some of these organisms may not be pathogenic when restricted to the large intestine, in the small intestine they may produce a number of detrimental effects which will cause diarrhea.!' in addition, a decrease in segmentation and sphincter tone allows a more rapid passage of fluid down the intestinal tract, particularly the colon, which could worsen diarrheal fluid losses. for these reasons, anticholinergics are not recommended in the treatment of the diarrheic calf. narcotic analgesics such as paragoric may, in contrast, be beneficial. these drugs decrease peristalsis and stimulate segmentation and sphincter tone which increase resistance to the flow of ingesta down the tract. these effects may decrease diarrheal fluid losses and aboral bacterial migration. however, these beneficial effects from narcotic analgesics have not been documented in the diarrheic calf. anticholinergics decrease intestinal secretion, an action that would be expected to be of benefit in the treatment of diarrhea. howev.er, because of their effect on intestinal motility, their use is not recommended. salicylates, such as are contained in pepto bismol and aspirin, may be beneficial in diarrheal diseases associated withe. coli or clostridium perfringens, whether these organisms are primary or secondary factors. these organisms induce diarrhea by secreting an enterotoxin which stimulates secretion from the intestinal epithelial cells. salicylates inhibit prostaglandin synthesis which decreases enterotoxin-induced intestinal secretion of water and electrolytes. however, the role of prostaglandins, if any, in bacterial or virus-induced diarrhea is not known at this time. in the rabbit, pepto bismol has been shown to bind vibrio cholerae and e. coli enterotoxins which were not previously bound to the intestinal wall, and to decrease intestinal secretion stimulated by these toxins by and per cent, respectively. there is no indication for the use of either corticosteroids or anti histamines in the treatment of diarrhea. the diarrheic calf already has an increased circulating level of endogenously produced corticosteroids. supplementation of this natural level of corticosteroids has not been shown to be of benefit. massive doses of corticosteroids ( mg per kg of dexamethasone) have been shown to be beneficial when given early in the treatment of hemorrhagic shock in the dog. in addition, prolonged glucocorticoid therapy is beneficial in endotoxic shock, which can be a sequela of diarrhea. however, a more beneficial response may be obtained from the diarrheic calf in h ypovolemic shock by correcting the hypovolemia with proper fluid therapy. therefore, corticosteroids, as well as drugs such as antihistamines and stimulants, are of doubtful value and are not recommended in the general treatment of the diarrheic calf. regardless of all other treatments given the diarrheic calf, the most important aspects are fluid therapy, care, and timing. the earlier in the course of the disease that treatment is initiated, the less therapy required and the greater the chance of recovery. treatment is not usually indicated in the lively, nursing calf with a loose, pasty stool unless prior experience in that herd indicates that this may lead to a more severe diarrhea. watery, voluminous feces, however, indicate the need for immediate therapy, even if the calf is not dehydrated, appears alert, and is nursing. the calf generally will not become depressed or decrease its food intake until the state of dehydration is greater than per cent (table ) . do not allow this to occur, since treatment becomes much more extensive with progressive disease. the owner should begin giving oral fluids as soon as diarrhea occurs. however, it should be emphasized that if the rate of absorption of oral fluids does not compensate for the rate ofloss of fluids from diarrhea, fluids must be given either subcutaneously, intravenously, or both. the calf should not be allowed to become severely dehydrated before fluids are given parenterally. in addition to fluid therapy, tender loving care is indicated. make sure that the calf is dry and warm. a heat lamp or electric blanket works nicely. also, do remember to consider the recumbent side of the calf. extensive loss of heat may occur as a result of an uninsulated cold floor or ground. bare concrete is particularly detrimental. plenty of bedding should be placed underneath the calf or, preferably, something such as an electrically warmed pad similar to that used for baby pigs.* following recovery, care must be taken to ensure that the calf does not overeat. if the calf is with the cow or will be returned to her, the cow should be milked out first. if this is not possible, the calf should be given several liters of the oral replacement fluid just prior to returning the calf to the cow. if this is not done, frequently the calf will ingest a large quantity of milk which may result either in a relapse or in sudden death from septicemia or endotoxemia. if the calfis to be fed by hand, dilute the milk by half with water or oral replacement fluid for the first one to two days. many calves are lost as a result of diarrhea during the first few weeks of life. prevention is the most important goal. when prevention fails, proper therapy will save many affected calves. the most important aspects of therapy are the administration of fluids to replace the extensive losses of water and electrolytes which have occurred, and tender loving care. if treatment is begun early, fluids may be given orally. this reduces the need for more intensive therapy that must be administered either subcutaneously or intravenously. although oral antimicrobial drugs are *protein plus labs, box , colfax, illinois; or osborne industry inc. , box , osborne, kansas. often given, there is little evidence to indicate that they are of benefit. however, systemic antimicrobial drugs are recommended in all severe cases of diarrhea to treat or prevent respiratory diseases. corticosteroids, antihistamines, stimulants, and anticholinergic drugs are not recommended. acute undifferentiated neonatal diarrhea of beef calves: the prevalence of enterotoxigenic e . coli, reo-like (rot a) virus, and other enteropathogens in cow-calf herds physiological responses to the administration of cold, room temperature , and warm balanced salt solutions in hemorrhagic shock in dogs role of enteric microorganisms in malabsorption bismuth subsalicylate inhibits activity of crude toxins of escherichia coli and vibrio chlorae dexamethasone treatment during hemorrhagic shock: blood pressure, tissue perfusion, and plasma enzymes pathophysiologic changes due to coronavirus-induced diarrhea in the calf veterinary pharmacology and therapeutics a clinical evaluation of antimicrobial agents and temporary starvation in the treatment of acute undifferentiated diarrhea in newborn calves key: cord- -my frz s authors: foster, derek; jacob, megan; stowe, devorah; smith, geof title: exploratory cohort study to determine if dry cow vaccination with a salmonella newport bacterin can protect dairy calves against oral salmonella challenge date: - - journal: j vet intern med doi: . /jvim. sha: doc_id: cord_uid: my frz s background: salmonellosis is a major cause of morbidity and mortality in neonatal calves, often occurring before preventative vaccines can be administered. hypothesis/objective: to evaluate the protective effect on calves of colostrum from cows vaccinated with a commercially available salmonella newport bacterin against a salmonella typhimurium challenge. animals: twenty holstein bull calves from a university dairy farm. methods: nonrandomized placebo‐controlled trial in which colostrum was harvested from cows that received doses of either salmonella bacterin or saline before calving. colostrum collected from each group was pooled and fed to groups of calves at birth. at approximately weeks of age, calves were challenged with salmonella typhimurium. clinical, hematologic, microbiological, and postmortem findings were compared between the groups. results: no differences in mortality, clinical findings, hematology results, blood and fecal cultures, or necropsy findings between the groups were observed. vaccinated cows had higher colostral titers, and calves fed this colostrum had higher serum titers (mean difference, . ; mean [se], . [ . ] for vaccinated versus . [ . ] for control calves). conclusions and clinical importance: transfer of colostral immunoglobulins from salmonella enterica serotype newport bacterin to neonatal calves was not sufficient to decrease mortality, clinical signs, sepsis, intestinal damage, or fecal shedding when exposed to a highly pathogenic salmonella isolate. a large‐scale randomized controlled clinical trial is needed to evaluate the efficacy of this bacterin when administered in the dry period for prevention of salmonellosis in neonatal calves. diarrhea remains the most common cause of morbidity and mortality in neonatal dairy calves despite efforts to decrease the incidence of diarrhea through colostral management, sanitation, and housing. diarrhea caused by salmonellosis can lead to substantially higher mortality in outbreaks, particularly in calves within the first few weeks of life. preventing these early-life salmonella infections is particularly difficult because no vaccines are labeled for use in young calves. extra-label use of commercially available vaccines has not been effective and is anecdotally associated with clinically relevant adverse effects. colostral immunity stimulated by vaccinating dry cows for escherichia coli, rotavirus, and coronavirus has been used extensively in the dairy and beef industries to protect calves against diarrhea challenge. , previous data also indicate that vaccination of dry cows can decrease fecal shedding of salmonella in young calves. [ ] [ ] [ ] however, the concept of using dry cow vaccination to decrease clinical disease caused by salmonella in calves has not been well examined. administration of a salmonella newport siderophore-receptor and porin (srp) subunit vaccine per label directions to cows during the dry period can substantially increase the salmonella-specific colostral titers. when fed to calves within the first hours of life, this colostrum led to a significant increase in the calves' salmonella newport antibody titers. this finding suggests that dry cow vaccination and subsequent colostrum administration may be a viable method to provide immunity against salmonella in neonatal calves, potentially decreasing the incidence, severity, and mortality of the infection. therefore, our objective was to evaluate the extent of protection from a salmonella typhimurium challenge in calves fed colostrum from cows vaccinated with a salmonella newport srp subunit vaccine. we hypothesized that administration of colostrum from cows vaccinated with a salmonella newport srp subunit vaccine would decrease mortality in calves fed with this colostrum and challenged with salmonella typhimurium. this study was approved by the nc state university institutional animal care and use committee (protocol # - a). thirty dry cows ( control and vaccinated) were given the srp salmonella newport bacterin (zoetis; parsippany, new jersey) or saline sc in the neck at cessation of lactation (dry off) and again - weeks later with . in, gauge needles. all cows were identified by a colored neckband and housed together on a grass lot with access to a total mixed ration. the cows were vaccinated between march and june at the nc state dairy education unit, a -cow dairy. all mature cows (no heifers were included) that were entering their dry period during this time were eligible to be included, but we ensured that no cows from our previous studies of salmonella vaccination , were included in either group. cows were enrolled - at a time into each group. two cows were eliminated and replaced because they calved before administration of the booster. at calving, colostrum from each enrolled cow was collected into gallon bags (perfect udder, dairy tech, inc., greeley, colorado) within hours of calving, labeled, and frozen at − c. a separate aliquot of colostrum from each cow was frozen at − c for determination of the colostral anti-salmonella titer. once colostrum from all cows was collected, the bags were gently thawed, mixed into either the control (ctl) or vaccinated (vax) pool, and realiquoted in gallon bags and refrozen. all holstein bull calves from single (not twin), normal, observed births on the nc state dairy education unit were eligible for inclusion in the study. any calf that met these criteria was enrolled into the study until calves were enrolled into each group. enrollment occurred between january and september of . calves were not randomly allocated to treatment groups before colostrum feeding. calves were fed l of either ctl or vax colostrum within hours of birth. randomization of treatments was achieved by mixing the bags of colostrum from the groups of cows in the freezer, and feeders would choose the next available bag when a calf was born. pooled ctl or vax colostrum was fed to calves and recorded by farm staff so that investigators who performed the inoculation and all assessments were blinded to the treatment. records of which calf was in each group were maintained on the farm of origin, away from the laboratory animal facilities where the inoculation and assessments took place. calves were transported to the bsl- laboratory animal facilities at - days of age, and fed l of a commercial milk replacer twice a day by bucket. each calf was housed in an individual stall in a common room separated by approximately - m to prevent contact among calves. all calves were infected with - colony-forming units (cfu, range: . × to . × ) of salmonella typhimurium between and days of age. the source was a clinical isolate originally obtained from a calf submitted to the wisconsin veterinary diagnostic laboratory. a single colony of the salmonella typhimurium was grown overnight in tryptic soy broth and incubated at c with gentle shaking. the next morning, a volume with the expected concentration of salmonella typhimurium was mixed with l of milk replacer and . g of sodium bicarbonate, . g of magnesium trisilicate, and . g magnesium hydroxide to alkalinize the abomasum. slight differences in inoculum dose occurred based on variability in the overnight growth of the isolate. the inoculum in milk was fed to the calf at the morning feeding on day . the calf then received the remainder of its feeding ( l). the milk replacer was mixed with deionized non-chlorinated water to prevent interference with infection because of chlorination. a ml aliquot of the overnight culture was diluted in sterile phosphate buffered saline -fold and plated in triplicate onto % columbia blood agar plates for confirmation of the starting bacterial concentration. the isolate, inoculum dose, and alkalization of the abomasum were chosen based on consistency of disease induction in a pilot study of normal colostrum-fed calves of a similar age. the primary outcome assessed was mortality in calves after infection. any calf that did not eat for hours or was moribund was euthanized for humane reasons. any calf that died or was euthanized before days after infection was counted toward the mortality in that group. secondary outcomes also were evaluated to determine if consumption of colostrum from vaccinated cows would impact the titers, clinical disease, or shedding of salmonella in calves after experimental infection. these secondary assessments are described below. blood samples were collected from each calf between and hours of age and centrifuged at g for minutes to collect serum to determine salmonella titers. serum and colostrum antibody titers for salmonella newport were determined as previously described by an elisa. personnel performing the elisa assays were not aware of group assignment for any of the animals. salmonella newport srp antigen was coated on elisa plates (immulon- elisa plates, dynatech laboratories, chantilly, virginia) at ng/well in carbonate coating buffer (ph . ), and samples were tested in duplicate. after incubation and washing, conjugate sheep anti-bovine immunoglobulin g (igg ) was added to each well. plates were covered and incubated at c for calves were assessed twice daily by blinded observers at the time of feeding to determine depression score, rectal temperature, fecal score, milk intake, and eyeball recession. all assessments were performed by a single investigator (derek foster) or research technician specifically trained to ensure consistency. the depression score was based on a - system as follows: -normal, no signs of depression; -noticeable depression without apparent signs of weakness, slower than normal calf but still raises head when approached and does not appear weak, actively follows observer's movements with a raised head; -marked depression with moderate signs of weakness without a substantially altered gait, stands with head lowered, will respond when approached but will return to depressed stance, moves slowly, may display signs of weakness such as incoordination; -severe depression with signs of weakness such as a substantially altered gait, obvious weakness, difficulty in moving, raises head only when approached closely; and -moribund, unable to rise. fecal scores were based on a - scale as follows: -normal; -semi-formed, pasty; -loose, but remains on top of bedding; -watery, seeps through bedding; and -watery with blood. blood samples were collected aseptically for a cbc and blood culture each morning, and feces were collected each morning for culture. any calf still alive on day was euthanized for sample collection. samples collected at necropsy for culture included ileal contents, ileal tissue, mesenteric lymph nodes, and bile. ileal tissue also was collected for histology, immunohistochemistry (ihc), and assessment of barrier function in ussing chambers. an appropriately filled ml edta collection tube was used for the cbc. all samples were checked for clots before being run through the advia (siemens healthineers, erlangen, germany). packed cell volume, plasma protein (pp) concentration, fibrinogen concentration, cell white blood cell differential, and slide review were performed by laboratory technical staff. to determine the fibrinogen concentration, microhematocrit tubes were filled and centrifuged (heraeus pico ; thermo fisher scientific, waltham, massachusetts) for minute. two microhematocrit tubes were placed into water bath ( c) for minutes, and all tubes then were centrifuged for additional minutes. the pp concentration from all tubes was measured using a digital refractometer (reichert technologies, depew, new york), and the fibrinogen concentration was calculated by subtracting the heated pp concentration from the unheated pp concentration. the final result was multiplied by . feces or gut contents obtained from calves were evaluated for the presence of salmonella typhimurium. briefly, a swab of fecal or gut contents was used to inoculate xlt- (remel; thermo fisher scientific) and hektoen enteric (he; remel) plates. additionally, g of feces was enriched in ml of tetrathionate broth with μl of iodine. the plates were incubated overnight at c, and the tetrathionate was incubated overnight at c. the next day, plates were observed for the presence of morphologically representative colonies (eg, black), and if observed, isolates were streaked for isolation and confirmed to be salmonella using maldi-tof mass spectrometry (vitek ms; biomerieux, marcy-l'etoile, france). if representative colonies were not observed on pre-enrichment plates, the tetrathionate enrichment was inoculated onto xlt- and he plates, which were incubated overnight at c and evaluated after hours. again, suspect colonies were streaked for isolation and confirmed. blood culture bottles were submitted to the nc state veterinary hospital microbiology & molecular diagnostic laboratory for processing according to standard operating procedures. five to ml of blood, collected directly into bd bactec plus aerobic media bottles (bd, franklin lakes, new jersey) were incubated in the bactec for up to days to assess growth. bacterial growth was assessed by automated detection of production of carbon dioxide associated with bacterial replication. if positive, the blood culture bottle was removed and evaluated by gram stain to confirm the presence of gram-negative rods. positive bottles also were subcultured onto % columbia sheep blood agar (remel), and isolates were confirmed to be salmonella. serial sections of formalin-fixed paraffin-embedded ileum were stained using hematoxylin and eosin. the sections were examined by light microscopy and scored in areas. the histologic scoring was as follows: villus blunting- = normal villus height, = mild-moderate blunting, = severe blunting; villus epithelial loss- = no denuded areas, = < % of villus denuded, = %- % of villus denuded, = %- % of villus denuded, = > % of villus denuded; subepithelial villus architecture- = normal, = some loss of architecture, = substantial loss of architecture. the sum of these scores was used to compare the ctl and vax groups. one investigator (derek foster) evaluated all slides, and was blinded to animal identity during slide evaluation. unstained, formalin-fixed paraffin-embedded ileal sections were stained using a mouse monoclonal anti-salmonella antibody and standard ihc techniques. a serial section was included on each slide in which the primary antibody was omitted and replaced with the blocking serum to assess nonspecific staining. sections were deparaffinized and rehydrated, then treated with a % h o / % meoh block followed by a protein block (#mp- : normal horse serum, vector, burlingame, california). for measurement of transepithelial electrical resistance (ter), ileal tissue was harvested at the time of euthanasia, opened on the antimesenteric border, and placed in oxygenated ringer's solution. the serosal and muscularis layers were stripped from the mucosa, and sheets of ileal mucosa were mounted in a . cm aperture chamber. the tissue was bathed with circulating, warmed, oxygenated ringer's solution containing glucose ( mmol/l serosal) and mannitol ( mmol/l mucosal) in an ussing chamber (physiologic instruments; san diego, california). tissue remained mounted for minutes. solutions were oxygenated and circulated by gas lift ( % o / % co ) and maintained at c by water-jacketed reservoirs. the spontaneous potential difference (pd) was measured using ringer-agar bridges, and the pd was shortcircuited using a voltage clamp that corrected for fluid resistance. if the spontaneous pd was between − . and . mv, tissues were current clamped at ± μa for seconds, and the pd was recorded. transepithelial electrical resistance (ΩÁcm ) was calculated from the spontaneous pd and short-circuit current (isc). automated measurements of pd and isc were taken every seconds over an hour. the mean ter for each tissue starting at minutes was calculated for each calf to account for changes associated with equilibration in the chambers. glucose was added to the mucosal surface at the conclusion of the hour to induce a current and confirm tissue viability at the conclusion of the hour. the study flow is outlined in figure table ). in the ctl group, / calves died or were euthanized before day , and / calves in the vax group died or were euthanized early ( figure and table ). neither the proportion of calves surviving nor the median survival time was significantly different between the groups (table ) . survival was not associated with time to colostral feeding, birth weight, or inoculum dose. the vax colostrum titers were significantly higher than the ctl titers (p = <. ). the ctl mean (se of the mean, sem) was . ( . ) and the mean (sem) of vax titers was . (p = . ; figure and table ). calf titers also were significantly different (p = <. ). the ctl mean (sem) titer was . ( . ) and the vax mean (sem) titer was . (p = . ; figure and table ). the median depression score for both groups was with a iqr of - (p = . ; table depression score of ≥ was . ( , ) days in the ctl group and ( , ) day in the vax group (p = . ; table ). the median (iqr) fecal score was ( , ) for both groups (p = . ), and the median (iqr) number of fecal scores ≥ was . ( . , -ctl; . , . -vax) days for both groups (p = . ; table ). the daily mean (sem) rectal temperature was not different between the groups (ctl = . [ . ] c, vax = . [ . ] c; p = . ; table ). at necropsy, ileal contents were positive for salmonella in / ctl and / vax calves (p = . , no ileal contents could be collected from calf in each group), and ileal tissue was culture positive for / ctl and / vax calves (p = . ; table ). mesenteric lymph nodes were culture positive in / ctl and / vax calves (p = . ; table ). bile was culture positive in / ctl and / vax calves (p = . , no bile could be obtained from vax calf; table ). mean total leukocyte counts were not different on any day of the study between the groups. similarly, no significant differences were found between the groups in mean fibrinogen concentration, segmented neutrophil count, or band neutrophil count on any day of the study. no table ). the mean (sem) ter of ctl calves was . ( . ) ΩÁcm and . ( . ) ΩÁcm (p = . ; table ) in vax calves, suggesting that there was no difference in epithelial barrier function between the groups. f i g u r e colostral titers from cows vaccinated twice during the prepartum period with a salmonella typhimurium subunit bacterin or saline control, and serum titers from calves fed pooled colostrum from these cows. s:p, sample-to-positive ratio. ***p < . and development of long-term carriers. increases in paracellular movement of ions. transepithelial electrical resistance has been used to assess epithelial cell loss and damage from a variety of causes including ischemia, bile injury, and infection. [ ] [ ] [ ] our finding that there was no difference in ter between the ctl and vax calves suggests similar severity of epithelial damage, which is supported by the histologic scoring. it is unclear why no differences were found for any measured outcomes despite increased titers in the vax group. little data exist about what constitutes a protective titer in calves, but potentially the titers in our study were not high enough to be protective. additionally, we did not measure titers at the time of infection, and the reported titers may not truly reflect the status of the calves at weeks of age. in most of the studies discussed above, salmonella challenge occurred between and days of age, and perhaps protection from colostral immunity had waned significantly by the time we challenged the calves in our study (days [ ] [ ] [ ] . the rate of decay for colostral antibodies does appear to vary for different pathogens, and the persistence of colostrum-derived salmonella immunoglobulins from this subunit vaccine is unknown. if rapid immunoglobulin elimination is the reason for the lack of protection, using passive transfer of immunity to prevent salmonellosis in neonatal calves is unlikely to be a successful strategy. we only assessed humoral immunity. however, cell-mediated immunity also is critical to protection from salmonella. although this subunit vaccine can stimulate cell-mediated immunity in mice, it is unknown if it is transferred to calves via colostrum. we chose to use pooled colostrum so that each calf would receive the same concentration of salmonella antibodies and limit potential variability. one disadvantage of this design is that it necessitated the use of frozen colostrum, which would negate any potential benefit from maternally derived t cells. furthermore, the absorption and activity of colostral leukocytes may be decreased when colostrum is from a cow other than the calf's dam, but this situation has not yet been fully studied. some evidence in swine indicates cell-mediated immunity was decreased in piglets immediately cross-fostered to other sows after birth. colostral leukocytes can be absorbed through the intestine of the calf primarily via the follicle-associated epithelium of peyer's patches. the majority of these cells are t-lymphocytes (both cd + and cd +) that once through the intestinal barrier can migrate along the lymphatics and recirculate to different organs including the liver and spleen. , although the exact function of these maternally derived lymphocytes is not clear, increasing evidence suggests that they are involved in the early immune function of the calf. , these cells are likely important in the development of neonatal monocytes, allowing more effective presentation of antigen as well as development and activation of neonatal lymphocytes. , a recent study also suggested that maternal colostral cells had long-term effects on the development of the calf's immune system. calves that received whole colostrum had stronger responses to vaccination to months post-colostrum feeding as compared to calves fed cell-free colostrum. this observation suggests that these maternal cells may play a role in "training" or "priming" the developing immune system of the calf. therefore, it is possible that the feeding of fresh colostrum to calves from cows vaccinated for salmonella newport would have produced better protection in this challenge model. finally, the failure of protection could be a consequence of the overwhelming infection. the isolate used was obtained from a necropsy sample, and was associated with substantial mortality in our study. potentially, some protection may have been seen had a less pathogenic isolate been used. furthermore, the dose of salmonella administered may be higher than that typically encountered in the clinical situation. unfortunately, other isolates and lower inoculation doses without abomasal alkalization did not reliably induce clinical disease in a small pilot study before this trial. the cycles of freezing and thawing of the colostrum could have decreased the igg content of the colostrum that was fed, and decreased any protective effect in the calves. regardless, the colostrum from vaccinated cows did induce a significant increase in serum titers in the vax group. we did not assess transfer of passive immunity in the calves. although every effort was made to optimize transfer of colostral antibodies in both groups, there could have been differences in the total igg transferred, which may have influenced our results. several limitations to this study limit the conclusions that can be drawn. first, the cows were not randomly allocated to vax or ctl groups. this could have inadvertently biased the overall colostral quality in group because this was not assessed. second, calves were not randomly allocated to treatment groups before the study. because treatments were randomly applied to the calves as they were born, this approach should have limited the bias introduced by this error, but we cannot rule out the possibility that this approach inadvertently influenced our findings. measurement of passive transfer of immunity in the calves would have provided some assurance that the lack of randomization did not bias our findings, but this measurement was not done. finally, the small sample size limits the conclusions that can be drawn because the study was underpowered to demonstrate differences in mortality. we erroneously calculated sample size by treating mortality as a continuous instead of a binary variable. because of this error, the likelihood of missing a difference in mortality when a difference truly exists is increased. the lack of appropriate randomization and sample size calculation preclude the application of these findings to clinical practice, but the results still are valuable to inform future investigations. administration of the salmonella newport bacterin to dry cows did not provide protective immunity to calves in our study despite increased titers. additional well-controlled blinded clinical trials on farms with substantial salmonella burdens will be necessary to determine if these titers are protective in clinical outbreaks of salmonellosis. this study was funded in part by zoetis. the authors also thank the epitopix company for running the salmonella newport serology. authors declare no off-label use of antimicrobials. approved by the north carolina state university iacuc. authors declare human ethics approval was not needed for this study. derek foster https://orcid.org/ - - - salmonella in calves efficacy of oral administration of a modified-live salmonella dublin vaccine in calves reduction of rotavirus-, coronavirus-and e. coli-associated calf-diarrheas in a largesize dairy herd by means of dam vaccination with a triple-vaccine passive immunity in calf diarrhea: vaccination with k antigen of enterotoxigenic escherichia coli and rotavirus passive protection of calves against experimental infection with salmonella typhimurium colostral immunity against salmonella infection in calves evaluation of an autogenous salmonella bacterin and a modified live salmonella serotype choleraesuis vaccine on a commercial dairy farm passive immunity stimulated by vaccination of dry cows with a salmonella bacterial extract short communication: characterization of the serologic response induced by vaccination of late-gestation cows with a salmonella dublin vaccine the spv genes on the salmonella dublin virulence plasmid are required for severe enteritis and systemic infection in the natural host effect of oral meloxicam on health and performance of beef steers relative to bulls castrated on arrival at the feedlot salmonella in dairy cattle bovine salmonellosis: experimental production and characterization of the disease in calves, using oral challenge with salmonella typhimurium morphologic and molecular characterization of salmonella typhimurium infection in neonatal calves characterization of intestinal invasion by salmonella typhimurium and salmonella dublin and effect of a mutation in the invh gene a guide to ussing chamber studies of mouse intestine ionic conductances of extracellular shunt pathway in rabbit ileum. influence of shunt on transmural sodium transport and electrical potential differences mechanisms of acid injury in porcine gastroesophageal mucosa prostaglandins i and e have a synergistic role in rescuing epithelial barrier function in porcine ileum proteasome inhibition of pathologic shedding of enterocytes to defend barrier function requires x-linked inhibitor of apoptosis protein and nuclear factor κb prevention of fatal salmonellosis in neonatal calves, using orally administered chicken egg yolk salmonella-specific antibodies maternally derived humoral immunity to bovine viral diarrhea virus (bvdv) a, bvdv b, bvdv , bovine herpesvirus- , parainfluenza- virus bovine respiratory syncytial virus, mannheimia haemolytica and pasteurella multocida in beef calves, antibody decline by half-life studies and effect on response to vaccination the comparison of cell-mediated immunity induced by immunization with porin, viable cells and killed cells of salmonella typhimurium effect of cross-fostering on transfer of maternal immunity to mycoplasma hyopneumoniae to piglets uptake of colostral leukocytes in the intestinal tract of newborn calves colostrum induced phenotypic and trafficking changes in maternal mononuclear cells in a peripheral blood leukocyte model for study of leukocyte transfer to the neonatal calf the influence of colostral leukocytes on the course of an experimental escherichia coli infection and serum antibodies in neonatal calves effect of maternal cells transferred with colostrum on the health of neonate calves transfer of maternal colostral leukocytes promotes development of the neonatal immune system i. effects on monocyte lineage cells transfer of maternal colostral leukocytes promotes development of the neonatal immune system part ii. effects on neonatal lymphocytes effect of feeding whole compared with cell-free colostrum on calf immune status: vaccination response exploratory cohort study to determine if dry cow vaccination with a salmonella newport bacterin can protect dairy calves against oral salmonella challenge key: cord- -pho miid authors: heine, j.; pohlenz, j. f. l.; moon, h. w.; woode, g. n. title: enteric lesions and diarrhea in gnotobiotic calves monoinfected with cryptosporidium species date: - - journal: j infect dis doi: . /infdis/ . . sha: doc_id: cord_uid: pho miid the pathogenicity of cryptosporidium species was studied by inoculation of two gnotobiotic calves with cryptosporidial oocysts that had been decontaminated by treatment with peracetic acid. two control calves were inoculated with similar material from which the oocysts had been removed by filtration. oocyst-inoculated animals shed cryptosporidium in their feces and developed depression, weakness, anorexia, and diarrhea. at necropsy five days after inoculation, endogeneous stages of cryptosporidium were found in association with epithelial cells throughout the small and large intestines of these animals. the parasites were most numerous in the lower small intestine. atrophic villi, disordered and degenerate villous epithelium, and hyperplastic crypt epithelium were associated with infection in the small intestine. control animals remained normal. extraneous agents were not detected in any of the calves. the results indicate that cryptosporidium can destroy intestinal epithelial cells and cause diarrhea in monoinfected gnotobiotic calves. the pathogenicity of cryptosporidium species was studied by inoculation of two gnotobiotic calves with cryptosporidial oocysts that had been decontaminated by treatment with peracetic acid. two control calves were inoculated with similar material from which the oocysts had been removed by filtration. oocyst-inoculated animals shed cryptosporidium in their feces and developed depression, weakness, anorexia, and diarrhea. at necropsy five days after inoculation, endogeneous stages of cryptosporidium were found in association with epithelial cells throughout the small and large intestines of these animals. the parasites were most numerous in the lower small intestine. atrophic villi, disordered and degenerate villous epithelium, and hyperplastic crypt epithelium were associated with infection in the small intestine. control animals remained normal. extraneous agents were not detected in any of the calves. the results indicate that cryptosporidium can destroy intestinal epithelial cells and cause diarrhea in monoinfected gnotobiotic calves. infections with the coccidian parasite cryptosporidium occur worldwide in the alimentary and respiratory tracts of numerous species of vertebrates. the infection is highly prevalent among cattle; it affects up to ; of calves during the first four weeks after birth [ ] [ ] [ ] [ ] [ ] . subclinical infection is common; however, infection is also frequently associated with diarrhea in calves and people [ ] [ ] [ ] [ ] [ ] [ ] . cryptosporidiosis has recently been recognized as a zoonosis [ ] [ ] [ ] . the infection is self-limiting in calves and people with normal immune systems. however, it can become persistent in immunologically compromised individuals, such as patients with the acquired immunodeficiency syndrome [ ] [ ] [ ] . clinically affected calves have atrophy of villi and hyperplasia of crypt epithelium (apparently as a result of the destruction of villous epithelium); those areas of the small intestine that are heavily infected with the parasite become inflamed [ , , ] . some calves also have epithelial damage and inflammation associated with numerous parasites in the large intestine [ ] . similar lesions occur in people with cryptosporidiosis [ ] [ ] [ ] . the diarrhea and intestinal lesions associated with cryptosporidiosis in calves and people are presumed to be caused (at least in some cases) by the parasite. however, in calves cryptosporidium frequently coexists with enteropathogenic viruses, bacteria [ ] , or other protozoa known to be independently capable of causing these signs and lesions. although the development of signs and lesions during intraspecies and interspecies transmission experiments with cryptosporidium, in the absence of other recognized enteropathogens, suggests that the parasite causes these changes [ , ] , the possibility of cotransmission of some unrecognized pathogens in such experiments cannot be excluded. moreover, experimental infections in conventional animals have not served to clarify whether or not the intestinal flora is required for colonization and lesion production by the parasite. on the other hand, if confirmed, the reported occurrence of diarrhea and intestinal lesions in gnotobiotic pigs infected with an inoculum treated in a manner that destroys infectious agents other than cryptosporidium [ ] provides strong evidence that the parasite can act as a primary enteropathogen in the absence of other enteric flora. the data reported here tend to confirm the report that cryptosporidium can cause diarrhea and intestinal lesions in monoinfected gnotobiotic animals. these studies extend the data to another host species and involve a different isolate of cryptosporidium, a different treatment for the destruction of extraneous agents, a comprehensive search for such agents, and the use of control animals exposed to putative, unrecognized viruses that might have survived the treatment intended to destroy these agents. calves. four gnotobiotic calves were delivered by cesarean section at - days of gestation and were maintained as described by matthews et al. [ ] . the calves were each fed liters of reconstituted condensed milk twice daily. the isolators were kept at c- c. inocula. feces from two calves experimentally infected with cryptosporidium of calf origin were suspended in two volumes of . potassium dichromate solution. this suspension was passed through a sieve (mesh size, jim) and stored for - weeks at c. the suspension contained . x cryptosporidial oocysts/ml, as determined by the counting technique reported previously [ ] . in preliminary tests cryptosporidial oocysts (in potassium dichromate suspensions of calf feces) that were treated with . % peracetic acid (vol/vol) and held at c for min retained infectivity for mice. oocysts were removed from peracetic acid by centrifugation ( g for min) and three aseptic washes (by centrifugation) of the sediment in pbs. such peracetic acid-treated oocyst preparations yielded no bacterial or fungal growth when cultured aerobically at c for one week in trypticase soy broth (tsb; bbl microbiology systems, cockeysville, md). in view of these results and the facts that peracetic acid is routinely used as a germicide in gnotobiotic procedures and is effective against a broad spectrum of microbes, this treatment was used for the destruction of putative mi- crobes other than cryptosporidium in the inocula given to gnotobiotic calves. each of two calves (no. and no. ) received the sediment (resuspended in ml of pbs) resulting from peracetic acid treatment of . ml of potassium dichromate-suspended feces (l oocysts per calf). inocula for two control calves (no. and no. ) were prepared from the same potassium dichromate suspension of feces ( ml of suspension per calf). the control inocula were passed twice through filters (pore size, . jim) for the removal of cryptosporidial oocysts before treatment with peracetic acid. the inocula were mixed with the milk fed to the calves at hr of age. aliquots of the oocyst-containing and control inocula were given to baby mice as a test for the presence of infective cryptosporidium and were cultured for aerobic bacteria at c in tsb. none of the inocula produced detectable growth in tsb, and only mice given the oocyst-containing inocula became infected with cryptosporidium. observations. all four calves were observed at -hr intervals for five days after inoculation. rectal temperature, appetite, strength, attitude, and character of feces were recorded at each interval. fecal samples were collected at each interval and examined microscopically for oocysts by the carbol fuchsin technique [ ] . the percentage of fecal dry weight was determined by drying of each sample to a constant weight at c. in addition, a daily fecal sample was cultured for aerobic bacteria at c for hr in tsb. a fecal sample obtained from each calf on day after inoculation was also cultured anaerobically at c for hr on sheep blood agar and in chopped meat broth. direct smears were made from the final fecal sample obtained from each calf. these smears were stained with gram stain and examined microscopically for bacteria. daily fecal samples were examined for bovine viruses (coronavirus, rotavirus, astrovirus, and breda virus by methods reported previously [ ] [ ] [ ] as well as by direct electron microscopy of negatively stained preparations. no viruses or bacteria were detected in any sample. blood samples were taken from each calf immediately before inoculation and , , and hr afterwards. the following values were determined by routine procedures: packed cell volume; erythrocyte count; total and differential leukocyte counts; concentrations of hemoglobin, plasma * sections jim thick were prepared hr after calves no. and no. were inoculated with cryptosporidial oocysts. the numbers under the headings "jejunum" and "ileum" represent centimeters distal to the duodenal site (jejunum) or proximal to the ileocecal valve (ileum). key: -== no demonstrable parasites; + == < parasites/mm of epithelial surface; + + == - parasites/mm; and + + + == > parasites/mm. protein, albumin, fibrinogen, calcium, inorganic phosphorus, urea nitrogen, na", k+, and ci-; and aspartate aminotransferase level. calves were anesthetized with barbiturate hr after inoculation. two ligated segments of intestine were created at each of the following sites: duodenum ( em distal to the pylorus), jejunum ( , , , and em distal to the duodenal site), ileum ( and em proximal to the ileocecal valve), cecum, apex of spiral colon, and descending colon. the ligated segments were instilled intraluminally with fixative ( formalin for one segment and % glutaraldehyde for the other), excised surgically, and placed in fixative. calves were killed after these segments were collected. portions of each formalin-fixed segment were embedded in paraffin, sectioned at urn, and stained with hematoxylin and eosin for the preparation of histological sections. portions of formalin-fixed small intestine were also stained by immersion in % methylene blue. "whole-mount" preparations of mucosa (one or two villi thick and four to villi long) were dissected from the methylene blue-stained segments with use of a dissecting microscope, a razor blade, and straight pins; the dissection was done underwater by a modification of the procedure of macdonald and ferguson [ ] . these preparations were mounted on glass slides in water, with the long axis of villi parallel to the surface of the slides. the lengths of the villi and depths of the crypts in the whole mounts were measured with a dissecting microscope equipped with an ocular micrometer. glutaraldehyde-fixed sections of the small intes-eluded) along segments of epithelium (villous epithelium in the small intestine, crypt and surface epithelium in the large intestine) and for determination of the lengths of the epithelial segments selected. clinical findings. calves no. and , which were inoculated with oocysts of cryptosporidium, had a decreased appetite, lost strength, and became depressed beginning and hr after inoculation, respectively. their feces subsequently became watery with clumps of mucus, and its color changed from brown to yellow-beige. fecal dry weights were initially % and for calves no. and , respectively; these values fell to % at hr and at hr and remained below % in both calves until the experiment was terminated at hr. the body temperature of calf no. increased by c (to . c) at hr; the body temperatures of calf no. and the two calves given the control inoculum remained at . c- . c throughout the experiment. no clinical signs developed in control calves. their feces remained formed, semisolid, and brown throughout the experiment. the initial dry weight of their feces was %, and this value gradually decreased to levels of % and %, respectively, by the end of the experiment. neither infected nor control animals underwent marked changes in any of the cellular or chemical hematologic parameters evaluated. parasitological findings. cryptosporidial cysts were first detected in the feces of calf no. at hr after inoculation and in the feces of calf no. at hr. both calves continued to shed cryptosporidium in their feces until the experiment was terminated. at necropsy, the parasites were associated with villous epithelium in the small intestine as well as with surface and crypt epithelium in the large intestine. the greatest numbers of parasites per unit (length) of epithelium tended to be in the lower small intestine (table ) . cryptosporidium was not detected in any fecal sample or histological section from the control calves. pathological findings. no histological or ultrastructural lesions were found in intestinal sections from control calves. in contrast, the villi throughout the jejunum and ileum of infected calves were significantly shorter than those in the jejunum and ileum of control animals (p < . ; figures and ). crypts from the jejunum and the ileum (i.e., the ileum em proximal to the ileocecal valve) of infected calves were significantly deeper than those from the same sites in controls (p < . ). the epithelium covering the atrophic villi in histological sections from infected calves was basophilic and usually low columnar to cuboidal, with disordered nuclei and an irregular surface (figure ). in contrast, the crypt epithelium from the small intestine of infected calves remained tall and columnar but contained more mitotic figures than that from controls. the villous epithelium and lamina propria in some sections were infiltrated with a variety of mononuclear and polymorphonuclear inflammatory cells. many villi from the infected calves were fused together. this fusion was apparent either as synechiae formed be- figure . transmission electron micrograph of epithelium from an atrophic villus from the ileum of a gnotobiotic calf infected with cryptosporidium (arrows). the epithelial cells are low columnar to cuboidal and have short, sparse microvilli and extensive cytoplasmic vacuolation. portions of two eosinophils can be seen beneath the epithelium; large electron-dense granules are evident at both the left and the right. tween the epithelium of adjacent villi or as villi with an increased width of the lamina propria and with multiple lacteals and central veins. in the large intestine some foci of parasitized epithelium consisted of low columnar to cuboidal cells with nonaligned nuclei and irregular surfaces. the fusion of some atrophic villi in infected calves was confirmed by scanning electron microscopy ( figure ) . transmission electron microscopy of epithelium from atrophic villi revealed parasites in various endogenous stages (figures and ). villous absorptive cells frequently had cytoplasmic vacuolation (figures and ). vacuolation was particularly marked in the ileum (figure ). occasional epithelial cells had electron-dense cytoplasm and nucleus (figure ). these "dark" cells were interpreted to be degenerate and shrunken absorptive or goblet cells. absorptive cell microvilli were irregular in length. they were short and sparse on many cells (figure ); however, those immediately adjacent to cryptosporidium were frequently longer than those further away from the parasite on the same or other absorptive cells ( figure ). occasionally, parasitized epithelial cells were observed, with disrupted apical plasma membranes and cytoplasmic protrusion into the intestinal lumen ( figure ). gnotobiotic calves inoculated with oocysts of cryptosporidium that had been treated with potassium dichromate and peracetic acid became infected with cryptosporidium and developed clinical signs and enteric lesions. in contrast, control gnotobiotic calves remained normal after the inoculation of similar material that had been passed through a filter for the removal of oocysts (but not of any putative viruses). we were unable to cultivate extraneous agents from the inocula or from the feces of any of the calves. we were also unable to demonstrate infectious agents other than cryptosporidium by direct light and electron microscopic examination of feces from the calves or by transmission electron microscopic study of sections of intestine from the animals. these results provide strong evidence that the calves inoculated with oocysts were mono infected with cryptosporidium and that cryptosporidium caused their enteric lesions and diarrhea. the data tend to confirm and extend a previous report that cryp- figure . transmission electron micrograph of villous epithelium from the jejunum of a cryptosporidiuminfected gnotobiotic calf. microvilli are irregular in length, and epithelial cell cytoplasm is finely vacuolated. several merozoites (crescentic, with electron-dense granules) have been released into the intestinal lumen at the center of the photograph. the residual body of the schizont and the parasitophorus envelope remain attached to a shrunken, electron-dense epithelial cell. the epithelial cell on the right has a disrupted apical plasma membrane with no visible microvilli, and cryptosporidium (arrow) is attached to a portion of the cell that is protruding into the lumen. tosporidium is pathogenic iii monoinfected gnotobiotic animals [ ] . both the enteric lesions (characterized by damage to and loss of epithelial cells, villous atrophy and crypt hyperplasia, and infiltration with a mixture of inflammatory cells) and the signs (diarrhea, depression, and anorexia) were similar to those associated with experimental and naturally acquired cryptosporidial infections in conventional animals [ , , ] and people ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . in aggregate, the evidence strongly suggests that cryptosporidium causes enteric disease during naturally acquired in-fection. subclinical infections are common. variations in signs and lesions associated with the infection are probably caused in part by variations in the dose (and possibly the virulence) of the cryptosporidium ingested. calves can shed as many as cryptosporidial oocysts/ml of feces [ ] . we assume that calves sometimes naturally encounter doses of oocysts as large as that used here (l ) because it seems reasonable that calves reared under conditions of poor hygiene could ingest ml of feces. there is also considerble variation in signs and lesions that is dependent on the species, age, and immune status of the host. in our experience, experimentally infected newborn calves usually develop diarrhea [ ] , but experimentally infected mice do not [ ] . presumably, the amount of mucosal damage is less or the compensatory capacity of the undamaged mucosa is greater in newborn mice than in calves. adult mice are cornparatively resistant to the infection, but immunodeficient (nude) newborn mice developed persistent infection and diarrhea, and some die [ ] . calves that recover from experimental cryptosporidial infection are resistant to a second challenge with the organism (author's unpublished data). we suspect that diarrhea occurs in infected animals because extensive damage to epithelial cells and villous atrophy result in malabsorption. the occurrence of hyperplastic crypt epithelium along with damaged villous epithelium and atrophic villi in infected calves represents evidence that the lesions develop as a result of accelerated destruction or loss (rather than decreased production) of epithelial cells. the mechanism(s) by which cryptosporidium destroys or accelerates the loss of epithelial cells is (are) unknown. some evidence suggests that villous atrophy can be induced by cell-mediated immune responses to protozoa and helminths [ , ] . however, the occurrence of villous atrophy in cryptosporidium-infected nude mice [ ] suggests that, although t lymphocytes are required for the development of villous atrophy in some enteric protozoan infections, this is not the case in cryptosporidiosis. perhaps cryptosp oridium damages epithelial cells directly through some toxic, metabolic, or physical effect. cryptosporidial infection in idaho dairy calves kryptosporidioza telat v obdobi mlecne vyzivy die bovine kryptosporidiose. diagnose und therapie der verlauf natiirlicher cryptosporidium-infektionen in vier rinderziichtbetrieben cryptosporidiosis as a probable factor in neonatal diarrhea of calves kryptosporidien-infektionen beim kalb. nachweis, vorkommen und experimentelle ubertragung fecal transmission of calf cryptosporidia between calves and pigs demonstration of cryptosporidia in calf feces: a comparative study bovine cryptosporidiosis: clinical and pathological findings in forty-two scouring neonatal calves cryptosporidiosis in animals and humans cryptosporidiosis in a veterinary student human cryptosporidiosis in immunocompetent and immunodeficient persons cryptosporidiosis: assessment of chemotherapy of males and acquired immune deficiency syndrome (aids) three-step stool examination for cryptosporidiosis in homosexual men with protracted watery diarrhea rubin ceo overwhelming watery diarrhea associated with a cryptosporidium in an immunosuppressed patient acute enterocolitis in a human being infected with the protozoan cryptosporidium intestinal cryptosporidiosis complicated by disseminated cytomegalovirus infection enterocolotis in piglets caused by cryptosporidium sp. purified from calf faeces derivation of gnotobiotic calves by an open cesarian method isolation of small viruses resembling astroviruses and caliciviruses from acute enteritis of calves antigenic relationships among some animal rotaviruses: virus neutralization in vitro and cross-protection in piglets studies with an unclassified virus isolated from diarrheic calves eine einfache nachweismethode fur kryptosporidien im kat hypersensitivity reactions in the small intestine. iii. the effects of allograft rejection and of graft-versus-host disease on epithelial cell kinetics persistent cryptosporidium infection with in congenitally athymic (nude) mice hypersensitivity reactions in the small intestine. i. thymus dependence of experimental 'partial villous atrophy coccidiosis: t-lymphocyte-dependent effects of infection with eimeria nieschulzi in rats key: cord- - mj isyl authors: chanter, n.; hall, g.a.; bland, a.p.; hayle, a.j.; parsons, k.r. title: dysentery in calves caused by an atypical strain of escherichia coli (s - ) date: - - journal: vet microbiol doi: . / - ( ) - sha: doc_id: cord_uid: mj isyl dysentery lasting – days was produced in five -day-old colostrum-fed calves, after inoculation with an atypical strain of escherichia coli s - ; peak excretion of s - occurred during the period of dysentery. two calves were killed when clinical signs were most severe and bacteria were seen attached to the surfaces of enterocytes in the large intestine; microscopic lesions were seen in these areas. the lesions were identical to those previously reported in a natural outbreak of dysentery in calves, from which e. coli s - was isolated, and to those seen in gnotobiotic calves experimentally infected with s - . reinfection of the three surviving calves – days later with s - and primary infection of two calves aged and days did not cause dysentery. four of coliforms isolated from field outbreaks of calf diarrhoea resembled the atypical strain s - . these four isolates and s - did not produce heat-stable enterotoxin, but all produced a toxin cytopathic for vero and hela cells. two of the four isolates were inoculated alone into -day-old gnotobiotic calves deprived of colostrum; neither calf developed dysentery but microscopic lesions identical to those produced by s - were detected in the large intestines of both animals. an atypical strain of escherichia coli (designated $ - ) was isolated from the faeces and intestinal contents of farm calves aged -- days old during an outbreak of dysentery (hall et al., ) and reproduced the disease after experimental infection of gnotobiotic calves . the distinctive characteristics of $ - included an atypical colony morphology on macconkey agar, production of urease and anaerogenicity, but it was identified as e. coli in particular by its abilities to produce acid in macconkey broth at °c and indole at °c. $ - was shown by an immunoperoxidase method to adhere to the mucosae of the large bowel in gnotobiotic and farm calves with dysentery, and microscopic lesions were seen in the colonic and rectal mucosae (hall et al., ) . these were identical to those seen in piglets (moon et al., ) , man (rothbaum et al., ) and rabbits (takeuchi et al., ) infected with e. coli which do not produce the classical enterotoxins and which are not invasive. the objectives of the experiments reported here were firstly to establish the pathogenicity of e. coli s - for conventional calves, secondly, to determine whether e. coli with atypical characteristics were an important cause of calf diarrhoea by examining a culture collection of isolates from field outbreaks of diarrhoea, and thirdly, to investigate by experimental infection of gnotobiotic calves, the association between the atypical characteristics of these isolates and pathogenicity. electron microscopy was used to examine more precisely lesions produced by atypical e. coli and to compare them with those described previously. experimental design five friesian cross calves were infected with e. coli $ - and compared with two uninfected calves for signs of dysentery. two of the five infected calves were killed when clinical signs were maximum to investigate the presence of lesions. immunity and age resistance to infection were investigated by allowing the other three calves to recover and then reinfecting them and comparing the results with two primarily infected calves, one age matched and one days older. the nine calves were left with their dams for h, then moved to a loose box, separate from other calves, and fed on a milk replacer diet. on the fourth day, the calves were moved to rooms in an isolation unit; they were fed sterilised canned milk. infected and control animals were housed in separate rooms. calves were inoculated orally after the morning feed; ml of sterilised canned milk (carnation ltd.) containing . x -- . x ° colony forming units (cfu) of e. coli grown on bovine blood agar at °c for h, was given by syringe. calves -- were infected when days old; calves and were killed on days when clinical signs were judged to be most severe. calves -- were allowed to recover and then reinfected when , and days old, respectively. calves and were infected for the first time at and days old respectively and were controls for the reinfection of calves -- . calves and were housed together until and days of age, respectively. faeces were sampled daily. blood samples were taken at days, at reinfection and daily from calves with dysentery. rectal temperatures were measured twice daily. the strain of atypical e. coli s - ( :k-:h-) was used . rotavirus and coronavirus were detected by enzyme linked immunosorbent assays of faecal samples taken daily and calicivirus by electron microscopic examinations of faeces taken on the first day of illness (bridger and hall, ) . smears of faeces stained with giemsa were examined for cryptosporidial oocysts on the first and last day of illness. e. coli were enumerated in scrapings of mucosa from the ileum, caecum, colon and rectum, and in faeces and intestinal contents as described previously . the mucosae were washed, scraped off with a glass slide and ground to make - suspensions in saline. one gram of the mucosal suspensions, intestinal contents or faeces were serially diluted fold in saline and . ml of appropriate dilutions spread on to macconkey agar plates (mackie and maccartney, } in triplicate before incubation at °c for h. e. coli g- were enumerated according to the dilution of sample and the number of lactose fermenting colonies per dilution. atypical e. coli were initially identified by their characteristic colonies on mac-conkey agar; these had a red centre, a clear outer zone and the surrounding medium was clear. isolates from experimental infections were occasionally tested for anaerogenicity and production of urease. faeces taken from calves aged and days and at onset of diarrhoea or dysentery were examined for salmonellae (jones et al., ) . tissues were fixed, processed and examined for the presence of lesions as described previously (hall et al., ) . paraffin sections of mercuric formol fixed tissue were stained by an indirect immunoperoxidase method with primary antisera to bovine rotavirus prepared in calves (parsons et al., ) or by a peroxidase-antiperoxidase method (sternberger et al., ) with primary rabbit antisera to live e. coli s - produced by the method of sojka ( ) . immunoglobulins were detected in calf serum taken at days of age by the zinc sulphate turbidity test (mcewan et al., ) . serum samples were serially diluted in . -ml volumes of isotonic saline in a u-bottomed microtitre tray. a suspension of e. coli s ° ( . ml equivalent to browns tube no. ) was added to each well and to wells con-taining saline alone. tests were incubated at °c for h followed by h at °c. rabbit antisera to live e. cpli s - was used as a positive control. during a survey of field cases of calf diarrhoea in southern england during the winters of / and / isolates that grew on macconkey agar (mackie and maccartney, ) at °c and fermented lactose, were subinoculated into vials of ferrous metabisulphate pyruvate (fbp) semi-solid medium (oxoid) and were frozen at - °c. stored cultures (total ) were revived by scraping frozen medium from vials onto trypticase glucose extract (tgx) agar (orskov et al., ) , subsequently incubated at °c for h. e. coli strains k and b ( :k ) were used as controls in characterisation tests. tests described by cowan and steel { ) were used. e. coli b was used as a typical control. serotyping of e. coli rabbit antisera to live e. coli s - , produced by the method of sojka ( ) , was used in a slide agglutination test. further typing of somatic antigens was kindly undertaken by dr. b. rowe at the central public health laboratory, colindale, london. the microtitre method of burrows et al. ( ) was used. tests were incubated at °c or °c in the presence or absence of % d-mannose. cytotoxin test e. coli isolates which were to be tested for production of cytotoxin were inoculated into ml of evans medium (evans et al., ) which was incubated at °c for h and shaken at r.p.m. a . -ml aliquot of this culture was subinoculated into a second evans medium and treated in the same way as the first. cultures were then centrifuged at x g for min and the supernatant fractions filtered through an . -pm millipore membrane and the filtrate tested for the presence of toxin. aliquots of pl of l s chinese hamster ovary (cho) or hela cells m - or × l s vero cells my were placed into all wells of a flat-bottomed microtitre tray (intermed, nunc); pl of filtrate was placed in a duplicate set of wells and serially diluted. microtitre trays were sealed and incubated in a co cabinet at °c for days. cells were stained with a mixture of equal volumes of . % (w/v) crystal violet and % (v/v) neutral buffered formalin for h, washed, dried at °c and examined. toxin was prepared in evans medium and tested in the suckling mouse test (guinee et al., ) . bacteria were washed from an overnight culture on either % sheep blood agar or tgx medium with phosphate buffered saline (ph . ) and dropped onto formvar/carbon-coated grids. after s, excess fluid was removed with filter paper. the grids were rinsed in distilled water and then stained with % (w/v) phosphotungstate (ph . ), excess stain removed, air-dried and examined in a philips electron microscope. experimental infection of gnotobiotic calves with atypical e. coli isolates / or / bacteria two out of four atypical isolates from the culture collection were selected for experimental infections. e. coli / had been isolated from a calf with diarrhoea from which no other enteropathogen was detected and e. coli / had been isolated from a calf which did not have diarrhoea. gnotobiotic calves were derived by the methods of dennis et al. ( ) . one was infected with e. coli / and another with e. coli / . infection of calves, pathology and immunoperoxidase staining methods these methods were as described for the experimental infection of conventional calves with e. coli s - . the occurrence of dysentery in calves and the excretion of enteropathogens are summarised in table i . all five calves ( -- ) infected with e. coli s - , at days of age, developed dysentery, whereas older calves ( -- ) infected for the first time or reinfected ( -- ), and control calves ( -- ) did not. the dysentery was characterised by the presence in faeces of bright, fresh blood (sometimes copious) and large amounts of clear or faeces-stained mucus. the faecal consistency was soft or semi-liquid. peak excretion of s - (> cfu g-i of faeces) occurred during signs of dysentery in all calves ( -- ) infected at days of age. in contrast only ( and ) of the older calves { -- , and ) excreted these numbers; this occurred on the first day after reinfection. rotavirus or coronavirus were excreted by calves -- , but not at times infection occurrence of excretion excretion excretion of dysentery of s - when dysentery was seen. calves and , infected at days of age and necropsied at the peak of dysentery ( days), were excreting rotavirus whereas control calves ( -- ) excreted rotavirus at times when mild diarrhoea was seen. salmonellae, cryptosporidia and calicivirus were not detected. pyrexia or bacteraemia did not occur consistently during the period of dysentery. prior to infection, the serum of calves contained widely different amounts of immunoglobulin ( -- zinc sulphate turbidity units). prior to infection the amount of serum immunoglobulins of calf was the lowest recorded. calf alone responded to infection by the production of agglutinins to e. coli s - (titre of ), and this calf had the longest period of dysentery and the longest period of excretion of $ - . in calves and , necropsied at the peak of dysentery, e. coli $ - colonised the colonic and rectal mucosae in approximately equal or greater numbers than in the contents. in contrast e. coli of other types colonised the mucosae in fewer numbers than in the contents (table ii) . the mean number of s - in these mucosae was log . g- whereas for other e. coli it was log~ . g- . the same distribution of e. coli was seen in the caecum of calf . e. coli of all types colonised the mucosae of the small intestine in lower numbers than in the contents. petechial haemorrhages were seen in the rectal mucosae of calves and ; all of the rectal mucosa of calf and the longitudinal folds of calf were markedly reddened. the same changes were seen in the colon of calf and were most noticeable over the gut-associated lymphoid tissue of the anterior colon and along the tops of longitudinal folds. in the caecal mucosa of calf there were areas of reddening -- cm in diameter. in the caecum, colon and rectum of calves and foci of bacteria were seen adherent to clumps of irregularly arranged and exfoliated enterocytes and there were neutrophils in the lamina propria. in the caecum, neutrophils were in foci in the lamina propria; in the colon and rectum, neutrophils were more numerous and were also present in foci in an exudate on the mucosal surface with mucus and exfoliated enterocytes. lesions were most severe in the colon. immunoperoxidase staining of sections of tissue with antiserum to e. coli s - revealed microcolonies of bacteria adherent to the colonic and rectal rugae of calves and . all adherent bacteria that were seen were immunostained. rotavirus was detected in the ileal enterocytes by immunoperoxidase staining and in the small intestine there were lesions of a viral enteropathy (stunted and fused villi covered by cuboidal enterocytes). of isolates, including from calves with diarrhoea, four had an atypical colony morphology on macconkey agar indistinguishable from that of e. coli s - , were anaerogenic and produced urease. on the basis of other tests these isolates were identified as e. coli (table iii) . the four isolates originated from different farms and three were isolated from calves with diarrhoea. coronavirus was isolated from one of these calves and sa/monella typhimurium from another; the third isolate from a normal calf was the results of further characterisation of the four atypical isolates and s - are in table iv . preliminary serotyping of e. coli / and / revealed that they produced the same somatic antigen ( ) as s - . in addition to the four atypical isolates, another isolate, which was aerogenic but otherwise indistinguishable from e. coli s - , produced a toxin active on hela and veto cells but not cho cells. fifty-five isolates were detected with either or of the atypical characteristics of e. coli s - ; of these were tested for the production of cytotoxins. they comprised four isolates which produced urease, five anaerogenic isolates, five which agglutinated in antiserum to e. coli s - , one with an atypical colony morphology and one with an atypical colony morphology which agglutinated in antiserum to e. coli $ - . one isolate produced a cytotoxin active on veto cells; it also produced urease. in a preliminary investigation, two gnotobiotic calves were infected with either e. coli / or / ; they did not develop dysentery, although the calf infected with e. coli / produced mucoid liquid faeces from to days after infection, when it was killed. e. coli / was isolated from a normal calf and the faeces of the gnotobiotic calf infected with / were unchanged until days after infection, when the animal was killed. this absorption of antibody to s - a fimbriae (f) or haemagglutinins (h) produced on suggested that isolate / was avirulent. calves excreted between . x and . x l° e. coli g- of faeces until they were killed. three days after infection, faeces of the gnotobiotic calf infected with e. coli / inoculated onto macconkey agar revealed e. coli with two different colony morphologies in approximately equal numbers. one was atypical and of the kind produced by e. coli $ - , and the other was typical of most isolates of e. coli. e. coli of both types, however, were anaerogenic, produced urease and agglutinated in antiserum to e. coli s - . at necropsy of the two gnotobiotic calves there were more e. coli in the caecal, colonic and rectal contents (~ . x cfu g-l) than in the respective mucosae ( . x -- . x cfu g-l) which indicated that colonisation of the large intestinal mucosae was less than that seen with e. coli s - infections of gnotobiotic calves . in the mucosae of the colon and rectum of both calves there was marked congestion and occasional small haemorrhages. neutrophils were numerous in the lamina and they were present as foci on the mucosal surface forming an exudate, together with mucous and exfoliated enterocytes. these lesions were apparently as severe as those seen with e. coli s - infections of gnotobiotic calves {hall et al., ) . scanning electron microscopy revealed many short, rod-shaped bacteria attached to the mucosal surface of the large intestines of both calves. enterocytes to which bacteria were attached, and adjacent enterocytes, lacked microvilli or microvilli were abnormally orientated or were shortened or lengthened. changes to microvilli were confirmed by transmission electron microscopy which also showed that the bacteria were closely associated with the enterocyte cell membrane. at the point of attachment, enterocyte cytoplasm was often cup-shaped or arranged as a pedestal. immunoperoxidase staining revealed microcolonies of e. coli adherent to the colonic and rectal rugae of both calves. e. coli s - was pathogenic for conventional calves; all animals infected at days of age developed dysentery similar to that previously seen in gnotobiotic and farm calves hall et al., ) . the calves excreted other enteropathogens, but in two these were not associated with dysentery, and in a third they were not excreted during the major episode of dysentery. in two calves, slaughtered at the estimated peak of clinical signs, rotavirus was detected in the ileum and was not associated with lesions that could have been a source of fresh blood in faeces. lesions in the large bowel, which appeared to be the source of blood in faeces, were consistently associated with adherent e. coli and were identical in nature to those produced in gnotobiotic calves and those seen previously in naturallyaffected farm calves (hall et al., ) . primary infection of two older calves did not reproduce dysentery, which suggested that the calves had acquired resistance to the effects of infection. in this respect, resistance might be similar to that acquired with age by calves to infection with k ÷ e. coli (smith et al., ) . detection of four atypical e. coli out of isolates collected from natural outbreaks of calf diarrhoea suggested that they were not an important cause of enteric disease in calves in that survey. one isolate with these properties has previously been associated with diarrhoea in a calf in france (de ryke et al., ) . two isolates of atypical e. coli ( / and / ) caused microscopic lesions indistinguishable from those produced by e. coli s - (hall et al., ) . however, one ( / ) did not cause disease and the other ( / ) caused mucoid diarrhoea. failure of these strains to cause dysentery may have been related to the extent of bacterial colonisation and the lesions caused. there were approximately -fold fewer e. coli / or / in the mucosae of the large intestine than were previously reported for e. coli $ - in gnotobiotic calves . the extent of microscopic lesions in the gnotobiotic calves was difficult to assess, particularly because of their focal nature, which was apparently similar to that seen in calves infected with e. coli s - . consequently, the virulence of these e. coli may vary not only with the ability to colonise but also the extent of the lesions they cause or, alternatively, it may differ with the production of an unknown pathogenic determinant. in man infected with different strains of verotoxic e. coli, which cause the same type of lesion, signs of disease vary from mild diarrhoea to dysentery (anon, ). an alternative explanation for the failure of e. coli / and / to cause dysentery may have been a variation of animals in their susceptibility. moon et al. ( ) reported considerable animal to animal variation in the response of pigs to infection with e. coli enteropathogenic for humans; identical lesions were seen in the colons of pigs with or without diarrhoea. historically, the term enteropathogenic e. coli or epec was first applied to strains of e. coli implicated in epidemic infantile diarrhoea of humans. some of these strains and others virulent for a number of species have been shown to cause highly characteristic microscopic lesions of the gut mucosa and to produce a cytotoxin detectable in vitro and have been called epec (anon, ) . the microscopic lesions and production of cytotoxin may be unifying features of an emerging sub-group of enteropathogenic e. coli for which the term epec is not sufficiently descriptive. takeuchi et al. ( ) suggested that these lesions in rabbits were induced by a cytotoxin which showed similarities to the shiga toxin produced by shigella dysenteriae (o'brien et al., ) . e. coli enteropathogenic for man have been shown to produce a toxin (konowulchuk et al., ) which is biologically and serologically related to the shiga toxin (o'brien et al., ) . the e. coli described here may form a distinctive sub-group in view of their isolation from cattle, their atypical characteristics and their distinctive properties in tests for fimbriae and haemagglutinins. these e. coli produced a mannose-resis-tant haemagglutinin in anaerobic but not aerobic conditions which was of particular interest; apparently the haemagglutinin was not associated with fimbriae. in aerobic conditions these bacteria produced fimbriae without detectable haemagglutinating activity which, although previously described (isaacson, ) , are nonetheless unusual. the unusual colony morphology produced on macconkey agar might usefully serve as a primary selective characteristic for potentially pathogenic e. coli of the calf. however, strain / produced in vivo a variant with a colony morphology indistinguishable from that produced by most e. coli. furthermore, if in vitro the common characteristic of the enteropathogenic e. coli is cytotoxigenicity then it is probable that e. coli which can cause enteric disease in the calf may be found with otherwise typical properties. mechanisms in enteropathogenic escherichia coli diarrhoea effects of a calicivirus-like agent (the newbury agent) on gnotobiotic calves haemagglutinating and adhesive properties associated with k antigen of bovine strains of escherichia coli dysentery in gnotobiotic calves caused by atypical escherichia coli identification of medical bacteria evidence ofescherichia coli with bacteraemic properties in mucoid enteritis of newborn calves a simplified apparatus for the microbiological isolation of calves identification of enterotoxigenic escherichia coli and serum antitoxic activity by the vascular permeability factor assay escherichia coli associated with neonatal diarrhoea in piglets and calves dysentery caused by escherichia coli (s - ) in calves: natural and experimental disease pili of enterotoxigenic escherichia coli salmonella saint-paul infection in two dairy herds properties of an escherichia coli cytotoxin handbook of practical bacteriology, edinburgh a turbidity test for the estimation of immune globulin levels in neonatal calf serum attaching and effacing activities of rabbit and human enteropathogenic escherichia coli in pig and rabbit intestines production of shigella dysenteriae type l-like cytotoxin by escherichia coli the establishment of k , a thermolabile, transmissible escherichia coli k antigen, previously called 'kco', possessed by calf and lamb enteropathogenic strains localisation of enteropathogens in paraffin embedded tissue by immunoperoxidase evaluation of elisa and electron microscopy for the detection of coronavirus and rotavirus in bovine faeces a clinico-pathologic study of enterocyte-adherent escherichia coli: a cause of protracted diarrhoea in infants observations by the ligated intestinal segment and oral inoculation methods on escherichia coli infections in pigs, calves, lambs and rabbits the unlabelled antibody enzyme method of immunohistochemistry : preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-anti-horseradish peroxidase) and its use in the identification of spirochaetes scanning and transmission electron microscopic study of escherichia coli ois (rdec- ) enteric infections in rabbits key: cord- -aqhyt jg authors: robertson, lucy j.; björkman, camilla; axén, charlotte; fayer, ronald title: cryptosporidiosis in farmed animals date: - - journal: cryptosporidium: parasite and disease doi: . / - - - - _ sha: doc_id: cord_uid: aqhyt jg cryptosporidiosis was first identified as a disease of veterinary, rather than human medical, importance, and infection of farmed animals with different species of cryptosporidium continues to be of veterinary clinical concern. this chapter provides insights into cryptosporidium infection in a range of farmed animals – cattle, sheep, goats, pigs, cervids, camelids, rabbits, water buffalo and poultry – presenting not only an updated overview of the infection in these animals, but also information on clinical disease, infection dynamics and zoonotic potential. although extensive data have been accrued on, for example, cryptosporidium parvum infection in calves, and calf cryptosporidiosis continues to be a major veterinary concern especially in temperate regions, there remains a paucity of data for other farmed animals, despite cryptosporidium infection causing significant clinical disease and also, for some species, with the potential for transmission of infection to people, either directly or indirectly. significant clinical disease and also, for some species, with the potential for transmission of infection to people, either directly or indirectly. farmed animals: overview farmed animals, also commonly referred to as livestock or domesticated animals, are those animals that are reared in an agricultural setting in order to produce various commodities -usually food (meat, organs, eggs, dairy products), and/or hair or wool. in some settings farmed animals are also used to supply labour, and the manure of domesticated animals is often used as fertilizer. animals were probably first farmed, that is their breeding and living conditions controlled by their human owners, around - bc during the first transitions from huntergatherer lifestyles to more settled agricultural living. the physiologies, behaviours, lifecycles of farmed animals generally differ quite substantially from those characteristics of the equivalent wild animals, and this difference impacts the interactions of these farmed animals with their parasites. farmed animals are exposed to different stresses than wild animals, are kept at different densities, and their lifecycles regulated to such an extent that a parasite-host interaction in a farmed animal may differ significantly from that in a wild animal. additionally, for infections that are of significant clinical importance, farmers may implement control measures (including treatment or prophylaxis) that alter the infection dynamics. with respect to cryptosporidium infection, for which a satisfactory chemotherapeutic cure or prophylaxis is not yet available, different species infect different species of farmed animal, and may or may not be of clinical relevance. table . provides an overview of the farmed animals included in this chapter, the species of cryptosporidium to which they are susceptible and brief notes on the clinical relevance. greater details are provided in the appropriate chapter sections. various categories of animals that are 'farmed', including mink, foxes, guinea pigs etc. are not included, largely because of a lack of information on cryptosporidium in these animals in the domesticated setting. additionally, farmed fish are not included in this chapter. c. parvum a common in pre-weaned calves -acute onset diarrhoea. intestinal location c. bovis common in post-weaned calves -less pathogenic than c. parvum c. andersoni older post-weaned calves, yearlings and adults-some failure to thrive. infects the gastric glands of the abomasum c. ryanae common in post-weaned calves a range of other species has been reported from cattle and other bovines. these seem to be unusual and are apparently of minor clinical significance small ruminants, including sheep (ovis aries) and goats (capra aegagrus hircus) c. parvum a relatively common in pre-weaned lambs, associated with diarrhoea c. xiaoi common in older lambs and sheep, often apparently asymptomatic c. ubiquitum a common in older lambs and sheep, often apparently asymptomatic pigs (sus scrofa domesticus) c. parvum a less common than in bovines and small ruminants; diarrhoea and vomiting c. suis relatively common, mild symptoms c. scrofarum relatively common, mild symptoms deer (cervids), including red deer (cervus elaphus) fallow deer (dama dama), elk/wapiti (cervus canadensis), white-tailed deer (odocoileus virginianus), and reindeer (rangifer tarandus) c. parvum a information on species detected amongst farmed deer is lacking; diarrhoea in young calves, possibly severe, but can also be asymptomatic c. meleagridis a appears to have a wide host range, including farmed poultry (and mammals). mostly infects the intestines and has been associated with generally mild clinical symptoms c. baileyi a a wide avian host range reported, including various farmed poultry species. detected in many different anatomical sites including digestive tract, respiratory tract, and urinary tract. has been associated with high morbidity and mortality (continued) of various cryptosporidium species (see table . ) means that public health may also be affected by infections in farmed animals. infection may be direct, from animal to human, or indirect, via a transmission vehicle. a large number of small outbreaks associated with c. parvum in calves and in veterinarians or veterinary students that have been exposed to calf faeces are documented in the literature (e.g. grinberg et al. ; gait et al. ; robertson et al. ) . in addition, a number of outbreaks have been documented associated with children visiting 'petting farms' or similar venues, where interaction with young animals such as lambs or calves is encouraged. less commonly, transmission between animals such as camels or alpacas and their carers has also been reported. drinking water, and less often food, has been associated with transmission of cryptosporidium infection from animals to human populations, with c. parvum from grazing cattle contaminating water supplies particularly implicated. the high densities of farmed animals in water catchment areas mean that implementation of catchment control measures, including preventing defecation into water courses, may have a significant effect on minimising the risk from this potential transmission pathway. there are various species of farmed bovines, with cattle and zebu (bos taurus and bos indicus, respectively) amongst the most important livestock worldwide. both provide meat, milk and other dairy products, and are also used as draught animals, with an estimated . billion head globally ( cattle for every people). a comprehensive overview of cryptosporidium infection in cattle has been published by and the information presented here is largely an update built on this solid basis. domesticated water buffalo (bubalus bubalis) is also important domestic livestock in the bovinae sub-family. domesticated buffalo consist of swamp buffalo and river buffalo. in , the world population of buffalo was estimated to approximately million animals, of which % were in asia (faostat ). the first report on bovine cryptosporidiosis was published in , when parasites were identified in an -month-old heifer with chronic diarrhoea (panciera et al. ) . since then, cryptosporidium infection in cattle has been documented in most countries worldwide. four major cryptosporidium species infect cattle: c. parvum, c. bovis, c. ryanae and c. andersoni (table . ; fayer et al. ; feng et al. ; santín et al. ; langkjaer et al. ) . cryptosporidium parvum has a broad host range and apparently has the ability to infect most mammals, including humans and cattle. in contrast, the other three species have almost exclusively been found in cattle. in addition to these four common species, sporadic natural infections with c. felis, c. hominis, c. scrofarum, c. serpentis, c. suis and c. suis-like genotype have been detected in cattle (bornay-llinares et al. ; geurden et al. ; langkjaer et al. ; santín et al. ; smith et al. ; chen and huang ) . the extent to which these findings reflect true infections or accidental carriage, i.e. ingested oocysts that pass intact through the gastrointestinal tract, remains to be clarified. cattle have also been experimentally infected with c. canis, but natural infection has not been reported ). many reports on cryptosporidium in cattle in different countries and settings have been published over the years, showing that cryptosporidium spp. infections are common worldwide. both dairy and beef cattle are infected and the prevalence estimates vary considerably among studies. reported herd level prevalences range from to % (olson et al. ; chang'a et al. ; maddox-hyttel et al. ; santín et al. ). infected animals have been reported from all age groups but infection is most common in preweaned calves. when calves up to months of age have been investigated in point prevalence surveys, - % of the calves shed oocysts (table . ; maddox-hyttel et al. ; santín et al. ; uga et al. ) . longitudinal studies performed in infected dairy herds showed that all calves in such herds shed oocysts at some time during their first months of life (o'handley et al. ; . the overall picture is that there is an age-related pattern in the species distribution. c. parvum is mostly found in preweaned, monogastric calves up to months of age where it is often the most prevalent species, responsible for more than % of cryptosporidium infections (brook et al. ; fayer et al. ; plutzer and karanis ; santín et al. trotz-williams et al. ) . in some areas, however, c. bovis is the dominating species found in preweaned calves (budu-amoako et al. a; wang et al. a; silverlås et al. b ). the prevalence of c. parvum is considerably lower in older calves and young stock, and there are few reports of c. parvum infection in adult cows langkjaer et al. ; silverlås et al. b; castro-hermida et al. a; khan et al. ; ondrácková et al. ; muhid et al. ; budu-amoako et al. a, b in older calves and young stock, c. bovis and c. ryanae are the most commonly found species fayer et al. ; muhid et al. ; langkjaer et al. ; silverlås et al. b) . c. andersoni is mainly found in young stock and adult cattle (enemark et al. ; wade et al. ; fayer et al. ; ralston et al. ) . older studies, based on microscopy alone, overestimated the c. parvum prevalence in weaned animals because the similarity in oocyst size makes it impossible to differentiate between c. parvum (~ .  . μm), c. bovis (~ .  . μm) and c. ryanae (~ .  . μm). molecular studies have revealed different genetic subtypes within the c. parvum and c. hominis species and dna sequence analysis of the kda glycoprotein gene is commonly used to further characterize the isolates. a number of c. parvum gp subtype families, designated iia-iio, have been described. of these, iic and iie are considered anthroponotic, whereas iia and iid are commonly found in both humans and animals. the other subtype families are uncommon and their zoonotic potential has not been determined. in cattle, c. parvum of the iia subtype family is especially common. in addition to iia, iid and occasionally iil (sometimes named iij) subtypes are found (table . ; xiao ). whether there is a difference in pathogenicity between subtypes is currently unknown, as most genotyping studies to date have focused on herds with a history of calf diarrhoea. it has been suggested that herd management strategies affect subtype distribution. studies from areas with closed herd management (limited animal movements between herds) have shown a high number of subtypes in the calf population, but only one subtype in each herd (brook et al. ; mišic and abe ; soba and logar ; ). it has also been shown that a unique gp subtype can persist over time in a closed dairy herd (björkman and mattsson ) . in contrast, only a few subtypes have been identified in areas with more animal movements between herds, but several subtypes could be present in a herd (brook et al. ; peng et al. ; trotz-williams et al. ). the information on the distribution of cryptosporidium infection in water buffalo is rather fragmentary. reported prevalences vary between % and % (table . ). an association between prevalence of infection and age of the animals, with the highest prevalence in young calves, has been found (helmy et al. ; maurya et al. ; nasir et al. ; bhat et al. ; díaz de ramírez et al. ). the first report on cryptosporidium species identification in water buffalo was published in , in which gómez-couso et al. ( ) used molecular tools to characterize cryptosporidium oocysts from an asymptomatic neonatal calf in a dairy buffalo farm in spain. sequence analysis of a fragment of the oocyst wall protein (cowp) gene revealed that the isolate was closely related to the cryptosporidium 'pig' genotype. a few years later, c. parvum was identified in water buffalo calves from italy (cacciò et al. ) and today c. ryanae, c. bovis and (table . ). most molecular investigations have been done in calves and thus it is not known if the species distribution differs between animals of different age. one of the few studies that include faecal samples from both calves and older animals was done in egyptian smallholder herds (helmy et al. ) . c. parvum was most common in calves younger than months but was detected in animals up to years of age. in another study, also from egypt but from another part of the country, c. parvum was only found in calves, whereas none of the sampled cows shed any cryptosporidium oocysts (amer et al. ) . when the c. parvum isolates were subtyped by sequence analysis of the gp gene, subtype families iid and iia were found, with a majority of iid in both studies. subtype iid is the dominating subtype family also in cattle in egypt (amer et al. ; helmy et al. ). cryptosporidium parvum and c. andersoni are the two species that have been associated with clinical disease in cattle. c. parvum infection is considered a major cause of diarrhoea in young calves (blanchard ; radostits et al. ). calves are often already infected during the first week of life (uga et al. ) and clinical cryptosporidiosis is mostly seen in calves up to weeks of age. the most prominent finding is pasty to watery diarrhoea, sometimes accompanied by lethargy, inappetence, fever, dehydration and/or poor condition. the calf most often recovers spontaneously within - weeks, but there is a large variation between individuals in how they respond to, and recover from, infection. in some cases the infection may be fatal (tzipori et al. ; fayer et al. ) . a decrease in growth rate may be seen in the weeks after the calves have recovered from the acute phase of the disease (klein et al. ), but no long-term effects on growth and performance have been reported. the pathogenesis of bovine cryptosporidiosis is not fully understood but the clinical signs are attributed to both malabsorption and an increase in fluid secretion in the ileum and proximal portions of the large intestine. for references and a brief overview see o'handley and olson ( ) . cryptosporidiosis may be seen in individual calves, but frequently it soon develops among the calves into a herd problem. concomitant infection with other pathogens, e.g. rotavirus, coronavirus and enteropathogenic escherichia coli (e. coli f +) can worsen the clinical signs and prolong the duration of illness (blanchard ) . a number of studies have reported an association between c. parvum infection and diarrhoea in young calves. many of these were published at the time when all cryptosporidium oocysts of around - μm in diameter were considered to be c. parvum. more recent investigations, applying molecular methods to analyse faecal samples from diarrhoeic calves, corroborate these earlier findings. when samples from young calves with diarrhoea were analysed, c. parvum is found to be the dominant species (quílez et al. b; imre et al. ; karanis et al. ; soba and logar ; plutzer and karanis ) . interestingly, this dominance of c. parvum in diarrheic calves was also seen in a recent swedish investigation of diarrheic calves ), although c. bovis is the predominant species in randomly selected calves in sweden. only one experimental trial has been performed with c. bovis (fayer et al. ). three calves under - weeks of age were orally inoculated with oocysts. this resulted in subclinical infection in of calves. both animals had, however, previously been infected with c. parvum and cross-protective immunity could not be excluded. calves with diarrhoea are significantly more likely to be infected with c. parvum than with c. bovis starkey et al. ; kváč et al. ) . based on these findings, and based on the fact that c. bovis is not common in calves, but is a widespread subclinical infection in older animals in most countries, c. bovis is commonly considered to be apathogenic to cattle. however, the pathogenic potential deserves further attention as high numbers of c. bovis oocysts in samples from diarrhoeic calves have been reported, even in the absence of c. parvum or other diarrhoeal agents (silverlås et al. a (silverlås et al. , b, . cryptosporidium ryanae was first described as a separate species by fayer et al. ( ) , and until then it was known as cryptosporidium deer-like genotype. an experimental trial was performed in two colostrum-deprived calves - days old. both calves started excreting oocysts days after inoculation, but neither of them showed any clinical signs ). there are several reports of the distribution of cryptosporidium deer-like genotype and c. ryanae. most studies found a predominance of the parasite in older calves and young stock. so far no association with clinical disease has been reported. in contrast to the other species, c. andersoni infects the abomasum. it does not cause diarrhoea, but c. andersoni infections have been associated with maldigestion. the infection may cause moderate to severe weight gain impairment in young stock and reduced milk production in cows (anderson ; esteban and anderson ; lindsay et al. ) . a major obstacle from a disease control perspective is the lack of effective means to control cryptosporidium infection and decrease the level of contamination of the environment with oocysts. preventive hygiene measures and good management are currently the most important tools to control cryptosporidiosis. reducing the number of oocysts ingested by neonatal calves may reduce the severity of infection and allow immunity to develop. a common recommendation is to ensure good hygiene in calf facilities and ascertain that all newborn calves ingest an adequate amount of colostrum during their first h of life. sick calves should be housed in a clean, warm, and dry environment and isolated to prevent spreading of the infection to other calves. acutely infected animals may need supportive care with fluid and electrolytes, and milk should be given in small quantities several times daily to optimise digestion and minimise weight loss. over the years, several substances have been tested for potential anticryptosporidial effects with limited success . halofuginone lactate has shown some beneficial effects such as milder clinical signs and reduced oocyst output when used as prophylactic treatment (de waele et al. ; silverlås et al. a ). this drug is approved in europe to treat calf cryptosporidiosis. however, the safety margin is narrow and the substance is toxic at only twice the therapeutic dose, so careful dosage is necessary. halofuginone lactate treatment should only be considered in herds with severe diarrhoeal problems strongly associated with c. parvum. when treatment is used, it should always be in conjunction with applying measures to reduce environmental contamination and risk of infection. a recent study investigated if an antibody-biocide fusion consisting of a monoclonal antibody "armed" with membrane-disruptive peptides (biocides) could be used for treatment of cryptosporidiosis in calves (imboden et al. ) . calves - h of age were challenged once with c. parvum oocysts and were simultaneously administered the antibody-biocide fusion mixed with milk replacer. the antibody-biocide fusion treatment was repeated - times. control calves were given milk replacer with placebo. calves receiving the antibody-biocide fusion had a significantly higher health score and shed fewer oocysts than control calves. these results suggest that this concept might be effective in cattle, but further testing is necessary (imboden et al. ) . vaccination is successfully used to control many infectious diseases in livestock. however, it takes weeks for a protective immune response to develop after a vaccine has been administered, and as calves may be exposed to cryptosporidium oocysts immediately after birth, vaccination of newborn calves is unlikely to be successful in preventing cryptosporidiosis. thus it has been suggested that the most feasible approach is likely to involve passive immunisation (innes et al. ) . dams are immunised in late gestation and their colostrum is fed to the calves. a recent study investigated antibody responses in calves fed colostrum from heifers vaccinated with a recombinant c. parvum oocyst surface protein (rcp / ). the calves had measurable quantities of the specific antibody in their serum. however, as the calves were not subsequently challenged with oocysts it remains to be seen whether this immunisation scheme can also prevent symptomatic infection and eliminate oocyst shedding (burton et al. ) . an association between cryptosporidium oocyst shedding and diarrhoea in buffalo calves has been reported from investigations performed in egypt, india and venezuela (mohanty and panda ; bhat et al. ; maurya et al. ; el-khodery and osman ; díaz de ramírez et al. ) suggesting the cryptosporidium infection is part of the calf diarrhoea syndrome in water buffalo, as it is in cattle. species identification was only performed in one of these studies, and c. parvum was the only species that was found (maurya et al. ). calves begin shedding c. parvum oocysts - days after infection and shedding continues for - days tzipori et al. ) . during the first weeks an infected calf can shed millions of oocysts uga et al. ) resulting in heavy environmental contamination, and efficient dissemination of the parasite within the herd and to the environment. in faecal samples obtained from symptomatic calves naturally infected with c. parvum - oocysts per gram faeces (opg) are often seen ). in herds with established c. parvum infection, most calves are excreting oocysts between and weeks of age (o'handley et al. ; uga et al. ) . when repeatedly sampled the same calves in a dairy herd from birth to years of age they found c. parvum oocysts in faeces of all individuals before they were weeks old, i.e. a cumulative prevalence of %. c. parvum oocysts were also found in samples collected from a calf at weeks of age and from another at months of age, indicating that oocysts can be shed intermittently over a long period after the initial infection. alternatively, these late-shedding individuals might not have developed a fully protective immunity after the first infection, and rather than this being a sign of prolonged infection, they had acquired new infections. in this study, molecular analyses indicated the same sub-genotype at the gp locus. however, this does not necessarily indicate prolonged infection, as re-infection with the same genotype in the environment may occur if the immunity is not protective. it has been suggested that an increase in c. parvum oocyst shedding may occur in adult cows around calving (so called periparturient rise), but to date there have been few reports to support this. in a recent study, however, dams in a suckler beef herd were found to shed low levels of c. parvum oocysts around the time of calving (de waele et al. ) . only one experimental infection for each of c. bovis and c. ryanae has been reported so far. regarding c. bovis, one calf shed oocysts from to days after infection and the other only for day (day ) (fayer et al. ). for c. ryanae, oocyst shedding started days after inoculation. both infected calves excreted oocysts during and consecutive days, respectively . shorter prepatent periods have been seen for both c. bovis and c. ryanae in natural infections (silverlås et al. b; silverlås and blanco-penedo ) . no oocyst excretion rate values were determined from the experimental infections, but in naturally infected calves to  opg and - , opg have been reported for c. bovis and c. ryanae, respectively (silverlås and blanco-penedo ) . young stock and adults may also be infected by the larger c. andersoni (oocyst size~ .  . μm) and may shed oocysts intermittently for many years (olson et al. ; ralston et al. ) . a periparturient rise in c. andersoni oocyst shedding, seen both as increase in prevalence and in number of oocysts in faeces, has been reported (ralston et al. ) . several studies have shown that age is associated with cryptosporidium infection and that young calves have the highest risk of being infected (maddox-hyttel et al. ; santín et al. ; sturdee et al. ; fayer et al. ). this is also the age group that is most often infected with c. parvum and suffers from clinical cryptosporidiosis. thus, from clinical and zoonotic perspective, knowledge on the epidemiology of cryptosporidiosis in young calves is highly valuable. when potential risk factors for cryptosporidium infection in pre-weaned calves have been explored, the results differ between studies. one factor that recurs in several studies is the type of flooring in the calf housing area. in spain, castro-hermida et al. ( ) found that straw on the floor or earth floors in the calf pens increased the risk for infection compared with cement flooring, and in malaysia calves kept in pens with slatted floors and sand floors had an increased risk compared with those in pens with cement floors (muhid et al. ) . a protective effect of cement floors was also reported from the usa (trotz-williams et al. ) . it was suggested that the reason for this protective effect of cement floors is that they facilitate thorough cleaning. this assumption corroborates the finding that a low frequency of cleaning of the calf pens increased the risk for infection (castro-hermida et al. ) . it is also consistent with the finding that the use of an empty period in the calf pen between introductions of calves was associated with a lower risk for infection in danish dairy herds (maddox-hyttel et al. ) . when cows as a cause of infection were investigated, a higher risk of infection was identified in calves that were housed separately from their dams (duranti et al. ), and a lower risk of infection in dairy calves kept with the cow for more than h after birth (silverlås et al. b) . in one of the few reports to investigate risk factors for infection with different cryptosporidium species in pre-weaned dairy calves to date (szonyi et al. ) , risk of infection with c. parvum differed to some extent from that of c. bovis. both c. parvum and c. bovis were more common in the younger calves, but herd size and hay bedding were associated with an increased risk for c. parvum infection, whereas jersey breed was a risk factor for c. bovis infection. experimental cryptosporidium infections in water buffalo have not been reported. however, oocyst shedding dynamics were investigated in naturally infected buffalo calves in a farm located in a tropical dry forest area in venezuela. twenty-five calves were sampled from birth to weeks of age. oocysts were detected from day and % of the calves shed oocysts before they were days (díaz de ramírez et al. ) . regarding risk factors for infection, there are some reports of seasonal variations in prevalence (bhat et al. there are numerous reports of cryptosporidiosis outbreaks in humans after contact with infected calves. these have often involved veterinary students and students at farm schools (see, e.g., gait et al. ; grinberg et al. ; pohjola et al. ; robertson et al. ; kiang et al. ), but also young children have fallen ill after visiting petting zoos or open farms (gormley et al. ; smith et al. ) . contact with cattle has been identified as a risk factor for disease also in casecontrol studies of sporadic human cryptosporidiosis (hunter et al. ; robertson et al. ; roy et al. ) . altogether, there is plenty of evidence to conclude that cryptosporidium can be transmitted from calves to humans by direct contact or by contaminated equipment. the risk for zoonotic transmission is likely to be highest in herds with cryptosporidium associated calf diarrhoeal problems, where oocyst contamination in the barn can reach high levels and where contact with naïve individuals is most likely to occur. key measures to prevent visitors becoming infected are to ensure good hygiene in the visitor area, providing suitable handwashing facilities and ensure that they are used when workers and visitors leave the premises. c. bovis infections have recently been detected in a few persons living or working on cattle farms (khan et al. ; ng et al. ) . it is not known if these were active infections and the implication of these findings is thus unclear. as molecular typing methods become more accessible, epidemiological studies can investigate c. parvum gp subtype distribution in cattle and human populations in different regions. the reports so far indicate that in many areas the subtypes that are most common in cattle are those most often found in humans. for example, c. parvum iiaa g r was the predominant subtype in both bovine and human infections in slovenia and portugal (soba and logar ; alves et al. ) . in new south wales, australia c. parvum iiaa g r dominated in both calves and people living on cattle farms (ng et al. ) , whereas iiaa g r was the predominant genotype identified in beef cattle and humans in prince edward island, canada (budu-amoako et al. c). further information is provided in the review by xiao ( ) . that the same subtypes are found in cattle and humans might be taken as an indication of zoonotic transmission. however, it is important to note that even when zoonotic c. parvum subtypes are identified in humans, cattle are not necessarily the source of the infection. these zoonotic subtypes can circulate and propagate in the human population in addition to the anthroponotic subtypes. the occasional finding of c. hominis chen and huang ) in cattle highlights the fact that cryptosporidiosis may be transmitted not only from cattle to humans, but also from humans to cattle. food-related cryptosporidiosis outbreaks have sometimes been associated with cattle. foodborne transmission was implicated in cases of children who had drunk unpasteurized milk (harper et al. ) or cider, made from apples collected in an orchard where calves from an infected herd had grazed (millard et al. ). other outbreaks in which cattle were suspected as the source involved vegetables that had been sprayed with water that could have been contaminated with cattle faeces. often there was only circumstantial evidence that cattle were the source of contamination, and it was not possible to exclude other potential sources (see e.g. cdc ; robertson and chalmers ) . outbreaks of cryptosporidiosis associated with drinking water have often been attributed to contamination of water catchments by cattle manure. the evidence implicating cattle has sometimes been substantial (bridgman et al. ; smith et al. ), but for others the evidence was not conclusive. grazing cattle or slaughterhouse effluent contaminating lake michigan were mentioned as two possible sources of cryptosporidium oocysts in the large outbreak in milwaukee, wisconsin in (mac kenzie et al , but retrospective analysis of clinical isolates revealed that it was caused by the anthroponotic species c. hominis . this was also the case in the most recent outbreaks in uk (compiled by chalmers ) and a large drinking waterborne outbreak in sweden in (anonymous ) . given that pre-weaned calves are the most likely age group to shed c. parvum oocysts, any measure to prevent waterborne zoonotic transmission should be directed towards this age group. protective measures could be to prevent young ruminants from accessing water catchments, and compost or spread calf manure on fields where runoff cannot occur. the manure of older ruminants is generally not a zoonotic concern with respect to cryptosporidium. the seemingly common occurrence of c. parvum in buffalo calves highlights the potential role of water buffalo in zoonotic transmission. thus the same precautions to prevent transmission of the parasite to humans, by direct contact or through food or water, are also applicable to water buffalo. sheep (ovis aries) and goats (capra aegagrus hircus) are important in the global agricultural economy -producing meat, milk and wool -both in developing countries such as india and iran, and industrialised countries such as australia and the united kingdom (de graaf et al. ; noordeen et al. ; robertson ). in , the world stocks were approximately one billion sheep and million goats (faostat a). asia has the largest populations of both species, with % and % of the total world populations, followed by africa (faostat a). according to a fao report, over % of the goat population can be found in developing countries (fao ) . sheep and goats tend to be managed differently to cattle, with flocks grazing large enclosures rather than being kept indoors. there have been fewer studies on cryptosporidium infection in sheep than in cattle, and even fewer studies have been performed on goats. nevertheless, it is known that these protozoans are economically important parasites in both ruminant species (noordeen et al. ; robertson ). infection and disease was first described in for sheep (barker and carbonell ) and in for goats (mason et al. ) . younger animals are more susceptible to infection than older ones, reflected in high shedding rates and diarrhoeal prevalences in lambs and kids up to month of age, whereas infection in older animals is usually subclinical with lower shedding rates (vieira et al. ). as for other animals, ovine and caprine cryptosporidium infection can be found throughout the world. the prevalence varies widely between studies, from % to % in sheep and from % to % in goats. all age groups are susceptible, but infection is more common in lambs and kids than in older animals (tables . and . ). study design factors other than age of sampled animals, such as whether only diarrhoeal animals were sampled or not, if a point prevalence study or a longitudinal study was performed and the diagnostic method(s) used, also can affect prevalence data. the effect of using different diagnostic methods is evident in, for example, giadinis et al. ( ; see also tables . and . ) and ryan et al. ( ) , where microscopy resulted in lower prevalences than detected by elisa and pcr, respectively. prevalence and species distribution for studies conducted on sheep dating back to are summarised in table . . specific data for studies on sheep published before can be found in "cryptosporidium and cryptosporidiosis" ; tables . and . ). prevalence rates and species distribution for all identified surveys of goats are summarised in table . . several species and genotypes have been identified in sheep, and the species distribution varies between studies and with age of the animals. cryptosporidium parvum, c. ubiquitum (previously cryptosporidium cervine/cervid genotype) and c. xiaoi (previously c. bovis-like genotype) are the most common species. sporadic infection with c. hominis, c. suis, c. andersoni, c. fayeri (previously marsupial genotype i), c. scrofarum (previously pig genotype ii), sheep genotype i, and unknown/novel genotypes have been identified (chalmers et al. ; giles et al. ; karanis et al. ; ryan et al. sweeny et al. b; wang et al. c) . species distribution differs between studies and between age groups within studies. for instance, c. parvum is commonly found in lambs in italy, romania, spain and the uk (díaz et al. a; imre et al. ; mueller-doblies et al. ; paoletti et al. ). in other studies, c. ubiquitum or c. xiaoi is the most common species (fiuza et al. a; geurden et al. ; robertson et al. ; sweeny et al. b) . for example, wang et al. ( c) identified c. ubiquitum in % of all analysed samples, and the species dominated in all age groups, whereas sweeny et al. ( b) found c. xiaoi to be the most common (table . ). cryptosporidium bovis has also been reported in sheep (mueller-doblies et al. ; soltane et al. ; wang et al. c ). whether c. bovis has actually been identified in sheep, or if it is the closely related species c. xiaoi is uncertain. for instance, soltane et al. ( ) reported isolates similar to c. bovis, and mueller-doblies et al. ( ) reported c. bovis, but a blast search of the genbank accession numbers identified these isolates as c. xiaoi. for the isolates reported as c. bovis by yang et al. ( ) , no genbank accession numbers are available, so the true identity of those isolates is uncertain. similarly, no genbank records are available from the study of ryan et al. ( ) reporting the "new bovine b genotype" in sheep. since c. bovis was first identified as the bovine b genotype, this could actually be the "c. bovis-like genotype", i.e. c. xiaoi. cryptosporidium andersoni has been identified in a few naturally infected adult sheep (wang et al. c ), but experimental infection in -month-old lambs failed (kváč et al. ) . a couple of apparently related surveys from mexico have been published, but because of lack of clarity in the data, they will not be reviewed in this text. two studies from brazil (sevá et al. ) and mongolia (burenbaatar et al. ) failed to identify cryptosporidium in any of the collected samples, but the number of sampled sheep was small - and animals, respectively. because of the small number of studies and isolates analysed, it is hard to draw any conclusion about the species distribution in goats. cryptosporidium parvum, c. xiaoi and a novel genotype have been identified in naturally infected goats (table . ). in addition, one report of natural infection with c. hominis is also available (giles et al. ). the identification of c. xiaoi in a number of samples is in contrast with a failed attempt to infect -week-old goats to determine the host range of c. xiaoi (fayer and santín ). because there is only scant information about cryptosporidiosis in goats, we do not know if an age-related resistance or immunity from a previous cryptosporidium infection could have affected this experiment, as natural infections indicate that c. xiaoi is infectious to goats. two studies from mongolia (burenbaatar et al. ) and the united kingdom failed to identify cryptosporidium in any of the collected samples, but the number of sampled goats was small - and animals, respectively. cryptosporidiosis has been associated with high morbidity and mortality rates in both lambs and goat kids (cacciò et al. ; chartier et al. ; de graaf et al. ; giadinis et al. giadinis et al. , johnson et al. ; munoz et al. ; paraud et al. ; vieira et al. ) . high mortality has been described both from natural infection and from experimental studies, where infection doses are generally high (chartier et al. ; giadinis et al. ; paraud et al. ) . in fact, it has been stated to be one of the most important pathogens associated with diarrhoeal disease and mortality in neonatal lambs and kids (quílez et al. a) . anorexia and apathy/depression are common symptoms, accompanied by abdominal pain and pasty to watery, yellow and foul-smelling diarrhoea (de graaf et al. ; snodgrass et al. ) . diarrhoea can last from a few days up to weeks (de graaf et al. ) . faecal consistency is correlated with oocyst excretion (de graaf et al. ; paraud et al. paraud et al. , , and a longer duration of diarrhoea is potentially associated with infection early in life (paraud et al. ) . body condition score and growth are affected (de graaf et al. ) , probably due to both anorexia and the intestinal damage, that can reduce nutrient uptake for weeks (de graaf et al. ; klein et al. ) . infection in animals older than month is usually subclinical, and even younger animals can be subclinically infected. however, the infection can still affect production, with reduced body condition score (sweeny et al. a (sweeny et al. , , reduced growth rate, and reduced carcass weight and dressing percentage at slaughter (sweeny et al. a ). as discussed above for cattle, before molecular methods were developed c. parvum was the only species considered to infect and cause disease in sheep and goats (chartier et al. ; de graaf et al. ; munoz et al. ) . cryptosporidium parvum infection has since been associated with diarrhoea in studies using molecular methods (caccio et al. ; díaz et al. a; drumo et al. ; imre et al. ; mueller-doblies et al. ) . however, c. xiaoi has also been associated with mild to severe diarrhoea and mortality (díaz et al. b; navarro-i-martinez et al. ; rieux et al. ) , and c. ubiquitum too has been found in a few diarrhoeal samples from lambs (díaz et al. a) , indicating that c. parvum is not the only pathogenic species in small ruminants. the prepatent period is - days in goat kids (paraud et al. ) and - days in lambs (de graaf et al. ). the patent period can last for at least days (paraud et al. ). shedding peaks a few days to a week into the patent period, and maximum shedding can be as high as  opg (rieux et al. ) . the length of the patent period and shedding intensity are determined by age, immune status and infection dose (de graaf et al. ) . a natural age-related resistance to infection seems to be present. in one study, the prepatent period increased and intensity of shedding decreased in lambs with increasing age at infection (ortega-mora and wright ). in another study, one naturally infected group of goat kids started shedding at days of age and excretion peaked at a mean of  opg - days later (rieux et al. ) , whereas another group of animals studied by the same authors started shedding at the age of days, with a mean peak of  opg - days later, indicating higher virulence, infection pressure or an age-related higher sensitivity in the latter group. factors such as hygienic conditions, milking practices, herd size (population density), season, climatic zone (within a country), and lambing/kidding season are factors that have been associated with prevalence of infection, prevalence of clinical cryptosporidiosis and intensity of oocyst shedding (alonso-fresan et al. ; bomfim et al. ; craig et al. ; delafosse et al. ; giadinis et al. ; maurya et al. ; noordeen et al. noordeen et al. , . however, factors associated with infection and shedding intensity are also impacted by different management systems and climatic conditions; results from small farms in, for example, india cannot be extrapolated to large-scale farming in, for example, the united kingdom. the significance of sheep and goats as reservoirs for zoonotic cryptosporidiosis is unclear (robertson ). the first case of suspected zoonotic transmission from sheep was described in (casemore ), but at that time diagnosis was based solely on microscopy and thus zoonotic transmission cannot be confirmed. however, since the introduction of molecular tools in diagnostics, a number of cases and outbreaks with suspected or confirmed zoonotic transfer from sheep have been described (cacciò et al. ; gormley et al. ). in the uk, where sheep farming is an important industry, a seasonal pattern with spring and autumn peaks of human cryptosporidiosis cases is observed, with the spring peak concurring with the lambing season (anonymous ; gormley et al. ; mclauchlin et al. ; nichols et al. ) . lambs in petting zoos seem to be a common infection source (chalmers et al. ; elwin et al. ; gormley et al. ; pritchard et al. ). the incidence of human cryptosporidiosis, especially due to c. parvum, dropped significantly during the foot-and-mouth outbreak in the spring and summer (hunter et al. ) when > million livestock animals (~ . million sheep; anonymous ) were slaughtered and there were restrictions in animal movements and farm visits, providing further evidence of the importance of zoonotic transmission in this region. in , an outbreak occurred in norwegian schoolchildren visiting a farm raising several animal species. cryptosporidium parvum oocysts of an identical and unusual gp subtype were identified in faecal samples from six human patients, two lambs and one goat kid. another human outbreak with the same c. parvum subtype had occurred at the same farm years previously, but at that time very few oocysts were detected in animal faecal samples and molecular analyses were not conducted (lange et al. submitted) . cacciò et al. ( ) described a case where a farmer's son fell ill with cryptosporidiosis, being infected with the same and unusual c. parvum subtype that caused high morbidity and mortality in the farm's lambs (table . ) . studies on c. parvum gp subtypes have been performed with sheep and goat isolates, and all isolates were found to belong to the iia and iid families (table . ) . several studies using multi-locus genotyping (mlg) have found evidence of specific host associated c. parvum populations (drumo et al. ; mallon et al. a, b; morrison et al. ). in the united kingdom, sheep mlgs clustered with human and bovine isolates (mallon et al. a, b) , indicating frequent zoonotic transmission, whereas only one of the mlgs identified in sheep/goats in italy was also identified in human samples (drumo et al. ) , indicating a low rate of zoonotic transmission. however, the latter study included very few human isolates. zoonotic transmission is commonly observed with c. parvum, but c. ubiquitum has also been identified in a number of sporadic human cases (cieloszyk et al. ; elwin et al. ; feltus et al. ; leoni et al. ; ong et al. ; soba et al. ; trotz-williams et al. ). oocysts of this species have been identified in storm water, wastewater, raw water and drinking water (jiang et al. ; liu et al. ; nichols et al. ; van dyke et al. ) . in scotland, c. ubiquitum was the third most common species in raw water and the most common species identified in drinking water (nichols et al. ) . thus, in areas where c. ubiquitum is common in sheep and goats, this species could be a more important cause of zoonotic infection than c. parvum. in addition, the relatively common presence of this species in water indicates a potential for waterborne outbreaks. natural infection with c. hominis has been reported in one goat and two sheep (giles et al. ; ryan et al. ) and in three lambs following experimental infection (ebeid et al. ; giles et al. ), but since animals are not natural hosts for this species, risk of zoonotic transmission with this species should be negligible compared with the risk of human-to-human transmission. it is important to note that because cryptosporidium infection can be subclinical, the zoonotic potential is not restricted to contact with diarrhoeic cryptosporidium-infected animals (pritchard et al. ). since domestication around bc in china, the pig has been an important food source (moeller and crespo ) . pigs are farmed worldwide, with the global swine inventory estimated at over million in . because asian countries are major consumers but do not produce sufficient pigs for their needs, there is a significant international trade in live and slaughtered pigs. china has the world's largest pig population, mostly small herds consisting of only a few animals, and is a net importer of pigs. the united states, european union, and canada are major exporters with relatively few but very large production units (moeller and crespo ) . the global trend is for fewer producers responsible for larger numbers of pigs, and more concentration within the swine industry. pigs are the primary host for c. suis ) formerly identified as cryptosporidium pig genotype i and for c. scrofarum (kváč et al. ) formerly identified as cryptosporidium pig genotype ii. farm pigs have also been found infected with c. parvum, c. muris, on one occasion with c. tyzzeri, a species common to mice, and with the novel genotype isolate eire . (kváč et al. ) . cryptosporidiosis occurs in pigs of all ages in countries on continents (table . ). before molecular methods were developed c. parvum was thought to infect species of mammals and to consist of several genotypes. consequently some early studies erroneously reported c. parvum infection in pigs based on the identification of oocysts in faeces by microscopy. subsequent use of molecular methods provided the necessary tools to identify and distinguish species. overall, prevalence data for locations, herds and age groups vary greatly and are not directly comparable because some data represent pooled samples (some from litters, others from fecal slurry), some data originate from single farms while other data come from multiple farms. some surveys have studied individual pigs at various ages, or only those pigs with diarrhoea, or simply specimens submitted to diagnostic laboratories from unspecified locations (table . ). even in comparable populations, such as preweaned pigs in the same country or indifferent countries, data differences are too great to draw any conclusions on prevalence. for example: in australia-reports of . % versus . % prevalence (johnston et al. vs. ryan et al. a ; in the czech republic-reports of . % versus . % prevalence (kváč et al. a vs. vitovec et al. ; or between serbia and spain -reports of versus % prevalence (mišić et al. vs. quílez et al. a ). some studies found significant association between the presence of a particular species and the pigs' age, with c. suis prominent in piglets and c. scrofarum prominent in weaners (enemark et al. ) . in contrast, others found no significant association between species and age or housing conditions (featherstone et al. b ). these prevalence data reflect vast differences in management practices from location to location with too many unknown factors to draw valid conclusions on cause and effect or location within the cited studies in table . that reported a prevalence of infection between . % and %. the only variable repeatedly associated with detection of cryptosporidium is age. most positive samples were from weaners and growers (table . ). generally, prevalence increased until pigs were weeks of age, then gradually declined. the first reports of cryptosporidiosis in pigs found one piglet among herds of nursing piglets with necrotic enteritis, but the significance of this finding was described as unknown (bergeland ) and cryptosporidium was found at necropsy in three pigs without clinical signs (kennedy et al. ) . although a higher prevalence of diarrhoea was found in cryptosporidium-infected pigs than in uninfected pigs (hamnes et al. ) , others found no significant relationship between infection and diarrhoea (quílez et al. b; guselle et al. ; maddox-hyttel et al. ; vitovec et al. ; suárez-luengas et al. ). cryptosporidium was detected histologically in the microvillus brush border of . % of , pigs from farms examined for routine diagnostic evaluation (sanford ) . most infected pigs were - weeks old. organisms were detected in the jejunum, ileum, caecum, and colon, but primarily in microvilli of dome epithelium in the ileum. twenty six percent of cryptosporidium-infected pigs had diarrhoea but most of these also had other primary agents capable of causing diarrhoea. similar observations have been made by others. whereas most infections are asymptomatic or cause only mild, non-specific colitis (higgins ) , pigs known to be naturally infected with c. suis or c. scrofarum have not been found with clinical signs of infection while pigs infected with c. parvum or co-infected other enteropathogens such as rotavirus, salmonella, or isospora have had associated diarrhoea and some have died (enemark et al. ; núñez et al. ; hamnes et al. ) . experimental infections with different species have helped to clarify the relationship of species with clinical disease. pigs experimentally infected with c. suis (enemark et al. ) or c. scrofarum (kváč et al. ) showed no clinical signs. the pathogenicity of c. parvum isolated from calves was demonstrated in early transmission studies to pigs (moon and bemrick ; tzipori et al. b tzipori et al. , argenzio et al. ; vitovec and koudela ; pereira et al. ) . experimental infection with the avian species, c. meleagridis, obtained from a human infection, consistently resulted in oocyst excretion and diarrhoea in pigs, although mucosal changes were milder than those described for c. parvum (akiyoshi et al. ) . piglets infected with c. suis had mild or no clinical signs despite excreting large numbers of oocysts, in contrast to those infected with c. parvum that had diarrhoea for a mean duration of . days and developed inappetence, depression and vomiting (enemark et al. ) . developmental stages of cryptosporidium have been observed throughout the intestinal tract. villous atrophy, villous fusion, crypt hyperplasia, and cellular infiltration of the lamina propria have been observed (kennedy et al. ; moon and bemrick ; tzipori et al. tzipori et al. , sanford ; vitovec and koudela ; argenzio et al. ; pereira et al. ; enemark et al. ; núñez et al. ; vitovec et al. ) . lesions caused by c. parvum were the most severe, as were clinical signs associated with that species. changes in the location of stages have been noted. in the first days of infection more stages were found in the proximal intestine, but later more stages were found in distal locations (tzipori et al. ; vitovec and koudela ) . extra-intestinal infections also have been reported in pigs. in two naturally infected piglets, the gall bladder was infected (fleta et al. ) . in experimentally immunosuppressed piglets, the gall bladder, bile ducts, and pancreatic ducts were found infected (healey et al. ) . infections in the trachea and conjunctiva were detected in experimentally infected normal piglets (heine et al. ). a survey of faecal slurry from swine finishing operations in ireland found c. suis, c. scrofarum and c. muris and concluded that cryptosporidium oocysts can persist in treated slurry and potentially contaminate surface water through improper discharge or uncontrolled runoff . hamnes et al. ( ) reported c. suis and c. scrofarum in faeces of suckling pigs in norway and reasoned that farrowing operations were sources of these parasites. additional data on oocyst concentrations, numbers of oocysts excreted, how long oocysts remain infectious under environmental condition, and modes of transmission of cryptosporidium species and genotypes are rare or non-existent. a year-long investigation was conducted at four types of swine operations (finishing, farrowing, nursery and gestation) in georgia, usa (jenkins et al. ) . mean oocyst concentrations ranged from to oocysts per ml of lagoon effluent; the nursery had the highest concentration of oocysts and the greatest percentage of viable oocysts ( . %), c. suis and c. scrofarum were the dominant species with some c. muris and c. parvum. experimental attempts to transmit c. scrofarum to adult scid mice, adult balb/c mice, mongolian gerbils, southern multimammate mice, yellow-necked mice, and guinea pigs were unsuccessful, suggesting that rodents are an unlikely source of transmission of this species under natural conditions (kváč et al. ). cryptosporidium suis was detected by immunofluorescence microscopy and rflp analysis of pcr products in stools from an hiv patient in peru (xiao et al. a ). the patient was not severely immunosuppressed and was asymptomatic. he had a dog but reported no contact with other animals or animal faeces, including pigs and pig faeces, so the source and method of transmission are unknown. many countries, including australia, canada, china, korea, norway, russia, sweden, uk, usa and vietnam, have thriving deer farming industries. new zealand, a country where deer are not native, has the world's largest and most advanced deer farming industry. although it is difficult to find estimates on the numbers of deer farmed worldwide, more than one million deer were being farmed in new zealand in (sources: statistics new zealand and deer industry new zealand http://www.deernz.org/about-deer-industry/nz-deer-industry) -compared with five million dairy cows -and there are over , deer farmers. more than % of the new zealand deer industry's products are exported, with approximately half of the export going to germany and benelux. species of deer which are commercially farmed varies regionally, but the following species are now being farmed in various parts of the world: red deer (cervus elaphus), wapiti or elk (cervus canadensis), fallow deer (dama dama), sika (cervus nippon), rusa deer (rusa timorensis), and reindeer (rangifer tarandus) (fao http://www.fao.org/docrep/ /x e/x e .htm). although farmed deer are an important resource in many countries, much of the published information on cryptosporidium in deer refers to studies on wild or free-ranging cervids (e.g. white-tailed deer in usa, roe deer in spain, caribou in canada, and moose, red deer, roe deer and reindeer in norway; rickard et al. ; castro-hermida et al. b; johnson et al ; hamnes et al. ) . while these studies on free-ranging cervids may give useful information regarding the species or genotype of cryptosporidium that might infect farmed deer (c. ubiquitum, c. parvum), as farmed deer probably differ quite substantially from their wild counterparts regarding exposures and stresses, extrapolation of prevalence data from wild to farmed deer may give an incorrect picture. indeed, a study in poland found that the prevalence of cryptosporidium was significantly higher in wild red deer than farmed red deer ( % compared with . %), and mean oocyst concentration was also five times higher in faecal samples from wild red deer (paziewska et al ) . however as the sample size was relatively small ( wild deer, farmed deer) and from only single locations and as age and symptoms were not indicated, it is not possible to determine the reason for these differences. the few studies on the prevalence of cryptosporidium infection in different farmed or domesticated cervids are summarised in table . . the lack of surveys for cryptosporidium infection in farmed deer is surprising, given the clear association of infection with clinical disease in farmed cervids. some of the first published studies on cryptosporidium infection in cervids are case reports of severe (high mortality) outbreaks among farmed red deer calves. in one outbreak in scotland, uk among artificially reared red deer calves, developed cryptosporidiosis and subsequently died; % of the calves with diarrhoea and % of apparently asymptomatic calves excreted oocysts and post mortem histopathological examination of the intestines demonstrated lesions similar to those seen in other species (tzipori et al. a) . another outbreak among new-born red deer calves in the uk also resulted in high mortality, with calves dying at - h of age. however, this outbreak was not characterized by diarrhoea, and terminal uraemia was proposed as the symptom leading to death (simpson ) . outbreaks of cryptosporidiosis in red deer calves have also been reported from new zealand, again with relatively high mortality ( out of calves dying within a few days of illness onset in one outbreak, and out of calves dying within days of illness in another outbreak) (orr et al. ) . severe subactute enteritis in the small and large intestine were reported in both outbreaks. information from other species of farmed deer is more scanty, but a retrospective study of neonatal mortality in farmed elk (pople et al ) identified cryptosporidium infection as one of the most important causes of enteritis leading to death ( cases out of of infectious enteritis from a total of cases in which had no specific cause of death identified). among these cases, were associated with an outbreak on a single farm (pople et al ) . unfortunately, information on the species of cryptosporidium infection associated with clinical disease in deer and cryptosporidium associated with asymptomatic infection is lacking. one outbreak on a scottish farm occurred when deer were put to graze on a pasture that had previously been grazed by a cryptosporidium-infected herd of cattle (angus ) , and therefore it seems probable that this might indicate infection with c. parvum; it might be speculated that infection of deer with deer-adapted c. ubiquitum is less likely to cause severe symptoms. published information about infection dynamics in farmed deer is minimal. however, a longitudinal study in asymptomatic farmed red deer in ireland (skerrett and holland ) provides some interesting data. asymptomatic low-level (< opg) oocyst shedding from adult hinds appeared to continue throughout the year, except during the calving season (may -june), when there was a -log increase in oocyst excretion rate; the highest level recorded was , opg. the authors speculate that this may be related to hormonal or immunological changes, or perhaps alterations in stress levels. the authors note that this preparturient rise in oocyst shedding results in contamination of the environment for the new born calves. however, in this study, although calves became infected, oocyst excretion was low (not exceeding opg, and usually less), and clinical disease was not observed. again, the species of cryptosporidium in these infections is unknown. both c. parvum and c. ubiquitum have zoonotic potential, but there appear to be no documented cases of proven zoonotic transmission from/to deer. those studies (usa and australia) that have investigated deer as sources of contamination in watersheds, have focused on wild deer only and provided contrasting results (cinque et al ; jellison et al camelids are members of the family camelidae, and include the tribe camelini (including dromedaries and bactrian camels), and the tribe lamini (llamas, alpacas, vicuñas, and guanacos). there are two species of camel. approximately million domesticated onehumped dromedaries (camelus dromedarius) are found in middle eastern countries including the sahel and horn of africa, as well as parts of southern asia where they provide people with milk, food, and transportation. nearly two million domesticated two-humped bactrians (camelus bactrianus) are native to the steppes of central asia the gobi and taklamakan deserts in mongolia and china. alpacas (vicugna pacos) and llamas (lama glama) exist only in the domesticated state and are found worldwide. however, both are native to south america and are raised primarily for fibre production although llamas were once used extensively as work animals. the young of both are called crias. camels and dromedaries: in the relatively few studies of dromedaries in northern africa and the middle east, the prevalence of cryptosporidiosis varied greatly (table . ). none of camels from iraq (mahdi and ali ) and none of camels on farms in tunisia were found positive for cryptosporidium (soltane et al. ) . cryptosporidium was detected in one of four camel calves in egypt (abou-eisha ). however, in an abattoir in yazd province in iran, microscopic examination of faecal specimens detected ( . %) positive for cryptosporidium and ( %) positive abomasal mucosa specimens. at an abattoir in isfahan province in central iran, of adult male and adult female dromedary camels examined, ( . %) were cryptosporidium-positive (razawi et al. ). in northwestern iran, of faecal samples from camels ( %) were positive for cryptosporidium-like oocysts (yakhchali and moradi ) . the prevalence was significantly higher ( %) in calves less than a year old. oocysts have also been recovered from wild and zoo-housed camels. faeces from a -year-old bactrian camel in the wild animals rescue centre of henan province in china were found positive for c. andersoni (wang et al. b) . oocysts from a zoo-housed bactrian camel (fayer et al. ) were infectious for mice (anderson ) and were identified as c. muris (xiao et al. ; morgan et al. ) ; those from camels in the czech republic were identified as c. andersoni. other zoo-housed camels have been found to be infected with cryptosporidium (abou-eisha ; gomez et al. ; gracenea et al. ) . alpacas and llamas cryptosporidium oocysts have been detected in both these species. however, in california none of llamas from facilities were found positive (rulofson et al. ) nor were alpacas on two farms in maryland . elsewhere in north america, europe, and australia small numbers of alpacas, llamas and guanacos have been examined and a few have been found positive for cryptosporidium (table . ). most examinations were conducted by microscopy, but those that utilized molecular methods identified only c. parvum (morgan et al. ; starkey et al. ; o'brien et al. ; twomey et al. ) . the exception is a study in which a cria was found infected with c. ubiquitum (gomez-couso et al. ) . a national survey of , - -day-old alpacas in andean herds in peru, the natural habitat for nearly % of the world's alpacas, found % of the youngest alpacas increasing to % of the oldest alpacas, infected with cryptosporidium spp., with an overall prevalence of % (lopez-urbina et al. ). more recently in peru, . % of alpacas from herds were found positive for cryptosporidium spp. (gomez-couso et al. ) . herd prevalence was . % ( / herds) for cryptosporidium. the highest prevalence ( %) was found in the -week-old group (gomez-couso et al. ) . camels and dromedaries few data are available on the subject of clinical illness associated with cryptosporidiosis in camels. of faecal samples, camels ( %) were positive for cryptosporidium-like organisms (yakchali and moradi ). the prevalence was significantly higher in camel calves (< years old) ( %) than other age groups, in which the diarrhoeic calves had a prevalence of %. alpacas and llamas not all alpacas and llamas infected with cryptosporidium show clinical signs of infection. of healthy crias and their dams % and %, respectively, were found excreting oocysts (burton et al. ) . oocysts of c. parvum were detected in of faecal samples from healthy crias and in one sample from a cria with diarrhoea (twomey et al. ) . cryptosporidium was observed in a post-operative neonatal llama with diarrhoea, cachexia, dehydration and electrolyte abnormalities (hovda et al. ). during days that intravenous fluids and nutritional support were provided, these signs were not observed. of cryptosporidium-infected alpaca crias with diarrhoea, exhibited weight loss and had a poor appetite (waitt et al. ). most were - days old when examined. additional potential gastrointestinal pathogens were found in of these crias. sixteen crias recovered after supportive therapy that included intravenous rehydration, with partial parenteral administration of nutrients, antimicrobials, oral nutrients, plasma, insulin and other palliative treatments. additional reports of diarrhoea associated with cryptosporidiosis have been reported in alpaca and llama crias (cebra et al. ; shapiro et al. ; whitehead and anderson ; starkey et al. ). three fatal cases ( with diarrhoea) of cryptosporidiosis were reported in alpaca crias less than days of age (bidewell and cantell ) . at necropsy, intestinal congestion and distension were noted, oocysts were detected in ziehl-neelsen stained smears, and no other significant organisms or toxins were detected. in south america, llama and alpaca husbandry is a vital economic activity and neonatal diarrhoea syndrome (nds) is the most common and costly enteric disease in newborn llamas and alpacas (lopez-urbina et al. ). however, the role of cryptosporidiosis in nds has not been clearly identified. camels and dromedaries only rare circumstantial data of zoonoses are available and the link is very tenuous. in yazd province in iran, of people in long-term contact with camels were found infected with cryptosporidium spp. (sazmand et al. ) . infection was higher in winter than summer ( / compared with / ). alpacas and llamas in new york, cryptosporidium parvum infection was identified in crias, of their caretakers were confirmed to have cryptosporidiosis, and three others were suspected to have cryptosporidiosis, suggesting zoonotic transmission (starkey et al. ). rabbit farming (cuniculture) for meat, wool, and fur production occurs in a variety of settings around the world, and mostly involves the european (or common) rabbit (oryctolagus cuniculus). small-scale backyard cuniculture is common in many countries (especially in africa and south america), but commercial operations on a larger scale are found in europe (particularly italy, spain and france) and asia (particularly china and indonesia). in the eu, rabbit meat production was estimated to be around , tonnes carcass-weight equivalent in (efsa-ahaw ). in addition, rabbits continue to be bred for biomedical purposes -but this type of rabbit breeding will not be considered further in this chapter. production and consumption of rabbit meat is relatively low in north america. different rabbit breeds are used for meat, wool, and fur -with the most commonly used meat breeds being new zealand, californian, florida white and altex, all having good growth rates and desirable reproductive characteristics. much of the information presented in this section is derived from a comprehensive review article from (robinson and chalmers ) . the majority of published prevalence information on cryptosporidium in rabbits refers to studies on wild rabbits. nevertheless, there have been several studies on the occurrence of infection in farmed rabbits and also in laboratory rabbits. the majority of these studies (involving both wild and domestic rabbits) are summarised in robinson and chalmers ( ) . in table . , selected prevalence studies (rather than case reports) from farmed rabbits only are summarized, including two recent studies from china. additionally, a further three studies from china and referenced in zhang et al. ( ) are not included in table . due to inaccessibility of the original publications. zhang et al. ( ) do not provide any details on these studies and it is not certain that they refer to farmed rabbits. although some surveys refer to cryptosporidium parvum, all those studies in which genotyping has been used (including from wild rabbits; e.g. nolan et al. ) suggest that the majority of natural infections in rabbits, if not all, are caused by c. cuniculus. nevertheless, experimental infections with other species of cryptosporidium have been established in rabbits, as summarised by robinson and chalmers ( ) . although the majority of surveys do not report symptoms associated with cryptosporidiosis in rabbits, experimental infections in preweaned rabbits have been associated with diarrhoea and high mortality (e.g. as reported by robinson and chalmers ; mosier et al ) and also as described by pavlásek et al. ( ) in farmed rabbits. however, even asymptomatic infection may result in some pathology, as noted by inman and takeuchi ( ) , who reported blunted villi, a decrease in villus-crypt ratio, and mild oedema in the lamina propria in an apparently asymptomatic adult rabbit. thus, even asymptomatic infection may reduce stock productivity. although no outbreaks of cryptosporidiosis in rabbit farms have been documented in the literature, acute outbreaks of diarrhoea with high mortality rates are frequently observed in rabbits (banerjee et al ) . although bacterial agents are frequently considered to be the aetiological agent, it seems probable that some may be due to undiagnosed cryptosporidiosis. for example, the parasitological techniques (direct microscopy and flotation) used for investigating epizootic outbreaks of diarrhoea, characterized by a high morbidity and mortality, in different commercial rabbit farms in mexico (rodríguez-de lara et al ) may have been insufficient for detecting cryptosporidium infection, particularly if the operators had little experience in diagnosing this infection. information on the dynamics of cryptosporidium infection in farmed rabbits is mostly lacking, although low oocyst excretion rates were reported in the majority of studies on rabbits in general (not just farmed rabbits). the studies from the czech republic provide some data, but, as the animals were not sampled individually, the data are difficult to interpret, and suggest that the source of infection for young rabbits may be low-level excretion of oocysts from mother rabbits at around parturition (pavlásek et al. ) . c. cuniculus is rarely, but sporadically, identified in human infections. in cryptosporidium-positive faecal samples submitted for routine typing in uk between and , ( . %) were identified as c. cuniculus, with both gp va and vb subtype families detected . however, the greatest evidence for c. cuniculus from rabbits having a significant zoonotic potential came from a waterborne outbreak of cryptosporidiosis in england in affecting people; c. cuniculus, subtype vaa was identified in eight patients, a water sample from the implicated supply, and from the colon of a carcass of a rabbit (presumably wild) that was found in a tank at the water treatment works . nevertheless, transmission of cryptosporidium to humans from farmed rabbits has not been recorded, and an investigation exploring associations between farm animals and human patients with cryptosporidiosis did not implicate rabbits as a source of infection . the world stock of birds in production in was estimated to  animals (faostat b). approximately % of the world stock was found in asia, whereas europe, north america and south america had approximately - % each of the population. the largest group was chickens, with % of the total stock. ducks, turkeys and geese/guinea fowls constituted . %, . % and . % respectively, and other birds (ratites, pigeons etc.) only constituted . % of the world stock. the main chicken, duck and goose/guinea fowl production is in asia ( %, % and % within each group respectively), and most of the turkey production in north america ( %), followed by europe ( %). for other birds, % of the reported production was located in asia, % in africa and % in europe. chickens (gallus gallus domesticus) are descendants of the red jungle fowl (gallus gallus), with some hybridization with the grey junglefowl (g. sonneratii). broilers are usually kept in intense systems and reach slaughter size at about weeks of age. organically bred broilers and broilers kept on free range grow a bit more slowly. laying hens can produce over eggs in their first production year, but after that production declines rapidly. domesticated ducks (anas platyrhynchos domesticus) are, except for the moscovy duck (cairina moschata), descendants of the mallard (anas platyrhynchos). the majority of domesticated geese (anser anser domesticus) descend from the greylag goose (anser anser), but the breeds chinese goose and african goose are derived from the swan goose (anser cygnoides). ducks and geese are bred for meat, eggs and down, and ducks, to a lesser degree, also for the production of foie gras. the domestic turkey (meleagris gallopavo) is a progeny of the wild turkey, which is found in the wild in the united states http://www.turkeyfed.com.au/turkey_info. php. turkeys are bred for meat production. the breed used is the white broad breasted turkey, introduced into commercial production in the s http://bizfil.com/turkeyraising-primer. as with commercial chicken broiler farming, turkey farming is intense. the poults are extremely fast-growing, and reach approximately kg at weeks of age if given proper nutrition http://bizfil.com/turkey-raising-primer/. the united states has the highest consumption of turkey meat per person, and they are also the largest turkey producer, with . billion pounds of turkey meat produced in http://www.agmrc.org/commodities__products/livestock/poultry/turkey. among ratites, mainly ostriches (struthio camelus) are farmed, but rheas (rhea americana) and emus (dromaius novaehollandiae) are also kept for production. ratites are bred for meat, egg, and feather and leather production. farming for feather production began already in the nineteenth century. partridges, such as the grey or english partridge (perdix perdix) and red-legged partridge (alectoris rufa), are gallinaceous birds used as game, and have been introduced in different parts of the world for this purpose. another gallinaceous bird is the helmeted guinea fowl (numida meleagris). they are used for pest control, eating ticks and other insects, and can be kept as an alarm system among other domesticated birds due to their loud and shrieking warning call. the meat is considered a delicacy. the japanese quail (coturnix japonica) is bred for meat and eggs. domestic pigeons (columba livia domestica) are the progeny of the world's oldest domesticated bird, the rock pigeon. pigeons are bred for meat, sporting competitions, homing, as exhibition birds or pets. cryptosporidium infection has been associated with large morbidity and mortality in different bird species (bezuidenhout et al. ; hoerr et al. ; pages-mante et al. ; penrith et al. ; ritter et al. ; santos et al. ) and can thus be of great economic importance. avian cryptosporidiosis was first described in chickens (tyzzer ) . the infection was subclinical and situated in the caecum. invasive stages looked identical to those of c. parvum, but no oocyst description was made, and no name was proposed. today, three valid species have been identified in poultry. in addition, five genotypes have been identified in wild ducks and geese, and five additional genotypes have been described from other birds. the cryptosporidium oocysts identified by slavin in were morphologically similar to c. parvum, described in mice in (tyzzer ) , and the infection site was the distal ileum. slavin identified this bird cryptosporidium as a unique species, c. meleagridis. when molecular methods were introduced as a means of species determination, it was verified that c. parvum and c. meleagridis were indeed different species (sreter et al. ) . a species with a larger oocyst, first identified in chickens, and infecting the intestine, bursa and cloaca, was described and named c. baileyi ). this species is also involved in respiratory cryptosporidiosis, infecting the epithelium of sinuses, air sacs, nasopharynx, trachea and bronchi (itakura et al. ; lindsay et al. ) . infection of the conjunctiva (chvala et al. ) and urinary tract, including the kidneys has also been shown trampel et al. ) . a third species, c. galli, infecting the proventriculus of chickens, was described by pavlásek in , and re-described in (pavlásek ryan et al. b) . the species was probably described in finches already in (blagburn et al. ) and later the name c. blagburni was proposed ). however, molecular analyses have shown that c. blagburni is the same species already described as c. galli, and thus the latter is considered to be the valid species name. in addition, isolates referred to as goose genotypes i-iv have been identified in canada geese and a duck genotype has been described in a black duck and canada geese. of the other genotypes described in birds (avian genotypes i-iv and the eurasian woodcock genotype), avian genotype ii has been detected in ostriches. two proposed species are today considered as nomen nudum due to lack of sufficient data. cryptosporidium tyzzeri in chickens was described in (levine ) and later c. anserinum, found in the large intestine of geese was described (proctor and kemp ) . based on s rdna phylogeny, c. galli and the woodcock genotype belong to the clade of gastric cryptosporidia together with c. andersoni, c. muris and c. serpentis, whereas c. meleagridis, c. baileyi, goose genotypes i and ii and the duck genotype belong to the intestinal clade . cryptosporidium meleagridis is closely related to the group including c. parvum and c. hominis; c. baileyi is closely related to the snake genotype, goose genotypes i, ii and the duck genotype cluster together and are closely related to c. scrofarum, c. bovis, c. ryanae and the deer genotype. goose genotypes iii-iv and avian genotypes i-iv were not included in the phylogenetic tree. in another publication, goose genotypes i (goose # , , , and ), ii (goose # ) and the duck genotype (goose # ) are closely related, whereas goose genotypes iii (goose # b) and iv (goose # ) are more distant (jellison et al. ) . the avian genotypes are more scattered. avian genotypes i and ii belong to the intestinal clade and are closely related to c. baileyi. genotypes iii and iv belong to the gastric clade, where genotype iii is closely related to the eurasian woodcock genotype and c. serpentis, and genotype iv is closely related to c. galli (ng et al. ) . prevalence data based on fecal examination could be affected by the time from sampling to analysis. this has been observed when oocyst numbers in chicken faeces dropped to approximately one third in samples stored for a week from first to second analysis, and where first analysis was performed on the day after sampling (c. axén, unpublished data). it is possible that oocysts die and are quickly degraded by detrimental effects (extreme ph) due to the high ammonium content of bird droppings. a flock prevalence of % ( / ), with - % within-flock prevalence, was reported for c. baileyi respiratory infection in broilers in the usa (goodwin et al. ) . in morocco, cryptosporidium sp. were found in ( %) of investigated flocks. within-flock prevalence ranged from % to %, and the highest prevalence ( %) was identified in broilers aged - days, with no infection prior to days of age (kichou et al. ) . diagnosis was based on histopathology. an overall cryptosporidium prevalence of . % for layer chickens and . % for broiler chickens was shown in a study of faecal samples from birds in china (wang et al. a ). the highest prevalence ( . %) was found in - -day old laying chickens, whereas prevalences in broiler chickens never exceeded %. dna analysis identified c. baileyi as the major species, with / investigated samples, and only samples were positive for c. meleagridis (wang et al. a) . in contrast, another recent study identified c. meleagridis as the major species in chickens (baroudi et al. ) . the overall cryptosporidium prevalence was . % by histopathology, and the highest prevalence ( . %) was identified in - -day-old chickens, which is in line with the results from kichou et al. ( ) and wang et al. ( a) . the majority of the birds were infected with c. meleagridis only (n ¼ ). cryptosporidium baileyi only was detected in four birds and a mixed c. meleagridis/c. baileyi infection was found in one bird. however, these chickens had died from diarrhoea, which could affect the outcome regarding cryptosporidium sp. a morbidity of - % due to sinusitis was reported for a flock where cryptosporidium sp. could be isolated from diseased poults (glisson et al. ) . it was stated that macroscopic post mortem examination of the infraorbital sinuses of healthy birds was normal compared with those of diseased birds, but it was not clearly stated whether cryptosporidium sp. was also identified in the healthy birds and thus the infection prevalence cannot be estimated. goodwin et al ( b) identified invasive cryptosporidium stages in turkey poults form a farm where the poults suffered from self-limiting diarrheal of unknown aetiology, but no prevalence estimation was given. prevalences of % in -day-old poults, % in -day-old and % in ! -day-old poults was found by woodmansee et al. ( ) . oocysts were identified as c. meleagridis based on morphology and infection site. a . % ( / ) prevalence in diarrhoeic or just unthrifty poults was reported in iran (gharagozlou et al. ) . prevalence was based on histological examination of intestinal, bursal and cloacal tissues. examination of faeces revealed that only % of the infected birds shed oocysts. infection was identified in - -week-old poults, whereas the uninfected poults all were older than weeks. dna analysis was not performed and oocyst size was not stated in the publication, but based on infection site, host species and symptoms the authors suggested that c. meleagridis was the species responsible. a . %, . % and . % pre-slaughter prevalence respectively (age - weeks) was detected upon faecal examination of three flocks from the same farm (mcevoy and giddings ). one of turkeys was positive at post-slaughter examination (age weeks). upon dna analysis, all six positive samples were identified as c. parvum. in a recent study, a . % prevalence of c. meleagridis was shown in deceased turkeys, with the highest prevalence ( . %) in poults aged - days (baroudi et al. ) . in one study, ( %) of ducklings and ( %) of goslings aged - days were infected with cryptosporidium (richter et al. ) . infection was present in both intestinal and respiratory tract, but oocyst morphology was not described. in a study on experimental infection with usutu virus in geese, cryptosporidium developmental stages in tissue samples were an accidental finding. this was further investigated by in situ-hybridization, and cryptosporidium infection was detected in % of conjunctival tissue samples and % of bursal tissue samples. dna analysis revealed presence of c. baileyi (chvala et al. ) . c. baileyi was also identified in two ducks in rio de janeiro (huber et al. ). ratites cryptosporidium infection in ostriches was first described in the early s (allwright and wessels ; bezuidenhout et al. ; gajadhar gajadhar , penrith et al. ; penrith and burger ) . infection was first identified in faecal samples from ( . %) of ostriches imported from africa to canada (gajadhar ) . penrith and burger ( ) identified invasive stages in a section of the small intestine of a -week old chick that has suffered from rectal prolapse, and allwright and wessels ( ) identified cryptosporidium in histology sections of the bursa, intestine and pancreatic ducts. in , gajadhar et al. characterized the isolated oocysts and investigated host specificity. the oocysts were morphologically similar to those of c. meleagridis, but attempts to infect suckling mice, chickens, turkeys and quail failed, indicating that this was probably another species. in addition, only faecal samples were investigated, so the infection site was not determined (gajadhar ) . as this study was conducted before molecular tools were commonly used for cryptosporidium species determination, the true identity of this isolate will remain unknown. a low prevalence, with only ( . %) of investigated samples from ostriches aged months- years being cryptosporidium positive, was found in greece (ponce gordo et al. ) . oocysts were of two sizes, .  . μm and .  . μm, indicating the presence of two different species. in contrast, in a spanish study a % cryptosporidium prevalence in adult rheas and ostriches was found (ponce gordo et al. ) . the authors reported an oocyst diameter of - μm, which is similar to the description provided by gajadhar ( ) . molecular analysis of the isolates was not performed. oliveira et al. ( ) found % prevalence in ostriches based on microscopy. oocysts were generally morphologically similar to c. baileyi and cryptosporidium avian genotype ii . however, the morphometric variation was so large that the authors suggested that more than one species had been identified (oliveira et al. ) , but this was not verified by molecular analysis. an isolate similar to c. baileyi in both oocyst morphology and pcr-rflp banding pattern was described from brazilian ostriches (santos et al. ) . the isolate was characterized as a sister taxon to c. baileyi by sequence analysis of the s rdna, hsp and actin genes (meireles et al. ) , and was named cryptosporidium avian genotype ii by another research group (ng et al. ) . experimental infection (oral or intratracheal) with the brazilian isolate in chickens failed (meireles et al. ) . the avian genotype ii has also been identified in vietnam. on a single ostrich farm ( . %) of samples were positive for cryptosporidium oocysts. the highest prevalence as well as the highest shedding intensity ( . %) was found in - month-old animals. of samples used for molecular characterization, all were found to be avian genotype ii (nguyen et al. ) . wang et al ( b) reported cryptosporidium infection in ( . %) of investigated ostrich samples. prevalence peaked at the age of - weeks with . %. no infection was detected in birds younger than week or older than months. molecular analysis of positive samples identified only c. baileyi. quails and partridges enteric cryptosporidiosis in quails, with oocysts similar to c. meleagridis, was first described in (hoerr et al. ; ritter et al. ) . early attempts at experimental infection of quail with c. baileyi isolated from chickens failed lindsay et al. ), but were later successful (cardozo et al. ) . natural infection was first documented in ). since then, natural c. baileyi infection has been described in two reports (murakami et al. ; wang et al. ) . one large survey of cryptosporidium infection in quails was performed in china . out of , faecal samples, ( . %) from ( . %) farms were positive. infection was most common among - -day old quails ( . %). dna analysis revealed c. baileyi in samples and c. meleagridis in two samples. one case of cryptosporidium infection in partridges was described (pages-mante et al. ) . pigeons there are a few reports of cryptosporidiosis in pigeons (ozkul and aydin ; qi et al. ; radfar et al. ; rodriguez et al. ) . radfar et al. ( ) describe an overall prevalence of . % in examined adult and nestling birds, with . % prevalence in adults and . % prevalence in nestlings. the other articles are case reports (ozkul and aydin ; rodriguez et al. ) and a study on pet birds in general, where c. meleagridis was found in one pigeon (qi et al. ). respiratory as well as intestinal and bursal cryptosporidium infections cause disease in chickens, but infection without clinical symptoms has also been observed (fletcher et al. ; taylor et al. ) . in spain, a % morbidity due to respiratory infection in one flock was caused by cryptosporidium sp. weekly mortality rates were . - . % (fernandez et al. ). infection was detected in the trachea and oesophagus. in another flock investigated in the same study, weight loss was the primary symptom, and bursal cryptosporidiosis was diagnosed (fernandez et al. ). goodwin et al. ( ) found a correlation between c. baileyi infection of the trachea and severity of tracheitis symptoms, airsacculitis and condemnation of birds. in respiratory cryptosporidiosis, co-infection with other pathogens has been identified in a number of studies. cryptosporidium sp. and concurrent adenovirus infection was identified in a large broiler flock with respiratory disease (dhillon et al. ) . in a retrospective study on post mortem diagnoses of respiratory cryptosporidiosis, it was found that co-infection with virus or bacteria was common (goodwin et al. a) . in another study, cryptosporidium sp. and aspergillus or bacteria were detected in the lungs of four layer chickens that died from pneumonia. cryptosporidium were also found in the ureters and kidneys (nakamura and abe ) . the effect of cryptosporidium infection alone on development of clinical symptoms in these cases cannot be estimated, but there is probably a synergistic effect of co-infections, increasing the severity. such a synergistic effect of co-infection with infectious bronchitis virus or escherichia coli has been reported (blagburn et al. ) . respiratory symptoms were reported from chickens that had been experimentally inoculated intra-tracheally with c. baileyi, whereas infection was successful but caused no symptoms in orally inoculated chickens (lindsay et al. ) . c. meleagridis infection was associated with diarrhoea and mortality in one study of algerian chickens (baroudi et al. ). experimental c. meleagridis infection of chickens has been observed to result in the chickens becoming indolent and having soiled feathers. growth retardation was reported, but compensatory growth occurred after a few weeks (tumova et al. ) . turkey was the first animal species in which clinical cryptosporidiosis was described (slavin ) . infection was associated with diarrhoea at - days of age, but other parasites (including histomonas, trichomonas and strongylides) were also detected. experimental infection (crop inoculation) with c. meleagridis produced infection of the ileum, caecum and bursa, but was not associated with clinical symptoms (bermudez et al. ). the isolate used was from symptomatic poults; however, these were simultaneously infected with reovirus (causing enteritis and hepatitis). co-infection with cryptosporidium and reovirus in turkeys with enteritis and hepatitis, leading to increased mortality, has also been shown in another study (wages and ficken ) . the presence of other pathogens in these studies could indicate a low to moderate primary pathogenicity of c. meleagridis. self-limiting diarrhoea (moderate to severe in character), a slower growth rate and growth deformities were reported from one farm where diseased poults were diagnosed with cryptosporidium infection (goodwin et al. b ). other pathogens were not excluded, as was also mentioned by the authors. in dead poults that had suffered from depression and diarrhoea (faeces adhered on the hind part of the body), necropsy revealed lesions in the small intestine. microscopic investigation identified cryptosporidium sp. in the respiratory tract and kidneys, as well as in the gastrointestinal tract (tacconi et al. ) . diarrhoea, emaciation, lethargy and reduced growth associated with natural c. meleagridis infection have been reported from iran, but the presence of other pathogens was not excluded (gharagozlou et al. ) . of diarrhoeal and/or unthrifty birds, ( . %) were identified as cryptosporidium positive by histology, and c. meleagridis was reported based on oocyst morphology. baroudi et al. ( ) identified c. meleagridis in ( %) of examined turkeys that died from diarrhoea, but infection with other pathogens was not investigated. respiratory cryptosporidiosis has also been described in turkeys (ranck and hoerr ; tarwid et al. ) . tarwid et al. ( ) identified cryptosporidium sp. in necropsied birds from two outbreaks of colibacillosis. colibacillosis is, according to the authors, a secondary disease in turkeys, and cryptosporidium sp. was identified as the primary pathogen. symptoms were frothy conjunctivitis and increased mortality. necropsy revealed pathological changes such as pericarditis, peritonitis and air-sacculitis in addition to the conjunctivitis that was observed in live birds. thirteen birds with respiratory disease were all positive for cryptosporidium sp. by histology (ranck and hoerr ) . microscopy of sinus and/or tracheal exudates revealed oval oocysts in some samples, but oocyst size was not described, and both c. baileyi and c. meleagridis can appear oval (length/width ratios of . - . and . - . respectively ). symptoms such as coughing, rattling, sneezing, frothy eyes and swollen sinuses were reported. other pathogens were present in all but two of the examined birds, and it is unclear whether the infection with cryptosporidium played a primary role in the pathogenesis or not. studies on cryptosporidiosis in turkeys, including clinical symptoms, are summarised in table . . clinical cryptosporidiosis in ducks and geese seems to be less common and milder (see table . ) than in other poultry. only mild respiratory symptoms resulted from experimental c. baileyi infection (both oral and intratracheal inoculation) in ducks (lindsay et al. ). respiratory and intestinal infection occurred for both infection routes, but symptoms (sneezing, rales, mild dyspnea) were only present in animals infected by the intratracheal route. mason ( ) described a case of conjunctival cryptosporidiosis. however, since only one of affected ducks was cryptosporidium positive, the author concluded that the parasite was not the cause of the disease. similarly, no symptoms occurred in geese in which cryptosporidium infection was detected in the conjunctivas and bursas (chvala et al. ); and richter et al. ( ) noted that enteritis and upper respiratory tract symptoms were equally present in infected and non-infected ducks and geese. mortality was not increased in the positive flocks (richter et al. ) . ratites cryptosporidium infection in ostrich chicks has been associated with cloacal and phallus prolapse, leading to high mortality (bezuidenhout et al. ; penrith et al. ; santos et al. ) . bezuidenhout et al ( ) found that prolapsed cloacas were heavily infected, whereas penrith et al. ( ) described heavy infection of both the bursa and cloaca in affected chicks, but healthy chicks were not infected. santos et al ( ) also identified cryptosporidium infection in the rectum, coprodeum, urodeum and bursa of two dead chicks with cloacal prolapse, both originating from a farm with high mortality rates in - -day-old chicks. however, the authors did not associate the problems with the infection, since changed management practices decreased clinical symptoms and mortality, although cryptosporidium infection was still present on the farm. enteritis was indicated by the presence of intestinal invasive stages and rectal prolapse in one chick examined by penrith and burger ( ) . because diarrhoea was not reported it is unknown whether the prolapse was caused by intense bowel movements or something else. cryptosporidium infection has also been associated with pancreatic necrosis (allwright and wessels ) . quails and partridges cryptosporidium infection has been shown in both diarrhoea and respiratory disease in quails (guy et al. ; hoerr et al. ; murakami et al. ; ritter et al. ). hoerr et al. ( ) reported high mortality rates from days of age in quails infected with cryptosporidium sp., and with no bacterial or viral pathogens detected. acute fatal diarrhoea with mortality rates of up to % in - -day-old birds was described by ritter et al ( ) . reovirus was also detected in necropsied birds, but another study reported that experimental infection with reovirus alone did not produce diarrhoea, whereas infection with cryptosporidium sp., either alone or simultaneously with reovirus, resulted in severe diarrhoea and mortality (guy et al. ) . a synergistic effect of co-infection was, however, shown, since oocyst shedding was higher and reovirus infection became systemic and liver necrosis occurred (guy et al. ) . muramaki et al. ( ) reported a daily mortality rate of . % in one farm, where birds suffered from upper respiratory tract disease and decreased egg production. respiratory symptoms were head swelling, nasal discharge and increased lacrimation, and necropsy revealed sinusitis, airsacculitis and egg peritonitis. co-infection of cryptosporidium sp., mycoplasma gallisepticum and other bacteria was shown. the authors concluded that m. gallisepticum was the primary pathogen, but that the mixed infections in conjunction with high ammonia concentrations in the air worsened the symptoms. the role of cryptosporidium infection in respiratory disease in quails thus remains unclear. wang et al. ( ) reported that no clinical symptoms were seen in , sampled quails, of which were cryptosporidium positive. c. meleagridis was the only pathogen identified in an outbreak of diarrhoea and cough in red-legged partridge chicks (pages-mante et al. ) . morbidity rates were - % and mortality more than %, indicating high pathogenicity. invasive stages were identified in both the respiratory and intestinal tract, suggesting that not only c. bailey might be associated with respiratory avian cryptosporidiosis. pigeons diarrhoea associated with cryptosporidiosis in pigeons has been described in four birds (ozkul and aydin ; rodriguez et al. ) . rodriguez et al. ( ) described a % morbidity of yellow watery diarrhoea, weight loss, dehydration and weakness in a farm with pigeons. mortality was % and necropsy of three birds revealed invasive stages of cryptosporidium in the small intestine, caecum, colon, cloaca, and bursa. no viruses or bacteria could be isolated. ozkul and aydin ( ) identified invasive stages in the small intestine of a pigeon that had been depressed and had evidence of diarrhoea in the form of faeces in its hind feathers. isolates of both c. baileyi and c. meleagridis derived from one domestic bird species have been successfully transmitted to other domestic birds lindsay et al. ) . c. galli has not been experimentally transmitted between different domestic birds, but has been shown in finches as well as chickens (blagburn et al. ; pavlásek pavlásek , ryan et al. b) , and thus has the potential to infect different bird species. the prepatent period of c. baileyi is approximately - days (hornok et al. ; lindsay et al. ; rhee et al. ; tumova et al. ) . however, in the first report on c. baileyi infection in chickens, a prepatent period of up to days was described ). older chicks have a slightly longer prepatent periods than younger ones (lindsay et al. ; rhee et al. ; taylor et al. ; tumova et al. ) . the patent period varies more. at oral inoculation of -day-old chicks, a patent period of days was seen, whereas it was - days in chicks inoculated at days of age, - days in chicks inoculated at days of age and < days in chicks inoculated at days of age (lindsay et al. ). with intratracheal inoculation, the same authors described patent periods of , - , - and < days in these age groups (lindsay et al. ). rhee et al. ( ) and tumova et al. ( ) observed a mean patent period of approximately days. oocyst excretion peaked on day and days - post inoculation, respectively (rhee et al. ; tumova et al. ) . taylor et al. ( ) showed shorter patent periods and lower total oocyst output in older than younger chickens. there was also an effect of infection dose, in that oocyst output was higher and declined more slowly with lower infection doses (taylor et al. ) . similar observations were made for -and -week old chicks (sreter et al. ) . in that study, the mean patent period for -week old chicks was days, but one chicken shed oocysts for days. c. meleagridis was shed in the faeces on days - post infection in two chickens experimentally infected at weeks of age (woodmansee et al. ). tumova et al. ( ) infected -day-old chicks. oocysts first appeared days later and the patent period lasted for - days. shedding rates were significantly lower than in chicks inoculated with the same number of c. baileyi oocysts. the prepatent and patent period of c. galli has been described to be and days respectively (pavlásek ) , but was later reported as unknown when c. galli was redescribed (ryan et al. b ). the prepatent period of c. meleagridis in turkeys inoculated at - days of age was - days (bermudez et al. ; sreter et al. ; woodmansee et al. ) . woodmansee et al. ( ) reported that oocysts were shed for only days; sreter et al. ( ) found the patent period to be - days, whereas bermudez et al. ( ) reported oocyst shedding and invasive stages still being present at day post inoculation. oocyst shedding rates were moderate (sreter et al. ) , and low to moderate (bermudez et al. ) . experimental infection with c. baileyi induced mild infection of the bursa ). lindsay et al. ( ) inoculated turkey poults via the intratracheal, oral and intracloacal route. all three experiments caused infection, but only poults inoculated via the trachea developed symptoms (lindsay et al. ). lindsay et al ( ) described a prepatent period of days and a possible patent period of - days in experimentally infected muscovy ducks, based on investigation of pooled faecal samples (lindsay et al. ). oocyst morphology was not described. the intestine, bursa and cloaca were positive for invasive stages, but these tissues can be infected by both c. baileyi and c. meleagridis (table . ). for quails, one study describes a prepatent period of days and a patent period of days for c. baileyi (cardozo et al. ) . otherwise, no data are available. only one of the species and genotypes commonly infecting birds -cryptosporidium meleagridis -has, so far, proved to be important in human cryptosporidiosis. this species is the third most common species in human cryptosporidiosis worldwide. in the industrialised world, c. meleagridis infection is usually associated with cryptosporidiosis cases in travellers to asia or africa (elwin et al. ; insulander et al. ; leoni et al. ), but autochthonous cases have also been described (elwin et al. ; leoni et al. ; silverlås et al. ) . studies on cryptosporidium prevalence and species distribution in humans in south america have identified c. meleagridis infection at about the same prevalence as c. parvum (cama et al. (cama et al. , . although this is a true zoonotic species, there is only one report in which the bird source has been identified, and in that case, chickens and not turkeys were involved (silverlås et al. ) . it is not known whether anthroponotic transmission occurs with this species, but it has been indicated by the fact that not all c. meleagridis-infected patients in an epidemiological investigation had had bird or animal contact (elwin et al. ) . c. meleagridis has the potential to infect other mammalian species as well, and experimental infection of mice, rats, rabbits, pigs and calves has been reported (akiyoshi et al. ; darabus and olariu ) . due to the wide host range and the close relationship of c. meleagridis to c. parvum and c. hominis, it has been proposed that this species originated as a mammalian cryptosporidium species, and later adapted to birds (xiao et al. b . one study has identified c. parvum in turkeys (mcevoy and giddings ), indicating that this species could play a role in zoonotic transmission. however, only one of birds post-slaughter was positive compared to . - . % of the - -week younger poults, which means risk of transmission via contaminated meat should be very small. the higher prevalence in poults should not pose a risk as long as the flocks are closed to the public. the shedding intensity was not reported, but prevalence indicates that infection rather than just intestinal passage was present. some studies have identified c. parvum, c. hominis and c. hominis-like isolates in canada geese (jellison et al. (jellison et al. , zhou et al. ). the authors conclude that these findings are probably not associated with infection and parasite proliferation, but rather transient carriage. nevertheless, this indicates that domesticated ducks and geese can potentially act as transmission vehicles for these species. infection with c. baileyi has been identified in one immunodeficient patient. diagnosis was based on oocyst morphology and biology -experimental infection of mice failed whereas inoculated chickens developed infection of the intestine, bursa and trachea (ditrich et al. ) . since this patient was immunodeficient and no other reports exist, this species should not be considered as a true zoonotic agent. ever since animals were first domesticated, and humans became dependent upon them for the commodities that they supply, particularly food and fibre, the infections that affect the health and productivity of livestock have been a concern. cryptosporidiosis was first identified as a disease of veterinary significance in the s (in turkeys) and then in the early s in calves, but major interest in cryptosporidiosis only developed with the first report of a human cases later that decade, and the recognition that cryptosporidium infection was also of medical importance. since then our knowledge on the veterinary significance of cryptosporidium infection has expanded enormously -particularly in the livestock sector most impacted by cryptosporidiosis -young calves. however, as demonstrated in this chapter, it should not be forgotten almost all farmed animals may be pathologically affected by at least one species of cryptosporidium, often causing clinical disease that in some instances may be fatal. for some cryptosporidium species in some farmed animal species, transmission may be anthropozoonotic. cryptosporidium is a hugely successful parasite, as demonstrated by its host range and wide geographic distribution, and its control has proved challenging. as long as humans raise and depend on animals, there will be a need to control the transmission of cryptosporidiosis amongst livestock species. renal cryptosporidiosis (cryptosporidium baileyi) in specific-pathogen-free chickens experimentally coinfected with marek's disease virus cryptosporidial infection in man and faro animals in ismailia governorate a longitudinal study on enteropathogenic infections of livestock in trinidad characterization of cryptosporidium meleagridis of human origin passaged through different host species cryptosporidium species in ostriches sheep management and cryptosporidiosis in central mexico distribution of cryptosporidium subtypes in humans and domestic and wild ruminants in portugal cryptosporidium genotypes and subtypes in dairy calves in egypt identity and public health potential of cryptosporidium spp. in water buffalo calves in egypt experimental infection in mice of cryptosporidium muris isolated from a camel cryptosporidiosis in bovine and human health mammalian cryptosporidiosis: a veterinary perspective foot and mouth disease online database. animal health and welfare: fmd data archive. department for environment, food and rural affairs (defra) the development of a national collection for oocysts of cryptosporidium. uk drinking water inspectorate (dwi) / / . marlow (uk), foundation for water research cryptosporidium in Ö stersund (in swedish). swedish institute for communicable disease control villous atrophy crypt hyperplasia cellular infiltration and impaired glucose-na absorption in enteric cryptosporidiosis of pigs prevalence of and associated risk factors for shedding cryptosporidium parvum oocysts and giardia cysts within feral pig populations in california cryptosporidium infection in non-human hosts in malawi acute diarrhoeal disease in rabbit: bacteriological diagnosis and efficacy of oral rehydration in combination with loperamide hydrochloride cryptosporidium agni sp.n. from lambs and cryptosporidium bovis sp.n. from a calf with observations on the oocyst common occurrence of zoonotic pathogen cryptosporidium meleagridis in broiler chickens and turkeys in algeria intestinal and bursal cryptosporidiosis in turkeys following inoculation with cryptosporidium sp. isolated from commercial poults prolapse of the phallus and cloaca in the ostrich (struthio camelus) prevalence of cryptosporidiosis in neonatal buffalo calves in ludhiana district of punjab, india cryptosporidiosis in young alpacas balantidium, cryptosporidium and giardia species infections in indoor and outdoor pig production units in croatia persistent infection in a dairy herd with an unusual genotype of cryptosporidium parvum cryptosporidium sp. infection in the proventriculus of an australian diamond firetail finch (staganoplura bella: passeriformes, estrildidae) pathobiology of cryptosporidiosis (c. baileyi) in broiler chickens diagnostics of dairy and beef cattle diarrhea natural infection by giardia sp. and cryptosporidium sp. in dairy goats associated with possible risk factors of the studied properties identification of cryptosporidium felis in a cow by morphologic and molecular methods outbreak of cryptosporidiosis associated with a disinfected groundwater supply molecular epidemiology of cryptosporidium subtypes in cattle in england giardia and cryptosporidium on dairy farms and the role these farms may play in contaminating water sources in prince edward island occurrence of cryptosporidium and giardia on beef farms and water sources within the vicinity of the farms on prince molecular epidemiology of cryptosporidium and giardia in humans on prince edward island, canada: evidence of zoonotic transmission from cattle occurrence of giardia and cryptosporidium in pigs on prince edward island prevalence and genotyping of cryptosporidium species from farm animals in mongolia antibody responses following administration of a cryptosporidium parvum rcp / vaccine to pregnant cattle fecal shedding of cryptosporidium oocysts in healthy alpaca crias and their dams molecular identification of cryptosporidium parvum and giardia duodenalis in the italian water buffalo (bubalus bubalis) a rare cryptosporidium parvum genotype associated with infection of lambs and zoonotic transmission in italy cryptosporidium species and genotypes in hiv-positive patients in lima peru differences in clinical manifestations among cryptosporidium species and subtypes in hiv-infected persons cryptosporidium species and subtypes and clinical manifestations in children peru cryptosporidium e isospora suis nel suino in italia experimental transmission of cryptosporidium baileyi (apicomplexa: cryptosporidiidae) isolated of broiler chicken to japanese quail (coturnix japonica) sheep as a source of human cryptosporidiosis prevalence of and risk factors involved in the spread of neonatal bovine cryptosporidiosis in galicia giardia duodenalis and cryptosporidium parvum infections in adult goats and their implications for neonatal kids occurrence of cryptosporidium parvum and giardia duodenalis in healthy adult domestic ruminants cryptosporidium spp. and giardia duodenalis in two areas of galicia prevalence of cryptosporidium and giardia in roe deer (capreolus capreolus) and wild boars (sus scrofa) in galicia (nw spain) potential pathogens in feces from unweaned llamas and alpacas with diarrhea cryptosporidiosis outbreak at a summer camp-north carolina waterborne outbreaks of cryptosporidiosis direct comparison of selected methods for genetic categorisation of cryptosporidium parvum and cryptosporidium hominis species cryptosporidium sp. rabbit genotype a newly identified human pathogen sporadic human cryptosporidiosis caused by cryptosporidium cuniculus, united kingdom unexpected results from large-scale cryptosporidiosis screening study in calves in tanzania prophylaxis using paromomycin of natural cryptosporidial infection in neonatal kids prevalence and molecular characterization of cryptosporidium spp. in dairy cattle from farms in china prevalence of cryptosporidium spp. in pigs in shanghai china cryptosporidium infection in domestic geese (anser anser f. domestica) detected by in-situ hybridization two cases of zoonotic cryptosporidiosis in spain by the unusual species cryptosporidium ubiquitum and cryptosporidium felis investigating public health impacts of deer in a protected drinking water supply watershed epidemiology of parasitic protozoan infections in soay sheep the life cycle of cryptosporidium baileyi n. sp. (apicomplexa cryptosporidiidae) infecting chickens the homologous and interspecies transmission of cryptosporidium parvum and cryptosporidium meleagridis a review of the importance of cryptosporidiosis in farm animals several enteropathogens are circulating in suckling and newly weaned piglets suffering from diarrhea in the province of villa clara control of cryptosporidiosis in neonatal calves: use of halofuginone lactate in two different calf rearing systems peri-parturient rise of cryptosporidium oocysts in cows: new insights provided by duplex quantitative real-time pcr herd-level risk factors for cryptosporidium infection in dairy-goat kids in western france respiratory cryptosporidiosis in broiler chickens dynamic of infections by cryptosporidum spp. and giardia spp. in buffaloes (bubalus bubalis) during the first three months of life genotype and subtype analysis of cryptosporidium isolates from calves and lambs in galicia identification of cryptosporidiumxiaoi in diarrhoeic goat kids (capra hircus) in spain the first finding of cryptosporidium baileyi in man evidence of host-associated populations of cryptosporidium parvum in italy cryptosporidial oocyst in faeces of water buffalo and zebu calves in india risk factors associated with cryptosporidium parvum infection in cattle infectivity of cryptosporidium parvum genotype i in conventionally reared piglets and lambs the impact of the current housing and husbandry systems on the health and welfare of farmed domestic rabbits cryptosporidiosis in buffalo calves (bubalus bubalis): prevalence and potential risk factors modification of a rapid method for the identification of gene-specific polymorphisms in cryptosporidium parvum and its application to clinical and epidemiological investigations the epidemiology of sporadic human infections with unusual cryptosporidia detected during routine typing in england and wales cryptosporidium andersoni from a danish cattle herd: identification and preliminary characterisation cryptosporidium parvum: infectivity and pathogenicity of the 'porcine' genotype results of parasitological examinations of faecal samples from horses, ruminants, pigs, dogs, cats, hedgehogs and rabbits between cryptosporidium muris: prevalence, persistency and detrimental effect on milk production in a dry-lot dairy detection and characterization of giardia duodenalis and cryptosporidium spp. on swine farms in ontario cryptosporidium xiaoi n. sp. (apicomplexa: cryptosporidiidae) in sheep (ovis aries) chronic cryptosporidiosis in a bactrian camel (camelus bactrianus) cryptosporidium parvum infection in bovine neonates: dynamic clinical parasitic and immunologic patterns cryptosporidium canis n. sp. from domestic dogs cryptosporidium bovis n.sp. (apicomplexa: cryptosporidiidae) in cattle (bos taurus) prevalence of species and genotypes of cryptosporidium found in - -year-old dairy cattle in the eastern united states prevalence of cryptosporidium species and genotypes in mature dairy cattle on farms in eastern united states compared with younger cattle from the same locations cryptosporidium ryanae n. sp. (apicomplexa: cryptosporidiidae) in cattle (bos taurus) species of cryptosporidium detected in weaned cattle on cow-calf operations in the united states cryptosporidium species in calves submitted for postmortem examination in england and wales cryptosporidium species infection in pigs in east anglia evidence supporting zoonotic transmission of cryptosporidium spp. in wisconsin wide geographic distribution of cryptosporidium bovis and the deer-like genotype in bovines common occurrence of a unique cryptosporidium ryanae variant in zebu cattle and water buffaloes in the buffer zone of the chitwan national park cryptosporidiosis in chickens from southern spain molecular characterization of cryptosporidium in brazilian sheep cryptosporidium pig genotype ii diagnosed in pigs from the state of rio de janeiro, brazil detection of cryptosporidium oocysts in extra-intestinal tissues of sheep and pigs cryptosporidiosis of the bursa of fabricius of chickens outbreak of cryptosporidiosis among veterinary students cryptosporidium species in imported ostriches and consideration of possible implications for birds in canada host specificity studies and oocyst description of a cryptosporidium sp. isolated from ostriches prevalence and genotyping of cryptosporidium in three cattle husbandry systems in zambia prevalence and molecular characterisation of cryptosporidium and giardia in lambs and goat kids in belgium intestinal cryptosporidiosis in turkeys in iran effect of halofuginone lactate on treatment and prevention of lamb cryptosporidiosis: an extensive field trial comparison of two techniques for diagnosis of cryptosporidiosis in diarrhoeic goat kids and lambs in cyprus experimental infection of a lamb with cryptosporidium parvum genotype cryptosporidium hominis in a goat and a sheep in the uk sinusitis in turkeys associated with respiratory cryptosporidiosis further report on cryptosporidium in barcelona zoo mammals characterisation of a cryptosporidium isolate from water buffalo (bubalus bubalis) by sequencing of a fragment of the cryptosporidium oocyst wall protein gene (cowp) presence and molecular characterisation of giardia and cryptosporidium in alpacas (vicugna pacos) from peru respiratory cryptosporidiosis in chickens diarrhea associated with intestinal cryptosporidiosis in turkeys respiratory coccidiosis (cryptosporidium baileyi) among northern georgia broilers in one company zoonotic cryptosporidiosis from petting farms transmission dynamics of cryptosporidium in primates and herbivores at the barcelona zoo: a long-term study giardia sp. cysts and infectious cryptosporidium parvum oocysts in the feces of migratory canada geese (branta canadensis) retrospective cohort study of an outbreak of cryptosporidiosis caused by a rare cryptosporidium parvum subgenotype biology of cryptosporidium parvum in pigs: from weaning to market experimental reproduction of enteritis in bobwhite quail (colinus virginianus) with cryptosporidium and reovirus prevalence of cryptosporidium and giardia in free-ranging wild cervids in norway occurrence of cryptosporidium and giardia in suckling piglets in norway outbreak of cryptosporidium linked to drinking unpasteurised milk bovine fallopian tube epithelial cells, adult c bl/ mice, and non-neonatal pigs as models for cryptosporidiosis experimental tracheal and conjunctival infections with cryptosporidium sp. in pigs prevalence of giardia and cryptosporidium and characterization of cryptosporidium spp. isolated from wildlife, human and agricultural sources in the north saskatchewan river basin in alberta molecular epidemiology of cryptosporidium in livestock animals and humans in the ismailia province of egypt surveillance for cryptosporidiosis fatal cryptosporidiosis in quail assessment of maternal immunity to cryptosporidium baileyi in chickens total parenteral nutrition in a neonatal llama genotypic characterization and phylogenetic analysis of cryptosporidium sp. from domestic animals in brazil foot and mouth disease and cryptosporidiosis: possible interaction between two emerging infectious diseases sporadic cryptosporidiosis case-control study with genotyping antibody fusions reduce onset of experimental cryptosporidium parvum infection in calves molecular characterisation of cryptosporidium isolates from pre-weaned calves in romania: is there an actual risk of zoonotic infections? zoonotic cryptosporidium parvum in romanian newborn lambs (ovis aries) spontaneous cryptosporidiosis in an adult female rabbit developing vaccines to control protozoan parasites in ruminants: dead or alive? molecular epidemiology and clinical manifestations of human cryptosporidiosis in sweden cryptosporidial infection in chickens prevalence of cryptosporidium parvum infections in weaned piglets and fattening porkers in kanagawa prefecture phylogenetic analysis of the hypervariable region of the s rrna gene of cryptosporidium oocysts in feces of canada geese (branta canadensis): evidence for five novel genotypes source tracking identifies deer and geese as vectors of human-infectious cryptosporidium genotypes in an urban/suburban watershed concentrations, viability and distribution of cryptosporidium genotypes in lagoons of swine facilities in the southern piedmont and in coastal plain watersheds of georgia distribution of cryptosporidium genotypes in storm event water samples from three watersheds in new york atypical outbreak of caprine cryptosporidiosis in the sultanate of oman prevalence of cryptosporidium genotypes in pre and post-weaned pigs in australia serum biochemistry, serology and parasitology of boreal caribou (rangifer tarandus caribou) in the northwest territories prevalence of cryptosporidium oocysts in livestock in trinidad and tobago molecular characterization of cryptosporidium from animal sources in qinghai province of china first description of cryptosporidium bovis in japan and diagnosis and genotyping of cryptosporidium spp. in diarrheic pre-weaned calves in hokkaido cryptosporidiosis in three pigs prevalence and molecular characterization of bovine cryptosporidium in qazvin province molecular characterization and assessment of zoonotic transmission of cryptosporidium from dairy cattle in west bengal recurrent outbreaks of cryptosporidiosis associated with calves among students at an educational farm programme natural cryptosporidium sp. infection in broiler chickens in morocco effect of cryptosporidium parvum infection on the absorptive capacity and paracellular permeability of the small intestine in neonatal calves preliminary communication on cryptosporidiosis of pigs in viet-nam isolation of cryptosporidium andersoni kawatabi type in a slaughterhouse in the northern island of japan occurrence of cryptosporidium and giardia in wild ducks along the rio grande river valley in southern new mexico failed attempt of cryptosporidium andersoni infection in lambs prevalence and age-related infection of cryptosporidium suis, c. muris and cryptosporidium pig genotype ii in pigs on a farm complex in the czech republic molecular characterization of cryptosporidium isolates from pigs at slaughterhouses in south bohemia molecular characterization of cryptosporidium spp. in pre-weaned dairy calves in the czech republic: absence of c. ryanae and management-associated distribution of c. andersoni, c. bovis and c. parvum subtypes cryptosporidium scrofarum n. sp. (apicomplexa: cryptosporidiidae) in domestic pigs (sus scrofa) prevalence and risk factors associated with intestinal parasites in pigs in chongqing submitted) second outbreak of infection with a rare cryptosporidium parvum genotype among schoolchildren associated with contact with lambs/goat kids at a holiday farm in norway langkjaer rb, vigre h, enemark hl, maddox-hyttel c ( ) molecular and phylogenetic characterization of cryptosporidium and giardia from pigs and cattle in denmark genetic analysis of cryptosporidium from humans with diarrhoea in england between and host specificity of cryptosporidium sp. isolated from chickens experimentally induced infections in turkeys with cryptosporidium baileyi isolated from chickens effect of broiler chicken age on susceptibility to experimentally induced cryptosporidium baileyi infection experimental infections in domestic ducks with cryptosporidium baileyi isolated from chickens cryptosporidium andersoni n. sp. (apicomplexa: cryptosporiidae) from cattle, bos taurus molecular identification and distribution of cryptosporidium and giardia duodenalis in raw urban wastewater in harbin, china prevalence of neonatal cryptosporidiosis in andean alpacas (vicugna pacos) in peru a massive outbreak in milwaukee of cryptosporidium infection transmitted through the public water supply cryptosporidium and giardia in different age groups of danish cattle and pigs -occurrence and management associated risk factors cryptosporidiosis among animal handlers and their livestock in basrah molecular characterization of cryptosporidium spp. in native breeds of cattle in kaduna state population structures and the role of genetic exchange in the zoonotic pathogen cryptosporidium parvum multilocus genotyping of cryptosporidium parvum type : population genetics and sub-structuring conjunctival cryptosporidiosis in a duck intestinal cryptosporidiosis in a kid goat prevalence and risk factors associated with cryptosporidium spp. infection in young domestic livestock in india cryptosporidium in commercially produced turkeys on-farm and postslaughter molecular epidemiological analysis of cryptosporidium spp. in the united kingdom: results of genotyping cryptosporidium spp. in , fecal samples from humans and fecal samples from livestock animals biological studies and molecular characterization of a cryptosporidium isolate from ostriches (struthio camelus) molecular characterization of cryptosporidium spp. in dairy calves from the state of são paulo, brazil an outbreak of cryptosporidosis from fresh-pressed apple cider subtype analysis of cryptosporidium parvum isolates from calves on farms around belgrade, serbia and montenegro using the kda glycoprotein gene sequences cryptosporidium infection in nursing, weaning and post-weaned piglets and sows in the belgrade district in: lal r (ed) agricultural sciences -volume . encyclopedia of life support systems prevalence of cryptosporidiosis in buffaloes in and around bhubaneswar fecal transmission of calf cryptosporidia between calves and pigs molecular characterization of cryptosporidium from various hosts molecular and biological characterisation of cryptosporidium in pigs molecular and phylogenetic analysis of cryptosporidium muris from various hosts molecular and phylogenetic characterisation of cryptosporidium from birds the population structure of the cryptosporidium parvum population in scotland: a complex picture experimental cryptosporidiosis in adult and neonatal rabbits distribution of cryptosporidium species in sheep in the uk prevalence of land management factors contributing to cryptosporidium sp. infection in pre-weaned and post-weaned calves in role of enteric pathogens in the aetiology of neonatal diarrhoea in lambs and goat kids in spain occurrence of conjunctivitis, sinusitis and upper region tracheitis in japanese quail (coturnix coturnix japonica), possibly caused by mycoplasma gallisepticum accompanied by cryptosporidium sp. infection respiratory (especially pulmonary) and urinary infections of cryptosporidium in layer chickens prevalence of cryptosporidium parvum infection in lahore (pakistan) and its association with diarrhea in dairy calves detection and molecular characterization of cryptosporidium bovis-like isolate from a newborn lamb in spain subtype analysis of cryptosporidium parvum and cryptosporidium hominis isolates from humans and cattle in iran identification of novel cryptosporidium genotypes from avian hosts evidence of cryptosporidium transmission between cattle and humans in northern prevalence and molecular characterization of cryptosporidium in ostriches (struthio camelus) on a farm in central vietnam cryptosporidiosis: a report on the surveillance and epidemiology of cryptosporidium infection in england and wales identification of cryptosporidium species and genotypes in scottish raw and drinking waters during a one-year monitoring period molecular detection of cryptosporidium cuniculus in rabbits in australia prevalence of cryptosporidium infection in goats in selected locations in three agroclimatic zones of sri lanka excretion of cryptosporidium oocysts by goats in relation to age and season in the dry zone of sri lanka coinfection by cryptosporidium parvum and porcine circovirus type in weaned pigs cryptosporidium gp genotypes from humans and domesticated animals in australia, north america and europe cryptosporidium infection in birds and mammals and attempted cross transmission studies giardiasis and cryptosporidiosis in ruminants duration of naturally acquired giardiosis and cryptosporidiosis in dairy calves and their association with diarrhea occurrence of cryptosporidium spp. oocysts (apicomplexa, cryptosporidiidae) in ostriches, struthio camelus l., (aves, struthionidae) reared in north and lowered coastline regions of the state of rio de janeiro giardia and cryptosporidium in canadian farm animals update on cryptosporidium and giardia infections in cattle prevalence and molecular characterization of cryptosporidium spp. in dairy cattle in south bohemia, the czech republic novel cryptosporidium genotypes in sporadic cryptosporidiosis cases: first report of human infections with a cervine genotype cryptosporidiosis in deer calves age-related resistance in ovine cryptosporidiosis: patterns of infection and humoral immune response small-intestinal cryptosporidiosis in a young pigeon an outbreak of disease associated with cryptosporidia on a red-legged partridge (alectoris rufa) game farm cryptosporidial infection in a calf immunoenzymatic analysis and genetic detection of cryptosporidium parvum in lambs from italy evaluation of oral tilmicosin efficacy against severe cryptosporidiosis in neonatal kids under field conditions control of cryptosporidiosis in neonatal goat kids: efficacy of a product containing activated charcoal and wood vinegar liquid (obionekk(r)) in field conditions cryptosporidia: biology diagnosis, host spectrum specificity and the environment findings of cryptosporidia in the stomach of chickens and of exotic and wild birds spontaneous cryptosporidium infection in weaned rabbits (article in czech) distribution of cryptosporidium and giardia spp. in selected species of protected and game mammals from north-eastern poland genetic diversity of cryptosporidium spp. in cattle in michigan: implications for understanding the transmission dynamics a cryptosporidium sp in an ostrich evidence for cryptosporidial infection as a cause of prolapse of the phallus and cloaca in ostrich chicks pathogenesis of human and bovine cryptosporidium parvum in gnotobiotic pigs genotype and subtype analyses of cryptosporidium isolates from cattle in hungary outbreak of cryptosporidiosis among veterinary students parasites from farmed ostriches (struthio camelus) and rheas (rhea americana) in europe a retrospective study of neonatal mortality in farmed elk cryptosporidium parvum infection in orphan lambs on a farm open to the public cryptosporidium anserinum sp. n. (sporozoa) in a domestic goose anser anser l, from iowa cryptosporidium spp. in pet birds: genetic diversity and potential public health significance comparison of oocyst shedding and the serum immune response to cryptosporidium parvum in cattle and pigs prevalence of cryptosporidium infections in pigs in aragón, northeastern spain cryptosporidium genotypes and subtypes in lambs and goat kids in spain cryptosporidium species and subtype analysis from dairy calves in spain biodiversity and prevalence of parasites of domestic pigeons (columba livia domestica) in a selected semiarid zone of south khorasan diseases of the newborn. in: veterinary medicine, th edn prevalence and infection pattern of naturally acquired giardiasis and cryptosporidiosis in range beef calves and their dams cryptosporidia in the respiratory tract of turkeys prevalence of cryptosporidium infection in camels (camelus dromedarius) in a slaughterhouse in iran isolation and identification of cryptosporidium from various animals in korea natural infections by cryptosporidium sp. in farm-raised ducks and geese the prevalence of cryptosporidium and giardia spp. in fecal samples from free-ranging, white-tailed deer (odocoileus virginianus) in the southeastern united states molecular characterization of cryptosporidium spp. in pre-weaned kids in a dairy goat farm in western france cryptosporidium and giardia in water buffaloes (bubalus bubalis) of the italian mediterranean bred giardia and cryptosporidium in water buffaloes (bubalus bubalis) intestinal cryptosporidiosis and reovirus isolation from bobwhite quail (colinus virginianus) with enteritis giardia and cryptosporidium infections in sheep and goats: a review of the potential for transmission to humans via environmental contamination foodborne cryptosporidiosis: is there really more in nordic countries? case-control studies of sporadic cryptosporidiosis in melbourne and adelaide a small outbreak of human cryptosporidiosis associated with calves at a dairy farm in norway the zoonotic potential of giardia and cryptosporidium in norwegian sheep: a longitudinal investigation of flocks of lambs the european rabbit (oryctolagus cuniculus), a source of zoonotic cryptosporidiosis intestinal cryptosporidiosis in pigeons (columba livia) studies on the evolution pathology, and immunity of commercial fattening rabbits affected with epizootic outbreaks of diarrhoeas in mexico: a case report risk factors for sporadic cryptosporidiosis among immunocompetent persons in the united states from to fecal shedding of giardia duodenalis, cryptosporidium parvum, salmonella organisms and escherichia coli o :h from llamas in california identification of a novel cryptosporidium genotype in pigs a redescription of cryptosporidium galli pavlasek, (apicomplexa: cryptosporidiidae) from birds identification of novel cryptosporidium genotypes from the czech republic cryptosporidium suis n. sp. (apicomplexa: cryptosporidiidae) in pigs (sus scrofa) sheep may not be an important zoonotic reservoir for cryptosporidium and giardia parasites cryptosporidium fayeri n. sp. (apicomplexa: cryptosporidiidae) from the red kangaroo (macropus rufus) porcine neonatal coccidiosis: clinical, pathological, epidemiological and diagnostic features enteric cryptosporidial infection in pigs: cases ( - ) livestock prevalence and age-related variation of cryptosporidium species and genotypes in dairy calves a longitudinal study of cryptosporidiosis in dairy cattle from birth, to years of age cryptosporidium infection in ostriches (struthio camelus) in brazil: clinical, morphological and molecular studies prevalence of cryptosporidium infection in goats maintained under semi-extensive feeding conditions in the southeast of spain the prevalence of cryptosporidium species in diarrhoeic lambs in kars province and potential risk factors prevalence of cryptosporidium spp. in camels and involved people in yazd province occurrence and molecular characterization of cryptosporidium spp. isolated from domestic animals in a rural area surrounding atlantic dry forest fragments in teodoro sampaio municipality, state of são paulo highlights of camelid diagnoses from necropsy submissions to the animal health laboratory, university of guelph from the identification of the cryptosporidium ubiquitum in pre-weaned ovines from aba tibetan and qiang autonomous prefecture in china studies on zoonotic cryptosporidiosisparvum in ismailia governorate cryptosporidium spp. in calves and cows from organic and conventional dairy herds systematic review and meta-analyses of the effects of halofuginone against calf cryptosporidiosis prevalence and associated management factors of cryptosporidium shedding in swedish dairy herds cryptosporidium infection in herds with and without calf diarrhoeal problems molecular characterisation of cryptosporidium isolates from swedish dairy cattle in relation to age, diarrhoea and region zoonotic transmission of cryptosporidium meleagridis on an organic swedish farm is there a need for improved cryptosporidium diagnostics in swedish calves? cryptosporidiosis in newborn red deer (cervus elaphus) asymptomatic shedding of cryptosporidium oocysts by red deer cryptosporidium meleagridis (sp. nov.) an outbreak of waterborne cryptosporidiosis caused by posttreatment contamination outbreaks of enteric infections caused by multiple pathogens associated with calves at a farm day camp natural cryptosporidium hominis infections in scottish cattle investigation of farms linked to human patients with cryptosporidiosis in england and wales experimental cryptosporidiosis in germfree lambs genetic classification of cryptosporidium isolates from humans and calves in slovenia molecular characterisation of cryptosporidium isolates from humans in slovenia prevalence of cryptosporidium spp. (eucoccidiorida: cryptosporiidae) in seven species of farm animals in tunisia pcr-rflp analysis of the cryptosporidium oocyst wall protein (cowp) gene discriminates between c. wrairi and c. parvum, and between c. parvum isolates of human and animal origin age-dependent resistance to cryptosporidium baileyi infection in chickens morphologic host specificity and molecular characterization of a hungarian cryptosporidium meleagridis isolate risk factors associated with cryptosporidium infection on dairy farms in a new york state watershed an outbreak of cryptosporidiosis among alpaca crias and their human caregivers survey of zoonoses recorded in scotland between long-term study of cryptosporidium prevalence on a lowland farm in the united kingdom molecular characterization of cryptosporidium isolates from pigs in zaragoza, northeastern spain differentiating human from animal isolates of cryptosporidium parvum cryptosporidium and giardia associated with reduced lamb carcase productivity longitudinal investigation of protozoan parasites in meat lamb farms in southern western australia impacts of naturally acquired protozoa and strongylid nematode infections on growth and faecal attributes in lambs evaluation of factors associated with the risk of infection with cryptosporidium parvum in dairy calves cryptosporidium in market pigs in southern california usa retrospective ultramicroscopic investigation on naturally cryptosporidial-infected commercial turkey poults cryptosporidiosis in the respiratory tract of turkeys in saskatchewan variations in oocyst output associated with cryptosporidium baileyi infections in chickens genotypes and subtypes of cryptosporidium spp. in neonatal calves in northern ireland urinary tract cryptosporidiosis in commercial laying hens genotype and subtype analyses of cryptosporidium isolates from dairy calves and humans in ontario association between management practices and within-herd prevalence of cryptosporidium parvum shedding on dairy farms in southern ontario detection of assemblage a giardia duodenalis and eimeria spp performance and oocyst shedding in broiler chickens orally infected with cryptosporidium baileyi and cryptosporidium meleagridis cryptosporidiosis in two alpaca (lama pacos) holdings in the south-west of england cryptosporidium parvum (sp. nov.), a coccidium found in the small intestine of the common mouse coccidiosis in gallinaceous birds diarrhea in young red deer associated with infection with cryptosporidium experimental infection of piglets with cryptosporidium enterocolitis in piglets caused by cryptosporidium purified from calf faeces experimental cryptosporidiosis in calves: clinical manifestations and pathological findings prevalence of cryptosporidium parvum infection and pattern of oocyst shedding in calves in japan identifying host sources, human health risk and indicators of cryptosporidium and giardia in a canadian watershed influenced by urban and rural activities outbreak of cryptosporidiosis in dairy goats in brazil cryptosporidium parvum in cattle, sheep and pigs in galicia (n.w. spain) incidence an risk factors of cryptosporidium sp. infection in water buffaloes confined in a communal management system in the philippines pathogenesis of intestinal cryptosporidiosis in conventional and gnotobiotic piglets prevalence and pathogenicity of cryptosporidium suis in pre-and post-weaned pigs prevalence of giardia sp. cryptosporidium parvum and cryptosporidium muris (c. andersoni) in dairy herds, in five counties of southeastern new york cryptosporidiosis and turkey viral hepatitis in turkeys cryptosporidiosis in alpaca crias molecular epidemiology spatiotemporal analysis and ecology of sporadic human cryptosporidiosis in australia molecular characterization of the cryptosporidium cervine genotype from a sika deer (cervus nippon temminck) in zhengzhou, china and literature review multilocus phylogenetic analysis of cryptosporidium andersoni (apicomplexa) isolated from a bactrian camel (camelus bactrianus) in china large-scale survey of cryptosporidium spp. in chickens and pekin ducks (anas platyrhynchos) in henan, china: prevalence and molecular characterization prevalence and molecular identification of cryptosporidium spp cervine genotype is the major cryptosporidium genotype in sheep in china prevalence of cryptosporidium baileyi in ostriches characteristics of cryptosporidium transmission in preweaned dairy cattle in henan cryptosporidium spp. in quails (coturnix coturnix japonica) in henan, china: molecular characterization and public health significance neonatal diarrhea in llamas and alpacas prevalence of enteropathogens in suckling and weaned piglets with diarrhoea in southern germany subclinical cryptosporidiosis of turkeys in iowa molecular epidemiology of cryptosporidiosis: an update prevalence of cryptosporidium and giardia infections on two ohio pig farms with different management systems phylogenetic analysis of cryptosporidium parasites based on the small-subunit r-rna gene locus identification of the cryptosporidium pig genotype in a human patient host adaptation and host-parasite co-evolution in cryptosporidium: implications for taxonomy and public health cryptosporidium taxonomy: recent advances and implications for public health prevalence and identity of cryptosporidium spp. in pig slurry prevalence of cryptosporidium-like infection in one-humped camels (camelus dromedarius) of northwestern iran prevalence and molecular characterisation of cryptosporidium and giardia species in pre-weaned sheep in australia prevalence of the cryptosporidium pig genotype ii in pigs from the yangtze river delta infection status of pigs with cryptosporidium parvum cryptosporidium cuniculus and giardia duodenalis in rabbits: genetic diversity and possible zoonotic transmission host-adapted cryptosporidium spp. in canada geese (branta canadensis) prevalence of cryptosporidium species in intensively farmed pigs in ireland key: cord- -e kuwf authors: nan title: opinion of the scientific panel on animal health and welfare (ahaw) on a request from the commission related with the risks of poor welfare in intensive calf farming systems date: - - journal: efsa j doi: . /j.efsa. . sha: doc_id: cord_uid: e kuwf nan summary efsa has been requested by the european commission to issue a scientific opinion on animal health and welfare aspects of intensive calf farming systems and their ability to comply with the requirements of the well-being of calves from the pathological, zootechnical, physiological and behavioural points of view. in particular the commission asked efsa to update the findings of the scientific veterinary committee (animal welfare section) report, on the welfare of calves of november , in the light of more recent data on this issue. where relevant the possible food safety implications of different farming systems should also be considered. in this report a risk assessment was made and the relevant conclusions and recommendations are forming the scientific opinion by the ahaw panel. the svc ( ) report contains information on measurements of welfare, needs of calves, descriptions of current housing systems, chapters on types of feed and feeding systems, weaning of calves, housing and pen design, climate, mananimal relationships, dehorning and castration. further chapters covered economical considerations of systems and for improving welfare. in the report conclusions were made on general management, housing, food and water and economics. the present report "the risks of poor welfare in intensive calf farming systems" is an update o the previous svc report with the exception of economical aspects which are outside of the mandate for this report. the various factors potentially affecting calves' health and welfare, already extensively listed in the report of the scientific veterinary committee animal welfare section (svc, ) , are updated and subsequently systematically determined whether they constitute a potential hazard or risk. to the latter end their severity and likelihood of occurrence in animal (sub) populations were evaluated and associated risks to calf welfare estimated, hence providing the basis for risk managers to decide which measures could be contemplated to reduce or eliminate such risks. in line with the terms of reference the working group restricted itself to (in essence a qualitative) risk assessment although it is agreed that welfare and health of calves can be substantially affected in the course of and as a result of transport and slaughter, this report does not consider animal health and welfare aspects of calves during transport and slaughter but such information can be found in a recently issued comprehensive report of the scientific committee on animal health and animal welfare (scahaw), on "the welfare of animals during transport (details for horses, pigs, sheep and cattle)" which was adopted on march (dg sanco, ) and in the efsa report "welfare aspects of animal stunning and killing methods" (ahaw / ). in relation with the food safety aspects, main foodborne hazards associated with calf farming are salmonella spp., human pathogenic-verotoxigenic escherichia coli (hp-vtec), thermophilic campylobacter spp., mycobacterium bovis, taenia saginata cysticercus and cryptosporidium parvum/giardia duodenalis. present knowledge and published data are insufficient to produce a universal risk assessment enabling quantitative food safety categorization/ranking of different types of calf farming systems. nevertheless, the main risk factors contributing to increased prevalence/levels of the above foodborne pathogens, as well as generic principles for the risk reductions are known. the latter are based on the implementation of effective farm management (e.g. qa, husbandry, herd health plans, biosecurity) and hygiene measures (e.g. gfp-ghp). in general, the conclusions made in the previous svc report remain. however, recent research has provided for some additional conclusions. the risk analysis is presented in the tables of annex . the graphics in this table are not intented to represent numerical relationships but rather qualitative relations. in some instances the exposure could not be estimated due to lack of data, in which cases the risks where labelled "exposure data not available". the following major and minor risks for poor animal health and welfare have been identified for one or several of the various husbandry systems considered: the hazards of iron deficiency and insufficient floor space are considered to be very serious, the hazard of inadequate health monitoring is considered to be serious and the hazards of exposure to inadequate hemoglobin monitoring, allergenic proteins and too rich diet are considered to be moderately serious. for these hazards, there is no consensus on the exposure of calves mainly due to lack of data and that is why it is recommended that further studies should be made to provide evidence for an exposure assessment. regarding castration and dehorning (and disbudding) without anaesthetic drugs, there is a variation in relation to national legislation why the risk of poor welfare in relation to castration and dehorning has a wide range between countries. council directive / /eec laying down minimum standards for the protection of calves as amended by council directive / /ec requires the commission to submit to the council a report, based on a scientific opinion, on intensive calf farming systems which comply with the requirements of the wellbeing of calves from the pathological, zootechnical, physiological and behavioural points of view. the commission's report will be drawn up also taking into account socio-economic implications of different calf farming systems. it should be noted that the scientific veterinary committee (animal welfare section) adopted a report on the welfare of calves on november which should serve as background to the commission's request and preparation of the new efsa scientific opinion. in particular the commission requires efsa to consider the need to update the findings of the scientific veterinary committee's opinion in light of the availability of more recent data on this issue. where relevant the possible food safety implications of different farming systems should also be considered. efsa has been requested by the european commission to issue a scientific opinion on animal health and welfare aspects of intensive calf farming systems and their ability to comply with the requirements of the well-being of calves from the pathological, zootechnical, physiological and behavioural points of view. in particular the commission requires efsa to update the findings of the scientific veterinary committee (animal welfare section) report on the welfare of calves of november in light of more recent data on this issue. where relevant the possible food safety implications of different farming systems should also be considered. the mandate outlined above was accepted by the panel on animal health and welfare (ahaw) at the plenary meeting, on / march . it was decided to establish a working group of ahaw experts (wg) chaired by one panel member. therefore the plenary entrusted a scientific report and risk assessment to a working group under the chairmanship of prof. bo algers. the members of the working group are listed at the end of this report. the scientific report is considered for the discussion to establish a risk assessment and the relevant conclusions and recommendations forming the scientific opinion by the ahaw panel. according to the mandate of efsa, ethical, socio-economic, cultural and religious aspects are outside the scope of this scientific opinion. . . the working group set out to produce a document in which the various factors potentially affecting calves' health and welfare [already extensively listed in the report of the scientific veterinary committee animal welfare section (svc, ) , are updated and subsequently to systematically determine whether these factors constitute a potential hazard or risk. to the latter end their severity and likelihood of occurrence in animal (sub) populations were evaluated and associated risks to calf welfare estimated, hence providing the basis for risk managers to decide which measures could be contemplated to reduce or eliminate such risks. it should be noted, however, that this does not imply that a hazard that has a serious effect on just a few animals should not be dealt with by managers on farm level as the suffering imposed on some animals constitute a major welfare problem for those individuals. in line with the terms of reference the working group restricted itself to (in essence qualitative) risk assessment, i.e. only one of three elements essential to risk analysis a risk assessment approach was followed, similar to the one generally adopted when assessing microbiological risks, i.e. along the lines suggested at the nd session of the codex alimentarius commission (cac, ) . incidentally, these guidelines have been characterized by the cac as 'interim' because they are subject to modifications in the light of developments in the science of risk analysis and as a result of efforts to harmonize definitions across various disciplines. cac's guidelines are in essence exclusively formulated for the purpose of assessing risks related to microbiological, chemical or physical agents of serious concern to public health. consequently -considering their disciplinary focus -the working group had to adapt the cac definitions to serve their purpose. these adapted definitions, have, in alphabethical order, been included in chapter (see risk analysis terminology) the objectives of this report are to review and report recent scientific literature on the welfare including the health of intensively reared calves, to report on recent findings as an update to the scientific veterinary committee's previous report, to make a qualitative risk assessment concerning the welfare of intensively kept calves. where relevant, food safety implications of different farming systems are also considered. the report is structured in five major parts. the first three follow the scientific veterinary committee's previous report "on the welfare of calves" with introductory chapters - on background, measurements and needs in relation to calf welfare, chapter describing housing, diet and management and chapter describing comparison of systems and factors. in chapter common disease and use of antibiotics is described. the other two parts involve aspects of meat quality and food safety (chapter ) and the risk assessment (chapters ). conclusions and recommendations from the previous svc document together with updated conclusions derived from recent research findings are presented in chapter . effect of transport and slaughter on calves' health and welfare although it is agreed that welfare and health of calves can be substantially affected in the course of and as a result of transport, this report does not consider animal health and welfare aspects of calves during transport because there is already a comprehensive recent report of the scientific committee on animal health and animal welfare (scahaw), on "the welfare of animals during transport (details for horses, pigs, sheep and cattle)" which was adopted on march (dg sanco, . the report takes into account all aspects related with transport that could affect the health and welfare of cattle and calves, including the direct effects of transport on the animals and the effects of transport on disease transmission. the loading methods and handling facilities for cattle, the floor space allowance, the relationships of stocking and the density requirements, the vehicle design, space requirements and ventilation for cattle transporters (see also the ahaw scientific opinion related to standards for the microclimate inside animal road transport vehicles, efsa-q- - ), the behaviour of cattle during road transport, the road conditions, long distance transport and the travel times are also reviewed. recommendations for all these aspects are also given in that report. feeding and housing systems, weaning strategies and quality of solid and liquid feed . . . feeding systems and weaning strategies recommendations without a fully functional rumen, calves will be unable to utilise nutrients provided in the post-weaning dry feed diet. attention must paid to type of forage and consistent of particle size of starter grain in order to achieve a proper rumen development. calf weaning should be based on the amount of dry feed calves ingest per day, not on their age or weight, and calf starter should be made available five to days after birth. a calf consuming . kg of dry feed or more on three consecutive days is ready for weaning. when calves are fed low levels of milk to encourage early consumption of dry food, weaning can be done abruptly. in contrast, if milk is given in large amounts, weaning may require two to three weeks of slow transition to avoid a setback in growth. the provision of solid feeds with adequate content and balance to veal calves is a prerequisite for the development of a healthy and functional rumen, the prevention of abnormal oral behaviours, and the stimulation of normal rumination activity. although some solid feeds may exacerbate problems with abomasal ulcers in milk-fed veal calves, properly balanced rations seem to moderate this effect. nutritional factors are clearly involved in the etiology of abomasal ulcers in veal calves. important elements include the consumption of large quantities of milk replacer and the interaction between a milk replacer diet and the provision of roughage. if vegetable proteins are not properly treated, milk replacers may cause hypersensitivity reactions in the gut, which may compromise calf welfare. it is recommended that solid feeds provided to veal calves, in addition to milk replacer, are adequately balanced in terms of the amount of fibrous material, which will promote rumination, and other components such as proteins and carbohydrates, which stimulate rumen development and support a healthy function of the digestive system. since milk replacer formulations are frequently changing, it is recommended to carefully and consistently examine allergenic properties and other possibly detrimental effects of all milk replacers before they are used on a large scale. if the concentration of haemoglobin in the blood of calves drops below . mmol l - , the ability of the calf to be normally active as well as lymphocyte count and immune system function are substantially impaired, and there is reduced growth rate. below . mmol l - , veal calves exhibit a number of adaptations to iron deficiency, including elevated heart rate, elevated urinary noradrenaline and alterered reactivity of the hpa axis. there is a lack of data on the variability in groups of calves. hence, when haemoblogin levels are found to be below . mmol l - in groups of young veal calves, it is field practice to give supplementary iron. for older calves, including those in the last four weeks before slaughter, efficient production is possible in individual calves whose haemoglobin concentration is above . mmol l - . if the concentration of haemoglobin in blood is not checked at all, there is a high risk of anaemia that is associated with poor welfare, for all calves fed a diet with a very low iron content. anaemia can be identified and quantified adequately if checks are carried out on veal production calves of - weeks, for example, when the calves are brought into a unit, between - weeks of fattening, and during the last four weeks before slaughter. if the concentration of haemoglobin in the blood of a group of calves during the last four weeks before slaughter is a mean of . mmol l - , some calves may have a concentration substantially lower than the group-mean, and hence their welfare may be poor. in order to avoid anaemia levels that are associated with poor welfare because normal activity is difficult or not possible and other functions are impaired, it is advisable that diets should be provided that result in blood haemoglobin concentrations of at least . mmol l - throughout the life of the calf. in order to avoid serious impairment of immune system function and hence poor welfare, no individual calf should have a blood haemoglobin concentration lower than . mmol l - . in most cases this is achieved by adjusting the concentration of iron in the diet and having an adequate checking system so that the above condition is avoided. other treatment may be needed for calves with clinical conditions which cause anaemia but which are not related to diet. since the lowest haemoglobin concentrations in the blood of veal calves are usually reached during the last four weeks before slaughter, these blood concentrations should be checked at this time. such controls would help to see if measures are necessary to be taken or not. a checking system using a mean level, but whose aim is to avoid the risk of a low haemoglobin concentration in any individual lower than . mmol l - would have to use a mean substantially higher than , mmol l - , probably mmol l - , and an appropriate sample size. in order to avoid poor welfare associated with anaemia, as explained in the conclusions (above), measurements of average blood haemoglobin concentration are not a satisfactory means of avoiding poor welfare but the use of a minimum level of . mmol l - for individual calves would achieve this. there is a lack of data on the haemoglobin levels and variation in groups in slaughtering calves. to gain more information as a basis for further actions and recommendations, it is advisable to perform sampling of calves at slaughter, by checking the haemoglobin level on a random basis in groups of calves. space and pen design recommendations space should be enough to allow animals to fulfill their needs for social behaviour, lying and grooming. as the pen shape affects the use of space by animals, pens should be rectangular rather than square and pen space should be divided into different usable areas. as the floor type affects the resting and lying postures of calves it should be comfortable. wet floors should be avoided due to thermal and resting problems. degree of social contact conclusions group housing can help calves to acquire social skills. some experience of mixing is important as calves that have been reared for a while in groups dominate calves that have always been in individual crates. when calves are mixed together in the first few days of life, and then kept for some weeks in a social group, there may be poor welfare because of the following risks: . especially when individuals are provided with inadequate access to teats and roughage in the diet, cross-sucking and other abnormal sucking behaviour may occur. . some individuals may be unaccustomed to the food access method, for example they may have only received food via a teat, and may find it difficult to drink from a bucket. . calves coming from different buildings, perhaps from different farms, may carry different pathogens and hence there is a risk of disease spread in all the calves that are put in the same airspace or are otherwise exposed to the pathogens. since calves are social animals, they should be kept in social groups wherever possible. these groups should be stable with no mixing or not more than one mixing. it is advisable for calves in the first two weeks of life not to be mixed with other animals. if calves from different buildings, perhaps different farms, are to be mixed in a pen or are to be put in different pens in the same airspace, quarantining animals for - weeks can reduce disease in the calves and hence prevent poor welfare. although cross-sucking can sometimes be minimised by provision of teats, water and roughage, if this is not possible, mixing into groups could be delayed for three to four weeks. calves fed by various means may require careful supervision after being put into groups in order that they learn how to feed effectively. temperature, ventilation and air hygiene calf rearing causes significant emissions of substances such as nitrate, phosphate, heavy metals and possibly antibiotics in manure and liquid effluents. in addition, there are odours, gases, dusts, micro-organisms and endotoxins in the exhaust air from animal houses. also in the handling of manure in storage and during application of manure and during grazing. these effluents can have distinct impacts on air, water, soil, biodiversity in plants, forest decay and also on animals and including humans. calf houses possess a high potential for emissions of ammonia and other gases. dust, endotoxins and micro-organisms are emitted in lower amounts than from pig or poultry production. odour, bioaerosols, ammonia, nitrogen, phosphorous and heavy metals may either have a local or a regional impact. gases such as methane and nitrous oxide contribute to global warming. respiratory disorders are the second largest reason for morbility and mortality in calf rearing. the most important causes are environmental conditions such as hygiene, management and the physical, chemical and biological factors in the environment. ventilation plays a decisive role in reducing the incidence of respiratory disease. temperatures below ºc can compromise lung function. ammonia concentrations of more than ppm seem to increase respiratory infections. relative humidity of more than % bear the risk of increased heat dissipation and can help bacteria to survive in airborne state. air velocities close to the animals of more than . m/s can significantly increase respiratory sounds in calves. sufficient air space in confined buildings can help to reduce the concentration of airborne bacteria. calf houses contain relatively high amounts of endotoxins ( eu) (eu: endotoxin unit, see scientific report, www.efsa.eu.int) there is concern that antibiotic residues may contribute to the development of bacterial resistance. local and regional environmental problems are enhanced by high animal densities and insufficient distances between farms and residential areas. the exact quantitative contribution of calf rearing to environmental pollution and its impact on water, air, soil vegetation and nearby residents is not yet well understood. when housing systems are compared, although dust emission levels will seldom pose problems for the health of calves, ammonia emission levels may be high enough to exacerbate calf disease, especially when calves are kept in slatted floor units. the development of low emission production systems should be encouraged including mitigation techniques, e.g. biofilters, bioscrubbers, covered manure pits and shallow manure application. in particular there is need to reduce ammonia emissions from slatted floor units or to reduce the usage of such systems. adequate and efficient feeding regimes are required with minimal wastage of nitrogen and phosphorous and limited use of growth promoters and drugs. there is an urgent need for cooperative research to design appropriate ventilation systems to improve health and welfare of calves kept in confined rearing conditions. temperatures for young calves should range between and ºc. ammonia concentrations should be kept as low as possible preferably not more than ppm. housing and management should aim a reducing dust, bacteria and endotoxin concentrations in the animal house air. minimum ventilation rates of m per kg live weight should be applied. human-animal relationships recommendation stockpersons should be appropriately trained so that they have sufficient skills in rearing calves. they should have a positive attitude towards animals and work with them in order to minimise stress and to maintain a high quality of health control. rough contact (e.g. use of painful device such as an electric prod, loud noises) should be avoided and gentle contacts (e.g. talking softly, stroking, offering food) should be encouraged. this sort of contact is of particular importance for calves in groups or with their dam that tend not to approach humans readily. dehorning and castration if cattle are to be dehorned, it is recommend to disbud young cattle rather than to dehorn older ones. disbudding by cautery is recommended over other methods. local anaesthesia (e.g. - ml lidocaïne or lignocaïne % around the corneal nerve) and analgesia with an nsaid (e.g. ml flunixin meglumine or - . mg ketoprofen % / kg body weight) should be given - min before disbudding. if cattle are to be castrated, it is recommended to castrate calves as early as possible (no later than . mo and preferably at wk of age), to use the burdizzo method, and to provide appropriate anaesthesia and analgesia (e.g. ml lignocaine % in each testicle through the distal pole and mg ketoprofen % / kg body weight injected intravenously both min before castration). prevention of typical calf diseases in the first months of life such as diarrhoea and enzootic bronchopneumonia requires a systematic approach by improving management and housing conditions, specifically the preparation of the cow, hygiene of the calving environment, including dry clean bedding and high air quality, immediate supply with maternal antibodies, no mixing with older animals and careful attention and a rapid response to any sign indicating disease. main foodborne hazards associated with calf farming are salmonella spp., human pathogenic-verotoxigenic escherichia coli (hp-vtec), thermophilic campylobacter spp., mycobacterium bovis, taenia saginata cysticercus and cryptosporidium parvum/giardia duodenalis. the prevalence-level of infection and/or contamination of calves with, and further spread of, foodborne pathogens on farms depend on the status and the inter-relationship of different contributing factors that are inherently highly variable. present knowledge and published data are insufficient to produce a universal risk assessment enabling quantitative food safety categorization/ranking of different types of calf farming systems. nevertheless, generic principles for risk reductions for the main foodborne pathogens at calf farm level are known and are based on the implementation of effective farm management (e.g. qa, husbandry, herd health plans, biosecurity) and hygiene measures based on gfp-ghp. for quantitative food safety risk categorization of farming systems individually, and/or their related ranking, further scientific information is needed. accordingly, related research should be encouraged. the conclusions of the scientific veterinary committee report on the welfare of calves are presented in table below together with additions relevant in the light of this update of the svc report. the best conditions for young rearing calves involve leaving the calf with the mother in a circumstance where the calf can suckle and can subsequently graze and interact with other calves.c agreed where the calf will be separated from its mother at an early age, evidence suggests that it is normally beneficial for the calf if the mother is allowed to lick the calf thoroughly for a few hours after birth.c agreed r whenever possible, cows should be given the opportunity to lick the calf during at least three hours after parturition. it is important that the calf should receive sufficient colostrum within the first six hours of life and as soon as possible after birth, in conditions which facilitate antibody absorption, preferably by suckling from the mother, so as to ensure adequate immunoglobulin levels in the blood. r where necessary, suckling assistance or additional colostrum should be provided for calves left to suckle from the dam.r agreed agreed calves need resources and stimuli which are normally provided by their mothers. all calves should be given adequate food and water, appropriate conditions of temperature and humidity, adequate opportunities to exercise, good lying conditions, appropriate stimuli for sucking during the first few weeks of life and social contacts with other calves from one week of age onwards. specific aspects of housing and management which fulfill these conditions are detailed. agreed young calves reared without their mothers should receive considerate human contact, preferably from the same stockperson throughout the growing period.r agreed stockpersons should be appropriately trained so that they have sufficient skill in the rearing of calves. they should have positive attitudes towards animals and to working with them in order to handle them while minimising stress and to maintain a high quality of health control. rough contacts (e.g. use of a painful device such as an electric prod, or loud noises) should be avoided and gentle contacts (e.g. talking softly, petting, offering food) should be encouraged. these contacts are of particular importance for calves in groups or with their dam that may tend not to approach humans easily. where calves cannot be kept with their mother, the system where welfare is best is in groups with a bedded area and an adequate space allowance available to them.c agreed. r see below. the welfare of calves is very poor when they are kept in small individual pens with insufficient room for comfortable lying, no direct social contact and no bedding or other material to manipulate. c agreed r as the floor affects the resting and lying posture of calves they should be useful to have a comfortable floor. wet floors should be avoided due to thermal and resting problems. tethering always causes problems for calves. calves housed in groups should not be tethered except for periods of not more than one hour at the time of the feeding of milk or milk substitute. individually housed calves should not be tethered. r calves are vulnerable to respiratory and gastro-intestinal disease and welfare is poor in diseased animals. better husbandry is needed to minimize disease in group housing conditions but results that are as good as those from individual housing can be obtained. c every calf should be able to groom itself properly, turn around, stand up and lie down normally and lie with its legs stretched out if it wishes to do so. r in order to provide an environment which is adequate for exercise ,exploration and free social interaction, calves should be kept in groups. calves should never be kept at too high stocking density. the following requirements are based on evidence of increasingly poor welfare as space allowance decreases. the space allowance should provide, especially for allowing resting postures, an area for each calf of at least (its height at the withers) x (its body length from the tip to its nose when standing normally to the caudal edge of the tuber ischii or pin bone x . ). the length measurements takes account of the forward and backward movements involved in standing up and lying down. this calculation takes account of differences in size amog breeds and with age. as a guideline, for holstein calves this area is . m at weeks, . m at weeks and . m at weeks. r r since calves are social animals, they should be kept in social groups wherever possible. these groups should be stable with no mixing or not more than one mixing. it is advisable for calves in the first two weeks of life not to be mixed with other animals. c when calves are mixed together in the first few days of life, and then kept for some weeks in a social group, there may be poor welfare because of the following risks: -especially when individuals are provided with inadequate access to teats and roughage in the diet, cross-sucking and other abnormal sucking behaviour may occur. -some individuals may be unaccustomed to the food access method, for example they may have only received food via a teat, and may find it difficult to drink from a bucket. -calves coming from different buildings, perhaps from different farms, may carry different pathogens and hence there is a risk of disease spread in all the calves that are put in the same airspace or are otherwise exposed to the pathogens. r if calves from different buildings, perhaps different farms, are to be mixed in a pen or are to be put in different pens in the same airspace a quarantine situation should be used in order to reduce disease in the calves and hence prevent poor welfare. for a given space allowance per calf, increasing group size results in a larger total area and hence better possibilities for exercise, social interaction and improved environmental complexity. c larger groups are preferred because of the better possibilities for providing an adequate environment but there are limits to the numbers of animals which should be in one building section and risks associated with mixing of calves from different sources should be considered. r agreed r the space provided for calves should be enough to allow animals to fulfill their needs for social behaviour, lying and grooming. space allowance per animal should be greater for groups of - animals and for feeding systems, and pen shapes or flooring materials that necessitate extra space availability. if the preferred system, group housing, is not possible then individual pens whose width is at least the height of the calf at the withers and whose length is at least the length of the calf from the tip of its nose when standing normally to the caudal edge of the tuber ischii or pin bone x . should be used. this space requirement is calculated on the basis of the space required for normal agreed r as the pen shape affects the use of space by animals, pens should maximize the perimeter and pen space should be divided into different usable areas. movements and evidence of increasingly poor welfare r appropriate bedding for example straw is recommended. bedding must be changed at appropriate intervals and every calf should have access to a dry lying area. slatted floors must not be slippery and must not be a cause of tail tip necrosis. r agreed see above buildings should be adequately ventilated taking into account of the number of animals present and the external conditions. the air space in the building should be m per calf up to weeks of age and an amount of air space which increases with age is needed for older calves.r agreed c -calf rearing causes significant emissions such as nitrate, phosphate, heavy metals and possibly antibiotics in manure and liquid effluents as well as odour, gases, dusts, micro-organisms and endotoxins in the exhaust air from animal houses, from manure storage facilities, during application of manure and during grazing. -these effluents can have distinct impacts on air, water, soil, and thus also on animals. -calf houses possess a high potential for emissions of ammonia and other gases. dust, endotoxins and micro-organisms are emitted in lower amounts than from pig or poultry production. -respiratory disorders are the second largest reason for morbidity and mortality in calf rearing. the most important reason are environmental conditions such as hygiene, management and the physical, chemical and biological factors of the aerial environment. -ventilation plays a decicive role in reducing the incidence of respiratory diseases. temperatures below c can compromise lung function. -ammonia concentrations of more than ppm seem to increase respiratory affections. relative humidity of more than % bear the risk of increased heat dissipation and can help bacteria to survive in airborne state. -air velocities close to the animals of more than . m/s can increase respiratory sounds in calves significantly. -sufficient air space in confined buildings can help to reduce the concentration of airborne bacteria. -calf houses contain relatively high amounts of endotoxins. -there is concern that antibiotic residues may contribute to the development of bacterial resistance. -environmental problems in calf houses are enhanced by high animal densities, insufficient distances between farms. -when housing systems are compared, although dust emission levels will seldom pose problems for the health of calves, ammonia emission levels may be higher enough to exacerbate calf disease, especially in slatted floor units. r -the development of low emission production systems should be encouraged including mitigation techniques, e.g. biofilters, bioscrubbers, covered manure pits and shallow manure application. in particular there is need to reduce ammonia emissions from slatted floor units or to reduce the usage of such systems. -adequate and efficient feeding regimes are required with minimal wastage of nitrogen and phosphorous and limited use of growth promoters and drugs. -there is an urgent need for cooperative research to design appropriate ventilation systems to improve health and welfare of calves kept in confind rearing conditions. -temperatures for young calves should range between and c. -ammonia concentrations should be kept as low as possible, preferably not more than ppm. -housing design and management procedures should aim to reduce dust, bacteria and endotoxin concentrations in the animal house air. -minimum ventilation rates of c per kg live weight should be applied. calves which lack specific nutrients, including iron, which are given poorly balanced diet, and which are not provided with adequate roughage in the diet after four weeks of age can have serious health problems, can show serious abnormalities of behaviour, and can have substantial abnormalities in gut development. c every calf should receive a properly balanced diet with adequate nutrients.r agreed r it is recommended that solids feeds provided to veal calves, in addition to milk replacer, are adequately balanced in terms of the amount of fibrous material, which will promote rumination, and other components such as proteins and carbohydrates, which stimulate rumen development and support a healthy function of the digestive system. c if the concentration of haemoglobin in the blood of calves drops below . mmol l - , the ability of the calf to be normally active as well as the lymphocyte count and immune system function are substantially impaired, and there is reduced growth rate. below . mmol l - , veal calves exhibit a number of adaptations to iron deficiency, including elevated heart rate, elevated urinary noradrenaline and alterered reactivity of the hpa axis. hence it is normal practice to identify young veal production calves with less than . mmol l - haemoglobin in plasma and to provide supplementary iron in addition to that normally included in the diet. for older calves, including those in the last four weeks before slaughter, efficient production is possible in individual calves whose haemoglobin concentration is above . mmol l - . if the concentration of haemoglobin in blood is not checked at all, there is a high risk of anaemia that is associated with poor welfare, for all calves fed a diet with very low iron content. anaemia can be identified and quantified adequately if checks are carried out on veal production calves of - weeks, for example, when the calves are brought into a unit, between - weeks of fattening, and during the last four weeks before slaughter. if the concentration of haemoglobin in the blood of a group calves during the last four weeks before slaughter is a mean of . mmol l - , some calves may have a concentration substantially lower than the group-mean, and hence their welfare may be poor. r in order to avoid anaemia levels that are associated with poor welfare because normal activity is difficult or not possible and other functions are impaired, it is advisable that diets should be provided that result in blood haemoglobin concentrations of at least . mmol l - throughout the life of the calf. in order to avoid serious impairment of immune system function and hence poor welfare, no individual calf should have a blood haemoglobin concentration lower than . mmol l - . in most cases this is achieved by adjusting the concentration of iron in the diet and having an adequate checking system so that the above condition is avoided. other treatment may be needed for calves with clinical conditions which cause anaemia but which are not related to diet, r since the lowest haemoglobin concentrations in the blood of veal calves are usually reached during the last four weeks before slaughter, these blood concentrations should be checked at this time. such controls would help to see if measures are necessary to be taken or not. a checking system using a mean level, but whose aim is to avoid the risk of a low haemoglobin concentration in any individual lower than . mmol l- , would have to use a mean substantially higher than , mmol l- , probably mmol l- . in order to avoid poor welfare associated with anaemia, as explained in the conclusions (above), measurements of average blood haemoglobin concentration are not a satisfactory means of avoiding poor welfare but the use of a minimum level of . mmol l - for individual calves would achieve this. some non-milk proteins are inappropriate for use in a milk substitute fed to calves because they produce allergenic reactions. some carbohydrates cannot be easily or properly digested by calves and they may cause digestive upset. no milk substitute should be fed to calves unless it can be easily digested and does not cause harmful reactions in the calves. r acidification of milk can reduce the incidence of diarrhoea, but any forms of acidified milk which are unpalatable to calves or which harm the calves should not be used. r every calf should be fed fermentable material, appropriate in quality and sufficient in quantity to maintain the microbial flora of the gut and sufficient fibre to stimulate the development of villi in the rumen. roughage, in which half of the fibre should be at least mm in length, should be fed to calves. they should receive a minimum of g of roughage per day from to weeks of age, increasing to g per day from to weeks of age but it would be better if these amounts would be doubled. the development of the rumen should be checked by investigating villi development in a proportion of calf guts after slaughter. r agreed r without a fully functional rumen, calves will be unable to utilise nutrients provided in the post-weaning dry feed diet. attention should paid to type of forage and consistent of particle size of starter grain in order to achieve a proper rumen development. calf weaning should be based on the amount of dry feed calves ingest per day, not on their age or weight, and calf starter should be made available five to days after birth. a calf consuming . kg of dry feed or more on three consecutive days is ready for weaning. when calves are fed low levels of milk to encourage early consumption of dry food, weaning can be done abruptly. in contrast, if milk is given in large amounts, weaning may require two to three weeks of slow transition to avoid a setback in growth. there are clear signs of increased disease susceptibility and immunosuppression in calves up to weeks of age, whose blood haemoglobin concentration is below . mmol/liter. however, in some studies the antibiotic treatment was not higher in calves whose haemoglobin was near to mmol/litre than in calves whose level was near to mmol/litreat weeks of age. studies of exercise in anaemic calves show that there can be problems during exercise at a level of , mmol/litre. c all calves should be fed in such a agreed see way that their haemoglobin level does not fall below a minimum of . mmol/litre. r where calves are fed a diet which is lower in iron than mg/kg, an adequate sample of animals should be checked at and weeks of age in order to find out whether the blood haemoglobin concentration is too low. r agreed see young calves have a very strong preference to suck a teat or teat-like object. it is preferable for calves to be fed milk or milk substitute from a teat during the first four weeks of life. calf welfare is improved if a non-nutritive teat is provided during the first four weeks of life especially if they are not fed from a teat. c when young group-housed calves are fed milk or milk substitute, the social facilitation effects of having a group of teats close together are beneficial. it is also advisable for several teats to be provided in groups of older calves. transponder controlled feeder systems have been found to work well. c the feeding to calves of large quantities of milk or milk substitute in a single daily meal can cause digestive problems. hence when calves are fed more than % of body weight in milk or milk substitute each day, this should be fed in at least two meals per day. r calves fed ad libitum, or close to this level should not be weaned off milk or milk replacer until they are consuming a minimum of g of concentrates per head per day in the week prior to weaning. where calves are fed restricted quantities of milk or milk replacer before weaning they should not be weaned until they are consuming a minimum of g of concentrates per head per day in the week prior to weaning. r calves which are diseased and calves which are in hot conditions often need to drink water as well as milk or milk substitute and all calves drink water if it is available. the provision of milk or milk substitute is not an adequate alternative for provision of water. hence calves should be provided daily with water to drink. it is recommended that drinkers be provided in all pens. r agreed r prevention of typical calf diseases in the first months of life such as diarrhoea and enzootic bronchopneumonia requires a systematic approach by improving management and housing conditions, specifically the preparation of the cow, hygiene of the calving environment, including dry clean bedding and high air quality, immediate supply with maternal antibodies, no mixing with older animals and careful attention and early reaction of all signs of any beginning diseases. dehorning calves between - weeks by cauterisation with adequate anaesthesia and analgesia (no precision given) castrate calves at months with adequate anaesthesia and analgesia (no precision given) r dehorning: if cattle are to be dehorned, it is recommended to disbud young cattle rather than to dehorn older ones. disbudding by cauterisation is recommended over other methods. local anaesthesia (e.g. - ml lidocaïne or lignocaïne % around the corneal nerve) and analgesia with a non steroidal anti-inflammatory drug ( ml flunixin meglumine or - . mg ketoprofen % / kg body weight) shall be performed - min before disbudding. r castration:if cattle are to be castrated, it is recommended to castrate calves as early as possible (no later than . mo and preferably at wk of age), to use the burdizzo method, and to provide appropriate anaesthesia and analgesia (e.g. ml lignocaine % in each testicle through the distal pole and mg ketoprofen % / kg body weight injected intravenously both min before castration). -main foodborne hazards associated with calf farming are salmonella spp., human pathogenic-verotoxigenic escherichia coli (hp-vtec), thermophilic campylobacter spp., mycobacterium bovis, taenia saginata cysticercus and cryptosporidium parvum/giardia duodenalis. -the prevalence-level of infection and/or contamination of calves with, and further spread of, foodborne pathogens on farms depend on the status and the inter-relationship of different contributing factors that are inherently highly variable. -present knowledge and published data are insufficient to produce a universal risk assessment enabling quantitative food safety categorization/ranking of different types of calf farming systems. -nevertheless, generic principles for risk reductions for the main foodborne pathogens at calf farm level are known and are based on the implementation of effective farm management (e.g. qa, husbandry, herd health plans, biosecurity) and hygiene measures based on gfp-ghp recommendations for future research . it is recommended that future research should be conducted within the following areas: -hemoglobin levels and iron deficiences of veal calves aged - weeks. -the monitoring of haemoglobin in groups of calves using representative samples -exposure to allergenic proteins -solid and liquid food balance. exposure to too rich diets and changes in feed composition. -space requirements -health monitoring systems and the effect of such on clinical health in calves -infection transmission (respiratory and digestive diseases) due to direct contact between calves in relation to social benefits of mixing -pain relief when disbudding, dehorning and castrating calves -design of appropriate ventilation systems for calves in confined rearing conditions -health and environmental effects of feeding minerals as antimicrobial agents -for quantitative food safety risk categorization of farming systems individually, and/or their related ranking, further scientific information is needed. accordingly, related research should be encouraged. references used in this scientific opinion are available and listed in the scientific report published at the efsa web (www.efsa.eu.int). the ahaw panel wishes to thank the members of the working group chaired by panel member summary efsa has been requested by the european commission to issue a scientific opinion on animal health and welfare aspects of intensive calf farming systems and their ability to comply with the requirements of the well-being of calves from the pathological, zootechnical, physiological and behavioural points of view. in particular the commission asked efsa to update the findings of the scientific veterinary committee (animal welfare section) report on the welfare of calves of november in light of more recent data on this issue. where relevant the possible food safety implications of different farming systems should also be considered. in this report a risk assessment was made and the relevant conclusions and recommendations are forming the scientific opinion by the ahaw panel. the svc ( ) report contains information on measurements of welfare, needs of calves, descriptions of current housing systems, chapters on types of feed and feeding systems, weaning of calves, housing and pen design, climate, mananimal relationships, dehorning and castration. further chapters covered economical considerations of systems and for improving welfare. in the report conclusions were made on general management, housing, food and water and economics. the present report "the risks of poor welfare in intensive calf farming systems" is an update o the previous svc report with the exception of economical aspects which are outside of the mandate for this report. the various factors potentially affecting calves' health and welfare, already extensively listed in the report of the scientific veterinary committee animal welfare section (svc, ) , are updated and subsequently systematically determined whether they constitute a potential hazard or risk. to the latter end their severity and likelihood of occurrence in animal (sub) populations were evaluated and associated risks to calf welfare estimated, hence providing the basis for risk managers to decide which measures could be contemplated to reduce or eliminate such risks. in line with the terms of reference the working group restricted itself to (in essence a qualitative) risk assessment although it is agreed that welfare and health of calves can be substantially affected in the course of and as a result of transport and slaughter, this report does not consider animal health and welfare aspects of calves during transport and slaughter but such information can be found in a recently issued comprehensive report of the scientific committee on animal health and animal welfare (scahaw), on "the welfare of animals during transport (details for horses, pigs, sheep and cattle)" which was adopted on march (dg sanco, ) and in the efsa report "welfare aspects of animal stunning and killing methods" (efsa, b) . in relation with the food safety aspects, main foodborne hazards associated with calf farming are salmonella spp., human pathogenic-verotoxigenic escherichia coli (hp-vtec), thermophilic campylobacter spp., mycobacterium bovis, taenia saginata cysticercus and cryptosporidium parvum/giardia duodenalis. present knowledge and published data are insufficient to produce a universal risk assessment enabling quantitative food safety categorization/ranking of different types of calf farming systems. nevertheless, the main risk factors contributing to increased prevalence/levels of the above foodborne pathogens, as well as generic principles for the risk reductions are known. the latter are based on the implementation of effective farm management (e.g. qa, husbandry, herd health plans, biosecurity) and hygiene measures (e.g. gfp-ghp). in general, the conclusions made in the previous svc report remain. however, recent research has provided for some additional conclusions. the risk analysis is presented in the tables of annex . the graphics in this table are not intented to represent numerical relationships but rather qualitative relations. in some instances the exposure could not be estimated due to lack of data, in which cases the risks where labelled "exposure data not available". the following major and minor risks for poor animal health and welfare have been identified for one or several of the various husbandry systems considered: the hazards of iron deficiency and insufficient floor space are considered to be very serious, the hazard of inadequate health monitoring is considered to be serious and the hazards of exposure to inadequate hemoglobin monitoring, allergenic proteins and too rich diet are considered to be moderately serious. for these hazards, there is no consensus on the exposure of calves mainly due to lack of data and that is why it is recommended that further studies should be made to provide evidence for an exposure assessment. regarding castration and dehorning (and disbudding) without anaesthetic drugs, there is a variation in relation to national legislation why the risk of poor welfare in relation to castration and dehorning has a wide range between countries. tables which clarify the risk assessment have been included in annex . calf a calf is a young bovine which is significantly younger and smaller in size than an adult of the same species and breed and which is not reproductively active. there is a gradual transition from a newborn animal, dependent on milk, to an animal with many adult characteristics. few people would use the term calf for domestic cattle of - months whilst most would call an animal of months or somewhat older a calf. in this report, calf is used for animals of up to months of age. however, in deciding on the end of the calf stage, any definition based on age or weight is arbitrary. the term calf is not normally restricted to animals that are unweaned or monogastric rather than having some degree of development of the rumen for its specialist function. the removal of the horn bud or the actual horn depending on the breed and the age of the animal. endotoxin unit (eu) endotoxin activity of . ng of reference endotoxin standard, ec- or eu/ng (fda). to convert from eu's into ng, the conversion is eu/ng. a process where water bodies receive excess nutrients that stimulate excessive plant growth (i.e. water pollution). intensively reared calf a calf which is not kept extensively at pasture. according to the council of europe european convention for the protection of animals kept for farming purposes, (chapter i, article ), "modern intensive animal farming systems are systems in which mainly technical facilities are used that are primarily operated automatically and in which the animals depend on the care of and supply from the farmer". nsaid non-steroidal anti-inflammatory drug. the process by which a mother mammal allows a young animal to obtain milk from its teats. odds ratio (or) the odds ratio is a measure of effect size particularly important in bayesian statistics and logistic regression. infection of the navel. meat produced from animals slaughtered at - weeks of age and supplied with roughage from at least months of age onwards. there is not any classification system for veal carcasses agreed across the eu. the only existing classification system would rather relate to a general beef carcass classification system, which comprises the following categories: however, these categories are valid for cattle having a live weight of more than kg. consequently, some member states have issued their own national schemes for veal carcass classification. in trade, there is agreement between importing and exporting countries that veal originates from calves which were fed predominantly milk replacers, and which displays a light colour. the age limit is around months. some countries such as the netherlands market meat of animals of the age of to months, as pink veal. the eu subsidies scheme represents an important incentive for pink veal production. the determination of the relationship between the magnitude of exposure of calves to a certain hazards and the severity and frequency of associated adverse effects on calf welfare. the quantitative and qualitative evaluation of the likelihood of hazards to welfare occurring in a given calf population. any factor, occurring from birth to slaughter, with the potential to cause an adverse effect on calf welfare. the qualitative and quantitative evaluation of the nature of the adverse effects associated with the hazard. considering the scope of the exercise of the working group the concerns relate exclusively to calf welfare. the identification of any factor, from birth to slaughter, capable of causing adverse effects on calf welfare. a function of the probability of an adverse effect and the severity of that effect, consequent to a hazard for calf welfare. the process of determining the qualitative or quantitative estimation, including attendant uncertainties, of the probability of occurrence and severity of known or potential adverse effects on welfare in a given calf population based on hazard identification, hazard characterisation, and exposure assessment. unaltered remain the following cac (codex alimentarius commission) definitions (note: for completeness all definitions used by cac -while not necessarily used in this document -have been included): a risk assessment that provides numerical expressions of risk and an indication of the attendant uncertainties (stated in the expert consultation definition on risk analysis). a risk assessment based on data which, while forming an inadequate basis for numerical risk estimations, nevertheless, when conditioned by prior expert knowledge and identification of attendant uncertainties, permits risk ranking or separation into descriptive categories of risk. a process consisting of three components: risk assessment, risk management and risk communication. a scientifically based process consisting of the following steps: i) hazard identification, ii) hazard characterisation, iii) exposure assessment and iv) risk characterisation. the interactive exchange of information and opinions concerning the risk and risk management among risk assessors, risk managers, consumers and other interested parties. output of risk characterisation. the process of weighing policy alternatives in the light of the results of risk assessment and, if required, selecting and implementing appropriate control options (i.e. prevention, elimination, or reduction of hazards and /or minimization of risks) options, including regulatory measures. a method to examine the behaviour of a model by measuring the variation in its outputs resulting from changes to its inputs. characteristics of a process where the rationale, the logic of development, constraints, assumptions, value judgements, decisions, limitations and uncertainties of the expressed determination are fully and systematically stated, documented, and accessible for review. a method used to estimate the uncertainty associated with model inputs, assumptions and structure/form. the process by which a young mammal obtains milk from the teat of its mother or another lactating female by sucking. the term veal refers to the meat produced from calves, principally those of the species bos taurus and bos indicus. there are several meat products from calves. they are generally distinguished by their colour: "pale" or "white" veal is generally produced from an animal under months of age and fed mostly milk or milk replacer; "pink" veal is generally produced from an animal of up to months fed larger amounts of solid foods and possibly weaned. meat from calves of - months is called young beef. weaning, weaned in mammals, weaning is a gradual process during which the young animal receives less and less milk from its dam and consumes more and more solid food. it is accompanied by changes in the dam-offspring relation. in farming, calves are often separated from their dams soon after birth and receive milk (or milk replacer) from humans or a machine. although separated from the dam, calves are considered as un-weaned as long as they are fed milk. suckler calves are left with their dam for some months and are generally weaned some time before the next calving by separating them suddenly from the dam. calves normally commence eating solid food at - weeks, although some start earlier, and they eat enough solid food for development of a functional rumen to start by about weeks of age. a weaned animal is one that no longer needs to suckle and so does not consume milk in any significant quantity indicating that the weaning process has finished. council directive / /eec laying down minimum standards for the protection of calves as amended by council directive / /ec requires the commission to submit to the council a report, based on a scientific opinion, on intensive calf farming systems which comply with the requirements of the wellbeing of calves from the pathological, zootechnical, physiological and behavioural points of view. the commission's report will be drawn up also taking into account socio-economic implications of different calf farming systems. it should be noted that the scientific veterinary committee (animal welfare section) adopted a report on the welfare of calves on november (svc, ) which should serve as background to the commission's request and preparation of the new efsa scientific opinion. in particular the commission requires efsa to consider the need to update the findings of the scientific veterinary committee's opinion in light of the availability of more recent data on this issue. where relevant the possible food safety implications of different farming systems should also be considered. efsa has been requested by the european commission to issue a scientific opinion on animal health and welfare aspects of intensive calf farming systems and their ability to comply with the requirements of the well-being of calves from the pathological, zootechnical, physiological and behavioural points of view. in particular the commission requires efsa to update the findings of the scientific veterinary committee (animal welfare section) report on the welfare of calves of november in light of more recent data on this issue. where relevant the possible food safety implications of different farming systems should also be considered. the mandate outlined above was accepted by the panel on animal health and welfare (ahaw) at the plenary meeting, on / march . it was decided to establish a working group of ahaw experts (wg) chaired by one panel member. therefore the plenary entrusted a scientific report and risk assessment to a working group under the chairmanship of prof. bo algers. the members of the working group are listed at the end of this report. this report is considered for the discussion to establish a risk assessment and the relevant conclusions and recommendations forming the scientific opinion by the ahaw panel. according to the mandate of efsa, ethical, socio-economic, cultural and religious aspects are outside the scope of this scientific opinion. in , the scientific veterinary committee of the european commission published the report on the welfare of calves. the svc ( ) report contains information on measurements of welfare, needs of calves, descriptions of current housing systems, chapters on types of feed and feeding systems, weaning of calves, housing and pen design, climate, mananimal relationships, dehorning and castration. further chapters covered economic considerations of systems and for improving welfare. in the report conclusions were made on general management, housing, food and water and economics. the present report "the risks of poor welfare in intensive calf farming systems" is an update of the previous svc report with the exception of economic aspects which are out of the mandate for this report. this report represents an update of the previous svc report ( ) with a risk assessment perspective. factors which are important for calf welfare include housing (space and pen design, flooring and bedding material, temperature, ventilation and air hygiene), feeding (liquid feed, concentrates, roughage) and management (grouping, weaning, human-animal relations). the measures used to assess welfare include behavioural and physiological measures, patho-physiological measures and clinical signs as well as production measures. as explained in the glossary, in this report young bovines are called calves up to a maximum of eight months of age and veal is the meat of a calf. countries with substantial production of veal are france, italy, the netherlands, belgium, spain and germany. significant veal production exists also in portugal, austria and denmark, the production of white veal, from calves that have been fed predominantly milk replacer and which has a light colour, takes place largely in france, the netherlands, belgium and italy. the eu subsidies scheme represents an important incentive for pink veal production. most calves produced for further rearing are in france, germany, uk, ireland and italy. the ways of keeping calves vary considerably from country to country and between breeds. most dairy calves are separated from their dam at birth and artificially fed whereas calves from beef breeds generally suckle their dam. according to eu statistics, in in the eu ( ) , , calves were reared for slaughter (table ) and , , calves were reared for other reasons than slaughter (table ). in total (table ) , tonnes of calf meat were produced in eu ( ) which probably implies that about , tonnes were produced in eu ( ) during . human consumption of meat from calves decreased slightly from to in eu ( ) ( table ). : item and element selected in the eurostat database to make the query and extract the data the working group set out to produce a document in which the various factors potentially affecting calves' health and welfare [already extensively listed in the report of the scientific veterinary committee animal welfare section (svc, ) , are updated and subsequently to systematically determine whether these factors constitute a potential hazard or risk. to the latter end their severity and likelihood of occurrence in animal (sub) populations were evaluated and associated risks to calf welfare estimated, hence providing the basis for risk managers to decide which measures could be contemplated to reduce or eliminate such risks. it should be noted, however, that this does not imply that a hazard that has a serious effect on just a few animals should not be dealt with by managers on farm level as the suffering imposed on some animals constitute a major welfare problem for those individuals. in line with the terms of reference the working group restricted itself to (in essence qualitative) risk assessment, i.e. only one of three elements essential to risk analysis a risk assessment approach was followed, similar to the one generally adopted when assessing microbiological risks, i.e. along the lines suggested at the nd session of the codex alimentarius commission (cac, ) . incidentally, these guidelines have been characterized by the cac as 'interim' because they are subject to modifications in the light of developments in the science of risk analysis and as a result of efforts to harmonize definitions across various disciplines. cac's guidelines are in essence exclusively formulated for the purpose of assessing risks related to microbiological, chemical or physical agents of serious concern to public health. consequently -considering their disciplinary focus -the working group had to adapt the cac definitions to serve their purpose. these adapted definitions, have, in alphabethical order, been included in chapter (see risk analysis terminology). the objectives of this report are to review and report recent scientific literature on the welfare including the health of intensively reared calves, to report on recent findings as an update to the scientific veterinary committee's previous report, to make a qualitative risk assessment concerning the welfare of intensively kept calves. where relevant, food safety implications of different farming systems are also considered. the report is structured in five major parts. the first three follow the scientific veterinary committee's previous report "on the welfare of calves" with introductory chapters - on background, measurements and needs in relation to calf welfare, chapter describing housing, diet and management and chapter describing comparison of systems and factors. in chapter common disease and use of antibiotics is described. the other two parts involve aspects of meat quality and food safety (chapter ) and the risk assessment (chapter ). conclusions and recommendations from the previous svc document together with updated conclusions derived from recent research findings are presented in the scientific opinion (www.efsa.eu.int). effect of transport and slaughter on calves' health and welfare although it is agreed that welfare and health of calves can be substantially affected in the course of and as a result of transport, this report does not consider animal health and welfare aspects of calves during transport because there is already a comprehensive recent report of the scientific committee on animal health and animal welfare (scahaw), on "the welfare of animals during transport (details for horses, pigs, sheep and cattle)" which was adopted on march (dg sanco, . the report takes into account all aspects related with transport that could affect the health and welfare of cattle and calves, including the direct effects of transport on the animals and the effects of transport on disease transmission. the loading methods and handling facilities for cattle, the floor space allowance, the relationships of stocking and the density requirements, the vehicle design, space requirements and ventilation for cattle transporters (see also the ahaw scientific opinion related to standards for the microclimate inside animal road transport vehicles; efsa, ), the behaviour of cattle during road transport, the road conditions, long distance transport and the travel times are also reviewed. recommendations for all these aspects are also given in that report. the following general requirements in relation to animal welfare were annexed as a protocol to the eu treaty of amsterdam in : "in formulating and implementing the community's agriculture, fisheries, transport, and internal market policies, the community and the member states shall pay full regard to the welfare requirements of animals, while respecting the legislative provisions and customs of member states relating to religious rites, cultural traditions and regional heritage." in the introduction to the proposed eu constitution, the following extended wording is included: "in formulating and implementing the european union's agriculture, fisheries, transport, internal market, research and technological development and space policies, the union and the member states shall pay full regard to the welfare requirements of animals, as sentient beings, while respecting the legislative provisions and customs of member states relating to religious rites, cultural traditions and regional heritage." this wording reflects the ethical concerns of the public about the quality of life of the animals. it also takes into account customs and cultural traditions. farm animals are subject to human imposed constraints and for a very long time the choice of techniques has been based primarily on the efficiency of production systems for the provision of food. however it is an increasingly held public view that we should protect these animals against mistreatment and poor welfare. in order to promote good welfare and avoid suffering, a wide range of needs must be fulfilled. these needs may require the animal to obtain resources, receive stimuli or express particular behaviours (hughes and duncan, ; jensen and toates, ; vestergaard, ) . to be useful in a scientific context, the concept of welfare has to be defined in such a way that it can be scientifically assessed. this also facilitates its use in legislation and in discussions amongst farmers and consumers. welfare is clearly a characteristic of an individual animal and is concerned with the effects of all aspects of its genotype and environment on the individual (duncan, ) . broom ( ) defines it as follows: the welfare of an animal is its state as regards its attempts to cope with its environment. welfare therefore includes the extent of failure to cope, which may lead to disease and injury, but also ease of coping or difficulty in coping. furthermore, welfare includes pleasurable mental states and unpleasant states such as pain, fear and frustration (duncan, ; fraser and duncan, ) . feelings are a part of many mechanisms for attempting to cope with good and bad aspects of life and most feelings must have evolved because of their beneficial effects (broom, ) . although feelings cannot be measured directly, their existence may be deduced from measures of physiology, behaviour, pathological conditions, etc. feelings cannot be directly measured and therefore care is necessary to avoid uncritical anthropomorphic interpretations (morton et al., ) . good welfare can occur provided the individual is able to adapt to or cope with the constraints to which it is exposed. hence, welfare varies from very poor to very good and can be scientifically assessed. measures which are relevant to animal welfare during housing, i.e. largely longterm problems, are described by broom and johnson ( ) and by broom ( broom ( , a . production criteria have a place in welfare assessment. however, although failure to grow, reproduce etc. often indicates poor welfare, high levels of production do not necessarily indicate good welfare. physiological measurements can be useful indicators of poor welfare. for instance, increased heart-rate, adrenal activity, or adrenal activity following acth challenge, or reduced heart-rate variability, or immunological response following a challenge, can all indicate that welfare is poorer than in individuals which do not show such changes. the impaired immune system function and some of the physiological changes can indicate the pre-pathological state (moberg, ) . in interpreting physiological measurements such as heart rate and adrenal activity it is important to take account of the environmental and metabolic context, including activity level. behavioural measures are also of particular value in welfare assessment (wiepkema, ) . the fact that an animal avoids an object or event, strongly gives information about its feelings and hence about its welfare (rushen, ) . the stronger the avoidance the worse the welfare whilst the object is present or the event is occurring. an individual, whom is completely unable to adopt a preferred lying posture despite repeated attempts will be assessed as having poorer welfare than one which can adopt the preferred posture. other abnormal behaviour which includes excessively aggressive behaviour and stereotypes, such as tongue-rolling in calves, indicates that the perpetrator's welfare is poor. very often abnormal activities derive from activities that cannot be expressed but for which the animal is motivated. for example, calves deprived of solid foods and hence lacking the possibility of nutritive biting, develop non-nutritive biting. whether physiological or behavioural measures indicate that coping is difficult or that the individual is not coping, the measure indicates poor welfare. studies of the brain inform us about the cognitive ability of animals and they can also tell us how an individual is likely to be perceiving, attending to, evaluating, coping with, enjoying, or disturbed by its environment so can give direct information about welfare (broom and zanella, ) . in studies of welfare, we are especially interested in how an individual feels. as this depends upon highlevel brain processing, we have to investigate brain function. abnormal behaviour and preferred social, sexual and parental situations may have brain correlates. brain measures can sometimes explain the nature and magnitude of effects on welfare. the word "health", like "welfare", can be qualified by "good" or "poor" and varies over a range. however, health refers to the state of body systems, including those in the brain, which combat pathogens, tissue damage or physiological disorder (broom and kirkden, ; broom, ) . welfare is a broader term than health, covering all aspects of coping with the environment and taking account of a wider range of feelings and other coping mechanisms than those associated with physical or mental disorders. disease, implying that there is some pathology, rather than just pathogen presence, always has some adverse effect on welfare (broom and corke, ). the pain system and responses to pain are part of the repertoire used by animals to help them to cope with adversity during life. pain is clearly an important part of poor welfare (broom, b) . however, prey species such as young cattle and sheep may show no behavioural response to a significant degree of injury (broom and johnson, ) . in some situations responses to a wound may not occur because endogenous opioids which act as analgesics are released. however, there are many occasions in humans and other species when suppression of pain by endogenous opioids does not occur (melzack et al., ) . studies of the brain inform us about the cognitive ability of animals and they can also tell us how an individual is likely to be perceiving, attending to, evaluating, coping with, enjoying, or disturbed by its environment so can give direct information about welfare (broom and zanella, ) . in studies of welfare, we are especially interested in how an individual feels. as this depends upon high-level brain processing, we have to investigate brain function. abnormal behaviour and preferred social, sexual and parental situations may have brain correlates. brain measures can sometimes explain the nature and magnitude of effects on welfare. the majority of indicators of good welfare which we can use are obtained by studies demonstrating positive preferences by animals (dawkins, ) . methods of assessing the strengths of positive and negative preferences have become much more sophisticated in recent years. the price which an animal will pay for resources, or pay to avoid a situation, may be, for example, a weight lifted or the amount of energy required to press a plate on numerous occasions. the demand for the resource, i.e. the amount of an action which enables the resource to be obtained, at each of several prices can be measured experimentally. this is best done in studies where the income available, in the form of time or energy, is controlled in relation to the price paid for the resource. when demand is plotted against price, a demand curve is produced. in some studies, the slope of this demand curve has been measured to indicate price elasticity of demand but in recent studies (kirkden et al., ) it has become clear that the area under the demand curve up to a particular point, the consumer surplus, is the best measure of strength of preference. once we know what animals strongly prefer, or strongly avoid, we can use this information to identify situations which are unlikely to fulfil the needs of animals and to design better housing conditions and management methods (fraser and matthews, ) . however, as pointed out by duncan ( duncan ( , , all data from preference studies must be interpreted taking account of the possibilities that, firstly, an individual may show a positive preference for something in the shortterm which results in its poor welfare in the long-term, and secondly, that a preference in a simplified experimental environment needs to be related to the individual's priorities in the more complicated real world. each assessment of welfare will pertain to single individual and to a particular time range. in the overall assessment of the impact of a condition or treatment on an individual, a very brief period of a certain degree of good or poor welfare is not the same as a prolonged period. however, a simple multiplicative function of maximum degree and duration is often not sufficient. if there is a net effect of poor welfare and everything is plotted against time, the best overall assessment of welfare is the area under the curve thus produced (broom, c) . . the needs and functioning of calves . . the concept of needs in assessing the needs and functioning of calves, many different approaches can be taken. one is to study, at a fundamental level, the physiology and behaviour of cattle and the ways in which they have evolved, in order to try to understand their causation and function. needs are in the brain but may be fulfilled by obtaining resources, physiological change, or carrying out a behaviour. in order to conclude that a need exists to show certain behaviour, it is necessary to demonstrate that the calves used in modern production systems are strongly motivated to show the behaviour and that, if the need is not provided for, there are signs of poor welfare such as abnormal behaviour or physiology or pathological effects (see chapter ). where the housing design allows the animals to show the behaviour that they need to show, this will promote the avoidance of poor welfare. a need is a requirement, which is a consequence of the biology of the animal, to obtain a particular resource or respond to a particular environmental or bodily stimulus. an animal may have a need that results in the existence at all times of mechanisms within the brain and abilities to perceive stimuli and respond appropriately. however, this does not mean that every individual at all times needs to carry out the response. for example, a calf has a need to avoid attack by a predator but it does not need to carry out anti-predator behaviour if no individual perceived as a predator is present. there are some needs which require urgent fulfilment, otherwise the body functioning will be impaired and in the medium or long term, the animal may suffer. for example, an adequate amount of an essential nutrient or avoidance of exposure to a serious disease. there are other needs which, if not fulfilled lead to frustration and excessive activities in an attempt to fulfil the need. the resulting poor welfare may be extreme and prolonged. needs to avoid predation and other danger mean that animals have a negative experience in some situations. close human presence and handling of animals may elicit physiological and behavioural anti-predator responses. the avoidance of such situations can also be considered as a need. calves require space to perform activities such as resting, feeding, exploring, interacting and escaping from perceived danger. to assess what risks of poor welfare are involved when the housing circumstances do not allow certain activities, it can be helpful to consider why the calves are intrinsically motivated to perform the activities. the selection criteria applied to modern cattle genotypes have resulted in changes in morphological phenotype. although these have not altered the categories of needs of calves, they may have altered rates of growth and energy partitioning so that the timing of problems and the probability that they will arise may be changed. the overall need of calves is to maintain bodily integrity while growing and preparing for adult life. in order to do this, calves have a series of needs that are relevant to the housing and management conditions imposed upon them by humans. the needs of calves are described in detail by broom ( broom ( , . in listing needs and in later consideration of how to provide for them, it is assumed that extreme human actions, such deliberately creating a large wound or infecting an animal with a dangerous pathogen, will not occur. the list of needs is not in order of importance. some of the needs mentioned here are discussed at greater length in the previous report. . . . to breathe calves need air that has sufficient oxygen and a low level of noxious gases in it. calves may be adversely affected by some of the gaseous products of the breakdown of animal faeces and they show preferences that help them to avoid any harm that they may cause. calves need to rest and sleep in order to recuperate and avoid danger. they need to use several postures which include one in which they rest the head on the legs and another in which the legs are fully stretched out (de wilt, ; smits, , ) . sleep disruption may occur if comfortable lying positions cannot be adopted or if there is disturbance to lying animals because they are trodden on or otherwise disturbed by other calves. exercise is needed for normal bone and muscle development. calves choose to walk at intervals if they can, show considerable activity when released from a small pen and have locomotor problems if confined in a small pen for a long period (warnick et al., ; dellmaier et al., ; trunkfield et al., ) . calves living in natural conditions would be very vulnerable to predation when young. as a consequence, the biological functioning of calves is strongly adapted to maximise the chance of recognition of danger and escape from it. calves respond to sudden events and approaches by humans or other animals perceived to be potentially dangerous with substantial sympathetic nervous system and hypothalamic-pituitary-adrenocortical (hpa) changes. these physiological changes are followed by rapid and often vigorous behavioural responses. fear is a major factor in the life of calves and has a great effect on their welfare. . . . to feed and drink . . . . sucking the calf needs to attempt to obtain nutrients at a very early stage after birth and shows behavioural responses that maximise this chance. as a consequence, from an early age, calves have a very strong need to show sucking behaviour and if a calf is not obtaining milk from a real or artificial teat, it sucks other objects (broom, (broom, , metz, ; hammell et al., ; jung and lidfors, ) . the need of the calf is not just to have the colostrum or milk in the gut but also to carry out the sucking behaviour on a suitable object (jensen, ) . further, the sucking is of importance for the release of gastrointestinal hormones. it has been shown in calves that oxytocin is released during milk ingestion. the amount released, however, was less in calves drinking their milk from a bucket compared with calves suckling the dam (samuelsson, ) . peripheral oxytocin stimulates the release of glucagon from the pancreas whereas central oxytocin increases hunger and the release of gastrointestinal hormones promoting growth (stock et al., ; björkstrand, ) . in the early days after birth, calves are motivated to suck and obtain milk. however, calves also have a need to obtain sufficient water and will drink water even when fed milk. if the temperature is high, calves will drink water if it is available and sick calves will also choose to drink water. if water is not available, over-heated calves and sick calves may become dehydrated. sick calves may become dehydrated even when water is offered. calves with acidosis with or without diarrhoea often lose their suckling reflex. this may also happen in calves with hypoglycaemia and septicaemia (berthold, pers. com). after the first few weeks of life, calves attempt to start ruminating. if they have received no solid material in their diet, calves still try to ruminate but cannot show the full rumination behaviour. in addition to the need to suck when young, calves need to manipulate material with their mouths. they try to do this whether or not they have access to solid material and they will seek out solid material that they subsequently manipulate. (van putten and elshof, ; webster, ; webster et al., ) . calves eat solid food better when water is offered simultaneously. certain rapidly digestible carbohydrates are necessary for the development of ruminal papillae with associated physiological development and fibrous roughage helps the anatomical development of the rumen. so it is clear that calves need appropriate solid food in their diet after the first few weeks of life; first, food that is digested rapidly and provide fatty acids; then fibrous foods. rumen development is enhanced when calves are fed with concentrates, water and roughage such as hay. . . . to explore exploration is important as a means of preparing for the avoidance of danger and is a behaviour shown by all calves (kiley worthington and de la plain, ; fraser and broom, ) . exploration is also valuable for establishing where food sources are located. calves need to explore and it may be that higher levels of stereotypes (dannemann et al., ) and fearfulness (webster and saville, ) in poorly lit buildings or otherwise inadequate conditions are a consequence of inability to explore. . . . to have social contact . . . . maternal contact the needs of young calves are met most effectively by the presence and actions of their mothers. in the absence of their mothers, calves associate with other calves if possible and they show much social behaviour. the need to show full social interaction with other calves is evident from calf preferences and from the adverse effects on calves of social isolation (broom and leaver, ; dantzer et al., ; friend et al., ; lidfors, ) . to minimise disease during the first few hours of life, the vigorous attempts of the calf to find a teat and suckle should result in obtaining colostrum from the mother. this colostrum includes immunoglobulins that provide passive protection against infectious agents. hence the needs of the calf have an evident function that is not just nutritional. calves also show preferences to avoid grazing close to faeces. they also react to some insects of a type which may transmit disease. if infected with pathogens or parasites, calves will show sickness behaviour that tends to minimise the adverse effects of disease (broom and kirkden, ) . young calves, less than four weeks of age, are not well adapted to cope with stressful events such as handling and transport, often suffering very high rates of mortality and the younger the calves are, the higher their mortality (staples and haugse, ; mormède et al., ) many succumbing to pneumonia or scouring, within four weeks of arrival at the rearing unit (staples and haugse, ). an inability to mount an effective glucocorticoid response, which is adaptive in the short term, may be a contributing factor to the high levels of morbidity and mortality which occur in young calves (knowles et al., ) as may neutrophilia (simensen et al., ; kegley et al., ) , lymphopaenia (murata et al., ) and suppression of the cell mediated immune response (kelley et al., ; mackenzie et al., ) . to groom grooming behaviour is important as a means of minimising disease and parasitism and calves make considerable efforts to groom themselves thoroughly (fraser and broom, ). calves need to be able to groom their whole bodies effectively. to thermoregulation calves need to maintain their body temperature within a tolerable range. they do this by means of a variety of behavioural and physiological mechanisms. . . . . selection of location when calves are over-heated, or when they detect that they are likely to become over-heated, they move to locations that are cooler. if no such movement is possible, the calf may become disturbed, thus exacerbating the problem and other changes in behaviour and physiology will be employed. responses to a temperature that is too low will also involve location change if possible. . . . . body position over-heated, or potentially over-heated, calves adopt positions that maximise the surface area from which heat can be lost. such positions often involve stretching out the legs laterally if lying and avoiding contact with other calves and with insulating materials. if too cold, calves fold the legs and lie in a posture that minimises surface area. . . . . water drinking over-heated calves will attempt to drink in order to increase the efficiency of methods of cooling themselves. to avoid harmful chemical agents calves need to avoid ingesting toxic substances and to react appropriately if harmful chemical agents are detected within their bodies. . . . to avoid pain calves need to avoid any environmental impact or pathological condition that causes pain. the text in this section refers to current situation in eu countries. calf housing in other countries may be different. replacement dairy calves . . . diet brief description of the diet of replacement heifer calves. this has not really changed since the report. following birth, calves receive (or should receive) colostrum and are than reared with whole milk or milk replacer. calves are weaned; weaning ages and weaning strategies may differ according to region or country. briefly mention current weaning strategies. calves receive starter and, for example, hay and maize silage to promote rumen development. according to the latest eu regulation on the housing of calves (council directive eu / /ec), group housing is compulsory for calves older than weeks, unless there is any need for isolation certified by a veterinarian. individual housing of rearing calves younger than weeks, is quite common in the european dairy industry. below, the most important housing systems for replacement heifer calves are briefly listed. . . . . hutches: partially closed, outside area hutches are made of plywood, plastic or fibre glass. if hutches are made from a synthetic opaque material, this prevents the greenhouse effect inside the hutch and reduces heat stress. if reflective material is used (light coloured), the sun rays are reflected which reduces the risk for overheating. the size of hutches may vary from . - . m width and . - . m length. a layer of sand, e.g. cm gravel or crushed stone can be placed under the calf hutch. litter may be provided preferably as straw, as it provides the warmest surface temperature (panivivat et al., ) , but also wood shavings, sawdust or newspapers are used and the layer should be thick enough to provide a comfortable and dry bed. calf hutches provide three different environments, as the inside is dry and protected from the weather and outside the calf is able to get limited exercise and sunlight. the calf can be also position itself half in and half out, getting sunlight and being protected from wind. hutches should be placed where they catch the most sunlight and avoiding hot, windy and wet locations. nevertheless, during hot summer conditions hutches should be placed in a shady area to avoid overheating. in the rear wall, a hole that can be closed provides better air ventilation within the hut in warm weather. in the hutches, the calf can be kept using wire panels in a building with an outdoor run, preferably of more than . m , enabling some contact to other calves. calves can also be fed outside using a milk bucket support, a dry feed recipient support and a hay rack. other hutch types locate feed and water pails inside the hutch. individual pens are situated in a roofed building. the area should be wellventilated so that the air is dry and fresh, but draught has to be avoided. separation from adult cows is advantageous with respect to disease prevention. pens are either made from hard material with concrete walls or dismountable with three solid sides (i.e. plywood) and an open front (see figure ). walls have to be perforated according to council directive eu / /ec in holdings with more than five calves, which allow at least limited social contact with other calves, one of the key needs of calves. the open front gets fresh air to the calf and makes them easier to feed through a bucket support provided on the front. hardwood is normally used for the floor, which is covered with a litter that is thick enough, dry and clean. totally slatted floors are in use also, made of wood, plastic or metal, but require more care for air temperature. the . - . m x . - . m pen can be put mm above the ground allowing for draining and the removal of urine. dismountable individual pens should be designed in such a way that they can be taken apart and stored when they are not needed, and also easily cleaned with a skid-steer loader or small bucket tractor. in case of cold weather, a plywood cover can be placed over the rear portion of the pen to preserve heat produced by the calf. in hot weather, a removable panel at the rear of the shelter can be opened to provide additional air exchange. collective hutches may house a group of between and calves. the hutches are made of synthetic materials or wood. the inside of the hutch is provided with litter and some hay may be put in a rack. roughage is distributed at a feeding barrier and anti-freeze drinking devices are needed if freezing temperatures may occur. with collective hutches fastened on concrete, a good outdoor run has a non-slippery surface. manure and bedding have to be removed manually or the collective hutch has to move over a few metres distance by means of a tractor and guide-blocks. as for the individual hutches, the location has to be chosen carefully to avoid overheating during summer and provide protection from wind and rain entering the hutch during cold seasons, but give as much sunlight as possible. when sufficient straw and proper ventilation is provided these are the most suitable facilities for young replacement heifer calves. if the calves stay there for several months it is necessary to provide a passage on slippery free concrete. if the floor of this passage is quite rough this will prevent slipping. the concrete floor may be replaced by a slatted floor provided that the spacing between slats agrees with the age of the animals. the lying area can be built in different ways and littered with different materials. in the deep litter system, the dung is removed at regular intervals from every few weeks to twice per year. . . . . group pens inside another common system for group housing of replacement heifer calves is group housing inside, in straw littered pens usually with - calves per pen. calves may enter such group housing already after weeks of individual housing. the regulatory change with regard to calf housing together with a general trend towards larger dairy farms has increased the interest in group housing systems for rearing calves during the milk feeding period (hepola, ; jensen, ) . in addition to systems with small groups of calves ( - animals per group) kept on straw and usually bucket-fed, calves are increasingly kept in larger groups ( up to about calves) with computer-controlled automatic milk feeders. an automatic milk feeder may contain two milking dispensers, and each milking dispenser can be used for about calves. to prevent hierarchic and health problems within the group, calves are grouped with a limited age difference between the animals. calves receive milk replacer according to their needs or ad libitum. when calves are fed according to their needs, a radio-frequency electronic identifier can be used, with a transponder inserted in the collar, in an ear tag, injected under the skin or inside a ruminal bolus swallowed by the animal. the diet of the vast majority of veal calves in the european union is determined by the market demand for "white meat", i.e. meat with low myoglobin content. the production of white veal meat comes from a tradition of fattening calves thanks to a diet based on milk, which is naturally poor in iron, and slaughtering the animals when they are young. nowadays most veal calves are fed milk replacers that contains a variable proportion of milk powder and which iron content is maintained at a low level. this results in relatively low blood haemoglobin levels. an average blood haemoglobin level at slaughter between . and . mmol/l is compatible with an acceptable meat colour. as haemoglobin levels increase, the number of animals whose meat is darker in colour increases. in order to prevent calves from having haemoglobin levels that are too low, early in the production phase, the iron supply in the milk replacer fed during the first - weeks of the fattening period (starter) is usually about ppm, whereas iron supply in the milk replacer fed during the remainder of the fattening period (fattener) is ppm. moreover, blood haemoglobin levels are generally monitored, most intensively upon arrival at the fattening unit, and calves with levels below age-dependent thresholds are treated with iron, either individually or group-wise. thus, blood haemoglobin levels usually gradually decline across the fattening period, and the lowest average levels are supposed to be reached during the last four weeks prior to slaughter. some veal calves are still fed raw milk. in case of dairy breeds, the cows are generally milked and the milk is given to calves in buckets. in case of beef breeds, the calves are led twice a day for suckling their dam or another cow. according to the latest amendment to the annex of council directive / /eec (commission decision / /ec) calves should receive sufficient iron to ensure an average blood haemoglobin of at least . mmol/l, and calves over two weeks old should be provided daily with some fibrous feed which should increase from to a minimum of grams per day from the beginning to the end of the fattening period. the main types of solid feed given to veal calves differ somewhat between the veal producing countries in europe. in france and italy solid feeds for veal calves usually consist of chopped straw or pelleted dry feed consisting of both fibrous (e.g. straw) and concentrate-like (e.g. cereal) materials. in the netherlands, maize silage is a popular roughage source for white veal calves, provided that the iron content is not too high (an upper limit of - pp/kg dry matter is generally imposed). maize silage is usually fed in relatively high amounts, with maximum daily amounts of up to . kg ( gr dry matter)/calf/day. other feeds used in the veal industry include chopped straw and rolled barley. white veal calves are fattened for approximately weeks in italy and the netherlands, and for - weeks in france. besides the production of white veal meat, several systems exist across europe that lead to the production of so-called "pink veal meat". the main differences from the more conventional production of white veal are that the calves are reared for a longer period and they receive higher amounts of solid foods. as a consequence the muscles have a higher content of myoglobin, hence the darker colour of the meat. in france, the calves are most often from suckler beef breeds; they are reared with their dam and may be weaned before the end of rearing. in the netherlands, pink veal meat is generally produced from calves of dairy breeds. pink veal calves are weaned at - weeks of age. after weaning, they receive a diet of ad lib roughage (frequently maize silage) and by-products. pink veal calves are not restricted with regard to dietary iron supply and, consequently, develop normal haemoglobin levels and the associated "red" (pink) meat colour. the age at slaughter can vary from calves of - months to young bred animals of - months with the slaughter age of individuals depending on the production rate. these products are labelled to help consumers to distinguish them from white veal meat. in line with the latest eu regulation (council directive eu / /ec), individual housing of veal calves has been officially abolished in the european union. already in the s extensive studies were initiated with the aim to develop a practically feasible husbandry system for group housing of veal calves. at present the systems involve both large and smaller groups. housing of calves is in groups of - animals, with a slight trend towards larger group sizes ( - calves per group). the floor can be bedded with straw or wood shavings but is more commonly made of wooden slats. wooden slats require less labour and straw or woodshavings easily become dirty and wet. calves are kept in individual pens, sometimes called "baby-boxes" for a period of - weeks upon arrival at the fattening unit to prevent overt preputial sucking thereafter and to be able to monitor more closely the health of calves. baby boxes are usually made of galvanised or wooden partitions placed inside the group pen. in these boxes, calves are bucket-fed individually. after - weeks, these temporary partitions are removed and calves are free to move around in the pen. calves are fed milk replacer in a trough or in individual buckets. a crucial management procedure associated with trough feeding is the regular re-grouping of calves, to maintain homogeneous groups in terms of calf weight and particularly drinking speed throughout fattening. experimental work confirmed the feasibility of this procedure in that calves could be repeatedly regrouped without effects on their health, growth rate and a number of physiological measures of stress . in this latter study, aggression between calves was rare, and calves seemed to habituate to repeated mixing. individual calves not thriving on milk replacer because of drinking problems, are provided with floating teats or with a teat-bucket. veal calves are sometimes kept in pairs. this type of housing results in less availability of space for movement and social opportunities than in larger groups of calves but is reported to have no disadvantages in health, weight gain and the occurrence of cross-sucking (chua et al., ) . suckling veal calves are generally accomodated in small groups. as in the rearing of dairy calves, automatic feeding systems have been extended to veal production systems, particularly since increasingly sophisticated computer technology is becoming available for sensor-aided recognition of individual animals, and to control feeding times and intake. calves are usually housed in large groups ( - calves) and receive milk replacer via an automatic feeding machine. with such feeders, calves suck to obtain their milk. the floor generally consists of wooden slats, or concrete in combination with wooden slats. some veal calves are kept on straw bedded floors, or have access to rubber mats or concrete covered by rubber. calf rearing and animal environmental pollution . . . general introduction in the report there was a short chapter on calf production and environmental pollution referring to gases (ammonia, nitrous oxide, carbon dioxide). manure resulting from calf production was seen as a fertiliser only. this chapter briefly describes in a condensed way the impact of modern animal calf production affects the environment of the animals. modern animal production is a source of solid, liquid and gaseous emissions which i.a. can be harmful to the animals. solid and liquid manure and waste water contain nitrogen and phophorus which are the most important plant nutrients, but are harmful when applied to agricultural land in excess amounts thereby leading to pollution of ground water by nitrates, surface water with phosphorous causing eutrophication and soil with heavy metals such as zinc and copper which are used as growth promotors in the feed stuff, all of which can affect the animals if returned to them. a third group of potentially hazardous effluents are drug residues, such as antibiotics, which may be present in the excreta of farm animals after medical treatment and which are passed to the environment during grazing or spreading of animal manure where they may conceivably contribute to the formation of antibiotic resistance in certain strains of bacteria. the same risk arises when sludge and waste water from sewage plants containing residues of antibiotics and other drugs from human consumption are discharged as fertiliser in the soil and water body of agricultural land. the most important aerial pollutants from calf rearing systems are odours, some gases, dust, micro-organisms and endotoxins, together also addressed as bioaerosols (seedorf and hartung, ) , which are emitted by way of the exhaust air into the environment from buildings and during manure storage, handling and disposal. aerial pollutants can give cause for concern for several reasons. e.g. an animal's respiratory health may be compromised by these pollutants. in fattening units, up to % of all calves may show signs of pneumonia, pleuritis or other respiratory disease within the first three weeks of housing when the calves come together from different herds (see chapter on temperature, ventilation and air hygiene). the travel distance of viable bacteria from animal houses via the air is presently estimated at m (müller and wieser, ) downwind why there is a possible transmission between animal houses. very little is known about the distribution characteristics of dust particles, endotoxins, fungi and their spores, in the air surrounding animal houses. recent investigations showed dispersion of staphylococcae sp. (bioaerosols) up to m (schulz et al., ) from a broiler barn. the contribution of calf production is presently unknown. it is estimated that calves produce about . kg fresh manure and . kg slurry per animal and day. this is a share of . % in the total amount of fresh manure produced in cattle farming (richter et al., ) . manure suspected to contain pathogens such as salmonella should be stored for at least months without adding or removing material and subsequently applied to arable land where it is ploughed in, or it is disinfected before any further use. the second area of concern is the emission compounds such as gases, odours, dust, micro-organisms and other compounds like endotoxins which are regularly present in calf house air where they can cause or exacerbate respiratory disorders in animal and work force. the quantities emitted from calf houses are summarised in seedorf et al. ( b) there are considerable emission amounts from calf husbandry. the emissions of micro-organisms are higher than from dairy or beef barns but distinctly lower than from pig or poultry production (seedorf et al., a) . the same is true for endotoxins which are one log lower in cow barns but distinctly higher in pig and poultry houses. the dust emissions can be times higher in piggeries and times higher in broiler barns . the ammonia concentration is usually lower than in piggeries or laying hen houses. however this depends greatly on the housing and manure management system. in a us study johnson et al. ( ) reported that cow-calf, stocker and feedlot phases contribute considerable amounts of nitrous oxide and methane to the emissions from cattle production. . comparison of systems and factors . . feeding and housing systems, weaning strategies and quality of solid and liquid feed . . . feeding systems the main potential problems associated with the housing of calves in large groups with automatic feeders include: cross sucking, i.e., non-nutritive sucking of parts of another calf's body (in particular the ears, mouth, navel, udder-base and, in case of bull calves, the scrotum and prepuce) (plath et al., ; bokkers and koene, ; jensen, ) , competition for access to the feeder (jensen, ; , and health problems, in particular a high incidence of respiratory disease (maatje et al., ; plath et al., ; svensson et al., svensson et al., , hepola, ; engelbrecht pedersen et al., ) . a number of factors have been identified that are likely associated with some of these problems, although conflicting results have been reported. cross-sucking is linked with the sucking motivation of calves and, hence, measures to reduce the motivation of calves for non-nutritive sucking may reduce the occurrence of cross-sucking (de passillé, ). an increased milk allowance also reduced non-nutritive sucking on a teat as well as cross-sucking in group-housed calves in one experiment (jung and lidfors, ), but did not affect cross-sucking in another (jensen and holm, ) . reducing the milk flow rate decreased nonnutritive sucking on a teat in individually housed calves (haley et al., ) , but failed to influence cross-sucking in group-housed ones (jung and lidfors, ; jensen and holm, ) . alternatively, it has been suggested that hunger may also control the level of non-nutritive sucking and possibly cross-sucking (jensen, ) . this idea is consistent with the observations that the duration of unrewarded visits to an automatic feeding station increased during gradual weaning (jensen and holm, ) , and that under practical farm conditions the frequency of cross-sucking among dairy calves around weaning is increased with decreasing availability or energy density of solid feeds (keil et al., ; keil and langhans, ) . in contrast to other calves, white veal calves are not weaned, receive large amounts of milk replacer and usually obtain only restricted amounts of solid food. these additional factors may also affect and perhaps exacerbate crosssucking in systems with an automatic feeder (jensen, ) . results by veissier et al. ( ) showing that bucket-fed group-housed veal calves show less cross-sucking than those fed by an automatic feeder again seem to implicate factors other than sucking motivation per se in the development and expression of cross-sucking. on the other hand, rearing calves in large groups with an automatic feeder allows more interactions between calves and offers calves the possibility to suck milk. competition for access to an automatic milk feeder was increased in groups of or calves in comparison with groups of or , respectively (herrmann and knierim, ; jensen, ) , and under dietary conditions of relatively low milk allowance and reduced milk flow rate (jensen and holm, ) . protecting calves from displacement at the feeder may also be accomplished by fitting a closed feeding stall to the station (weber and wechsler, ) . in comparison with the usual setup, this modification increased the duration of visits to the feeder as well as the duration of non-nutritive sucking on the teat after milk ingestion, and significantly reduced the frequency of cross-sucking within minutes after milk ingestion. however, the incidence of cross-sucking performed without prior milk ingestion was not affected by the design of the feeder (weber and wechsler, ) . in a recent comprehensive review, jensen ( ) observes that there is a lack of knowledge on the effect of different weaning methods on cross-sucking. she also concludes that future research should focus on preventive measures to reduce cross-sucking and problems with aggression in automatically fed calves, including the establishment of appropriate numbers of calves per feeder. the apparent increase in health problems of calves kept in large groups with automatic feeders might be related to group size rather than to feeding system. a comparison of two different group sizes of calves fed by an automatic milk feeder showed that calves housed in groups of - had a higher incidence of respiratory illness and grew less than calves housed in groups of - (svensson and liberg, ) . similarly, placement of preweaning heifer calves in groups of or more was associated with high calf mortality in a large scale epidemiological survey (losinger and heinrichs, ) . interestingly, in a study by kung et al. ( ) , group-housed calves fed by an automatic feeding system for milk supply had fewer days of medication than those kept individually in separate calf hutches. these authors also emphasize the importance of good management and frequent observations of calves as an integral part of a successful rearing program. likewise, howard ( ) specifically links good and correct management practices with the prevention of disease and successful group housing of dairy calves. natural weaning in cattle takes place when young animals are around - months of age. depending on productive system, weaning can usually occur between and months of age. dairy calves are usually reared away from their dams and they are given milk or milk replacer until weaning at to weeks of age. however holstein calves can be weaned at to weeks of age (early weaning). beef calves are usually weaned at to months of age depending on season of birth. early weaning of beef calves may be considered as a management practice in poor climate conditions and where forage quality is poor later in the grazing season. several studies have shown that it is possible to wean calves at very young ages based on concentrate intake (svc, ) . however, regardless of the productive system, weaning is effective and does not cause health and welfare problems to calves when it occurs as a smooth transition from an immature to mature ruminant with an adequate size and development of the reticulo-rumen for efficient utilisation of dry and forage based diets. at birth, the reticulum, rumen, and omasum of the calf are undeveloped, nonfunctional and small in size compared to the abomasum and rumen remains underdeveloped during the first - months of age. calves being ruminant animals require a physically and functionally developed rumen to consume forages and dry feeds. however, the rumen will remain undeveloped if diet requirements for rumen development are not provided. solid feed intake stimulates rumen microbial proliferation and production of microbial end products, volatile fatty acids, which initiate rumen epithelial development . solid feeds are preferentially directed to the reticulo-rumen for digestion, however they differ in efficacy to stimulate rumen development. recent studies have shown that addition of yeast culture ( %) increased calf grain intake, but did not affect rumen development in young calves ; while papillae length and rumen wall thickness were significantly greater in week old calves fed calf starters containing steamflaked corn over those fed dry-rolled and whole corn when these corn supplements made up % of the calf starter showing that the type of grain processing can influence rumen development in young calves. forages seem to be the primary stimulators of rumen muscularization development and increased rumen volume (zitnan et al., ) . large particle size, high effective fibre content, and increased bulk of forages or high fibre sources physically increase rumen wall stimulation, subsequently increasing rumen motility, muscularization, and volume coverdale et al., ) . besides, solid feeds other than forages or bulky feedstuffs can be effective in influencing rumen capacity and muscularization. coarsely or moderately ground concentrate diets have been shown to increase rumen capacity and muscularization more than finely ground or pelleted concentrate diets, indicating that extent of processing and/or concentrate particle size affects the ability of concentrates to stimulate rumen capacity and muscularization (beharka et al., ; greenwood et al., ) . therefore, it seems that concentrate diets with increased particle size may be the most desirable feedstuff for overall rumen development, due to their ability to stimulate epithelial development, rumen capacity, and rumen muscularization . calf weaning should be based on the amount of dry feed calves ingest per day, not on their age or weight, and calf starter should be made available five to days after birth. but, as pointed out from recent research attention must paid to type of forage and consistent of particle size of starter grain in order to achieve a proper rumen development. a calf consuming . kg of dry feed or more on three consecutive days is ready for weaning. when calves are fed low levels of milk to encourage early consumption of dry food, weaning can be done abruptly. in contrast, if milk is given in large amounts, weaning may require two to three weeks of slow transition to avoid a setback in growth. early weaning systems should not be used if the animals are in a negative energy balance. . . . quality of solid and liquid feed . . . . solid feed: concentrates and roughage traditionally, veal calves were fattened on a diet consisting exclusively of milk replacer. calves fed in this manner show a number of welfare problems (reviewed in the previous report), including abnormal behaviours and disease associated with lack of rumen development. to better safeguard the welfare of calves, provision of (some) solid feed to veal calves has become compulsory according to the latest amendment to the annex of council directive / /eec (commission decision / /ec). however, provision of roughage to veal calves fed a regular milk replacer diet, has clearly been demonstrated to increase the incidence of abomasal ulcers, in particular in the pyloric part (which connects to the duodenum) (wensink et al., ; welchman and baust, ; breukink et al., ) . thus, recent studies have largely focussed concurrently on the effects of provision of roughage on calf behaviour, abomasal lesions and rumen development, in an attempt to identify feeds that may benefit veal calf welfare without compromising abomasal integrity. in a comprehensive eu-funded project, a range of different types of roughage/solid feeds (straw, maize silage, maize cob silage, rolled barley and beet pulp) in different amounts ( versus gr dry matter) and of different particle sizes and physical characteristics (i.e., chopped versus ground, dried versus fresh, un-pelleted versus pelleted) were given to veal calves in addition to milk replacer in large-scale multifactorial trials (chain management of veal calf welfare, ; cozzi et al., ; mattiello et al., ) . control treatments consisted of milk replacer only, and milk replacer with ad lib access to hay. another control group consisted of bull calves reared in a similar way to normal dairy calves, i.e. the animals received ad libitum hay and concentrates and were weaned at weeks of age. in comparison with milk replacer only, those types of roughage that were richest in fibrous material, i.e. straw (regardless of amount and physical structure) and hay, significantly reduced the level of abnormal oral behaviours (composed of tongue rolling, tongue playing and compulsive biting/sucking of substrates), and concomitantly increased the level of rumination. weaned calves exhibited no abnormal oral behaviours. higher levels of rumination in veal calves as a function of the fibre content of the solid feed were also reported by morrisse et al. ( morrisse et al. ( , . in line with these findings, veissier et al. ( ) , observed reduced levels of biting at substrate and more chewing behaviour in veal calves provided with straw compared with un-supplemented controls. previously, it has been suggested that a sucking deficit causes abnormal oral behaviours in calves (sambraus, ) . more recent data, however, clearly identify the lack of appropriate roughage as a major determinant of abnormal oral behaviours in veal calves. correspondingly, bokkers and koene ( ) found no differences in abnormal oral behaviours between group-housed veal calves fed either by bucket or by an automatic feeder. results obtained in veal calves are also fully consistent with data in cows (redbo et al., ; redbo and nordblad, ) and other ruminants such as giraffes (baxter and plowman, ) , which all link increased levels of abnormal oral behaviours with feeds poor in fibre. in agreement with previous data, most roughages provided to milk-fed veal calves significantly increased the incidence of abomasal lesions, particularly ulcers in the pyloric region, in comparison with the feeding condition without additional roughage (chain management of veal calf welfare, ; . incidences of abomasal ulcers (expressed as the percentages of calves with one or more lesions) among weaned bull calves, calves fed milk only, and veal calves given supplemental roughages were , between - , and between - %, respectively. this suggests that the interaction between roughage and a milk replacer diet rather than roughage per se, is involved in the etiology of abomasal ulcers in veal calves. these findings support the hypothesis that pyloric ulcers in milk-fed veal calves may be caused by local ischaemia followed by focal necrosis as a consequence of strong contractions of the pyloric wall when large volumes of milk are consumed. provision of roughage, in turn, would then exacerbate an existing problem in that roughage particles exert a mechanically abrasive effect on a sensitive abomasal mucosa, and delay the healing of any lesions already present (unshelm et al., ; dämmrich, ; krauser, ; welchman and baust, ; breukink et al., ) . this explanation may also fit the observations that veal calves fed either hay or a combination of concentrates and straw exhibited similar incidences of abomasal ulcers to those fed milk replacer only (chain management of veal calf welfare, ; veissier et al., ) . these roughages represent more balanced feeds, accompanied by better rumen fermentation. this may have improved ruminal digestion of fibres, thereby preventing sharp undigested particles entering the abomasum. other factors proposed or examined in relation to the pathogenesis of abomasal ulcers in calves include stress, infection with bacteria, trace mineral deficiencies, and prolonged periods of severe abomasal acidity (lourens et al., ; mills et al., ; jelinski et al., ; de groote et al., ; palmer et al., ; ahmed et al., ahmed et al., , ahmed et al., , . however, so far none of these factors have been convincingly related to abomasal ulcers in veal calves. calves fed milk only, showed a high incidence of ruminal hairballs. in different experiments between - % of milk-fed veal calves had hairballs (chain management of veal calf welfare, ; cozzi et al., ) . feeding roughage gave a profound reduction of hairballs; depending on the type of roughage the incidence varied between - %. similarly, morisse et al. ( ) reported a marked reduction of ruminal hairballs in calves fed pelleted straw and cereals. this reduction was thought to result from a continuous elimination of ingested hair by improved ruminal motility. however, it may well be that abnormally high self-licking behaviour is reduced when roughage is provided. it is suggested that further optimising the composition of roughage in terms of adequate rumen development and rumen function, may eventually result in feeds that promote rumination and reduce abnormal oral behaviours without damaging the digestive apparatus (chain management of veal calf welfare, ; morisse, ; mattiello et al., ) for all newborn calves, receiving an adequate amount of high quality colostrum is essential for their health and survival. in comparison with mature milk, colostrum contains greater concentrations of total solids and of fat, protein, vitamins and minerals. most importantly, colostrum provides the calf with immunoglobulins (igg), which are vital for its early immune protection. in addition, colostrum contains a range of other non-nutrient and bioactive components including various types of cells, peptide hormones, hormone releasing factors, growth factors, cytokines and other bioactive peptides, oligosaccharides and steroid hormones. these factors modulate the microbial population in the gastrointestinal tract, have profound effects on the gastrointestinal tract itself (e.g. cell proliferation, migration, differentiation; protein synthesis and degredation; digestion, absorption, motility; immune system development and function), and in part exert systemic effects outside the gastrointestinal tract on metabolism and endocrine systems, vascular tone and hemostasis, activity and behaviour, and systemic growth (waterman, ; blum, ) . the highest quality colostrum, or true colostrum, is obtained from the very first milking after parturition. thus, provision of first colostrum to newborn calves is one critical factor for successful calf rearing. the timing of provision of colostrum is also crucial since the ability of the calf's small intestine to absorb large proteins such as igg decreases rapidly following birth. consumption of sufficient colostrum within the first h of life is needed not only for an adequate immune status but also to produce the additional important and favourable effects on metabolic and endocrine traits, and on vitality. finally, colostrum should be regularly provided for a sufficient length of time, preferably for the first three days after birth (hadorn et al., ; waterman et al., ; rauprich et al, ) . although the importance of colostrum for calf health and survival is generally recognized, actual practices in calf rearing do not always favour adequate colostrum intake in newborn calves, and may therefore pose a risk for their welfare. after the period of colostrum feeding, calves can be switched to whole milk or a high quality milk replacer. in the case of rearing calves, both sources of liquid feed are used, although the majority of dairy calves are currently reared on a milk replacer diet. milk replacers are usually less costly than saleable whole milk, and the feeding of raw waste milk may pose several health and contamination risks, including the transfer of infectious diseases to the calf, and problems with antibiotic residues or overdoses (wray et al., ; selim and cullor, ; waltz et al., ) . at present, good quality milk replacers may provide comparable performance to whole milk. however, pasteurization of waste milk prior to feeding it to calves may also represent an effective and viable alternative for minimizing health risks (stabel et al., ) . results from a recent clinical survey by godden et al. ( ) even suggested that dairy calves fed pasteurized waste milk have a higher growth rate and lower morbidity and mortality rates than do calves fed conventional milk replacer. with the exception of production systems involving suckler cows, veal calves are generally fattened on milk replacer diets. over time, formulations of commercially available milk replacers for veal calves (as well as those for dairy calves) have become more and more sophisticated. at the same time, economic pressures continuously prompt the industry to reduce feeding costs and to consider alternative components and raw materials. originally, proteins in milk replacers were milk-based, and skim milk powder constituted the major protein source. subsequently, milk replacers based on whey powder became available. approximately during the last two decades, attempts have been made to replace animal-based proteins in milk replacers by vegetable proteins, mainly from soybean and wheat and, to a lesser extent, pea and potato. initially, some of these attempts met with little success because of health problems in the calves. for example, compared to calves fed diets based on skim milk powder, calves fed milk replacers containing heated soybean flour developed severe immunemediated gut hypersensitivity reactions characterized by partial atrophy of the small intestinal villi, malabsorption, diarrhoea, and large infiltrations of the small intestine by immune cells, accompanied by the presence of high antibody titres against soy antigens in plasma and intestinal mucous secretions (lalles et al., a (lalles et al., , b (lalles et al., , (lalles et al., , dreau et al., ; dreau and lalles, ) . however, the nutritional utilization of vegetable proteins can be improved by a variety of technological treatments including, for example, heating, protein hydrolysis, and ethanol extraction. such treatments reduce anti-nutritional factors and antigenic activity, and increase protein digestibility by denaturing three-dimensional structures (lalles et al., c (lalles et al., , d . at present a number of processed plant proteins are successfully applied in combination with milkbased protein sources in milk replacers for (veal) calves, including hydrolysed soy protein isolate and hydrolysed wheat gluten. recent research in the area of plant proteins in milk replacer formulas is focussed on understanding mechanisms underlying the flow of proteins in duodenal digesta, and the interaction of dietary peptides with the gut, in particular at the level of the mucus layer (montagne et al., (montagne et al., , (montagne et al., , . results of this type of work may further enhance the use of plant proteins in milk replacers for calves. in addition to an enhanced risk for gut problems, low quality milk replacers may also cause dysfunction of the oesophageal groove reflex, which may result in ruminal acidosis. in this respect, temperature is also an important quality feature; a too cold drinking temperature of the milk replacer attenuates the oesophageal groove reflex (gentile, ) . if vegetable proteins are not properly treated, milk replacers may cause hypersensitivity reactions in the gut, which may compromise calf welfare. low iron dietary supply is a prerequisite for the production of white veal. the blood haemoglobin level in veal calves towards the end of fattening (between . and . mmol/l), is generally considered a threshold below which iron deficiency anaemia occurs (bremner, ; sprietsma, a, b; postema, ; lindt and blum, a) , although some authors have argued that this level is already below a critical value (welchman et al., ) . when calves were forced to walk on a treadmill, those with a mean haemoglobin level of . mmol/l consumed more oxygen and exhibited higher cortisol levels after walking than calves whose haemoglobin level was . . or . mmol/l (piguet et al., ) . on the other hand, blood lactate after transport was not significantly different between groups of calves with average haemoglobin levels of . and . . mmol/l, respectively (lindt and blum, b) . there is a large body of evidence showing that iron deficiency anaemia may compromise immunocompetence, in particular cellular immune function, in a range of species including laboratory rodents and humans (dallman, ; dhur et al., ; galan et al., ; latunde-dada and young, ; ahluwalia et al., ) . in human children, iron-deficiency states have been epidemiologically associated with increased morbidity due to respiratory infection and diarrhoea (keusch, ; de silva et al., ; levy et al., ) . this justifies the question of whether dietary iron supply and associated haemoglobin levels are sufficient to guarantee adequate health in white veal calves. previous results concerning the relationship between clinical health and anaemia in veal calves are scarce, and were inconclusive. using very small numbers of calves, möllerberg and moreno-lopez ( ) found no difference between iron anaemic and normal calves in the clinical response to infection with an attenuated parainfluenza- virus strain, whereas sárközy et al. ( ) reported a depressed immune response as reflected in significantly lower antibody levels in anaemic calves compared with controls following inoculation with a live adenovirus. in a study by gygax et al. ( ) , cellular immune function was depressed, and disease incidence, especially of respiratory infections, was increased in calves fed low amounts of iron. however, in this particular study, haemoglobin levels dropped considerably below the value of . mmol/l. a more recent study (van reenen et al., ) , therefore, aimed to examine immunocompetence in a bovine herpes viral (bhv ) infection model in white veal calves with blood haemoglobin levels maintained at all times above or just at . mmol/l. calves daily supplemented with extra iron exhibited normal haemoglobin levels across the entire experiment (average approximately . mmol/l), whereas white veal calves had average haemoglobin levels at the time of bhv infection and at slaughter of approximately . and . mmol/l, respectively. dietary iron supply did not affect the reactions of calves to bhv infection (clinical signs, viral excretion in nasal fluid, antibody reponse), white blood cell and lymphocyte counts, and growth rate. by contrast, in comparison with calves with high haemoglobin levels, white veal calves exhibited a higher heart rate during milk intake, had consistently elevated levels of urinary noradrenaline, and showed enhanced plasma acth and reduced plasma cortisol responses in a number of hpa axis reactivity tests. these latter findings concur with increased heart rate and catecholamines in urine, and altered responsiveness of the hpa axis in iron-deficient or anaemic humans and laboratory rodents (voorhess et al., ; dillman et al., ; dallman et al., ; groeneveld et al., ; saad et al., ) . these physiological changes are part of an elaborate adaptive response to iron deficiency (beard, ; rosenzweig and volpe, ) , which also involves alterations in glucose metabolism (blum and hammon, ) . veal calves with blood haemoglobin levels clearly below . mmol/l demonstrated reduced growth rates as well as a large depression in white blood cell and lymphocyte counts (reece and hotchkiss, ; gygax et al., ) . thus, it is suggested that maintaining blood haemoglobin in individual veal calves over . mmol/l induces a number of physiological adaptations which seem universal for iron-deficient mammals in general, but do not harmfully compromise biological capacities in terms of growth and immunocompetence. in actual practice, however, the haemoglobin threshold of . mmol/l is currently considered at the group rather than at the individual level. for example, an average haemoglobin level of . mmol in a group of finished veal calves is assumed to be exactly at the lower threshold value. however, depending on the variation between individuals, if a group of calves has an average haemoglobin level of . mmol/l, then some individuals within that group may have levels well below this lower threshold value. in fact, based on an analysis of the variation between calves in blood haemoglobin levels, it has been argued that the haemoglobin threshold for anaemia of a group of veal calves should be higher than that of an individual calf, i.e. an average level of . rather than mmol/l (van hellemond and sprietsma, a) . in order to prevent anaemia during fattening, blood haemoglobin levels are monitored to some extent in white veal calves, and animals are treated with supplemental iron according to age-dependent haemoglobin thresholds. however, systematic monitoring generally occurs only on two occasions: within the first - weeks upon arrival at the fattening unit, in all animals, and between - weeks of fattening, in a sample of calves. outside these instants, individual calves may receive iron supplementation in the presence of clinical signs of iron deficiency. but once clinical signs are apparent, haemoglobin levels are usually well below . mmol l - (blaxter et al., ; bremner et al., ) . since blood haemoglobin levels are not routinely monitored in veal calves beyond the th week of fattening, there is a likelihood of too low haemoglobin levels occurring in part of the animals, in particular towards the end of fattening, when low haemoglobin levels are most likely to occur. general housing calves kept indoors are housed in an environment where several important factors interact such as space, pen design, social contacts, flooring and bedding material as well as climate. in experimental studies, usually one or a few of these factors are varied and the others controlled for. however in larger epidemiological studies many of these factors vary and their interaction can be measured. in a study of heifer calves in swedish dairy herds the effect of draught, cleanliness of the animals, hygiene level of the farm, placing of the calf pens, nature of the pen walls, air volume per animal, management factors such as status of the caretaker and feeding routines was evaluated by means of a two-level variance component logistic model. the placing of calf pens along an outer wall was significantly associated with the risk of diarrhoea (odds ratio (or): . , p< . ), the risk for respiratory disease was significantly associated with an ammonia concentration below ppm (or: . , p< . ) while the or for moderately to severely increased respiratory sounds was significantly associated with draught (or: . , p< . ) (lundborg et al., ) . odds ratios for respiratory disease were increased in calves housed in large-group pens with an automatic milk-feeding system (or: , ) . the report highlights that the housing systems of calves and the available space affect the development and determine which behaviours the animals are able to perform. the report (svc, ) recommends the minimum space for both single crate and group pen and it points out how lack of space can affect health and welfare of reared calves (maatje and verhoeff, ; dantzer et al., ; friend et al., ) . the report also suggests that shape of the pen can be important to the animal. recent studies confirmed that the space available can affect both behavioural and physiological traits and productive performances of cattle. however, the majority of them compare behaviour, production or other indicators of calves reared in individual crates versus group pens (vessier et al., ; andrighetto et al., ; jensen, ; verga et al., ; cozzi et al., ; bokkers and koene, ) or tethered or single pen (terosky et al., ; wilson et al., ) which were already discussed in chapter . little research has been done to directly compare behavioural and physiological indicators of welfare in calves reared in pens of various space allowances. in dairy calves it has been shown that spatial environment stimulated play: calves in small group pens performed less locomotory play that the ones kept in larger pens (jensen et al., ; jensen and kyhn, ) . it has been reported in a preliminary study that dairy calves kept, from birth to month of life in larger stalls ( . m x . m) showed a higher percentage of lying behaviour and grooming than calves kept in smaller stalls ( . m x . m); besides, lymphocyte proliferation was significantly higher in calves reared in large stalls (ferrante el al., ) . it is known that cattle prefer to use the perimeter of pens rather than the central area (stricklin et al., ; hinch et al., ; fraser and broom, ) . the ratio between the number of corners in the pen and number of animals seems to influence the individual space, the space that calves try to keep to other calves, as showed by simulation models (stricklin et al., ) . therefore pen shapes maximising the perimeter to area ratio might be preferable for cattle (jóhannesson and sørensen, ) . for this reason it has been pointed out that measurements such as pen perimeter, the number of corners and the diagonal distance of the pen could be important for dairy cattle (jóhannesson and sørensen, ) . however there is a lack of knowledge on this topic on calves. in a study on veal calves most of the animals lying next to the wall, the quieter and drier part of the pen, stood more on the side of the far pen and eliminated in the feeding area (stefanowska et al., ) . calves kept in a large group ( animals) and fed using an automatic milk replacer showed an elevated use of the area around the partition of the pen and they spent little time in the centre of the area. (morita et al., ) , this use of the pen space could lead to a pen design functionally divided into a walking and feeding area and a lying area. the report concludes that slatted floors must not be slippery, it also recommends appropriate bedding, for example straw, and that every calf should have access to a dry lying area. the report highlights that housing and management conditions can affect the posture adopted when lying and resting in calves. . . . recent findings regarding importance of floor and bedding materials slatted floors have been used for many years as convenient for intensive housing for beef cattle but concerns have been expressed about their effects on animal welfare (scahaw, ) . the type of surface not only affects the movements of getting up and lying down, lying and resting behaviour of the fattening animals but also other behavioural traits and physiological indicators of stress (scahaw, ) . moreover when cattle can choose between different floor types they prefer deep litter to slatted floor especially for resting. many studies were conducted in order to analyse the floor comfort in the lying area in dairy and in beef cattle (for a list of references see tuyttens, ; scahaw, ) . the group pens for veal calves do not have separate lying areas and therefore the animals spend all their time on the same surface. if the floor is too hard for lying or too slippery, discomfort, distress and injury may result. a suitable floor is very important for calves as adequate rest is essential for the good welfare of young growing animals, moreover a positive correlation between the amount of rest and growth rates has been observed for growing cattle (mogensen et al., ; hanninen et al., ) . adequate resting is important both for sleep and temperature regulation. veal calves are often housed on slatted floors, commonly made of hardwood, a product that is controversial because it often comes from unsustainable forestry in tropical countries (stefanowska et al., ) , or on concrete floors due to the fact that bedding material is costly and requires more labour and can cause problems in manure handling systems. wooden slatted floors can absorb liquid from manure and a wet surface is not comfortable for moving and lying (verga et al., ) . even if straw bedding provides better floor comfort to animals than slatted or concrete floors, suitable alternatives to reduce or eliminate the use of straw bedding are available for cattle (tuyttens, ) . recent studies have investigated the effect of the texture (how soft) and the thermal properties of floor on lying postures and resting behaviour of calves. in cool or drafty floors calves spent less time resting on the side and rest curled up in order to conserve heat (hanninen et al., ) . in contrast with adult dairy cows which rested longer and lay down more frequently on softer floors, there was no effect of type of floor (concrete floor or rubber mats) on resting behaviour of dairy calves (hanninen et al., ) . in another experiment where veal calves could choose to use a hardwood slatted floor surface or a synthetic rubber coated floor surface the calves preferred the wooden floor for lying (stefanowska et al., ) . moreover the animals rested in the drier part of the pen (stefanowska et al., ) . from these studies it seems that the texture of the floor is not as important to calves as to older animals, whereas thermal comfort seems to affect lying and lying postures. panivivat et al. ( ) investigated growth performance and health of dairy calves bedded with five different types of materials (granite fines, sand, rice hulls, long wheat straw, wood shavings) for days during august to october from birth. overall average daily gain and dry matter intake of calves did not differ with bedding type, although during week , calves housed on rice hulls had the greatest dry matter intake and those housed on wood shavings had the lowest. during week , calves housed on granite fines and sand were treated more often for scours, and calves housed on long wheat straw received the fewest antibiotic treatments (week by bedding material interaction). granite fines formed a harder surface than other bedding, and calves housed on granite fines scored the dirtiest. long wheat straw had the warmest surface temperature, and rice hulls and wood shavings were warmer than granite fines and sand. serum cortisol, alpha ( )-acid glycoprotein, immunoglobulin g concentrations, and the neutrophil:lymphocyte ratio were not affected by bedding type. on day , coliform counts were greatest in rice hulls. after use, coliform counts were greatest in long wheat straw (week by bedding material interaction). growth rates of calves bedded for d with bedding types did not differ; however, the number of antibiotic treatments given for scours was greatest on granite fines and sand; coliform counts in the bedding were highest in rice hulls before use and in long wheat straw after days of use. degree of social contact the report recommends that calves are cared for by their dam after birth so that they are licked and receive colostrum and that calves are not deprived of social contact, especially with other calves because ) calves for social contacts; ) calves isolated from other calves express more abnormal activities (e.g. excessive grooming, tongue rolling), are hyper-reactive to external stimuli and their subsequent social behaviour is impaired; and ) in combination with restricted space or lack of straw, individual housing induces a chronic stress state as assessed through enhanced responses to an acth challenge. . . . recent findings regarding contacts with the dam the bond between dam and calf is likely to develop very soon after birth: calves separated from their dam at h can recognise the vocalizations of their own dam one day later (marchant-forde et al., ) in their review about early separation between dairy cows and calves, flower and weary ( ) conclude that, on the one hand, behavioural reactions of cows and calves to separation increase with increased contacts but, on the other hand, health and future productivity (weight gain for the calf, milk production for the cow) are improved when the two animals have spent more time together. calves reared by their dam do not develop cross-sucking while artificially reared calves do so (margerison et al., ) . the provision of milk through a teat, a long milk meal, and the possibility to suck a dry teat can decrease non-nutritive sucking in artificially reared calves but do not abolish it (review by jensen, ; lidfors and isberg, ; veissier et al., ) . the presence of adult cows other than the dam do not help calves to get accustomed to new rearing conditions, as observed by schwartkorf-genswein et al. ( ) for calves submitted to feedlot conditions. . . . recent findings regarding contacts with other calves recent studies confirmed that calves are motivated for social contact. such a motivation was shown using operant conditioning by holm et al. ( ) ; furthermore calves that are housed individually engage in more contacts with their neighbours than calves housed in pairs (raussi et al., ) . the presence of a companion can reduce emotional responses of calves. this, for instance, is the case when group housed calves are exposed to a novel situation like a novel object (boivin et al., ) , a novel arena (jensen et al., ) , a sudden event (veissier et al., ) , or a lorry (lensink et al., ) . humans are not a good substitute for social contacts. individually housed calves interact more with their neighbours compared with pair-housed calves, even when they receive additional contacts from the stockperson (e.g. stroking, letting suck fingers, speaking softly) (raussi et al., ) . (see section on humananimal relationships). . . . comparison between individual housing vs. group housing individual housing can be stressful to calves as measured by adrenal responses to acth (raussi et al., ) . group housed calves are generally more active than individually housed calves as far as gross activity is concerned (more time spent moving or eating, less time spent idling or lying) (babu et al., ; raussi et al., ) . group housing can benefit production: xiccato et al. ( ) found that calves housed in fours put on more weight than calves tethered in individual crates. however, this seems not to be the case when the calves are not tethered in individual crates (veissier et al., ) . group housed calves are less easy to handle. human contact is thus essential for them to become accustomed to humans and to react less to handling (lensink et al., ; mogensen et al., ) . group housing can help calves acquiring social skills (boe and faerevik, ) . some experience of mixing is of particular importance: calves that have been reared for a while in a group dominate calves that have always been in individual crates (veissier et al., ) . by contrast, it is not clear whether repeated mixing would be beneficial or harmful to calves veissier et al., ) . recent research (e.g. svenson et al., and svensson and liberg, ) suggests that transfer from individual pen to group-housing during the second week of life is disadvantageous for health reasons (see chapter ) and, that a delay in mixing until the calf is weeks old may be preferable. additional research seems necessary to establish what mixing age would be preferable from a health and welfare perspective. . . temperature, ventilation and air hygiene the importance of the aerial environment inside a calf house for the health status of the animals was stressed in the report, and it still seems to be one of the major factors which cause morbidity and mortality (svensson et al., ) . bioaerosols (micro-organisms, dust), low air temperatures together with high air humidity, gases such as ammonia, draught, insufficient air space and poor ventilation form a complex environmental situation which can be detrimental particularly for the respiratory health of young calves (lundborg et al. ; svc, ) . . . . temperature and relative humidity a healthy calf consuming a sufficient amount of feed has a wide zone of thermal neutrality. there is no difference in the performance of healthy, normal eating calves at temperatures ranging between ºc and ºc provided it is dry and not exposed to draughts. above ºc conditions in confined calf houses can start to become uncomfortable. moran ( ) suggests that the ideal temperature and relative humidity for calves are ºc and %, respectively. however, there is a large number of influencing factors to consider which can alter the situation for a calf substantially. lower critical temperatures for a calf in calm air and full feed are different whether it stands (- ºc) or is lying on dry concrete (- ºc) or on dry straw (- ºc) (thickett et al. ). the younger the animal the higher is its demand for the thermal environment. by week of age, the lower critical temperature in still air is approximately ºc (webster, ) . this temperature can be significantly changed by draught, wet coat and feeding level. young calves start to shiver at ºc when they are exposed to draught even if their coat is dry and they are fed sufficient feed. when fed on maintenance only level shivering starts at ºc. if their coat is wet and they are exposed to draught shivering starts at ºc when on full feed and ºc when fed on a low level (moran, ) . no signs of shivering are observed at ºc when the coat is dry with no draught and feeding is at a normal level. cold stress in calves can be prevented by providing dry lying areas, appropriate feeding and draught free ventilation. dry bedding such as straw significantly improves thermal comfort for the lying calf. in summer situations reduced feeding (but with sufficient water supply!) or feeding calves in the cooler evening or at a reduced animal density per pen and increased ventilation rates can help to lower heat stress. heat stress can also be reduced through constructing sheds with insulated roofs and well ventilated walls. calf houses with a solid wall construction and a high capacity to store energy combined with an efficient ventilation system can also contribute to create comfortable environmental temperatures for young calves kept indoors all year (din , ) . the preferred environmental temperature for calves is not fixed, it largely depends on management and other environmental factors such as wind speed and humidity of the air. the generally accepted range of relative humidity for calf barns is between and % with an optimum around % which is not too humid to dissipate excess heat and not too dry to dry out the respiratory pathways predisposing the mucous membranes to infectious and noxious agents present in the inhaled air. air humidities of more than % can lead to condensation on the walls and ceiling increasing the risk of wetting the animals by water dripping off these surfaces. high relative humidities can also impair the insulation properties of the walls increasing heat losses. cold and humid air at high velocities can considerably increase the heat loss of animals. lundborg et al. ( ) showed that draughts greater than . m/s measured close to the animal, significantly increased the odds ratio for moderate to severe respiratory sounds. the higher the humidity the higher the risk of wet skin and cooling and shivering. high air humidity increases the probability that bacteria survive in an airborne state and are transmitted between animals in the same pen and between animal pens (wathes, ) . there are existing numerous reports from s onwards of the survival of bacteria and viruses employing simple regression models to describe the loss of viability of microbes over time. however, these models lack an insight into biophysical and biochemical mechanisms of cell and virus death. as long as we do not understand these mechanisms, the measures to reduce the air pollutants are limited to either increased ventilation or increased air space. the smaller the air space per animal the more sophisticated the ventilation system must be. the influence of air space was demonstrated by wathes ( ) showing that doubling of the air space in a calf barn from to m³ per calf had the same effect on the concentration of airborne bacteria as a six fold increase in ventilation rate (air exchange rate). an air space of to m³ per calf was recommended by hilliger ( ) from experience. . . . air quality aerial pollutants in confined animal houses are widely recognised as detrimental for respiratory health. primary and opportunistic microbial pathogens may cause directly infectious and allergic diseases in farm animals, and chronic exposure to some types of aerial pollutants may exacerbate multi-factorial environmental diseases, such as enzootic bronchopneumonia. the factors can be inadequate environmental conditions, e.g. too low temperatures, high ammonia concentrations and poor ventilation resulting in low air quality. poor air and surface hygiene in calf buildings are nearly always associated with intensive systems of husbandry, poor standards of management and high stocking densities (wathes ) . the most common aerial pollutants in calf housing are summarised in table . . gases such as ammonia (nh ), hydrogen sulphide (h s), carbon dioxide (co ), and more than hundred trace gases form an airborne mixture of bioaerosols composed of about % organic compounds and can also contain endotoxin, antibiotic residues and further trace components. significantly high amounts of endotoxins were found in calf house air while bacteria and dust are relatively low compared with pig and poultry houses and suggest that a high number of gram-negative bacteria are present in the air. the average concentration of ng/m³ endotoxin given in table . represents about eu (endotoxin units) according to the new nomenclature. it seems rather high in comparison to a formerly proposed occupational threshold of eu for humans at the work place (rylander and jacobs, ) . it can be assumed that high endotoxin concentrations in calf house air may substantially contribute together with the other bioaerosol compounds and the physical environment to the high level of respiratory disorders in young calves up to days (assie et al., ) . in general, there is little detailed knowledge on the specific composition of bioaerosols in calf keeping systems and which factors cause respiratory diseases. assie et al. ( ) found e.g. a tendency to higher repiratory disorders in non-weaned calves reared in loose-housing yards compared with tied-cow stalls. the highest incidence rates of cases were observed between november and january, while daily meteorological conditions obviously did not influence incidence rates. one of the most detrimental gases in calf barns is ammonia which is formed by bacterial degradation of nitrogen containing compounds in urine and faeces. it is the most widespread naturally occurring alkaline gas in the atmosphere and a strong irritant in animals and humans. concentrations in the air of more than ppm can impair the proper functioning of the mucous membranes of the respiratory tract and predispose to infection. in a recent study lundborg et al. ( ) found that the risk for respiratory disease was significantly associated with an ammonia concentration below ppm (or: . ; p< . ). high concentrations of hydrogen suphide can be released in high amounts from stored liquid calf manure when it is stirred up before removal from the slurry pit of the barn. concentrations of about ppm are acute fatal. the composition of the inhalable and respirable particles in animal houses is associated with compounds such as dried dung and urine, skin dander and undigested feed. the majority of bacteria found in shed airspace have been identified as gram-positive organisms, with staphylococci spp. predominating (cargill et al., ) . a survey by heinrichs et al. ( ) showed the importance of good ventilation which removes dust and other respirable particles as well as noxious gases and is essential for calf health. adequate ventilation is seen as vital to help to reduce the incidence of respiratory disease. air inlets should be above calf height and the outlet at least . m above the inlet (howard, ) . however, heating and ventilation in combination with an air filtration system significantly improved the environment in a calf house but did not completely eliminate pneumonia (bantle et al., ) . this may have to do with other factors such as ambient temperature. in a recent study by reinhold and elmer ( ) some compromise in lung function (compared with controls) was seen in calves exposed to an ambient temperature of ºc. . . . . light in the past, veal calves were often kept in dark to reduce muscle activity but the requirements for light have increased over the last years from to lux (bogner and grauvogel, ) , to over lux (irps, ) to lux for at least h and according to daylight circadian rhythm. (tierschutz-nutztierhaltungsverordnung germany, ) . there is wide agreement that calves need light for orientation in their boxes or pens and for social contact. a precise threshold has not been determined. there is a need for air movement around calves to supply fresh air and to remove excessive heat, moisture and air pollutants (gases, dust, microorganisms). good ventilation systems provide this exchange. however, high air speeds close to the animals can lead to draughts and should be avoided. draughts happen when part of an animal is hit by an air stream with a higher velocity than the ambient air movement and which has a substantially lower temperature than the surrounding air, causing a feeling of cold and physiological reaction in that particular part of the body. draught can lead to poor welfare and disease when it continues and the animal cannot escape, e.g. when it is tethered. it is generally recommended that wind speeds around animals should be between . m/s in winter (least value) and about . m/s in summer. these values strongly depend on relative humidity and temperature of the air and whether the skin of the animals is dry or wet, full fleece or shorn. in confined buildings this complex relationship between the various factors is strongly influenced by the ventilation system and the ventilation rate which is necessary for the number of animals kept in the animal house. it seem useful to develop a more comprehensive model for the interaction of the different air quality compounds and the air exchange rate to improve our understanding of the welfare and health impacts arising from the air environment. . . . ventilation ventilation plays an essential role in improving air quality in calf barns. this applies to free ventilated and forced ventilated houses. calculations of ventilation rates are usually based on heat removal in summer and moisture removal in winter, and give some guideline temperatures and humidities of the air which should not be exceeded (e.g. cigr, ; din , ) . ventilation rates in calf barns can only be calculated satisfactorily for confined buildings. minimum ventilation rates around m³ per kg live weight should be sufficient to keep the air quality within acceptable limits if the air distribution system ensures an even air exchange in all parts of the building. such guidelines (cigr) and norms (din , ) cannot guarantee healthy calves but they can substantially help in designing confined calf houses. it seems useful to standardise the air quality and ventilation requirements for confined calf houses in europe in order to reduce respiratory disorders, suffering of the animals and economic losses. the report highlights two aspects of human-animal relationships: the skills and motivations of caretakers to raise healthy calves, which are of particular importance for indoor calves and in large groups and are linked to the health status of calves; the physical contacts between caretakers and calves to improve subsequent reactions of calves to humans. it recommends careful monitoring (by the same person throughout rearing) and careful handling to habituate calves to human contacts. recent studies confirmed that the stockpersons have a great impact on both the productivity (e.g. growth) and the welfare of farm animals (stress responses, fear reactions during handling) (boivin et al., ; reviewed by hemsworth and coleman, ) . the effect of stockmanship is two-fold: good stockmanship leads to healthy animals and less stressful human-animal interactions. stockman skills are associated with positive attitudes towards work and towards animals. in calf production, a better health status is observed on farms where the caretaker (also the owner) believes that calves are sensitive animals and he/she has a positive attitude towards farming tasks in calf production (lensink et al., b) contacts given by stockpersons to animals depend also on human attitudes. stockmen that are positive towards gentle contacts with calves (e.g. stroking, talking) are more likely to provide calves with such contacts (lensink et al., a) . it is not only the duration of contact but also its nature that plays a role. gentle contacts (e.g. stroking, talking, letting a calf suck fingers, offering food) lead to calves approaching humans as they have less fear of handling (boivin et al., ; jago et al., ; lensink et al., b) . whereas rough contacts (e.g. hitting with a stick, use of nose tongs or an electric prod) lead to fear reactions in presence of humans (rushen et al., ) . the electric prod seems particularly stressful to calves (croney et al., ) and noises (metal clanging, shouts by humans) will also increase stress during handling (waynert et al., ) . during transport to slaughter, less fear responses to handling (e.g. due to regular previous experience of gentle contact) not only improves the welfare of calves but also improves meat quality (lower ph and lighter colour) (lensink et al., c; lensink et al., a) . cattle are able to distinguish between familiar and unfamiliar persons (rybarczyk et al., ; taylor and davis, ) . among familiar persons, they distinguish between those who have been rough with them and those who have been gentle (e.g. stroking, brushing, and offering food) (de passillé et al., ) . compared with individually housed calves, calves housed in groups tend not to approach humans and are more difficult to handle (lensink et al., b) . the presence of the dam can lower the effectiveness of gentle contacts with animals (boivin et al., ) . contact early after birth can be more effective that contact provided later; however, regular contact is necessary to maintain a lower level of fear responses to humans (boivin et al., ) . the report recommended: to dehorn calves between - weeks by cauterisation with adequate anaesthesia and analgesia (no details given) to castrate calves at months with adequate anaesthesia and analgesia (no details given) . . . dehorning dehorning means the removal of horns while disbudding (on young animals) corresponds to the removal of horn buds. disbudding can be performed by cautery, or by rubbing or covering the horn buds with a chemical (naoh, koh or colloidon), or by amputation with a specifically designed sharp tool, a scoop. recent publications confirmed that disbudding and dehorning are painful to cattle (stafford and mellor, ) . the existence of pain is deduced from observations of an increase in blood cortisol for several hours after dehorning and from specific pain related behaviour: head shaking, ear flicking (faulkner and weary, ) disbudding without anaesthesia or analgesia is painful to calves, even when young, and dehorning with a wire-saw is painful to cows even if anaesthesia is carried out (taschke and folsch, ) . disbudding by cautery (hot iron, electric tool) and chemical disbudding (naoh) are less painful than disbudding with a scoop (stilwell g. et al., a; stilwell g. et al., b; sylvester et al., ) . local anesthesia ( - ml lidocaine or lignocaine % around the corneal nerve - min before disbudding) can abolish the pain that immediately follows cautery and largely diminishes the pain caused by disbudding by other methods; the effects last for the few hours and when the nerve block has lost its effect, pain ensues stilwell et al., b; sutherland et al., b) . local anesthesia plus analgesia with a non-steroidal anti-inflammatory drug (nsaid)(e.g. ml flumixin meglubine or - . mg/kg ketoprofen ( %) min before disbudding) abolish pain caused by cautery but only reduces it in the case of disbudding with a scoop (unless it is followed by cautery) (sylvester et al., ; faulkner and weary, ; sutherland et al., a; stilwell et al., b) . in their review, stafford and mellor ( ) concluded that cautery is the less painful method for disbudding and that optimal pain relief is obtained with xylazine sedation, local anaesthesia and analgesia with a non-steroidal antiinflammatory drug. the report recommend that when cattle are to be castrated this should be done at around months of age and under appropriate anaesthesia and analgesia. methods to castrate cattle are: clamping (generally with a burdizzo clamp), constriction of the blood supply with a rubber ring, and surgical removal (cutting of the scrotum then traction on the testes and spermatic cords or cutting across the spermatic cross). calves are castrated as early as week up to over months (see review of practices used in uk by kent et al., ) . castration is painful whatever the method used and whatever the age of the calf (molony et al., ; robertson et al., ) . acute pain is deduced from the observation of increases in blood cortisol and abnormal postures (immobility), and behaviours such as foot stamping and kicking. chronic pain is deduced from the observation of activities targeting at the site of castration (e.g. licking, head turning, alternate lifting of the hind legs, and slow movements of the tail) as well as abnormal standing. burdizzo clamping and surgery induce acute pain for at least h (molony et al., ; obritzhauser et al., ; robertson et al., ) . burdizzo is less painful than surgery but may also cause pain for longer (at least h) due to scrotal inflammation (stilwell, pers. comm.) . castration with a rubber ring causes both acute and chronic pain for at least . mo (molony et al., ) castration is less painful for wk old calves than for - wk old calves (robertson et al., ) and it is less painful at . months than at older ages (ting et al., ) . local anesthesia ( ml lignocaine % into each testicle through the distal pole) abolishes the acute pain associated with castration by a ring or a band (stafford k.j. et al., ) . it reduces but does not abolish acute pain associated with castration by surgery or clamping (fisher et al., ; stafford et al., ) . analgesia with a nsaid drug (e.g. ketoprofen % mg/kg body weight) reduces the pain associated with clamping (ting et al., ) . some analgesics (e.g. caprofen, , mg/kg body weight) are effective for longer than h and are thus more likely to provide more effective pain relief (stilwell, comm. pers.) . local anaesthesia plus analgesia appears to eliminate the acute pain due to castration by surgery or clamping . the most important diseases in young calves are diarrhoea and respiratory disease (olsson et al., ; sivula et al., ; virtala et al., a , donovan et al., lundborg, ) . a prospective study was carried out on randomly selected beef herds in the midi-pyrenees region in france (bendali et al., ) . the objective was to describe diarrhoea and mortality in beef calves from birth to days of age. calves ( , ) were followed from december to april , and a total of visits allowed records of herd management practices, individual data and environmental conditions to be collected. the incidence rate for diarrhoea during the neonatal period was . %, and varied markedly between herds. eighteen herds did not suffer from diarrhoea, while five herds had an incidence of more than %. results indicate that % of diarrhoea appears during the first week and only % after the second week of life. the greatest risk of diarrhoea for a calf was during the first and second weeks of life ( . and . times, respectively). the month of birth was also significantly associated with morbility, the highest incidence was observed in december and march ( . % and . %, respectively) . the global mortality rate was . % and was two-times higher in december than in other months. forty per cent of herds did not exhibit mortality, and % had mortality rates greater than %. in a study of calf health in cow-calf herds in switzerland, busato et al. ( ) found that of calves included in the study % of the calves had been treated by a veterinarian. of those, % of the treatments were given because of diarrhoea and % because of respiratory disease. another swiss study (frei et al., ) showed that in swiss dairy herds, the incidence density (id) per animal-years of diarrhoea, omphalitis (infection of the navel), respiratory diseases and other diseases were . , . , . and . respectively. in a study of nine herds and calves with swedish red and whites, swedish holsteins and some crossbreeds the effect of group size on health was studied (svensson and liberg, ) . after transfer to group pens (at - days of age) . % of the calves had diarrhoea, . % had omphalophlebitis/umbilical abscess, . % had a clinical respiratory-tract disease and . % had weak calf syndrome. of all calves, in . % there was associated general condition impairment. in . % of the diarrhoea cases antibiotics were used as treatment and of the clinical respiratory-tract cases % were treated with antibiotics. several factors have been associated with an increased risk of infectious disease during the first days of life, particularly factors affecting serum immunoglobulin concentration. in a study of dairy herds in south-west sweden, svensson et al. ( ) clinically monitored the health of heifer calves from birth until days of age. % of the calves developed one or more diseases during this period. most of the diarrhoea cases were mild ( %) whereas of the cases of respiratory disease % were severe. the total morbidity was . cases per calf-month at risk and the incidence rates of arthritis, diarrhoea, omphalophlebitis, respiratory disease and ringworm were . , . , . , . and . cases per calf-month at risk respectively. odds ratios were calculated for severe diarrhoea in calves born in the summer (or: . ) and receiving colostrums through suckling instead of a bucket or nipple (or: . ). it has been shown that calves left with their mothers have a delayed/longer time to ingest colostrum and often fail to ingest adequate volumes (rajala and castrén, ) . svensson and liberg ( ) found that the health status of the mother cow - days before calving, length of dry period, retained placenta and somatic cell count were predisposing risk factors for respiratory disease in the calf. svensson et al. ( ) were also able to demonstrate that the odds ratios for respiratory disease and increased respiratory sounds were increased in calves housed in large group pens with an automatic milk-feeding system (or: . and . ). similar results have been reported by maatje et al. ( ) and plath ( ) . there was a decreased odds ratio for respiratory disease if calving was supervised (or: . ) . if birth was taking place in individual maternity pens or in tie stalls instead of in cubicle or group maternity pen, the odds ratio for increased respiratory sounds was . or . respectively. % of the diarrhoea cases were treated with antibiotics whereas % of the respiratory cases were treated using antibiotics. in another study of nine farms, svensson and liberg ( ) found that in pens for six to nine calves there was a significantly reduced risk of clinical respiratory tract disease (or: . - . ) compared with pens with - calves and there was also an association with the age at transfer to the group pen. the risk of diarrhoea was not affected by housing the calves in differently sized groups. however, calves housed in large sized groups grew significantly less quickly (approximately g/day) than calves housed in groups of six to nine. serological responses to respiratory viruses (e.g. bovine respiratory syncytical virus, parainfluenza virus, corona virus and viral diarrhoea virus) showing that animals within a herd are usually either all seropositive or all seronegative, indicate that infections spread to all calves in the herd when introduced or activated (hägglund et al., in press) and hence that aerosol is an important means for the spread of viruses. however, an infected animal is not equivalent to a diseased animal. it has been shown that calves housed in adjacent pens can maintain quite different levels of disease. svensson and liberg ( ) reported that calves in a group of had a significantly higher incidence of clinical respiratory disease that calves in an adjacent pen kept in groups of . engelbrecht ( ) reported calves transferred to group pens in a batchwise manner had significantly higher prevalence of diarrhoea and respiratory disease than calves in adjacent pens that were transferred to and from the group pen continuously. in both studies calves had no direct contact with calves in adjacent pens. these results indicate an important role of direct contact for the transmission of respiratory disease and hence the importance in disease control of decreasing direct contact between calves within the same building by means of solid walls. svensson and liberg ( ) also reported that the age at transfer from single pens to group pens was associated with the risk of respiratory disease, indicating that delaying transfer to after two weeks of age might be preferable for health reasons. enteritis is the most common disease in calves less than a month old (virtala et al., b; wells et al., ; radositis et al., ) . diarrhoea is caused by dietary factors or caused by infections due to viruses, bacteria or parasites. routines in distributing colostrum to the calf are crucial for transferring immunoglobins to the calf and to obtain a good health. (rajala and castrén, ; liberg and carlsson, ) . enteritis is clinically recognized by the observation of faeces with a looser consistency than normal. colour as well as smell of the faeces might be affected. diseased animals exhibit fever and may be inactive usually as a result of dehydration and possibly acidosis (radositis et al., ) . usually it is not possible to differentiate between different agents causing the diarrhoea by clinical findings. rotavirus is worldwide a major cause of diarrhoea and it is an often detected agent among young calves with enteritis (e.g. björkman et al., ) . rotavirus affects calves usually between and weeks of age and the diarrhoea can vary from very mild to lethal (de leeuw et al., ) . the virus is excreted through faeces of infected animals and is very resistant for several months, that is why cleaning of pens is necessary to break the infectious path (saif and theil, ) . bovine coronavirus is most commonly seen in calves at about week of age (fenner et al., ) . escherichia coli k + may cause diarrhoea in young calves although it is a part of the normal intestinal flora. poor routines for transferring colostrum to the calf, stress etc. might trigger a diarrhoea outbreak (wray and thomlinson, ) . severity of the disease may vary but with a high proportion of mortality (radositis et al., ) . only amoxicillin is recommended for the treatment of diarrhoea caused by e. coli bacteria associated by systemic illness. in calves with diarrhoea and no systemic illness (normal appetite for milk, no fever), the health of the calves should be monitored carefully and no antibiotics should be administered (constable, ) . bovine viral diarrhoea may occur at any age. the infection causes an immunosuppression in infected animals which may lead to infections with other intestinal or respiratory pathogens (elvander et al., ; de verdier klingenberg, ) and it may increase the mortality rate in the herd (ersböll et al., ) . salmonella spp, mainly s dublin and s typhimurium can affect calves usually between and weeks of age. the pathogen is introduced into the herd via infected feed, water, pastures, cattle or humans or via other animals entering the herd. calves are infected orally and clinical signs are fever and bloody diarrhoea (carter and chengappa, ) . clostridial infections in the gastrointestinal tract are sometimes a problem in calves. usually, the calf, less than days of age, develops haemorrhagic, necrotic enteritis and enterotoxemia, often associated with clinical abdominal pain. affected calves exhibit tympany, hemorrhagic abomasitis and ulcerations in abomasum (songer and miskimins, ) . as yet, relatively little is known about the etiology aside of the participation of c. perfringens type a. overfeeding or feeding which decreases gut motility is suggested to contribute to the occurrence of the disease (songer and miskimins, ) eimeria spp. are frequently present in cattle in europe (bürger, ) . predominantly e. ellipsoidalis was found in housed calves in east germany (hiepe et al., ) and the distribution may differ from country to country. svensson ( ) found a predominance of e. alabamensis in swedish dairy calves. clinically, signs are rarely seen but diarrhoea can occur usually as a result of exposure at the first grazing season in areas contaminated with oocysts (svensson, ) . there is evidence that infection rates have increased since the prohibition of tethering (berthold, pers. com.). emeria bovis and emeria zuernii are other intracellular protozoan parasites belonging to the same group and with a worldwide distribution (urquhart et al., ) . it is often seen in calves between - months of age (holliman, ) . the disease is triggered by stress such as very cold or hot climate (urquhart et al., ) . cryptosporidial infection in calves less than days old is significantly associated with the risk of infection in the dairy herd. the risk increases when animals are grouped together and when hygiene and management practices are deficient (attwill et al., ; mohammed et al., ) . factors associated with a decreased risk of infection in preweaning calves were shown to be use of ventilation in calf rearing areas, daily addition of bedding, feeding of milk replacer, daily disposal and cleaning of bedding and use of antibiotics. in addition, postweaning moving of animals was also associated with a decreased risk of infection with c. parvum (mohammed et al., ) . perryman et al. ( ) showed that with appropriate supply of immune colostrum, diarrhoea can be prevented. two species are distinguished: c. parvum and c. andersoni, although only c. parvum has been shown to be associated with diarrhoea . cryptosporidia parvum is an intracellular protozoan parasite belonging to coccidiae. in the uk c. parvum has been considered to be one of the most common agents in neonatal diarrhoea in calves (reynolds et al., ) . in denmark it was found mixed with other enteropathogens in % of diseased calves (krogh and henriksen, ) . in two recent swedish studies it was found in % and % respectively of calves with diarrhoea (de verdier klingenberg and svensson, ; björkman et al., ) . the most common form of respiratory disease affecting young calves is enzootic pneumonia (ames, ; radositis et al., ) . it is considered to be a multifactorial disease with causative agents, individuals and environmental factors as important components (ames, ) . enzootic pneumonia usually affects calves between and months of age (radositis et al., ) . the signs usually found are fever, nasal discharge, coughing and increased respiratory sounds when lung auscultation is performed. secondary bacterial infections may occur which might increase the fever. diagnosis of etiological factors may be achieved from serological examinations, viral examinations from nasal discharge or at autopsy. bovine respiratory syncytical virus (brsv) is a worldwide present agent with seasonal peaks during autumn and winter (baker and frey, ) . the virus is thought to be transmitted from infected animals, by transmission of humans or by airborne transmission (van der pohl et al., ; elvander, ) . morbidity can be high but mortality is usually low . another virus with a milder course of disease is para-influenza- virus but the virus can cause immunosuppression predisposing to secondary bacterial infections (adair et al., ) . the most common bacterial pathogens in calves with respiratory disease are pasteurella multocida and manheimia hemolytica (mosier, ; bengtsson and viring, ) . these agents are usually found in the bovine nasopharynx and may, as a result of viral disease proliferate and colonise the lungs of the calf (kiorpes et al., ) . haemophilus somnus was shown to be commonly present in danish calves (tegtmeier et al., ) where no such agents were found in swedish calves (bengtsson and viring, ) . arcanobacterium pyogenes and staphylococcus aureus (carter and chengappa, ) as well as mycoplasma spp. (ames, ) are other agents found in immune depressed calves with other infections. infections may occur in the umbilical cord (radositis et al., ) of newborn calves. various bacteria are found and through a bacteraemia infection may spread to to joints, meninges and internal organs (radositis et al., ) . omphalitis is painful in response to palpation of the umbilicus. arthritis is often secondary to an umbilical infection and usually affects the calf during its first month resulting in warm and swollen joints, fever and lameness (radositis et al., ) . the effect of environmental factors on the risk of diarrhoea and respiratory disease was studied by lundborg et al. ( ) in the same farms and calves as previously reported by svensson et al. ( ) . they found that the placing of calf pens along an outer wall was associated with a significantly higher risk of diarrhoea (or: . ). an ammonia level below ppm was significantly associated with the risk of respiratory disease (or: . ) but variations of ammonia levels were low, while the odds ratio for increased respiratory sounds was associated with a bvdv infection in the herd (or: . ) and draught (or: . ). absence of draught was associated with the risk for infectious diseases other than diarrhoea and respiratory disease (or: . ), a finding which could not be explained by the authors. an increased calf mortality rate in herds with a bvdv infection has also been reported by ersböll et al. ( ) and the eradication of bvdv infection in a dairy herd has been demonstrated to decrease the incidence of calf diarrhoea (de verdier klingenberg et al., ) . typically, clinical experience is that the incidence and prevalence of infectious respiratory disease is much higher in rearing systems where the calves have been bought and transported from several farms where they were born to a specific rearing farm than if they are reared on the farm they were born on. calves reared indoors commonly develop complex respiratory diseases. bergmann ( ) reported that % of calves of a large fattening unit with several thousand calves suffered from bronchopneumonia within the first six month of their life. similar figures were reported from herrmann ( ) with a prevalence of to %, lämke et al. ( ) % and busato et al. ( ) %. the disease seems to be continuously present and does not come or go in form of isolated outbreaks. therefore kielstein et al. ( ) called it enzootic pneumonia. it is a typical multifactorial disease caused by a variety of different types of micro-organisms which are always present but becoming a nuisance only when additional factors contribute (grunert, ) . the most prominent reasons for losses of calves in the first weeks of life are respiratory and digestive disorders (katikaridis ; girnus, ) . losses can reach to per cent in the first six months of rearing (berchtold et al. and others). estimations show that the financial losses are reaching million €/year in germany (biewer, ) . this sum does not cover the costs of veterinary treatment and reduced growth rates of the calves. there are several epidemiological studies on the different diseases in calves in the first couple of weeks of life (katikaridis, ; biewer, ; girnus , svenson et al., . heinrichs ( ) reported that % to % of , fallen calves coming for post-mortem dissection showed digestive disorders. calves weeks of age died predominantly of respiratory diseases ( % to %). an investigation of , calves less than months shows that enzootic bronchopneumonia can already start with the age of weeks (buhr, ) . the calves were not older than months. % displayed abomasum enteritis. % of the animals suffered from pneumo-abomasal enteritis. only . % of the fallen animals suffered from a distinct pneumonia. an epidemiological survey of calf losses in free range and suckling cow herds showed that the percentage of calf losses is increased with herd size. % of herds with less than suckling cows had a calf mortality of less than %. in herds with more than cows, these were % of all investigated farms, calf losses were higher than % (laiblin and metzner, ) . main disorders were again diseases of the digestive tract ( %) and respiratory tract ( %). the authors calculated that the disease risk for calves born from cows that were housed during the calving season was . times higher compared with cows kept the whole year on free-range. the epidemiological data from the vast majority of investigations suggest considerable differences in morbidity and mortality of calves among different farms. this implies that the management and housing conditions greatly influence heath, welfare and survival of calves in the first months of their life. the situation was not substantially improved by vaccination of cows against a cocktail of infectious agents causing diarrhoea. there are no experimental studies available to indicate whether or not there is any advantage to calf welfare of preventing individuals in separate pens from social contact as opposed to a disadvantage to calf welfare of greater spread of disease with housing where such social contact is possible. in general, disease spread occurs in buildings with continous air space and contact is not a clearly identified factor. however, recent results indicate an important role of direct contact for the transmission of respiratory disease and hence the importance in disease control of decreasing direct contact between calves within the same building by means of solid walls (see chapter / / ). antibiotic resistance although the use of antibiotics as growth promoters is being progressively restricted through eu regulations, they are still used in large quantities in calf rearing for both prophylactic and therapeutic purposes. in those instances where calves are not reared on site but transported to other locations and mixed in groups, the incidence of clinical illness is high and the use of antibiotics is frequent. in a study of antibiotic resistance, berge et al. ( ) found high levels of multiple resistance in calf commensal faecal escherichia coli both on farms with calf production and on dairy farms. the investigators found that escherichia coli from calves on dedicated calf-rearing facilities was more likely to be multiple-resistant than e. coli from dairy-reared calves (or: . ) (berge et al., a) . in her phd thesis, berge ( ) showed that both prophylactic use of antibiotics in milk replacer and individual antibiotic therapy increased the resistance of faecal e. coli in calves. e. coli isolates from calf ranches were the most resistant, with in order of decreasing levels, isolates from feedlots, dairies and beef cow-calf farms. on organic dairies fewer resistant e. coli isolates were found in comparison with conventional dairies. e. coli isolates from beef cow-calf farms were less resistant if the farms were on remote locations compared to those on locations close with dairy intense areas. the use of antibiotics to treat clinical illness will increase the welfare of the animal given that the drug has a beneficial clinical effect. however, the frequent use of antibiotics results in increasing resistance in bacteria such as e. coli and thus poses a threat to the welfare of calves in a longer perspective as well as to man (aarestrup and wegener, ) . in a clinical trial on a calf ranch in california, it was shown that the most important factor for decreasing morbidity and mortality was to ensure adequate passive transfer through colostrum (berge et al., c) . thereafter, the ability to use antibiotics for clinical treatment of disease was important to decrease morbidity and mortality. the use of antibiotics in the milk replacer had a minor protective effect on calf health. the authors concluded that in order to minimize prophylactic use of antibiotics, adequate passive transfer of colostrum needs to be assured. furthermore measures need to be taken to optimize nutrition, decrease environmental stress and pathogen load on the farms. in the same study, the antibiotic resistance patterns of the commensal faecal e. coli of calves receiving antibiotics in the milk-replacer, antibiotics for clinical disease, and no antibiotic therapy were compared (berge et al., ) . the study showed the emergence of highly multiple resistant e. coli in the calves receiving antibiotics in the milk-replacer. the commensal faecal e. coli were predominantly resistant to at least of antibiotics tested. the resistance covered the antibiotics available for clinical therapy. antibiotic treatments for clinical disease resulted in a transitory shift to more resistant faecal e. coli, but the effect was not detectable approximately days post-treatment. the effect of clinical therapy with antibiotics was similarly assessed in steers in south dakota. in a feedlot study a single dose injectable florfenicol to steers resulted in transitory shifts to increasing levels of multiple resistant e. coli in the faeces. the e. coli from the treated steers were not only more resistant to chloramphenicol (same antibiotic group as florfenicol), but were increasingly resistant to several other antibiotics in other antibiotic classes. (berge et al., b) in dairy cattle it has been estimated (kelton et al., ) that between and % of all lactations include a mastitis infection. most of these cases are treated with antibiotics. milk must be withheld from sale during the treatment and for the compulsory withdrawal period. such "waste milk" is often fed to calves as it is the most economical alternative from the farmer's perspective. earlier studies have previously given various results on how antibiotic resistance develops as a result of the use of this procedure. recently, a controlled, multiple-dose experiment by langford et al. ( ) found an increasing resistance of gut bacteria to antibiotics with increasing concentrations of penicillin in milk fed to dairy calves. in a multi-farm study in california (berge et al., ) including dairies, no association was found between increasing levels of antibiotic resistance in calf faecal e. coli and the consumption of waste milk. it should, however, be noted that mastitis in these dairies are predominantly treated with intra-mammary antibiotics (cephalosporins) and injectable antibiotics are rarely used for mastitis treatments (berge, non-published data) . the use of rearing systems for calves that increase the incidence of disease and thus the use of antibiotics for either preventive or clinical purposes should be avoided. further, there is a risk that the use of "waste milk" for calves will increase antibiotic resistance in gut bacteria in calves. . food safety aspects of calf farming foodborne hazards that can be present on calf farms include biological and chemical hazards. biological hazards associated with calf farming include following main examples: a) bacterial foodborne pathogens salmonella spp., human pathogenic vtec (hp-vtec), thermophilic campylobacter spp., and mycobacterium bovis; and b) parasitic foodborne pathogens tania saginata cysticercus and cryptosporidium/giardia. on-farm control of chemical foodborne hazards is outside the scope of this chapter and will not be considered. faecal shedding of foodborne pathogens can occur in calves and adult bovines without symptoms of disease; but the shedding pattern may differ between the two age categories. in the conventionally reared animal the intestinal tract becomes colonised from birth by combinations of bacterial species until the characteristic and complex flora in the adult animal is achieved. in the early stages of the process infections with bacterial pathogens are common. once the indigenous flora is established it resists colonization by pathogens and other 'foreign' strains by competitive exclusion . the gut flora of the bovine species changes with ruminal development and the population of faecal coliforms of the adult differ markedly, both qualitatively and quantitatively, from that of the young; particularly that of veal calves fed milk replacer (smith, some early studies (gronstol et al., a; gronstol et al., b) , based on experimental salmonella infection of calves, described virulence, spread of infection and the effects of stress on the carrier status. hinton et al. ( hinton et al. ( , a hinton et al. ( , b determined the incidence of salmonella typhimurium (dt and dt ) excretion by veal calves fed milk replacer and report that, while initially low on in-take at around days of age, its incidence rose to a peak by days. the level of salmonella contamination of the environment also affects the incidence of infection in housed animals (hinton et al., a (hinton et al., , b . provided calves are reared in separate fattening units and slaughtered on separate slaughterlines the incidence of salmonellae in calves can be maintained at a very low level (guinée et al., ) . during meat inspection clinical salmonellosis is sometimes diagnosed in a herd of veal calves; however, the prevalences are usually of the order of magnitude of per thousand and the strains isolated are generally restricted to salmonella typhimurium (occasionally) and more commonly, salmonella dublin (up to %). although salmonellae may not be isolated from faeces, significant proportions of calves slaughtered commercially ( . - . %) have contamination involving hepatic lymph nodes, liver, mesenteric lymph nodes and, because of cross contamination, they may ultimately also be isolated from the carcass surface (nazer and osborne, ; wray and sojka, ) . studies conducted in the netherlands in the late 's indicate that microorganisms may be released from lymph nodes through transport stress and may appear in the faeces. this results in young veal calves being cross-infected in transit and at markets; however, in dutch studies faecal samples from no more than . % of the animals were found to contain salmonellae on arrival at the fattening units (van klink and smulders, ) . moreover, within weeks of arrival, faeces samples become negative again (van zijderveld et al., ) . subsequent studies by the same workers (van zijderveld et al., unpublished, cited by van klink and smulders, ) indicate that faecal samples from calves which had survived clinical salmonellosis also become culture-negative, albeit only after weeks. these findings suggest that, provided stressful transport conditions are avoided and sufficient hygienic care is taken to avoid cross infection during transport to the abattoir, the extent of introduction of salmonellae to the veal slaughterline is indeed extremely low. this is substantiated by repeated failure to isolate salmonellae from carcass surfaces of veal calf populations, and from their livers and offal meats (van klink and smulders, ) . as with other bacterial foodborne pathogens, antimicrobial resistance in salmonella shed by calves represents an additional food safety risk. numerous studies have shown that use of antimicrobials in food producing animals selects for resistance in non-typhoid salmonella spp. and that such variants have been spread to humans (who, ; walker et al., ; fey et al., ) . in general, antimicrobial resistance in s. typhimurium isolates from bovines in the eu was widespread in , with highest prevalence of resistance to ampicilin, sulfonamide, tetracycline and streptomycin (efsa zoonosis report, b), but the data does not relate specifically to calves. vtec is a group of e. coli that produces one or more verocytotoxins (vt) also known as shiga toxins (stx), but not all members of this group cause foodborne disease in humans. in the opinion on verotoxigenic e. coli (vtec) in foodstuffs (scvph, c) , vtec that have been associated with causing human disease were referred to as human pathogenic vtec (hp-vtec). foods of bovine origin (e.g. beef, milk) have been implicated in a number of foodborne outbreaks caused by hp-vtec (borczyck et al., ; chapman et al., ; martin et al., ; pennington, ; scvph, a) ; these include several serotypes (e.g. o , o , o , o and o ). when adult cattle were inoculated with vtec o , they showed no outward signs of infection and the organism was cleared from the gastrointestinal tract within two weeks (wray et al., ) . the organism seems to be a constituent of their naturally-occurring microflora, and longitudinal studies show most cattle occasionally carry e. coli o in their faeces (hancock et al., ; lahti, ) . however, the prevalence of infection with hp-vtec of, and the shedding patterns in, cattle can vary due to variable factors including age, immunocompetence status, husbandry conditions, season and geographical areas. prevalence of vtec o is usually higher in younger animals (synge, ; cray and moon, ; hancock et al., ; mechie et al., ; van donkersgoed et al., ) . in calves, the prevalence of e. coli o :h can range from zero to . % prior to weaning, and often increases after weaning (bonardi et al., ; laegried et al., ) . calves normally show no, or little, sign of infection, perhaps only some excess faecal mucus (myers et al., ; synge and hopkins, ; brown et al., ; richards et al., ; wray et al., ) ; the shedding rate can fall rapidly in the first two weeks after inoculation and continue intermittently for several weeks. in the first three months of life on contaminated farms, faecal prevalence can increase from around % to %, possibly due to the decline in maternal immunity (busato et al., ) . fasting showed little effect on the carriage and excretion of e. coli o in calves (harmon et al., ) . less information is available on non-o hp-vtec in calves that have potential to cause enterohaemorrhagic disease in humans, so establishing indicators for virulence and clarifying the epidemiology of such serotypes is needed. thermophilic campylobacter spp. according to the biohazard scientific report on campylobacter in animals and foodstuffs (efsa, a) , the most important species of campylobacter are the thermophilic species c. jejuni ssp. jejuni, c. coli and c. lari; other species which are known to cause human illness are c. upsaliensis, c. fetus ssp. fetus and c. jejuni ssp. doylii. the most common species recovered in human disease is c. jejuni. campylobacter spp. can be found throughout the intestine of cattle, but the level of the organism in the rumen is significantly lower than that found in the small intestine and in faeces (stanley et al., ) . the class of cattle also has an effect on the level of campylobacter spp. found in the faeces; faeces may contain around cfu/g in calves, around cfu/g in beef cattle and around cfu/g in adult dairy cattle. campylobacter spp. are more often found in the faeces and intestine than in the rumen, while campylobacter jejuni prevalence is much less than that of campylobacter hyointestinalis (ataby and corry, ; grau, ) . campylobacter jejuni has been found in calves in % of beef farms, campylobacter coli in % and campylobacter hyointestinalis in % (busato et al., ) . within the herds, zero to over % of the calves may be excreting campylobacter spp.; % of the calves may be positive for campylobacter spp. in herds without evidence of diarrhoeal disease (myers et al., ) . in this study, % of the isolates were c. jejuni. in a study of veal calves at slaughter, c. jejuni was found in % of calf rumen samples (in low numbers; < /g) and in % of calf faecal samples (grau, ) , whilst c. hyointestinalis was found in % of calf rumen samples (in numbers > /g) and in % of faecal samples. the coats of the calves were also contaminated, as % were positive for c. jejuni and % for c. hyointestinalis. as with other bacterial foodborne pathogens, antimicrobial resistance in thermophilic campylobacter spp. shed by calves represents an additional food safety risk. in , although the total number of isolates was relatively small and the data are not related specifically to calves, some eu member states reported relatively high prevalence of resistance to quinolones, fluoroquinolones and tetracyclines in campylobacter spp. including c. jejuni from bovines (efsa zoonosis report, ) which can be an emerging public health concern. . . mycobacterium bovis efsa a, opinion on the risk assessment of a revised inspection of slaughter animals in areas with low prevalence of cysticercus); therefore, the parasite will not be further considered here. cryptosporidium parvum and giardia duodenalis are protozoan parasites that have caused disease in humans primarily via contaminated water or foods (e.g. salads), but also via chicken salad and milk drinks. high prevalences of cryptosporidium and giardia in veal calves (the age group - weeks) have been reported (van der giessen et al., ) . however, in this study, all isolates from the former group belonged to the pathogenic cryptosporidium parvum genotype , whilst only few isolates from the latter group showed similarities with giardia isolates from humans. other authors also reported presence of these protozoan parasites, cryptosporidium (de visser et al., ) and giardia (trullard ; mcdonough et al., ) in veal calves. risk evaluation and principles of food safety assurance at calf farm level the prevalence-level of infection and/or contamination of calves with, and further spread of, foodborne pathogens at calf farms depend on a large number of risk factors that are inherently variable even at single-farm level. the complexity of the problem is further exacerbated by the existence of a number of different farming systems for veal calves in the eu; and even within each of the main farming categories (e.g. intensive vs extensive) a large number of "epidemiological" subcategories exists that differ with respect to one or more risk factors. therefore, presently, both knowledge and published data are insufficient to produce a universal risk assessment enabling quantitative categorization of different types of calf farms and/or their quantitative comparison/ranking with respect to main foodborne pathogens. nevertheless, the role of some main factors contributing to an increase in prevalence and/or in levels of foodborne pathogens in food animals on farms (including calves) are reasonably well understood, as are the generic principles of their control. they are indicated in a condensed form in table . . it is logical that calves from farming systems in which fewer of the contributing factors exist and where the controls are more complete/efficient will represent lower foodborne pathogen-risk than calves from farming systems having opposite contributing factors-controls situation. therefore, future food safety risk categorization of individual farming system, or related between-systems comparisons, would be dependent upon obtaining and analysing quantitative information on: a) status/levels of contributing factors; b) status/levels of hazards of main concern; and c) existence and effectiveness of their controls. increased "importation" and spread of pathogens via animals "asymptomatic excretors" animal supply only from known, epidemiologically "equivalent" sources; "all in-all out" system presence of animal diseases spread of zoonotic agents in calves global disease control programmes; heard health plans *good farming practice-good hygiene practice . risk assessment . . introduction to risk assessment approach when the ahaw panel of efsa was confronted with the tasks of updating the report (svc, ) , the working group members were asked to make it on the basis of a risk assessment , and particularly to consider the possible effects on the calf and, where relevant, on food safety. it appeared entirely feasible for the working group members to follow this part of a risk analysis approach where risks were defined as those concerning the welfare of calves. the risk of concern in this report is that the welfare of the calves will be poor. this may involve an increased risk of injury, of disease, of negative feelings or of failure to cope. the time span of such poor welfare might vary from short to long and severity can vary from low to high. a member experienced in risk assessment procedures was included in the working group from the start. initially, the procedure adopted for the risk assessment was identified and presented to the participants of the whole working group. when identifying the hazards, it has been assumed that the managers of the farm and animal keepers have a basic knowledge, that they have undergone training, that they are aware that the particular constraints on the farm do not hamper their work (e.g. lack of facilities on a farm). however, it is pointed out that under practical conditions hazards may interact, e.g. inadequate air flow may interact with poor air quality, inadequate clinical health monitoring may interact with inadequate haemoglobin monitoring, etc. the identification of hazards and consequential risks to welfare, as well as the risk assessment approach, were agreed by the working group. steps of the risk assessment a. multidisciplinary approach the expert working group it was selected on the basis of having expertise in animal science, ethology, veterinary medicine, risk assessment and food safety. b. listing of potential hazards, hazard characterization and exposure assessment the first step was to describe the needs of calves (see chapter and listed below). then, hazards that might compromise those needs were identified (table . ) and related to each specific need (table . ). the hazards were characterized in relation to the impact they have on the animal. the exposure to the hazard might vary between different rearing systems. for this purpose a set of different rearing categories was developed (table . ) as well as scoring categories for the hazard characterisation, exposure assessment and risk evaluation (table . ). b.a. small groups, bucket fed (not suckling) + solid foods, weaned at - months b.b. groups with an automatic feeding system (not suckling) + solid foods, weaned at - months b.c. feed lots (high density groups within outside pens) b.d. hutches outside, bucket fed (not suckling) + solid foods, weaned at - months beef calves c.a. suckler calves in small groups kept inside, led twice a day to the dam for suckling up to - months the hazards were identified and characterized, as well as, an estimate of the probable exposure. however, to ensure that these estimates of exposure correspond with current practice in various european calf production systems, a group of veterinarians, experts in clinical practice in calf production, named the "consultation group", was identified. criteria for invitation were the following; predominantly engaged in clinical practice; extensive clinical experience in calf medicine; and covering various geographical areas where calf production is significant in the eu. another important criterion was that the consultant should not be affiliated with the calf production industry. in total veterinarians accepted an invitation to assist in the exposure assessment. the experience of the individuals covered the various husbandry systems and important veal producing countries in europe. the consultation group prompted that for the exposure assessment, a quintile distribution (i.e. five classes of % increments) of exposure classes be adopted. in some instances the estimates of the wg and the consultation group on exposure did not agree in which case the opinion of the consultation group was interpreted to represent the factual situation. in other instances the exposure could not be estimated due to lack of data, in which cases the risks were labelled "uncertain". for hazards characterized as moderately to very serious, this uncertainty is highlighted. table in annex show the agreed scoring between the wg members and the "consultation group" for the hazard characterisation, exposure assessment and risk evaluation. inadequate colostrum intake -quantity b) inadequate colostrum intake -quality c) inadequate colostrum intake -duration ) iron deficiency resulting in haemoglobin levels below . mmol/l ) deficiency of other minerals (cu, se) ) insufficient access to water (not milk) (especially during warm season) ) allergenic proteins ) insufficient appropriately balanced solid food ) overfeeding (too rich diet) ) underfeeding ) too low temperature of milk or milk replacer ) exposure to excessively contaminated feed that results in pathology ) no access to natural teat or artificial teat housing ) high humidity and too high or low a temperature ) indoor draughts ) inadequate ventilation, inappropriate airflow, and air distribution within the house, airspeed, temperature ) poor air quality (ammonia, bio-aerosols and dust)) ) poor air quality ( major risk ; is considered not applicable c. assessment of whether hazards pose risks (substantiation by scientific evidence) as a consequence of the hazard characterisation and exposure assessment, the risk for poor animal welfare and health was assessed by integrating the hazard character with exposure according to the table . below. the risk was assessed as "major" if the hazard was judged to have a very serious effect and the exposure was frequent or very frequent or if the hazard was serious and exposure was very frequent. the risk was assessed to be "minor" if the hazard was very serious and exposure was rare, if hazard was moderately serious and exposure was moderately frequent or if hazard was adverse and exposure was very frequent. the hazards of iron deficiency and insufficient floor space is considered to be very serious, the hazard of inadequate health monitoring is considered to be serious and the hazards of exposure to inadequate hemoglobin monitoring, allergenic proteins and too rich diet is considered to be moderately serious. for these hazards, there is not enough information on the exposure of calves mainly due to lack of data why it is recommended that further studies should be made to provide evidence for an exposure assessment. regarding the hazard ) castration and dehorning without anesthetic and analgesic drugs, there is a variation in relation to national legislation as to why the risk of poor welfare in relation to castration and dehorning is widely different between countries. further, there is a variation in the use of analgesia during the time after the surgery is carried out which also affects the welfare of the calf. the ahaw panel wishes to thank the members of the working group chaired by panel member the needs and functioning of calves the needs of calves hutches: partially closed, outside area individual pens: open front structure straw yard with bedded lying area . . . group pens with automatic milk feeder . calf rearing and animal environmental pollution . . the quantitative share of calf production in the pollution problem . calf diseases and use of antibiotics human pathogenic-verotoxigenic escherichia coli (hp-vtec) on calf farms risk evaluation and principles of food safety assurance at calf farm level listing of potential hazards, hazard characterization and exposure assessment assessment of whether hazards pose risks (substantiation by scientific evidence) annex . hazard characterisation and exposure assessment the effects of antibiotic usage in food animals on the development of antimicrobial resistance of importance for humans in campylobacter and escherichia coli cytotoxic interactions between bovine parainfluenza type virus and bovine alveolar macrophages immune function is impaired in iron-deficient, homebound, older women effect of orally administered cimetidine and ranitidine on abomasal luminal ph in clinically normal milk-fed calves effect of feeding frequency and route of administration on abomasal luminal ph in dairy calves fed milk replacer effect of orally administered omeprazole on abomasal luminal ph in dairy calves fed milk replacer dairy calf pneumonia. the disease and its impact effect of type of housing on veal calf growth performance, behavior and meat quality incidence of respiratory disorders during housing in non-weaned charolais calves in cow-calf farms of pays de la loire (western france) age, geographic, and temporal distribution of fecal shedding of cryptosporidium parvum oocysts in cow-calf herds effect of individual versus group rearing on ethological and physiological responses of crossbred calves bovine respiratory syncytical virus bovine respiratory syncytical virus ventilation system to reduce pneumonia in a warm calf house: a case study the effect of increasing dietary fibre on feeding, rumination and oral sterotypies in captive giraffes (giraffa camelopardalis) neuroendocrine alterations in iron deficiency effects of form of the diet on anatomical, microbial, and fermentative development of the rumen of neonatal calves pattern of diarrhoea in newborn beef calves in south-west france respiratory infections -project, panorama and treatment strategies kälberkrankheiten spatial, temporal and management-specific factors influencing antibiotic resistance and carbohydrate fermentation patterns in bovine enteric escherichia coli and the clinical consequences of limiting antibiotic use in pre-weaned calves assessing antibiotic resistance in fecal escherichia coli in young calves using cluster analysis techniques animal and farm influences on the dynamics of antibiotic resistance in faecal escherichia coli in young dairy calves assessing the effect of a single dose florfenicol treatment in feedlot cattle on the antimicrobial resistance patterns in fecal escherichia coli a clinical trial evaluating prophylactic and therapeutic antibiotic use on health and performance of calves a field trial evaluating the influence of prophylactic and therapeutic antibiotic administration on antibiotic resistance in fecal escherichia coli in dairy calves cryptosporidium parvum, giardia intestinalis and other enteric pathogens in swedish dairy calves role of oxytocin in glucose homeostasis and weight gain iron-deficiency anaemia in calves endocrine and metabolic aspects in milk-fed calves nutritional physiology of neonatal calves grouping and social preferences in calves, heifers and cows emotional reactivity affected by chronic stress: an experimental approach in calves submitted to environmental instability is gentling by people rewarding for beef calves? early contact with peers or a stockperson influences later emotivity and social behaviour of dairy calves the maternal environment limits the effect of early human nursing and contact on lambs' socialisation to the stockperson stockmanship and farm animal welfare activity, oral behaviour and slaughter data as welfare indicators in veal calves: a comparison of three housing systems isolation of verocytotoxin-producing escherichia coli o :h from cattle at slaughter in italy bovine reservoir for verotoxin-producing escherichia coli o :h . the lancet(i anaemia and veal calf production abomasal ulcers in veal calves: pathogenesis and prevention husbandry methods leading to inadequate social and maternal behaviour in cattle indicators of poor welfare needs and welfare of housed calves. in: new trends in veal calf production scientific research on veal calf welfare welfare, stress and the evolution of feelings welfare assessment and problem areas during handling and transport the use of the concept animal welfare in european conventions, regulations and directives. food chain evolution of pain coping, stress and welfare welfare behaviour and welfare in relation to pathology the effects of group-rearing or partial isolation on later social behaviour of calves approaching questions of stress and welfare effects of disease on farm animal welfare bem-estar animal: conceito e questões relacionadas -revisão brain measures which tell us about animal welfare experimental escherichia coli o :h carriage in calves untersuchung von todesursachen bei kälbern in schleswig-holstein in den jahren eimeria-infektionen beim rind calf health in cow-calf herds in swotzerland prevalence and infection risks of zoonotic enteropathogenic bacteria in swiss cow-calf farms principles and guidelines for the conduct of microbiological risk assessment. document cac/gl the influence of air quality on production increases associated with all-in/all-out management essentials of veterinary bacteriology and cattle as a possible source of verocytotoxin-producing escherichia coli o infections in man effects of pair versus individual housing on the behavior and performance of dairy calves antimicrobial use in the treatment of calf diarrhoea effects of various levels of forage and form of diet on rumen development and growth in calves somministrazione di un mangime solido a vitelli a carne bianca stabulati in gabbia individuale the provision of solid feeds to veal calves: i. growth performance, forestomach development, and carcass and meat quality experimental infection of calves and adult cattle with escherichia coli o :h effects of handling aids on calf behavior iron deficiency and the immune response the pituitary-adrenal response to stress in the iron-deficient rat indicators relevant to animal welfare: a seminar in the c.e.c. programme of coordination of research on animal welfare the behaviour of calves under four levels of lighting the effect of different housing conditions on behavioural and adrenocortical reactions in veal calves from an animal's point of view: motivation, fitness, and animal welfare phylogenetic characterization of 'candidatus helicobacter bovis', a new gastric helicobacter in cattle rotavirus infections in calves in dairy herds sucking motivation and related problems in calves dairy calves discrimination of people based on previous handling iron supplementation improves iron status and reduces morbidity in children with or without upper respiratory tract infections: a randomized controlled study in colombo, sri lanka enhancement of clinical signs in experimentally rotavirus infected calves by combined viral infections group a rotavirus as a cause of neonatal calf enteritis in sweden incidence of diarrhea among calves after strict closure and eradication of bovine viral diarrhea virus infection in a dairy herd enteric infections in veal calves: a longitudinal study on four veal calf units behaviour and welfare of veal calves in relation to husbandry systems comparison of four methods of calf confinement. ii report of the scientific committee on animal health and animal welfare. health and consumer protection, directorate-general, directorate c -scientific opinions iron status, immune capacity and resistance to infections planungs-und berechnungsgrundlagen für geschlossene zwangsbelüftete ställe (thermal insulation for closed livestock buildings. thermal insulation and ventilation forced. part : principles for planning and design for closed ventilated livestock buildings). deutsches institut für normung e assosiations between passive immunity and morbidity and mortality in dairy heifers in florida, usa contribution to the study of gut hypersensitivity reactions to soybean proteins in preruminant calves and early-weaned piglets igg, iga, igg and igg specific responses in blood and gut secretion of calves fed soyabean products the interpretation of preference tests in animal behaviour animal right-animal welfare. a scientist's assessment measuring preferences and the strength of preference animal welfare as defined in terms of feelings. acta agriculturae scandinavica section a opinion of the scientific panel on biological hazards of the european food safety authority on standards for the microclimate inside animal transport road vehicles opinion of the scientific panel on biological hazards of the european food safety authority on "the risk assessment of a revised inspection of slaughter animals in areas with low prevalence of cysticercus welfare aspects of the main systems of stunning and killing the main commercial species of animals opinion of the scientific panel on biological hazards of the european food safety authority on "campylobacter in animals and foodstuffs the community summary report on trends and sources of zoonoses, zoonotic agents and antimicrobial resistance in the european union in opinion of the scientific panel on biological hazards of the european food safety authority on "an assessment of the public and animal health risks associated with the adoption of a visual inspection system in veal calves raised in a member state (or part of a member state) considered free of tuberculosis an experimental study of a concurrent primary infection with bovine respiratory syncytical virus (brsv) and bovine viral diarrhoea virus (bvdv) in calves a study of bovine respiratory syncytical virus infections in swedish cattle performance and health of group-housed dairy calves fed milk automatically versus manually virkning av forskellige insaettelsestrategier på tillvaekst og sundhed hos gruppeopstaldede kalve increased mortality among calves in danish cattle herds during bovine virus diarrhoea infection reducing pain after dehorning in dairy calves veterinary virology the effect of the size of individual crates on the behavioural and immune reactions of dairy calves ceftriaxone-resistant salmonella infection acquired by a child from cattle effect of castration method and the provision of local anesthesia on plasma cortisol, scrotal circumference, growth, and feed intake of bull calves farm animal behaviour and welfare farm animal behaviour and welfare preference and motivation testing pleasures, "pains" and animal welfare: towards a natural history of affect the effects of early separation on the dairy cow and calf the production system and disease incidence in a national random longitudinal study of swiss dairy herds comparison of four methods of calf confinement. i interleukin production in iron-deficient children rumional acidosis in milk fed calves. large animal veterinary rounds inzidenz und verlauf von neugeborenendurchfall bei kälbern in einem praxisgebiet in oberbayern economic analysis of feeding pasteurized nonsaleable milk versus conventional milk replacer to dairy calves campylobacter jejuni and campylobacter hyointestinalis in the intestinal tract and on the carcasses of calves and cattle a new method of measuring diet abrasion and its effect on the development of the forestomach urinary catecholamines in iron-deficient rats at rest and following surgical stress experimental salmonella infection of calves. . the effect of stress factors on the carrier state experimental salmonella infection of calves. . virulence and the spread of infection concentrations and emissions of ammonia in livestock buildings in northern europe zur Äthiologie und prophylaxe der perinatalen mortalität beim kalb collegium veterinarium xxiv salmonella bij gezonde runderen en kalveren in nederland immune functions of veal calves fed low amounts of iron delaying colostrum intake by one day has important effects on metabolic traits and on gastrointestinal and metabolic hormones in neonatal calves dynamics of virus infections involved in the bovine respiratory disease complex in swedish dairy herds. the vet effects of resistance to milk flow and the provision of hay on non-nutritive sucking by dairy calves sucking behaviour of dairy calves fed milk ad libitum by bucket or teat a longitudinal study of escherichia coli o in fourteen cattle herds resting behaviour, growth and diarrhoea incidence rate of young dairy calves housed individually or in groups in warm or cold building the effect of flooring type and social grouping on the rest and growth of dairy calves fecal shedding and rumen growth of escherichia coli o :h in fasted calves feeding the newborn dairy calf calf and heifer rearing : principles of rearing the modern heifer from calf to calving the national dairy heifer evaluation project: a profile of heifer management practices in the united states human-livestock interactions: the stockperson and the productivity and welfare of intensively farmeed the relationship between the presence of helicobacter pylori, clostridium perfringens type a, campylobacter spp, or fungi and fatal abomasal ulcers in unweaned beef calves effects of confinement on rebound of locomotor behaviour of calves and heifers, and the spatial preferences of calves the effects of feeding method, milk allowance and social factors on milk feeding behaviour and cross-sucking in group housed dairy calves computer-controlled milk feeding of dairy calves: the effects of number of calves per feeder and number of milk portions on use of feeder and social behaviour who needs 'behavioural needs'? motivational aspects of the needs of animals play behaviour in group-housed dairy calves, the effect of space allowance the effect of milk flow rate and milk allowance on feeding related behaviour in dairy calves fed by computer controlled milk feeders effect of single versus group housing and space allowance on responses of calves during open-field tests play behaviuor in domestic calves kept in pens: the effect of social contact and space allowance evaluation of welfare indicators for social environment in cattle effect of amount of milk, milk flow and access to a rubber teat on cross-sucking and non-nutritive sucking in dairy calves epidemiologische erhebungen zur kälberdiarrhoe in einem praxisgebiet in oberbayern effect of shipping and chromium supplementation on performance, immune response, and disease resistance of steers the development of intersucking in dairy calves around weaning factors associated with intersucking in swiss dairy heifers whole blood leukocytes vs separated mononuclear cell blastogenesis in calves. time-dependent changes after shipping recommendations for recording and calculating the incidence of selected clinical diseases in dairy cattle castration of calves: a study of methods used by farmers in the united kingdom onderzoek naar de uit ethologisch oogpunt minimaal gewenstle boxmaten voor vleeskalveren met een gewicht van tot kg spatial requirements of individually housed veal calves of to kg nutritional effects on response of children in developing countries to respiratory tract pathogens: implications for vaccine development problems on the etiological importance of pasteurella, mycoplasma and parainfluenza- -virus in enzootic pneumonia of calves the behaviour of beef suckler cattle enzootic pneumonia in calves: clinical and morphological features theoretical comparison of the consumer surplus and elasticities of demand as measures of motivational strength effects on calves less than one month old of feeding or not feeding tham during road transport of up to hours untersuchungen zur pathogenese der pylorusulzera beim mestkalb bovine cryptosporidiosis in denmark. ii. cryptosporidia associated with neonatal calf diarrhoea an evaluation of two management systems for rearing calves fed milk replacer prevalence of escherichia coli o :h in range beef calves at weaning cattle and reindeer as possible sources of escherichia coli o infection in humans aktuelle probleme der tierärztlichen betreuung von mutterkuhherden mother-young behaviour in cattle prevention of microbial contamination of red meat in the ante mortem phase; epidemiological aspects small intestinal motility disorders in preruminant calves chronically fed a diet based on antigenic soya : characterization and possible mediators influence of soya antigen levels in milk replacers on the disruption of intestinal motility patterns in calves sensitive to soya systemic and local gutspecific antibody responses in preruminant calves sensitive to soya feeding heated soyabean flour increases the density of b and t lymphocytes in the small intestine of calves hydrolysed soy protein isolate sustains high nutritional performance in veal calves antigenicity and digestive utilization of four soya products by the preruminant calves antibiotic resistance in gut bacteria from dairy calves: a dose response to the level of antibiotics fed in milk iron deficiency and immune responses the relationship between farmers' attitude and behaviour towards calves, and productivity of veal units reducing veal calves' reactivity to people by providing additional human contact the influence of farmers' behavior towards calves on animals' responses to transport and quality of veal meat the farmers' influence on calves' behaviour, health and production of a veal unit the impact of gentle contacts on ease of handling, welfare, and growth of calves and quality of veal meat reactions of calves to handling depend on housing condition and previous experience with humans effects of supplemental yeast culture on rumen development, growth characteristics, and blood parameters in neonatal dairy calves effects of corn processing on growth characteristics, rumen development, and rumen parameters in neonatal dairy calves anemia as a risk factor for infectious diseases in infants and toddlers: results from a prospective study colostrum in swedish dairy cows: quality and effects on passive immunity in the calves cross-sucking in group-housed dairy calves before and after weaning off milk intersucking in dairy cattle--review and questionnaire cryptosporidium andersoni n. sp. (apicomplexa: cryptosporidiidae) from cattle, bos taurus occurrence of iron deficiency in growing cattle growth performance, haematological traits, meat variables, and effects of treadmill and transport stress in veal calves supplied different anounts of iron prevention of microbial contamination of red meat in the ante mortem phase; epidemiological aspects management practices associates with high mortality among preweaned dairy heifers the gastric mucosal barrier and abomasal ulcers in veal calves housing, management and health in swedish dairy calves. doctoral thesis herd-level risk factors for infectious diseases in swedish dairy calves aged - days automated feeding of milk replacer and health control of group-hiused veal calves automated feeding of milk replacer and health control of group-housed veal calves effect of transportation and weaning on humoral immune responses of calves responses of dairy cows and calves to each other's vocalisations after early separation cross-sucking and other oral behaviours in calves, and their relation to cow suckling and food provision isolation of escherichia coli o :h from dairy cattle associated with two cases of haemolytic uraemic syndrome the provision of solid feeds to veal calves: ii. behavior, physiology, and abomasal damage enteric pathogens in intensively reared veal calves a fifteen month study of escherichia coli o :h in a dairy herd a comparison of catecholamine and cortisol responses of young lambs and calves to painful husbandry procedures acute pain in an emergency clinic regulation of sucking behaviour of calves laboratory findings associated with abomasal ulcers/tympany in range calves biological response to stress: key to assessment of animal well-being? association between resting behaviour and live weight gain in dairy heifers housed in pens with different space allowance and floor type long-term effect of housing method during the first three months of life on human-animal relationship in female dairy cattle risk factors associated with cryptosporidium parvum infection in dairy cattle in southeastern new york state the response of normal and iron anemic calves to nasal infection with an attenuated strain parainfluenza- virus assessment of acute and chronic pain after different methods of castration of calves intestinal digestion of dietary and endogenous proteins along the small intestine of calves fed soybean or potato influence of dietary protein level and source on the course of protein digestion along the small intestine of the veal calf effect of diet on mucin kinetics and composition: nutrition and health implications calf rearing a practical guide, natural resource and environment influence of dry feed supplements on different parameters of welfare in veal calves the effect of four fibrous feed supplementations on different welfare traits in veal calves behavioural investigation of group rearing calves in automatic milk replacer feeding system effect of transportation on blood serum composition, disease incidence, and production traits in young calves. influence of the journey duration critical anthropomorphism, animal suffering and the ecological context bacterial pneumonia dust and microbiol emissions from animal production influence of truck transportation of calves on their cellular immune function prevalence of enteric pathogens in the feces of healthy beef calves salmonella infection and contamination of veal calves: a slaughterhouse survey comparison of two castration methods in cattle: plasma cortisol levels, leukocyte count and behavioral changes calf diseases and mortality in swedish dairy herds abomasal ulcers in captive white-tailed deer (odocoileus virginianus) growth performance and health of dairy calves bedded with different types of materials factors involved in recent outbreaks of escherichia coli o :h in scotland and recommendations for its control protection of calves against cryptosporidiosis with immune bovine colostrum induced by a cryptosporidium parvum recombinant protein treadmill exercise of calves with different iron supply, husbandry, and work load beurteilung verschiedener tränketechniken und betreuungsmassnahmen hinsichtlich ihrer auswirkungen auf die oralen aktivitäten, den gesundheitszustand und die mastleitung über zwei bis acht wochen alter mastkälber in gruppenhaltung. dissertation. institut für tierhygiene und tierschutz der tierärztlichen hochschule hannover group housing for two to eight week old fattening calves veterinary and zootechnical aspects of veal production veterinary medicine. a textbook of the diseases of cattle, sheep, pigs, goats and horses serum immunoglobulin concentrations and health of dairy calves in two management systems from birth to weeks of age influence of feeding different amounts of first colostrum on metabolic, endocrine, and health status and on growth performance in neonatal calves the impact of social and human contacts on calves' behaviour and stress responses stereotypies om heifers are affected by feeding regime feeding level and oral stereotypies in dairy cows blood studies and performance among calves reared by different methods consequences of short term fluctuations of the environmental temperatures in calves--part : effects on the health status of animals within three weeks after exposure microbiology of calf diarrhea in southern britain studies on the presence of verocytotoxic escherichia coli o in bovine faeces submitted for diagnostic purposes in england and wales and on beef carcases in abattoirs in the united kingdom effect of different methods of castration on behaviour and plasma cortisol in calves of three ages iron, thermoregulation, metabolic rate the validity of behavioural measures of aversion: a review fear of people by cows and effects on milk yield, behavior, and heart rate at milking recognition of people by dairy calves using colour of clothing endotoxins in the environment: a criteria document reduced cortisol secretions in patients with iron deficiency viral diarrhea in man and animals mouth-based anomalous syndromes the influence of management routines on endocrine systems involved in the control of lactation in dairy cattle immune response in anemic calves the welfare of cattle kept for beef production. directorate general health and consumer protection, directorate c -scientific health opinions, unit c -management of scientific committees staphylococci as an indicator for bacterial emissions from a broiler house the use of trainer cows to reduce stress in newly arrived feedlot calves scvph (scientific committee on veterinary measures relating to public health) scvph (scientific committee on veterinary measures relating to public health) scvph (scientific committee on veterinary measures relating to public health) bioaerosole in und aus der landwirtschaftlichen nutztierhaltung (vorläufiger titel). kuratorium für technik und bauwesen in der landwirtschaft (ktbl, hrsg.) concentrations and emissions of airborne endotoxins and microorganisms in livestock buildings in northern europe a survey of ventilation rates in livestock buildings in northern number of viable bacteria and presumptive antibiotic residues in milk fed to calves on commercial dairies effects of transportations, a high lactose diet and acth injections on the white blood cell count, serum cortisol and immunoglobulin g in young calves descriptive epidemiology of morbidity and mortality in minnesota dairy heifer calves the development of the bacterial flora of the faeces of animals and man: the changes that occur during aging clostridial abomasitis in calves: case report and review of the literature destruction of mycobacterium paratuberculosis, salmonella spp., and mycoplasma spp effects of local anaesthesia or local anaesthesia plus a non-steroidal anti-inflammatory drug on the acute cortisol response of calves to five different methods of castration dehorning and disbudding distress and its alleviation in calves seasonal variation of thermophilic campylobacters in lambs at slaughter losses in young calves after transportation reaction of claves to two flooring materials offered simultaneously in one pen effects of three different methods of dehorning on cortisol levels and behaviour of calves evaluation of the effect of local anaesthesia and local anaesthesia associated with analgesia on the levels of cortisol after hot-iron, chemical or scoop dehorning effects of in vivo release of insulin and glucagon studied by microdialysis in rat pancreas and audiographic evidence for ( h)-oxytocin binding sites within the islets of langerhans some theoretical and observed relationships of fixed and portable spacing behavior of animals selfish animats and robot ethology: using artificial animals to investigate social and spatial behavior effect of local anaesthetic combined with wound cauterisation on the cortisol response to dehorning in calves cortisol responses to dehorning of calves given a -h local anaesthetic regimen plus phenylbutazone, ketoprofen, or adrenocorticotropic hormone prior to dehorning scientific veterinary committee, animal welfare section. directorate generale for agriculture, vi/bii. . adopted the effect of group size on health and growth rate of swedish dairy calves housed in pens with automatic milk-feeders bovine coccidiosis with special reference to eimeria alabamensis infections in grazing calves morbidity in swedish dairy calves from birth to days of age and individual calf-level risk factors for infectious diseases health status of dairy calves kept in individual pens or in group pens with or without automatic milk feeder acute cortisol responses of calves to scoop dehorning using local anaesthesia and/or cautery of the wound verotoxigenic escherichia coli o in scottish calves verocytotoxin-producing escherichia coli: a veterinary view concentrations and emissions of airborne dust in livestock buildings in northern europe ethological, physiological and histological aspects of pain and stress in cattle when being dehorned individual humans as discriminative stimuli for cattle (bos taurus) pathological and microbiological studies on pneumonic lungs from danish calves effects of individual housing design and size on special-fed holstein veal calf growth performance, hematology, and carcass characteristics calf rearing verordnung zum schutz landwirtschaftlicher nutztiere und anderer zur erzeugung tierischer produkte gehaltener tiere bei ihrer haltung (tierschutz-nutztierhaltungsverordnung -tierschnutztv)*) vom geändert durch die erste verordnung zur Änderung der tierschutz -nutztierhaltungsverordnung vom effects of age of holstein-friesian calves on plasma cortisol, acute-phase proteins, immunological function, scrotal measurements and growth in response to burdizzo castration effect of ketoprofen, lidocaine local anesthesia, and combined xylazine and lidocaine caudal epidural anesthesia during castration of beef cattle on stress responses, immunity, growth, and behavior etude de la prévalence de l'infection des veaux par giardia duodenalis en pays de la loire effects of housing on responses of veal calves to handling and transport. in new trends in veal calf production the importance of straw for pig and cattle welfare: a review behavioural and physiological studies on rearing and veal calves molecular epidemiology of cryptosporidium parvum and giardia duodenalis to study the potential for zoonotic transmission dynamics of bovine respiratory syncytical virus: a longitudinal epidemiological study in dairy herds the prevalence of verotoxins, escherichia coli o :h and salmonella in the feces and rumen of cattle at processing haemoglobin and haematocrit levels in veal calves is anaemia of veal calves normochromic or hypochromic a comparison of different enrichment media for the isolation of salmonella dublin from livers, kidneys and muscles of salmonella-positive veal calves zusatzfuttering von stroh an mastkälber the effect of housing system and dietary iron supply on stress physiological measures and resistance against an experimental infection with bovine herpesvirus (bhv ) in veal calves enkele epidemiologische aspecten van salmonellose bij mestkalveren in nederland calves' responses to repeated social regrouping and relocation does nutritive and non-nutritive sucking reduce other oral behaviors and stimulate rest in calves nonnutritive oral activities and stress responses of veal calves in relation to feeding and housing conditions providing social contacts and objects for nibbling moderates reactivity and oral behavior in veal calves the effects of rearing in individual crates on subsequent social behaviour of veal calves somministrazione di un mangime solido a vitelli a carne bianca stabulati in gabbia individuale o in box di gruppo. . parametri comportamentali, fisiologici e patologici behaviour and performance of veal calves under different stabling conditions assessing animal welfare: the significance of causal studies of behaviour at the motivational level epidemiologic and pathological characteristics of respiratory tract disease in dairy heifers during the first three months of life morbidity from nonrespiratory diseases and mortality in dairy heifers during their first months of life iron deficiency anemia and increased urinary norepinephrine excretion decreased susceptibility to ciprofloxacin in outbreak-associated multiresistant salmonella typhimurium dt otitis media in preweaned holstein dairy calves in michigan due to mycoplasma bovis effects of group, individual, and isolated rearing of calves on weight gain and behavior colostrum -the beginning of a successful calf raising program. dairy feed facts air and surface hygiene the response of beef cattle to noise during handling reduction of cross-sucking in calves by the use of a modified automatic teat feeder calf husbandry, health and welfare rearing of veal calves the effect of different rearing systems on the development of calf behaviour a survey of abomasal ulceration in veal calves haematology of veal calves reared in different husbandry systems and the assessment of iron deficiency health status of preweaned dairy heifers in the united states the effect of feeding pellets of different types of roughage on the incidence of lesions in the abomasum of veal calves st joint fao/oie/who expert workshop on non-human antimicrobial usage and antimicrobial resistance: scientific assessment on the significance of ethological criteria for the assessment of animal welfare effects of individual housing design and size on behaviour and stress indicators of special-fed holstein veal calves feeding antibiotic-contaminated waste milk to calves--effects on physical performance and antibiotic sensitivity of gut flora reviews of the progress of dairy science: bovine salmonellosis factors influencing occurrence of colibacillosis in calves natural and experimental infection of normal cattle with escherichia coli o rearing veal calves with respect to animal welfare: effects of group housing and solid influence of dietary concentrate to forage ratio on the development of rumen mucosa in calves the members of the working group which authored this report were: animals. cab international, oxon/new-york pp.hepola, h., . milk feeding systems for dairy calves in groups: effects on feed intake, growth and health. applied animal behaviour science : - .herrmann, j., knierim, u., hinton, m., linton, a.h., . antibacterial drug resistance among escherichia coli isolated from calves fed on a milk substitute diet, vet. rec., , - .hinton, m., ali, e. a., allen, v., linton, a. h., . the excretion of salmonella typhimurium in the faeces of calves fed on a milk substitute diet. j. hyg. camb. , - .hinton, m., hedges, a. j., linton, a. h., a . the ecology of escherichia coli in calves fed on a milk substitute diet. j. appl. bacteriol. , - .hinton, m., linton, a. h., hedges, a. j., b . the ecology of escherichia coli in calves reared as dairy-cow replacers. j. appl. bacteriol. , - .hinton, m., rixson, p. d., allen, v., linton, a. h., a . the persistence of drugresistant escherichia coli strains in the majority faecal flora of calves, j. hyg. camb., , - .hinton, m., suleyman, i. o., allen, v., linton, a. h., b . further observations on the excretion of salmonella in the faeces of calves fed milk substitute, j. hyg. camb., , - .holliman, a., . overview of coccidiosis -recent observations. cattle practice ; - .holm, l., jensen, m. b., jeppesen, l. l., key: cord- -fgvu wcx authors: schnyder, m.; kohler, l.; hemphill, a.; deplazes, p. title: prophylactic and therapeutic efficacy of nitazoxanide against cryptosporidium parvum in experimentally challenged neonatal calves date: - - journal: vet parasitol doi: . /j.vetpar. . . sha: doc_id: cord_uid: fgvu wcx diarrhoea caused by cryptosporidium parvum is a major problem in calves younger than weeks of age. to date only a few compounds have been approved for prophylactic and none for therapeutic use. nitazoxanide (ntz) has proven its efficacy in vitro against c. parvum and is approved by fda for the treatment of human cryptosporidiosis. in a first experimental study, uninfected calves were treated with ntz and pharmacokinetics was followed through blood samples. serum samples of uninfected treated calves contained both ntz metabolites (tizoxanide and tizoxanide glucuronide) and oral administration at h intervals was considered as optimal. three groups of three calves ( – days old) were then each inoculated with × ( ) oocysts of c. parvum (cattle genotype): the prophylactic group received mg/kg body weight ntz twice daily orally in milk from day before to days postinoculation (dpi). the therapeutic group received the same dosage of ntz for days from the appearance of diarrhoea (between and dpi). the control group was left untreated. all calves were monitored daily from day − to dpi and faecal samples were collected for evaluation of consistency and for determination of oocyst numbers per gram (opg) of faeces. diarrhoea was observed in all calves within the first week. neither prophylactic nor therapeutic use of ntz improved the clinical appearance and calves of the therapeutic showed a longer diarrheic episode (p < . ) with strong altered faecal consistence compared to the untreated control group. the number of days with oocyst excretion did not differ significantly between the groups. in out of infected and treated calves oocyst excretion stopped only after discontinuation of treatment. in the prophylactic and in the control group mean values of the sum of the daily opg per calf ( . × ( ) and . × ( ), respectively) and of the mean daily number of opg ( . × ( ) and . × ( ), respectively) were similar, while the therapeutic group showed significantly lower values ( . × ( ) and . × ( ), respectively, p < . ). however oocyst determinations in this group may have been altered by the severe diarrhoea, diluting oocyst densities in the analysed faecal samples. in conclusion, these preliminary results about the first prophylactic and therapeutic use of ntz in calves did not show the expected positive effect on the course of the cryptosporidium-infection, neither on reducing the clinical severity, nor on oocyst excretion. diarrhoea caused by cryptosporidium parvum is a major problem in calves younger than weeks of age. to date only a few compounds have been approved for prophylactic and none for therapeutic use. nitazoxanide (ntz) has proven its efficacy in vitro against c. parvum and is approved by fda for the treatment of human cryptosporidiosis. in a first experimental study, uninfected calves were treated with ntz and pharmacokinetics was followed through blood samples. serum samples of uninfected treated calves contained both ntz metabolites (tizoxanide and tizoxanide glucuronide) and oral administration at h intervals was considered as optimal. three groups of three calves ( - days old) were then each inoculated with  oocysts of c. parvum (cattle genotype): the prophylactic group received mg/kg body weight ntz twice daily orally in milk from day before to days postinoculation (dpi). the therapeutic group received the same dosage of ntz for days from the appearance of diarrhoea (between and dpi). the control group was left untreated. all calves were monitored daily from day À to dpi and faecal samples were collected for evaluation of consistency and for determination of oocyst numbers per gram (opg) of faeces. diarrhoea was observed in all calves within the first week. neither prophylactic nor therapeutic use of ntz improved the clinical appearance and calves of the therapeutic showed a longer diarrheic episode (p < . ) with strong altered faecal consistence compared to the untreated control group. the number of days with oocyst excretion did not differ significantly between the groups. in out of infected and treated calves oocyst excretion stopped only after discontinuation of treatment. in the prophylactic and in the control group mean values of the sum of the daily opg per calf ( .  and .  , respectively) and of the mean daily number of opg ( .  and .  , respectively) were similar, while the therapeutic group showed significantly lower values ( .  and .  , respectively, p < . ). however oocyst determinations in this group may have been altered by the severe diarrhoea, diluting oocyst densities in the analysed faecal samples. in conclusion, these preliminary results about the first prophylactic and therapeutic use of ntz in calves did not show the expected positive effect on the course of the cryptosporidium-infection, neither on reducing the clinical severity, nor on oocyst excretion. ß elsevier b.v. all rights reserved. (olson et al., ) . bovine cryptosporidiosis occurs worldwide with prevalences of - % in young calves in different european countries (appelbee et al., ) . studies performed in the united states showed that particularly calves at weeks of age were excreting c. parvum oocysts (santin et al., ) with prevalences of . % in calves of - weeks of age. prevalences in milking cows were significantly lower and mainly not concerning c. parvum, representing the only zoonotic species and also the most pathogenic species for cattle (fayer et al., ) . although c. parvum infection is often detected in combination with other enteropathogens such as rotavirus, coronavirus, escherichia coli k or salmonella species, many experimental studies and investigations in the field have shown that cryptosporidium may act as a primary pathogen (tzipori et al., ; naciri et al., ) . more than substances have been tested against cryptosporidiosis (o'donoghue, ; stockdale et al., ) and some such as halofuginone and decoquinate are reported to exhibit promising effects (lefay et al., ; lallemand et al., ) . prophylactic administration of paromomycin inhibited oocyst shedding and reduced the number of days with diarrhoea in experimentally infected calves (fayer and ellis, ) , but in a field trial oocyst shedding and diarrhoea started with withdrawal of the drug and no reduction of the disease was obtained (grinberg et al., ) . reports on the outcomes of halofuginone treatment are controversial: in some studies, calves showed significantly reduced oocyst excretion and decreased diarrhoea (joachim et al., ) preventing mortality (naciri et al., ) . in another study treatments with halofuginone or decoquinate had no effect on the levels of diarrhoea or dehydration, but halofuginone significantly reduced the excretion of oocysts and decoquinate increased the average daily weight gain of calves (lallemand et al., ) . in a further study the administration of halofuginone during the first days of life inhibited oocyst excretion until weeks of age, but the incidence of diarrhoea was only delayed for . days and there was no significant difference compared to the control group concerning weight gain (jarvie et al., ) . halofuginone is the only drug currently available for the treatment of bovine cryptosporidiosis, but it is known to be relatively toxic and care must be taken not to exceed the therapeutic dose. nitazoxanide (ntz) is an antimicrobial substance that has been applied for antiparasitic treatments in dogs, cats, sheep and goats (euzeby et al., ; gookin et al., ; viel et al., ) . ntz has been fdaapproved for human use for the treatment of cryptosporidiosis and giardiasis in children and adults, and also for the treatment of equine protozoan myeloencephalitis caused by sarcocystis neurona. the goal of the present pilot study was to investigate the efficacy of ntz for the prophylaxis and treatment of cryptosporidiosis in calves. animal experiments were approved and supervised by the cantonal veterinary office of zü rich and the respective animal experimentation ethics committee. the study was carried out during may-december in experimental barns which are part of the veterinary hospital of the university of zurich, switzerland. calves were purchased from surrounding farms - days after birth and all calves had received colostrum. they were housed in pens ( . - . m ) in groups of up to animals with a litter of straw over a concrete base. the pens were cleaned daily with hot vapour pressure. calves were fed with a commercial milk replacer supplied by an automatic milk deliverer; animals with reduced appetite were additionally hand-fed with milk and/or electrolyte solution. water was supplied ad libitum and from the third week hay was also available. calves were neither vaccinated, nor did they receive any supplementary vitamins during the experiment. all animals were checked twice daily clinically including faecal consistency and appetite. weight was recorded daily. at the first manifestation of diarrhoea, faeces from all calves were tested once for antigens of bovine coronavirus (fastest bcv), rotavirus (fastest rota), e. coli main pilar k (fastest e. coli-k ) and against c. parvum (fastest crypto), all from megacor, austria. calves were euthanized between and dpi. as part of the postmortem analysis, they were additionally examined by antigen detection in skin biopsies (hilbe et al., ) for the presence of bovine virus diarrhoea (bvd). this was to exclude persistent and primary postnatal infections with bvd virus resulting in immunosuppression, thereby enhancing the vulnerability of the calves to secondary infections (potgieter, ) . the cryptosporidium-isolate used in this study originated from an affected outpatient calf of the animal hospital. oocyst production was achieved in an experimentally inoculated calf (  oocysts). faeces from this calf had been collected by means of a bag that was attached through an appliance around the belly of the animal. oocysts were purified on ficoll gradients, and c. parvum ''cattle genotype'' was identified (ward et al., ) . these oocysts were stored for a maximum of days at c in phosphate buffered saline until to be used for infection experiments. at first ntz was fed to three calves, representing the uninfected treatment group, for a period of days starting from day after their arrival at the experimental farm for the determination of ntz metabolites. afterwards, upon arrival at the experimental barns, calves were randomly assigned to three experimental groups. on day animals were inoculated with  c. parvum oocysts. in a first group, the prophylactic group, ntz was administered prior to inoculation (day À ) and then daily for a period of days. ntz (kindly provided by cambrex profarmaco landen nv; belgium) was fed through a suckling bottle with milk replacer at mg/kg bodyweight (bw) twice daily. the three calves of a second group, the therapeutic group, received ntz starting at the time point of the first manifestation of diarrhoea and then daily for a period of days. the three calves of the third group, the infection control group, were left untreated. blood samples were taken prior to drug treatment and at , , , , , , and h after the first administration of ntz from calves of the uninfected treatment group. samples were sent to sgs life science services, wavre, belgium, for the determination of the active metabolites tizoxanide and tizoxanide glucuronide as previously described (stettler et al., ) . faecal samples were directly collected in containers of about dl from the rectum from all calves every day; each sample was labelled, sealed and transported to the laboratory on the day of collection. a single faecal smear was prepared from each mixed sample, stained with ziehl-neelsen (eckert et al., ) for visualisation of oocysts and entirely examined by microscopy. if oocysts were present, samples were analysed by a modified method of grinberg et al. ( ) to determine the number of cryptosporidial oocyst per gram (opg) of faeces. briefly, g of the mixed faecal sample was mixed with ml of tap water, passed through a -mesh sieve, the suspension centrifuged and the sediment resuspended in ml of normal saline. afterwards, ml of this suspension was poured as a drop on a slide, air-dried and stained with ziehl-neelsen. the entire area of smear was examined with a  objective lens and oocysts counted. the opg was then calculated by multiplying the result by (or , if there were too many oocysts to be counted and the sample had previously been diluted -fold). results were not corrected for dilution in diarrhoeic stools. the mean opg for each calf was the arithmetic mean of daily opg from day À to dpi. the number of oocysts shed was the sum of the daily opg of each calf. to determine the effective total number of oocysts shed would have required the collection of all faeces on all days, this was not possible. faecal consistency was based on scores: faeces solid, brownish and with adhesion at the plastic container, faeces semi-solid, yellowish and without adhesion at the plastic container (sample spread across the bottom of the container but not liquid), and liquid diarrhoea, as previously described (grinberg et al., ) . differences in oocyst excretion intensities were statistically compared by maximum likelihood estimation based on the negative binomial distribution (torgerson et al., ) and were considered significant with values p < . . the number of days with diarrhoea or semi-solid faecal consistency was compared between treatment and control groups using the student's t-test. blood samples from uninfected ntz treated calves contained both ntz metabolites (fig. ) . tizoxanide (tiz) represents the deacetylated primary metabolite, and was first detected within - h after administration (limit of traceability: . mg/ml). highest tiz-levels were recorded between and h ( . - . mg/ml), followed by a decrease until the next administration. tizoxanide glucuronide (tig), was first detected slightly after tiz (limit of traceability: . mg/ml) and highest levels were also seen between and h ( . - . mg/ml). tig was no longer detected after h. after oral administration in humans, maximum plasma concentrations of both metabolites had also been observed within - h, but reached levels up to mg/ml (broekhuysen et al., ) . oral administration with milk or milk replacer at h intervals seemed to be an appropriate way for ntz administration. cryptosporidium-infection took place in all infected animals. none of the calves, including the uninfected ntz treated animals, was positive for viral or bacterial agents tested (c. parvum, rota-and coronaviruses, e. coli k ), so that other infectious agents potentially causing diarrhoea could be excluded. skin biopsies postmortem were negative for bvd virus. altered faecal consistency and particularly diarrhoea, main symptom of cryptosporidiosis in calves, was observed in all animals, but there were differences in duration and severity (table ). in the therapeutic group, animals showed a significantly higher (p < . ) number of days with liquid diarrhoea if compared with the infected but untreated control group. bloody faeces were present for . and . days in the prophylactic and the therapeutic group respectively, while the infected untreated control group had a mean of days with bloody faeces, but differences were not significant. also calves from the uninfected group receiving ntz developed diarrhoea for a mean duration of . days. blood was present in faeces from one calf on days and of treatment, from the second calf on days and and from the third calf on days and , for a mean of days per animal. thus the diarrhoea in this uninfected group was possibly caused by ntz treatment. in the untreated control group conspicuous liquid diarrhoea was detected in only . % ( . / . days) of the days with altered faecal consistency. this indicated less pathology compared to the treatment groups. the percentages in the uninfected, the prophylactic and the therapeutic group were twice as much: . % ( . / . ), . % ( / . ) and . % ( / ), respectively. additionally, % ( / days) and respectively . % ( . / days) of the days with liquid diarrhoea in the prophylactic and the therapeutic group occurred during the ntz administration, suggesting a cumulative negative effect of ntz in combination with parasite infection. results of daily opg determination of infected calves are presented in table and the dynamic of oocyst excretion is shown in fig. . oocyst excretion started in all infected animals within - dpi (days postinoculation) and highest excretion of oocysts took place at - dpi. in both infected treatment groups faecal consistency changed just before the start of oocyst excretion. after discontinuation of the medication in the prophylactic and the therapeutic group, the mean duration of oocyst excretion was . and day, respectively. consequently calves of these two groups excreted oocysts mainly during the treatment days ( . % and . %, respectively). the number of oocysts shed and the daily mean opg value was significantly lower in the therapeutic group. however, oocyst counts in this group may be altered by the severe diarrhoea, diluting oocyst densities in the faecal samples. one chronic carrier was noted in each the prophylactic and the control group and none in the therapeutic group. however, the number of excreted oocysts from dpi on in chronic carriers was very low. also the faecal consistency of these two animals was not altered from dpi. after dpi all other calves were no longer excreting oocysts, except one animal excreting until dpi. nevertheless these animals continued having altered faecal consistency for several days. horses treated for s. neurona infection receive . mg/ kg bw for days, then . mg/kg bw for days once a day (see u.s. food and drug administration, navigator , idexx laboratories). in previous experiments other animals such as mice, rats, pigs and cats had been treated with higher dosages of ntz, such as - mg/kg bw for days (blagburn et al., ) , - mg/kg bw for days (li et al., ) , - mg/kg for days (theodos et al., ) and - mg/kg bw for - days (gookin et al., ) . euzeby et al. ( ) described the use of ntz in adult dogs, cats and sheep with dosages from to mg/kg bw and observed that general tolerance was excellent. however, in all species a softened faecal consistency with almost sub-diarrhoeic patterns appeared - h after administration of ntz. this mild diarrhoea lasted for approximately - h. no enteric lesions have been observed during postmortem analysis, with the exception of a mild catarrhal inflammation in sheep. the oral administration of ntz with a dosage of mg/kg bw bid to the calves in this study was twice the usual dosage table diarrhoea in calves experimentally infected with cryptosporidium oocysts (p-, t-and c-group) and/or treated with nitazoxanide during the first days of the experiment (uninfected ntz-, p-and t-group for human patients with protozoan infections ( . mg/kg bw every h for consecutive days as recommended by romero cabello et al., ) , but seemed to be an appropriate dosage for this preliminary study. in clinical studies with paediatric patients (alinia , nitazoxanide , www.mosbysdrugconsult.com/drugconsult/ .html), the most frequent adverse events reported, regardless of causality assessment, were abdominal pain ( . %) and diarrhoea ( . %), followed by vomiting ( . %) and headache ( . %). nevertheless, symptoms in children were mild and transient, and did not differ significantly from the placebo-group. initially the study groups had been designed to consist of animals each. however, the experiment was terminated with groups of animals since these preliminary results did not show the expected positive effect on the course of the cryptosporidium-infection. on the contrary, the data from the uninfected but treated group suggested that ntz had a direct negative effect on mucous membranes of gastrointestinal tracts of calves, a problem which was also seen in piglets (theodos et al., ) and cats (gookin et al., ) . our results were supported by viel et al. ( ) , reporting about the use of ntz (navigator , equine paste, %, idexx laboratories) in - -day-old kids experimentally infected with c. parvum. an acute toxicity and similar results concerning oocyst excretion and clinical outcome were observed in an experiment with totally animals. the drug was described to be also active against microaerophilic and anaerobic bacteria of the gastrointestinal flora (euzeby et al., ) . this could therefore cause significant problems in the gastrointestinal tract of ruminants, which in turn could be more serious than the disease itself (fox and saravolatz, ; hemphill et al., ) . it has been speculated that gastrointestinal problems could be due to the action of the enzyme pyruvate ferredoxin oxidoreductase (pfor) or other nitro-reductases which have been hypothesized to reduce the thiazole ring-associated nitro group, and thus kill the intestinal bacterial flora through the production of free radicals (sisson et al., ; hoffman et al., ; reviewed in hemphill et al., ) . a concomitant c. parvum-infection or other infectious agents damaging the superficial intestinal epithelium may enhance the potential toxicity of treatments substances due to augmented resorption. as a consequence the severity of diarrhoea may increase, augmenting also dehydration and mortality of the animals, particularly of this delicate age group. this is the first report about the use of ntz against cryptosporidiosis in calves. in conclusion, neither prophylactic nor therapeutic use of ntz in new born calves infected with c. parvum improved clinical appearance in this preliminary experiment or diminished intensity or duration of oocyst excretion. treatment with ntz did not show the expected anti-cryptosporidial effect and appeared to fail. histopathology would be required to support the hypothesis that ntz has a negative impact on the integrity of the intestinal epithelium or on the normal flora in the calves, but in consideration of animal welfare reasons the results of this study were not sufficiently encouraging to justify the prosecution of experiments. table number of days with oocyst excretion, mean opg based on daily faecal samples ( - dpi) and number of oocysts shed (sum of the daily opg per calf) on days - postinfection for each calf of the prophylactic, the therapeutic and the control group. number of days with oocyst excretion mean opg excreted number of oocysts shed prophylactic group a .  .  .  .  .  .  mean (s.d.) ( . ) .  ( .  ) .  ( .  ) therapeutic group .  .  .  .  .  .  mean (s.d.) . ( . ) .  b ( .  ) .  b ( .  ) control group a .  .  .  .  .  .  mean (s.d.) . ( . ) .  ( .  ) .  ( .  ) a chronic carrier. b p < . : maximum likelihood estimate based on negative binomial distribution. in statistics. the study was conducted according to ethical guidelines and was approved by the ethics committee and the cantonal veterinary office from the canton of zü rich, permission number / . giardia and cryptosporidium in mammalian wildlife-current status and future needs comparative efficacy evaluation of dicationic carbazole compounds, nitazoxanide, and paromomycin against cryptosporidium parvum infections in a neonatal mouse model nitazoxanide: pharmacokinetics and metabolism in man ordnung cryptosporiida expé rimentation des proprié té s anthelminthiques de la nitazoxanide chez le chien, le chat et les ovins paromomycin is effective as prophylaxis for cryptosporidiosis in dairy calves prevalence of cryptosporidium species and genotypes in mature dairy cattle on farms in eastern united states compared with younger cattle from the same locations nitazoxanide: a new thiazolide antiparasitic agent experimental infection of cats with tritrichomonas foetus controlling the onset of natural cryptosporidiosis in calves with paromomycin sulphate nitazoxanide, a broad-spectrum thiazolide anti-infective agent for the treatment of gastrointestinal infections immunohistochemical diagnosis of persistent infection with bovine viral diarrhoea virus (bvdv) on skin biopsies antiparasitic drug nitazoxanide inhibits the pyruvate oxidoreductases of helicobacter pylori, selected anaerobic bacteria and parasites, and campylobacter jejuni effect of halofuginone lactate on the occurrence of cryptosporidium parvum and growth of neonatal dairy calves prevalence and control of bovine cryptosporidiosis in german dairy herds field study of the efficacy of halofuginone and decoquinate in the treatment of cryptosporidiosis in veal calves efficacy of halofuginone lactate in the prevention of cryptosporidiosis in suckling calves long-lasting anticryptosporidial activity of nitazoxanide in an immunosuppressed rat model role of cryptosporidium parvum as a pathogen in neonatal diarrhoea complex in suckling and dairy calves in france the effect of halofuginone lactate on experimental cryptosporidium parvum infections in calves cryptosporidium and cryptosporidiosis in man and animals update on cryptosporidium and giardia infections in cattle immunology of bovine viral diarrhoea virus nitazoxanide for the treatment of intestinal protozoan and helminthic infections in mexico a longitudinal study of cryptosporidiosis in dairy cattle from birth to years of age enzymes associated with reductive activation and action of nitazoxanide, nitrofurans and metronidazole in helicobacter pylori secondary and primary murine alveolar echinococcosis: combined albendazole/nitazoxanide chemotherapy exhibits profound antiparasitic activity prophylaxis and chemotherapy efficacy of nitazoxanide against cryptosporidium parvum in cell culture and in animal models modelling the transmission dynamics of echinococcus granulosus in cattle and sheep in kazakhstan experimental cryptosporidiosis in calves: clinical manifestations and pathological findings efficacy of nitazoxanide against experimental cryptosporidiosis in goat neonates detection of eight cryptosporidium genotypes in surface and waste waters in europe the authors thank hanspeter mü ller for his intensive care of the animals and dr. paul torgerson for his support key: cord- -rtepmxt authors: heller, meera c.; chigerwe, munashe title: diagnosis and treatment of infectious enteritis in neonatal and juvenile ruminants date: - - journal: vet clin north am food anim pract doi: . /j.cvfa. . . sha: doc_id: cord_uid: rtepmxt common causes of infectious enteritis in neonate and juvenile ruminants include viral, bacterial, and protozoal pathogens. the most common presenting sign in ruminants with infectious enteritis is diarrhea. diagnosis of the cause of enteritis has important zoonotic and herd health implications. severity of clinical signs with similar pathogens may differ between calves and small ruminants. treatment of enteritis involves supportive care to correct fluid and electrolyte imbalances, provision of nutritional support for neonates, prevention and treatment of endotoxemia or sepsis, and pathogen-specific treatments when relevant and available. include maldigested disaccharides, and increased d-lactate levels from bacteria fermentation of unabsorbed nutrients that enter the colon. secretory specific pathogens, such as enterotoxigenic escherichia coli (etec), stimulate cyclic amp, thus increasing secretion of chloride (cl), sodium (na), and potassium (k) into the intestinal lumen, thereby drawing water into the intestinal lumen. in addition, some pathogens denude the intestinal surface and cause villous blunting, resulting in maldigestion and malabsorption. this damage to the villous leads to proliferation of secretory crypt cells and increased secretory capacity of the intestinal wall. abnormal intestinal motility decreasing intestinal transit time may lead to maldigestion and malabsorption because of inadequate time for digestion and absorption of the ingested feed material. this process further contributes to osmotic retention of fluid in the intestinal tract. increased hydrostatic pressure disease conditions, including heart failure, renal disease, and liver disease, may result in increased hydrostatic pressure within the intestinal tract causing movement of water from extracellular tissues into the intestinal lumen, resulting in diarrhea. inflammation of the gi tract or the peritoneum (peritonitis) can exacerbate all of the above mechanisms of diarrhea. increasing intestinal permeability or increasing hydrostatic pressure within the intestinal wall can increase fluid loss into the lumen. in addition, prostaglandin production stimulates fluid secretion into the lumen. infiltration of the intestinal wall by inflammatory cells can also disrupt intestinal motility, increase intestinal secretion, and decrease absorptive function. diarrhea often results in fluid and electrolyte losses for the patient. as long as the ruminant neonate can compensate for losses, it will remain hemodynamically stable, and continue to nurse. however, if losses exceed intake, systemic effects will be observed on clinical examination. fluid loss from the vascular compartment leads to hypovolemia (dehydration), hypotension, and shock. metabolic acidosis develops as a result of intestinal and fecal loss of sodium bicarbonate, increased l-lactate from hypoperfused tissues, and increased absorption of l-lactate and d-lactate produced by bacterial fermentation in the intestinal tract. as dehydration and acidosis worsen, clinical signs progress, leading to weakness, loss of suckle reflex, and recumbency. vascular collapse and electrolyte imbalances can lead to heart failure, whereas death can also result from malnutrition and hypoglycemia in neonates. in addition, endotoxemia from gram-negative bacterial infection, such as salmonella or e coli, can directly cause circulatory failure. patient history should include information regarding the age and use of the animal (eg, dairy, beef, show animal), history of colostrum ingestion, duration and progression of diarrhea, age, and number of animals affected or dead in the herd. assessment of housing, management, feeding, sanitation practices, and preventive health measures is also important. on-farm standard operating procedures regarding treatment protocols are important to obtain and review, especially when approaching outbreaks of diarrhea. it is also important to ascertain whether there have been any recent dietary or husbandry changes (weaning), transportation, on-farm treatments, or addition of new animals. in clinic settings, ruminant patients should be examined in an area that can be isolated from other patients according to infectious disease control protocols. in farm settings, care should be taken to minimize cross-contamination between animals and particularly minimizing exposure to younger animals. in either scenario, the facility should be cleaned and disinfected following the examination. the examiner should wear personal protective equipment (eg, gloves, boots that can be disinfected, and coveralls) that are cleaned or discarded after handling the patient. although it is necessary to perform a complete physical examination in ruminant patients with enteritis, this article focuses on the techniques that are specific for organ examination in ruminants with enteritis. these techniques include the following: assessment of hydration status: tacky or dry mucous membranes, decreased skin turgor, and eyeball recession (sunken eyes) indicate dehydration. parameters for assessing dehydration in neonates are presented in table . also, a previous issue (march ) covers this topic as well. fig. shows a calf with eyeball recession caused by dehydration. signs of endotoxemia or septicemia: assess mucous membranes for color and capillary refill time. assess the sclera for injected, dilated blood vessels. septicemic calves may show evidence of hypopyon (fig. ) in the anterior chamber of the eyes, swollen joints, omphalophlebitis, or meningitis on physical examination. evidence of sepsis negatively affects prognosis. posture: the posture of the patient may indicate evidence of abdominal pain; for instance, abdominal distension, arching of the back (kyphosis), treading of the hind feet, and lying down with hind legs outstretched. in cases of overt abdominal pain, the possibility of surgical conditions should be investigated and ruled out. in primary cases of neonatal enteritis that do not show evidence of septicemia or endotoxemia, the attitude and posture of the animal can provide evidence of dehydration and metabolic acidosis. recumbent animals with greater mentation deficits in general have more severe metabolic acidosis. rectal temperature: body temperature may be either pyrexic, normal, or hypothermic in neonates with enteritis. hypothermia generally indicates that the patient's body is decompensating. it may also be an indication of hypoglycemia. the presence of hypothermia is often a poor prognostic indicator. body condition: poor body condition may indicate chronicity or malnutrition, which could be a compounding factor and worthy of further herd investigation with the client. oral examination: examination for oral ulceration and hypersalivation (ptyalism) is important because some viral causes of enteritis can also cause oral lesions. the presence of a suckle reflex is important and helps to determine the most appropriate fluid therapy strategy. the presence of a suckle reflex makes oral nutritional support much easier and allows a more cost-effective treatment plan. abdominal palpation, auscultation, percussion and succussion: abdominal palpation may help identify evidence of pain or allow palpation of abdominal viscera. simultaneous auscultation and percussion (pinging) on the left and right abdominal walls aids in identification of viscera filled with fluid and air under pressure. intestinal structures that may ping in a neonate include the abomasum, small intestine, and cecum. succussion of the abdomen is used to evaluate for the presence of excessive fluid in abdominal viscera, including the abomasum, small intestine, cecum, and rumen. the presence of sloshing fluid sounds in the abdomen (succussion splash) is an indication of fluid distension of the viscera caused by decreased intestinal motility and fluid accumulation. characteristics of the diarrhea: patients should be evaluated for color, odor and volume of feces, presence of tenesmus, blood, and mucus. important differential diagnoses for infectious enteritis can be broadly classified into bacterial, viral, and protozoal. there is a strong correlation between age and the observation of particular pathogens. common differential diagnoses associated with age in calves and small ruminants are shown in table . for an in-depth discussion of clostridial enteritis, please see simpson km and colleagues' article, "clostridial abomasitis and enteritis in ruminants," in this issue; nematodiasis can be seen in craig tm article, "gastrointestinal nematodes, diagnosis and control," in this issue; coccidiosis can be seen in keeton stn and navarre cb's article, "coccidiosis in large and small ruminants," in this issue; and herd assessment and control of salmonella can be seen in holschbach cl and peek sf's article, "salmonella in dairy cattle," in this issue. this article provides an overview of the salient features of the various infectious enteritis diseases in ruminant neonates (less than months of age). enterotoxigenic escherichia coli fimbria (pili) confer the ability of etec to attach to immature enterocytes. fimbria antigens identified in pathogens causing disease in livestock include f (k ), f (k ), f ( p), f , f , f , f , and f . enterotoxins produced by etec stimulate increased secretion by intestinal crypt cells, causing a severe secretory diarrhea. expression of the enterocyte surface proteins that allow for etec pili attachment decreases with age. thus, affected ruminants are usually less than days of age and often less than days of age. necropsy findings include fluid-filled intestines with a lack of mucosal damage or hemorrhage. diagnosis is through fecal culture followed by immunoassay for the fimbria antigen or polymerase chain reaction (pcr) to detect the specific fimbria virulence factor gene. pcr can also be used to identify the specific toxin virulence factor genes. attaching and effacing /shiga toxin-producing, enterohemorrhagic escherichia coli belong to the "o" labeled serogroups. cause cytotoxic damage to the intestinal mucosa, producing a mucohemorrhagic colitis. calves experience diarrhea, dysentery, and abdominal pain. clinical signs generally occur in calves days to weeks of age; however adult cattle may have subclinical disease and serve as a source of infection. common necropsy findings include mucosal damage, mucosal petechiation, and gi hemorrhage. diagnosis is through fecal culture followed by pcr for bacterial virulenceassociated genes. there are many subtypes of salmonella species. cattle disease is mainly caused by serogroups b, c, d, and e. infection can result in a wide range of clinical disease, from subclinical shedders to acute bacteremia, endotoxemia, and death. the most common clinical signs in young ruminants are fever, diarrhea, anorexia, depression, and dehydration. affected animals frequently show signs of endotoxemia or sepsis. infection results in severe inflammation of the mucosa. rapid emaciation occurs in clinically affected animals because of cachexia stimulated by systemic inflammation coupled with malabsorption and protein loss from the inflamed gut. septic calves require more intensive treatment, including systemic antibiotics. diagnosis is based on fecal culture with increased sensitivity achieved by using an enrichment broth, followed by pcr. necropsy findings usually include fibrinous to fibrinonecrotic enteritis, and there may be signs of bacterial embolization to other organs such as the kidneys. some salmonella serotypes are zoonotic (eg, salmonella typhimurium, salmonella newport). salmonella dublin is host adapted in cattle, meaning that a nonclinical carrier status can exist in infected cattle. for further details, please refer to holschbach cl and peek sf's article, "salmonella in dairy cattle," in this issue. clostridia species are present in the soil and can be cultured from the intestinal tract of normal livestock. clostridium perfringens proliferates quickly postmortem, making definitive diagnosis difficult. diagnosis is usually based on history, rapidly progressing clinical signs, necropsy findings, culture results, and the presence of toxins. c perfringens types a, b, c, d, and e can cause abomasitis or enteritis in young ruminants. table lists common diseases caused by each type and the specific toxins produced. the different types produce different toxin profiles, and the actions of these various toxins largely dictate the clinical signs observed. c perfringens type a causes hemorrhagic abomasitis and enteritis in calves. clinical signs include pasty feces, which may progress to hemorrhagic diarrhea, accompanied by a painful distended abomasum. clinical signs progress quickly and calves can be found dead without previously observed clinical signs. the alpha toxin is a phospholipase that causes endothelial damage, resulting in hemorrhagic and necrotic intestinal lesions. normal abomasal ph and gi motility reduce the likelihood of clostridial overgrowth. bacterial proliferation and toxin production are usually associated with decreased abomasal motility and increased abomasal ph. c perfringens types b and c both cause severe necrotic enterocolitis and enterotoxemia in lambs, kids, calves, piglets, and foals. the disease is characterized by diarrhea that swiftly becomes hemorrhagic and contains sloughed gi mucosa. systemic signs progress rapidly from weakness, dehydration, and depression to toxemia and death. progression may be so swift that overt signs of diarrhea may not be observed. types b and c produce beta toxin, which is highly pathogenic but readily inactivated by proteolytic enzymes present in the intestinal tract, such as trypsin. neonates are predisposed because of the presence of trypsin inhibitors in colostrum. the disease is generally seen in neonatal ruminants most commonly less than days of age but can occur up to month of age if trypsin inhibitors are still present in the diet. diagnosis should be suspected based on rapidity and severity of disease, necropsy findings that include severe mucosal ulceration of the small intestine and colon, and identification of the bacteria in intestinal contents. beta toxin assays are not generally available from diagnostic laboratories in the united states. c perfringens type d is associated with overeating disease and is most commonly seen in fast-growing lambs and kids. bottle-raised animals and animals consuming large amounts of readily fermentable carbohydrates are most susceptible. the epsilon toxin may cause local intestinal necrosis but is also absorbed and results in systemic disease related to disruption of endothelial cells in affected organs. please refer to simpson km and colleagues', "clostridial abomasitis and enteritis in ruminants," in this issue. clinical signs associated with peracute bvd include severe diarrhea, pyrexia, thrombocytopenia, hemorrhagic disease, and death. persistently infected calves may have severe enteritis as part of mucosal disease syndrome or may be more susceptible to diarrhea caused by other diarrheal agents because of their immunocompromised status. other zoonotic viruses that may be associated with diarrhea in calves are norovirus (bovine norovirus) and torovirus (bovine torovirus [btov]) norovirus is a major cause of acute gastroenteritis in humans; however, the virus isolated from cattle is phylogenetically distinct from human viruses, which may indicate that it has a low zoonotic potential. experimental infections in newborn calves showed that the virus infects small intestinal epithelial cells causing villous blunting. norovirus has also been detected in the feces of clinically healthy calves. toroviruses cause acute enteric infection in piglets and children, and btov has been found in feces of diarrheic calves. it causes a mild to moderate diarrhea in calves less than weeks of age. the virus causes damage to villous and cryptic enterocytes in the upper small intestine. the virus isolated from cattle has antigenic cross reactivity with human toroviruses. many other viruses have been associated with enteric disease in small ruminants, including adenoviruses, astroviruses, bunyaviruses, and paramyxoviruses. however, the relevance of these viruses to clinical practice in north america is unknown. there are currently species of cryptosporidium with varying degrees of host specificity. multiple genotypes of cryptosporidium parvum have been identified and can cause enteritis in sheep, goats, and humans. although it can infect all ages of ruminants, clinical signs are typically seen in preweaning animals. cryptosporidium hominis infects humans, and cryptosporidium andersoni has been found in the abomasum of cattle but its clinical significance is unknown. infection occurs via ingestion of an encysted sporulated oocyst. the parasite invades intestinal epithelium but remains extracytoplasmic, residing in the cell membrane cleft. epithelial destruction causes mild to moderate villous atrophy, resulting in a malabsorptive diarrhea. small infective doses can result in prolonged infection and high parasite burdens because of the phenomenon known as autoinfection, in which the parasite replicates within the host and is directly reinfective without exiting the body. the organism is extremely hardy in the environment and is resistant to most chemical disinfectants, including bleach and alcohol. ammonia-based or peroxide-based products are effective. care should be taken to avoid contamination of watersheds, which can result in significant environmental and public health issues. diagnosis is through fecal smear or sugar floatation; however, the parasite is very small and may be missed by less experienced technicians. acid-fast staining of samples increases sensitivity. in areas where acid-fast stain may not be readily available, there is also a technique for using more typical ziehl-neelsen stain to identify cryptosporidium oocysts. on-farm control of cryptosporidiosis is difficult because of the extremely high levels of oocytes shed and the environmental hardiness of oocytes resulting in a high environmental burden. the infective dose is small because of the replication and autoinfection that take place within the host. several drugs have been tested and found to have limited activity against cryptosporidium, including paromomycin, decoquinate, and halofuginone. of these, halofuginone is considered the most promising but is not available or labeled in all countries and has limited efficacy in cases of multipathogen diarrhea complex. giardia duodenalis (also called giardia lamblia, giardia intestinalis) is classified into different assemblages based on genotypes. assemblages a and b are zoonotic, and assemblage e is livestock associated. all assemblages have been reported in cattle. young calves are most often affected within the first months of life, and infection is often asymptomatic or subclinical. however, giardia infection can cause acute or chronic diarrhea, reduced weight gain, and general ill thrift in young calves. giardia is commonly found in coinfections with coccidia or cryptosporidium. infection occurs via ingestion of cysts from the environment. after ingestion, each cyst releases trophozoites in the upper small intestine. the trophozoites can either attach to epithelial cells via their ventral disk or live freely in the intestinal lumen. trophozoites multiply in the lumen by binary fission. exposure to bile salts causes encystation. excreted cysts are immediately infective (entire cycle in humans, hours). the prepatent period in ruminants ranges from to days. diagnosis is through fecal smear or floatation; however, fecal examination for giardia requires relevant expertise. diagnosis can also be achieved by antigen detection in feces via indirect fluorescent antibody test, enzyme-linked immunosorbent assay, or snap test, or by pcr. in areas where diagnostic laboratories are difficult to access, staining fecal smears with a romanowsky stain can aid in identifying organisms. coccidia are fairly host specific, and most do not cause clinically relevant disease. ruminants are affected by species of the genus eimeria. at least eimeria species infect cattle, infect sheep, and have been reported in goats. only a few eimeria species are pathogenic and cause significant disease. in cattle, lifethreatening disease is most commonly caused by eimeria bovis and eimeria zuernii. infected animals shed unsporulated oocysts, which sporulate in the environment to become infective. oocysts are resistant to environmental changes and persist, especially in cool, moist environments. once ingested, the oocysts are degraded to allow excystation to occur. schizonts and gamonts replicate within cells of the lower ileum, cecum, and large intestine, rupturing the host cells. the life cycle length differs depending on species, but in general the prepatent period ranges from to days. disease is usually seen in older calves, kids, and lambs, and is usually associated with a stressor such as weaning. typical clinical signs are diarrhea with tenesmus. mucus and blood may be observed in the feces. in some cases, severe bloody diarrhea is a significant cause of blood loss. neurologic signs (nervous coccidiosis) are also possible and are associated with toxin production by the coccidia in the gi tract. animals raised in contaminated environments can experience chronic reinfections. chronic subclinical infections can present as ill thrift and poorly growing juvenile animals that are susceptible to other diseases. these animals may also show anemia and hypoproteinemia. diagnosis is through fecal floatation, mcmaster, or modified stoll techniques. for detailed information, please refer to keeton stn and navarre cb's article, "coccidiosis in large and small ruminants," in this issue. differential diagnoses of noninfectious causes of diarrhea in neonatal and juvenile ruminants include the following: improper mixing of milk replacer improper handling of milk or milk replacer grain overload (lactic acidosis) laboratory diagnostic tests are important for guiding therapy, but they are poor predictors of prognosis in calves with diarrhea. physical examination findings are more sensitive predictors of outcome and should be the primary consideration when making clinical decisions. packed cell volume (pcv) and serum total protein (stp) aid in the assessment of the level of anemia and hypoproteinemia, respectively. pcv and stp can be determined using a hematocrit centrifuge and refractometer. assessment of these parameters is useful in guiding the initiation and continuation of fluid therapy. increased pcv and stp levels generally indicate dehydration at the time of initial presentation. however, pcv and stp levels decrease in severely dehydrated neonates treated with large volumes of intravenous fluids and can be detrimental to the survival of the patient if not monitored so that fluid therapy can be adjusted appropriately. low stp level is associated with protein loss via the gi tract or failure of passive transfer of immunity in neonatal animals. in cases of infectious enteritis, low hematocrit can be caused by blood loss from the gi tract or anemia of chronic disease. in cases of blood loss from the gi tract, both hematocrit and stp are decreased simultaneously. coccidiosis is a frequent cause of whole-blood loss via the gi tract. diarrheal diseases also causing blood loss in neonates include salmonellosis, enterohemorrhagic e coli, and clostridial enteritis. blood gas analysis provides an assessment of blood ph and acid/base status. metabolic acidosis is common in neonatal and juvenile ruminants with enteritis. debilitated animals who are not ventilating adequately because of weakness may also have evidence of respiratory acidosis. portable point-of-care blood analyzers (i-stat, epoc) are available for use in ruminants and can perform blood gas analysis. these blood analyzers frequently include major electrolytes in their analysis, allowing assessment of potassium, sodium, and chloride status. in acidotic patients, it is important to consider the extracellular shift of potassium when assessing blood potassium levels. hyperkalemia may be observed in patients with metabolic acidosis, increasing the risk of cardiac arrhythmias and potential cardiac arrest. hyperkalemia generally resolves on initiation of appropriate fluid therapy. blood lactate levels can be helpful in assessing systemic perfusion. severely dehydrated calves can have markedly increased blood l-lactate levels. note that most lactate analyzers only report l-lactate and not d-lactate. d-lactate is produced by microbial flora in the colon and is exacerbated in conditions resulting in maldigestion and malabsorption. d-lactate is an important cause of metabolic acidosis in calves with enteritis and contributes to the weakness and decreased mentation often observed in these patients. a complete blood count provides information to appropriately classify the anemia present (smear evaluation) and assess inflammation (leukocytes with differential counts, and fibrinogen). leukopenia characterized by neutropenia with a left shift, and the presence of cellular toxic changes are evidence of systemic sepsis and might be the result of systemic endotoxemia, bacteremia, or salmonellosis. leukopenia and thrombocytopenia may be observed with acute bvdv type ii infection. marked leukopenia may also be observed with bvdv mucosal disease. serum biochemical analysis assesses concentrations of albumin and globulin, identifies electrolyte derangements, and provides evidence of organ dysfunction secondary to the infectious agent. portable serum biochemical analyzers may be useful in identifying electrolyte imbalances but may not be equipped to assess albumin, globulin, and organ enzyme activities. infectious enteritis causes diarrhea and associated fluid and electrolyte losses. thus, fluid therapy is an important part of management of infectious enteritis. oral fluid therapy, if instituted early in the disease process, can be highly successful and costeffective in treating animals with enteritis and diarrhea. oral electrolyte solutions should be evaluated for their sodium composition, ph buffering capacity, energy content, and osmolarity. guidelines for electrolyte replacers are provided in table . in animals with severely compromised intestinal motility, intravenous fluid therapy can be more effective at correcting the electrolyte imbalances and fluid loss than oral administration. physical examination findings and diagnostic results should be used to guide treatment decisions, and some published algorithms exist to help clinicians in the decision process. blood and protein loss should also be considered and treated accordingly. please refer to the march, issue for an in-depth discussion of fluid therapy in calves. these include . % sodium bicarbonate, . % sodium chloride, and balanced electrolyte solutions such as lactated ringer or plasma-lyte. the choice of the crystalloids may depend on the test results of a serum biochemical analysis. when serum biochemical analysis test results are not available, a balanced electrolyte solution such as lactated ringer or plasma-lyte should be considered for intravenous fluids. both the level of dehydration at presentation and ongoing fluid losses caused by diarrhea must be considered when calculating fluid administration rates. administration rates greater than maintenance are often necessary to treat hypovolemic shock caused by dehydration and account for continued losses from diarrhea. the patient's stp status must also be considered. intravenous fluids should be administered with caution in patients with albumin levels less than g/dl. initial treatment of shock with intravenous fluid replacement may be indicated in severely compromised patients. a typical shock fluid therapy plan is to provide ml/kg of intravenous fluids at a maximum rate of to ml/kg/h. slower rates should be used for small ruminants and in animals with low total protein levels. signs of appropriate response include improved mentation and activity, decreased skin tent or eyeball recession, improved suckle response, decreased capillary refill time, and improved peripheral perfusion noted by warming of distal extremities. signs of fluid overload include wet cough, harsh lung sounds, increased respiratory rate, and edema. general maintenance fluid rates for juvenile ruminants range from to ml/kg/h ( - ml/kg/d). additional fluid losses from diarrhea may increase fluid needs by % to %. thus, total fluid rates in ruminants with active diarrhea are in the range of . to times maintenance. any oral fluid administration also contributes to the daily fluid requirement and should be taken into account when calculating fluid volume to correct dehydration and when calculating maintenance requirements. most neonates with enteritis have decreased nutritional intake and absorption. they benefit from additional intravenous dextrose supplementation. dextrose may safely be added to intravenous fluids at a concentration of . % to % when administered at a maintenance fluid rate. this concentration should be decreased proportionally when increasing fluid rate to more than table recommendations for electrolyte, carbohydrate, buffering capacity, and osmolality of oral electrolyte replacement fluids used to treat enteritis and diarrhea in neonatal ruminants maintenance, or if blood glucose measurements are greater than the normal range ( . ae . mmol/l, ae . mg/dl). , note that neonatal ruminants are effectively monogastrics and normally have higher blood glucose levels than adults, so adult reference ranges should not be applied to them. a reasonable goal for blood glucose management in calves is to keep blood glucose within to mg/dl. correction of hyperkalemia can be accomplished by administering small volumes of hypertonic ( . %) sodium bicarbonate, followed by oral electrolytes. hypertonic sodium bicarbonate can safely be administered at a rate of . ml/kg body weight (equivalent to . meq hco -/kg body weight) as a bolus over minutes in calves with diarrhea and evidence of metabolic acidosis. a recent issue (march, ) covered fluid therapy in calves extensively. plasma transfusion should be considered in ruminants with severe hypoproteinemia (albumin levels < . g/dl) on serum biochemical analysis. dosage rates for plasma administration range from to ml/kg. blood transfusion (particularly in coccidiosis) should be considered when pcv is less than % with associated clinical signs of compromise, including tachycardia, tachypnea, weakness, and poor appetite. following plasma or blood transfusion administration, fluid therapy may be continued with crystalloid fluids. enteritis can predispose neonates to bacteremia and secondary infections because of compromised gut barrier and bacterial translocation. broad-spectrum antibiotics, with special attention to adequate gram-negative coverage, should be instituted in patients showing signs of endotoxemia or sepsis. although the choice of antibiotics should be based on susceptibility of the isolate cultured, broad-spectrum antibiotics may be initiated while awaiting these results. susceptibility of salmonella to tetracyclines, ampicillin, and amoxicillin is variable, whereas resistance to penicillin, erythromycin, and tylosin is highly likely. florfenicol should be considered for treatment of salmonellosis. use of aminoglycosides should be avoided because of prolonged tissue residues in animals intended for food. cephalosporins and fluoroquinolones may not be used in an extralabel manner in the united states. tetracyclines should be avoided in dehydrated neonates until fluid hydration and renal perfusion are restored to minimize the risk of nephrotoxicity. use of nonsteroidal antiinflammatory drugs (nsaids; eg, flunixin meglumine) may be considered to control pyrexia and inflammation. flunixin meglumine ( . mg/kg intravenously) or meloxicam ( . mg/kg intravenously or subcutaneously) were reported to improve outcome in calves with nonspecific diarrhea. , the use of nsaids should be restricted in dehydrated ruminants and only administered once the patient has been sufficiently hydrated. metaphylactic use of antimicrobials can only be recommended for outbreaks caused by a specific bacterial pathogen. prophylactic treatment with antimicrobials has been shown to increase the risk of diarrhea in neonatal calves. appropriate colostrum handling and administration are instrumental in preventing neonatal diarrhea in ruminants. few clinicians dispute a link between inadequate colostrum consumption and an increased risk for diarrhea in neonates. supplementing calves with colostrum orally past the traditional hours after birth decreases diarrhea and diarrheal treatments in preweaned calves , a minimum of to g of immunoglobulin g (igg) in colostrum or a colostrum replacer should be fed to calves within the first hours (chigerwe and colleagues, ) to provide adequate transfer of passive immunity. the concentration of igg in colostrum can be estimated before feeding calves using a hydrometer or a brix refractometer. evaluation of the passive transfer status of neonates provides valuable risk assessment, husbandry, and epidemiologic information in both individual cases and herd outbreaks. passive transfer status can be assessed using stp, sodium sulfite precipitation, immunocrit test, or radial immunodiffusion. other factors associated with an increased risk of diarrhea include hygiene of the maternity area and neonate housing, stocking density, and disinfection practices. attention should be paid to maternal health prepartum as well. vaccination of the dams before parturition with a k e coli, rotavirus, coronavirus product can reduce the diarrhea associated with that pathogen. , vaccination for other diarrheal agents is of varying efficacy. in beef operations, a method known as the sandhills calving system can be used to prevent calf diarrhea in a herd or to break a herd of a recurring neonatal diarrhea problem. in this method, cows are continually calving on new ground, reducing contamination and reducing mixing of calves of different ages. causes of infectious enteritis in adult ruminants are bacterial, viral, and parasitic. the most consistent clinical sign of infectious enteritis is diarrhea. specific causes of infectious enteritis in adult ruminants cannot be distinguished easily based on clinical examination alone. laboratory diagnostic tests are required to differentiate the causes. most of the causes have herd implications, thus identification of the cause is recommended. management of infectious enteritis in adult ruminants includes administration of oral electrolyte fluids or intravenous fluids such as crystalloids and colloids, nsaids, antibiotics, anthelmintics, and anticoccidiosis treatments. investigations of d-lactate metabolism and the clinical signs of d-lactataemia in calves clinical and laboratory assessment of hydration status of neonatal calves with diarrhea treatment of calf diarrhea: oral fluid therapy diseases of the alimentary tract, in veterinary medicine: a textbook of the diseases of cattle, horses, sheep, pigs and goats bovine noroviruses: a missing component of calf diarrhoea diagnosis case-control study of microbiological etiology associated with calf diarrhea enteric viral infections in lambs or kids update on cryptosporidium and giardia infections in cattle evaluation of modified ziehl-neelsen, direct fluorescent-antibody and pcr assay for detection of cryptosporidium spp. in children faecal specimens controlling the onset of natural cryptosporidiosis in calves with paromomycin sulphate prophylactic use of decoquinate for infections with cryptosporidium parvum in experimentally challenged neonatal calves utility of halofuginone lactate for the prevention of natural cryptosporidiosis of calves, in the presence of co-infection with rotavirus and salmonella typhimurium validation of romanowsky staining as a novel screening test for the detection of faecal cryptosporidial oocysts clinical signs, profound acidemia, hypoglycemia, and hypernatremia are predictive of mortality in , critically ill neonatal calves with diarrhea d-lactic acidosis in calves construction and validation of a decision tree for treating metabolic acidosis in calves with neonatal diarrhea treatment of calf diarrhea: intravenous fluid therapy reference values of blood parameters in beef cattle of different ages and stages of lactation changes in the blood biochemical and haematological profile of neonatal calves with age effect of intravenous small-volume hypertonic sodium bicarbonate, sodium chloride, and glucose solutions in decreasing plasma potassium concentration in hyperkalemic neonatal calves with diarrhea update on the use of blood and blood products in ruminants large animal internal medicine th edition nonsteroidal anti-inflammatory drug therapy for neonatal calf diarrhea complex: effects on calf performance evaluation of flunixin meglumine as an adjunct treatment for diarrhea in dairy calves targeting therapy to minimize antimicrobial use in preweaned calves: effects on health, growth, and treatment costs evaluation of the effects of oral colostrum supplementation during the first fourteen days on the health and performance of preweaned calves comparison of four methods to assess colostral igg concentration in dairy cows vaccines for preventing enterotoxigenic escherichia coli infections in farm animals use of biologics in the prevention of infectious diseases strategies for controlling neonatal diarrhea in cow-calf herds-the sandhills calving system key: cord- -jdu h e authors: dubourdieu, dan title: colostrum antibodies, egg antibodies and monoclonal antibodies providing passive immunity for animals date: - - journal: nutraceuticals in veterinary medicine doi: . / - - - - _ sha: doc_id: cord_uid: jdu h e passive immunity can be provided to animals by several sources of antibodies including from colostrum, avian eggs, and monoclonal sources. these antibodies have been shown protect production and companion animals from a number of pathogens. this chapter reviews the immune system for the principles of immune response to antigens and the synthesis of immunoglobulins of the five classes of antibodies in the body. colostrum antibodies are described for passive immunity protection in animals such as calves. chicken egg antibodies are another source of antibodies for passive immunity. therapeutic monoclonal antibodies are also used to provide passive immunity in the veterinary field. the use of antibodies by veterinarians to maintain the health of animals has a long history. fundamentally, when it comes to the immune system health of production and companion animals, there are little absolute differences in the intended purpose of the immune system. mammals have the same basic immune system with minor differences between the species. even the differences between birds and mammals are not so great since the purpose of the immune system is to keep infectious microorganisms, such as certain bacteria, viruses, and fungi, out of the body and to destroy any infectious microorganisms that do invade the body. how veterinarians take advantage of the immune system to maintain health can roughly be defined as taking advantage of the body's inherent method of maintaining health through adaptive immunity provided by vaccinations to generate antibodies inside the animal or by administering preformed antibodies to an animal through a process called passive immunity. evolution has produced an amazing immune system in animals that utilizes various cell types and proteins to protect them from invasive organisms. this system has two broad categories: nonadaptive and adaptive. the nonadaptive immune system is mediated by cells that respond in a nonspecific manner to foreign substances. this response includes phagocytosis by macrophages, secretion of lysozymes by lacrimal cells, and cell lysis by natural killer cells. the adaptive immune response is mediated by lymphocytes that produce a set of proteins called antibodies that are either secreted by or found on the surface of the lymphocyte. when the antibodies themselves are created within the animal following vaccination or from exposure to pathogens, the process is called adaptive immunity. when preformed antibodies from a host animal are given to another recipient animal, such as it offspring or even a completely different species animal, the process is called passive immunity. scientists have long taken advantage of the adaptive immune system by using vaccines. vaccines for animal diseases were the first to result from laboratory-based scientific investigation. french chemist louis pasteur developed a vaccine for chicken cholera in , and one for anthrax of sheep and cattle in . the major goals of veterinary vaccines are to improve the health and welfare of companion animals, increase production of livestock in a cost-effective manner, and prevent animal-to-human transmission from both domestic animals and wildlife. these diverse aims have led to different approaches in the development of veterinary vaccines from crude but effective whole-pathogen preparations to molecularly defined subunit vaccines, genetically engineered organisms or chimeras, vectored antigen formulations, and naked dna injections (meeusen et al. ). it has also resulted in various guidelines for vaccinations of companion animals such as for dogs (ford et al. ) and production animals such as swine (alabama and auburn ) , in poultry (stewart-brown ), cow/calf (missouri ) , and other production animals. successful veterinary vaccines have been produced against viral, bacterial, protozoal, and multicellular pathogens, which in many ways have led the field in the application and adaptation of novel technologies. passive immunity whereas active immunity refers to the process of exposing the individual to an antigen to generate an adaptive immune response, passive immunity refers to the transfer of antibodies from one individual to another (marcotte and hammarström ) . passive immunity provides only short-lived protection, lasting from several weeks to up to - months, but is immediate. nature intended passive immunity to occur when maternal antibodies are transferred to the fetus through the placenta or from breast milk to the gut of the infant. however, it can also be produced artificially when antibody preparations are derived from sera or secretions of immunized donors and are delivered via oral or systemic routes to nonimmune individuals. passive immunization is a new approach to providing protection to animals against pathogens because of the emergence of new and drug-resistant microorganisms, diseases that are unresponsive to drug therapy and individuals with an impaired immune system who are unable to respond to conventional vaccines. the immune system can respond specifically to millions of different molecules and is constantly challenged by huge numbers of antigens. a major feature of the immune system is that it can synthesize a vast number of antibodies. each of these antibodies can bind to a different antigen. this binding is the basis for the molecular specificity of the immune response. antibodies are proteins produced by a type of terminally differentiated b lymphocytes. b cells take the b name from chicken bursa cells where they were first discovered (gitlin and nussenzweig ) . antibodies are produced in response to the presence of foreign molecules in the body. the antibodies circulate throughout the blood and lymph where they bind to the foreign antigens. once bound, these antibody-antigen complexes are removed from circulation, primarily through phagocytosis by macrophages. this is the basis for antibodies protecting the animal against pathogens. antibodies are a large family of glycoproteins that share key structural and functional features. from a structure standpoint, antibodies look like a y-shaped molecule (fig. ) . each y contains four polypeptides. two of the polypeptides are identical and called heavy chain. the other two, also identical, are called light chain and are connected by disulfide bonds. there are five classes of antibodies, igg, igm, iga, ige, and igd, that are classified based on the number of y-like units and the type of heavy-chain polypeptide they contain (fig. ). igm is the largest antibody, and it is the first to appear in response to initial antigen exposure. the spleen, where plasma cells responsible for antibody production reside, is the major site of specific igm production (capolunghi et al. ; marchalonis et al. ) . igg is the main type of antibody found in blood and extracellular fluid allowing it fig. antibody structure as drawn, by protein model and by electron microscopy to control infection within body tissues. approximately % of all antibodies in humans and companion animals are of the igg class. immunoglobulin a (iga) plays a crucial role in the immune function of mucous membranes. the amount of iga produced in association with mucosal membranes is greater than all other types of antibodies combined (fagarasan and honjo ; holmgren and czerkinsky ; snoeck et al. ) . igd was initially thought to be a recently evolved antibody class because it was only detected in primates, mice, rats, and dogs and not guinea pigs, swine, cattle, sheep, and frogs (preud'homme et al. ) . however, recent discoveries of igd in ancient vertebrates suggest that igd has been preserved in evolution from fish to humans for important immunological functions (chen and cerutt ) . immunoglobulin e (ige) has only been identified in mammals. ige's main function is immunity against parasites such as helminths (erb ) like schistosoma mansoni, trichinella spiralis, and fasciola hepatica (watanabe et al. ; pfister et al. ) . ige also has an essential role in type i hypersensitivity (gould et al. ) which manifests in various allergic diseases, such as allergic asthma, most types of sinusitis, allergic rhinitis, food allergies, and specific types of chronic urticaria and atopic dermatitis (mueller et al. ) . antibodies bind antigens at the upper tips of the y molecule. the region of antigen where binding occurs is called the epitope (fig. ). antibodies can bind to a wide range of chemical structures and can discriminate among related compounds. how well the antibody binds to an antigen is known as affinity. this affinity can range from low to high. colostrum and passive immunity mammals are born without a fully functional adaptive immune system even though the basic elements are present. when a mammal is born, it emerges from the sterile uterus into an environment where it is immediately exposed to a host of microorganisms. the gastrointestinal tract (git) acquires a complex microbial flora within hours. if it is to survive, the newborn animal must be able to control this microbial invasion. in practice, the adaptive immune system takes some time to become fully functional, and innate mechanisms are responsible for the initial resistance to infection. in some species with a short gestation period, such as mice, the adaptive immune system may not even be fully developed at birth. in animals with a long gestation period, such as domestic mammals, the adaptive immune system is fully developed at birth but cannot function at adult levels for several weeks. the complete development of active immunity depends on antigenic stimulation. the proper development of b cells and b-cell receptor diversity requires clonal selection and antigen-driven cell multiplication. thus, newborn mammals are vulnerable to infection for the first few weeks of life. they need assistance in defending themselves at this juncture. temporary help is provided by the mother in the form of colostrum, which contains antibodies. the passive transfer of immunity from mother to newborn is essential for survival. calves are born without an active immune system and rely on the consumption of antibodies for protection from disease such as scours and pneumonia. the cow provides its calf with nutrients for growth and development during gestation, but the cow cannot directly provide the calf with antibodies to protect it from diseases. fortunately, immunoglobulins form an important component of the immunological activity found in milk and colostrum. while humans have a large amount of iga in their colostrum, the colostrum from most other animals contains a high percentage of igg (hurley and theil ) (fig. ) . immunoglobulins found in mammary secretions arise from systemic and local sources. in the case igm iga ige igd of igg in milk, the major portion comes from the serum (mayer et al. ) . while plasma cells producing igg may occur within the mammary tissue, their contribution to the igg in colostrum is minor compared with the igg derived from serum. the other major classes of immunoglobulins transported into colostrum and milk are iga and igm. immunoglobulin a (iga) is the major immunoglobulin in human colostrum and milk; however, it is also present in milk of most other species. colostrum and milk iga and igm are found in the form of secretory iga, or siga, and sigm. much of this is produced by plasma cells in the mammary tissue. the plasma cells are part of the gut-associated lymphoid tissue (galt), the largest immune organ of an organism, which includes the peyer's patches, lymphoid and myeloid cells in the lamina propria, and intraepithelial lymphocytes (ishikawa et al. ) . interestingly enough, more than % of the immune system is located in the gastro intestinal tract, the site where many oral pathogens first interact with an animal (vighi et al. ) . galt is a part of the mucosa-associated lymphoid tissue and works in the immune system to protect animals from invasion of pathogens in the gut. one of the physiological functions of the mucosa in the gut is for food absorption. however, of equal importance of the galt is in the body's defense, due to its large population of plasma cells whose number exceeds the number of plasma cells in the spleen, lymph nodes, and bone marrow combined (nagler-anderson ) . lymphocytes from the galt system will move to the mammary gland and provide a direct link between the antigen exposure response in the mother's mucosa system and the secretory immunoglobulins of the mammary gland (brandtzaeg ) . as such, this means that maternal colostrum and milk will contain antibodies specific for pathogens that may be encountered by the neonate's intestine and other mucosal tissues. this provides a rationale for the observations that bovine colostrum from nonimmunized cows may also afford passive immune protection against human pathogens in both humans and animals (li-chan et al. ; yolken et al. ) and opens the door to new technology to provide veterinarians another way to protect animals from pathogens that does not involve antibiotics. antibodies must be obtained by drinking colostrum within the first couple of hours after birth as part of the passive immunization system in order to maximize antibody absorption (pakkanen and aalto ) . like other animals, antibodies are generated by healthy cows as a result of every day exposure to infectious agents. antibodies can also be the result of specific vaccination programs. the cow's natural antibodies to these infectious agents are passed from the cow to the calf through colostrum. the level of antibodies transmitted from the cow through the colostrum can be elevated by a pre-calving vaccination program (thomas ) . when the calf drinks colostrum, the maternal derived antibodies are absorbed from the calf's git into the blood stream. some of the immunoglobulins also remain in the gut where they can neutralize pathogenic bacteria and help prevent the development of diarrhea. the absorption of antibodies from the git into the bloodstream is called passive transfer. failure of passive transfer (fpt) in dairy calves is defined by a blood igg level of < mg/ml - h after birth (stilwell and carvalho ) . calves that experience fpt are more likely to become sick or die in the first months of life than calves with adequate immunity. many factors can contribute to fpt, but colostrum and the management of colostrum feeding are often involved. to successfully obtain passive transfer and provide the calf with protection from diseases, it is thought that the calf needs to consume a minimum of - g of immunoglobulins (meganck et al. ) . the pathway between the gastrointestinal tract and the bloodstream is only open for a short window of time. research shows that this pathway starts to close shortly after birth, and after - h, approximately % of the calf's ability to absorb colostrum antibodies is gone. it has therefore been recommended to feed calves as much colostrum as they want by bottle within - h after birth and at h of age to substantially reduce the probability of fpt (chigerwe et al. ; trotz-williams et al. ) . colostrum also provides the calf with protein, energy in the form of fat and sugar, and vitamins (quigley and drewry ) . some vitamins do not cross the placental barrier, and colostrum is the primary source of these nutrients for the calf after birth. energy is required for all metabolic functions including maintenance of body temperature. one of the leading causes of death in dairy calves is failure to initiate breathing and metabolic processes in the first hours of life. the newborn calf only has a few hours of energy reserves in stored fat and therefore needs the energy from colostrum. research also confirms that the sooner a calf consumes colostrum, the more maternal antibodies it can utilize. the quality of colostrum is a major issue that the dairy industry faces on a regular basis. generally speaking, quality of colostrum is related to the amount of antibody that is present. colostral igg concentration is an important factor that affects whether calves receive sufficient passive immunity (godden et al. ) . unfortunately, the amount of igg in maternal colostrum varies dramatically among cows (< - g/l) with . - . % of samples that do not reach the desired amount of g igg/l (gulliksen et al. ) . colostral quality is difficult to estimate by the farmer based on produced volume or appearance of the colostrum. there are many variables that impact colostrum quality, including nutrition, the time the cow is milked, heat stress, and stage of lactation. a study from iowa state university suggests that a minimum % of dairy calves in the usa are currently being fed colostrum classified below industry standards for igg content and are at a greater risk of fpt, mortality, and morbidity (morrill et al. ) . other production mammals such as piglets face the same issues as calves for colostrum quality. studies have shown that on average % of pigs with a colostrum intake below g usually die. in pigs with a colostrum intake below g, mortality was as high as % (devillers et al. ) . giving spray-dried bovine colostrum to other animals such piglets has been shown (sty et al. ) to help protect against gut dysfunction and inflammation. it may be possible that using bovine colostrum for piglets could help supplement sow colostrum. research has also been done in foals with an enhanced bovine colostrum supplementation (fenger et al. ). since up to % of cows have colostrum with an igg level below mg/ml, which will not prevent fpt, a variety of programs and protocols have been implemented by dairy producers. high-quality maternal colostrum is still the gold standard for feeding newborn calves. however, colostrum supplement and replacer products can be valuable tools to increase calf immunity when colostrum supplies are limited or disease eradication is desired. colostrum products that contain igg are regulated by the usda center for veterinary biologics and are available in bolus, gel, and powder formats. supplement products are unable to raise the blood concentration of igg above the species standard, which is mg/ml for calves. any product that is able to raise serum igg concentration above mg/ml may be called a colostrum replacer (penn state ). supplements do not contain sufficient quantities of antibodies to raise the blood igg level in calves beyond what average quality colostrum will do. colostrum replacer contains greater levels of igg and other nutrients and provides an effective, convenient method of providing passive immunity to calves when maternal colostrum is not available. colostrum supplements available today are made from dried bovine colostrum or serum and contain - g of igg per dose ( - % globulin protein). the fat content of these products ranges from . to %. spray-dried colostrum with high concentrations of immunoglobulin may be produced economically and used as an effective and convenient colostrum replacer in newborn calves (chelack et al. ) . numerous products designed to replace colostrum are now on the market. these products are made from bovine colostrum or serum and contain - g of igg per dose. these products also provide fat, protein, vitamins, and minerals needed by the newborn calf, although the amount varies between products. a summary (pennsylvania state university ) of treatment means from published studies investigating colostrum products indicated that replacer products provided an average of g of igg, with an absorption efficiency of %, and serum igg of mg/ml. supplement products (fed in addition to colostrum) provided g of igg with % absorption efficiency and resulted in serum igg of mg/ml. it has been recognized that while antibodies found in colostrum can certainly reduce diseases in animals via passive immunity when given to a newborn, they will only work if the colostrum donor animal has been exposed either naturally to the disease or given a vaccine to the disease, in order to have specific antibodies produced. bovine colostrum that is typically spray-dried for supplements or replacer will contain only the antibodies that the cow may have encountered naturally. therefore, the colostrum used may not have specific antibodies against particular diseases that a producer might be interested in. the animal industry has recognized this issue and has developed methods to produce specific antibodies in high titer against specific diseases that can be delivered in colostrum products. this is achieved by hyperimmunizing animals such as cows against specific animal diseases, collecting colostrum and processing it into powders such as by freeze-drying methods and then giving to a newborn animal in gels or boluses. a number of diseases including bacterial diseases like e. coli (selim et al. ) and viral diseases such as rotavirus and coronavirus (combs et al. ) or coccidial diseases such as giardia or cryptosporidia (graczyk et al. ; fayer et al. ; naciri et al. ) have been researched for hyperimmunized colostrum efficacy. the hyperimmunized colostrum is collected, processed, and given to newborn calves. to varying degrees of success, these hyperimmunized colostrum antibodies have been proven to be successful in providing passive immunization. besides calves, research has been done in a variety of production animals using bovine or other sources of colostrum. for example, lambs have been supplemented with ewe colostrum as well as hyperimmunized serum from sheep against e. coli (pommer ) . other research has examined vaccination of cows with clostridial antigens and passive transfer of clostridial antibodies from bovine colostrum to lambs (clarkson et al. ) . piglets have also been given passive protection against porcine epidemic diarrhea by hyperimmune bovine colostrum (shibata et al. ) . specific antibodies in a hyperimmunized colostrumderived product can be used while complementing early colostrum feeding and can be delivered at the same time as colostrum. there are some advantages to this strategy as specific immunoglobulins immediately fight at the gut level to protect against diseases that destroy the intestinal lining while also allowing for antibodies to be absorbed into the bloodstream. it's important to protect the intestinal lining because if the cells that line the digestive tract become damaged, milk cannot be digested or absorbed by the calf. besides the quality of the colostrum itself, researchers first believed that calves could not be vaccinated effectively while they had circulating maternal antibodies from the colostrum in their system. preweaning calves can respond to vaccination stimulation as early as month of age. the maternal antibodies absorbed from colostrum, however, cannot distinguish between the antigens of a natural challenge and the antigens in a vaccine. therefore, colostrum antibodies can interfere with the immune response to a vaccination (niewiesk ) . work continues to be done to develop ways to circumvent maternal antibody interference. vaccination of calves in the face of maternal antibodies (ifoma) often does not result in seroconversion as maternally derived immunity interferes with the activation of adequate antibody responses to vaccination. however, it can prime t-and b-cell memory responses that protect calves against clinical disease when maternal immunity has decayed. the activation of b-and t-cell memory responses in calves vaccinated ifoma varies and is affected by several factors, including age, level of maternal immunity, type of vaccine, and route of administration. these factors influence the adequate priming of humoral and cell-mediated immune responses and the outcome of vaccination. failure to adequately prime immune memory after vaccination ifoma could result in lack of clinical protection and an increased risk of viremia and/or virus shedding (chamorro et al. ). there is obviously some controversy about whether newborn calves should be vaccinated (cook et al. ) . it is thought that the process of the calf mounting an immune response to a vaccine requires energy that could better be used to fight off disease and gain weight and the response could actually be detrimental to the early health of that calf. on the other hand, while maternal antibodies can block response to vaccination, sometimes they do not (woolums ) . the exact immunologic outcome in calves vaccinated ifoma can vary, and this variation likely depends on many factors. these factors are not well characterized but likely include the nature of the vaccine administered, number of doses administered, age of the calf and level of maternal antibody present in the calf, and the means by which a protective response is defined. guidelines for vaccinating newborn animals such as calves require additional research to clarify the ifoma vaccination reactions. fortunately, producers also have the option of using antibody products in order to generate immediate protection in situations where colostrum quality is poor. antibody products complement colostrum feeding because they can be fed at the same time. these products are available in bolus, gel, and powder form. they also are included in some colostrum replacer and supplement formulas for added value. typically, the antibody products are from hyperimmunized cows, and the specific antibody is found in the colostrum. the colostrum is processed and typically fractionated into a semi-purified preparation of antibody that is freeze-dried and put into capsules or boluses. because antibody boluses can be fed in conjunction with colostrum, they can be a tool to help the calf not only achieve adequate passive transfer but also provide enough specific antibodies to protect against the most common early calf hood diseases. antibody products do not require the calf to react to a vaccine in order to develop antibodies. rather specific antibodies against various diseases are already present, measured, and verified to be at a high enough level to protect the calf from scour-related diseases, and they can be fed as close to birth as possible. united states department of agriculture (usda)-approved antibody products are available on the market that can be fed in conjunction with colostrum and provide the calf with immediate immunity. these antibodies go to the gut to immediately bind and neutralize diarrhea antigens while also being absorbed into the blood stream for extended protection (combs et al. ; chamorro et al. ) . another advantage of this approach is that providing specific antibody can potentially avoid vaccine stimulation. avoiding vaccine stimulation can allow a calf to conserve its minimal supply of fat and nutrients that are critical to get the calf through its first few days of life. passive immunity by egg antibodies cows pass their immunity to their offspring by colostrum, and various colostrum products are on the market to achieve passive immunity. additional sources of antibody products can be utilized in a similar manner to achieve passive immunity. these are from avian sources such as chickens. in birds, passive transfer of immunity occurs through the egg. by hyperimmunizing chickens over a period of time with inactivated multivalent bacterial or viral vaccines, this procedure results in the production of polyclonal immunoglobulins of the igy class (specific to avians) directed against the stimulating organisms. oral consumption of the "immune" eggs containing specific igy antibodies protects the animal against the specific organism(s) with which the hen was stimulated. unfortunately, the eggs really cannot be cooked since heat dentures the antibodies found in the eggs. other physical parameters such as an acidic ph will also destroy the antibodies to a certain extent. however, enough orally administrated igy may survive passage through the git and, after excretion, still retain a great deal of its antigen-binding ability indicating that orally administrated igys are useful for passive immunity. eggs as a natural source of immunoregulatory factors just as immune protection is transferred in utero in mammals or passively by a lactating mother via colostrum, hens passively transfer protection to their young by secreting immunoglobulin and other immune factors into their eggs for use by the hatching chick. the transfer of chicken immunoglobulins from the hen's serum to the yolk and from the yolk to the chick is analogous to cross-placental transfer of igg from the mammalian mother to its offspring. while igm and iga are found in chicken eggs, the principle immunoglobulin is igy which is found in the yolk of the egg (hamal et al. ) . igy (y stands for yolk) is an immunoglobulin class specific to avians and analogous in function to that of mammalian immunoglobulins. igy has a similar structure as mammalian igg with some minor differences in the heavy chains (fig. ) . both eggs and milk (including breast milk) contain naturally occurring antibodies, and there are reports of immunomodulatory factors in milk as well (li et al. ) . however, immunoglobulin levels in eggs can be significantly higher than levels found in serum or milk (woolley and landon ) . this may not be surprising since mammals have a considerably longer time of weeks or months during which they may passively transfer immunoglobulin and immune factors, while the hen has a single opportunity (the egg) to transfer all necessary survival components to its offspring. all the aspects that the chick needs to survive must be in the egg. because egg products are a common source of protein in human diets and eggs contain antibodies and immune factors, it was obvious to utilize egg antibodies to provide passive immunity to animals. as such, this has resulted in a number of commercial egg antibody products on the market for production animals as well as companion animals to prevent various diseases. with few exceptions, oral consumption of specific antibodies has been reported (diraviyam et al. ) to protect both humans and animals. furthermore, in vitro studies with specific igy antibodies have been found to inhibit processes associated with bacterial growth, adhesion to intestinal cells, and toxin production (sugita-konishi et al. ) . meta-analysis has demonstrated the beneficial effects of igy (diraviyam et al. ) for a variety of animals. this fig. structure of mammalian igg compared to avian igy analysis supports the opinion that igy is useful for prophylaxis and treatment. currently, the oral passive immunization using chicken igy has been focused as an alternative to antibiotics for the treatment and control of diarrhea. igy has been demonstrated to be effective in controlling and preventing diarrhea in humans and in animals including piglets (cui et al. ; vega et al. ) , mice (buragohain et al. ) , poultry (el-ghany ; farooq et al. ) , and calves (cook et al. ; germine et al. ; vega et al. ) . most commercially available chickens are immunized at birth to protect them from avian diseases. the only difference between such supermarket eggs and "immune" eggs is that the latter are from chickens that have received additional proprietary vaccinations with other inactivated pathogens known to be the etiologic agents of animal infections. a wide range of bacteria, viruses, and coccidia have been used in vaccines for producing commercial igy products. a partial list includes shigella dysenteriae, staphylococcus epidermidis, escherichia coli, e. coli k , salmonella enteritidis, and s. typhimurium, pseudomonas aeruginosa, klebsiella pneumoniae, haemophilus influenzae, species of streptococcus, salmonella dublin, salmonella anatum, clostridium perfringens type a and type c toxoids, rotavirus types and , coronavirus, reovirus, parvovirus, and cryptosporidium parvum (schade et al. ) . only the chicken (not the egg) is exposed to inactivated pathogens. immunoglobulins and other immune factors are passively transferred to the egg from the serum for use by the chick and, more importantly, for passive immunity for other animals. after appropriate times following vaccination, eggs are collected from the specially designated chicken flocks, washed and broken, and the yolk and egg white are typically dried to a fine proteinaceous powder. various processes have been developed to help minimize heat damage to egg antibodies and immunoregulatory factors during the spraydrying procedure. however, spray-drying eggs can denature the igy to an extent, due to at least some heating during the process. keeping the egg away from heat is an optimal way of maintaining igy titer levels and efficacy. while freezedrying is an expensive option for maintaining antibody titer levels, simply feeding fresh immunized eggs is another method which exists. this is to keep the immunized eggs in a stabilized liquid (dubourdieu ). this stabilized liquid format is a practical and inexpensive delivery method that allows the specific igy to be delivered in watering systems to production animals or incorporation into other delivery formats such as soft chews that can be readily given to companion animals (fig. ) . the effective mechanism by which avian igy's work is effective is by the same mechanism that mammalian igg antibodies work in the animal to provide passive immunity. for example, bacteria e. coli strain k typically causes problems in production animals that result in diarrhea and possible death. therefore, a vaccination program is used in chickens to create specific anti-e. coli k igy antibodies. these specific antibodies found in the yolk of the egg are given orally to animals to prevent e. coli k from causing disease. this occurs when specific anti-e. coli k igy antibodies bind to the pathogenic bacteria. this binding blocks the ability of the pathogen to bind to the mucin layer obtain egg yolk containing specific igy against antigen spray dry into powder containing specific igy stabilized into liquid containing specific igy fig. vaccination for specific antibodies and processing egg yolk strategies in the gi tract of the animal. the pathogens are flushed out of the gi tract with the feces since they have been rendered unable to bind. the total immunoglobulin content of eggs from hyperimmunized hens is identical to the total level of immunoglobulins found in conventional table eggs. however, the quantities of immunoglobulins to selected antigens are different in the two varieties of eggs. additionally, both the table egg and the "immune" egg contain immunoregulatory factors, but eggs from hyperimmunized chickens may contain many times greater concentrations of individual factors as compared to regular eggs. besides immunoglobulins, eggs contain a number of bioactive components, including phospholipids, cholesterol, lutein, zeaxanthin, and proteins, that possess a variety of proand/or anti-inflammatory properties. two major categories of immune components are found in "hyperimmune" egg: ( ) the immunoglobulins with neutralizing specificities against the stimulating pathogens and ( ) the immunoregulatory factors that modulate cellular functions. the immunoglobulins provide local protection against gastrointestinal intoxication. the immunomodulatory mediators act directly on gastrointestinal surfaces and circulate systemically, affecting every immune, physical, metabolic, and neuroendocrine pathway in the body. these may have important implications for the pathophysiology of numerous chronic diseases and immune responses to acute injury (andersen ) . given the essentiality of pro-inflammatory responses in normal immune defense against pathogens, further research into the role of egg intake on immunity is warranted and may lead to further commercial uses of eggs and igy technology. biological medicine in humans, an intervention pioneered in the last years, is now on the horizon for companion animals. this strategy includes the use of monoclonal antibodies (mabs) to selectively target proteins such as cellular receptors or soluble molecules involved in disease pathogenesis. such treatment holds the potential for targeted therapies of chronic diseases such as osteoarthritis, atopic dermatitis, or lymphomas in dogs and cats. monoclonal antibodies are antibodies that are made by identical immune cells that are all clones of a unique parent cell. monoclonal antibodies have monovalent affinity, in that they bind to the same epitope. in contrast, polyclonal antibodies bind to multiple epitopes and are usually made by several different plasma cell lineages (fig. ) . given almost any substance, it is possible to produce monoclonal antibodies that specifically bind to that substance; they can then serve to detect or purify that substance. this has become an important tool in biochemistry, molecular biology, and medicine. therapeutic mabs can be used medically to block disease-relevant proteins (e.g., cytokines or receptors on cells) and cancer and have gained significant use in humans. they can also be used to target viruses or bacteria and aid in their destruction and elimination. veterinary medicine is only now incorporating this tool. despite the success of mabs in human medicine, there are considerably fewer in the pipeline for veterinary medicine. this may be due to uncertain regulatory guidance in both europe and the usa. these regulatory documents are written from the perspective of human medicine-based risk assessment and development. it is recognized that this may present a challenge to veterinary mabs manufacturers to achieve the extent of characterization/quality control testing typically required for human mabs. (ema ) . regardless, the pet medicine industry is making strides to put mabs into the market through basic research. early human therapeutic mabs contained a high proportion of mouse-derived sequences (fully mouse or mouse/ human chimeric mabs) that were recognized by the human immune system as foreign. this immune response triggered production of anti-mabs, leading to reduced therapeutic efficacy. subsequently, the design of humanized and fully human mabs has resulted in a vast reduction in their immunogenicity, although most therapeutic mabs may have some remaining immunogenicity that is not followed by apparent adverse clinical manifestations. these same issues hold true antigen y y polyclonal antibody antigen monoclonal antibody fig. polyclonal antibody compared to monoclonal antibody binding of antigens for making veterinary mabs and utilizing canine mabs for dogs that helps efficacy. it has been found that caninized anti-ige mabs reduce ige hypersensitivity in mite-sensitized beagles (gearing et al. ) . caninized anti-nerve growth factor mab (webster ) significantly reduced pain scores (webster et al. ) in dogs, and mabs that neutralize the pruritogenic cytokine il- in dogs reduced the pruritic response for weeks after injection (dunham et al. ) . one of the aspects regarding commercializing therapeutic antibodies involves regulation from the usda. in , two usda-approved monoclonal antibody treatments for b-cell and t-cell lymphomas in dogs were granted. these mabs fight lymphoma by targeting the protein cd , which is commonly expressed in b-cell lymphoma (ogilvie et al. ; bulman-fleming et al. ) . while these particular mabs were not commercially successful, they are part of a new approach for using therapeutic antibodies in veterinary medicine. other companies are developing veterinary mabs for cancer, allergies, and chronic inflammatory disease such as atopic dermatitis and for the control of pain associated with osteoarthritis in dogs and cats (webster et al. ). the first mab approved for veterinary use was in the european union (bmj ). it treats the clinical signs of atopic dermatitis in dogs, including itch and inflammation, for up to month. the mab treatment works by mimicking the activity of natural antibodies to selectively bind to and neutralize interleukin- (il- ), a key protein involved in cell communication which triggers itching associated with atopic dermatitis in dogs. because it neutralizes il- , it has been demonstrated not to interfere with the immune response, meaning that it does not induce unintended immunosuppression or enhancement. most therapeutic mabs are delivered via intravenous, intramuscular, or subcutaneous injection. this is because antibodies can't be delivered orally because of breakdown in the stomach by acids or other factors found there. various encapsulation methodologies are required in order to overcome this basic issue, and these methods should be forthcoming. once injected, most therapeutic mabs, like natural antibodies, have a long half-life (about days). the absolute half-life for each is unique, depending upon its concentration, distribution of its target, and, if the mab is directed to a cellsurface receptor, clearance and elimination of the target receptor. therapeutic mabs have two main safety advantages: ( ) they have very specific targets, and ( ) they don't have intracellular activity. as a result, there are few anticipated side effects and reactions although they can occur (catapanoab and papadopoulosc ) . mabs are eventually eliminated via intracellular catabolism in the lysosome, where they are broken down into peptides or amino acids that can be either reused for synthesis of new proteins or excreted via the urine. concluding remarks and future directions antibodies for veterinary use have great potential for the future. passive antibody therapy in the treatment of infectious diseases is a concept which dates back more than years, to the s, when the use of serum from immunized animals provided the first effective treatment options against infections with clostridium tetani and corynebacterium diphtheriae (hey ) . however, due to the discovery of penicillin by fleming in , and the subsequent introduction of the much cheaper and safer antibiotics in the s, serum therapy was largely abandoned. but in more recent times, the broad and general use of antibiotics in human and veterinary medicine has resulted in the development of multiresistant strains of bacteria with limited or no response to existing treatments and thus a need for alternative treatment options. this situation can be partially attributed to the overuse of antibiotics as growth promoters for production animals and other indiscriminate use. the combined specificity and flexibility of antibody-based treatments in providing passive immunity makes them very valuable tools for designing specific antibody treatments to infectious agents. these attributes have already caused a revolution in new antibody-based treatments in oncology and inflammatory diseases, with many approved products for human use. however, only very few mabs are approved for veterinary use. mabs therapies are expensive, and this has been a barrier for their development in the presence of inexpensive antibiotics. the use of antibiotics as growth promoters came to an end in in the usa with the rest of the world already limiting their use for this purpose manner (fda ) . this opens the door to new technologies and antibodies from monoclonal sources, chicken eggs, cow colostrum, or other sources to be among the chief contenders for limiting diseases in a safe manner and potentially to act as growth promoters. for that purpose, antibodies and antibodyderived treatments offer very attractive tools and attributes to neutralize infectious agents or modulate the immune system to enable effector cells to escape immunosuppressed conditions and contribute to the elimination of infections. the ability to raise antibodies to any target, and the ability to modulate effector functions, half-life, and size of the treatment units, makes antibodies ideal for tailoring treatments for specific infectious agents. however, more research into the use of antibodies as growth promoters will need to occur. one area that researchers are looking to make better use of antibody treatments for veterinary use includes more use of mabs. it has been predicted that minimal amino acid changes are needed to adapt an antibody from one species to another to avoid immune rejection. using libraries of genetic information and algorithms to make sure that key amino acid sequences are recognized as "self" or "native" by the target species' immune system can reduce the chance of undesirable immune reactions. this will increase the advantages typical of mabs for potency, safety, and a prolonged elimination half-life. therefore, these second-and third-generation mabs will be at the forefront of veterinary antibody technology along with igy technology. molecular targets for therapeutic mabs, igy, and colostrum igg in animals should ( ) be involved in clinical signs or disease mechanism and ( ) not have redundant pathways compensating for blockade of the intended target. the validity of blocking a molecule or eliminating a cell type must also be weighed against the importance of this protein or cell for desirable normal body functions. it is possible to speculate on uses of these antibodies in companion animals. these might include immune-mediated hemolytic anemias/ thrombopenias, myasthenia gravis, and autoimmune blistering diseases such as pemphigus, among other conditions. for cancer, the use of mab or igy therapy targeting b-lymphocytes will be valuable for b-cell lymphomas in dogs and cats. in allergic diseases, the use of mabs to inhibit production of ige via its promoting cytokines such as interleukins il- /il- , their cytokine receptors, or ige itself might be beneficial in dogs and cats with ige-mediated atopic dermatitis or food allergies. the itch sensation itself could be altered, at least theoretically, by antibodies targeting itch-promoting cytokines such as il- , nerve growth factor, thymic stromal lymphopoietin, or neuromediators involved in itch transmission. in arthritis therapeutic mabs that inhibit pro-inflammatory cytokines (tnf-alpha, il , etc.) or their receptors are likely to be of benefit in treating dogs and cats with arthritis. the usefulness of anti-ngf mabs as an analgesic must be confirmed. in autoimmune diseases, the use of mabs specific for b-lymphocyte surface proteins could theoretically lead to reduced production of autoantibodies. veterinary vaccines have had, and continue to have, a major impact not only on animal health and production but also on human health, through increasing safe food supplies and preventing animal-to-human transmission of infectious diseases. the continued interaction between animals and human researchers and health professionals will be of major importance for adapting new technologies, providing animal models of disease, and confronting new and emerging infectious diseases. one area of research where more information is needed is on factors that limit efficacy of vaccination ifoma, particularly in calves < month old. this is a complex issue. however, research continues to evolve in the area of newborn calf vaccinations. passive immunity provided by chicken egg antibodies will gain increasing use in production animals. there is no doubt that chicken abs can be produced and used, with minor modifications, in similar ways to mammalian abs. it can also be said that, depending on the circumstances, the use of igy abs often has significant advantage over the use of mammalian abs. however, from a realistic point of view, igy abs probably will not be able to completely replace the use of igg abs in diagnostic systems in the near future (schade et al. ) . chickens have the potential to be used to complete the spectrum of animals that have been used for ab production. however, a prerequisite is to make igy technology more popular and to convince the scientific community of its significant advantages. an interesting possibility for the future is the production of chicken mabs. these would combine the advantages of mabs with the advantages of chicken abs. it is to be expected that studies on the therapeutic or prophylactic use of igy abs will be intensified in the future. in particular, because of the increasing resistance of microorganisms to antibiotics, research on all aspects related to the development of specific igy against pathogenic microorganisms will have to be intensified. in conclusion, the next decade will see continued development of therapeutic mabs, igys, and colostrum antibodies for production and companion animals in both treatment and prevention. these highly specific molecules are likely to prove beneficial to uniquely target disease mechanisms without the side effects associated with broad-spectrum pharmacotherapy. while vaccines will continue to play a very important role in maintaining the health of animals by active immunity, antibodies that provide passive immunity will be an increasing part of the arsenal available to veterinarians to promote growth in production animals in a safe manner and to maintain health in companion animals. vaccinations for the swine herd bioactive egg components and inflammation first antibody therapy in veterinary medicine launched for dogs in the uk the mucosal immune system and its integration with the mammary glands treatment of canine b-cell lymphoma with doxorubicin with or without an anti-cd monoclonal antibody: an open-label pilot study evaluation of hyperimmune hen egg yolk derived anti-human rotavirus antibodies (antihrvigy) against rotavirus infection why do we need igm memory b cells? the safety of therapeutic monoclonal antibodies: implications for cardiovascular disease and targeting the pcsk pathway comparison of levels and duration of detection of antibodies to bovine viral diarrhea virus , bovine viral diarrhea virus , bovine respiratory syncytial virus, bovine herpesvirus , and bovine parainfluenza virus in calves fed maternal colostrum or a colostrum-replacement product vaccination of calves against common respiratory viruses in the face of maternally derived antibodies (ifoma) evaluation of methods for dehydration of bovine colostrum for total replacement of normal colostrum in calves the function and regulation of immunoglobulin d evaluation of factors affecting serum igg concentrations in bottle-fed calves vaccination of cows with clostridial antigens and passive transfer of clostridial antibodies from bovine colostrum to lambs protection of neonatal calves against k -e. coli and corona virus using a colostrumderived immunoglobulin preparation conjugated linoleic acid enhances immune responses but protects against the collateral damage of immune events orally administered anti-escherichia coli o : h chicken egg yolk antibodies reduce fecal shedding of the pathogen by ruminants study and application of the hyperimmunized yolk antibodies against tgev and pedv in piglets influence of colostrum intake on piglet survival and immunity effect of chicken egg yolk antibodies (igy) against diarrhea in domesticated animals: a systematic review and meta-analysis stabilized liquid egg material for extended shelf life. united states patent evaluation of anti-il- monoclonal antibodies in a model of il- -induced pruritus in beagle dogs comparison between immunoglobulins igy and the vaccine for prevention of infectious bursal disease in chickens committee for medicinal products for veterinary use (cvmp). questions and answers on monoclonal antibodies for veterinary use european medicines agency allergic disorders and ige-mediated immune responses: where do we stand? intestinal iga synthesis: regulation of front-line body defenses passive immunization in infectious bursal disease virus infected birds using chemically purified immune yolk immunoglobulins (igy) efficacy of hyperimmune bovine colostrum for prophylaxis of cryptosporidiosis in neonatal calves enhanced bovine colostrum supplementation shortens the duration of respiratory disease in thoroughbred yearlings food and drug administration ( ) food and drug administration center for veterinary medicine veterinary feed directive guidance for industry # american animal hospital association canine vaccination guidelines a fully caninised anti-ngf monoclonal antibody for pain relief in dogs field evaluation of egg yolk antibodies in prevention and treatment of enteric colibacillosis in calves immunology: fifty years of b lymphocytes heat-treated colostrum and reduced morbidity in preweaned dairy calves: results of a randomized trial and examination of mechanisms of effectiveness the biology of ige and the basis of allergic disease hyperimmune bovine colostrum treatment of moribund leopard geckos (eublepharis macularius) infected with cryptosporidium spp risk factors associated with colostrum quality in norwegian dairy cows maternal antibody transfer from dams to their egg yolks, egg whites, and chicks in meat lines of chickens history and practice: antibodies in infectious diseases mucosal immunity and vaccines perspectives on immunoglobulins in colostrum and milk development and function of organized gut-associated lymphoid tissues immunomodulatory constituents of human breast milk and immunity from bronchiolitis survey of immunoglobulin g content and antibody specificity in cow's milk from british columbia structural, antigenic and evolutionary analyses of immunoglobulins and t cell receptors chapter -passive immunization: toward magic bullets expression of the neonatal fc receptor (fcrn) in the bovine mammary gland current status of veterinary vaccines advances in prevention and therapy of neonatal dairy calf diarrhoea: a systematical review with emphasis on colostrum management and fluid therapy programs for the cow/calf operation nation-wide evaluation of quality and composition of colostrum fed to dairy calves in the united states allergens in veterinary medicine treatment of experimental ovine cryptosporidiosis with ovine or bovine hyperimmune colostrum man the barrier! strategic defences in the intestinal mucosa maternal antibodies: clinical significance, mechanism of interference with immune responses, and possible vaccination strategies treatment of canine b-cell lymphoma with chemotherapy and a canine anti-cd monoclonal antibody: a prospective double-blind, randomized, placebocontrolled study growth factors and antimicrobial factors of bovine colostrum colostrum and replacer ige production in rat fascioliasis sheep antiserum as an antibody supplement in newborn lambs structural and functional properties of membrane and secreted igd nutrient and immunity transfer from cow to calf pre-and post-calving chicken egg yolk antibodies (igy-technology): a review of progress in production and use in research and human and veterinary medicine passive immunotherapy in neonatal calves -i. safety and potency of a j escherichia coli hyperimmune plasma in neonatal calves passive protection against porcine epidemic diarrhea (ped) virus in piglets by colostrum from immunized cows the iga system: a comparison of structure and function in different species vaccination programs in poultry clinical outcome of calves with failure of passive transfer as diagnosed by a commercially available igg quick test kit spray dried, pasteurized bovine colostrum protects against gut dysfunction and inflammation in preterm pigs immune functions of immunoglobulin y isolated from egg yolk of hens immunized with various infectious bacteria precalving vaccination programs for cows passive immunity in ontario dairy calves and investigation of its association with calf management practices egg yolk igy: protection against rotavirus induced diarrhea and modulatory effect on the systemic and mucosal antibody responses in newborn calves igy antibodies protect against human rotavirus induced diarrhea in the neonatal gnotobiotic piglet disease model allergy and the gastrointestinal system ige: a question of protective immunity in trichinella spiralis infection canine brief pain inventory scores for dogs with osteoarthritis before and after administration of a monoclonal antibody against nerve growth factor comparison of antibody production to human interleukin- (il- ) by sheep and chickens vaccinating calves: new information on the effects of maternal iimmunity antibody to human rotavirus in cow's milk key: cord- -kdeynhzq authors: opgenorth, julie; sordillo, lorraine m.; lock, adam l.; gandy, jeff c.; vandehaar, michael j. title: colostrum supplementation with n- fatty acids alters plasma polyunsaturated fatty acids and inflammatory mediators in newborn calves date: - - journal: j dairy sci doi: . /jds. - sha: doc_id: cord_uid: kdeynhzq calves may experience increased oxidative stress at birth through activation of metabolic and respiratory processes. reducing oxidative stress may enhance calf viability in early life. our objective was to determine the dose response to fish and flaxseed oil when supplemented in colostrum on concentrations of plasma fatty acid (fa), fa metabolites, and index of oxidative stress during the critical first week of life in calves to understand how supplementing n- fa may decrease oxidative stress. we hypothesized that n- fa supplemented in colostrum in a linear dose-dependent fashion would associate with increased plasma n- fa concentrations and decreased oxidative stress. twenty-four male and female holstein calves were randomly assigned to receive , , , or ml of a : fish to flaxseed oil supplement in colostrum. all calves received . l of previously frozen colostrum (≥ % brix) with their respective treatment within h after birth. blood was sampled before first feeding after birth and on d , , , , and d of age to assess oxidant status and plasma free pufa, phospholipid fa, and oxylipid concentrations. health indicators were observed daily. indicators of general health and growth were unaffected by treatment. supplemented calves exhibited greater concentrations of n- fa in plasma as free and phospholipid fa and some n- and n- fa-derived oxylipids in the first week of life in a linear fashion with increasing supplemental dose. fish and flaxseed oil treatments did not alter oxidant status but overall decreased isoprostane concentrations in plasma, indicating oxidative stress was decreased. together, these responses indicate that the fish and flaxseed oil supplement was antiinflammatory. in conclusion, supplementing colostrum with , , and ml of a : mixture of fish and flaxseed oil linearly increased plasma concentrations of n- fa and metabolites and decreased biomarkers of oxidative stress, but did not alter oxidant status or affect health or growth. our findings suggest neonatal calves may benefit from n- fa supplementation in colostrum to encourage a greater antiinflammatory state. parturition and the associated physiological changes that accompany it are stressful to the dam and calf (ling et al., ) , contributing to an increased risk of immune dysfunction and oxidative stress. though acute inflammation aids normal parturition (hansen et al., ) , unresolved inflammation and oxidative stress can lead to increased disease incidence in the cow (bradford et al., ) and decreased calf vitality (abuelo et al., ) . oxidative stress occurs in the neonate just after birth as oxygen concentrations entering tissues increase rapidly, resulting in production of reactive oxygen and nitrogen species (rons; frank and sosenko, ) . the overproduction of rons and associated free radicals may prove toxic to newborn tissues if antioxidant defense mechanisms are overwhelmed (buonocore et al., ) . additionally, dystocia can decrease neonatal plasma concentrations of vitamins with antioxidant properties such as vitamins a and c and β-carotene, along with a concurrent increase in cortisol (civelek et al., ) . these changes further affect the balance between free radical production and the capacity of antioxidants to mitigate them. supplementation of calves with fish or flaxseed oils, both of which are enriched for n- fatty acids (fa), seems to improve health and growth of milk-fed calves (ballou and depeters, ; hill et al., ; karcher et al., ) . ballou and depeters ( ) found that fish oil reduces signs of disease and the acute phase response during an endotoxemic challenge. karcher et al. ( ) compared fish and flaxseed oils and found both oils decrease inflammatory cytokine gene expression, but flaxseed oil enhances feed efficiency in early life and reduces fever after vaccination. hill et al. ( ) fed a diversified fat supplement containing flaxseed oil to improve feed efficiency and decrease disease incidence. in each of these studies, calves received their n- fa supplement as part of their milk replacer feeding program, starting several days after birth. however, the best time to supplement n- fa might be at birth when oxidative stress is likely to occur. compared with milk at mo after parturition, colostrum collected within the first h has more fat ( . vs. . %) with a smaller n - : n - fa ratio ( vs. ; contarini et al., ) . provision of n- fa to cows before calving does little to increase n- fa concentrations in blood of calves (moallem and zachut, ) . thus, supplementation of n- fa in colostrum might be a reasonable approach. we recently found that supplementing calves with a ml of : mixture of fish: flaxseed oil along with mg of α-tocopherol in colostrum decreased oxidant status index (osi) during the first week of life . oxidant status is the ratio of the concentrations of rons to antioxidants, so a decreased oxidant status indicates that supplemented calves had less oxidative stress. our supplement contained n- fa and α-tocopherol, which both have antioxidant properties, so whether the benefit of our treatment was due to n- fa-enriched oils, α-tocopherol, or both was not known. because calves on both treatments received α-tocopherol, albeit in different dosages, we postulated that the major benefit was from the fish and flax oils. in the current study, we supplemented calves with n- fa without additional α-tocopherol. in addition, to examine possible mechanisms, we measured blood concentrations of inflammatory markers and oxylipids. oxylipids, which are produced from enzymatic oxidation of n- fa, promote resolution of inflammation and repair of oxidative tissue damage (raphael and sordillo, ) . therefore, the objective of our current study was to determine effects of doses of a : blend of fish and flaxseed oils (without extra α-tocopherol) on plasma fa composition and osi, on the concentration of several oxylipids and inflammatory markers in blood, and on health and growth. we hypothesized that an n- fa supplement would increase plasma n- fa concentrations in a linear, dose-dependent manner, leading to an increase in the biosynthesis of antiinflammatory n- fa-derived oxylipids and decreased oxidative stress. the study was conducted from june to july of at the michigan state university (msu) dairy teaching and research center (east lansing) and was ap-proved by the msu animal care and use committee (approval no. / - - ). calves were born between june and july and housed in outdoor hutches under shade. throughout the course of the study, outside temperature averaged °c and the minimum and maximum temperatures were °c and °c, respectively. twenty-four holstein ( male, female) calves were assigned to of treatment groups in a randomized block design by sex. treatments followed a pattern of varying amounts of a : mixture of fish: flaxseed oil, which also included polysorbate- at . ml/ml of oils to emulsify the oils and enhance absorption. treatments were assigned as follows: ( ) control: no supplement in colostrum (con), ( ) ml of : fish: flaxseed oil blend in colostrum (ff ), ( ) ml of : fish: flaxseed oil blend in colostrum (ff ), and ( ) ml of : fish: flaxseed oil blend in colostrum (ff ). colostrum from cows in the msu herd was frozen in pouches of . l for later use if it scored ≥ % on the brix scale (indicating ≥ g/l immunoglobulins: bielmann et al., ) . each calf received a randomly assigned pouch. the pouches were thawed and sampled for immunoglobulin concentration. the supplement for treatment calves was then added to . l of colostrum, but control calves did not receive any supplement. calves were fed colostrum within h after birth (average was . h); if a calf did not consume by suckling, the colostrum was delivered by intragastric tube. after birth, all calves were removed from the dam immediately and received a bovine rotavirus and coronavirus vaccine and intramuscular supplements of ml of vitamin a and d vetone, equivalent to kiu of vitamin a and kiu of vitamin d (mwi animal health, boise, id) and ml bo-se (merck animal health, madison, nj), which contains mg of α-tocopherol and mg of selenium per ml. a second colostrum feeding of . l was given to h after birth, but no supplement was added to the second feeding for any calves. calves were housed outside after their first day of life in individual calf hutches bedded with sand and were isolated to prevent physical contact with other calves. they were provided with ad libitum access to water and calf starter grain (ampli-calf as starter p r , % protein; land o'lakes, arden hills, mn) and fed daily with milk replacer (cow's match warmfront milk replacer, % protein and % fat; land o'lakes) at approximately , , and h. each liter of milk replacer contained % solids. milk replacer was given times per day at , , and h. age to wk calves received . l at the morning and opgenorth et al.: colostrum supplementation and oxylipid profiles afternoon feeding and . l in the evening. calves to wk of age received . l at the morning and afternoon feeding and . l in the evening. at to wk, calves received . l in the morning only. health was scored for wk after birth. medication, milk replacer refusal feedings, and disease incidence were recorded throughout the preweaning period. we measured bw with a calibrated calf cart scale (digi-star, fort atkinson, wi) and wither height and heart girth at birth, wk of age, and weaning. health was assessed daily for wk after birth by trained researchers blind to treatment before the am milk feeding. health scores for feces, eyes, nose, and ears were based on methods by mcguirk, university of wisconsin, on a to scale where scoring was described as fecal: = normal to = watery, discolored, or bloody; eyes: = no discharge to = excessive discharge; nose: = normal discharge to = excessive discharge; ear: = normal ear alertness to = tilted head or blatant ear droop (university of wisconsin madison, ). twenty microliters of an antioxidant-reducing agent of % methanol, % ethanol, and % water with . mm of butylated hydroxytoluene, . mm edta, . mm triphenylphosphine, and . mm indomethacin, as described in kuhn et al. ( ) , was added to µl of thawed colostrum. samples underwent lipid hydrolysis via the addition of µl of koh and incubating for min at °c. once samples cooled to room temperature, they were centrifuged at , × g for min at °c. the hcl at m was added to the removed supernatant in increments of µl until the supernatant ph was decreased to or less. a mixture of internal standards of µl was added to each sample mixture as well, consisting of . µm (s)-hydroxyeicosatetraenoic-d , . µm ( )-epoxyeicosatrienoic acid-d , . µm prostaglandin e -d , and . µm , -dihydroxyeicosatrienoic acid-d . samples underwent solid-phase extraction with oasis hlb cc lp extraction columns (waters, milford, ma) via a biotage extrahera (biotage, charlotte, nc), further described in putman et al. ( ) . samples were then dried in a savant speedvac (thermo fisher scientific, waltham, ma) and reconstituted in . : methanol: hplc water. after filtration, samples were placed in glass vials with inserts and stored at − °c until liquid chromatography/ms analysis. blood was collected from the jugular vein of calves on d (before first colostrum feeding), ( ± h), ( ± h), (± d), (± d), and (± d) after birth. analysis of d samples were used as a covariate in the statistical model, but if not significant, were not included in results. serum was harvested to determine serum total protein using a digital brix refractometer and was sent to saskatoon colostrum company for analysis of immunoglobulins (saskatoon, sk, canada). serum was harvested and immediately flash frozen in liquid nitrogen and placed in dry ice during transit to a − °c freezer for later analysis of osi. plasma was harvested immediately and stored at − °c until analysis of fa phospholipid profiles. another aliquot of plasma was immediately flash frozen with liquid nitrogen and stored at − °c for future analysis of oxylipid and free pufa concentrations. the concentrations of rons and of antioxidants in serum were measured in the same sample concurrently to calculate osi. the rons were measured using the cell biolabs inc. oxiselect in vitro ros/rns assay kit (cell biolabs inc., san diego, ca). free radicals in samples convert a dichlorodihydrofluorescein fluorogenic probe to ', '-dichlorodihydrofluorescein to detect fluorescence intensity to quantify total free radical concentration as described previously (putman et al., ) . antioxidant potential (aop) of the same samples was quantified through the units of trolox equivalence, a synthetic analog of α-tocopherol, where values of aop were compared with a photometric plate reader. , '-azino-bis- -ethylbenzothiazoline- -sulfonic acid was used as a radical cation to be reduced by antioxidants as described previously (putman et al., ) . the reduction potential of each sample, compared as trolox equivalence, was compared by a photometric plate reader. this decolorization assay is further described in re et al. ( ) . sample preparation. extraction and analysis of plasma for analysis of free pufa, oxylipid, and isoprostane concentrations followed methods modified from mavangira et al. ( ) . in brief, ml of plasma was thawed on ice and ml % formic acid and µl/ ml of an antioxidant-reducing agent to protect samples from lipid peroxidation during processing (o'donnell et al., ) was added to plasma. the antioxidantreducing agent was % methanol, % ethanol, and % water with . mm butylated hydroxytoluene, . mm edta, . mm triphenylphosphine, and . mm indomethacin, as described in kuhn et al. ( ) . a mixture of internal standards of µl was added to each sample mixture as well, consisting of . µm (s)-hydroxyeicosatetraenoic acid-d , . µm (s)-hydroxyeicosatetraenoic acid-d , . µm ( )-epoxyeicosatrienoic acid-d , . µm prostaglandin e -d , and . µm , -dihydroxyeicosatrienoic acid-d . waters oasis prime hlb cc solid-phase extraction columns were used for solid phase extraction performed by biotage extrahera (biotage). after samples were loaded onto columns and excess infranatant was discarded with nitrogen, columns were washed with ml of % methanol and then . ml of : acetonitrile: methanol eluted samples. leftover solvents were evaporated with a savant speedvac. a mixture of . : methanol: hplc water brought the fa back into solution before filtering and dispensing into glass chromatography vials with inserts. quantification of oxylipids. in short, the quantification of metabolites was accomplished on a waters xevo-tq-s tandem quadrupole mass spectrometer using multiple reaction monitoring. chromatography separation was performed with an ascentis express c hplc column (sigma-aldrich, st. louis, mo), held at °c and autosampler held at °c. mobile phase bottle a was water containing . % formic acid and mobile phase bottle b was acetonitrile; the flow rate was . ml/min. liquid chromatography separation took min per sample with linear gradient steps programmed as follows (a: b ratio): time to . min ( : ), to ( : ) at . min; to ( : ) at . min; to ( : ) at . min; . min held at ( : ) until min . ; then return to ( : ) at . min, and held at this condition until . min. all oxylipids were detected using electrospray ionization in negative-ion mode. cone voltages and collision voltages were optimized for each analyte using waters quanoptimize software and data analysis was carried out with waters masslynx software. quantification of isoprostanes. quantification of isoprostanes was accomplished with a waters xevo tq-s tandem quadrupole mass spectrometer using multiple reaction monitoring. chromatography separation was performed with a waters acquity uplc utilizing a beh c . µm ( . × mm) column, held at °c, and autosampler held at °c. mobile phase bottle a was . % acetic acid and mobile phase bottle b was acetonitrile, mobile phase bottle c was methanol, and the flow rate was . ml/min. the gradient initial phase a:b, : to min changing to a:b:c, : : , to min changing to a:b:c, : : , to . changing back to initial phase and holding until min. all oxylipids were detected using electrospray ionization in negative-ion mode. cone voltages and collision voltages were optimized for each analyte using waters quanoptimize software and data analysis was carried out with waters masslynx software. quantification of free polyunsaturated fatty acids. briefly, reverse-phase liquid chromatography/ ms on a waters acquity uplc employing a beh c . µm ( . × mm) column with a flow rate of . ml/min at °c was utilized. the quadrupole ms was in electrospray negative ionization mode and voltage was − kv with the turbo ion spray source temperature at °c. the gradient mobile phase was programmed in the following manner (a/b/d ratio): time to . min ( / / ), to ( / / ) at . min, to ( / / ) at . min, to ( / / ) at . min, and held until . min, then return to ( / / ) at . min, and held at this condition until . min. in this gradient mobile phase a = acetonitrile, b = methanol, and d = . % formic acid. fatty acids were quantified by matching mass- and retention time with corresponding deuterated internal standard abundance and calibrated to a linear -point standard curve (r > . ) using waters empower software. phospholipids were analyzed using methods adapted from folch et al. ( ) and kramer et al. ( ) . in brief, total lipids were extracted from an aliquot of ml of plasma by shaking samples in ml of methanol and ml of chloroform. a solution of % sodium chloride in water was then added. the chloroform layer with lipids was filtered out, dried, and weighed for total lipid content. next, the phospholipid fractions were isolated using solid phase extraction. columns were initially washed with . ml of : acetone: water and eluted with ml of hexane before loading lipid samples dissolved in . ml of hexane: methyl tertbutyl ether: acetic acid ( : : . ). cholesterol esters were first eluted with ml of hexane, and free fa eluted with ml of hexane: chloroform: ethyl acetate ( : : ). columns were washed with ml of : chloroform: isopropanol. next, the triglyceride fraction was collected via ml of chloroform: methanol: acetic acid ( : : ), and phospholipids eluted via ml of methanol: chloroform: water ( : : ). the phospholipid fraction was then washed with ml of % sodium chloride in water and phospholipids in the chloroform layer were collected. only the phospholipid fraction was analyzed in the interest of relevance to experiment objectives. after phospholipid content was calculated, . ml of the internal standard, c : -heptadecenoic acid in toluene, was added. phospholipids were then methylated into fame as previously described (lock et al., ) . hexane reconstituted fame in solvent to produce a % solution for glc analysis with a gc- plus gas chromatograph (shimadzu, kyoto, japan). conditions for glc analysis are further described in lock et al., ( ) . plasma phospholipid fa concentrations were calculated as a percentage of total phospholipids recovered from plasma in g/ g. all values were calculated with average response factor of from external standards. due to sample number constraints per analysis, samples collected d , , , and were analyzed separately from d and and were run consistently with randomized blocks. data were analyzed using sas version . (sas institute inc., cary, nc) with a mixed procedure. fixed effects were sex, treatment, and day. random effects were block within sex and calf within block, sex, and treatment. contrast coefficients were assigned for each treatment to test linear, quadratic, and cubic effects as follows: linear − , − , , and ; quadratic , − , − , and ; cubic − , , − , and for con, ff , ff , and ff , respectively. when analyzing statistics for bw and adg up to weaning, the variable week replaced day. average daily gain for the first wk of life, health scores, and d , , , and of blood variables were analyzed with the mixed model. blood variables from d were included in figures but not in statistical analysis, as we expected most variables to return to baseline by d . all p-values or data expressed in tables are results from analyzing d , , , and after birth only. after birth and before the first colostrum feeding, blood was sampled and analyzed to use as a potential covariate in the model. if the covariate was not significant and did not correlate with day, it was not included in the model. assays were conducted by block. the order on the well plate of the rons concentration assay significantly affected resulting values, and therefore, was included in the model for osi analysis as order within block. effect of block was confounded with order. treatment was not confounded with order, as treatments were random with respect to order of wells. preweaning adg, general health observations, colostrum immunoglobulin count, and brix measures were analyzed with the general linear model anova procedure. normality was assumed if a variable's general linear model procedure's bartlett homogeneity of variance test indicated p > . . data were log-transformed if p ≤ . and transformed least squares means were backtransformed from the model for interpretation in tables and figures. differences were considered significant if p ≤ . and a tendency if . < p ≤ . . differences were considered significant if interaction p ≤ . and a tendency if . < p ≤ . . during the first wk of life, calves were treated for diarrhea. calves typically showed initial symptoms of diarrhea to d after birth. across treatment groups, the average number of medication doses for diarrhea per calf was . during the first wk of life, and differences between treatments were not significant (p = . ). if a calf did not complete a meal of milk replacer, it was recorded as one refusal. the average number of refusals per calf was . during the first wk and was similar across treatment groups (p = . ). one calf died at d of age, presumably from heat stress; all available data for this calf were included in the data set. average health scores over the first wk of life were . , . , . , and . for fecal, eye, nasal, and ear scores, respectively, and were not altered by treatments (p > . ). despite the fact that calves were given treatments by farm staff blinded to treatment, the time of feeding first colostrum was . , . , . , and . h for the con, ff , ff , and ff groups, respectively, and the overall treatment effect was significant (p = . ). however, the concentrations of immunoglobulins and total protein in serum in calves in the first week were not altered by treatment and were > and ≥ g/l, respectively, indicating all calves received colostrum of acceptable quality in a timely fashion for antibody absorption in the bloodstream (calloway et al., ; godden, ) . treatment did not alter serum total protein (mean of g/l) or immunoglobulin concentration (mean of g/l) during the first week of life (p > . for overall treatment effect for both variables). mean colostrum n - : n - fa ratios before addition of supplements for con, ff , ff , and ff were . , . , . , and . , respectively, and did not differ across treatment groups. treatment did not alter growth rates in the first wk of life or during the preweaning period (p > . ). average wither height, heart girth, and bw gain during the first wk were . cm/d, . cm/d, and . kg/d and were not altered by treatment (p > . ). treatment also did not alter bw gain through the entire preweaning period ( . , . , . , and . kg/d for con, ff , ff , and ff , respectively; p = . ). fish and flaxseed oil (ff) treatments increased free concentrations of the n- fa α-linolenic acid (ala), eicosapentaenoic acid (epa), docosahexaenoic acid (dha), and docosapentaenoic acid in plasma on d , , , and after birth in a linear fashion (table , p < . ). this corresponded well with the fa found in fish and flaxseed oil. dha was significantly increased in plasma free pufa concentrations with ff treatments where con, ff , ff , and ff averaged , , , and nm dha (p < . ) during the first week after supplementation. likewise, epa also increased with increasing ff supplement volume ( . , . , . , and . nm, respectively; p < . ). the ala significantly differed in concentration among con and ff treatment calves during the first week of life ( , , , and nm, respectively; p < . ) as well. all primary n- fa concentrations were linearly increased by ff treatments (p ≤ . ) on d , , and after birth. by d after birth, concentrations either returned to normal or tended to be elevated (p ≥ . ) with ff, and by d , n- fa concentrations all returned to baseline (figure ) . linoleic acid constitutes % of flaxseed oil and is a precursor to arachidonic acid (ara). plasma concentrations of free linoleic acid averaged ~ , nm in calves given ff treatments, compared with , nm for con, but these were not different. additionally, treatments did not alter concentrations of free ara in plasma (mean nm, p = . for treatments). the concentration of n- fa remained constant for all calves, whereas primary n- fa linearly increased in ff treatments. thus, during the first week of life, the ratio of free n - : n - fa in plasma was decreased with ff in a linear fashion (p = . ; , , , and for con, ff , ff , and ff , respectively). the linear trend of increased free n- fa and decreased free n - : n - fa ratio was also associated with similar changes in the plasma phospholipid fa fraction (supplemental table s , https: / / doi .org/ . / jds . - ). as expected, supplementing ff increased (p < . ) the concentrations of ala, epa, and dha in the first week of life ( figure ; table ). the dha increased linearly (p < . ) with increasing ff dose, whereas ala and epa increased quadratically (p < . and p = . , respectively). treatment did not alter the content of linoleic acid and ara in phospholipids (both p > . ), but linoleic acid had a quadratic effect (p = . ). the content of total n- fa and total pufa in phospholipids increased with ff supplementation in linear and quadratic fashions (p = . ). however, the concentration of total n- fa did not change with treatments, although a quadratic upward trend was observed (p = . ). the ratio of n- to n- fa in plasma phospholipid decreased as calves were fed more ff ( . , . , . , and . ) in a linear fashion (p < . ). the ff treatments linearly decreased monounsaturated plasma phospholipid fa and quadratically altered sfa (p = . , p = . , respectively). for all phospholipid pufa that were altered by treatment, the concentration in phospholipids decreased over time in calves fed ff treatments (p ≤ . for day; figure ). treatments (n = ): con = control, no supplement added to colostrum; ff = ml of a : ratio fish and flaxseed oil blend added to colostrum; ff = ml of a : ratio fish and flaxseed oil blend added to colostrum; ff = ml of a : ratio fish and flaxseed oil blend added to colostrum. l = linear polynomial contrast. q = quadratic polynomial contrast. c = cubic polynomial contrast. overall p-value = treatment effect p-value. average concentrations of oxylipids and isoprostanes for each treatment during wk are reported in supplemental tables s and s , respectively (https: / / doi .org/ . / jds . - ). concentrations of oxylipids and isoprostane in plasma were altered by ff treatments (figure ). calves given ff treatments had linearly increased (p < . ) epa-derived , -dihydroxy-eicosatetraenoic acid ( , -dihete) and quadratically increased , -dihete (p = . ) and , -dihete (p = . ) with supplement dose. the ff treatments increased dha-derived , -dihydroxy-docosapentaenoic acid ( , -dihdpa) by , , and % of con, respectively (p = . in quadratic fashion). the n- -derived leukotriene b (ltb ) increased with ff treatments , , and % of con (p = . in quadratic fashion), and -iso-prostaglandin-a decreased by , , and % of con (p = . overall and p = . cubic). the , and total n - : total n- fa ratio (d) predicted from the model. the ala, epa, and dha are backtransformed for interpretation with adjusted se. during wk , fish and flaxseed oil treatments increased ala, epa, and dha and decreased the n - : n - fa ratio (p < . ). treatments (n = ): con = control, no supplement added to colostrum; ff = ml of a : ratio fish and flaxseed oil blend added to colostrum; ff = ml of a : ratio fish and flaxseed oil blend added to colostrum; ff = ml of a : ratio fish and flaxseed oil blend added to colostrum. , -lipoxin a (lxa ) increased (p = . in linear fashion) with ff treatments by , , and % of con. three enzymatic complexes produce the n- and n- fa-derived oxylipids analyzed in this experiment. oxylipids produced from cytochrome p epoxygenase (cyp) and lipoxygenase (lox) were primarily affected by supplementation. no oxylipids analyzed that derived from cyclooxygenase (cox) pathways were altered in concentration by ff treatments. the ff treatment did not decrease osi during the first week of life (p = . ). the rons concentrations and aop remained constant across treatment groups (p = . and p = . , respectively). least squares means of indicators of oxidative stress, including -isoprostaglandin-a , are shown as treatment by sampling day in figure . increasing doses of , , and ml of : fish: flaxseed oil blend supplemented in colostrum did not affect health or growth in calves, decreased phospholipid n- fa: n - fa profile, increased free and phospholipid n- fa concentrations, and increased several oxylipids deriving from n- fa during the first week of life. though osi was unaltered by ff treatments, concentrations of an n- fa-derived isoprostane, -iso-pga , were reduced, indicating oxidative stress was decreased in ff calves the first week of life. our previous experiment, which supplemented ml of a : ratio fish: flaxseed oil blend with an additional mg of α-tocopherol , observed increased free ala, epa, and dha in plasma similar to the current study. we also analyzed phospholipid fa content, which is of particular interest because, once incorporated in phospholipid membranes, phospholipid pufa and esterified forms cleaved by phospholipase a become available for enzymatic oxidation. oxylipids become products of oxidation and are able to mediate inflammation and oxidative stress (raphael and sordillo, ) . with an increase in the n- fa in plasma phospholipids of ff calves, we observed an increase in some n- fa-derived oxylipid concentrations. raphael et al. ( ) proposed that manipulation of oxylipids may be feasible via dietary pufa, and we found that with one n- fa supplementation, several oxylipid concentrations were altered. though we observed oxylipids in plasma, contreras et al. ( ) similarly found that increasing n- fa in the phospholipid profile of endothelial cells leads to increased beneficial n- fa-derived oxylipid concentrations. they also observed a decrease in reactive oxygen species (contreras et al., ) , though we did not find a corresponding rons decrease in plasma in our experiment. oxylipids are products of pufa substrates of enzymes such as lox, cox, and cyp. these are cell signaling molecules able to mediate inflammation and its resolution through a variety of mechanisms. oxylipids that increased in concentration by ff , ff , and ff were primarily end products of metabolism of epa and dha. the , , and , , -dihdpa from dha increased linearly with increased n- fa supplementation. of the aforementioned oxylipids, all are end products of cyp enzymatic activity. the n- fa are more favorable substrates to lox and cyp enzymes when compared with n- fa substrates (zhang et al., ) , but cox favors n- fa (wada et al., ) . this may explain why no oxylipids formed from cox pathways were altered by increasing n- fa supplementation. dietary supplementation of linoleic acid causes an observed increase in cox activity (marchix et al., ) , and dha decreases cox expression (massaro opgenorth et al.: colostrum supplementation and oxylipid profiles figure . fish and flaxseed oil treatments depicted as a lsm percentage of control (con) ± sem for n- fatty acid (fa)-derived oxylipids (a) and n- fa-derived oxylipids (b) that differed from con concentrations during the first week of age. oxylipids were dihydroxy-eicosatetraenoic acid (dihete), dihydroxy-docosapentaenoic acid (dihdpa), lipoxin (lx), and leukotriene (lt). overall treatment p-values were as follows: , -dihete: p < . ; , -dihete: p < . ; , -dihete: p = . ; , -dihdpa: p = . ; , -lxa : p = . ; ltb : p = . . treatments (n = ): con = no supplement added to colostrum; ff = ml of a : ratio fish and flaxseed oil blend added to colostrum; ff = ml of a : ratio fish and flaxseed oil blend added to colostrum; ff = ml of a : ratio fish and flaxseed oil blend added to colostrum. et al., ) . though we did not analyze gene expression, it is possible ff treatments may decrease cox expression due to an increase in dha concentrations and a decrease in the ratio n - : n - fa. many painrelieving drugs such as aspirin or meloxicam function to inhibit cox activity (brune and patrignani, ) , which is the very reason they are considered antiinflammatory. the only cox-produced oxylipid that linearly decreased with increasing ff supplementation was thromboxane- , which has proinflammatory functions, though no overall difference (p = . ) in concentration among treatments was observed. thus, further analysis of cox gene expression may be warranted to provide further evidence of the antiinflammatory effects of ff treatments in neonatal calves. the oxylipids ltb and lxa also increased with ff supplementation. interestingly, ara, the source of these oxylipids, did not increase as plasma free pufa the rons, osi, and -iso-pga means are back-transformed from the model for interpretation with adjusted se. the fish and flaxseed oil treatments did not alter rons, aop, or osi, but did decrease -iso-pga (p = . ) during the first week of age. treatments (n = ): con = control, no supplement added to colostrum; ff = ml of a : ratio fish and flaxseed oil blend added to colostrum; ff = ml of a : ratio fish and flaxseed oil blend added to colostrum; ff = ml of a : ratio fish and flaxseed oil blend added to colostrum. rfu = relative fluorescent units; te = trolox equivalents; au = arbitrary units. or phospholipid pufa in ff-supplemented calves compared with con. the lxa is antiinflammatory and ltb has some known proinflammatory functions. the lxa functions to enhance epithelial cell wound healing in rodents (gronert et al., ) and initiate remodeling of phospholipids in humans (nigam et al., ) among many more actions (gabbs et al., ) . contrastingly, ltb helps instigate neutrophil chemotaxis in bovines (heidel et al., ) . our study is perhaps the first to describe oxylipid and isoprostane concentrations during the first week of life of any neonate mammalian species. oxylipid concentrations differ among cows in varying stages of lactation, and concentrations are not always necessarily indicative of physiological changes (kuhn et al., ; putman et al., ) . though calf oxylipid concentrations were found to be much smaller than adult cows (nm vs. µm), the periparturient stage of the dairy cow can yield some interesting parallels to neonatal calves. kuhn et al. ( ) suggested lxa is preferentially produced in periparturient cows, perhaps as a mechanism to reduce the systemic inflammatory state observed after parturition. we found supplementing calves with n- fa in their first meal tended to increase lxa concentrations, which is curious considering lxa is derived from ara. supplementation with n- fa in rainbow trout and female rats decreases lxa and other ara-derived lipoxygenase products (ashton et al., ; poulsen et al., ) , which was consistent with the idea that ara and n- fa compete for lipoxygenase binding (schmitz and ecker, ) . however, calves receive a greater n - : n - fa ratio in the diet, and supplementing n- fa in a short period of time in the current study is likely not enough to alter n- fa phospholipid content, and thus their oxylipid products, in all tissues. nevertheless, lxa concentrations tended to increase with increasing n- fa supplementation, indicating preferential production of this metabolite similar to what has been observed in periparturient cows (kuhn et al., ) during a time when reducing systemic inflammation is a key homeostatic goal. differing from oxylipids, isoprostanes are direct biomarkers of oxidative stress because their production results from rons-induced peroxidation and damage to the phospholipid membrane and thus cellular components (montuschi et al., ) . a decrease in isoprostane concentration indicates reduced lipid peroxidation and thus oxidative damage that compromises normal cellular functions (van 't erve, ). alternatively, osi, the concentration of rons to aop, is considered a measure of redox balance (kuhn et al., ) ; therefore, we speculate ff treatments decreased oxidative stress due to decreased -iso-pga in plasma, even though osi remained unchanged. some evidence suggests neonate immune processes gravitate toward a proinflammatory state at birth (braekke et al., ; boro et al., ) . the placenta favors prooxidant isoprostanes derived from n- fa pathways; in humans, -iso-prostaglandin-f α , a prominent and well-studied isoprostane known to indicate increased free radical concentrations, is highly concentrated in the umbilical vein (braekke et al., ) . placental production of this biomarker of oxidative stress may be regulated by hormones, though explanations as to why this phenomenon occurs are still unclear (hermenegildo et al., ) . the placenta may require a certain degree of inflammation to detach normally (boro et al., ) , and inflammation plays important and necessary roles in the calving process (bradford et al., ) . however, oxidative stress can affect calves well beyond the acute response at birth (abuelo et al., ) , and the additional supplementation of antioxidant and antiinflammatory nutrients after birth may be beneficial for resolving these pathways quickly to avoid decreased calf viability and a disadvantaged immune system. a : fish: flaxseed oil supplement in colostrum at , , and ml linearly increased plasma concentrations of n- fa and decreased n - : n - fa ratios in both free fa and phospholipid fa fractions. in addition, ff supplements decreased concentrations of isoprostane -iso-pga , a direct biomarker of lipid peroxidation during oxidative stress. however, ff supplements did not alter plasma oxidant status, health, or growth, regardless of the n- fa dose level. we conclude that supplementing n- fa in colostrum promoted an antiinflammatory state in the first week after birth, but that growth up to weaning was not enhanced. we suggest neonatal calves may benefit from n- fa supplementation in colostrum to encourage a greater antiinflammatory state and that effects on health should be tested with more calves per treatment. and the michigan state university dairy cattle teaching and research center (east lansing) for calf care. the authors have not stated any conflicts of interest. oxidative stress index as a new tool to assess redox status in dairy cattle during the transition period effects of dietary fatty acids on eicosanoidgenerating capacity, fatty acid composition and chemotactic activity of rainbow trout (oncorhynchus mykiss) leucocytes. biochim supplementing milk replacer with omega- fatty acids from fish oil on immunocompetence and health of jersey calves an evaluation of brix refractometry instruments for measurement of colostrum quality in dairy cattle expression of short chain fatty acid receptors and pro-inflammatory cytokines in utero-placental tissues is altered in cows developing retention of fetal membranes invited review: inflammation during the transition to lactation: new adventures with an old flame oxidative stress and antioxidant status in fetal circulation in preeclampsia new insights into the use of currently available non-steroidal anti-inflammatory drugs free radicals and brain damage in the newborn comparison of refractometers and test endpoints in the measurement of serum protein concentration to assess passive transfer status in calves effects of dystocia on plasma cortisol and cholesterol levels in holstein heifers and their newborn calves bovine colostrum: changes in lipid constituents in the first days after parturition enhanced n- phospholipid content reduces inflammatory responses in bovine endothelial cells a simple method for the isolation and purification of total lipids from animal tissues development of lung anti-oxidant enzyme system in late gestation: possible implications for the prematurely born infant advances in our understanding of oxylipins derived from dietary pufas colostrum management for dairy calves a role for the mouse / -lipoxygenase pathway in promoting epithelial wound healing and host defense a pronounced uterine pro-inflammatory response at parturition is an ancient feature in mammals in vivo chemotaxis of bovine neutrophils induced by -lipoxygenase metabolites of arachidonic and eicosapentaenoic acid estradiol reduces f α-isoprostane production in cultured human endothelial cells fatty acid intake alters growth and immunity in milk-fed calves comparison of supplementation of n- fatty acids from fish and flax oil on cytokine gene expression and growth of milk-fed holstein calves evaluating acid and base catalysts in the methylation of milk and rumen fatty acids with special emphasis on conjugated dienes and total trans fatty acids differences in the oxylipid profiles of bovine milk and plasma at different stages of lactation production of -f-isoprostane as an assessment of oxidative stress in dairy cows at different stages of lactation maternal late-gestation metabolic stress is associated with changes in immune and metabolic responses of dairy calves feeding a c : -enriched fat supplement increased the yield of milk fat and improved conversion of feed to milk excessive dietary linoleic acid induces proinflammatory markers in rats the omega- fatty acid docosahexaenoate attenuates endothelial cyclooxygenase- induction through both nadp(h) oxidase and pkc inhibition polyunsaturated fatty acids influence differential biosynthesis of oxylipids and other lipid mediators during bovine coliform mastitis the effects of supplementation of various n- fatty acids to late-pregnant dairy cows on plasma fatty acid composition of the newborn calves isoprostanes: markers and mediators of oxidative stress lipoxin a and lipoxin b stimulate the release but not the oxygenation of arachidonic acid in human neutrophils: dissociation between lipid remodeling and adhesion eicosanoids: generation and detection in mammalian cells colostrum supplementation with n- fatty acids and α-tocopherol alters plasma polyunsaturated fatty acid profile and decreases an indicator of oxidative stress in newborn calves identification of inflammatory and proresolving lipid mediators in bone marrow and their lipidomic profiles with ovariectomy and omega- intake oxylipid profiles of dairy cattle vary throughout the transition into early mammary gland involution changes in biomarkers of nutrient metabolism, inflammation, and oxidative stress in dairy cows during the transition into the early dry period association between polyunsaturated fatty acid-derived oxylipid biosynthesis and leukocyte inflammatory marker expression in periparturient dairy cows dietary polyunsaturated fatty acids and inflammation: the role of phospholipid biosynthesis antioxidant activity applying an improved abts radical cation decolorization assay. free radic strategies to decrease oxidative stress biomarker levels in human medical conditions: a meta-analysis on -iso-prostaglandin f α enzymes and receptors of prostaglandin pathways with arachidonic acid-derived versus eicosapentaenoic acid-derived substrates and products stabilized epoxygenated fatty acids regulate inflammation, pain, angiogenesis and cancer sordillo https: / / orcid vandehaar https: / / orcid colostrum supplementation and oxylipid profiles this project was supported by the michigan alliance for animal agriculture (#aa - ), michigan milk producers association, and michigan state university agbioresearch. the authors appreciate lynn worden and the saskatoon colostrum company (saskatoon, sk, canada) for their contributions to sample analysis key: cord- -a vxxzh authors: quigley, j. d.; wolfe, t. a.; elsasser, t. h. title: letter to the editor: a response to the comments of borderas et al. ( ) date: - - journal: journal of dairy science doi: . /jds. - sha: doc_id: cord_uid: a vxxzh nan we appreciate the opportunity to respond to the letter to the editor by borderas et al. ( ) in response to our paper published in the journal of dairy science (quigley et al., ) . in their letter, the authors offer an alternative interpretation of our data and offer criticisms of our methodology. responses to their comments are below. the authors propose that increased morbidity and mortality in our study was caused by administration of refused calf milk replacer (cmr) by esophageal feeder. they wrote "we suggest that the increased incidence of illness observed in the calves fed the higher levels of milk replacer was due to sick calves being force-fed." they further suggested that feed refusals were a manifestation of anorexia caused by expression of proinflammatory cytokines. to determine the role of proinflammatory cytokines in any observed anorexia, we measured concentrations of tumor necrosis factor-α (tnf-α) in blood samples on d , , , , and . regarding the interaction of cytokines, anorexia, and illness, we wrote (p. ) "although we recorded temporal changes in tnf-α concentrations in blood, these changes were unrelated to dietary treatment. . . there were no differences in proportion of calves refusing cmr or in the number of refusals recorded. additionally, there were no effects of feeding regimen on concentrations of plasma tnf-α, suggesting that feeding method per se had no effect on anorexia or the calf's ability to use ingested nutrients." the authors wrote " quigley et al. ( ) did not report the number of calves that were fed with the esopha-geal feeder, but the likelihood of force-feeding was greater for the calves receiving the higher quantities of milk, due to both increased intakes and the longer milk-feeding period ( vs. d)." we reported that there were no differences in proportion of calves refusing cmr or in amount of cmr refused (p. ). further evaluation of our data (determined after publication) showed that calves voluntarily consumed . , . , and . % of the cmr for calves fed fixed amount of cmr (con), variable amount of cmr in an accelerated feeding program (var), or var plus dietary supplement (gam) treatments, respectively. during the study, , , and % of calves refused ≥ ml of cmr on at least d during the study for con, var, and gam treatments, respectively. the authors' contention that force-feeding was greater for calves fed more cmr is not supported by the data presented here or in our article. the authors criticized our decision to administer refused cmr by esophageal feeder. it is understood that administration of cmr by esophageal feeder entails significant risk, but we determined that the practice was necessary to minimize variation in nutrient intake among calves during the study. administration of cmr into the rumen may induce ruminal and metabolic acidosis, particularly if large amounts of milk ( l/d) are repeatedly administered into the rumen (> d) by esophageal feeder (gentile et al., ) . it should be noted that, in our study, calves were tubed with less cmr than that reported by gentile et al. ( ) . in addition, in most cases (> % of all instances), calves were administered cmr by esophageal feeder for d only; thereafter, they consumed all cmr offered. the authors also wrote "clearly, force-feeding sick animals should not be (and never has been) recommended." first, we disagree with the implication that all feed refusals occurred when calves were sick. most incidences of administration of refused cmr were isolated incidents. furthermore, we believe that adminis-tration of refused cmr by esophageal feeder is a common practice. for example, mcguirk and ruegg ( ) stated "sick calves must be able to stay clean and dry. . . . offer milk/milk replacer at the usual dilution and temperature but reduce volume (to liter, for example) and feed more frequently ( times/day, if needed). what liquid feed isn't consumed, administer by an esophageal feeder that has been cleaned, disinfected, and rinsed between calves." the authors criticized our paper for including the use of fecal scoring in calves fed varying amounts of cmr and wrote "although this system has been widely used to evaluate diarrhea in limit-fed calves, the authors provide no evidence that this measure was repeatable or a valid indicator of any underlying pathology." the authors further suggested that ". . . this increased intake of liquids likely decreased fecal consistency in a way that was not related to pathology." if this were the case, others should have reported increases in fecal scores associated with increased cmr feeding. however, bartlett et al. ( ) and cowles et al. ( ) reported no difference in fecal scores between groups of calves fed conventionally or in an accelerated feeding program. khan et al. ( ) reported that calves fed at % of bw to d had lower fecal scores during w and compared with calves fed at % of bw to d. jasper and weary ( ) reported no difference in "diarrhea" [defined as a score of or using the fecal scoring system of larson et al. ( ) ] when calves were fed milk at % of bw or offered milk for ad libitum consumption. conversely, diaz et al. ( ) and nonnecke et al. ( ) reported that calves fed accelerated feeding programs had higher fecal scores compared with calves fed conventionally. the data suggest that there is not necessarily an increase in fecal scores associated with accelerated cmr feeding programs, as was implied by the authors. fecal scoring is a widely used and reported tool for determination of incidence of diarrhea in young milkfed calves and has been used by many researchers, including authors of this letter (tikofsky et al., ; jasper and weary, ; von keyserlingk et al., ) . several of these published studies used fecal scoring with calves fed in accelerated or ad libitum liquid feeding programs. the authors criticized our manuscript for including abrupt changes in the amount of cmr offered. our feeding rates, including abrupt changes, were selected journal of dairy science vol. no. , to exactly mimic those being recommended in the industry at the time the research was conducted. at that time (as now), commercial milk replacer companies in the united states recommend increasing the amount of cmr in accelerated programs by to % from wk to wk , as we did in our study. we do not consider our selection of feeding rates to constitute a methodological problem. the authors wrote "furthermore, body temperatures were only measured in calves that showed high fecal scores rather than being done systematically for all calves. thus, the method of detecting fever was likely biased towards calves fed higher amounts of milk replacer." although we reported (p. ) that rectal temperatures were recorded on all calves with fecal score > , we recorded rectal temperatures when calves showed clinical signs of disease, including anorexia and dehydration, according to normal good management practice. this was reported on p. . the authors wrote that our use of contaminated bedding was uncontrolled and potentially lacked equal distribution among treatment groups. use of contaminated bedding to introduce an immunological challenge is not without precedent. others (coffey and cromwell, ; williams et al., ; bassaganya-riera et al., ) have used contaminated bedding to compare "clean" and "dirty" housing environments and found significant differences in growth and responses to immunological support in young pigs. wray and sojka ( ) used contaminated bedding as a means of infection of calves with salmonella dublin. although we accept the criticism of the authors that the coronavirus exposure was not controlled, we believe that it represented a situation that could normally be found on commercial calf facilities in the united states and, therefore, contributed to the overall goals of the research. the evaluation of our work is appreciated. however, we conclude that many of their criticisms overlooked information available in the published article or from other sources. their alternative hypothesis does not provide a more accurate interpretation of the data. we continue to believe that the paper is an important contribution to the understanding of feeding calves a higher plane of nutrition and we stand by the methods and interpretations of the data. growth and body composition of dairy calves fed milk replacers containing different amounts of protein at two feeding rates effects of dietary conjugated linoleic acid in nursery pigs of dirty and clean environments on growth, empty body composition, and immune competence the effects of force-feeding sick dairy calves: a comment on the impact of environment and antimicrobial agents on the growth response of early-weaned pigs to spray-dried porcine plasma growth characteristics of calves fed an intensified milk replacer regimen with additional lactoferrin composition of growth of holstein calves fed milk replacer from birth to -kilogram body weight d-lactic acidosis in calves as a consequence of experimentally induced ruminal acidosis effects of ad libitum milk intake on dairy calves pre-and postweaning performance of holstein female calves fed milk through stepdown and conventional methods guidelines towards more uniformity in measuring and reporting calf experimental data calf diseases and prevention composition and functional capacity of blood mononuclear leukocyte populations from neonatal calves on standard and intensified milk replacer diets effects of additional milk replacer feeding on calf health, growth, and selected blood metabolites in calves effect of varying carbohydrate and fat content of milk replacer on body composition of holstein bull calves effects of continuous versus periodic milk availability on behavior and performance of dairy calves effect of chronic immune system activation on the rate, efficiency, and composition of growth and lysine needs of pigs fed from to kg salmonella dublin infection of calves: use of small doses to simulate natural infection on the farm key: cord- -wchxkdtj authors: lofstedt, jeanne; dohoo, ian r.; duizer, glen title: model to predict septicemia in diarrheic calves date: - - journal: j vet intern med doi: . /j. - . .tb .x sha: doc_id: cord_uid: wchxkdtj the difficulty in distinguishing between septicemic and nonsepticemic diarrheic calves prompted a study of variables to predict septicemia in diarrheic calves, days old that were presented to a referral institution. the prevalence of septicemia in the study population was %. variables whose values were significantly different (p < . ) between septicemic and nonsepticemic diarrheic calves were selected using stepwise, forward, and backward logistic regression. variables identified as potentially useful predictors were used in the final model‐building process. two final models were selected: based on all possible types of predictors (laboratory model) and based only on demographic data and physical examination results (clinical model). in the laboratory model, variables retained significance: serum creatinine > . mg/dl (> μmol/l) (odds ratio [or] = . , p = . ), toxic changes in neutrophils ≥ (or = . , p = . ), failure of passive transfer (or = . , p = . ), presence of focal infection (or = . , p = . ), and poor suckle reflex (or = . , p = . ). four variables retained significance in the clinical model: age ≤ days (or = . , p = . ), presence of focal infection (or = . , p = . ), recumbency (or = . , p = . ), and absence of a suckling reflex (or = . , p = . ). the hosmer—lemeshow goodness‐of‐fit chi‐square statistics for the laboratory and clinical models had p‐values of . and . , respectively, indicating that the models fit the observed data reasonably well. the laboratory model outperformed the clinical model by a small margin at a predictabilty cutoff of . , however, the predictive abilities of the models were quite similar. the low sensitivities ( % and %) of both models at a predicted probability cutoff of . meant many septicemic calves were not being detected by the models. the specificity of both models at a predicted probability cutoff of . was . %, indicating that . % of nonsepticemic calves would be predicted to be nonsepticemic by the models. the positive and negative predictive values of the models were – %, which indicated the proportion of cases for which a predictive result would be correct in a population with a prevalence of septicemia of %. t he mortality risk of live-born neonatal calves Ͻ month of age has been reported to range from to %. [ ] [ ] [ ] [ ] the majority of deaths are attributable to infectious diseases; diarrhea, pneumonia, and septicemia are the most common. [ ] [ ] [ ] [ ] the predominant pathogen cultured from calves with septicemia is eschericia coli, but other gramnegative, gram-positive, and mixed bacterial infections have been documented. [ ] [ ] [ ] [ ] important risk factors for the development of septicemia in calves include decreased passive transfer of colostral immunoglobulins and exposure to invasive bacterial serotypes. neonatal diarrhea may also predispose calves to septicemia. in separate studies conducted on a california veal operation, blood cultures revealed bacteremia in % and % of calves with signs of diarrhea, depression, and/ or weakness. , septicemia in these calves was attributed to intestinal mucosal damage caused by bacterial, viral, or parasitic gastrointestinal infections, which allowed opportunistic gut pathogens to enter the systemic circulation. the early signs of septicemia in neonatal foals and calves are vague and nonspecific and are often indistinguishable from signs of noninfectious diseases or those of focal in-fections such as diarrhea. , positive blood cultures are required for a definitive antemortem diagnosis of septicemia, but results are not reported for - hours, and false-negative culture findings are common. , no single laboratory test has emerged as being completely reliable for the early diagnosis of septicemia in farm animal neonates, , therefore, various scoring systems and predictive models using easily obtainable historical, clinical, and clinicopathologic data have been developed for this purpose. , [ ] [ ] [ ] the goal of these mathematical models is to identify septicemic neonates early in the course of disease when appropriate therapeutic intervention would most likely result in a favorable outcome. for a period of time, routine blood cultures were performed on all diarrheic calves presented to the atlantic veterinary college teaching hospital regardless of whether the clinical or clinicopathologic findings indicated a diagnosis of septicemia. results of this exercise indicated that septicemia was more common in diarrheic neonatal calves than we had anticipated and that it increased the cost of treatment while decreasing the prognosis for survival. a model capable of predicting sepsis in diarrheic neonatal calves presented to the hospital for treatment would assist the clinicians in distinguishing between calves with undifferentiated diarrhea and calves with diarrhea complicated by sepsis. in the case of a relatively low-value calf with diarrhea and predicted sepsis, the farmer may be advised not to initiate treatment because of the expense involved and the poor prognosis. in contrast, the owner of a valuable diarrheic calf that was predicted to be septic could be informed that early initiation of costly but appropriate antimicrobial and supportive therapy may result in an improved outcome. models to predict septicemia in calves have been published. , study populations in these reports consisted of veal calves, day to months of age, with signs of serious illness (diarrhea, depression, and/or weakness) and calves Ͻ days of age that were presented to a veterinary teach- medical records of calves Ͻ days of age presented to the atlantic veterinary college teaching hospital between the years and with a primary complaint of diarrhea were retrieved. pretreatment data were extracted from the medical records of these calves. independent variables included demographic information and physical examination findings and clinicopathologic values for hematology, venous blood gases, serum chemistry, and immunoglobulins (table ) . immunoglobulin concentration in serum was determined quantitatively or qualitatively using a variety of procedures, including the quantitative zinc sulfate turbidity test ( calves), sodium sulfite precipitation test ( calves), gluteraldehyde coagulation test ( calves), and radial immunodiffusion test ( calves). failure of passive transfer of immunoglobulins was defined as an igg concentration of Յ mg/dl by any of the above mentioned tests, a globulin concentration of Յ g/ dl (Յ g/l), or total serum protein of Յ g/dl (Յ g/l) ( table ). other information extracted from the medical records of the calves in the study population were fecal culture, fecal electron microscopic examination, and fecal floatation results. logistic regression assumes that the relationship between any independent variable recorded on a continuous scale (eg, respiratory rate) has a linear relationship with the logit of the probability of sepsis. for each such independent variable, this assumption was assessed by evaluating the distribution of the independent variable, categorizing the independent variable in or more groups and cross-tabulating the new variable with sepsis, or running unconditional logistic regressions with the variable in question and evaluating the hosmer-lemeshow goodness-of-fit statistics. the most appropriate form for each independent variable was then chosen (table ) . categorical variables were all entered into the predictive models as a series of dummy variables. the dependent variable of interest was a diagnosis of septicemia. a diagnosis of septicemia in diarrheic calves was based on the following antemortem criteria: ( ) positive blood culture, ( ) culture of the same bacterial agent from Ն body fluids, or ( ) culture of a bacterial agent from a single joint in a calf with joint effusion involving multiple joints. blood for culture was collected aseptically from the jugular vein and inoculated into the oxoid signal blood culturing system (oxoid canada, inc, nepean, on, canada). a number of criteria contributed to a postmortem diagnosis of septicemia, including ( ) morphologic changes such as multiple disseminated abscesses of similar size, purulent vasculitis and intravascular identification of bacteria, or fibrin in multiple body cavities; ( ) bacterial isolation from heart blood; or ( ) recovery of the same bacterial organism from Ն body tissues (excluding intestine). other outcome variables recorded were days hospitalized and calf survival. means for continuous variables were compared between septicemic and nonsepticemic calves using student's t-test. categorical variables were analyzed using contingency table chi-square analysis. variables that were significantly different (p Ͻ . ) between septicemic and nonsepticemic calves were grouped into of categories: physical variables, hematological variables, and chemistry variables, which included measures of passive transfer. variables in each group were selected for logistic regression models using stepwise, forward, and backward selection procedures. variables identified as possibly useful predictors by the various selection procedures were used in the final model building process. this process involved comparing a number of possible models and selecting those that maximized the area under the receiver operating characteristic (roc) curve but that did not include any variables whose coefficients were not significant (p Ͻ . ). two final models were selected: one based on all possible types of predictors (the laboratory model) and one based only on demographic data and physical examination results (the clinical model). odds ratios (or) derived from the models were interpreted as measures of increased risk of disease. because septicemia was not a rare condition, the or were not precise measures of increased risk, but the approximation was reasonable and was utilized to clarify the presentation of results. once final models were selected, their sensitivity and specificity were determined at predictive probability cutoff points of . and . (the points that roughly balanced the sensitivity and the specificity). the goodness of fit of the models was evaluated using the hosmer-lemeshow goodness-of-fit chi-square statistic with the data divided into groups. for each model, the pearson residuals, the standardized pearson residuals, and the ⌬␤ values were computed for all covariate patterns to determine if any specific covariate pattern had an undue influence on the model. because evaluating the fit of a model using the same data that were used to build the model is likely to overestimate the predictive ability of the model, a split sample goodness-of-fit evaluation was also carried out. the data were randomly (using a computer based random number generator) divided into separate databases with % and % of the observations in each. the models were refit using the database containing % of the observations by forcing in all of the independent variables included in the final models described above. the predictive ability of the resulting models was assessed using the database containing % of the observations. analyses were performed on a personal computer using the statistical software package stata (stata corp, college station, tx). two hundred fifty-four calves met the criteria for inclusion in the study. however, calves were excluded from analysis because they could not be classified as either septicemic or nonsepticemic based on criteria established for the study; campylobacter-like organisms were seen on gram stains of their blood, but the organisms were not recovered from blood culture. the mean age of diarrheic calves at presentation was . days. crossbred beef calves ( . %) and holstein calves seventy-eight ( %) of the calves met the study criteria for a diagnosis of septicemia. positive blood cultures were used to diagnose cases ( . % of all blood cultures were positive). a single organism was cultured from most calves but of the blood cultures yielded isolates each. e coli accounted for ( %) of the isolates from blood. an additional bacterial agents were cultured from the blood, including campylobacter spp. ( ) ( ), and clostridium spp. ( ) . culture of the same pathogen from Ն body fluids antemortem were used to diagnose cases, and a positive antemortem joint culture from a calf with multiple enlarged joints was used to diagnose case. the same bacterial agent was cultured from Ն tissues at necropsy in calves, postmortem heart blood culture was positive in calf, and morphologic lesions such as multiple abcessation, purulent vasculitis, intravascular identification of bacteria, and fibrin exudation in multiple body cavities, were present in calves. e coli was also the most frequent isolate in antemortem body fluid and postmortem tissue samples, accounting for / ( %) and / ( %) isolates, respectively. because individual calves often satisfied multiple criteria for a diagnosis of septicemia, the totals for all the above criteria are greater than the total number of septicemic calves. one hundred forty-one of the nonsepticemic calves ( %) were discharged from the hospital (survived) compared with only of septicemic calves ( . %). the mean hospital stay for nonsepticemic calves that lived was . days compared with . days for septicemic calves that survived. variables identified as potentially useful predictors (p Ͻ . ) are described in table . septicemic calves had significantly higher (p Ͻ . ) values for respiratory rate, packed cell volume, band neutrophil count, and venous pco than did nonsepticemic calves. mean values for rectal temperature and total plasma and serum protein, globulin, calcium, and glucose concentrations were significantly lower (p Ͻ . ) in septicemic than in nonsepticemic calves. a significantly larger (p Ͻ . ) proportion of septicemic calves was Յ days of age, unresponsive or comatose, and in sternal or lateral recumbency at presentation when compared with nonsepticemic calves. the proportion of septicemic calves exhibiting a weak or absent suckling reflex, scleral injection, and hyperemic or cyanotic oral mucous membranes was also significantly greater (p Ͻ . ). elevated serum creatinine concentration, toxic changes in neutrophils Ն ϩ, and failure of passive transfer of colostral immunoglobulins were present more frequently in septicemic than in nonsepticemic calves (p Ͻ . ). two final models, the laboratory model and the clinical model, were selected based on the maximal area under the roc curve (table ) . variables included in the laboratory model were serum creatinine concentration, toxic changes in neutrophils Ն ϩ, failure of passive transfer, focal infection, and a poor suckle reflex. the clinical model identified age of Յ days, focal infection, recumbency, and a poor suckle reflex as predictors of septicemia. the laboratory model indicated that calves with moderately increased serum creatinine concentration were twice as likely to be septicemic (or ϭ . ; % ci ϭ . - . ), whereas those with creatinine concentration of Ͼ . mg/dl (Ͼ mol/l) were times more likely to be septicemic (or ϭ . ; % ci ϭ . - . ). similarly, evidence of toxic changes in their neutrophils (Ն ϩ), failure of passive transfer, or evidence of focal infections all increased the risk of septicemia by . - . times. calves with a poor suckle reflex were times as likely to be septic. the clinical model (ie, model containing only data from the physical examination and history) showed that each of the following was a factor that increased the risk of being septicemic by approximately . - -fold: Ͻ days of age, signs of focal infection, recumbent on admission, and poor suckle reflex. the hosmer-lemeshow goodness-of-fit chi-square statistics for the laboratory and clinical models had p-values of . and . , respectively, indicating that the models fit the observed data reasonably well. evaluation of residual patterns did not identify any outlining observations, and no specific group of calves (ie, calves with a common covariate pattern) exerted a large influence on the model. we concluded that the models fit the data reasonably well. the predictive abilities of the models are presented in table . when evaluated at a cutoff value of . (ie, a calf was predicted to be septicemic if the predicted probability was Ն . ), all models had quite low sensitivity and very high specificity. a cutoff of . roughly balanced the sensitivity and specificity and has been presented for comparison purposes. the laboratory models outperformed the clinical models, but only by a small margin. similarly, the models based on the full data set outperformed those in which the model was constructed on % of the data and its predictive ability was evaluated in the other %. however, the differences were small. overall, using a probability cutoff of . to define predicted sepsis, the model sensitivities ranged from to % and the specificities ranged from to %. the low sensitivities mean that many septicemic calves would not be detected by the model. however, the positive and negative predictive values ranged from to %, which indicates the proportion of cases in which the predicted result was correct (for a population with a prevalence of septicemia of %). at a cutoff of . , the models had sensitivities and specificities in the - % range, which means that a much higher proportion of septicemic calves would have been detected by the model. however, a positive prediction was only associated with a - % probability (positive predictive value) that the calf was truly septic. however, a negative prediction was associated with an - % probability (negative predictive value) that the calf was not septic. thirty-one percent of the diarrheic calves in this study were diagnosed with septicemia based on results of blood cultures, antemortem or postmortem tissue or fluid cultures, and morphologic changes at postmortem. the prevalence of septicemia in this study was identical to that reported for calves with diarrhea, depression, and/or weakness on a veal raising facility, which suggests that the predictive values of the models developed herein may be relevant to other calf populations. the risk of contamination of the blood sample for culturing probably was low, so it is unlikely that many truly nonsepticemic calves were misclassified as septicemic. however, because of the low sensitivity of blood cultures , and the fact that blood was not cultured for some surviving calves, some truly septicemic calves may have been misclassified as nonsepticemic if they survived and were not subjected to postmortem examination. however, this misclassification probably was not common because ( ) the calves survived, and the probability of survival was low if the calf was truly septicemic; and ( ) the effect of misclassification of truly septicemic calves on the model would likely been a reduction in specificity, but low specificity was not a problem with either model. e coli was the bacterial agent cultured with greatest frequency from septicemic calves in this study, a finding that agrees with results of previous investigations , and may be attributed to the fact that certain strains of e coli possess virulence factors that promote systemic invasion. in addition to e coli, a variety of noncoliform bacteria were isolated. of particular interest was the recovery of campylobacter fetus subsp. fetus from blood of diarrheic calves ( % of the isolates from blood); there is only previous report of this agent being cultured from a septicemic calf. possible explanations for the frequent isolation of campylobacter spp. in this study were that the oxoid signal blood culture system was more effective than other blood culture systems for growing this agent or that there were unique predisposing factors leading to campylobacter bacteremia in calves in our geographic location. two models, the laboratory model and the clinical model, were ultimately selected to predict septicemia in diarrheic neonatal calves presented for treatment. the laboratory model, which included all possible variables, was intended for use in a hospital setting where the clinician would have access to laboratory facilities. the clinical model, which contained only demographic and physical examination variables, was developed for use in the field. clinicopathologic parameters identified by the laboratory model as being associated with an increased risk of septicemia were moderate ( . - . mg/dl) ( - mol/ l) and marked (Ͼ . mg/dl) (Ͼ mol/l) increases in serum creatinine concentration, moderate to marked toxic changes in neutrophils (Ն ϩ), and failure of passive transfer (igg concentration Յ mg/dl, globulin Յ g/dl [Յ g/l]), and total serum protein Յ g/dl (Յ g/l). because azotemia secondary to shock and dehydration was anticipated in both septicemic and nonsepticemic diarrheic calves presented for treatment, it was interesting to note that moderate and marked increases in serum creatinine concentration in this study increased the risk of a calf being septicemic by -and -fold, respectively. moderate to severe azotemia in septicemic calves was attributed to endotoxin induced tubular necrosis and embolic nephritis, in addition to decreased renal perfusion associated with dehydration. toxic changes in neutrophils, which are induced by inflammatory mediators and endotoxins, are frequently encountered in septicemic calves ; therefore, inclusion of this variable in the laboratory model was not unexpected. because the relationship between septicemia and failure of colostral antibody transfer has been documented in previous studies, , the increased risk of septicemia in calves with failure of passive transfer was also anticipated. both the laboratory model and the clinical model indicated that the risk of septicemia in calves with identified sites of focal infection (omphalitis, arthritis, meningitis, uveitis) was more than double that of calves without evidence of focal infection. this association was biologically plausible because one of the initial sites of bacterial invasion in septicemic calves is the umbilicus, and hematogenous spread of infection to the meninges, synovial lining, and uveal tract are known to occur. inclusion of poor suckling reflex by both models and recumbency by the clinical model as predictors of septicemia in a population of diarrheic calves presented for treatment was unforseen. the fluid, blood gas, and electrolyte derrangements associated with the diarrhea could have caused recumbency and disinterest in suckling in the majority of calves in the study. however, multiple organ invasion by bacterial pathogens and the cascade of inflammatory reactions initiated by these infecting agents were more likely responsible for the increased frequency of recumbency and poor suckling reflex in septicemic calves. the clinical model indicated that being Ͻ days of age increased the risk of septicemia by . fold. this contrasts with results of a study conducted in veal calves where age Ͼ days significantly increased the risk of being blood culture positive. the reason for this discrepancy between the studies is unclear. because most calves in the present study were presented for treatment in the winter months when all calves presented to the hospital would be born inside and there is increased stocking density in maternity pens, they could have been heavily exposed to pathogens early in life. the theory that enteritis predisposes calves Ͼ week of age to septicemia was not supported by results of this study. although the laboratory model outperformed the clinical model by a small margin at a probability cutoff of . , the predictive abilities of the models were remarkably similar (table ) . thus, the practitioner in the field, without immediate access to a laboratory, could use the clinical model to predict septicemia in a diarrheic calf with almost the same accuracy as that of the clinician at a referral institution, who would be using the laboratory model for the same purpose. the low sensitivities ( % and %) of both mod- els at a probability cutoff of . meant that many septicemic calves were not being detected by the models. one explanation is that other useful predictors of septicemia were not included in the models. however, with the exception of historical data pertaining to calving and management practices, most variables previously identified as risk factors in calves and foals were evaluated. , , there may be no group of variables unique to the septicemic calf, partly because septicemia is a dynamic process and laboratory and clinical parameters can vary widely depending on when in the disease course the calf is evaluated. the specificity of both models at a cutoff of . was Ͼ %, indicating that Ͼ % of nonsepticemic calves would be predicted to be nonsepticemic by the models. when the predictive probability cutoff value was set to . , the laboratory model had a positive predictive value of %. this cutoff could be used when the clinician wants to be relatively certain that a calf predicted to be septicemic would, in fact, be septicemic. for example, when dealing with a relatively low-value calf, a decision to euthanize the calf may be made given the poor prognosis for septicemic calves. in this situation, a high positive predictive value would be desirable. when the predictive probability cutoff was set to . , the positive predictive value decreased but the negative predictive value rose to %. consequently, a clinician could be relatively certain that a calf that tested negative was, in fact, nonsepticemic. this would be a desirable situation when treating a valuable calf with diarrhea in which ancillary treatments for septicemia (broad spectrum antimicrobials and plasma) would only be omitted from the treatment plan if the clinician was relatively confident that the calf was nonsepticemic. the predictive values for any test depend on the sensitivity and the specificity of the test and the prevalence of the disease. all of the predictive values discussed above are based on the assumption that the prevalence of septicemia was %. if the prevalence of septicemia in a population of calves presented for treatment were Ͻ %, the negative predictive value of the test would rise but the positive predictive value would drop off dramatically (fig ) . the relatively small reduction in the predictive ability of the models when they were evaluated using a split sample approach was encouraging and suggests that the models may perform reasonably well in predicting future observations. however, there may still be a slight upward bias in the assessment of the performance of these models because the variables chosen for inclusion in the split sample assessment were those obtained from the analysis of the whole data set. in conclusion, the predictive models fit the observed data reasonably well and had moderate predictive ability. the main limitation in the models seemed to be a low sensitivity. many of the septicemic calves may have been early in the course of the disease and may not have had any distinguishing features that could be used to identify them as septicemic. reassessment of these calves as the disease progresses may clarify their septicemia status but would not help in making initial decisions about therapy. calf mortality management factors associated with calf mortality in south carolina dairy herds dairy calf mortality rate: influence of management and housing factors on calf mortality rate in tulare county, california factors influencing dairy calf mortality in michigan descriptive epidemiology of calfhood morbidity and mortality in new york holstein herds factors affecting susceptibility of calves to disease an epidemiologic study of calf health and performance in norwegian dairy herds. iii. morbidity and performance: literature review dairy calf management, morbidity and mortality in ontario holstein herds. iii. association of management with morbidity blood culture from calves and foals bacteriological culture of ill neonatal calves pasteurella multocida septicaemia in two calves neonatal septicemia in calves: cases ( - ) septicaemic colibacillosis and failure of passive transfer of colostral immunoglobulin in calves use of a clinical sepsis score for predicting bacteremia in neonatal dairy calves on a calf rearing farm brewer bd, koterba am. bacterial isolates and susceptibility patterns in foals in a neonatal intensive care unit development of a scoring system for the early diagnosis of equine neonatal sepsis comparison of empirically developed sepsis score with a computer generated and weighted scoring system for the identification of sepsis in the equine neonate bovine neonatal sepsis score quantitation of bovine immunoglobulins: comparison of single radial immunodiffusion, zinc sulfate turbidity, serum electrophoresis, and refractometer methods primer of biostatistics applied logistic regression basic principles of roc analysis bacteremia: pathogenesis and diagnosis comparison of bacteriological culture of blood and necropsy specimens for determination of the cause of foal septicemia: cases ( - ) escherichia coli from calves with bacteremia key: cord- -ewowkq o authors: ollivett, t.l.; mcguirk, s.m. title: salt poisoning as a cause of morbidity and mortality in neonatal dairy calves date: - - journal: j vet intern med doi: . /jvim. sha: doc_id: cord_uid: ewowkq o nan cause of increased morbidity and mortality in calves < days of age on a large commercial dairy farm. historically, affected calves suffered from anorexia, diarrhea, abnormal gait, seizures, and death between and days of age. serum chemistry determinations and measurement of the sodium content of milk replacer and water used on the farm identified salt poisoning as the cause of the clinical signs. the use of high salinity water to mix milk replacer, mixing of oral electrolyte powder directly into liquid feed, adding additional milk replacer powder to the same volume of water for daily cold weather feedings, and limited access to water contributed to the salt poisoning. morbidity and mortality rates decreased after switching to an entirely whole-milk diet, mixing oral electrolyte powder in water according to label instructions, and providing adlibitum access to normal salinity water. sodium is the major cation in extracellular fluid, and under normal conditions serves as a marker of total body water. its osmotic effect is important in establishing concentration gradients that drive fluid shifts between the extracellular and intracellular fluid compartments. active and passive transport mechanisms in the body facilitate these processes. maintenance of sodium homeostasis is critical to maintaining cellular hydration and preventing rapid fluxes of water between compartments. , sodium imbalances may be caused by dysfunction in either sodium or water regulation. in dairy calves, milk replacer or milk sodium content and water consumption are the major dietary contributors to serum sodium concentration. less common contributors may be high salinity water, oral electrolyte powders, and sodium-containing feed additives or medications such as lasalocid that also might provide a substantial amount of sodium to the calf. inappropriately high concentration of the milk replacer solution, milk replacer mixing errors, lack of access to water, dehydration from disease, or a combination of these factors also can result in hypernatremia. [ ] [ ] [ ] regardless of the cause, hypernatremia causes inhibition of neuronal cell glycolysis and results in central nervous system (cns) depression. , hypernatremia also causes plasma osmolality to increase relative to tissue osmolality. this is particularly important with nervous tissue in which neurons effectively shrink as water is lost to the extracellular space in an attempt to equilibrate the osmotic gradient between the intracellular and extracellular space. a partially protective mechanism occurs with accumulation of amino acids, sugars, and alcohols in neuronal cells in an attempt to balance the intra-and extracellular osmolality. , correcting hypernatremia can be difficult because the animal is predisposed to additional neuronal damage as fluid shifts into the intracellular space creating cellular edema and possible brain herniation through the foramen magnum. , the current report describes acute and chronic salt toxicity in neonatal dairy calves on a wisconsin dairy from november to february in which abnormally high concentrations of sodium were detected in sera collected from these animals and in the water in which a commercial milk replacer powder was being mixed. the dairy that is the subject of this report milks approximately holstein, jersey, and holstein-jersey mixed breed cows. the calves are born at the dairy, separated from the dam, moved to an individual pen in the calf barn, and fed colostrum. calves are fed whatever volume of colostrum that they voluntarily suckle, up to a maximum volume of quarts for the jersey and quarts for the holstein calves. colostrum force-feeding occurs only when the calf does not suckle at all. water by itself is not provided until month of age. whole milk is fed for the first - days of life followed by milk replacer, which is fed until - weeks of age, when weaning occurs. the primary complaint by the farm owner that led to this herd investigation was high mortality in calves between and days of age, starting in november . the farm had lost % of its newborn calves in the interim period (approximately months) for a total of calves. calves that did not die had a tendency to develop diarrhea at - days of age with recovery approximately days later. abnormal gaits and seizures had occurred in several of the calves. since the onset of the problem, attempts at treatment included addition of electrolyte powder to the liquid feed, administration of sodium lasalocid to the liquid feed (which already contains lasalocid from the manufacturer) and with multiple antibiotics to no avail. upon further questioning, it was found that the owner began increasing the amount of milk replacer powder provided to the calves in late fall to provide additional energy during the winter months. additional history indicated that water quality had been a concern. the depth of the well had been increased and gallons of sodium hypochlorite had been added to the well water supplying the calf barn to improve water quality. the clinical signs reported by the owner included scours, ataxia, seizures, and lethargy before death. in the week before the farm visit, calf health seemed to have improved. the only management change at that time was switching the calves to an entirely wholemilk diet. upon investigation, of calves were showing signs of decreased mentation, base-wide stance, and mild ataxia. seventeen of ( %) calves had respiratory disease, seven of which had or more signs of respiratory disease (fever, cough, abnormal nasal discharge, ocular discharge, or otitis media) and were under no treatment. thirteen of ( %) calves had scours. based upon the history and clinical signs observed, hypernatremia was considered the primary differential diagnosis. other considerations were meningoencephalitis, lasalocid toxicity, bovine virus diarrhea (bvd) infection, enteric colibacillosis, salmonellosis or other causes of diarrhea, and associated metabolic derangements or toxemia. blood samples were collected for measurement of serum electrolyte concentrations from lethargic animals that stood with abnormal extension of the head and neck and were reluctant to move. fecal samples were obtained for salmonella enterica culture and acid fast stain for cryptosporidium spp. from each of untreated calves, five of which had diarrhea at the time of sampling. two milk replacer samples from the first and last calves fed for each of successive feedings after the farm visit were analyzed for total solids and sodium concentration. milk replacer total solids were determined by a brix refractometer a after construction of a standard curve, with known milk replacer concentrations. milk replacer sodium concentrations were determined by a vitros â , fs chemistry system b after the milk replacer solution was agitated, centrifuged, and an aliquot of the supernatant obtained for analysis. milk replacer powder c also was collected, to be mixed with university water for sodium analysis and to serve as quality control standards for total solids calculations by a brix refractometer. the farm protocol was to mix ounces ( g) of milk replacer in quarts ( . l) of water. results of analyses are presented in the tables as follows: milk replacer (table ) and well water ( table ) . postmortem reports from calves that died previously were reviewed. antemortem testing disclosed severe hypernatremia in the calves with neurologic signs that were sampled. serum sodium concentrations were , , and meq/l (reference range: - meq/l), respectively. chloride concentrations also were increased at , , and meq/l (reference range: - ), respectively. only of the calves had an increased anion gap of mmol/l (reference range: - mmol/l). extremely high salinity well water (farm water, , ppm; safe level, < , ppm ) used to mix the milk replacer on the farm resulted in extremely high milk replacer sodium concentrations, compared with considerably lower milk replacer sodium concentrations in milk replacer mixed according to farm protocol but by university water (table ) . fecal diagnostic testing showed no evidence of pathogenic or toxin-producing e. coli or salmonella enterica. one of ( %), of ( %), and of ( %) of the calves were shedding rotavirus, coronavirus and cryptosporidium spp., respectively. no postmortem testing was performed on the day of the farm visit but the reports from previously examined calves indicated no gross lesions and no evidence of meningoencephalitis, bvd infection, or salmonellosis. no lung or intestinal pathogens were identified, including toxinproducing e. coli. salt toxicity outbreaks have been reported in cattle on pasture [ ] [ ] [ ] [ ] [ ] and in a few calves, , but none have involved a herd problem associated with dairy calves. access to a salt lick without free-choice water created signs of salt toxicity, including diarrhea, abdominal pain, anorexia, convulsions, muscle twitching, paresis, and death in a group of -to -month-old holstein beef cattle. clinical signs seen in cattle grazing on coastal pastures with high salinity water ( , ppm) and marsh grass ( , ppm) included diarrhea and excitability. in another report, mature beef cows showed signs of recumbency, seizures, opisthotonus, caudal ataxia, and hypersensitivity after excessive consumption of a protein supplement containing % sodium chloride after breaking into a salt storage shed. severe neurologic signs including mentation changes (excitability or lethargy), head pressing, ataxia, blindness, recumbency, and death were noted in several adult steers that had been deprived of water after allowing access to salt after a period of salt restriction. the calves in this report exhibited many of these signs, including diarrhea, convulsions, anorexia, salt poisoning of calves ataxia, and death. lastly, the calves of this report were given approximately mg/kg lasalocid between the liquid medicated suspension and the medicated milk replacer, which is less than reported toxic dosage of mg/kg in neonatal calves. blood, liquid feed, and water analysis confirmed the primary differential diagnosis of severe hypernatremia secondary to high salinity water, lack of water availability, unusually high milk replacer total solids concentration (table ) , and history of adding additional sodium lasalocid to the milk replacer. the volume of the extracellular fluid (ecf) is determined by the total body sodium content, whereas the osmolality and sodium concentration of ecf are determined by sodium and water balance. sodium, glucose, and serum urea nitrogen (sun) are solutes that affect plasma osmolality. all conditions associated with hypernatremia reflect hyperosmolality and hypertonicity of the ecf. these conditions are caused by a deficit of pure water, such as lack of water access; loss of hypotonic fluid, such as occurs during osmotic diuresis; or gain of sodium, such as during salt poisoning which may result in either relative or absolute dehydration. hypernatremia caused by the gain of sodium results in expansion of the ecf at the expense of the intracellular fluid (icf), that is, relative dehydration in an attempt to return plasma osmolality to normal. hypernatremia because of deficient free-water intake or hypotonic fluid loss is associated with absolute dehydration and reduction in total body water content. characterization of dehydration is important when determining treatment options. it is likely that the calves tested in this report suffered predominately from relative dehydration because gain of salt was the primary factor predisposing to hypernatremia. specifically in cattle, the pathogenesis of hypernatremia involves excessive intake of sodium as a result of mixing errors, excessive water loss, or prolonged administration of iv isotonic crystalloids without access to free-choice water. water salinity of , ppm on this farm contributed . meq/l of sodium from water added to the milk replacer powder. additional sodium from the milk replacer powder (approximately meq/l), electrolyte powder ( % sodium), and times the recommended lasalocid concentration fed to calves with no access to free-choice water likely contributed to the early onset of clinical signs and death. no deaths occurred in calves fed colostrum and whole milk exclusively. multiple factors contributed to salt poisoning in the neonatal dairy calves on this farm. the importance of the availability of free-choice water, appropriate mixing of milk replacer and electrolyte solutions, as well as water salinity testing are evident. two of the calves with hypernatremia died. the surviving calf was given no access to water. instead, it was fed whole milk and offered a progressively more dilute oral electrolyte solution over a course of days. water was offered on day eight after the farm visit. treatment of sodium balance disorders. water intoxication and salt toxicity disorders of sodium and water balance in hospitalized patients a case of presumptive salt poisoning (water deprivation) in veal calves hypernatremia in calves hypernatremia in neonatal elk calves: cases ( - ) treatment of hypernatremia in an acidotic neonatal calf effects of hypernatremia on organic brain osmoles practical toxicologic diagnosis. mod vet prac water deprivation-sodium ion toxicosis in cattle acute salt poisoning in cattle salt poisoning in beef cattle sodium chloride poisoning in cattle salt poisoning in beef cattle on coastal pasture on prince edward island abutarbush sm, petrie l. treatment of hypernatremia in neonatal calves with diarrhea conflict of interest declaration: authors disclose no conflict of interest. key: cord- -s s awtn authors: apperson, k. denise; vorachek, william r.; dolan, brian p.; bobe, gerd; pirelli, gene j.; hall, jean a. title: effects of feeding pregnant beef cows selenium-enriched alfalfa hay on passive transfer of ovalbumin in their newborn calves date: - - journal: journal of trace elements in medicine and biology doi: . /j.jtemb. . . sha: doc_id: cord_uid: s s awtn abstract intestinal absorption of immunoglobulins is critical for health and survival of newborn calves because there is no transfer of immunoglobulins in utero. the objective of this study was to determine if feeding beef cows se-enriched alfalfa hay during the last trimester of gestation improves passive transfer of ovalbumin (ova), a surrogate protein marker for igg absorption. control cows (n = ) were fed non-se-fortified alfalfa hay ( . mg se/head daily) plus a mineral supplement containing inorganic se ( mg se/head daily). med-se (n = ) and high-se cows (n = ) were fed se-biofortified alfalfa hay ( . and . mg se/head daily, respectively); both groups received mineral supplement without added se. calves were randomly assigned to receive orally administered ova at , , or h of age. calves that received their oral dose of ova at h of age had higher serum ova concentrations across the first h of life if born to high-se cows compared to calves born to control cows (p = . ), with intermediate values for calves born to med-se cows. our results, using ova as a model for passive transfer, suggest that if calves do not receive adequate colostrum to reach maximum pinocytosis, then supranutritional se supplementation in beef cattle may improve passive transfer in their calves, if calves receive colostrum within the first h of age. in cattle, as in other bovid species, immunoglobulins (ig) are transferred from colostrum of the dam to the newborn calf by passive transfer of ig across intestinal epithelium as there is no transfer of ig across the syndesmochorial placenta of the cow in utero [ ] . calves that fail to suckle adequately possess low concentrations of ig in their serum. therefore, intestinal absorption of a sufficient amount of ig is a key factor in neonatal health and survival. passive immunity provided by maternal ig protects neonates from enteric disease, diarrhea, and respiratory disease for several weeks after birth [ ] . if failure of passive transfer occurs, calves are predisposed to infection, sepsis, and death [ , ] . immunoglobulin igg is the predominant igg subclass in colostrum of cows and is derived from serum of the dam [ , ] . calves are born with a digestive tract that facilitates absorption of colostral proteins instead of degrading them [ ] . the level of proteolytic activity is low in the abomasum and small intestine, and further decreased by trypsin inhibitors in colostrum [ ] . three distinct processes must occur in gut epithelia of the neonate for igg absorption to be successful: pinocytosis of large proteins, transcellular transfer of proteins across the enterocyte, and exocytosis of proteins into the lymphatic system [ ] . epithelial cells of the small intestine absorb colostral igg via pinocytosis. proteins pass through enterocytes in vacuoles and are released via exocytosis into lacteals [ , ] . from the lacteals, colostral proteins enter the systemic circulation, which allows newborn calves to receive maternal igg by passive transfer [ , ] . closure of the intestine to absorption is defined as cessation of absorption of macromolecules from gut to blood in neonates [ ] . it is thought that the process of closure occurs in a retrograde manner, in that the basal cell membrane ceases to release the vacuolated proteins, transport ceases, and then uptake by the pinocytosis tubular system ceases [ ] [ ] [ ] [ ] . the period during which the intestine is permeable to igg varies in t calves, but is highest immediately after birth, declines after h, and drops to relatively low levels by h, signaling gut closure to passive transport [ ] . peak serum igg concentrations are normally reached h postpartum [ ] . after absorption ceases, serum concentrations of passively acquired igg begin to decline through normal catabolic processes [ ] . we have previously shown that supplementing dairy cows with supranutritional se-yeast during the dry period, or spiking colostrum with na-selenite, improved igg status in their calves [ ] ; kamada et al. [ ] showed similar results with spiking colostrum with na-selenite. the underlying mechanism for improved passive transfer is unknown. selenium has been postulated to act directly on intestinal epithelial cells to activate pinocytosis [ ] . another hypothesis is that supranutritional concentrations of se delay enterocyte replacement by a more mature population of intestinal epithelial cells, which have been shown to be incapable of passive transfer [ , ] . based on our previous findings in dairy cows, the objective of this study was to determine if feeding se-replete beef cows se-enriched alfalfa (medicago sativa) hay during the last trimester of gestation delays turnover and replacement of fetal intestinal epithelial cells in their calves, thus extending the period of time during which large proteins can be transferred from intestinal lumen to neonatal blood. we previously reported that feeding se-biofortified hay to these pregnant beef cows improved se status in cows and their offspring, as well as the igg concentrations in colostrum compared to cows receiving control hay [ ] . in this paper, to test the hypothesis that passive transfer is enhanced in calves born to cows fed se-biofortified alfalfa hay in the last trimester of pregnancy compared to calves born to control cows fed the usda-approved upper limit of na-selenite in salt [ ] , we administered g oral ovalbumin (ova) to calves at , , or h after birth, and measured serum ova concentrations to estimate the time of gut closure. we chose ova with , da molecular weight (mw) as a surrogate protein for igg (the bovine igg molecule is approximately , da mw) because human albumin with , da mw and human igg with , da mw have been previously shown to be absorbed equally in relation to total dose in calves [ ] . if passive transfer was prolonged by enhanced se intake in dams, we expected ova concentrations in serum of newborn calves to be higher at all dosing time points compared to control calves. the experimental protocol was reviewed and approved by the oregon state university animal care and use committee (acup number: ) and in accordance with the national institutes of health guide for the care and use of laboratory animals (nih publications no. , revised ) . this was a prospective clinical trial of - wk (average wk) duration involving pregnant angus and anguscross beef cows. multiple bulls had been utilized initially to establish pregnancy by artificial insemination; several bulls were then used to cover non-pregnant cows. the study was conducted at the hogg animal metabolism barn on the oregon state university campus (corvallis, or, usa). the cows ranged in age from to yr (mean ± sd, . yr ± . ) and originated from the oregon state university beef ranch. all cows had calved at least once previously. body weights at mo of pregnancy ranged from to kg (mean ± sd, . kg ± . ), and bcs ranged from to ( to scale). routine farm management practices included the following vaccinations during the study design consisted of treatment groups, with pens of cows each (n = ) per treatment. before calving, cows were blocked by bw, cow age, and projected calving date and assigned to treatment group using a randomized complete block design. ear tags were used to identify cows. cows were housed in dry barn lots with continuous access to water, feed bunks, and shelter. pens provided m /cow of concrete flooring in open lots that were strip cleaned once weekly, m /cow of shavings in loafing area, and cm of feeder space/cow as concrete bunks. all measurements exceeded recommended beef housing requirements [ ] . all cows received hay at a rate of . % bw/d, of which % was alfalfa hay ( % cp; . % tdn) and % was grass hay [ . % cp; predominantly schedonorus phoenix (scop.) holub and agrostis capillaris] to achieve a ration cp of . % (table s ). cows were fed alfalfa hay once daily in the evening. alfalfa hay was readily consumed such that by the next morning feed bunks were empty. there was no wastage as alfalfa hay was kept swept into reach. grass hay was fed in the morning. mineral supplement was added as a top dressing on the alfalfa hay in the evenings. control cows received mineral supplement containing mg/kg se (us fda regulations) from na-selenite, at a calculated amount to provide mg se/head daily. treatment cows received the same amount of mineral supplement top dressing that did not contain na-selenite. the mineral supplement (dm basis) was in loose granular format and contained . to . g/kg calcium; . g/ kg phosphorus; to g/kg sodium chloride; . g/kg magnesium; mg/kg cobalt; mg/kg copper; mg/kg manganese; mg/kg iodine; mg/kg zinc (wilbur-ellis company, clackamas, or). prior to the start of this experiment, cows had free-choice access to the same mineral supplement containing mg/kg se from na-selenite. after calving, calves were left with their mothers in individual pens for h. ear tags were used to identify calves. body weights of all calves were recorded within h of calving, and after each nursing for h. calves were placed in a large weigh sling (nasco; fort atkinson, wi) and weighed using a max digital weight indicator (western scale co.; port coquitlam, b.c., canada) for precise mobile weighing. within each treatment group an equal number of calves were randomly assigned to receive an oral dose of ova at , , or h after birth. calves were given their oral dose of ova ( g albumin from chicken egg-white powder dissolved in ml water; sigma, st. louis, mo; - % purity) using a dosing syringe. the soil was enriched with se by mixing inorganic na-selenate (retorte ulrich scharrer gmbh, röthenbach, germany) with water and spraying it onto the soil surface of an alfalfa field at application rates of , . , or . g se/ha immediately after the second cutting of hay in july . the application rates were chosen based on a previous study [ ] . third-cutting alfalfa hay was harvested d after se application and then analyzed for nutrient and se content (table s ). alfalfa yield was approximately . t/ha. a penn state forage sampler was used to take cores from random bales in each hay source ( , . , or . g se/ha). samples were collected prior to beginning the feeding trial for each alfalfa hay source. core samples were blended and representative samples were selected for analysis. alfalfa hay samples were submitted to commercial laboratories for analysis. hay samples were prepared for se analysis as described [ ] , and se was determined using inductively coupled argon plasma emission spectroscopy (icp-ms; elan , perkin elmer, shelton, ct). quantification of se was performed by the standard addition method, using a four-point standard curve. a quality-control sample (in similar matrix) was analyzed after every five samples, and analysis was considered acceptable if the se concentration of the quality-control sample fell within ± % of the standard/reference value for the quality control. colostrum was collected from cows prior to the first nursing. whole blood samples were collected from the jugular vein of calves within h of birth (before colostrum feeding) into evacuated ml edta tubes (final edta concentration g/l; becton dickinson, franklin lakes, nj). blood samples were also collected at , , , and h after birth. colostrum and blood samples were frozen at − °c until further analyses were performed. selenium concentrations in colostrum and whole blood (wb) were determined by a commercial laboratory (utah veterinary diagnostic laboratory, logan, ut) using an inductively coupled argon plasma emission spectrometry (elan , perkin elmer, shelton, ct) method as previously described [ ] . results are reported as ng/ml se. jugular venous blood was also collected from calves within h of birth and at , , , and h after birth into evacuated tubes without edta ( ml; becton dickinson) for subsequent harvesting of serum. the tubes were centrifuged at x g for min at room temperature; serum was collected, transferred into . ml screw cap selfstanding micro tubes (isc bioexpress, kaysville, ut) and stored at - °c until further analyses were performed. concentrations of igg in colostrum and calf serum were quantified using a direct sandwich elisa procedure as previously reported [ ] . results are reported as mg/ml igg . the blood collection times ( , , , and h after birth) ensured that blood was collected at uniform time intervals after oral dosing of calves with ova. concentrations of ova in calf serum were quantified using a direct sandwich elisa procedure. the protocol was adapted from a commercially developed assay (bethyl laboratories, montgomery, tx). in brief, -well microplates (thermo scientific™ pierce™, fisher scientific, pittsburgh, pa) were coated with μl of antiserum containing . μg of anti-chicken egg albumin antibody produced in rabbit (whole antiserum; mg/ml; sigma) diluted in . m carbonate-bicarbonate buffer (ph . ) and incubated for h at room temperature. after incubation, plates were washed times in tween-tris buffered saline (t-tbs; mm tris, . m nacl, . % tween- ; ph . ). the plates were then incubated for min with μl t-tbs, and then washed again times with t-tbs. for the standard, albumin from chicken egg white (sigma) was diluted in t-tbs from ng to . ng. calf serum samples from all time points were diluted in t-tbs at : , : , and : . the standards and samples were plated in duplicate at μl per well, and allowed to incubate for h at room temperature. after incubation, plates were washed times in t-tbs. anti-ovalbumin antibody complexed with horseradish peroxidase [rabbit anti-ovalbumin (ova) antibody (hrp); mg/ml; antibodies-online inc., atlanta, ga) was added at μg in μl/well and allowed to incubate for h at room temperature. plates were then washed times in t-tbs, and μl of , ', , '-tetramethyl benzidine (tmb; sigma) was added to each well. plates were kept in the dark at room temperature and read at nm until an absorbance of at least . optical density was reached in the most concentrated standard well. the tmb reaction was then stopped by adding μl of n h so and the plate was read at nm. results are reported as milligrams ova per deciliter plasma. the inter-assay cv for ova was . %. statistical analyses were performed using sas, version . (sas, inc; cary nc) software. the experimental design was a dose-response trial with control and two treatment levels of se-biofortified alfalfa hay fed to cows. for outcome variables (except for colostrum), repeated measures over time were collected in calves. for ova measurements, we additionally accounted for time of ova administration. we tested for homogeneity of variance among treatments and for normal distribution of residuals. the effects of agronomic se fortification on colostral se and igg concentrations were measured using proc glm. fixed effects in the models were se application rate (control, med-se, high-se), calf gender, and age group of cow ( - years, > years). for repeated measures within animals (calf wb-se concentrations, serum igg concentrations, serum ova concentrations), we modeled repeated measurements within animals using an unstructured variance-covariance structure. the unstructured variance-covariance matrix provided the most parsimonious matrix based on the lowest value by the aikaike information criterion. fixed effects in the models were se application rate (control, med-se, high-se), blood sampling time, and the interaction of blood sampling time and se application rate. all statistical tests were two-sided. data are reported as least-square means ± sem. statistical significance was declared at p ≤ . and a statistical tendency was declared at . < p ≤ . . as previously reported [ ] , fertilizing fields with increasing amounts of na-selenate increased the se concentration of third-cutting alfalfa hay from . mg se/kg dm (non-fertilized control) to . and . mg se/kg dm for na-selenate application rates of . and . g se/ha, respectively. calculated se intake from dietary sources was . (including mg se/head daily from na-selenite), . , and . mg se/ head daily for cows consuming % alfalfa hay and % grass hay with se concentrations of . to . and . mg se/kg in the alfalfa hay dm, respectively. as previously reported and shown in fig. s , colostral se concentrations collected from cows within h of parturition increased linearly with agronomic se biofortification (p < . ). colostrum seconcentrations were , , and ng/ml respectively for cows assigned for control, med-se, and high-se treatments (sem = ng/ ml). in calves, wb-se concentrations at birth also increased linearly (p < . ) with agronomic se biofortification from ± for control to ± for med-se, and ± ng/ml for high-se treatments. during the first h, wb-se concentrations decreased in calves to ± for control, ± for med-se, and ± ng/ml for high-se treatments. there were significant time (decreased from birth; p = . ), treatment (p = . ), but not time by treatment interaction effects (p = . ). as previously reported and shown in fig. s , igg concentrations of colostrum collected from cows within h of parturition increased linearly with agronomic se biofortification (p < . ). control cows had mg igg /ml (mean), med-se cows had mg igg /ml, and high-se cows had mg igg /ml (sem = ). most calves in the study consumed their first colostrum within to h of birth and calves consumed an average of . l (range: . to . l; of calves consumed more than l) within the first h. serum igg concentrations in calves differed within the first h of life (p < . ). before colostrum ingestion, igg was not detectable, but by h, igg was detectable in all calves. the igg concentrations increased from h to h (p < . ), remained similar at h (p = . ), and tended to decrease at h (p = . ). there was no effect of se treatment (p = . ) nor se treatment by time interaction (p = . ). if ova is administered orally to newborn calves within the first h of life, there is greater passive transfer in calves whose dams received seleniumbiofortified alfalfa hay during pregnancy as shown in fig. a using ova as a surrogate protein marker for igg in newborn calves, we evaluated the hypothesis that supranutritional se supplementation of the dam delays turnover and replacement of fetal epithelial cells by a more mature population of intestinal epithelial cells that are incapable of passive transfer. calves born to high-se dams that had ova administered orally at h of age had the highest serum ova concentrations at and h of age, whereas no differences between treatment groups were observed in calves that had ova administered orally at either or h of age. we suggest that an enhanced se status at birth improves passive transfer at or before h of age, but the beneficial effect disappears if the surrogate protein marker is administered when calves are older ( and h of age). pinocytosis is a non-selective process by which macromolecules, including ova, are taken up by fetal intestinal epithelial cells. once taken up by pinocytosis, macromolecules are repackaged by the golgi apparatus and transferred across the basal membrane of the cell into the lymphatic system. movement of macromolecules out of gut epithelial cells in neonatal calves continues after pinocytosis stops, consistent with the observation that maximum concentration of serum igg occurs after gut closure [ , ] . after fetal epithelial cells are replaced with cells of the adult phenotype, all of the processes associated with absorption of large molecules cease. from our data, it is not possible to determine which of these processes in passive transfer is affected by se supplementation of dams. our results are consistent with either or both increased pinocytosis and a delay in turnover of fetal epithelial cells in calves born to high-se dams. we previously reported that concentrations of igg in colostrum increased in a linear relationship to the amount of se ingested by the dam [ ] . the mechanism by which high-se supplementation increases colostral igg concentration is unknown, and multiple factors may be involved. it is possible that increased concentrations of selenoproteins acting on mammary gland epithelium alter the number of specific nutrient transporters or growth and vascularization of mammary tissues as is hypothesized for intestinal tissues [ ] . because timing of colostrum formation also has an effect on igg concentrations, high-se supplementation may also increase the duration of colostrum synthesis attributable to endocrine regulation or genetic variation of the transporter (s) [ ] . because igg concentrations were higher in colostrum of high-se dams, their calves may have consumed more igg ; however, this was not reflected in short-term (≤ h) higher serum igg concentrations in calves [ ] . we previously hypothesized that calves reached the physiologic limitation for mass of ig that can be passively transferred from a given volume of colostrum [ ] . in besser et al. [ ] , calves consumed more than l of colostrum, a threshold volume that stott et al. [ ] proposed as necessary to maximize pinocytosis of all available fetal enterocytes. this suggests that reaching thresholds for mass of igg consumed or volume of colostrum consumed are the primary factors in achieving effective passive transfer in se-replete calves [ ] . our new results, using ova as a model for passive transfer, suggest that if calves do not receive adequate colostrum to reach maximal pinocytosis, then high-se treatment of dams may improve passive transfer if calves receive colostrum within the first h of age. these results are consistent with the findings of stott et al. [ ] who showed that age at initial feeding had little influence on passive transfer up to h postpartum, and that in general the highest amount of passive transfer, based on serum ig concentrations, occurred during the first h after initial feeding of colostrum and was dependent on the amount of colostrum fed. calves fed l did not have a further increase in passive transfer if a second feeding was administered h later. stott et al. [ ] showed that peak serum igg concentrations occurred during the first h after the time of initial colostrum ingestion. immunoglobulin g , albumin, and the neonatal form of the ig receptor, fcrn, may be secreted into colostrum as a large complex [ ] . despite its name, this receptor does not play a role in absorption of igg in the neonate. the main role of this receptor is to recycle igg back into the intestinal lumen [ ] , thus prolonging the availability of igg for absorption by pinocytosis. our hypothesis that passive transfer is enhanced in calves born to cows fed se-biofortified alfalfa hay in the last trimester of pregnancy was confirmed if calves received colostrum within the first h of age. maternal nutritional plane and se supply during gestation in sheep have been shown to affect total proliferating small intestinal cells in their lambs at days of age [ ] . lambs from ewes fed % of nrc nutritional requirements as well as high dietary se had greater percent proliferation of small intestinal cells compared to lambs from ewes receiving adequate se [ ] . total small intestinal cells were also affected by nutritional plane during gestation [ ] . thus, it is possible that small intestinal growth and development can be affected by maternal se nutrition in cattle during the third trimester of gestation, thereby altering passive transfer capacity in small intestinal epithelial cells of newborn calves. gut closure is a gradual process, beginning around h post-partum and ending around h post-partum [ , ] . moretti et al. [ ] observed vacuoles containing colostral ig in jejunal epithelial samples taken from goat kids at and h of age, but not in samples taken at and h of age. the later samples were collected after both pinocytosis and transcellular transfer of large molecules ended. turnover of gut epithelial cells from fetal to adult phenotypes is probably the most important component of this gradual change. in some species such as the sheep and pig, the time of gut closure is affected by the time of first colostrum feeding, such that delaying the first feeding delays the time of gut closure [ ] . this also appears true in calves, although the difference in time of gut closure in calves given an early feeding and calves given a later feeding was only - h [ ] . thus, the time of the initial feeding is important. serum concentrations of igg in calves given colostrum at h or later were not as high as those fed before h [ ] . wallace et al. [ ] noted a trend of increasing concentrations of igg in calf serum between and h for all treatment groups, but no further increase after h. we chose to provide control cows with the maximum fda recommended level of se ( mg se/head daily) from na-selenite, as this is common practice in se-deficient areas in the united states. current fda recommendations limit the amount of dietary se supplementation to . mg/kg (as fed), or mg per cow daily without taking into account the amount of se already present in feed sources or the chemical form of se [ ] . consequently, calculated se intake from dietary sources was . mg se/head (including mg se/head daily from na-selenite) for control cows. this is reflected by the increase in whole blood se concentrations in control cows from ± ng/ml (mean ± sem) at baseline to ± ng/ml at parturition [ ] . in comparison, treatment groups receiving se-biofortified hay consumed . ( . mg se/head daily) and . -fold ( . mg se/head daily) higher dietary levels of se compared with control cows. we have previously shown in ruminant species (sheep, dairy cattle, and beef cattle) that there are production and immune function benefits of supplementing organic se at supranutritional rates (i.e., lower concentrations for extended time periods, and higher concentrations for shorter periods) in se deficient regions [ ] . no adverse effects were observed in this study or our previous studies using na-selenite supplementation at current fda recommended levels or supranutritional organic se supplementation. our data suggests that increases in calf wb-se concentrations were attained in utero as concentrations were increased at birth prior to ingestions of colostrum. although se concentrations in colostrum were enriched in cows that received supranutritional se supplementation, subsequent ingestion of colostrum did not further increase wb-se concentrations in their calves [ ] . we also observed a significant decrease in serum ova concentrations over time within treatment groups. because of its lower mw compared to igg , ova is likely cleared from the blood by the kidneys over to h [ ] . thus, igg serum concentrations remained nearly the same at h compared to h, whereas ova concentrations had decreased significantly by h. in summary, we have demonstrated that calves born to high-se dams and fed ova at h of age had higher serum ova concentrations at and h of age compared to control calves. this suggests that supranutritional se supplementation of dams can improve passive transfer by increasing the amount of protein transferred. it is possible that neonatal small intestinal development can be affected by maternal se nutrition in cattle during the third trimester of gestation, thereby altering passive transfer capacity in newborn calves. enhancing the se status of the neonatal calf can be achieved by feeding the dam se-biofortified alfalfa hay during the last weeks of pregnancy. in addition, feeding se-fortified alfalfa hay to the pregnant beef cow increases igg concentrations in colostrum. based on these results from ova administration, we suggest that early feeding (≤ h of age) will also increase igg concentrations in calves from high-se dams receiving similar volumes/concentrations of igg colostrum compared to control calves. jah and gjp conceived and designed the experiments; kda, wrv, bpd, gb, jah performed the experiments; gb, jah, bpd analyzed the data; all authors contributed to data interpretation and manuscript writing. none absorption of colostral immunoglobulins in newborn calves factors affecting duration of intestinal permeability to macromolecules in newborn animals passive transfer of colostral immunoglobulins in calves bovine immunoglobulins: an augmented review colostrogenesis: mass transfer of immunoglobulin g into colostrum proteolytic activity during the absorption of [ i]γ-gamma-globulin in the new-born calf concentrations of trypsin-inhibitor and immunoglobulins in colostrum of jersey cows colostral immunoglobulin transfer in calves i. period of absorption effect of dietary regimen on cessation of intestinal absorption of large molecules (closure) in the neonatal pig and lamb the ultrastructure of neonatal calf intestine and absorption of heterologous proteins fine structure of duodenal absorptive cells in the newborn pig before and after feeding of colostrum the jejunal absorptive cell of the newborn pig: an electron microscopic study maternal transport of immunoglobulins to the calf nutrient and immunity transfer from cow to calf preand postcalving neonatal immunity and immunisation in early age: lessons from veterinary medicine effect of supranutritional maternal and colostral selenium supplementation on passive absorption of immunoglobulin g in selenium-replete dairy calves selenium addition to colostrum increases immunoglobulin g absorption by newborn calves electron-microscopic studies of the jejunal epithelium from neonatal pigs fed different diets epithelial cell renewal and antibody transfer in the intestine of the foetal and neonatal lamb effects of feeding pregnant beef cows selenium-enriched alfalfa hay on selenium status and antibody titers in their newborn claves fda, title -food and drugs: food additive permitted in feed and drinking water of animals. section cfr. selenium mwps- , beef housing and equipment handbook effect of feeding selenium-fertilized alfalfa hay on performance of weaned beef calves toxicokinetics and pathology of plant-associated acute selenium toxicosis in steers maternal nutritional plane and selenium supply during gestation impact visceral organ mass and intestinal growth and vascularity of neonatal lamb offspring effect of colostral immunoglobulin g and immunoglobulin m concentrations on immunoglobulin absorption in calves colostral immunoglobulin transfer in calves ii. the rate of absorption colostrogenesis: igg( ) transcytosis mechanisms goat kids' intestinal absorptive mucosa in period of passive immunity acquisition selenium supplementation strategies for livestock in oregon intestinal permeability to proteins in the newborn herbivore funded by a grant from the agriculture research foundation (jah and gjp, principal investigators), oregon state university, corvallis, oregon, united states of america. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.jtemb. . . . key: cord- -h p kmss authors: follet, jérôme; guyot, karine; leruste, hélène; follet-dumoulin, anne; hammouma-ghelboun, ourida; certad, gabriela; dei-cas, eduardo; halama, patrice title: cryptosporidium infection in a veal calf cohort in france: molecular characterization of species in a longitudinal study date: - - journal: vet res doi: . / - - - sha: doc_id: cord_uid: h p kmss feces from animals were collected on farms in the region of brittany, france. each sample was directly collected from the rectum of the animal and identified with the ear tag number. animals were sampled three times, at , and weeks of age. after dna extraction from stool samples, nested pcr was performed to amplify partial s-rdna and kda glycoprotein genes of cryptosporidium. the parasite was detected on all farms. one hundred out of calves ( . %) were found to be parasitized by cryptosporidium. amplified fragments were sequenced for cryptosporidium species identification and revealed the presence of c. parvum ( . %), c. ryanae ( . %), and c. bovis ( %). one animal was infected with cryptosporidium ubiquitum. the prevalence of these species was related to the age of the animal. c. parvum caused . % of cryptosporidium infections in -week-old calves but only . % in -week-old animals. the analysis of the results showed that animals could be infected successively by c. parvum, c. ryanae, and c. bovis for the study period. c. parvum gp genotyping identifies iia subtypes of which . % were represented by iiaa g r . this work confirms previous studies in other countries showing that zoonotic c. parvum is the dominant species seen in young calves. cryptosporidium is a genus of protozoan parasites infecting a wide range of hosts [ ] . all groups of vertebrates are susceptible to cryptosporidium infection worldwide. this parasite is the etiological agent of cryptosporidiosis, which is mainly characterized by diarrhea in humans and livestock. massive outbreaks of enteritis in people such as in milwaukee, wisconsin (usa) have increased public awareness of this parasite [ ] . in humans, the prevalence and severity of infection increase in immunodeficient individuals such as aids patients. in immunocompetent patients, the disease is self-limited [ ] . no drug therapy is yet available and the high resistance of oocysts to environmental conditions and chemical treatment make cryptosporidiosis difficult to control [ ] . cattle have been considered to be a primary reservoir for cryptosporidium oocysts for zoonotic c. parvum [ ] . these animals could be a risk factor via environmental contamination from their manure being spread on farmland or their grazing on watersheds [ ] . on farms, transmission of cryptosporidium spp. can result from ingestion of contaminated food or water, by direct transmission from host to host, or through insect vectors [ ] . in cattle, infection by cryptosporidium spp. was first reported in [ ] . since vaccines have become commercially available against escherichia coli k , rotavirus, and coronavirus, cryptosporidium has emerged as the main neonatal diarrheic agent in calves [ ] . in farm animals, the economic impact is related to morbidity, mortality and growth retardation [ ] . among the species previously described (if the three fish species are accepted without complete genetic characterization) [ , [ ] [ ] [ ] , c. parvum, c. bovis, c. ryanae and c. andersoni usually infect cattle. c. parvum has zoonotic potential and is a frequent cause of human cryptosporidiosis [ ] . c. bovis and c. ryanae have not been found in humans and there is only one description of c. andersoni in a patient [ ] . recent reports have described an age-related distribution of these aforementioned species in dairy cattle on the east coast of the united states [ ] [ ] [ ] , india, china, georgia [ ] , malaysia [ ] , and denmark [ ] . the most prevalent species were c. parvum in preweaned calves, c. ryanae and c. bovis in postweaned calves and c. andersoni in adult cows [ , ] . in france, previous studies on the prevalence of cryptosporidium in cattle were based on microscopic determination [ ] or coproantigen detection using elisa [ ] . these studies on dairy calves reported a within herd prevalence of cryptosporidium without identifying species or the relation to the host's age. the present study was conducted in farms in brittany, france to determine the prevalence of cryptosporidium in veal calves. we used genotyping and subtyping for the molecular study of cryptosporidium isolates. follow-up of the same animal allowed us to determine the outcome of the infection and the age distribution of cryptosporidium species. fifteen fattening units in brittany (france) were included in this work. they belonged to three industrial veal producers representative of integrators in france and did not present any known history of cryptosporidium infection. these farms were located in four administrative regions ( figure ): côtes d'armor (ca -ca ), morbihan (mo ), ile-et-vilaine (iv -iv ), and mayenne (ma -ma ). during the summer and autumn of , all farms were visited three times and fecal samples were taken from animals exhibiting diarrhea at the age of weeks old. calves arrived in fattening units at the age of weeks old and were confined in small groups from four to six animals per pen. because of a concomitant welfare study [ ] , calves had to stay to weeks without any external stress despite the farmer's presence. at the age of weeks old, calves were finally sent to the slaughterhouse. consequently, sampling was done at the ages of weeks, weeks, and weeks (table ). these points of sampling corresponded to the beginning, the middle and the end of the fattening period. fecal samples were collected and shipped by a veterinarian. collectors respected the following criteria: use of a single pair of latex gloves per animal, a single plastic sterile cup per animal, and collection of at least g of feces per sample. feces were collected directly from the rectum of each animal and stored at °c in potassium dichromate ( . % wt/vol) until processed. cups were capped, labeled with the animal's ear tag number, and accompanied by a form recording the date of sampling, the animal's sex, breed, identification number, and the mean age of the herd. after washing steps in water to eliminate potassium dichromate from the samples, dna was extracted according to the method previously described [ ] without the cetyl trimethylammonium bromide (ctab) and polyvi-nylpyrrolidone (pvp) treatment steps. an s rna gene fragment was amplified by nested pcr according to xiao et al. [ ] . the partial gp gene was amplified according to gatei et al., [ ] . pcr products were analyzed on % agarose gel and visualized by ethidium bromide staining. to ensure purity and limit the presence of pcr inhibitors, all pcr-negative samples were reprocessed. samples were treated for oocyst purification by immunomagnetic separation (dynabeads ® anti-cryptosporidium, invitrogen ™, norway) according to the manufacturer's instructions. these samples were finally processed as previously for dna extraction and pcr amplification. cryptosporidium species identification pcr products were purified on an ultracel ym membrane (microcon, millipore, bedford, ma, usa) according to the manufacturer's instructions. dna sequencing reactions were performed using internal primers of the nested pcr with the abi prism big dye terminator cycle sequencing kit (applied biosystem, foster city, ca, usa). capillary electrophoresis was performed by genoscreen (lille, france). sequences were analyzed using blast at ncbi [ ] . the prevalence of cryptosporidium infection on farms from four administrative regions in brittany (france) was studied ( figure ). all cryptosporidium-positive specimens generated the expected ssu-rna products in nested pcr and revealed that no farm was free of cryptosporidium. the molecular analysis of fecal samples revealed that ( . %) were positive for cryptosporidium. as shown in table , the overall prevalence of infected animals was . % ( / ) and ranged from % on a farm in morbihan (mo ) to % on farms in ile-et-vilaine (iv , iv ) and in mayenne (ma ). amongst the specimens sampled from -week-old and -week-old animals, cryptosporidium prevalence was . % and . %, respectively (range, %- . %). in -week-old calves, the prevalence decreased to . % (range, %- . %). the prevalence of infection decreased as the age of the calves increased. for species identification, the positive nested pcr products were sequenced. sequence analysis from readable electrophoregrams revealed the presence of c. parvum, c. bovis, and c. ryanae. one additional cryptosporidium genotype showing % identity with cryptosporidium ubiquitum (eu ) (previously identified as * a calf is considered to be positive if at least one out of the three samples is positive. **the number of animals is because one calf died between the age of and weeks. cryptosporidium cervine genotype [ ] ) was detected in one calf. this sequence was deposited in genbank under the accession number gu . sixty ( . %) samples were identified as c. parvum as follows: forty-six sequences had % identity with the genbank af nucleotide sequence, had % identity with the af nucleotide sequence and three had % identity compared to both references. these sequences were deposited in genbank under the accession numbers gu to gu . for the other positive specimens, ( . %) were identified as c. ryanae (previously described as cryptosporidium deer-like genotype). thirtyone of these had % identity with the ay sequence [ ] and eight were % identical to this reference. these nucleotide sequences were deposited in gen-bank under the accession numbers gu to gu . for the last positive samples, ( %) had an identical nucleotide sequence with c. bovis (genbank accession number, ay ) formerly known as the cryptosporidium bovine b genotype. within these sequences, had % identity to the reference deposited in genbank, three sequences had % identity. these last sequences were deposited in genbank under the accession numbers gu to gu . prevalence of c. parvum, c. ryanae, and c. bovis in relation to calf age the distribution of cryptosporidium species identified in animals at the age of , , and weeks is shown in figure . the prevalence of each species changed with the age of the calves. c. parvum prevalence was . % in the -week-old calves and decreased to . % in -week-old animals. this species was not identified in -week-old calves. c. ryanae and c. bovis were identified in -weekold calves in . % and . % of the specimens, respectively. the prevalence of these species in -week-old animals increased to . % and . %, respectively. this prevalence evolved to % and % in -week-old animals. the presence of one, two, or three species of cryptosporidium was determined in each animal (n = ) for which the sequences were readable in all positive samples. three calves positive for c. parvum at the age of weeks were excluded because cryptosporidium species could not be identified in all of the following samples collected in these animals. as shown in in the time lapse of this study, % of the animals ( / ) were found to excrete two different species of cryptosporidium successively. indeed, . % ( / ) produced c. parvum and c. ryanae, . % ( / ) excreted c. parvum and c. bovis, and . % ( / ) excreted c. ryanae and c. bovis. finally, . % ( / ) of the animals studied were detected to produce c. parvum, c. ryanae, and c. bovis. the subtyping analysis was performed on c. parvum positive specimens. from targeted samples, could be used for sequence analysis. as shown in table , all alleles identified belong to the iia family. the most common subtype iiaa g r ( % identity with reference strain ab ) was found in out of samples ( . %). six samples ( . %) were typed as subtype iiaa g r ( % identity with reference strain gq ), three samples ( . %) as subtype iiaa g r ( % identity with reference strain dq ) and two samples ( . %) as subtype iiaa g r ( % identity with reference strain dq ). finally one sample ( . %) was subtyped as iiaa g r ( % identity with reference strain dq ) and another one ( . %) as subtype iiaa g r ( % identity with reference strain dq ). discussion calves under month of age are frequently infected with cryptosporidium sp [ ] which results in economic loss [ ] . in france, up to date, the prevalence of cryptosporidium in diarrheic calves has been studied only by elisa and microscopic strategies [ , , ] . no data are available on a molecular basis to study cryptosporidium species in calf herds in that country. the present study based on s rdna and gp gene analysis is the first in france to include molecular characterization to describe the prevalence and the host age related susceptibility to different cryptosporidium species after a follow up of the same animal. our results showed that all fifteen farms were contaminated with cryptosporidium. the parasite prevalence on farms ranged from % to % of the sampled animals. this observation was in accordance with results in michigan (usa) where this parameter ranged from % to % [ ] . the prevalence of . % obtained in this work tended toward the upper end of the scale compared to other investigations done in france which ranged from . % in beef herds [ ] to % in suckling calves [ ] and in other european countries where prevalence ranged from . % to % [ , ] . however, the sampling program did not allow the study of animals under weeks of age. indeed, the animals arrived in these structures at the age of to weeks and farmers did not allow sampling before two complete resting weeks for each animal. therefore, our results could underestimate the real prevalence as huetink et al. showed that the percentage of parasite excreting animal declines after the third week of age [ ] and that the first peak of prevalence is at the age of days [ ] . in our study, the higher prevalence of cryptosporidiosis was observed in calves weeks old ( . %) and the lowest ( . %) in the -week-old animals. this observation shows that prevalence of cryptosporidium infection decreases with increasing age of the cattle in france as in many other countries [ , , [ ] [ ] [ ] [ ] [ ] [ ] . additionally, our data confirmed the presence in france of a host age-related susceptibility to the infection with different cryptosporidium species. c. parvum was predominantly detected in -week-old calves ( . %) compared to c. ryanae or c. bovis detected in . % and . % of the positive samples respectively. it is noteworthy that these results are very similar to data obtained in ireland on calves under days of age with %, . %, and . % of prevalence of the same species, respectively [ ] and in the uk on animals over weeks old with % c. parvum, % c. bovis, and % c. ryanae [ ] . in contrast to previous studies [ , ] , c. ryanae and c. bovis were found with similar prevalence predominantly in week and week old calves. this association between the age of the cattle and the cryptosporidium species identification has been supported by several studies [ , , , , ] but different reports suggest that cryptosporidium species repartition regarding the age of the host could be due to a technical artifact. despite the fact that the methodological strategy based on pcr using genus specific primers and partial direct sequencing of the s rdna is commonly used to identify cryptosporidium species [ ] , this molecular tool is limited in the case of mixed infections. feng et al., [ ] suggested that the important shedding of c. parvum in preweaned calves had probably masked the concurrent infection of these animals by c. bovis or c. ryanae. furthermore, previous reports suggested that a dominant cryptosporidium species in a sample can be preferentially amplified by pcr [ , ] . it is noteworthy that this situation of mixed cryptosporidium species infection in farm animals would be more prevalent than originally believed [ ] [ ] [ ] . mixed cryptosporidium species could also explain sequencing difficulties encountered in this work. the simultaneous presence of several species in the same sample could lead to amplification and sequencing of different genetic fragments leading to unreadable superimposition of electrophoregrams. consequently, in our work based on the utilization of cryptosporidium generic primers, the amplification of a single fragment with a single sequence is not conclusive evidence that the sample contains only a single species. however, based on our results, it is possible to confirm the predominance of different species of cryptosporidium by group of age among the calves. particularly, our data showed that animals can be sequentially infected with c. parvum, c. ryanae and c. bovis as well as c. parvum, c. bovis and c. ryanae. this observation provides evidence that a previous infection with c. parvum did not protect calves against an infection with other cryptosporidium species. fayer et al. suggested that the peak of cryptosporidiosis prevalence in young calves could reflect the immaturity of the immune status [ ] . it was also suggested that the low excretion of c. parvum oocysts in older calves might be related to the development of immunity that also protected the animal against a secondary challenge [ ] . it has been reported that immunity arises in the first two weeks after infection [ ] . interestingly, fayer et al. [ ] described that calves previously challenged with c. parvum were able to excrete oocysts after a second challenge with c. bovis but not with c. parvum. the authors concluded that immunity to c. parvum was not extended to c. bovis. consistently, in our study, the presence in the same animal during sequential sampling of c. parvum, c. bovis and c. ryanae suggests that immunity against c. parvum and against c. bovis did not extend to c. ryanae. furthermore, the observation that one animal excreted sequentially c. parvum, c. ryanae and c. bovis suggests that immunity against c. ryanae did not extend to c. bovis as well. finally, the risk to human health posed by cryptosporidium infected cattle in france was assessed. the detection of c. ubiquitum (a rare infectious agent detected in humans [ ] ), c. ryanae and c. bovis (which are mainly specific for cattle) led to consider that the -week-old calves are not likely a public health concern. however, the major detection of c. parvum, a prevalent zoonotic species, in -week-old calves was in agreement with the report of atwill et al., who considered that the contribution of cattle to human cryptosporidiosis is limited to calves under months of age [ ] . to determine c. parvum subtypes, the sequence analysis of a fragment of the gp gene was done. our results show that in the region of brittany, all identified c. parvum gp subtypes belonged to the iia family which was previously found in both animals and humans [ ] . particularly, human infections with the iia subtype are commonly seen in areas with intensive animal production [ ] . among the gp subtypes formerly described in cattle [ ] , only six were identified in this work, being iiaa g r the most commonly found. this subtype has been widely reported in calves and humans in different countries such as in portugal [ ] , slovenia [ ] and the netherlands [ ] . this observation confirms previous works and suggests a zoonotic transmission of the parasite also in this region. it is noteworthy that the three predominant subtypes (iiaa g r , iiaa g r , and iiaa g r ) found in this work were also described in cattle with an equivalent distribution in the netherlands [ ] and england [ ] . thus, the subtype iiaa g r was found in . % of the samples in this work, . % in the netherlands and . % in england. the iiaa g r was identified in . % of the samples in this report, . % in the netherlands and . % in england. the iiaa g r determined in . % of our samples, was characterized in . % in the netherlands and . % in england. it is remarkable that subtypes, iiaa g r , iiaa g r and iiaa g r were equivalently underrepresented in these three countries. this observation could suggest that the proportion of a gp subtype would not be randomly represented in a population. finally, the zoonotic transmission assessment of c. parvum in france would require a comparative investigation of variable genetic loci both in human and animal samples. this is the first report on the molecular identification of cryptosporidium species or genotypes in veal calves in france. according to data reported previously in many countries, a sequential distribution of species is observed in cattle according to age. c. parvum was mainly observed in the youngest calves, while c. ryanae and c. bovis became predominant in stool specimens collected in older animals. in some cases, several cryptosporidium species were successively detected in the same calf, suggesting that the immune defense against c. parvum is not efficient against c. ryanae or c. bovis. finally, the major identification of the iiaa g r subtype in france suggests that -week old calves could be a reservoir for zoonotic parasites transmissible to humans. fayer r: taxonomy and species delimitation in cryptosporidium a massive outbreak in milwaukee of cryptosporidium infection transmitted through the public water supply the cell biology of cryptosporidium infection advances in the epidemiology, diagnosis and treatment of cryptosporidiosis zoonotic cryptosporidiosis in the uk -challenges for control cryptosporidium and giardia-zoonoses: fact or fiction? involvement of insects in the dissemination of cryptosporidium in the environment cryptosporidial infection in a calf cryptosporidiosis in neonatal calves: cases ( - ) a review of the importance of cryptosporidiosis in farm animals apicomplexa: cryptosporidiidae) in sheep (ovis aries) re-description of cryptosporidium cuniculus inman and takeuchi, (apicomplexa: cryptosporidiidae): morphology, biology and phylogeny cryptosporidium ubiquitum n. sp. in animals and humans epidemiology of anthroponotic and zoonotic human cryptosporidiosis in england and wales genetic analysis of cryptosporidium from humans with diarrhoea in england between and prevalence of species and genotypes of cryptosporidium found in - -year-old dairy cattle in the eastern united states prevalence and agerelated variation of cryptosporidium species and genotypes in dairy calves a longitudinal study of cryptosporidiosis in dairy cattle from birth to years of age wide geographic distribution of cryptosporidium bovis and the deer-like genotype in bovines prevalence of and management factors contributing to cryptosporidium sp. infection in pre-weaned and post-weaned calves in johor maddox-hyttel c: molecular and phylogenetic characterization of cryptosporidium and giardia from pigs and cattle in denmark prevalence of cryptosporidium infection in calves in france role of cryptosporidium parvum as a pathogen in neonatal diarrhoea complex in suckling and dairy calves in france prevalence of gastrointestinal disorders recorded at postmortem inspection in white veal calves and associated risk factors molecular characterization of cryptosporidium isolates obtained from humans in france identification of species and sources of cryptosporidium oocysts in storm waters with a smallsubunit rrna-based diagnostic and genotyping tool development of a multilocus sequence typing tool for cryptosporidium hominis basic local alignment search tool prevalence of cryptosporidium and giardia infections in cattle in aragon (northeastern spain) pattern of diarrhoea in newborn beef calves in south-west france genetic diversity of cryptosporidium spp. in cattle in michigan: implications for understanding the transmission dynamics risk factors associated with cryptosporidium parvum infection in cattle cryptosporidium and giardia in different age groups of danish cattle and pigs-occurrence and management associated risk factors epidemiology of cryptosporidium spp. and giardia duodenalis on a dairy farm molecular characterization and assessment of zoonotic transmission of cryptosporidium from dairy cattle in west bengal prevalence of cryptosporidium parvum infection in punjab (india) and its association with diarrhea in neonatal dairy calves high prevalence of cryptosporidium bovis and the deer-like genotype in calves compared to mature cows in beef cow-calf operations molecular characterization of cryptosporidium spp. in native breeds of cattle in kaduna state genotypes and subtypes of cryptosporidium spp. in neonatal calves in northern ireland molecular epidemiology of cryptosporidium subtypes in cattle in england cryptosporidium genotype and subtype distribution in raw wastewater in shanghai, china: evidence for possible unique cryptosporidium hominis transmission molecular epidemiology of cryptosporidiosis: an update cryptosporidium parvum mixed genotypes detected by pcr-restriction fragment length polymorphism analysis identification of genotypically mixed cryptosporidium parvum populations in humans and calves new view on the agespecificity of pig cryptosporidium by species-specific primers for distinguishing cryptosporidium suis and cryptosporidium pig genotype ii a multiplex polymerase chain reaction assay to simultaneously distinguish cryptosporidium species of veterinary and public health concern in cattle characteristics of cryptosporidium transmission in preweaned dairy cattle in henan cryptosporidium parvum infection in bovine neonates: dynamic clinical, parasitic and immunologic patterns resistance of calves to cryptosporidium parvum: effects of age and previous exposure cryptosporidium parvum in calves: kinetics and immunoblot analysis of specific serum and local antibody responses (immunoglobulin a [iga], igg, and igm) after natural and experimental infections apicomplexa: cryptosporidiidae) in cattle (bos taurus) genotype and subtype analyses of cryptosporidium isolates from dairy calves and humans in ontario age, geographic, and temporal distribution of fecal shedding of cryptosporidium parvum oocysts in cow-calf herds distribution of cryptosporidium subtypes in humans and domestic and wild ruminants in portugal genetic richness and diversity in cryptosporidium hominis and c. parvum reveals major knowledge gaps and a need for the application of "next generation" technologies-research review genetic classification of cryptosporidium isolates from humans and calves in slovenia molecular epidemiology of cryptosporidium in humans and cattle in the netherlands submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution this study was supported by the catholic university of lille through the "projet grande campagne ensemble innovons" genotyping program. we would like to thank the veal unit managers who participated in this study. authors' contributions jf and kg participated in the conception and design of the study, carried out the experiments and drafted the manuscript. hl designed the sampling strategy and collected samples on farms. jf, kg and afd designed the protocol for molecular assay and participated in the analysis result. ohg carried out molecular assays. edc, gc and ph participated in the coordination of the study and helped draft the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -mukc mxi authors: philippe, p.; alzieu, j.p.; taylor, m.a.; dorchies, ph. title: comparative efficacy of diclazuril (vecoxan(®)) and toltrazuril (baycox bovis(®)) against natural infections of eimeria bovis and eimeria zuernii in french calves date: - - journal: vet parasitol doi: . /j.vetpar. . . sha: doc_id: cord_uid: mukc mxi a blinded, randomized, controlled, multi-centric field study was conducted on french dairy farms (n = ) to evaluate the long term efficacy of metaphylactic, single oral treatments with either mg/kg body weight (bw) of diclazuril (vecoxan(®)), or mg/kg bw of toltrazuril (baycox(®)) against natural infections with eimeria zuernii and/or eimeria bovis, compared to untreated control animals. a total of calves from nine commercial farms aged between and days old at the start of study were included and randomly allocated to one of three groups. calves on all farms were observed for a period of days post treatment, using both parasitological (oocyst excretion), and clinical parameters (faecal score and body weight). the assessment of efficacy was based on both control of oocyst excretion, and on the average daily weight gains throughout the study. during the whole study period, the mean number of days with diarrhoea (≥ ) was similar ( . days) between treated groups. excretion in the untreated group peaked at days after treatment. in both the diclazuril and toltrazuril-treated groups, mean oocyst excretion decreased dramatically in the five days following treatment. thereafter, particularly towards the end of the study period, oocyst counts and percentage levels of e. zuernii were highest in the toltrazuril-treated group. in pooled data from all trial sites, the average daily weight gain was significantly (p = . ) higher (+ . kg/day) in the diclazuril group when compared to the toltrazuril group, and the average body weight gain of the diclazuril treated group was . kg higher than the toltrazuril group. on eight of the nine trial sites, the average daily gain was greater in the diclazuril group than in the toltrazuril group. this study demonstrates that, over an extended observation period of days, metaphylactic treatment with both diclazuril and toltrazuril reduces the impact of coccidiosis, but greater performance benefits based on average daily weight gains, were achieved following the use of diclazuril. historically, bovine coccidiosis was considered to be a severe infection only during the calf weaning period and for a few weeks thereafter. in the weeks prior to weaning, rotavirus, coronavirus, escherichia coli and other pathogens were considered to be the most common causes of diarrhoea and few considered coccidiosis as a severe concern for calves younger than month of age. more recently, several surveys have shown that very young calves (from birth to months old) were infected by eimeria bovis and eimeria zuernii, the most pathogenic coccidia of cattle (daugshies and najdrowski, ) , and that poor hygiene, overcrowding and stressful situations (transport, weaning etc.) increases the risk of contracting coccidiosis. during the days following birth, calves can be infected with cryptosporidium parvum and sometimes, during the first week of life, by giardia intestinalis and strongyloides papillosus. by the end of the first month, coccidia are prevalent and numerous oocysts are expelled in the faeces and recycle quickly (alzieu et al., ) . maximum intensity of coccidiosis occurs between week and week after birth (jäger et al., ; sánchez et al., ) . several recent papers have stressed that bovine coccidiosis is a global problem, spreading infection equally in dairy and beef calves. intestinal damage is common with subsequent symptoms of decreased weight gain, weakness and secondary bacterial infection frequently reported (daugshies and najdrowski, ; von samson-himmelstjerna et al., ; bangoura and daugschies, ) . coccidiosis is generally diagnosed by faecal examination in order to detect oocysts and it is greatly recommended that the pathogenic species are identified if present. for reporting purposes, most parasitologists consider the presence of e. zuernii and/or e. bovis oocysts to be more important than their numbers (jolley and bardsley, ) ; as there is no strong correlation between the numbers of oocysts shed and severity of clinical signs . practitioners must distinguish between viral, bacterial and parasitic agents involved in the development of gut diseases of very young calves . haemorrhagic diarrhoea in coccidiosis is not very common but mild infection by the most pathogenic coccidia, e. zuernii and e. bovis, impairs nutrient absorption and causes dramatic changes in the villous architecture with a reduction in epithelial cell height and a decrease in extent of the brush border (mundt et al., ) . these lesions result in a reduction in surface area available for absorption and consequently a decreased feed efficiency (taylor et al., ) . infected animals develop an effective protective immunity to homologous re-infection (sühwold et al., ) . increased levels of interferon-gamma in primary infection, appears to be a key feature of coccidiosis control (taubert et al., ) . the economic consequences of coccidiosis of calves must be controlled. unfortunately, it is too late to intervene after the appearance of first clinical signs as significant tissue damage has already occurred. timing of treatment with respect to the impact on growth is therefore critical. as well as this, infected animals spread millions of oocysts and heavily contaminate their environment, spreading infection to younger calves. anticoccidial products are often used for controlling coccidiosis in calves. however, only a few drugs (the benzene acetonitriles -diclazuril, toltrazuril) have great efficacy against pre-existing infection with pathogenic eimeria spp. the desired scenario would be to kill the parasite but also to allow the development of natural immunity (agneessens et al., ; taylor et al., ) . several studies have confirmed the efficacy of these drugs on bovine coccidiosis by trials using either diclazuril (daugshies and najdrowski, ; daugschies et al., ; romero et al., ) , or toltrazuril (bohrmann, ; mundt et al., ; epe et al., ; mundt et al., ) ; or comparing both (mundt et al., ; veronesi et al., ) . in all but one study (veronesi et al., ) , the monitored periods were comparatively short, typically less than days. the aim of the present study was to investigate the effectiveness of metaphylactic treatments with diclazuril (vecoxan ® ) and toltrazuril (baycox bovis ® ) against natural infections with e. zuernii and/or e. bovis in dairy calves over a longer ( day) period. between august and march , nine commercial dairy farms in brittany, france were selected based on their previous history of coccidiosis outbreaks. more detailed investigations were then conducted as blind, randomized, controlled, field trials in accordance with "good clinical practice" (vich guideline gl ) and national animal welfare requirements. on each trial site, three groups of calves were randomly assigned to three different preventive treatments for coccidiosis, in separate pens (table ) : • group a: diclazuril treated once mg/kg body weight (vecoxan ® . % suspension, elanco animal health) • group b: toltrazuril treated once with mg/kg body weight (baycox bovis ® % suspension, bayer animal health) • group c: untreated controls. calves in groups a and b were treated close to the time of expected observation of clinical coccidiosis, usually weeks after the grouping of animals in collective pens, where coccidiosis is endemic. treatments were given on study day (sd ), i.e. days after grouping in collective pens. a total of one hundred and ninety nine healthy calves (primarily holstein and montbeliard) from the brittany area (france) were randomly selected from nine dairy farms ( - calves per farm). twenty two sequences were conducted in total. between and calves were included in each sequence, and these were divided into three groups (a, b and c) containing the same numbers of treated animals, with one untreated calf as a sentinel control in the ratio : : ; or : : ; or : : depending on the number of calves per farm. before the inclusion into a sequence, calves were housed in individual pens. two weeks before treatment, they were moved to collective pens. on study day (sd ), calves in each treatment group were randomly selected according to the criteria of age, sex, body weight and breed. from sd to the end of study, the calves were housed in collective pens, separated according to treatment groups. all calves were individually monitored from sd until sd (i.e. the end of the trial). none of the calves had received anti-coccidial treatment prior to the beginning of the trial. at the beginning of the study (sd ), diclazuril and toltrazuril-treated groups (a and b) were not statistically different with regards to age, oocyst excretion, body weight, sex or faecal score (p > . ) ( table ). all calves were submitted to three complete health checks with individual weights recorded on sd , sd and sd . individual body weights were quantified by weighing the calves on a weighing scale, calibrated at each use. from sd to sd , individual rectal faeces samples were taken twice weekly, and faecal consistency was evaluated immediately on site, according to a faecal score (fs) using a scoring system ( = no diarrhoea, normal faeces; = pasty faeces to semi-liquid; = watery faeces; = watery faeces with blood; = watery faeces with blood and tissues). diarrhoea was present when fs was ≥ . oocyst counts were performed using a modified mcmaster method, mixing g of faeces per ml of saturated nacl solution (specific gravity = . ). the faecal oocyst counts were evaluated and species identified directly, according to the size and the shape of nonsporulated oocysts (taylor et al., ) . for pathogenic coccidia, e. bovis and e. zuernii, faecal excretion of opg or more is usually accepted as the threshold indicating coccidia-induced diarrhoea (mundt et al., ) . data collected at the individual trial sites were pooled and analyzed descriptively and statistically. nonparametric statistical tests were used to perform comparisons between groups. all qualitative data (qualitative oocyst excretion, incidence and prevalence of diarrhoea) were tested with fisher's exact test. the quantitative data (quantitative oocyst excretion, body weight, age) were tested with the mann-whitney test. growth performance was analyzed by calculating weight gain between the end of the study period (sd ) and the day of treatment (sd ). a multiple linear regression model with mixed effect was used to compare average daily gain between groups. effects were considered significant at an error level of % (p = . ) or lower. in all of the trial sites, all control calves expelled oocysts at each faecal examination. highest mean counts in this group were observed between sd to sd ( fig. ) , and then decreased. oocyst differentiation of samples taken from the control calves indicated that e. bovis predominated during the initial period but then declined. the pattern of e. zuernii oocyst excretion followed that of e. bovis, but fluctuated in higher numbers and percentage presence throughout the study period ( figs. and ) . oocyst excretion for other (non-pathogenic) eimeria species also fluctuated throughout, suggesting that there was exposure to several different species over time. total oocyst, e. bovis and e. zuernii output for the two treatment groups are also given in figs. and , with the of eimeria species present shown in fig. . the percentages table observational findings for treatment groups at the start of the trials (sd ). group of calves excreting oocysts of the two pathogenic coccidia species (e. zuernii and e. bovis > opg) in both treatment and control groups, are given in fig. . although it was apparent that less than % of treated calves were infected on sd , the numbers of calves excreting oocysts in the two treated groups dramatically decreased in the five days following treatment. from sd to sd , the percentage of calves excreting oocysts in group b (toltrazuril), was significantly (p < . ) lower than for calves in group a (diclazuril). from sd to sd , less differences were observed, except for sd , sd , sd and sd when the percentage of calves in group a (diclazuril) excreting oocysts were significantly (p < . ) lower than for group b calves (toltrazuril). two months after treatment (sd ), almost % of toltrazuril-treated calves were shedding oocysts of the pathogenic coccidia species (e. zuernii + e. bovis > opg), the largest proportion of which were e. zuernii (figs. - ) . during the entire study period, the mean number of days with diarrhoea (faecal score ≥ . ) in the treated groups was low (< %). there was similarity ( . days, p = . ) shown between treated groups but the untreated controls displayed higher ( . days) days of diarrhoea with a faecal score greater than . . in the diclazuril, and toltrazuril-treated groups, the percentage of calves showing diarrhoea decreased from sd to sd (fig. ). there were no statistical differences between the treatment groups. for all trial sites, the average body weight gain of group a calves (diclazuril) was . kg higher than the group b calves (toltrazuril). using a multiple linear regression model with mixed effect (data normally distributed, shapiro-wilk normality test, p-value = . ), the diclazuril treated calves had an average daily gain (adg) of . kg/day, significantly (p = . ) higher compared to calves in the toltrazuril group over the weeks post-treatment observation period. between sd and sd , the adg was not significantly different between the two treatment groups, whereas adg of the group a (diclazuril) calves was higher ( g/day) between sd and sd when compared to the group b (toltrazuril) calves (fig. ) . the adgs differed noticeably between trial sites, which were most likely due to differing breeds and farm management practices (fig. ). in eight out of nine trial sites, a higher adg was recorded for diclazuril-treated calves in comparison to toltrazuril-treated calves. statistical analysis was not significant at the individual farm level, due to the limited number of calves included in each trial site. published studies dealing with the control of bovine coccidiosis confirm that benzene acetonitrile compounds are effective against various life cycle stages of eimeria and consequently, and are well suited for metaphylactic treatment (bohrmann, ) . the timing of treatment is very important, particularly for dairy calves because they typically spend their first or weeks of life in individual pens before being gathered with other calves into infected yards, often on different premises. at this time, calves are exposed to multiple stresses and subsequently are prone to bacterial, viral and parasitic infections. it is well known that coccidiosis treatment at such a time is only curative and retrospective. by the time of treatment, it is too late to avoid the development of extensive intestinal lesions. consequently, most practitioners will administer metaphylactic treatment between and days after movement of calves on dairy farms. in this study, the inclusion of sentinel, control calves confirms a high level of oocyst excretion at sd , i.e. days after introduction onto their respective farms, and the presence of oocyst counts > opg comprising approximately % pathogenic eimeria species (fig. ) . these results fully justify the timing of metaphylactic treatment with one of the benzene acetonitrile anticoccidial products. ideally, administration of these drugs after the first infection should allow the development of immunity protecting against new severe coccidia infections. four main criteria allow the comparison of trial results of benzene acetonitriles (bohrmann, ; mundt et al., mundt et al., , mundt et al., , daugshies and najdrowski, ; epe et al., ; daugschies et al., ; veronesi et al., ; romero et al., ) : ( ) the duration of the study, ( ) fig. . average daily weight gain (kg/day) at each trial site. oocyst excretion of pathogenic species, ( ) diarrhoea score and ( ) body weight gain or/and the adg. most previous studies have considered that the goal of treatment is suppression of oocyst excretion, i.e. parasitological criteria, which cannot be evaluated directly in the field. in this study, clinical criteria (diarrhoea and growth performance) have also been considered as additional useful indicators for monitoring treatment efficacies. compared to previous reported studies, this study brings the results of a longer monitoring period ( days post treatment) along with higher numbers of calves included in the trial. the mean age of calves at the beginning of the trial was days and at the end of the trial over days; i.e. during the period of highest susceptibility to clinical coccidiosis (taylor and catchpole, ). this has not been the case in previously described studies comparing toltrazuril and diclazuril. one study gives a very short follow-up period; only days (mundt et al., ) ; the other study (veronesi et al., ) had two groups of calves treated with both drugs, and a non-treated control group monitored over a period of weeks, but with no data provided on the kinetics of oocyst excretion. the results of this trial cannot therefore be easily compared with other, shorter duration trials due to the fact that calves are reportedly, typically susceptible to clinical coccidiosis between weeks to six months in the field (taylor and catchpole, ) and the fact that it is very difficult to attribute the effects of a single metaphylactic treatment weeks later. kinetics of the comparative studies of mundt et al. ( ) and those of this trial are very different because of the respective study periods. in this study, oocysts disappeared from faeces for five days post-treatment, and re-appeared more rapidly for the group a (diclazuril) calves compared to the group b (toltrazuril) calves. however, during the second part of the study (sd to sd ), the levels of oocyst excretion for group b (toltrazuril) calves then increased when compared to the group a (diclazuril) treated calves. during the initial challenge period, the predominant pathogenic species present was e. bovis, which then declined, only to be superseded by e. zuernii. levels of the non-pathogenic eimeria species fluctuated throughout. towards the end of the study period, however, oocyst counts and the percentages of pathogenic species with opg > opg were higher in the toltrazuril-treated group, with e. zuernii predominating. we summarise that the higher levels of protection provided immediately post-treatment, had prevented the development of species-specific immunity to e. zuernii in the toltrazuril-treated group leading to sub-clinical coccidiosis and an impact on growth. due to the published persistency of toltrazuril, these observations are highly unlikely to have been observed in previously published trials of shorter duration. farmers are used to treating calves as soon as diarrhoea is observed in a group of young calves. similar to other studies (daugshies and najdrowski, ; mundt et al., ) , there was no significant correlation shown between diarrhoea and oocyst excretion in this trial. the present study reports a mean duration of diarrhoea of only . days after treatment. this result is similar to those reported by mundt et al. ( ) in all trial groups without showing any significant difference. this criterion is one of the most critical from a clinical perspective, allowing an objective evaluation (clinical criteria) of the consequences of metaphylactic treatment. in this -day study, the diclazuril-treated group (group a) had a mean body weight of . kg greater than the toltrazuril-treated group (group b). unlike the findings of mundt et al. ( ) , we demonstrate that adg is significantly higher (+ . kg/day (p = . )) in the diclazuril-treated group (group a) compared to the toltrazuril-treated group (group b). this finding is most probably the result of the longer study period; the main effect of metaphylactic treatment being observed one month after administration. the most important factor for farmers is a higher growth rate. here, it must be stressed that these results was observed within the period of reported highest susceptibility of calves to coccidiosis ( weeks to months of age) when reared under commercial farm conditions. interestingly, it is possible to differentiate between two periods in this study: a) between sd and sd : despite a higher percentage of calves excreting oocysts in the diclazuril-treated group a calves, the adg and the faecal score were similar between the two groups. b) between sd and sd : when the percentage of calves excreting oocysts increases in the toltrazuril-treated group b; and the adg is higher for the diclazuril-treated group a calves than for the toltrazuril-treated group b calves. this latter period comprises the total difference in adg between both groups. it may appear surprising with particular regard to the concept of metaphylactic treatment in controlling coccidiosis in older calves that the suppression of oocyst excretion over a longer period of time (in the toltrazuril-treated calves) does not correlate with higher growth performance or lower diarrhoea. epidemiological studies (jäger et al., ; sánchez et al., ; alzieu et al., ) show that younger calves are more susceptible to coccidiosis, with a peak average of excretion prevalence between and days of age. neonate calves are immunologically naïve at birth; the development of the immune system in calves progresses from conception to maturity at approximately months after birth. in order to enhance the adaptive immune response, newborn calves need to "experience" pathogens (chase et al., ) . consequently, persistent or longer-acting treatments against coccidiosis in young calves could delay development of the immune response; and in this respect the toltrazuril-treated calves in this study displayed a peak of excretion prevalence days after treatment, suggesting a detrimental effect on growth performance. it has previously been surmised that treatments with a benzene acetonitrile compound may have an effect on the development of immunity to coccidial infection in cattle and that if a treatment did interfere with the acquisition of immunity following subsequent reexposure, then severe disease may follow (jonsson et al., ) . a study conducted by taylor et al. ( ) with diclazuril given at mg/kg bw (recommended dose) to lambs, demonstrated that at this dose level the treatment was effective in preventing coccidiosis in lambs, but at higher dose levels ( . and . mg/kg bw) it appeared to completely clear the lambs of coccidia rendering them susceptible to re-infection. due to differences in pharmacokinetic properties between diclazuril and toltrazuril, we can hypothesize that differences in dose rate (diclazuril mg/kg; toltrazuril mg/kg) and subsequent periods of activity, could explain why toltrazuril-treated calves appear to be more susceptible to infection from days post treatment compared with diclazuril-treated calves, as demonstrated by the differences in growth performance and increased levels of oocyst excretion. this study confirms that benzene acetonitrile drugs are effective at controlling the pathogenic coccidia, e. bovis and e. zuernii. the choice of correct timing for the beginning of preventive treatment must be carefully determined based on history and farm management. for most calves, cases of coccidiosis typically develop days after stressful events or movement on, or between farms. oocyst counts on their own are insufficient in evaluating the efficacy of metaphylactic treatments, and speciation should also be undertaken. in this study, monitoring adg over a longer post-treatment period identified an advantageous effect for metaphylatic treatment of dairy calves with diclazuril. the shorter duration of action appeared to allow a better degree of exposure to sufficient numbers and species of eimeria for subsequent species-specific protective immunity to develop. the study reported herein was funded by elanco animal health (neuilly sur seine, france). with the exception of dr philippe, who is a paid employee of elanco animal health, the remaining authors have no conflicting interests that may have biased the work, and were involved with the study design, study conduct, data analysis and review of the manuscript. build up of immunity after a diclazuril (vecoxan ® ) treatment in calves prevalence of strongyloides papillosus and coccidia in young calves in france: concurrent infections are a serious threat for the health parasitological and clinical parameters of experimental eimeria zuernii infection in calves and influence on weight gain and haemogram treatment with toltrazuril in natural outbreak of coccidiosis in calves neonatal immune development in the calf and its impact on vaccine response eimeriosis in cattle: current understanding the effect of a metaphylactic treatment with diclazuril (vecoxan ® ) on the oocyst excretion and growth performance of calves exposed to a natural eimeria infection parasitologie clinique des bovins efficacy of toltrazuril as a metaphylactic and therapeutic treatment of coccidiosis in first-year grazing calves endoparasites of beef cattle herds: management systems dependent and genetic influences efficacy of toltrazuril % suspension against eimeria bovis and eimeria zuernii in calves and observations on the associated immunopathology efficacy of toltrazuril against artificial infections with eimeria bovis in calves pathology and treatment of eimeria zuernii coccidiosis in calves: investigations in an infection model control of coccidiosis du to eimeria bovis and eimeria zuerni in calves with toltrazuril under field conditions in comparison with diclazuril and untreated controls metaphylactic effect of diclazuril . % in suckling beef calves, during a coccidiosis outbreak in extensive farming dynamics of eimeria oocyst excretion in dairy calves in the province of buenos aires (argentina), during their first months of age tcell reactions of eimeria bovis primary and challenge-infected calves antigeninduced cytokine production in lymphocytes of eimeria bovis primary and challenge infected calves dose response effects of diclazuril against pathogenic species of ovine coccidian and the development of protective immunity coccidiosis of domestic ruminants long-term effect of toltrazuril on growth performances of dairy heifers and beef cattle exposed to natural eimeria zuernii and eimeria bovis infections clinical and epidemiological characteristics of eimeria infections in first-year grazing cattle the authors thank magali melon and jean-loïc martin, from elanco france, for their support during the study. samplings and clinical examination were performed by local french practitioners, faecal oocysts count and species identification were tested by laboratoire vétérinaire départemental de l'ariège, rue de las escoumes, foix cdis (france). key: cord- -nux gc authors: de graaf, dirk c; vanopdenbosch, emmanuel; ortega-mora, luis m; abbassi, hayet; peeters, johan e title: a review of the importance of cryptosporidiosis in farm animals date: - - journal: int j parasitol doi: . /s - ( ) - sha: doc_id: cord_uid: nux gc cryptosporidium species are coccidian parasites with a large capacity to reproduce and to disseminate. several species are known to infect farm animals, although the economic importance of cryptosporidiosis is highly host species dependent. this paper reviews the impact of cryptosporidial infections in livestock and poultry. for different farm animals, the cryptosporidium spp. that occur, as well as their clinical and pathological features, and their interactions with other pathogens, are described. in addition, data concerning the prevalence, the transmission and the epidemiology of the disease are mentioned and a description of the economic losses associated with cryptosporidiosis in each of the hosts is given. cryptosporidiosis seems to be mainly a problem in neonatal ruminants. cryptosporidium parvum is considered to be an important agent in the aetiology of the neonatal diarrhoea syndrome of calves, lambs and goat kids, causing considerable direct and indirect economic losses. avian cryptosporidiosis is an emerging health problem in poultry, associated with respiratory disease in chickens and other galliformes, and with intestinal disease in turkeys and quails. because of limited availability of effective drugs, the control of cryptosporidiosis relies mainly on hygienic measures and good management. the genus cryptosporidium was named at the beginning of this century, but was only recognised as a potential cause of disease in , when it was found to be associated with diarrhoeic turkeys [ ] . although cryptosporidium was subsequently found in a broad range of farm animals, its impact was neglected until the early s when it was found to be a common, serious primary cause of outbreaks of diarrhoea in certain farm mammals [ , ] . the fact that cryptosporidium was found to infect humans [ ] , and could cause a life-threatening disease in immunode®cient people, especially aids patients [ ] , as well as the association of cryptosporidium with waterborne-related human outbreaks of diarrhoea [ ] , has certainly given the parasite a more widespread recognition. it has encouraged the scienti®c work on cryptosporidium in many domains, such as in the veterinary ®eld, to some extent because animal husbandry is seen as a threatening source of infection for humans by the release of tremendous numbers of resistant oocysts in surface waters. recent studies have revealed the heterogeneity of the cryptosporidium sp. infecting both humans and animals, making the causal connection between animal and human cryptosporidiosis far more complex [ ] . nevertheless, the interest for cryptosporidiosis in the veterinary ®eld arises even more from the fact that it concerns a harmful, dicult to control disease of many farm animals, that results in signi®cant economic losses. this point will be addressed in this review. the life cycle of cryptosporidium is direct and monoxenous and it follows the patterns described for other enteric coccidia, which include a merogonic cycle with two generations of meronts, a gametogonic cycle with macrogametes, microgametes and zygotes and a sporogony (outlined in [ ] ). compared with eimeria spp., the life cycle of cryptosporidium is characterised by a number of peculiarities, some of them of major importance for the establishment and spread of the infection and for the treatment of the disease: (i) exposure of cryptosporidium oocysts to reducing conditions, pancreatic enzymes and bile salts, results in a high percentage of excystation. however, in contrast to most other coccidia, cryptosporidium oocysts can liberate their sporozoites in warm aqueous solutions without any of the aforementioned special stimuli [ ] . this spontaneous excystation explains, in part, the ability of cryptosporidium to infect tissues other than the intestine, such as the conjunctiva of the eye [ ± ] and the respiratory tract [ , ± ]; (ii) the parasite develops inside the epithelial cell of the digestive or respiratory tract, although on the edge of the host cell cytoplasm and separated from it by a feeder organelle membrane. this intracellular extracytoplasmatic location is unique for the coccidia and might play a major role in the failure of many antimicrobial agents to inhibit the growth of cryptosporidium [ ] ; (iii) two stages can cause auto-infection: the recycling type i meronts and the thin-walled oocysts. consequently, in the absence of a protective immune response cryptosporidium may persist inside a single host, even without further exposure to exogenous oocysts; (iv) the thick-walled oocysts are already fully sporulated when they leave the body with the faeces and are therefore immediately infectious. thus, cryptosporidium seems to have an extraordinary reproductive ability. in addition, the oocysts can travel a considerable distance following runo [ ] , can survive for a relatively long time in an aqueous environment [ , ] , and are infectious to a wide range of animals, thus having many potential excretors [ ] . as a result, this parasite undoubtedly has an exceptional capacity to disseminate. infection by cryptosporidium spp. in cattle were ®rst reported in the early s [ ± ]. however, because of the association with other viral or bacterial enteropathogens, the role of cryptosporidium spp. as primary enteropathogens was uncertain until , when tzipori et al. [ ] attributed an outbreak of neonatal diarrhoea to cryptosporidial infection alone. in the following years methods to free the infective oocysts from other contaminating pathogens became available, which permitted the experimental demonstration that cryptosporidium was capable of causing clinical diarrhoea in calves [ , ] . in cattle two species of the genus cryptosporidium can be distinguished: cryptosporidium parvum infecting the distal small intestine, and cryptosporidium muris infecting the abomasum. substantial dierences in the size and shape of c. parvum ( . mm . mm and sperical [ , ] ) and c. muris ( . mm . mm and ovoid [ ] ) oocysts enables the two species to be distinguished readily on microscopical examination. only c. parvum has been associated with neonatal diarrhoea. cryptosporidium muris is much less prevalent and was only found in weaned calves or adult cattle [ ± ] and c. muris infection is considered to be clinically mild, aecting weight gain [ ] and milk production [ ] . the kinetics of oocyst shedding of experimentally c. parvum infected neonatal calves, revealed a prepatent and a patent period ranging from ± and ± days, respectively [ ] . in practice, oocyst excretion has been described as early as days of age, which means that calves are already susceptible for infection during or shortly after birth [ ± ] . calves raised in isolation from cryptosporidium remain susceptible to infection at older age, but the clinical signs become less severe [ ] . in neonates, a great variability was observed in the severity and duration of diarrhoea due to cryptosporidiosis, even when the animals were exposed to similar conditions. in most calves diarrhoea has already began ± days p.i., and lasted from to days [ ] . in fattening units, where the prevalence of c. parvum is known to be high ( %), up to % of the calves developed a liquid diarrhoea, that could be attributed to cryptosporidiosis [ ] . cryptosporidial diarrhoea is associated with the excretion of tremendous numbers of oocysts [ ] . high mortality due to cryptosporidiosis has been reported, even in the absence of other enteropathogens [ ] , and this would occur more often in the belgian blue±white, and the french limousin and charolais meat breeds [ ] . cryptosporidium infections are mainly concentrated in the distal small intestine but lesions were also found in the caecum and colon [ , ] , and occasionally in the duodenum [ ] . the pathological ®ndings associated with cryptosporidium are a mild to moderate villous atrophy, villous fusion, and changes in the surface epithelium (reviewed in [ ] ). further, in®ltration of mononuclear cells and neutrophils was seen in the lamina propria [ ] . after primary exposure, a/b t-cells, both cd + and cd + , and g/d t-cells accumulated in the intestinal villi, while in challenged immune animals only an increase in the number of cd + t-cells was found [ ] . respiratory cryptosporidiosis has been reported [ ] , but can be considered to be of less economic importance than the enteritic form. the neonatal diarrhoea syndrome, both in large and small (see section . ) ruminants, is a clear example of a multifactorial disease governed by a wide range of factors related to the animal, conditions of the environment and husbandry, and a variety of viruses, bacteria and protozoan parasites. in calves, enterotoxigenic escherichia coli (etec), with thermolabile and/or thermostable enterotoxins and with colonisation factors [ ] , are recognised as a common cause of diarrhoea in calves under days old. this age-dependence is caused by a diminished adherence of etec to enterocytes after the ®rst few days of life [ ] . between days and weeks of age digestive problems can mostly be attributed to c. parvum and/or a variety of viruses, with rotavirus, coronavirus and bovine viral diarrhoea (bvd) virus being the most important. data from the veterinary and agrochemical research centre in brussels, on the proportional occurence of viral enteropathogens and c. parvum in samples from diarrhoeic calves (fig. ) , highlighted the increasing importance of cryptosporidium. during the period july ±june cryptosporidium infections made up only . % ( . % as a single pathogen, . % in association) of the digestive problems, whereas the impact of cryptosporidiosis was considerably greater in ± . indeed, almost % of the diarrhoeic calves shed c. parvum; in . % of the cases cryptosporidium was the only pathogen present, and in . % it was found in association with a viral infection. for comparison: rotavirus, coronavirus and bvd-virus were found as a single pathogen in, respectively, . , . and . % of the diagnosed samples. the decreased number of cases of rotavirus, shown in fig. , is probably due to the use of commercialised rotavirus vaccines that became available halfway during the studied period (vanopdenbosch, personal communication). the proportional occurrence of these pathogens may dier according to geographic parameters and the studied period, but in the last decade several studies described c. parvum as the most commonly detected enteropathogenic agent in calves [ ± ]. attaching and eacing e. coli have been implicated in diarrhoea and dysentery in -to -weekold calves [ , ] , with a calculated mean age of weeks [ ] . verotoxigenic e. coli, a group of bacteria that produces potent cytotoxins known as verocytotoxins, were found to be occasionally associated with calf diarrhoea [ ] . they were detected in . and % of diarrhoeic calves in germany [ ] and spain [ ] , respectively. there are reports of viruses other than the aforemen-tioned ones, populating the intestine of young calves (picobirnavirus [ ] , calici-and astrovirus [ ] , adenovirus [ ] , enterovirus [ ] and parvovirus [ ] ), but their pathogenicity is still uncertain. giardia infections have also been found to be exceptionally frequent in suckling and weanling calves, although the role of this protozoan parasite in the aetiology of diarrhoea in calves remains unclear [ ] . finally, the problems related to clinical salmonellosis are only minor in dairy, beef [ ] and veal units [ ] . cryptosporidium parvum oocysts were detected in bovines ranging from days old to adults, although the prevalence was signi®cantly higher in suckling calves [ , ± ] . in a spanish epidemiological study on the prevalence and age distribution of c. parvum infections, as many as . % of calves aged ± days were infected, but infection rates peaked ( . %) at ± days of age. prevalence was also high in weanling calves aged . ± months ( %), fattening calves and heifers ± months old ( . %) and adults ( . %). however, cryptosporidium infection was only statistically associated with diarrhoea in suckling calves [ ] . the transmission occurs by the faecal-oral route. the majority of adult cattle can be described as excretors of c. parvum oocysts when sensitive detection methods are used. nevertheless, their importance in the transmission of the disease remains questionable, since oocyst excretion by adult cattle was similar in herds with serious problems of cryptosporidial neonatal diarrhoea and in those without [ ] . so far, in cattle, no increased output of c. parvum oocysts around parturition has been observed [ ± ]. however, these ®ndings should be considered with great caution, as it was shown that more frequent samplings and more sensitive detection methods in sheep revealed a periparturient rise in oocyst score (requejo-fernandez ja, pereira-bueno j, pilar-izquierdo m, rojo-vazquez fa, ortego-mora lm. periparturient rise in ovine cryptosporidiosis. in: proc cost wg meeting. brussels, , p. ). nevertheless, infected newborn calves excrete oocyst numbers of the order of to g À of faeces [ ] and were considered to be a more dangerous source of infection. infection can rapidly spread from calf to calf when animals are communally housed and overcrowded, or from cow to calf via the udders when they are contaminated with infected calf faeces in the lying area of the dams [ ] . this might explain the association between cryptosporidial diarrhoea and the farm type and/or its speci®c hygienic conditions. cryptosporidium infections were more common in single or multiple suckler beef herds [ ] and dairy farms with multiple-cow maternity facilities [ ] . in the so-called fattening units, where calves are purchased through markets, almost % of the animals become infected during transit from the market to the rearing unit or soon after their arrival [ ] . other potential risk factors are herd size [ ] and season. in a canadian study of beef calves, higher prevalence was found in winter and spring, the period related to calving season and consequently the period with the greatest number of calves in the high risk group ( -to -weeksold) [ ] . however, in american dairy farms, where calvings tend to be year-round, and environmental contamination level is less subjected to¯uctuations, cryptosporidiosis was more prevalent in the summer [ ] . little information is available on dierences in infectivity, excretion patterns, virulence or immunogenicity among dierent isolates of c. parvum from cattle or other sources [ ] . this is at least partially due to the lack of single oocyst cloned isolates since all reported isolates are heterogenic mixtures. the economic losses due to cryptosporidial infections of neonatal calves are related to diarrhoea: dehydratation, growth retardation and to a lesser extent mortality [ ] . diarrhoeic problems of calves demand special care: feeding of electrolyte solutions, i.v.¯uid therapy, drug administration, hygienic measures, etc. which are costly as well as labour and time-consuming. in belgium, mortality due to the neonatal diarrhoea syndrome is estimated between ± % (vanopdenbosch, personal communication). cryptosporidium parvum is considered as the most commonly found enteropathogen in calves during their ®rst weeks of life [ ± ]. the parasite frequently acts alone, but the losses are more severe when concurrent infections occur (vanopdenbosch, personal communication) , as has been demonstrated for viral and bacterial enteropathogens [ ] . in sheep, infection by cryptosporidium was ®rst described in australia in -to -week-old lambs with diarrhoea [ ] . its role as a primary aetiological agent was con®rmed in the early s in studies on experimental infections in the absence of other enteropathogenic agents [ , ] . since then, cryptosporidium has been attributed an increasingly important role in neonatal diarrhoea syndrome in this domestic species and is currently associated with high morbidity rates and, depending on environmental conditions and the presence of other intestinal pathogens, mortality [ , , ] . in goats, infection by this agent was also ®rst described in australia, in a -week-old kid with diarrhoea [ ] . since then, the infection has been diagnosed in outbreaks of diarrhoea in goat kids in several european countries [ ± ] and is now considered to be one of the principal enteropathogens in these animals [ ] . only one of the two species of the genus cryptosporidium described in domestic ruminants, c. parvum, has been associated with diarrhoea in small ruminants. in a recent work, a bovine isolate of c. muris did not produce infection in goats [ ] . in lambs, the prepatent period oscillated between and days [ ] and around days in goat kids [ ] . this period increases with a reduction in the dose of the infectious agent [ ] or with an increase in the age of the animal [ ] . the main clinical manifestations of cryptosporidiosis in neonate small ruminants are: apathy and depression, anorexia, abdominal pain, and mainly diarrhoea accompanied by the shedding of a large number of oocysts [ , , ± ] . the faeces are usually yellow, have a soft or liquid consistency and give o a strong unpleasant odour. in experimental infections in lambs [ ] the dry weight of the faeces can drop from to %. in milder cases of the disease the animals have diarrhoea for to days and in more severe cases for to weeks. the diarrhoea usually coincides with the period of oocyst shedding. the duration of the oocyst shedding depends on factors such as the age or immune status of the animals. the kinetics of the shedding is usually similar in experimentally and naturally infected animals. after the ®rst or days of the patent period there is a progressive increase in the number of oocysts shed which reaches a maximum ± days p.i. and drops sharply between days and p.i. [ , , ] . in addition to the diarrhoea and oocyst shedding the animals character-istically manifest anorexia [ , , ] which results in weight loss and retarded growth during the ®rst few weeks of life [ ] . studies on the development of the lesions over the incubation period and the clinical course of the disease [ , ] have shown that, over this period, the parasite mainly proliferates in the jejunum and the ileum. after the start of oocyst shedding ( days after the start of infection) the lesions can spread to other parts of the small and large intestine. a recent study showed that the most frequent aetiologic agent involved in outbreaks of diarrhoea in lambs was c. parvum ( % of the outbreaks and % of the individuals) followed by e. coli potentially pathogenic ( % of the outbreaks and % of the individuals). other agents such as rotavirus were less important ( % of the outbreaks and % of the individuals). in goat kids, c. parvum was present in % of the outbreaks and % of the individuals, followed by e. coli ( and %), rotavirus ( and %), clostridium perfringens ( and %) and salmonella spp. ( and %) [ ] . in a parallel study, it has been shown that e. coli strains isolated from diarrhoeic lambs and kids older than days (mainly between and days old) are not generally toxigenic and belong to a large number of serogroups [ , ] . in small ruminants older than weeks, infections by eimeria spp. are increasingly important accompanied by dietary changes and situations of stress [ ] . the role of other intestinal protozoa such as giardia sp. is controversial and although infection by this parasite is often diagnosed during the ®rst month of life [ ] it is not clearly associated with the production of diarrhoea [ , ] . the prevalence of infection by c. parvum in lambs and goat kids has been studied in both outbreaks of diarrhoea and in randomly selected farms (table ) , although more research has been done on cattle. in outbreaks of diarrhoea, morbidity can be very high in lambs [ , ] and goat kids [ , ± ] . mortality increases when the disease is associated with concurrent infections or de®ciencies in nutrition or husbandry [ , , ] . in goat herds in france and hungary, c. parvum was considered to be the predominant aetiological agent in neonate goat kids with diarrhoea [ , ] . in spain, in a study on sheep farms and goat farms, all randomly selected, corresponding to a total of lambs and goat kids under weeks old, ock prevalence was and % and individual prevalence was and % for lambs and goat kids, respectively [ ] . infection occurs mainly by the ingestion of oocysts previously eliminated with the faeces of infected neonates or asymptomatic adult carriers [ ] . daily excretion of oocysts by infected lambs can exceed  and more than oocysts are shed during the patent period [ , ] . moreover, adult sheep can act as asymptomatic carriers shedding small numbers of oocysts to the environment which was shown to increase in number in the perinatal period and contribute to maintaining the infection between lambing periods. between and % of lambs born in this environment become infected in the ®rst few weeks of life [ ] . in a recent study, the viability of oocysts shed was shown to be greater between days and p.i. than between days and p.i. [ ] . other possible sources of infection, such as farm rodents, should also be considered [ ] . the in¯uence of the dose of the infectious agent on the course of the disease has been studied by several authors. there do not seem to be any dierences between natural or experimental infections with respect to clinical symptoms, oocyst shedding or serum antibody responses [ ] . however, the prepatent period is a few days longer in experimental infections with low infectious doses [ ] . in gnotobiotic lambs the minimum infectious dose can even correspond to a single oocyst and the average infectious dose is around ®ve oocysts [ ] . at present, no intraspeci®c dierences in pathogenicity have been found between isolates obtained from domestic ruminants. however, in the laboratory of one of the authors (ortega-mora, personal communication), two dierent isolates obtained from diarrhoeic ruminants showed signi®cant dierences in the number of oocysts shed and the severity of the diarrhoea after experimental infection in lambs. however, further research is necessary to con®rm this ®nding. in both natural and experimental infections the clinical process that accompanies infection has been reported to be more common in lambs and goat kids under days old [ , , ] . extension of the prepatent period and reduction of oocyst shedding occur as the age of the lambs at infection increases. in a recent study, diarrhoea was only present in lambs infected at days old and in a small percentage of lambs infected at days old, but not in lambs infected at days old. the size of the speci®c serum (igg and iga) and faecal (iga) humoral response was similar and apparently independent of the age, suggesting the participation of mechanisms other than the humoral response in the development of protection [ ] . however, a greater intestinal mucus production and increased glucoprotein concentration in the mucus was observed in animals infected at or days old. the kinetics of the iga in the intestinal mucus response varied with the age of the animal at infection, the most rapid responses and highest titres occurred in animals which were older when infected (quintanilla-gozalo a, wright s, pereira-bueno j, rojo-vazquez fa, ortega-mora lm. changes in the intestinal mucus during infection by cryptosporidium parvum in lambs. in: proc cost annual workshop. prague, , p. ). in natural infections, the majority of the infected lambs and kids were also less than weeks old and the proportion of infected animals underwent a pronounced decrease in animals more than weeks old [ , , ] . the economic losses associated with this disease are not only due to the resulting mortality, but also to the retarded growth of the animals, the cost of drugs, veterinary assistance and the increased labour involved. in the absence of other enteropathogens, mortality is higher in lambs than in calves [ ] and morbidity can reach % [ , ] . the anorexia is very pronounced at the start of the process in both lambs [ , , ] and goat kids [ , ] . in lambs naturally infected with c. parvum, there was a mean weight dierence of kg at weeks old compared with non-infected control lambs of this age (ortega-mora, personal communication). first reports of a cryptosporidium sp. in avian species were from tyzzer [ ] . he found a parasite in the caecal epithelium of chickens with structural similarities to c. parvum, found in mice. at present, only two cryptosporidium spp. are recognised as valid in avian hosts: (i) cryptosporidium meleagridis, ®rst reported in turkeys [ ] ; and (ii) cryptosporidium baileyi, isolated from broiler chickens [ ] . cryptosporidium meleagridis and c. baileyi can be distinguished by morphological dierences between their oocysts (length and width) [ ] . there is possibly a third species from bobwhite quail [ , ] and fourth species from ostrich [ ] . infection by cryptosporidium spp. has been detected in over species of birds including domesticated chickens, turkeys, ducks, geese, quails, pheasants, peacocks, and a wide variety of wild and captive birds [ , ] . cryptosporidium meleagridis may infect the intestinal tract, bursa of fabricius (bf) and cloaca of turkeys [ ] and chickens [ ] , but infection was only associated with illness, including diarrhoea and moderate mortality in turkeys [ ] . cryptosporidium baileyi may infect the respiratory tract (larynx, trachea, primary and secondary bronchi, air sacs), bf and cloaca of chickens [ , ] , turkeys [ ] and ducks [ ] . it is the most prevalent cryptosporidium sp. in poultry and the most commonly associated with disease in chickens. following experimental in-oculation, c. baileyi can develop infections in many anatomic sites of its avian hosts, and it seems that the route of oocyst administration strongly determines the site where the infection ®nally establishes (reviewed in [ ] ). a cryptosporidium sp. was responsible for intestinal disease, including diarrhoea, in quails [ , ] . sometimes, the disease can be severe and mortality can reach % in young quails [ ] . naturally occurring cryptosporidiosis in chickens usually manifests as respiratory disease, and only occasionally as intestinal or renal disease [ ] . symptoms (depression, anorexia, emaciation, coughing, sneezing, dyspnoea), pathological consequences (excessive mucoid exudate, local in¯ammation, airsacculitis, deciliation, epithelial hypertrophy and hyperplasia), as well as increased mortality are most often associated with respiratory cryptosporidiosis [ , , ] . the primary respiratory disease potential of c. baileyi was proven in the absence of other detected pathogens, and the negative eect of cryptosporidiosis on growth performance and carcass pigment was clearly shown [ ] . severe air sac disease [ , ] which resulted in heavy carcass condemnation at processing, places c. baileyi among the agents to be considered in the respiratory disease complex [ ] . the infection of immunocompetent birds is self-limiting. two-week-old broiler chicks inoculated orally with c. baileyi oocysts and capable of clearing the endogenous stages from the cloaca and/or bf, were resistant to subsequent oral challenge [ ] . furthermore, there is an age-related resistance to clinical disease. older chickens are less susceptible to c. baileyi infection, exhibiting a longer prepatent and a shorter patent period [ ± ]. unlike younger ( -day-old) chicks [ ] , food conversion and body weight gain were not in¯uenced by c. baileyi infection in -day-old broilers [ ] . in practice, infections with cryptosporidium are known to occur only in chickens less than weeks of age [ ] , and not in adult birds [ , ] . when appropriate diagnostic tools are used, cryptosporidium spp. appear to be present wherever avian hosts are raised commercially [ ] . cryptosporidium baileyi has been reported in domestic chickens from asia, australia, europe, north america [ ] and north africa [ ] . the prevalence of cryptosporidium spp. in chickens and other galliformes has been studied both in outbreaks of respiratory and/or intestinal disease and in randomly selected¯ocks ( table ) . histological observations demonstrated a similar incidence of cryptosporidium infection in broilers and commercial egg-layer-type pullets [ ] . however, limited epizootiological data suggest that the infection in the latter [ ] is underreported compared with that in broilers [ ] . goodwin et al. [ ] found that % ( / ) of the northern georgia broiler¯ocks had c. baileyi tracheitis. parasitism rates among c. baileyi-infected¯ocks ranged from a low of % to a high of %. cryptosporidium baileyi tracheitis was very highly correlated to severity of tracheitis, negatively correlated with average body weight, and correlated with airsacculitis and condemnations. this study indicated that c. baileyi infection rates in georgia were much higher than previously suspected. avian cryptosporidiosis has been reported in concurrence with immunosupressive viruses like reovirus [ ] , marek's disease virus (mdv) [ ± ], infectious bursal disease virus [ , ] , and chicken anemia virus [ ] . in chickens, the synergistic eect of c. baileyi and these viruses was experimentally con®rmed (reovirus [ ] [ ] , chicken anaemia virus [ ] ). marek's disease virus may induce the establishment of the parasite in inhabitual sites ( [ ] , abbassi h, coudert f, cherel y, brugere-picoux j, naciri m. experimental reproduction of renal cryptosporidiosis (c. baileyi) in spf chickens after oral inoculation of parasite. in: aaap meeting. baltimore, , pp. ± ). the severity of respiratory disease induced by c. baileyi can be enhanced by other respiratory pathogens, such as infectious bronchitis virus and e. coli ( , hoerr fj, blagburn bl, lindsay ds, giambrone jj. interactions of cryptosporidium with other infectious pathogens in chickens. in: proc of the th annual meeting of the am vet med assoc, chicago, usa, , p. ). moreover, c. baileyi infection aects the development of humoral immunity to heterologeous antigens [ , , ] , and can diminish cellmediated immunity, as shown by decreased delayed hypersensitivity indices of infected chickens [ ] . chickens can become infected by ingestion or inhalation and/or aspiration of oocysts present in the environment (litter, faeces, water, breeding materials, dust, etc.). as few as oocysts can result in intestinal or respiratory infections [ ] . since c. baileyi can infect a variety of wild birds they must be considered as possible sources of infection. although c. baileyi does not infect rodents (mice and rats) or insects, they may serve as mechanical carriers of oocysts [ ] . hygienic conditions and management strongly in¯uences the incidence and persistance of avian cryptosporidiosis. the disease was shown to be more prevalent in¯ocks with defective hygiene [ ] . goodwin et al. [ ] , suggested that the increase of intestinal and respiratory cryptosporidiosis infection from % ( / ) during ± to % ( / ) during ± , could be explained in part by the relatively widespread practice of cleaning out poultry houses less frequently. the increased use of`built-up litters' could increase exposure to and infection of chicks by cryptosporidium [ ] . in quails, rigorous clean up and disinfection with hypochlorite acid was eective in preventing continued infection, morbidity and mortality attributed to cryptosporidium sp. [ ] . furthermore, outbreaks of cryptosporidiosis seem to be related to climatologic parameters. it was found that the percentage of cryptosporidiosis cases in winter was signi®cantly lower than in other seasons [ ] . dierences in the incidence between geographic locations were related to the number of days of frost [ ] . for a long time, avian cryptosporidiosis was considered to be an opportunistic disease, but it is now recognised as a ®rst order disease [ , , ] . the economic losses associated with this disease are due to poor¯ock performance (growth retardation and increased consumption index) and/or mortality [ , , , ] . in addition, cost of therapy, although most drugs are inecient [ , , , ] , and carcass condemnation at processing due to air sacculitis [ , ] , should also be taken into account. when c. baileyi occurs concurrently with immunosuppressive or other respiratory pathogenic agents, the consequences are more serious, and the losses more important [ , , ] . vaccination with the attenuated serotype mdv can enhance the severity of subsequent cryptosporidial respiratory disease, resulting in unexpected losses (abbassi h, couder f, cherel y, brugere-picoux j, naciri m. eect of cryptosporidium baileyi on the development of vaccinal immunity to marek's disease in spf chickens. in: th avian immunology research group meeting. turku, , p. . ). thus, the role this parasite plays in the pathogenesis of respiratory disease and the related production losses could be unexpect-edly large. therefore, avian cryptosporidiosis should not be neglected or overlooked during diagnosis. cryptosporidial infection was ®rst reported in pigs by bergeland [ ] and kennedy et al. [ ] . thereafter, naturally occurring cryptosporidiosis in pigs has been described worldwide [ ] . experimental infections of piglets resulted only in diarrhoeic problems when the animals were inoculated before weeks of age [ ± ]. in older piglets, experimental cryptosporidium infection did not cause clinical manifestations [ ± ] . the prevalence of cryptosporidiosis was reported to be very low in nursing piglets [ , , ] . several studies demonstrated that naturally occurring cryptosporidiosis is delayed until after weaning [ , , ± ] . the infections are mostly asymptomatic and usually of low intensity, and no statistical association between infection and clinical symptoms has been found [ , ] . previous studies have con®rmed that diarrhoea in suckling and weaned piglets is usually a multifactorial problem, where mixed infections between c. parvum and e. coli or rotavirus are frequent. it has been suggested that cryptosporidium is not an important primary agent of diarrhoea in piglets but it may be a copathogen in the multifactorial aetiology [ ] . recently, genetic analysis of the cryptosporidium isolates from pig herds revealed that the animals harboured two distinct genotypes: a porcine genotype and a bovine genotype with distinct virulence in nude mice [ ] . the zoonotic potential of the porcine genotype is still uncertain and requires further study. in commercial rabbits, digestive disorders are the predominant cause of mortality. mainly weaned rabbits of to weeks of age are aected, although another peak may occur in suckling rabbits of to days old [ ] . experimental cryptosporidium infection of rabbits resulted in high mortality and liquid diarrhoea in suckling -day-old rabbits, but no mortality and very discrete diarrhoea in weaned animals [ ] . the parasite was found in ± % of weaned diarrhoeic belgian rabbits [ ] . in general, ®eld outbreaks of cryptosporidiosis in suckling rabbits are rarely detected and in weaned rabbits the parasite causes only subclinical enteritis. when concurrent infections with immunosuppressive agents or respiratory pathogens occur, the impact of cryptosporidium infection can be more signi®cant (peeters, personal communication). aetiological treatment of cryptosporidiosis has only recently been made possible. drugs demonstrated to be partially eective in the treatment and prophylaxis of cryptosporidiosis in ruminants include halofuginone lactate, paromomycin and decoquinate (table ) . however, the commercial availability of some of these products is still a problem in many countries. in poultry, so far none of the tested drugs showed a satisfactory anti-cryptosporidial activity, unless at toxic dosage [ , , , ] . recently, it was shown that the oral infection of hens with c. baileyi at the beginning of their laying period, resulted in partial protection of their progeny [ ] . the importance of colostrum in protecting neonatal ruminants against infection by c. par-vum is a very valuable point to address. in ®eld conditions, passively acquired antibodies did not protect calves [ , ] and lambs against naturally acquired infection [ ] . however, both calves [ ] and lambs [ ] fed with colostrum from immunised mothers with high titres of speci®c antibodies were partially protected against infection. immunoprophylaxis of cryptosporidiosis is more thoroughly discussed in another contribution of this special issue of the international journal for parasitology [ ] . from a perspective of disease control, preventive hygiene measures are the most important tools in the struggle against cryptosporidiosis in farm animals, the objective being to destroy external forms of the parasite and to prevent their transmission among animals and from the environment to the host [ ] . in ruminant husbandry, the destruction of oocysts in the pens and buildings used for parturition by applying moist heat and/or chemical disinfectants, the use of abundant clean straw beds, avoidance of high stocking rates in the parturition area and the separation of healthy and ill animals during outbreaks of diarrhoea, in addition to the administration of appropriated supplies of colostrum to neonates, all help to prevent outbreaks of cryptosporidiosis and to minimise mortality and morbidity in infected herds. [ ] * per kg body weight. cryptosporidiosis is mainly a problem in neonatal ruminants. cryptosporidium parvum is the most commonly found enteropathogen during the ®rst weeks of the life of calves, lambs and goat kids and is considered to be an important agent in the aetiology of the neonatal diarrhoea syndrome. the parasite frequently acts alone, but the losses are more pronounced when concurrent enteropathogens are present. economic losses associated with cryptosporidiosis are retarded growth and mortality, and a number of hard to estimate costs resulting from interventions necessitated by diarrhoeic problems. especially in small ruminants, the direct losses due to mortality caused by cryptosporidiosis alone was reported to be high. because of the limited availability of eective drugs, hygienic measures and good management are the most valid weapons in the struggle against this disease. avian cryptosporidiosis is an emerging health problem. in chickens and other galliformes, cryptosporidiosis is mostly manifest as respiratory disease, although in turkeys and quails enteric forms of the disease were also reported to be responsible for morbidity and mortality. it was alarming to ascertain the unexpectedly high prevalence of c. baileyi in chicken¯ocks and the enhanced pathogenicity when immunosuppresive or other respiratory pathogens are present, or when birds are subjected to vaccination against other pathogens. in pigs and rabbits, cryptosporidium spp. were reported to cause only clinical disease when administered experimentally to nursing neonates, but in these farm animals naturally occurring cryptosporidiosis is mostly asymptomatic. cryptosporidium meleagridis (sp.nov.) an outbreak of calf diarrhoea attributed to cryptosporidial infection intestinal lesions in spf lambs associated with cryptosporidium from calves with diarrhoea acute enterocolitis in a human being infected with the protozoan cryptosporidium human cryptosporidiosis in immunocompetent and immunode®cient persons. studies of an outbreak and experimental transmission a massive outbreak in milwaukee of cryptosporidium infection transmitted through the public water supply genetic heterogeneity and pcr detection of cryptosporidium parvum general biology of cryptosporidium the eects of reducing conditions, medium, ph, temperature, and time on in vitro excystation of cryptosporidium experimental tracheal and conjunctival infections with cryptosporidium sp. in pigs experimental cryptosporidium baileyi infections in chickens and turkeys produced by ocular inoculation of oocysts conjunctivitis in pheasants associated with cryptosporidial infection cryptosporidiosis of the conjunctiva in siv-infected rhesus monkeys pulmonary cryptosporidiosis in acquired immuno de®ciency syndrome respiratory cryptosporidiosis in broiler chickens sinusitus in turkeys associated with respiratory cryptosporidiosis cryptosporidium as a cause of laryngotracheitis in an infant cryptosporidiosis in quails respiratory cryptosporidiosis in a bovine natural history and biology of cryptosporidium parvum use of a novel soil tilting table apparatus to demonstrate the horizontal and vertical movement of the protozoan pathogen cryptosporidium parvum in soil small system control of cryptosporidium survival of cryptosporidium parvum, escherichia coli, faecal enterococci and clostridium perfringens in river water: in¯uence of temperature and autochthonous microorganisms cryptosporidial infection in a calf cryptosporidium agni sp. n. from lambs and cryptosporidium bovis sp. n. from a calf with observations on the oocyst cryptosporidiosis in a calf experimental cryptosporidiosis in calves: clinical manifestations and pathological ®ndings enteric lesions and diarrhea in gnotobiotic calves monoinfected with cryptosporidium species cryptosporidium parvum (sp. nov.), a coccidium found in the small intestine of the common mouse the species of cryptosporidium (apicomplexa: cryptosporidiidae) infecting mammals cryptosporidium spp. in cattle detection of cryptosporidium muris oocysts in the faeces of adult dairy cattle in scotland cryptosporidium muris in dairy cattle in brazil abomasal cryptosporidiosis in cattle cryptosporidium muris: prevalence, persistency and detrimental eect on milk production in a drylot dairy cryptosporidium parvum infection in bovine neonates: dynamic clinical, parasitic and immunologic patterns cryptosporidia associated with rotavirus and escherichia coli in an outbreak of calf scour infection patterns of cryptosporidium and giardia in calves prevalence of cryptosporidium and giardia infections in cattle in aragon (northeastern spain) resistance of calves to cryptosporidium parvum: eects of age and previous exposure ecacy of halofuginone lactate against cryptosporidium parvum in calves bovine cryptosporidiosis: clinical and pathological ®ndings in forty-two scouring neonatal calves cryptosporidium parvum in calves: kinetics and immunoblot analysis of speci®c serum and local antibody responses (immunoglobulin a (iga), igg and igm) after natural and experimental infections cryptosporidiosis as a probable factor in neonatal diarrhea of calves cryptosporidiosis in ruminants localization of a/b and g/d lymphocytes in cryptosporidium parvum-infected tissues in naõÈ ve and immune calves enterotoxins, colonization factors and serotypes of enterotoxigenic escherichia coli from humans and animals the aetiology and diagnosis of calf diarrhoea cryptosporidiosis in neonatal calves: cases ( ± ) enteric pathogens in intensively reared veal calves proportional morbidity rates of enteropathogens among diarrheic dairy calves in central spain dysentery in gnotobiotic calves caused by atypical escherichia coli association between the eacing gene (eae) and the shiga-like toxin-encoding genes in escherichia coli isolates from cattle prevalence and molecular typing of attaching and eacing escherichia coli among calf populations in belgium role of verocytotoxigenic escherichia coli in cattle and bualo calf diarrhoea shiga toxinproducing escherichia coli strains from bovines: association of adhesion with carriage of eae and other genes verotoxin-producing escherichia coli (vtec) and eae-positive non-vetec in ± -days-old diarrhoeic dairy calves bovine birna type virus: a new etiological agent of neonatal calf diarrhoea? isolation of small viruses resembling astrovirus and caliciviruses from acute enteritis of calves necrotizing enteritis in a calf infected with adenovirus isolation and characterization of bovine enteric viruses diarrhoea in dairy calves reduced by feeding colostrum from cows vaccinated with rotavirus microbiology of diarrhoea in young beef and dairy calves in argentina enteric infections in veal calves: a longitudinal study on four veal calf units detection of oocysts and igg antibodies to cryptosporidium parvum in asymptomatic adult cattle excretion of cryptosporidium parvum by a herd of beef suckler cows an epidemiological study of cryptosporidium parvum in two herds of adult beef cattle evaluation of periparturient dairy cows and contact surfaces as a reservoir of cryptosporidium parvum for calfhood infection microbiology of calf diarrhoea in southern britain potential risk factors for cryptosporidium infection in dairy calves infection with cryptosporidium spp. in humans and cattle in manitoba neonatal calf diarrhoea: a complex etiology experimental cryptosporidiosis in germ-free lambs epidemiologia de la criptosporidiosis en el ganado ovino y caprino de la montana de leo n role of enteric pathogens in the aethiology of neonatal diarrhoea in lambs and goat kids in spain intestinal cryptosporidiosis in a kid goat les cryptosporidies en france. techniques usuelles d'identi-®cation et re sultats pre liminaires d'enqueà tes e pide miologiques studies on cryptosporidial infection of goat kids prevalencia de la infeccion por cryptosporidium parvum en corderos, cabritos y terneros en la provincia de leo n infectivity of cryptosporidium muris isolated from cattle diarrhea due to cryptosporidium infection in arti®cially reared lambs experimental cryptosporidiosis in kids infective dose size studies on cryptosporidium parvum using gnotobiotic lambs age-related resistance in ovine cryptosporidiosis: patterns of infection and humoral immune response diarrhea in goat kids attributed to cryptosporidium infection cryptosporidiosis in idaho lambs: natural and experimental infections an outbreak of diarrhoea associated with cryptosporidiosis in naturally reared lambs analysis of kinetics, isotype and speci®city of serum and coproantibody in lambs infected with cryptosporidium parvum cryptosporidiose expe rimentale du cheÁ vreau. in¯uence de la prise du colostrum. essais de traitements toxins and antibiotic resistance of escherichia coli strains isolated from diarrhoeic lambs in spain toxins and antibiotic resistance of escherichia coli strains isolated from diarrhoeic goat kids in spain coccidiosis and cryptosporidiosis in sheep and goats giardia infection in farm animals giardia and cryptosporidium in canadian farm animals eect of nomada shepherds and their sheep on the incidence of cryptosporidiosis in an adjacent town diagnosi di criptosporidiosi in alcuni allevamenti dell'italia centrale participacion de cryptosporidium parvum en brotes de diarrea en corderos en al no de castilla y leon prevalence of cryptosporidium oocysts in livestock in trinidad and tobago diagnosis of cryptosporidium on a sheep farm with neonatal diarrhea by immuno¯uorescence assays surveillance of cryptosporidia in a veterinary diagnostic laboratory enqueà te e pide miologique sur les diarrhe es ne onatales des chevreaux dans les e levages de touraine. in: yvore p, perrini g, editors. les maladies de la cheÁ vre cryptosporidiosis in kids of dairy goats outbreak of cryptosporidiosis in dairy goats in brazil periparturient rise in the excretion of giardia sp. cysts and cryptosporidium parvum oocysts as a source of infection for lambs cryptosporidium parvum: oocyst excretion and viability patterns in experimentally infected lambs infectivity of cryptosporidium sp. isolated from mice for calves and mice serum antibody response in lambs naturally and experimentally infected with cryptosporidium biopathological data of goat kids with cryptosporidiosis coccidiosis in gallinaceous birds the life cycle of cryptosporidium baileyi n. sp. (apicmplexa cryptosporidiidae) infecting chickens morphometric comparison of the oocysts of cryptosporidium meleagridis and cryptosporidium baileyi from birds intestinal cryptosporidiosis and reovirus isolation from bobwhite quail (colinus virginianus) with enteritis experimental reproduction of entiritis in bobwhite quail (colinus virginianus) with cryptosporidium and reovirus host speci®city studies and oocyst description of a cryptosporidium sp. isolated from ostriches cryptosporidium and cryptosporidiosis in man and animals cryptosporidium and cryptosporidiosis intestinal and bursal cryptosporiosis in turkeys following inoculation with cryptosporidium sp. isolated from commercial poults diarrhea associated with intestinal crypotosporidiosis in turkeys experimental cryptosporidium infections in chickens: oocyst structure and tissue speci®city experimental cryptosporidiosis in broiler chickens experimental induced infections in turkeys with cryptosporidium baileyi isolated from chickens experimental infections in domestic ducks with cryptosporidium baileyi isolated from chickens cryptosporidiosis in birds fatal cryptosporidiosis in quail cryptosporidial infection in chickens respiratory cryptosporidiosis in chicken development of an serologic evaluation of acquired immunity to cryptosporidium baileyi by broiler chickens eect of broiler chicken age on susceptibility to experimentally induced cryptosporidium baileyi infection variations in oocyst output associated with cryptosporidium baileyi infections in chickens age-dependent resistance to cryptosporidium baileyi infection in chickens eect on cryptosporidium baileyi on broilers infected at days of age cryptosporidium sp. and cryptosporidiosis histologic incidence and distribution of cryptosporidium sp. infection in chickens: cases in diseases of poultry cryptosporidiosis in birdsÐa review infection naturelle de cryptosporidium sp. chez le poulet de chair au maroc prevalence of cryptosporidium antibodies in animal species cryptosporidiosis of the bursa of fabricius and trachea in broilers cryptosporidia in the bursa of fabriciusÐa correlation with mortality rates in broiler chickens serologic incidence of cryptosporidium in delmarva broiler ocks cryptosporidial infection in broiler chikens in greece cryptosporidia-positive rates of avian necropsy accessions determined by examination of auramine o-stained fecal smears subclinical cryptosporidiosis of turkeys in iowa the relationship of cryptosporidium sp. infection on the bursa of fabricius, intestinal tract, and respiratory system of chickens in georgia isolation and identi®cation of cryptosporidium from various animals in korea. i. prevalence of cryptosporidium in various animals incidence of respiratory cryptosporidiosis in georgia broilers: ± respiratory coccidiosis (cryptosporidium baileyi) among northern georgia broilers in one company association cryptosporidiose et maladie de marek chez des poulets nains cryptosporidiosis of the bursa of fabricius of chickens small-intestinal cryptosporidiosis in a chicken concurrent cryptosporidiosis and chicken anemia virus infection in broiler chickens interaction of reovirus and cryptosporidium baileyi in experimentally infected chickens experimental cryptosporidiosis and infectious bursal disease virus infection of speci®c pathogen-free chickens interaction of chickens anaemia virus and cryptosporidium baileyi in experimentally infected chickens esophageal and proventricular cryptosporidiosis in a chicken pathobiology of cryptosporidiosis (c. baileyi) in broiler chickens eect of cryptosporidium baileyi infection on antibody response to srbc in chickens immunosuppresive eect of cryptosporidium baileyi infection on vaccination against newcastle disease in chicks potentiation of ionophorous anticoccidials with duokvin: battery trials against cryptosporidium baileyi in chickens necrotic enteritis in nursing piglets cryptosporidiosis in three pigs cryptosporidiosis in pigs and horses experimental infection of piglets with cryptosporidium fecal transmission of calf cryptosporidia between calves and pigs pathogenesis of intestinal cryptosporidiosis in conventional and gnotobiotic piglets enterocolitis in piglets caused by cryptosporidium spp. puri®ed from calf faeces cryptosporidium sp. and isospora suis in pigs in italy the occurrence and eect of cryptosporidium species on livestock in ile-ife intestinal cryptosporidiosis of diarrhoeic piglets in our environment prevalence of cryptosporidium infections in pigs in aragon (northeastern spain) cryptosporidium in market pigs in southern california, usa prevalence of cryptosporidium oocysts in livestock in trinidad and tobago prevalence and epidemiology of selected enteric infections of livestock in trinidad molecular and biological characterisation of cryptosporidium in pigs recent advances in intestinal pathology of rabbits and further perspectives pouvoir pathogeÁ ne de cryptosporidium sp. chez les lapereaux avant et apres sevrage. in: e mes journe es de la recherche cunicole en france ecacite du lactate d'halofuginone dans le traitement de la cryptosporidiose chez l'agneau the eect of halofuginone lactate on experimental cryptosporidium parvum infections in calves speci®c serum and local antibody responses against cryptosporidium parvum during medication of calves with halofuginone lactate paromomycin is eective as prophylaxis for cryptosporidiosis in dairy calves chemoprophylaxis of cryptosporidium parvum infection with paromomycin in kids and immunological study the eect of varying levels d. eccox on experimental cryptosporidia infections in holstein bull calves evaluation of decoquinate to treat experimental cryptosporidiosis in kids assessment of maternal immunity to cryptosporidium baileyi in chickens eect of colostral antibody on susceptibility of calves to cryptosporidium parvum infection ecacy of hyperimmune bovine colostrum for prophylaxis of cryptosporidiosis in neonatal calves treatment of experimental ovine crytosporidiosis with ovine or bovine hyperimmune colostrum speculation on whether a vaccine against cryptosporidiosis is a reality or fantasy key: cord- -ky mq y authors: velasquez-munoz, ana; manriquez, diego; paudyal, sushil; han, hyungchul; callan, robert; ryan, elizabeth p.; pinedo, pablo title: effect of prebiotic supplementation with stabilized rice bran in milk of pre-weaned organic holstein calves date: - - journal: bmc vet res doi: . /s - - - sha: doc_id: cord_uid: ky mq y background: the first month of life possess significant challenges for dairy calves due to high susceptibility to digestive diseases. the objective of this study was to evaluate the effect of prebiotic supplementation with stabilized rice bran (srb) in milk on health, immunity, and performance of pre-weaned organic dairy calves. holstein heifer calves (n = ) were enrolled at ± days old and monitored for days, from july to august . calves were randomly assigned to a control (ctr; n = ) or a treatment group (srb; n = ). the ctr group received milk alone and the srb group received g of srb per day in milk to achieve a % w/w dose of the total calories. daily health evaluations were conducted to score health status and disease severity (healthy, slightly affected, moderately or severely sick) of calves, through integrated assessment of diarrhea, dehydration, attitude, and milk intake. body weights and fecal iga quantification were completed on the first and last day of the study. results: overall, weight gain and fecal iga concentrations were not affected by the dietary addition of srb. the total number of calf-days classified as healthy or sick were not different between treatment groups. similarly, the number of calf-days categorized as slightly affected, moderately sick, or severely sick did not differ between treatment groups. time to event analyses indicated a tendency for a treatment effect in the time to the first moderate case of diarrhea (p = . ), as well as in the time to recovery from diarrhea (p = . ), favoring control calves. conclusions: these results indicated that the dietary addition of srb in milk did not have an effect in health, immunity or performance of pre-weaned dairy calves. rearing healthy calves that maintain adequate growth rates is essential for the success of dairy operations. however, during the first month of life, calves face multiple stressors while the immune system is still developing, resulting in a high susceptibility to digestive diseases [ ] . during the first weeks of life of dairy calves, diarrhea is the most prevalent health disorder, as well as the main cause of death. a recent report in the us indicated that . % of calf mortality was a consequence of diarrhea and animals less than weeks old were the most affected [ ] . in , % of pre-weaned calves presented diarrhea and % of all pre-weaned calves were treated with antimicrobials [ ] . rehydration and antibiotic therapy are common treatments for calves with neonatal diarrhea. however, due to consumer concerns, regulations for the use of antibiotics in food animals are becoming more restrictive. consequently, research focused on alternatives to the use of antimicrobials, including strategies to prevent disease is required. prebiotics are defined as non-digestible feed ingredients that stabilize the intestinal microbiota, stimulating the growth of beneficial bacteria and inhibiting the colonization by pathogens [ , ] . prebiotic-probiotic interactions have been shown to improve immune responses [ , ] , contrasting with the action of antibiotics that eliminate and restrict the growth of detrimental and beneficial microorganisms with no distinction. the use of prebiotics has been studied in young ruminants as a prophylactic strategy to prevent disease and as an alternative to antibiotics and one of the most common products is mannanoligosaccharides (mos), a derivative of the cell wall of the yeast saccharomyces cerevisiae. however, the effects of prebiotics on performance, health, and immunity of calves has not been consistent. for example, the supplementation of mos resulted in a reduction in almost point on the severity of neonatal diarrhea in a to scale [ ] and decreased the number of days with high diarrhea scores [ ] . contrary, other studies reported no differences in diarrhea cases after the supplementation prebiotics [ ] [ ] [ ] in pre-weaned dairy calves. furthermore, some studies indicated no differences in weight gain [ , , ] , while greater gains were reported by others [ , , ] after a prebiotic supplementation in pre-weaned calves. heat stabilized rice bran (srb) contains prebiotics that have been tested in mice, chickens, pigs, horses, dogs and humans. this is a natural product that has been heat stabilized to prevent rancidity. as other prebiotics used in calf health, srb is a carbohydrate. however, srb contains ɣ-oryzanol (omega - ), antioxidants (tocopherols, tocotrienols, polyphenols, phytosterols), vitamin e and b, amino acids (tryptophan, histidine, methionine, cysteine, arginine) and micronutrients (magnesium, calcium, phosphorus, manganese), which may have the potential to enhance the host health, not only through a symbiotic effect with the probiotic bacteria in gi tract [ ] . previous research indicated that this product had positive effects reducing the presentation and duration of diarrhea from human rotavirus and human norovirus in pigs [ ] , increasing the production of local and systemic iga and enhancing the immune system in mice and pigs [ ] [ ] [ ] [ ] . the effect of srb has not been previously studied in young ruminants and its potential as a supplement or additive in whole milk of pre-weaned dairy calves has not been explored. we hypothesized that the addition of srb in milk of pre-weaned calves would reduce the presentation and severity of neonatal diarrhea, improving the immune response and consequently the overall calf performance. therefore, our specific objective was to determine the effect of srb on average daily gain (adg), fecal iga concentration, presentation of diseases, time to recovery from disease, and animal removal. overall, calves were included for the final analyses, as calves in the srb group did not consume the milk with added srb. all calves had baseline total serum protein (tsp) measurements above . g/dl, indicating no failure in passive immune transfer [ , ] . however, calves had tsp measurements above . g/dl and from these, presented diarrhea at the time of enrolment. consequently, % of the enrolled animals might have presented some degree of dehydration that could alter to some extent the values of tsp. no significant difference (p = . ) was found for the proportion of tsp above . g/dl between control (ctr) and srb calves (or = . , % ci = . - . ). additionally, at enrollment, ( %) calves presented signs of slight disease (diarrhea), which may also explain the high tsp level and possible dehydration. no differences (p = . ) were found in the odds ( % ci) of diarrheal disease at enrollment (or = . [ . - . ]) for ctr calves in comparison with the srb group. for the overall day study, total calf-days classified as "healthy" and "sick" ("slight", "moderate" or "severe") for calves were and , respectively. these cumulative days were analyzed according to disease severity and dietary treatment group ( table ). the repeated measures analyses for a binary response did not indicate a significant effect for treatment in the number of "healthy" or "sick" days for any of the disease categories ( table ). no differences between treatment groups were found for the time to the first "moderate" disease event (p = . ). the survival curve demonstrated a pronounced slope (fig. a) during the first days of study, with about % of srb calves and % of ctr calves presenting the first "moderate" disease status within this period. when the health status at enrollment was included as a covariate in the analysis, a tendency was determined in the survival function for the effect of health at day (p = . ; fig. b ). day "healthy" ctr and srb calves presented the first moderate subsequent diarrhea episode in ± days and ± days respectively. ctr control group not exposed to heat stabilized rice bran in the diet, srb group receiving a daily dose of g of stabilized rice bran corresponding to % of the daily calories contrary, d "sick" ctr and srb presented the first moderate diarrhea episode in ± and ± , respectively. the time to recovery from a "moderate" disease status to a "slight" or "healthy" status, indicated a significance trend for treatment group in the kaplan meier analysis (p = . ). calves in ctr group recovered from a "moderate" status in . ± . days, while srb calves recovered in . ± . days (fig. a) . importantly, when health at enrollment was added as a covariate, there were no longer differences found in time to recovery (p = . ). the survival curve considering health status at enrollment indicated that ctr calves classified as "healthy" at d recovered from a moderate diarrhea episode in . ± . days; d "sick" ctr calves recovered in . ± . days; d "healthy" srb calves in . ± . days; and d "sick" srb calves recovered in . ± days (fig. b) . all the fecal samples collected at enrollment and at the end of the study submitted for detection of coronavirus and rotavirus (n = ) were negative. treatment groups presented a similar adg in the days of study (ctr = . ± . kg; srb = . ± . kg. p = . ) and the concentrations of fecal iga did not differ between treatment groups and health status at enrollment (p = . ). mean iga concentrations for ctr and srb were . ± . ng/ml and . ± . ng/ml, respectively. seventy nine calves completed the days period of the study; out calves enrolled died (crt = , srb = ) and were culled (ctr = , srb = ). the odds of leaving the study due to death or culling did not differ between ctr and srb group (or = . [ . - . ]; p = . ). additionally, no differences were found in the time that calves left the study due to death or culling (p = . ). overall, (ctr = , srb = ) calves received organic certified treatment for at least one disease event during the follow up period. ten calves (ctr = , srb = ) presented more than disease event between d in study and weaning. no significant difference (p = . ) was found in the odds of presenting more than events of disease between treatment groups (or = . , % ci = . - . ). in addition, the time to a first disease event after completion of srb addition was similar in both treatment groups (p = . ). in total, out of calves were lost during the post treatment follow up period, between the end of the days study period and weaning. eleven calves were sold (ctr = , srb = ) and calves died (ctr = , srb = ). no differences were found in the odds of leaving the study by treatment group (p = . , or = . , % ci = . - . ). additionally, time to death or culling did not differ between groups (p = . ). the addition of prebiotics via srb into milk starting at - days of age was assessed for effects on health and performance of pre-weaned organic dairy calves over a days period. overall, this study resulted in no treatment differences in the number of days calves were sick or in the number of days by category of disease severity. notably, the addition of srb was tested in a challenging calf population, as the compromised health status of some calves was apparent at enrollment. the beginning of this study coincided with nutritional management adjustments made by the farm that resulted in high incidence of neonatal diarrhea. total serum protein determination in calves is a commonly used tool to measure passive immune transfer and, consequently, new born and colostrum management practices at farms [ ] . all the enrolled calves had tsp measurements above . g/dl. it has been described that concentrations ≥ . g/dl in healthy calves and ≥ . g/dl in clinically ill calves is considered a measure of adequate passive transfer of immunity [ , ] . however, the concentration of tsp in calves might be affected by dehydration and, although a cut-off point for high tsp readings in calves has not been established, readings above . have been linked to dehydration [ ] . notably, % of our calves presented tsp concentrations above . g/dl at enrollment, with close to % of the population showing signs of clinical disease (diarrhea or slight dehydration). however, no differences were found between treatment groups, indicating that both groups started in similar immune and health conditions. in addition, considering this issue, health at enrollment was included in the statistical models as a covariate. supporting our results, a previous study reported that the use of an oral electrolyte containing rice, promoted diarrhea in young calves less than weeks old [ ] . pre-ruminant calves lack the production of enzymes to digest maltose and starch from rice and this situation might lead to osmotic diarrhea when it is provided in milk replacers or in oral electrolytes [ , ] . this fact might explain the increase in the days srb calves spent in the moderate and severely sick categories in our study. published studies using prebiotics as a prophylactic or treatment therapy in pre-weaned calves are limited and there is not consensus on their effect on health and diarrhea presentation in young dairy calves. although positive effects were reported in the reduction of disease presentation or diarrhea scores by some authors [ , , , ] , other studies did not find significant differences [ , ] . the decision of analyzing the time to a first "moderate" status of disease was made considering the health situation of the study population at enrollment. calves presented a first "moderate" health condition as a result of diarrhea in the first days in study, when they were to days old. treatment groups did not differ in the time to a first "moderate" status and, as it was expected, calves that were sick at enrollment showed a tendency to present the first "moderate" health status before than calves that were healthy at that time point. contrary to our expectations, srb calves that were healthy at enrollment presented a moderate health status earlier than healthy ctr calves. even though these differences were not significant, we attribute this finding to a possible osmotic effect of srb on the large intestine of young animals [ ] . our results contrast with neonatal animal model research, where srb had a protective effect in the presentation of disease through the stimulation of the immune response and increases of probiotic bacteria in the gastrointestinal tract [ ] . a tendency for different times to recovery from a "moderate" to a "slight" health status between treatment groups was established; srb calves required more days to recover than ctr calves. this information is valuable and suggests that srb may have a potentially detrimental effect in young calves, explained by the incapacity to digest carbohydrates and starches from rice [ ] . control calves and srb calves had a similar adg during the days in study. published data is not consistent on resulting adg in calves fed with prebiotics. no differences in adg has been reported [ , , ] . conversely, some studies found a greater adg in pre-weaned calves fed with prebiotic (mos) in milk for days [ , ] . interestingly, rice protein has been used as a replacement of whey protein in milk replacers and the results are not conclusive in the effects on performance of pre-weaned calves [ , ] . a negative impact on adg was reported when calves were fed with rice protein replacing % and more of the whey protein in the milk replacer, these animals had a reduction of to % in body weight, although health parameters were not collected [ ] . conversely, no effects on adg or growth were reported in calves when % rice protein was added in the milk replacer of pre-weaned calves [ ] . immunoglobulin a is the major immunoglobulin class found in mucosal secretions and prevents mucosal infections by agglutinating pathogens [ ] . our study found similar iga concentrations in feces from the two treatment groups. the immunomodulatory response of dietary srb has been described in animal models. it was reported an increased production of mucosal iga in to weeks old mice fed % of the daily calories for days [ ] . in that study, rice bran enhanced the growth of lactobacillus ssp. and other beneficial bacteria that might have increased the iga concentration in intestine. similarly, an increase in the serum titer of iga in gnotobiotic pigs fed srb was found [ ] . however, previous reports are not consistent on the immunomodulatory response of prebiotic fed to pre-weaned calves and the quantification of fecal iga. no difference in fecal and salivary iga was reported when newborn calves were fed for days with a commercial prebiotic in milk replacer (prebio support, meiji feed co., ltd. tokyo, japan) [ ] . conversely, the same product had an effect increasing fecal iga of pre-weaned calves at specific time points [ ] . although the ctr group had twice as many calves leaving the study as the srb group, the odds of leaving the farm due to death or culling were not significantly different in our two groups. contrary to the expectations, the follow up period until weaning indicated that a similar number of animals were lost in each group (ctr = vs srb = ). additionally, a similar proportion of calves was treated for more than disease episode in the follow up period. consequently, the days of addition of srb in the milk of calves did not have influence in health outcomes in the pre-weaned life. our daily dose of srb was greater than that of published studies testing prebiotics on pre-weaned calves, where authors worked with commercial products in doses no greater than g/d [ , [ ] [ ] [ ] ] . we offered srb in its natural form in a dose of g/d (only heat stabilized to prevent rancidity) and one difficulty observed in this trial was the necessity of an intense mixing to suspend the srb dose in milk. furthermore, if milk was not served soon after mixing, srb started to decant in the bottom of the bottle, which was also reported in other study using a different product [ ] . the major finding from this study was that the addition of srb in the milk of newborn calves for days did not enhance performance, health, or immunity during the first month of life, a period characterized for the presentation of digestive diseases. furthermore, no differences were found from birth to weaning in the presentation of diseases or death and removal. further research is encouraged in older calves to investigate the potential beneficial effects of srb at more advanced stages of life. the study was conducted in a commercial certified organic dairy calf rearing facility located in northern colorado. calves were owned by this farm that provided consent for their inclusion in this study. pre-weaned holstein calves were managed during the study in accordance to the guidelines set by the institutional animal care and use committee of colorado state university (protocol id: - a). ninety pre-weaned holstein heifer calves, ± days old, were enrolled in this research. calves were monitored for days to assess the effect of srb addition in milk. after the days feeding period, a follow up period until weaning (around d of life) was completed to evaluate health outcomes based on farm records. the first stage of the study began in july and ended in august . the second stage was completed in october . after completion of this study, calves returned to the regular management for calves in this dairy farm. a detailed description of the calves' management at birth and in the rearing facility (housing, feeding, dehorning and vaccination program) was published [ ] . in general, calves were immediately separated from their dam at birth, fed . l of colostrum during the first hour of life and at and h of life. colostrum quality was at least mg/ml igg. after h of life, calves arrived in the rearing facility and they were housed in rows of individual hutches (agri-plastics, stoney creek, on, canada) with sand bedding and a wire panel pen attachment of . m . calves had visual but no physical contact with other animals until weaning. milk was provided in . l bottles (e-z nurse™) three times per day. during the study period the feeding schedules were : am, : pm, and : pm. milk collected from the hospital pen, and organic sealable milk delivered each day from an organic processing plant was pasteurized for calf feeding. also, organic certified powder milk was provided, following preparation instructions. milk composition was analyzed weekly during the study period. average ± sd fat, protein, lactose, and total solids were . ± . %, . ± . %, . ± . %, and . ± . %, respectively. organic certified calf starter was offered to the calves from day of life in clean buckets ( % organic calf starter, feedex companies, llc. south hutchimsin, ks) and water was offered ad libitum since the arrival of calves. total serum proteins were measured by trained personal to evaluate passive transfer of immunity. a ml blood sample was collected from the jugular vein in calves to days old in a tube without anticoagulant. the sample was allowed to clot before centrifugation. serum was analyzed in an optical engine digital refractometer (palm abbe™ , solon, oh) and all readings were kept in the farm recording system. the completion of the step-down weaning process took three weeks and it was based on calf starter consumption ( . to . kg per day) and fully weaned calves stayed during one week in the individual hutches to monitor health before transferring to collective pens. trained personnel had the responsibility to perform daily health evaluations to all the calves in the facility, with the objective of detecting and monitoring sick animals to apply treatments established in the farm standard operating procedures (sop). as the study farm is an organic certified dairy, calves that were not immediately responsive to initial treatment were sold to a conventional calf operation, where animals can receive antibiotic therapy. a paired comparison design with treatment groups was performed. calves were randomly assigned to a control (ctr, n = ) or a treatment group (srb, n = ) and a clinical examination was completed to determine the health status of each calf at enrolment. all calves were weighted at enrollment and at day using a mobile platform digital scale (caf-cart. raytec llc, ephrata, pa). this procedure was performed after the morning feeding. a subsample of calves from each group was randomly selected for fecal samples collection at enrollment and at day of the study, after the morning feeding. twenty grams of fecal matter were obtained by rectal stimulation with a gloved finger and stored in two separate sterile containers. one set of samples was submitted fresh to colorado state university, veterinary diagnostic laboratories for coronavirus and rotavirus screening. the second sample was frozen at − °c for subsequent iga analysis (iga bovine elisa kit, abnova corporation, taipei, taiwan.). a daily health assessment was performed for each calf every morning after the milk feeding. the calf health scoring chart by university of wisconsin [ ] was modified to assess fecal score. the scoring was categorized as healthy or for normal feces, as abnormal or for loose and pasty feces and as severe or for watery feces. dehydration status was assessed daily using a calf dehydration chart [ ] . the scores were assigned as for non-dehydrated animals (< % water body loss) with a normal attitude, strong suckle reflex, appetite, no eyeball retraction into the orbit and skin tent lower than s. score was described as moderate dehydration ( to % of water body loss) were the calf was depressed with weak suckle reflex, dropped ears, dry and slightly recessed eyes into the orbit and skin tent duration of to s. score was described as severe dehydration (> % of water body loss), when the calf showed signs of depression no suckle reflex, skin tent > s, dry and recessed eyes into the orbit and recumbency. calf attitude was assessed daily in conjunction with the health assessment. a depression scoring system to determine sickness [ , ] was modified. score corresponded to non-depressed animals. score corresponded to calves with noticeable depression and moderate signs of weakness but without altered gait. score corresponded to calves with severe depression marked signs of weakness and altered gait, in addition calves in recumbency were included. approximate milk intake was recorded after the am and pm feedings for all the calves that participated in the study. the intake was divided in categories, depending on milk refusal ( %; %; %; %; %) and an average daily intake was calculated. each animal was assigned with a daily health severity score, based on the combined morning health assessment (diarrhea score, dehydration score, attitude score) and the average milk intake. a status of "healthy" was determined when all the scores were (normal) and milk refusal was ≤ %. a "slight" disease status was applied to all the calves that had a milk refusal below % and at least one health score of . in the case of diarrhea, a score was also considered "slight" when the calf was not dehydrated and its attitude was not compromised. a "moderate" disease status was applied to the calves that presented more than two health scores of (or diarrhea score or ) and milk refusal above %. a "severe" disease status sick was given to calves in recumbency with more than two health scores in and milk refusal above %. organic certified jasmine stabilized rice bran was provided from urmatt thailand as a gift from rice bran technologies, sacramento, ca ( table ). the dose of srb was calculated to achieve % of the daily total calorie intake during the first weeks of life ( cal). this dose was calculated based on research with monogastric animals [ , , ] that indicated this level of inclusion as optimal. due to the milk feeding routine in this large rearing facility, reducing this % of the daily calories for the treatment group was not a possibility. the daily dose was divided into two feeding periods and mixed in the milk of the morning and the night feedings, as a higher milk intake was observed at these times compared with noon feeding. study personnel were responsible for the mixing and feeding of treatment calves. data were analyzed using sas statistical software ( . , sas institute inc., cary, nc usa). calf was considered the experimental unit of analyses. treatment group and health status at enrollment were included in the models unless otherwise specified. logistic regression analysis (proc logistic) was performed to determine differences between treatment groups in the frequency of events at enrollment, during the study period, and in the follow up until weaning. total sp measurements were categorized in two levels to detect failures in passive immune transfer or dehydration: < . g/dl and ≥ . g/dl. in addition, health status at enrollment was categorized as "healthy" or "diseased" and group differences were analyzed. these analyses were performed to assess the initial health condition of the treatment groups. additionally, logistic regression analysis was used to determine differences in frequencies of animal removal (death and culling aggregated in one variable) between treatment groups and to analyze differences in presentation of disease (< vs ≥ or more diseases) within the follow-up period. the association between the number of days sick (categorized by severity of disease) and treatment group was analyzed by use of repeated measures analysis for a binary response (proc genmod), assuming an exchangeable correlation structure. total calf days "healthy" were compared with total days "sick" (combining "slight", "moderate" and "severe"). in addition, total days calves spent with a "slight" disease condition were compared with the combination of "moderate" and "severe" days. finally, days in "severe" condition were compared combining days with "slight" and "moderate" condition. time to event analysis kaplan meier (proc lifet-est) was performed to evaluate differences in time to presentation and time to recovery from the first "moderate" case of disease between the groups. additionally, time to event analysis was used to evaluate differences in the time animals were removed and to evaluate differences in time to first disease after the end of the addition of srb. the wilcoxon test was used to determine statistical significance. least square means (proc glm) were calculated for adg and iga concentration. iga results were firstly log normalized. statistical significance was defined at p < . . tendency was defined at . < p < . . stress, immunity and the management of calves part iii: health and management practices in the u.s. dairy operations dietary modulation of the human colonic microbiota: introducing the concept of prebiotics prebiotics: why definitions matter prebiotics and resistance to gastrointestinal infections influence of dietary supplementation of prebiotics (mannanoligosaccharide) on the performance of crossbred calves fecal and saliva iga secretion when feeding a concentrated mannanoligosaccharide to neonatal dairy calves effects of a prebiotic supplement on health of neonatal dairy calves the effects of a prebiotic supplement (prebio support) on fecal and salivary iga in neonatal dairy calves effects of supplemental mannanoligosaccharides on growth performance, faecal characteristics and health in dairy calves evaluation of essential oils and prebiotics for newborn dairy calves effects of probiotic and prebiotic on average daily gain, fecal shedding of escherichia coli, and immune system status in newborn female calves body weight gain, feed efficiency, and fecal scores of dairy calves in response to galactosyl-lactose or antibiotics in milk replacers dietary rice bran protects against rotavirus diarrhea and promotes th -type immune responses to human rotavirus vaccine in gnotobiotic pigs consumption of rice bran increases mucosal immunoglobulin a concentrations and numbers of intestinal lactobacillus spp bioactive food components and health properties of rice bran dietary rice bran supplementation prevents salmonella colonization differentially across varieties and by priming intestinal immunity passive transfer of colostral immunoglobulins in calves managing the production, storage and delivery of colostrum tools to assess colostrum management. pennstate extension tolerance of a rice-based oral rehydration solution given to normal calves utilization of carbohydrates by the young calf effects of using wheat gluten and rice protein concentrate in dairy calf milk replacers effects of protein sources for milk replacers on growth performance and serum biochemical indexes of suckling calves effect of aluminized reflective hutch covers on calf health and performance calf health scoring chart heifer raising -birth to weaning. neonatal diarrhea. babcock institute for international dairy research and development clinical trial design in feedlots using calf depression score the authors also wish to express gratitude to rice bran technologies (sacramento, ca) for the donation of all the organic certified stabilized rice bran used in the study.funding usda-nifa orei award number - - funded the proposal "enhancing animal care strategies on organic dairy farms", providing resources for the completion of this study through graduate student support. the datasets generated and/or analyzed during the current study are not publicly available due to the extension of the daily health assessment but are available from the corresponding author on reasonable request. authors' contributions av: experimental design, data collection, laboratory analysis, statistical analysis, manuscript preparation. dm: data collection, laboratory analysis, statistical analysis. sp: data collection. hh and rc: manuscript preparation. epr: experimental design, srb procurement, manuscript preparation. pp: experimental design, statistical analysis, manuscript preparation. all authors have read and approved the manuscript.ethics approval and consent to participate animals were managed during the study in accordance to the guidelines stablished by the institutional animal care and use committee of colorado state university (protocol id: - a). a written consent, detailing all the animal related procedures, to use the cows in this study was provided by the owner. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -xyhh uky authors: oliveira, victor h.s.; agnol, alais m. dall; fritzen, juliana t.t.; lorenzetti, elis; alfieri, amauri a.; alfieri, alice f. title: microbial diversity involved in the etiology of a bovine respiratory disease outbreak in a dairy calf rearing unit date: - - journal: comp immunol microbiol infect dis doi: . /j.cimid. . sha: doc_id: cord_uid: xyhh uky the etiological agents involved in a bovine respiratory disease (brd) outbreak were investigated in a dairy heifer calf rearing unit from southern brazil. a battery of pcr assays was performed to detect the most common viruses and bacteria associated with brd, such as bovine viral diarrhea virus (bvdv), bovine respiratory syncytial virus (brsv), bovine alphaherpesvirus (bohv- ), bovine coronavirus (bcov), bovine parainfluenza virus (bpiv- ), mannheimia haemolytica, pasteurella multocida, histophilus somni, and mycoplasma bovis. bronchoalveolar lavage fluid (balf) samples were taken from heifer calves (symptomatic n = ; asymptomatic n = ) that, during the occurrence of the bdr outbreak, were aged between and days. at least one microorganism was detected in . % ( / ) of the balf samples. mixed infections were more frequent ( . %) than single infections ( . %). the interactions between viruses and bacteria were the most common in coinfections ( . %). the frequencies of brd agents were . % for brsv, . % for bvdv, . % for bcov, . % for p. multocida, . % for m. bovis, and % for h. somni. bohv- , bpiv- , and m. haemolytica were not identified in any of the balf samples. considering that balf and not nasal swabs were analyzed, these results demonstrate the etiological multiplicity that may be involved in brd outbreaks in dairy calves. the present study reports a molecular diagnostic survey for multiple etiological agents during an outbreak of brd in heifer calves in a brazilian dairy calf rearing unit. the brd outbreak occurred in a dairy calf rearing unit located in parana state, southern brazil. the region has a humid subtropical climate with hot, humid summers and mild winters with an average temperature of °c. the rearing unit maintained approximately mixed-breed heifer calves obtained from small dairy cattle herds for household milk production that were associated with a dairy cooperative. data on housing, feeding, and management of the calves were collected through an interview with the veterinarian in charge. calves arrive at the rearing unit at - days of age and are housed in group pens ( x m). twenty to calves are grouped in each pen until approximately days of age. calves are fed in an automatic feeder system for each pen with calf milk replacer in a common nipple, and concentrates on pelleted calf feed containing % crude protein are provided ad libitum. thus, heifer calves from diverse origins and with distinct health and immunological status are grouped together in the same rearing unit. data about the sanitary status of the original herds of the calves were unknown, except for the compulsory sanitary management practices against bovine brucellosis, tuberculosis, and foot and mouth disease, according to the national program for the control and eradication of these diseases. none of the dairy herds that provided heifer calves for the rearing unit vaccine cows to improve the colostrum quality and thus provide passive protection to calves against the major brd-causing pathogens. the j o u r n a l p r e -p r o o f efficiency of the transfer of passive immunity is also not monitored. in the rearing unit, the calves also do not receive vaccines for brd control. first, asymptomatic and symptomatic heifer calves with brd were identified by clinical examination. typical clinical signs of respiratory disease were not present in calves classified as asymptomatic. calves classified as symptomatic showed clinical signs of coughing and copious nasal discharge in association with at least two of the following clinical manifestations: rectal temperature above . °c, prolonged capillary refill time, pale mucous membranes, heart rate above beats/min, respiratory discomfort, and respiratory rate above breaths/min [ , ] . symptomatic calves with brd signs were not separated from asymptomatic animals, even during clinical treatment. the treatment of brd-affected calves was performed with broad-spectrum antibiotics (spectinomycin and tulathromycin) and anti-inflammatory drugs. after clinical examination, bronchoalveolar lavage fluid (balf) samples were collected from asymptomatic (n = ) and symptomatic (n = ) untreated calves following the collection procedures previously described [ ] . the calves of the calf rearing unit were divided into groups based on age, between - days, - days and over days. at least four balf samples per age group were collected at random including asymptomatic and symptomatic calves. the collection procedures of balf samples were conducted by veterinarians at the universidade estadual de londrina, paraná, brazil, including a trained veterinary surgeon, in a single visit to the rearing unit. the samples were placed in sterile tubes, shipped on ice baths and stored at - °c until processing. j o u r n a l p r e -p r o o f nucleic acids were extracted from -μl aliquots of balf samples pretreated with sodium dodecyl sulfate (sds) and proteinase k incubated at °c for min at a final concentration of % (v/v) and . mg/ml, respectively. balf samples were then processed following a silica/guanidine isothiocyanate protocol [ ] . the extracted nucleic acid was eluted in μl of ultrapure nuclease-free diethylpyrocarbonate-treated sterile water and stored at - °c until used for molecular analysis. molecular diagnostic assays (pcr, rt-pcr, and nested pcr) were performed for the detection of the main infectious agents associated with brd. the techniques were performed separately to amplify each of the infectious agents, a product with bp was amplified of the bvdv ' utr gene [ ] , bp of the brsv g gene [ ] , bp of the bcov n gene [ ] , bp of the bpiv- hn gene [ ] , bp of the bohv- d gene [ ] , bp of the p. multocida orf clone kmt [ ] , bp of the h. somni s gene [ ] , bp of the m. haemolytica lkta-artj intergenic region [ ] , and bp of the m. bovis s- s intergenic region [ ] . aliquots of sterile ultrapure water were included as negative controls in all procedures. samples previously known as positive for each of the pathogens investigated in this study were included as positive controls as follows: prototype los angeles, nadl, a , sf / , and mebus strains cell culture (mdbk) adapted for bohv- , bvdv, brsv, bpiv- , and bcov, respectively; nucleic acid from previous reports for h. somni [ ] ; and housekeeping samples for p. multocida, m. haemolytica, and m. bovis [ ] were also included as positive controls. similarity searches were performed with nucleotide sequences deposited in the genbank database using the blast highly similar tool software (http://blast.ncbi.nlm.nih.gov/blast.cgi). the brd outbreak analyzed in this study lasted approximately days, from the first clinical signs were observed until their complete remission. the morbidity rate of calves with clinical manifestations of respiratory distress reached %. calves aged up to days were more frequently affected than older calves, and in the last week of the brd outbreak, seven calves died. the frequencies of microorganism detection in this study were . % ( / ) for bvdv, . % ( / ) for brsv, . % ( / ) for bcov, . % ( / ) for p. multocida, % ( / ) for h. somni, and . % ( / ) for m. bovis. all these respiratory pathogens were detected in asymptomatic and symptomatic calves ( table ). the detection of viruses and bacteria occurred in calves of all age groups ( - days; table ). single infections were more frequently associated with bacteria ( / , . %) than with viruses ( / , . %). associations between viruses and bacteria were the most frequent / ( . %). other mixed infections presented a frequency of / ( . %) ( table ). the identities of the respective detected microorganisms were confirmed by their similarities with other nucleotide sequences deposited in a database using blast software. all obtained dna sequences were clearly readable and have been deposited quadruple bvdv + brsv + bcov + p. multocida total to the best of our knowledge, the current study is the first molecular-based investigation of the main pathogens involved in brd during an outbreak in a dairy heifer calf rearing unit. this study also represents the first south american report of molecular detection of brd mixed infections in calves with clinical signs respiratory disease. the results obtained in the current study add to what was described on pathogens associated with brd in dairy calves [ ] , since most studies of brd infections were conducted in feedlot cattle [ , , , ] . bpi- , bohv- , and m. haemolytica were not detected in balf samples these calves evaluated. in brazilian cattle herds, bpi- was detected in few studies, isolated in a single animal in south of the country [ ] and another study using immunohistochemical it was detected in four animals [ ] , but in the most brd studies was not found [ , , , ] . possibly, bpi- circulates with low frequency in brazilian cattle herds. the seroprevalence of bohv- infection in cows of dairy cattle herds in the geographical region of the calf rearing unit is high [ ] . brd associated with bohv- usually occurs in older animals. thus, it is likely that maternal antibodies acquired by calves against bohv- provided adequate protection in the first months of age [ ] . m. haemolytica is a natural inhabitant of the upper respiratory tract of the bovine species, occasionally it may develop brd outbreaks [ ] . in this study, no balf sample analyzed was positive for this bacterium. our results are consistent with another study also carried out in rearing unit, with calves from different dairy herds of origin, in which a low number of animals infected by m. haemolytica was observed ( %), however a greater number of positive for bacterial agents, such as mycoplasma sp. and p. multocida, and viral agents such as brsv and bcov [ ] . considering that the upper airways of asymptomatic calves may be colonized by a variety of bacterial pathogens [ ] , the present study was performed using balf as clinical specimens. these samples of the lower respiratory tract are suitable to achieve a more reliable result of microorganisms associated with the etiology of brd [ ] . in addition, the molecular diagnosis adopted in the current study permitted a large number of species-specific tests for each pathogen after molecular assays were standardized. brd cases in brazil usually include pathological examination, serology, bacterial cultures and/or virus isolation [ , , , ] . however, these techniques may be appropriate for one agent but not for the other due to different sensitivity values [ ] . additionally, serology tests in young cattle may interfere with colostral antibodies and cross-reactions of pathogens with other commensal microorganisms, making interpretation of test results difficult [ ] . classical virus and bacterial isolation methods are laborious and time-consuming [ ] . among the antemortem diagnostic procedures applicable to brd-affected calves, molecular techniques may be considered the most appropriate due to their fast and reliable results, facilitating actions to define preventive and therapeutic strategies in brazilian herds [ ] . in comparison to pathogens detected in balf from diseased and healthy calves, a higher frequency of viral detection was observed in animals with clinical signs of brd. calves housed together were the main reservoir of infectious agents to susceptible young cattle [ ] ; thus, possibly asymptomatic and symptomatic calves may have contributed to pathogen dissemination among the heifer calves. evaluating bacterial pathogens, there was no increase in detection in disease animals compared to clinically healthy animals in this study. however, a study conducted with a larger number of samples and herds found a higher bacterial frequency in diseased animals [ ] , this difference in our study may have been due to the small number of samples used. in brazil, there are few reports about brd, and these studies have focused on efforts to detect the involvement of respiratory tract organs by gross and microscopic lesions, serological evaluations, and characterization of specific pathogens [ , , - , , , , , ] . a retrospective study of years was performed by a university in the south of brazil, herds with outbreaks of brd were evaluated and animals that died, the age of the calves varied from day to months. a high morbidity rate was observed reaching up to % of the animals and the mortality rate reached % [ ] . however, preventive and control measures are not usually performed in most brazilian dairy cattle herds, and may have contributed to the high rates of morbidity and mortality [ ] . in our study, we found a morbidity rate that reached %; the high rate of infected animals j o u r n a l p r e -p r o o f may be due to the non-separate disease calves, increasing load of pathogens in the environment and favoring the infection of susceptible calves [ ] . in this study, the findings of . % positive balf samples for at least one of the pathogens associated with brd highlight the importance of this disease in the calf rearing unit, especially of the mixed infections that were more frequent ( . %) than single infections ( . %). the most frequent mixed infections were between viruses and bacteria ( . %), which shows the synergism of these infectious agents in brd, similar to severe pneumonia in children and puppies [ , ] . single viruses associated with brd are rarely lethal to cattle. severe pneumonia usually occurs when commensal bacteria from the nasopharynx invade the lower airways of cattle after stressful conditions and viral infections. these predisposing factors affect the host defense mechanisms by altering mucosal surface components and decreasing the activity of innate immune system cells, such as t lymphocytes, b-lymphocytes, monocytes, and macrophages, thereby increasing the exposure to pathogens [ ] . brsv had the highest prevalence of this outbreak ( . %). this result is in keeping with previous studies conducted in demark and finland, which observed that this virus is the most important in brd of dairy calves [ , ] . the bcov described in this brd outbreak was the second report of this virus associated with respiratory symptoms in cattle in brazil. the first case was detected in a beef cattle feedlot [ ] . the role of bcov in brd has been recognized recently with the dual enteric and respiratory tropism of some strains [ ] . brd related to bcov is a potential threat to calves in calf rearing units, especially if the peak of virus shedding is highly short, occurring up to days after arrival at facilities for rearing practices [ ] . the immunosuppressive effect of bvdv in the host organism is considered the main factor associated with the development of brd in cattle of affected herds due to j o u r n a l p r e -p r o o f an increased risk of infections by other pathogens [ , ] . multiple strains of bvdv have been reported in cattle from the same geographic region of the calf rearing unit, such as bvdv a, bvdv b, and bvdv d, associated with reproductive failure in cows [ ] . within these subgenotypes, bvdv b was suggested be predominantly associated with brd in calves [ ] . however, the phylogenetic analysis performed using 'utr sequences of bvdv strains in the balf samples of the current brd outbreak clustered with the bvdv d subgenotype. furthermore, susceptible calves do not become ill until weeks of age after exposure to bvdv virulent strains when passive immunity is acquired, even supposing that low virus replication may occur in these animals [ ] . however, in the present study, bvdv was detected in balf samples of calves in the first weeks of age. this finding may be due a failure in the transfer passive antibodies to the newborn calves due to colostrum deprivation, low intake or even the lack of immunity of the cow with the different strains of bvdv, this can make the calves susceptible the infection occurs in the first weeks of life. also, infection of these calves with different bvdv strains of their herds of origin, since cross-protection between different bvdv subgenotypes is incomplete [ , ] . among the four bacterial pathogens in brd cases evaluated in this study, only at the time of sampling, the brd outbreak in the calf rearing unit was not controlled. antimicrobial drugs used in calves (spectinomycin and tulathromycin) are commonly used in brazil for brd treatment. however, these drugs were not used as metaphylaxis and/or therapy in the early stages of brd, which increased the success of treated calves [ , ] . in association with this condition, potential risk factors for brd previously described were detected at the rearing unit [ , , ] . this unit includes automatic milk feeders with a common nipple, large group pens (> calves), older calves housed with younger calves, and calves from various sources with unknown sanitary status. bovis isolated from young cattle in france revealed that % of recent strains are resistant to common antibiotics used to control brd [ ] . thus, it may be suggested that when calves are challenged with so many etiological agents and in the presence of risk factors, simple maternal immunity and broad-spectrum antimicrobial drugs may not be sufficient to control brd in calves. however, an essential tool for prevention is the use of commercial vaccines against the main pathogens that cause respiratory, bacterial, and viral diseases. these vaccines should be administered mainly to cows in the final period of pregnancy, to promote the transfer of immunoglobulins by colostrum. in addition, it is essential to ensure the intake of an adequate amount of colostrum soon after the calves are born. finally, all preventive measures together can help to reduce the number of susceptible animals and the excretion of microorganisms, in addition to reducing the risk of brd occurring mainly in the critical phases, which are transport and grouping in the calf rearing unit [ , ] . in conclusion, the findings of this study revealed the presence of several microorganisms, viruses and bacteria, associated with brd in a dairy heifer calf rearing unit from brazil that differs from previous reports carried out with lower respiratory the authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. an economic model to calculate farm-specific losses due to bovine respiratory disease in dairy heifers morbidity in swedish dairy calves from birth to days of age and individual calf-level risk factors for infectious diseases bovine respiratory disease complex associated mortality and morbidity rates in feedlot cattle from southeastern brazil preweaning cost of bovine respiratory disease (brd) and cost-benefit of implementation of preventative measures in calves on california dairies: the brd k study risk factors associated with exposure to bovine respiratory disease pathogens during the peri-weaning period in dairy bull calves respiratory disease in calves: microbiological investigations on transtracheally aspirated bronchoalveolar fluid and acute phase protein response respiratory infections in norwegian dairy calves lower respiratory tract microbiome and resistome of bovine respiratory disease mortalities pathogenesis and pathology of bovine pneumonia diseases and pathogens associated with mortality in ontario beef feedlots pathogens, patterns of pneumonia, and epidemiologic risk factors associated with respiratory disease in recently weaned cattle in ireland factors associated with morbidity, mortality, and growth of dairy heifer calves up to months of age morbidity in - -month-old dairy calves in south-western sweden, and risk factors for diarrhoea and respiratory disease dairy calf rearing unit and infectious diseases: diarrhea outbreak by bovine coronavirus as a model for the dispersion of pathogenic microorganisms etiology of respiratory disease in non-vaccinated, non-medicated calves in rearing herds prevalence of respiratory pathogens in diseased, non-vaccinated, routinely medicated veal calves bovine respiratory disease in feedlot cattle: environmental, genetic, and economic factors molecular survey of infectious agents associated with bovine respiratory disease in a beef cattle feedlot in southern brazil management factors associated with bovine respiratory disease in preweaned calves on california dairies: the brd study the epidemiology of bovine respiratory disease: what is the evidence for predisposing factors? bacterial pathogens and cytology or tracheobronchial region in calves during neonatal period respiratory diseases in calves in southern rio grande do sul: study of outbreaks mycoplasma bovis and viral agents associated with the development of bovine respiratory disease in adult dairy cows control, management, and prevention of bovine respiratory disease in dairy calves and cows association between antimicrobial drug class for treatment and retreatment of bovine respiratory disease (brd) and frequency of resistant brd pathogen isolation from veterinary diagnostic laboratory samples rapid and simple method for purification of nucleic acids pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis development of nested pcr assays for detection of bovine respiratory syncytial virus in clinical samples improved detection of bovine coronavirus n gene in faeces of calves infected naturally by a semi-nested pcr assay and an internal control isolation and genetic characterization of bovine parainfluenza virus type from cattle in china bovine herpesvirus type abortions detected by a semi-nested pcr in brazilian cattle herds development of pcr assays for species-and type-specific identification of pasteurella multocida isolates development of a pcr test for identification of haemophilus somnus in pure and mixed cultures a nested-pcr strategy for molecular diagnosis of mollicutes in uncultured biological samples from cows with vulvovaginitis histophilus somni-induced infections in cattle from southern brazil histophilus somni is a potential threat to beef cattle feedlots in brazil lung pathology and infectious agents in fatal feedlot pneumonias and relationship with mortality, disease onset, and treatments isolation of bovine parainfluenza virus type in rio grande do sul, brazil mycoplasma bovis and viral agents associated with the development of bovine respiratory disease in adult dairy cows seroprevalence and risk factors of bovine herpesvirus infection in cattle herds in the state of parana, brazil maternally derived humoral immunity to bovine viral diarrhea virus (bvdv) a, bvdv b, bvdv , bovine herpesvirus- , parainfluenza- virus bovine respiratory syncytial virus, mannheimia haemolytica and pasteurella multocida in beef calves, antibody decline by half-life studies and effect on response to vaccination mannheimia haemolytica in bovine respiratory disease: immunogens, potential immunogens, and vaccines innate immunology of bovine respiratory disease laboratory test descriptions for bovine respiratory disease diagnosis and their strengths and weaknesses: gold standards for diagnosis, do they exist? investigation of polymerase chain reaction assays to improve detection of bacterial involvement in bovine respiratory disease brazilian strain of bovine respiratory coronavirus is derived from dual enteric and respiratory tropism bacterial pathogens of the lower respiratory tract of calves from brazilian rural settlement herds and their association with clinical signs of bovine respiratory disease phylogenetic relationships of brazilian bovine respiratory syncytial virus isolates and molecular homology modeling of attachment glycoprotein impact of bacterial and viral coinfection on mycoplasmal pneumonia in childhood communityacquired pneumonia bacterial pneumonia in dogs and cats dual enteric and respiratory tropisms of winter dysentery bovine coronavirus in calves bovine respiratory coronavirus bovine viral diarrhea viral infections in feeder calves with respiratory disease: interactions with pasteurella spp., parainfluenza- virus, and bovine respiratory syncytial virus bovine viral diarrhea virus: global status the diversity of bvdv subgenotypes in a vaccinated dairy cattle herd in brazil bovine viral diarrhea virus (bvdv) b: predominant bvdv subtype in calves with respiratory disease bvdv genotypes and biotypes: practical implications for diagnosis and control association of bovine respiratory disease with clinical status and acute phase proteins in calves detection of mycoplasma bovis with an improved pcr assay bovine respiratory disease: pathogenesis, clinical signs, and treatment in lightweight calves calf mortality in norwegian dairy herds overall decrease in the susceptibility of mycoplasma bovis to antimicrobials over the past years in france effects of vaccination prior to transit and administration of florfenicol at time of arrival in a feedlot on the health of transported calves and detection of mannheimia haemolytica in nasal secretions the brazilian institutes cnpq, capes, finep, and fap/pr provided funding for this study. aa alfieri and af alfieri are recipients of the national council for key: cord- -a ia kxf authors: bellinzoni, r. c.; blackhall, j.; baro, n.; auza, n.; mattion, n.; casaro, a.; la torre, j. l.; scodeller, e. a. title: efficacy of an inactivated oil-adjuvanted rotavirus vaccine in the control of calf diarrhoea in beef herds in argentina date: - - journal: vaccine doi: . / - x( ) - sha: doc_id: cord_uid: a ia kxf abstract we have assessed the potency of an inactivated oil-adjuvanted rotavirus vaccine in beef herds in argentina. two different vaccine trials were conducted. in a small-scale experimental trial, involving pregnant cows ( vaccinated and eight unvaccinated controls), a significant increase in neutralizing antibody titres against different serotypes of bovine rotaviruses was found in both the colostrum and serum of vaccinated cows compared with that of unvaccinated controls. seven days after birth, half of the calves born to vaccinated dams or to control cows were challenged with live virulent virus whereas the other half of both groups were left in contact with the infected calves in order to mimic a natural field challenge. although no statistically significant differences in the rate of protection were observed among the different groups of animals, a larger number of vaccinated calves were protected in comparison with their controls, particularly where animals in contact with infected calves were concerned. secondly, a large-scale field trial was carried out in beef herds involving a total of vaccinated pregnant cows. in farms morbidity and mortality in calves from vaccinated cows were compared with historical data from the previous years at the same locations. in the other six herds, control groups were used to compare data of the same year: cows were vaccinated and were left as controls. taking into account the previous and current incidence of diarrhoea, morbidity and mortality were significantly reduced in of the beef herds tested. vaccine effectiveness was also evident in farms where other enteropathogens such as cryptosporidium and coronaviruses were present, together with rotavirus. diarrhoea in newborn calves is a syndrome in which several causative agents (acting separately or associated in different ways) may be involved. surveys carried out in different countries have shown that rotaviruses, coronaviruses, enterotoxigenic escherichia coli, salmonella spp. and cryptosporidium are the pathogens chiefly responsible for outbreaks of calf diarrhoea - . the control of diarrhoea caused by different pathogenic microorganisms should be possible by the development of preventive strategies through the use of appropriate vaccines , in addition to the general measures currently used, such as management, nutrition and chemotherapeutics. to be effective, these vaccines must be designed rationally and based on the impact that the different enteropathogens have in a definite geographical region; therefore single agents or appropriate combinations of them should be components of these vaccines. other relevant factors in achieving effective vaccination are the type of vaccine (live attenuated or inactivated virus) the formulation, and the route and timing of immunization(s). calves can be protected against rotavirus and enterotoxigenic e. coil by passive immunization, taking advantage of the lactogenic immunity stimulated by maternal vaccination or directly by feeding calves with hyperimmune colostrum obtained from vaccinated dams ' . in contrast, attempts to stimulate active immunity in newborn calves by oral vaccination with live attenuated virus have been unsuccessful so far ' . with regard to the formulation of the vaccine, the most relevant factor is which type of adjuvant to use. when the target for vaccination is the pregnant cow, it has been demonstrated that oil-based adjuvants are more effective than alhydrogel to enhance colostrum and milk antibody titres, at least against e. coli, rotavirus and coronavirus °, . in argentina, neonatal diarrhoea is responsible for important economic losses in beef and dairy herds; morbidity can reach - % whereas mortality ranges between % and %. rotavirus is the major agent associated with diarrhoeal problems in argentinian beef herds . for that reason, after several years of epidemiological studies, it was decided to develop and test an inactivated oil-adjuvanted vaccine with the aim of controlling diarrhoea in beef and dairy herds in argentina. we now report the results of two trials performed with the experimental vaccine. the first trial was a challenge experiment performed in confinement and involving cows; the second was an open field trial involving vaccinated cows in different beef herds. the results showed that the oil-adjuvanted rotavirus vaccine tested was effective in the control of calf neonatal diarrhoea in argentina. the following rotavirus strains were used throughout this work: strain t . this was isolated from a diarrhoeic calf in argentina ( ) and was adapted to grow in ma- cells; it was cloned four times by limited dilution after the sixth passage. according to its antigenic characteristics this strain was classified as serotype (see table ). strain t . this strain, isolated from a diarrhoeic calf in argentina (in ), was adapted to grow in ma- cells and was cloned three times. it was selected for this work because it is one of the few local strains that does not cross-react with the bovine prototype strain, uk. strain uk. this was kindly provided by dr d.r. snodgrass (moredum research institute, edinburgh, scotland, uk). it was passaged times in ma- cells, cloned three times and classified as serotype (ref. ) . strain b . this bovine strain, isolated in iowa, usa and kindly provided by dr g. woode (texas, usa), was passaged nine times in ma- cells and cloned three times. like the local strain t , this strain did not cross react in neutralization assays with the ncdv prototype strain ~ . hyperimmune antisera were prepared in seronegative guinea pigs by injecting intramuscularly #g of the corresponding purified virus emulsified with an equal volume of freund's complete adjuvant. two more injections were given subsequently with the same quantity of virus but mixed with freund's incomplete adjuvant. sera were collected one week after the last inoculation and monitored by elisa and neutralization assays. the assays were carried out as described by gerna et al. ~ briefly, pl virus, diluted with medium to give focus-forming units (f.f.u.) per well, were mixed with # twofold serial dilutions of the antisera to be tested. mixtures were incubated for min at °c and inoculated on top of confluent monolayers of ma- cells growing in -well microtitre plates. the plates were incubated for - h. after fixation with acetone, cells were stained by the ipa technique . the neutralizing titre of each serum was expressed as the reciprocal of the highest dilution that gave % reduction in the number of the stained cells compared with control wells. homologous control viruses were included in the test for each serum. the vaccine was produced with the local rotavirus strain, t , the virus was grown in monolayers of ma- cells. after complete cytopathic effect, cellular fluids containing debris and virus were clarified by centrifugation ( for rain), inactivated with . % formalin ( h at °c), and emulsified with an equal volume of oil adjuvant comprising a mixture of % marcol and % montanide (trademarks of seppic, france). the innocuity of each inactivated antigen preparation was tested by observation of any posible cytopathic effect after three serial passages in ma- cell monolayers. thirteen pregnant hereford heifers were vaccinated twice subcutaneously with ml of the oil vaccine and days before expected delivery. eight heifers remained as unvaccinated controls. calves were nursed by their mothers throughout the experiment; on the seventh day of life half of the calves of both groups were injected orally with tcidso of the t strain. the uninoculated calves were kept in close proximity to those inoculated in order to evaluate the possible occurrence of natural transmission of challenge virus, as might happen in the field ('field exposure'). calves were examined daily for days and were considered diarrhoeic if animals excreted loose or watery faeces for at least days . daily faeces samples were examined for rotavirus, coronavirus, cryptosporidium, enterotoxigenic e. coli and salmonella as described by snodgrass et al. a. cows were bled before first and second vaccination, at calving and at days after delivery. colostrum and milk samples were collected at calving and at , and days postcalving. rotavirus-neutralizing antibodies were assayed in each sample, as described previously . neutralizing antibodies against the uk and t strains (serotype ), the b strain and the t local strain, were determined in colostrum (first milking). in addition, neutralizing antibodies against the t strain were determined in the milk on various days post-calving. seventeen beef herds were selected to perform the field trials, the criterion being presentation of calf diarrhoea problems during ~> years before this study. the beef herds selected for the vaccine trial were located in the provinces of buenos aires and c rdoba, comprising a considerable area of the principal breeding region of the country. these herds had previously been part of an extensive epidemiological study (involving a total of herds) performed by our laboratory to determine the aetiology of the high incidence of neonatal diarrhoea in this region (r.c. bellinzoni et al., unpublished results) . in herds the results were compared with historical controls and in the other six herds approximately two-thirds of the cows were left as unvaccinated controls. a total of cows were vaccinated once, as described above, month before the onset of calving. in four of those herds, unvaccinated cows were separated from the vaccinated animals. in the other two herds, both groups were mixed; in this case, the animals in the unvaccinated group were selected randomly. all the cows involved in the experiment were correctly identified and each one was feeding its own calf. as shown in table , in the vaccinated group only two of seven challenged calves, and none of those kept in close contact with them, developed diarrhoea, whereas in the control group, three of four inoculated animals and two of four uninoculated animals developed diarrhoea. virus excretion did not differ in the inoculated calves from both groups (vaccinated and unvaccinated), but differences were observed in close-contact controls ( table ). all rotaviruses isolated from positive faeces showed identical genomic rna patterns to the t strain used for vaccine production and challenge (data not shown). the results shown in table suggest a higher rate of protection in vaccinated animals compared with unvaccinated controls; however, because of the small number of animals involved, no significant differences were noted on statistical analysis of the data. other difficulties with this type of experiment are, first, assessment of the correct dose of virus to be used for the challenge, and second, the furthermore, management of the herds was not disturbed by participation in the trial. the experiment was carefully controlled by trained veterinarians in close cooperation with farm personnel. during weekly visits to the herds, information about diarrhoeic animals was recorded. the trial lasted until each calf was days old. in all cases, diarrhoea was defined as loose or watery faeces, a situation that usually called for treatment of the diseased animals. in some herds, faecal samples were taken from diarrhoeic calves the year before the trial ( ) and during the present trial ( ), and were examined for rotavirus, coronavirus, cryptosporidium, salmonella and etec as described previously . inability to ensure that all the unchallenged animals kept in close proximity to the infected calves received a pathogenic dose of the virus . coronavirus, cryptosporidium or etec were not detected in any of the diarrhoeic samples analysed. aspects of the immunological status of vaccinated animals were also analysed. as shown in figure , compared with controls, vaccinated cows showed significantly higher neutralizing antibody levels against rotavirus in serum, colostrum and milk until at least days after calving. in addition, the titres of colostrum neutralizing antibodies were sufficiently high to neutralize other strains of rotavirus as well. as can be seen in table , colostrum from cows vaccinated with a strain belonging to serotype also showed neutralizing activity at similar titres against heterologous serotypes. table summarizes the results obtained from the large-scale field trial. in the case of herds a-f, morbidity and mortality data obtained from the last years were compared with data obtained during the current trial. no significant differences were detected in the incidence of diarrhoea and mortality in the contemporary control vaccine, vol. , june aln farms a-f, data relating to the incidence of diarrhoea and mortality in calves born from vaccinated cows were compared with data obtained from an unvaccinated control group as well as with data from the last years at the same farms. in two farms (e and f), controls were mixed (m) with vaccinated animals; in the other four farms (a-d), control animals were separated (s) from the vaccinated areas. in farms c_,-q =, data were compared only with the data of the last years at the same farms; bmean of the last years; centeropathogens detected the year before vaccination: r, rotavirus; co, coronavirus; cr, cryptosporidium; as, controls kept apart from vaccinated animals; m, control and vaccinated animals kept in close proximity groups ( . and . % respectively) when compared with the data of the last years ( and . % respectively) suggesting that, in general, the epidemiological situation did not change significantly in those herds. thus (apart from herd f which showed a low previous incidence of diarrhoea and mortality), vaccination of the cows led to an important reduction in the incidence of diarrhoea and mortality ( . and . %, respectively). in four herds (a-d), vaccinated and control animals were kept apart and in the other two (e, f), controls were mixed with vaccinated animals. in one case (see table , herd f) the incidence of diarrhoea and mortality in the control group was lower than that previously recorded. however, in the other herd kept under similar conditions (table , herd e) the values for the control group were similar to the mean of the last years. thus it is not possible from these results, to reach any conclusions about the effect of maintaining vaccinated animals together with, or separated from, non-vaccinated animals. in the remaining herds (table , herds g-p) the incidence of diarrhoea and mortality in calves born to vaccinated animals was compared with the data of the previous years. the results show again that in of those herds (g-o) diarrhoea and mortality in calves from vaccinated cows ( . and . % respectively) were far less than previously recorded in the same herds ( . and . %, respectively). in nine of the herds involved in this study, the presence of other enteropathogens was studied the year before vaccination. in addition to rotaviruses, coronavirus and/or cryptosporidium was detected. in three herds (table : ll, n and p) animals were sampled during the year of the trial and the same pathogens were found (data not shown). in this context, it should be emphasized that, in eight of the nine herds in which other enteropathogens as well as rotavirus were present, the vaccine was also effective in decreasing morbidity and mortality due to diarrhoea. a growing body of evidence ' indicates that in order to guarantee adequate protection of newborn calves against enteropathogens it is necessary to supply them with continuous and sutticient amounts of neutralizing antibodies against the specific agent. this can be achieved by immunization of the dams, before delivery, with inactivated oiladjuvanted vaccines that will induce the production and excretion of rotavirus antibodies in colostrum and postcolostral milk ' . in this study an oil-adjuvanted inactivated vaccine was assayed in two different experiments. the main purpose of the preliminary small-scale trial, which involved pregnant cows and was conducted with the animals confined and under careful veterinary control, was to evaluate the immune response of such cows after vaccination. the efficacy of this vaccine in protecting calves from experimental challenge was also assayed in this trial. in concordance with previous reports . , the results of this experiment show that the level of neutralizing antibodies against homologous and heterologous rotaviruses in sera and colostrum was significantly enhanced on vaccination of the pregnant cows. under the conditions of this trial the incidence of diarrhoea in calves born from vaccinated cows was lower than those born from unvaccinated controls. an interesting observation was the finding that all four uninoculated control calves were actively excreting rotavirus, indicating that the natural exposure used in this experiment was a valid method of challenge. this finding is important because the dose of challenge virus used in a direct inoculation probably would not be representative of the field conditions in which spontaneous infections are continuously occurring. in this regard, difficulties (overwhelming or inadequate doses of virus) in evaluation of the efficacy of immunization by direct inoculation of calves, have been reported , . the results of the small experimental trial showed that the experimental vaccine was able to induce, in the milk and sera of vaccinated cows, an adequate level of neutralizing antibodies against homologous and heterologous rotaviruses; in addition, the results of the challenge were encouraging. it was therefore decided to test the vaccine in a large .field trial in order to evaluate the protection afforded by the vaccine against circulating field rotavirus. in the field trial the efficacy of the vaccine was assessed by two different approaches: by comparing the incidence of diarrhoea and mortality between vaccinated and unvaccinated groups of animals and by comparison of these data with previous values of diarrhoea and mortality (mean of the last years) at the same farms. the use of previous data to compare the results of vaccination can be problematical because of the possibility that the incidence of the various enteropathogens may change from year to year; on the other hand, when data are compared with contemporary controls that are in close contact with the vaccinated group, the incidence of diarrhoea and mortality in these controls may be underestimated because of herd immunity . in order to avoid this problem, control and vaccinated groups can be separated during the trial. the experiment shown in table was designed to test the comparison methods. as judged by the results obtained, it was clear that the vaccine was effective in protecting calves born from vaccinated dams independently of the method used for comparison (historical or actual values of incidence of the disease). it is likely that the higher level of protection against rotavirus infection of calves born to vaccinated dams, compared with those born to control cows, was acquired through colostral immunity. all the herds selected for this work had experienced problems with calf diarrhoea during at least the three years before the trial. nine of the herds studied were sampled the year before the trial and, besides rotavirus, coronavirus and/or cryptosporidium were detected; in three of those herds the same enteropathogens that had been found the year before were detected again at the moment of the trial (see results) , suggesting that there were no significant changes in the epidemiological situation at the trial locations. this observation is relevant because the vaccine was equally effective in herds where rotavirus alone, or where rotavirus and the other two enteropathogens, were present. these results are in contradiction to those reported by snodgrass who found that, in two herds in which rotavirus was associated with cryptosporidium, rotavirus vaccination was effective in diminishing the excretion of rotavirus but not in controlling diarrhoea. the reason(s) for such discrepancy are at present obscure and further work with a larger number of herds and animals is necessary to elucidate this problem. however,judging by the results obtained in this work, it is tempting to speculate that under the particular conditions of cattle breeding in our country, rotaviruses could be the agents that play a major part in determining diarrhoea in newborn calves. in this regard, it is likely that biological, nutritional, environmental or management factors could influence the persistence of a given pathogen in the field, or could well influence the degree of pathogenicity of a given infectious agent or different combinations of agents . the results of this work have confirmed and extended the results of snodgrass and colleagues regarding the effectiveness of an oil-adjuvanted inactivated rotavirus vaccine in controlling (in this case) diarrhoea in beef herds in argentina. these data, obtained from vaccinated cattle in different herds, represent the largest field trial so far for this type of vaccine. pathological and microbiological observations made on spontaneous cases of acute neonatal calf diarrhoea infectious agents associated with neonatal calf disease in south western idaho and eastern oregon aetiology of diarrhoea in young calves microbiology of calf diarrhoea in southern britain vaccination against enteric rota and coronaviruses in cattle and pigs: enhancement of lactogenic immunity evaluation of a combined rotavirus and enterotoxigenic escherichia coli vaccine in cattle passive immunity to bovine rotavirus in newborn calves fed colostrum supplements from immunized or nonimmunized cows intestinal antibody response after vaccination and infection with rotavirus of calves fed colostrum with or without rotavirus antibody laboratory experiments or viral vaccination of calves against rotavirus or coronavirus induced diarrhoea. zentralbl. veterinaermed passive immunity in calf rotavirus infections: maternal vaccination increases and prolongs immunoglobulin g antibody secretion in milk evolution des anticorps anti-rota dans le lait de vaches traitees en fin de gestation soit par le vaccine anti-rota complet, soit par i'adjuvant seul incidence of rotavirus in beef herds in argentina serotypic similarity and diversity of rotaviruses of mammalian and avian origin as studied by plaque-reduction neutralization antigenic relationships among some bovine rotaviruses: serum neutralization and cross-protection in gnotobiotic calves serotyping of cell culture adapted subgroup human rotavirus strains by neutralization bovine rotavirus serotypes and their significance for immunization bovine milk immunogtobulins for passive immunity to infantile rotavirus gastroenteritis the authors are indebted to the veterinary surgeons and farmers who participated in this trial and to andros bellinzoni and alvina lorensi for technical assistance. this work was supported by consejo nacional de investigaciones cientificas y t~cnicas (conicet), secretaria de ciencia y trcnica (secyt) and the swedish agency for research cooperation with developing countries (sarec). key: cord- -ci onndm authors: de waele, v.; speybroeck, n.; berkvens, d.; mulcahy, g.; murphy, t.m. title: control of cryptosporidiosis in neonatal calves: use of halofuginone lactate in two different calf rearing systems date: - - journal: prev vet med doi: . /j.prevetmed. . . sha: doc_id: cord_uid: ci onndm to date there is no effective treatment for bovine cryptosporidiosis. this study describes the use of halofuginone lactate in preventing cryptosporidiosis in naturally infected neonatal calves on a dairy farm with a high prevalence of infection. the animals were kept in two different calf rearing systems. a randomized double-blind trial was carried out with naturally infected calves, divided into four groups. the two prophylactic halofuginone lactate treated groups were kept in either individual or group pens. similarly, the animals receiving the placebo were housed in either individual pens or together in a large pen. a total of ten faecal samples were collected periodically during the days study from each calf and tested for the presence of cryptosporidium spp. using microscopic and molecular methods. generalized estimating equations models were used to determine if the effects of the various treatments and/or rearing systems on the presence of diarrhoea and infection were statistically significant. further analysis (classification trees models) was carried out to explore possible risk factors for cryptosporidiosis and interactions between treatments and rearing systems. halofuginone lactate was shown to be effective in reducing clinical signs of cryptosporidiosis and environmental contamination. however, the treatment did not delay the onset of diarrhoea and did not reduce the risk of infection amongst calves reared together in a highly contaminated environment. the use of halofuginone lactate in combination with good hygienic measures, such as rearing animals in clean individual pens, was the most effective method to reduce the risk of cryptosporidiosis amongst – days old calves. it was concluded that the control of the parasite could be achieved by the combination of using effective preventive drugs, such as halofuginone lactate and good animal husbandry procedures. to date there is no effective treatment for bovine cryptosporidiosis. this study describes the use of halofuginone lactate in preventing cryptosporidiosis in naturally infected neonatal calves on a dairy farm with a high prevalence of infection. the animals were kept in two different calf rearing systems. a randomized double-blind trial was carried out with naturally infected calves, divided into four groups. the two prophylactic halofuginone lactate treated groups were kept in either individual or group pens. similarly, the animals receiving the placebo were housed in either individual pens or together in a large pen. a total of ten faecal samples were collected periodically during the days study from each calf and tested for the presence of cryptosporidium spp. using microscopic and molecular methods. generalized estimating equations models were used to determine if the effects of the various treatments and/or rearing systems on the presence of diarrhoea and infection were statistically significant. further analysis (classification trees models) was carried out to explore possible risk factors for cryptosporidiosis and interactions between treatments and rearing systems. halofuginone lactate was shown to be effective in reducing clinical signs of cryptosporidiosis and environmental contamination. however, the treatment did not delay the onset of diarrhoea and did not reduce the risk of infection amongst calves reared together in a highly contaminated environment. the use of halofuginone lactate in combination with good hygienic measures, such as rearing animals in clean individual pens, was the most effective method to reduce the risk of cryptosporidiosis amongst - days old calves. it was concluded that the control of the parasite could be achieved by the combination of using effective preventive drugs, such as halofuginone lactate and good animal husbandry procedures. © elsevier b.v. all rights reserved. the zoonotic apicomplexan cryptosporidium parvum is considered the most common enteropathogen of neonatal calves (de la fuente et al., ; . infected calves can exhibit clinical signs ranging from asymptomatic infection to profuse diarrhoea and dehydration (fayer et al., ; thompson et al., ) . these animals readily contaminate their immediate environment as total oocysts - /$ -see front matter © elsevier b.v. all rights reserved. doi: . /j.prevetmed. . . output per infected calf can be up to over a week (fayer et al., ) . a major problem concerning c. parvum is the lack of an effective means for controlling infection and decreasing environmental contamination with oocysts. because oocysts are highly resistant to environmental stresses and to many disinfectants, hygienic measures on their own are not sufficient to avoid infection and long term contamination of calf rearing facilities (o'donoghue, ) . in addition, many drugs and vaccines have been evaluated as potential therapeutic or prophylactic agents for cryptosporidiosis but with little success . halofuginone lactate is a synthetic quinazolinone with cryptosporidiostatic activity on the sporozoite and merozoite stages of c. parvum (jarvie et al., ) . it has been recommended for both therapeutic and prophylactic use as it delays the onset of infection, reduces shedding of oocysts, and decreases the severity of cryptosporidiosis in calves (joachim et al., ; jarvie et al., ) . its effectiveness as a prophylactic treatment has not been evaluated for the various calf rearing systems used in ireland. the primary objective of this study was to evaluate the effect of halofuginone lactate in decreasing the number of diarrhoeic calves kept in two rearing systems on a dairy farm with a high prevalence of cryptosporidiosis amongst neonatal calves. the secondary objective was to test the effect treatment and rearing systems may have on the onset of diarrhoeic signs and oocysts shedding, as well as on the number of calves excreting oocysts and the level of this excretion. a randomized double-blind trial was carried out during the period march to may in a dairy herd of cows situated in co. westmeath, ireland. the herd was selected on the basis of a previous study in which the prevalence of c. parvum was estimated by bayesian analysis to be % (credibility interval: - ) in -week-old calves (unpublished data) . in this herd, calving occurred throughout the year. cows were moved to a calving pen approximately week before calving and re-introduced to the milking herd h after calving. the straw bedding of the maternity pen was changed every weeks. calves were fed l of their dam's colostrum and separated from their mother within h of birth. commercial vaccines were not administered to cows or calves. all holstein friesian calves born during the -week period, march th-april th, , were included in the experiment, with the exception of one calf that died a few hours after birth. newborn calves (n = ) were allocated to their respective experimental groups using a table of random numbers. the first group consisted of calves treated with halofuginone lactate (halocur, intervet ireland ltd.) and maintained in individual calf pens; the second group was also treated with halofuginone lactate but the calves were allowed to mix freely in a large loose box; the third group consisted of calves treated with a placebo and maintained in individual calf pens; and, the fourth group was treated with a placebo and the animals were allowed to mix freely in a large loose box (table ). during the time calves were allocated to their respective experimental groups, the number of calves in the loose boxes increased from one initially to a final population of six and eight in the placebo and halofuginone lactate treatment groups, respectively. the individual calf pens were made of aluminum with a slatted wooden base. the loose boxes had concrete floors. both were washed with disinfectant (hyperox, dupont, uk) before introduction of the calves. these floors were covered with fresh straw every day. every weeks, the old bedding was removed; the floor was washed with disinfectant and covered with fresh straw. the placebo solution was prepared according to the procedure described by jarvie et al. ( ) and was similar in consistency, color and composition to the commercial halocur without the active ingredient (halofuginone lactate). an equal volume of either halofuginone lactate ( g/kg) or placebo was administered orally to the calves in the morning just before feeding for the first days of their life. the first dose of halofuginone lactate or placebo was given within h of birth and after colostrum was fed. the animal handlers were not informed of the various treatments administered to calves until all the data had been collected. each calf received . l of whole milk twice daily. water was supplied ad libitum with a ration containing soya, wheat and citrus pulp which was mixed on the farm. serum was collected on one occasion from each -weekold calf. this was analyzed for the transfer of maternallyderived immunoglobulins using the zinc sulfate test (zst) (mcewan et al., ) . a total of ten faecal samples ( g) were taken from each calf. the calves were sampled on days and , and thereafter every second day on days , , , , and . a further two samples were collected on days and . the consistency of the faeces was recorded at the time of collection using the following scoring system: for solid or pasty sample, for liquid sample and for watery table cumulative geometric mean and range of the oocysts per gram (opg) of faeces excreted by untreated control calves and calves treated with halofuginone lactate during their first weeks and reared in either individual or group pens. sample. the mean faecal score for each experimental group was calculated by taking the mean of the faecal scores of the animals of each group at each sampling point. diarrhoeic faeces (faeces scored as liquid or watery) were also tested for the presence of other enteropathogens, i.e. escherichia coli k , salmonella species, rotavirus and coronavirus. a faecal sample ( g) was collected from each dam within h of calving. dust and straw samples ( g) were collected from five different locations on the floor of the calving pen once a week for the duration of the experiment. prior to the start of the experiment, five straw samples ( g) were collected in the two group pens; in addition, dust, faeces and other debris ( g) were scraped from five different areas of the walls of the group pens. at the same time, dust, faeces and other debris ( g) were collected from the wooden floors and aluminum walls of all individual calf pens. all these environment samples were collected as aseptically as possible by scraping the surfaces with sterile scalpels. concentration of oocysts was initiated by washing sequentially g of calf faeces through a series of stainless steel sieves with diminishing pores sizes until a final mesh size of m. oocysts were further concentrated using a diethyl-ether sedimentation method described by bukhari and smith ( ) . after the last washing step, distilled water was added to the pellet to give a final volume of ml of faecal suspension. in the case of cow samples, the starting amount of faeces was g and this was divided in two . g aliquots and processed as described for calf faeces. the environmental samples were initially diluted ( : ) with phosphate buffered saline (ph . ); and the suspension stirred for min using a magnetic stirrer. oocysts were then concentrated using the same protocol as described for faeces. a l aliquot of the concentrated faecal suspension was added to a well (diameter: mm) on a microscope slide and stained with l of fluorescein isothiocyanate (fitc) conjugated anti-cryptosporidium monoclonal antibody (cellabs pty ltd., australia) using the procedure described by mcevoy et al. ( ) . the smears were examined at × magnification using a fluorescent microscope (olympus, japan) containing a filter cube with an emission of nm and an excitation wavelength of nm. a slide was considered positive if at least one cryptosporidium oocyst was identified. for each positive slide, the approximate number of oocysts per gram of faeces (opg) was calculated using the mean number of oocysts present in ten randomly chosen fields at × magnification and corrected for the total surface of the well and the dilution of the original sample. no correction was made for the consistency of diarrhoeic samples. the trapezium rule was applied between the successive sampling episodes to estimate the number of oocysts excreted by calves during the period of the experiment (whittaker and robinson, ) . for each calf, the values obtained for each interval of sampling were added to give the cumulative amount of oocysts over the entire period of the experiment. the cumulative amounts of oocysts obtained for each calf were used to calculate the geometric mean cumulative amount of oocysts per gram excreted by the four experimental groups of calves during the period of the study. dna was extracted with a commercial kit (fastdna spin kit for soil, qbiogene inc., usa) from l of the faecal suspension obtained after concentrating the oocysts. cryptosporidium spp. dna was detected using a nested pcr that amplified a segment of the small subunit rrna gene of approximately b.p. (xiao et al., ) . secondary pcr products of seven randomly chosen positive faecal samples and all positive environmental samples were sequenced to identify the genotype of the isolates present on the farm. in addition, these samples were further tested using another nested pcr that amplified a b.p. fragment of the -kda glycoprotein gene (alves et al., ) . similarly, secondary pcr products from this assay were also sequenced to identify the sub-genotype of the c. parvum isolates. a positive cryptosporidium dna template and a negative control were included in every pcr assay. purification and sequencing of the amplicons were carried out by a commercial company (mwg biotech ag, germany). the amplified pcr products were sequenced in both directions using forward and reverse primers of the secondary pcr. sequences were assembled, edited and aligned with reference sequences from genbank using lasergene software (dnastar, inc., madison, usa). the recently proposed nomenclature was used in labelling the c. parvum subtypes (sulaiman et al., ) . the results were analyzed with stata/mp . software (stata corporation, college station, texas, usa). at each sampling point, correlation between categorical variables (e.g. diarrhoea, infection, treatment, location and sex) was assessed by the fisher exact test, while correlation between continuous variables, e.g. value of the zst and level of oocysts excretion, was assessed by the spearman correlation coefficient. the non-parametric method kaplan-meier method was used to create the survivor function to visually compare the groups of calves for the onset of diarrhoea and oocyst shedding (kaplan and meier, ) . four statistical tests, i.e. the log-rank test, the wilcoxon test, the tarone-ware test and the peto-peto-prentice and harrington-flemming tests, were conducted to test for significant differences (p < . ) between the survivor functions. the differences among the four tests depended on the weights used to combine the estimates derived at each point in time (dohoo et al., a) . further, generalized estimating equations models (gee) were used to test for any significant effect (p < . ) that treatment and rearing systems may have on the number of diarrhoeic calves and calves shedding oocysts and also on the level of oocyst excretion, taking into account repeated observations for each animal (dohoo et al., b) . for dichotomous outcomes, binomial family, logistic link and exchangeable correlation matrix were assumed; while for continuous outcomes, poisson family, log link and exchangeable correlation matrix were selected (johnson et al., ) . an alternative analytical non-linear and nonparametric approach described by duc thang et al. ( ) was also conducted using the classification and regression trees (cart) software to explore possible risk factors for cryptosporidiosis and identify interaction between treatment and calf-rearing regimes. a cart model is fitted by binary recursive partitioning of a multidimensional covariate space, in which the dataset is successively split into increasingly homogeneous subsets until a specified criterion is satisfied (duc thang et al., ) . the one-standard error rule was applied to select the best tree. the cart model was used only as an indicator of interaction between treatment and calf-rearing regimes, as it does not take into account the clustering effect within calves. a total of calves were used in the study and ( %) of them were male. the results of the zinc sulfate turbidity test were above units in all calves indicating that they had received a sufficient quantity of colostrum. all the animals were negative for cryptosporidium sp. at birth. the results of the microscopy and sequence analyses indicated that the majority of calves were infected with c. parvum subtype a g r at some stage during the experiment, the exception being one halofuginone treated calf kept in an individual pen that remained free of infection for the duration of the study. there was no adverse reaction to the medication in any of the halofuginone lactate treated calves, i.e. no mortality or clinical findings, such as dehydration, prostration, presence of mucus or visible blood in faeces, were recorded from the calves treated with halofuginone lactate during the study. e. coli was detected in the faeces of three calves aged week old in the placebo groups, one reared in an individual pen and two others reared in the loose pen. these were also infected with c. parvum. three placebo treated calves over weeks old, one reared in individual pen and two others reared in loose pen, died during the experiment. these animals were subjected to post-mortem examination to determine the cause of dead. based on gross pathological lesions, two calves had died as a result of respiratory infection and one due to acute enteritis with a concentration of × cryptosporidium oocysts per gram of faeces. no other common enteropathogen was detected from this animal. the pcr assays carried out on faecal samples collected from cows were negative for cryptosporidium spp. dna. in addition, there was no evidence that the cows were contaminating the environment with oocysts as the environmental samples from the calving pen were also negative. however, all the environmental samples from the floor and two from the walls of the loose calf pens were positive for cryptosporidium spp. dna in a pcr assay. in contrast, the majority of samples collected from the individual calf pens were negative with the exception of two pens. these two pens had been blindly assigned to calves of the halofuginone lactate treatment group. the calves in these two pens only excreted oocysts at days of age. sequencing of these environmental isolates identified them as c. parvum subtype a g r . the majority of the calves receiving the placebo were infected within the first week of age (range - days) while the mean age for onset of infection in calves treated with halofuginone lactate was days (range - days) (p ≤ . ). as illustrated by the kaplan-meier survival estimates, calves receiving the placebo became diarrhoeic earlier than calves receiving halofuginone lactate at a mean age of days (range - days) and days (range - days), respectively (fig. ) . this difference was statistically confirmed by the four tests, i.e. log-rank, wilcoxon, tarone-ware, peto-peto-prentice and harrington-flemming, used to test whether the overall survivor functions in the groups are equal (p < . ). for the event of diarrhoea, the peto-peto-prentice and harrington-flemming test was the most appropriate test when comparing the kaplan-meier curves of the treated and the control groups of animals because this test takes into account different censoring patterns between the groups and, in this case, diarrhoea was not detected for five calves treated with halofuginone lactate while all placebo calves had diarrhoea at some stage of the experiment (table ) . only one calf was censored for the event of infection; therefore, the log-rank test that assigns equal weight at each time point, was selected (table ) . a significant correlation between diarrhoea and infection was found when comparing the experimental groups treated with halofuginone lactate and placebo at the age days (p = . ). in addition, correlations between infection and treatment received were found for the calves aged days (p < . ), days (p = . ) and days (p < . ). analysis of the pcr and ifa results using gee models indicated that halofuginone lactate significantly reduced the number of diarrhoeic calves, infected calves and oocysts excreted by these calves (p < . , table ). fig. a illustrates the pattern of the mean faecal scores for the groups of treated and placebo calves. peak oocysts production (mean circa , oocysts per gram of faeces) amongst the untreated calves occurred at days of age. in addition, the cumulative geometric means of the number of oocysts per gram of faeces was higher for the placebo calves than the treated calves (table ) . however, at days, more of halofuginone treated calves ( %) were shedding, albeit low numbers of oocysts, than the untreated calves ( %) fig. . comparison of the mean faecal scores for calves maintained in either individual pens or group pens and treated with either halofuginone lactate or a placebo. (fig. a) . at this age, only one of the infected treated calves was diarrhoeic. the halofuginone lactate significantly reduced the number of diarrhoeic calves and oocysts excreted by the calves in the group pens (table and fig. b) . however, the time of onset of diarrhoea was not statistically different between treated and placebo calves. one of the treated calves excreted oocysts while on the medication. all the calves were found to shed oocysts at some stage during the experiment when the nested-pcr assay was used as a detection method (fig. b) . correlation between the number of calves excreting oocysts and the treatment received was found for the calves aged days (p = . ). the cumulative geometric means of the number of oocysts per gram of faeces was higher for the placebo calves than the treated calves (table ) . when comparing the two placebo groups, no significant differences in the onset of clinical signs and infection and in the number of infected and diarrhoeic calves were observed between the two rearing systems (table ) . one calf from the group pen excreted oocysts as early as days old. in addition, the amount of oocysts excreted by the placebo calves in the group pen was significantly greater than the amount shed by calves in individual pens (p < . , table ) . when comparing the two halofuginone lactate groups, the time of onset of oocysts excretion was reduced in the calves kept in the individual pen (p < . , table ). maintaining treated calves in individual pens significantly reduced the number of animals that were infected with cryptosporidium and the amount of oocysts they excreted (p < . , table ). the gee model indicated a significant interaction of halofuginone lactate treatment and individual pen in reducing the proportion of infected calves (p = . ) and the number of oocysts excreted (p < . , table ). according to the overall discriminatory power in the cart analysis, age was the strongest overall risk factor for cryptosporidiosis, followed by treatment and housing system. the cart model suggested that, among the treated calves aged between and days old, the rearing system was the most important risk factor (fig. ) . an abridged version of this study has been published previously and the abbreviated results were used in a recent review and meta-analyses of the effects of halofuginone lactate in treating and preventing neonatal cryptosporidiosis (de waele et al., ; silverlas et al., ) . the present study evaluated the prophylactic benefits of halofuginone lactate against cryptosporidiosis in two different calf rearing systems on the same infected farm. results were analyzed using kaplan-meier and gee models. in addition, the cart model was used to highlight interactions between various risk factors associated with neonatal cryptosporidiosis. the primary source of infection for the experimental calves can only be surmised. however, the possibility of initial exposure to infection in calving pen appeared unlikely as oocysts were not detected in any of the faecal and environmental samples taken from the cows and calving pen, respectively. this is in contrast from previous studies which suggested cows as a source of infection for calves due to a peri-parturient rise in oocyst excretion and resultant contamination of the calving pen (garber et al., ; castro-hermida et al., ) . the results confirmed previous reports that, when calves are reared in good hygienic conditions, such as disinfected individual calf pens, halofuginone lactate was effective in delaying the onset of cryptosporidium infection and diarrhoea, in reducing the number of calves that become infected and exhibit signs of enteritis, and decreasing the level of oocysts excretion lefay et al., ; jarvie et al., ; klein, ) . delaying the onset of diarrhoea is important as newborn calves are susceptible to dehydration and acid-base balance disturbances. since these risks decrease with age, the preventive use of halofuginone lactate may delay infection and the onset of clinical signs to an age when animals are better able to cope with the pathogenic affects of the parasite. additional benefits of halofuginone lactate treatment are reduction in the number of infected animals exhibiting signs of enteritis and in the amount of oocysts they excrete (joachim et al., ; lallemand et al., ) . as shown in the current study, the efficacy of halofuginone lactate is decreased when calves are maintained in less than optimal conditions such as mixed together in group pens. the benefits of good husbandry were also evident when the placebo groups were compared. calves reared in the individual pens excreted significantly less oocysts than calves kept in the group pen. at weeks, the majority of treated calves excreted oocysts albeit at low level. this so-called "rebound phenomenon" has been reported by previous workers lallemand et al., ) . as observed in this study, it is generally considered that it has limited clinical consequences and does not markedly increase the cumulative amount of oocysts produced by treated calves. explanations for its occurrence might include re-infection, activation of an auto-infection phase with thin-wall oocysts and/or re-activation of inhibited stages (villacorta et al., ; naciri et al., ) . another possibility is that the older calves may have been infected with different cryptosporidium species. however, the sequencing of s gene and gp gene fragments carried out on isolates from faecal samples collected from some calves exhibiting the rebound phenomenon indicated that they were c. parvum subtype a g r , the prevailing cryptosporidium strain amongst the experimental calves. the calves, that received the placebo, did not show any evidence of a recrudescence of oocysts excretion at days. this may be attributable to some degree of acquired resistance from the initial infection . all the statistical models used to analyze the experimental data highlighted the need for an integrated control programme combining good animal husbandry measures with prophylactic use of halofuginone lactate to reduce the risk of neonatal cryptosporidiosis. hence, as indicated by the cart model, treated calves aged between seven and days were more at risk of infection when reared in group pens than in individual pens. in addition, the cart model identified the age of the calf as the strongest discriminating risk factor for bovine cryptosporidiosis. calves over days old were more likely to be infected than younger animals whether or not they received halofuginone lactate. silverlas et al. ( ) came to the same conclusion after their metaanalysis of data from a number of previous studies. other workers have also reported a similar age predisposition in the susceptibility of calves to cryptosporidiosis (kvac et al., ; . therefore, it is recommended to separate and rear calves in age-groups based on their level of susceptibility. thus, newborn calves should not be mixed with calves between seven and days old. the size of the experimental groups was based on the number of calves born in the farm during the allocation period, feasibility of completing all the laboratory tests in a reasonable time period and also on the number of animals used in previous studies on the treatment of calves with halofuginone lactate peeters et al., ; jarvie et al., ) . the limited number of calves in each experimental group did not affect the statistical power in detecting a beneficial effect of halofuginone lactate treatment. the husbandry practices, including calf rearing facilities on the farm in which this experiment was carried out, were typical of those on other large dairy herds in ireland. there was a history of neonatal enteritis and the prevalence of cryptosporidiosis amongst young calves had been estimated over % (unpublished data). throughout the design and implementation of the study, every effort was made to ensure it would be possible to extrapolate from the results to calf rearing practices on the majority of irish dairy farms and that this study could form the basis of a larger study on a number of farms in the future. in conclusion, halofuginone lactate was shown to be effective in reducing clinical signs of cryptosporidiosis and environmental contamination with cryptosporidium oocysts. however, control of the parasite can only be achieved by integrated measures such as not mixing animals of different ages, placing newborn calves in an uncontaminated environment and the prophylactic use of drugs such as halofuginone lactate. an effective on-farm control programme in addition to benefiting animal health directly also indirectly benefits public health as the concentration of oocysts in slurry and run-off water from calf houses will be markedly lower thus reducing the risk of surface water contamination with oocysts of zoonotic cryptosporidium species (fayer et al., ) . subgenotype analysis of cryptosporidium isolates from humans, cattle, and zoo ruminants in portugal effect of three concentration techniques on viability of cryptosporidium parvum oocysts recovered from bovine feces a study of cryptosporidiosis in a cohort of neonatal calves proportional morbidity rates of enteropathogens among diarrheic dairy calves in central spain control of cryptosporidiosis in neonatal calves: the use of halofuginone lactate modelling survival data generalised estimating equations malaria in central vietnam: analysis of risk factors by multivariate analysis and classification tree models cryptosporidium parvum infection in bovine neonates: dynamic clinical, parasitic and immunologic patterns zoonotic protozoa: from land to sea potential risk factors for cryptosporidium infection in dairy calves effect of halofuginone lactate on the occurrence of cryptosporidium parvum and growth of neonatal dairy calves prevalence and control of bovine cryptosporidiosis in german dairy herds poisson distribution nonparametric estimation from incomplete observations preventive and therapeutic efficacy of halofuginone-lactate against cryptosporidium parvum in spontaneously infected calves: a centralised, randomised, double-blind, placebo-controlled study age-related and housingdependence of cryptosporidium infection of calves from dairy and beef herds in south bohemia field study of the efficacity of halofuginone and decoquinate in the treatment of cryptosporidiosis in veal calves efficacy of halofuginone lactate in the prevention of cryptosporidiosis in suckling calves the prevalence and characterization of cryptosporidium spp. in beef abattoir water supplies a turbidity test for the estimation of immune globulin levels in neonatal calf serum the effect of halofuginone lactate on experimental cryptosporidium parvum infections in calves cryptosporidium and cryptosporidiosis in man and mammals specific serum and local antibody responses against cryptosporidium parvum during medication of calves with halofuginone lactate livestock a longitudinal study of cryptosporidiosis in dairy cattle from birth to two years of age systematic review and metaanalyses of the effects of halofuginone against calf cryptosporidiosis unique endemicity of cryptosporidiosis in children in kuwait the public health and clinical significance of giardia and cryptosporidium in domestic animals efficacy of halofuginone lactate against cryptosporidium parvum in calves the trapezoidal and parabolic rules identification of types of cryptosporidium parasites in children in lima the financial support for this project was provided by the research stimulus fund of the department of agriculture, fisheries and food of ireland. the assistance and cooperation of the dairy farmer is gratefully acknowledged. the technical assistance of mrs henrietta cameron and mr martin hill is also appreciated. key: cord- -eym p authors: katsoulos, panagiotis d.; karatzia, maria a.; dovas, chrysostomos i.; filioussis, george; papadopoulos, elias; kiossis, evangelos; arsenopoulos, konstantinos; papadopoulos, theologos; boscos, constantin; karatzias, harilaos title: evaluation of the in-field efficacy of oregano essential oil administration on the control of neonatal diarrhea syndrome in calves date: - - journal: res vet sci doi: . /j.rvsc. . . sha: doc_id: cord_uid: eym p the objective of this study was to evaluate under field conditions, whether daily administration of oregano essential oil is effective in preventing and/or diminishing the severity of neonatal diarrhea syndrome in calves aged less than days. ninety-one newborn calves from three dairy farms were assigned into two groups; “eco” group (n = ) calves were drenched with greek oregano (origanum vulgare ssp. hirtum) essential oil (ecodiar® liquid %) at the dose of . mg/kg body weight once per day until the age of days. “conts” group (n = ) calves were left untreated and served as controls. all animals were monitored daily for the incidence of diarrhea until the age of days and their fecal score was recorded. fecal samples were collected on days , and for microbiological and parasitological evaluation. average fecal score throughout the experiment, incidence of diarrhea, duration and severity of diarrhea episodes were significantly lower in eco group compared to the controls. daily administration of oregano essential oil in calves for the first days of their life effectively diminishes the severity of naturally acquired diarrhea under field conditions and, under certain hygiene practices, possess a preventive effect against neonatal diarrhea syndrome. neonatal calf diarrhea is the most common cause of illness and mortality in calves and a major cause of economic loss to cattle herds (barragry, ; de la fuente et al., ) . economic losses, on top to mortality, is associated with treatment, veterinary and labor costs as well as with reduced growth rates of the affected calves (anderson et al., ; ok et al., ) . the most important infectious agents associated with the disease are enterotoxic escherichia coli k /f (etec), rotavirus a (rva), bovine coronavirus (bcov), and cryptosporidium spp. with rva and cryptosporidium spp. being most frequently identified in fecal samples from young calves (gulliksen et al., ; bartels et al., ; silverlås et al., ) . co-infections with more than one of these pathogens are frequently detected in diarrheic calves in clinical practice and are associated with increased morbidity and mortality rates (blanchard, ) . it is well documented that diarrheic calves, regardless of the causative agent or agents of diarrhea, often have small intestinal overgrowth of e. coli bacteria. this overgrowth increases the severity of diarrhea, retards the recovery and increases the risk of bacteremia and death (constable, ) . in respect to this, oral administration of antibacterial agents that inhibit coliform bacterial overgrowth in calves with diarrhea might have beneficial effects on the disease outcome by decreasing the duration and the severity of the disease and by preventing the development of bacteremia. oregano essential oil could be used as such an antibacterial agent providing an antibiotic alternative for this purpose. it has been proven in many experiments in vitro that oregano essential oil and its main constituents, carvacrol and thymol, have strong antibacterial activity against gram negative bacteria and especially e. coli (elgayyar et al., ; si et al., ; nazzaro et al., ) . apart from its antibacterial properties, oregano essential oil was also found to have antiviral (pilau et al., ) and anticryptosporidial (gaur et al., ) effects in vitro. based on these properties it could be hypothesized that the administration of oregano essential oil in newborn calves might have beneficial effects not only in treating but also in preventing diarrhea syndrome. in the available literature there is not any relevant controlled research evaluating potential prophylactic or treatment effects of oregano essential oil on diarrhea in calves aged < days. in the only available study it was observed that the administration of oregano leaves had comparable effects with neomycin on the duration of diarrhea in diarrheic calves aged to days without however using control group (bampidis et al., ) . the objective of the present study was to evaluate under field conditions whether daily administration of oregano essential oil is effective in preventing and/or diminishing the severity of neonatal diarrhea syndrome in calves aged < days. prior to the onset of the experiment the minimum required total sample size was calculated using general linear multivariate model with wilks likelihood ratioprocedure at the glimmpse software (http://glimmpse.samplesizeshop.org/). the desired power was set at . , the type i error rate at . , the desired detectable difference of days with diarrhea at days with standard deviation ; the means scale factor was set at . and the variability scale factor at . the results of the analysis revealed that a minimum sample size of calves ( per group) was required (power = . ). ninety-one newborn holstein calves ( females and males) from three dairy farms were finally used in the study. they were all born to dams that were vaccinated against rotavirus, coronavirus and e. coli f (k )/f antigens (lactovac c; zoetis, hellas) one month prior to the expected day of calving. the calves were randomly alternately assigned into one of two groups according to their birth date in each farm until the total number of calves per farm was reached. in farm however calves were finally used due to a twin birth at the last calving. the experiment was run in all farms simultaneously from september th to november th . the animals of eco group (n = ) were orally drenched with greek oregano (origanum vulgare ssp. hirtum) essential oil (ecodiar® liquid %; ecopharm hellas s.a., kilkis, greece) at the dose of . mg/kg body weight once per day for the first days of their life whereas those of conts group (n = ) were left untreated and served as controls. the experiment started at the day of calving (day ) and lasted until day . each calf was separated from its dam after calving, weighed, its navel was disinfected using an antibiotic spray (terramycin™ aerosol spray; zoetis, hellas) and was offered its first colostrum meal. the calves of eco group were orally drenched immediately afterwards with the respective amount of oregano essential oil that was diluted with normal saline up to the volume of ml with the aid of a feeding syringe of equal volume. the following days and until day , oregano essential oil was administered in the calves of this group at the same way after the morning feeding. the same procedures were followed in all farms. feces were scored every day throughout the experiment after the morning feeding by the same person who was blinded to group allocation using a three point scale with = normal, = intermediate and = watery and the score was recorded. calves with fecal scores ≥ were considered diarrheic. based on these records, number of days with diarrhea (fecal score ≥ ) was determined and diarrhea index (di) was calculated: di = number of days with diarrhea × average fecal score on these days. fecal samples were collected on days , and of the experiment for microbiological and parasitological evaluation. the samples were obtained directly from the rectum using sterile latex gloves, were separated into aliquots in sterile containers and were transferred refrigerated to the laboratories for analysis. on the same days, the calves of eco group were weighed and the daily dosage of oregano essential oil was modified accordingly. on day a blood sample was also collected via jugular vein-puncture ( g) into evacuated glass tubes from all calves and transferred refrigerated in the laboratory for the evaluation of passive immunity transfer. all calves were routinely clinically examined by the same person who was blinded to group allocation at the fecal sampling days (days , and ) and at the end of the experiment (day ). in cases of diarrhea the animals were clinically evaluated daily until recovery. diarrheic calves in all farms continued milk feeding and were receiving an extra meal per day with oral electrolytes (diaproof k®; virbac, hellas). in cases of dehydration or inappetence the calves were receiving lactated ringers solution intravenously and, if blood serum glucose concentration was lower than mmol/l (as determined on farm with a handheld glucose meter; freestyle precision, abbott, uk), a dextrose % solution was also administered intravenously. calves with inappetence, hypoglycemia, depression and fever or hypothermia were also treated with antibiotics, based on antimicrobial susceptibility tests the last six months (farm : enrofloxacin mg/kg sc, baytril®, bayer animal health gmbh, germany; farms and : ceftiofour . mg/kg sc, excenel® rtu, zoetis, hellas) and non-steroid anti-inflammatory drugs (carprofen . mg/kg sc, rimadyl® cattle, zoetis, hellas). in calves of eco group that were still diarrheic after day , administration of oregano essential oil continued until recovery. newborn calves in farm , were housed in individual pens in a separate building. windows ( m by , m) served as ventilation inlets, situated m above the pens and oil heaters were used for heat supplementation when ambient temperature was below °c. each individual pen had a concrete floor area of m , with visual access to neighboring pens. long stem wheat straw bedding was layed at a density of kg/m . wet bedding was removed and replaced twice a week. all rails, gates, partitions, walls, feeders and floors were cleaned on a weekly basis and after each calf was removed from the building. a pressure water system and a broad spectrum disinfectant were used and adequate time for drying was provided. farm housed newborn calves outdoors, in individual polyethylene hutches with an outside run for the calves to move around. hutches were situated on free draining concrete and were placed , m apart, enabling visual contact between calves. each individual hatch had a concrete floor area of . m . newborn calves in farm were open housed in group pens partially enclosed under a metallic shed. each group pen had a concrete floor area of m for approximately calves. clean, dry straw bedding which was disposed of after each batch of calves, was used in all farms. concrete feed troughs on the external side of the pens and water bowls mounted inside the pens were used in farm . feed and water buckets were secured outside individual pens and hutches in farms and , respectively. newborn calves were bottle-fed fresh colostrum from their respective mothers in all farms. each calf was fed l of colostrum within h after birth and another l after h. at days two and three of their lives, all calves were fed colostrum/milk from their respective dams, at % of their bodyweight. at four days old and afterwards, calves in all farms consumed milk replacer meal. the nutrient analysis of the milk replacer used is presented in table . milk replacer was offered at °c, the optimal drinking temperature, twice a day. calves were fed % of their bodyweight in milk replacer. along with that, all farms offered fresh water, which was available at all times and was replaced daily. the same calf starter (table ) was also offered starting at day five in all farms. approximately g of calf starter per day were offered in a shallow bucket. the amount increased gradually, as calves started consuming all of the feed. all buckets were emptied and refreshed every h with clean feed. the fecal samples were diluted ( : ) with tap water and passed through a sieve (no. ) in a centrifuge tube, centrifuged at g for min and smears of μl of the sediment were dried, stained using the acid-fast ziehl-neelsen technique (henriksen and pohlenz, ) , and examined at × magnification for cryptosporidium spp. oocysts. oocyst shedding was estimated semiquantitatively according to the average number of oocysts in random fields. calves found positive at at least one sampling were considered as positive for cryptosporidium spp. ten percent suspensions of each fecal sample were prepared in phosphate-buffered saline (pbs), and rna was extracted according to rna extraction method c described by pappi et al. ( ) . rna extracts were stored at − °c until testing. two different real-time quantitative reverse transcription-pcr (qrt-pcr) assays were applied for the detection of rva (freeman et al., ) and bcov (decaro et al., ) . calves found positive even at one sapling were considered as positive for the respective virus. to enumerate the population of e. coli in fecal samples viable plate count method was used. initially g of the sample was diluted in subsequent ten-fold dilutions in phosphate buffer saline. one hundred microliters of each diluent was plated on tbx agar medium (merck kgaa; darmstadt, germany) and incubated aerobically at °c. plates with to typical x-glucuronide positive colonies (blue in color) were calculated. each colony was represented as colony forming unit (cfu) and measured with the formula shown below in cfus/ml: = × cell density number of colonies dilution factor of the plate counted furthermore colonies per plate were selected and subcultured on nutrient agar plates ( merck kgaa; darmstadt, germany). total dna of the e. coli isolates was further analyzed using a multiplex pcr system for the detection of enterotoxigenic, attaching, effacing, and shiga toxin producing genes (franck et al., ) . blood samples were allowed to clot and serum was separated by low speed centrifugation ( g for min) and transferred into plastic vials. serum total protein concentration was determined refractometrically with a temperature compensated refractometer (reichert ts meter refractometer, model a, reichert scientific instruments buffalo, ny) according to the instructions of the manufacturer. passive immunity transfer was considered as adequate when serum total protein values were ≥ g/l (tyler et al., ; poulsen et al., ) . all procedures were done according to the ethical standards in the helsinki declaration of , as revised in , as well as the national law and the guidelines of our institutional animal care and use committee. according to the regulations of our institution, a formal approval from the ethical committee was not required as long as samples were acquired for diagnostic or monitoring purposes under informed farmer consent. data were analyzed using the statistical program spss® . normality of data distribution was assessed with kolmogorov-smirnov test and homogeneity of variances was evaluated with levene test. chisquare test was used to determine whether the percentage of calves with adequate passive transfer, the incidence of diarrhea, the percentage of calves that were positive to the evaluated infectious agents in total and in diarrheic animals as well as the percentage of animals that needed treatment were significantly different among groups and between farms. the effects of the fixed factors group, farm, infectious agents detected (pathogens), as well as of their interactions on the fecal score, the number of days with diarrhea, the fecal score at the days with diarrhea and the diarrhea index were tested with univariate analysis of variances. repeated measures anova was run to evaluate the effect of sampling day, of oregano essential oil administration (group) and their interactions (group × day) on the fecal output of e. coli and on the ct values obtained from the rva real-time rt-pcr. in cases of negative results a ct value of was set in order to facilitate the quantitative analysis. in both aforementioned models, the main effects of the fixed factors and of their interactions were tested with bonferroni test. friedman test was also run in order to determine the significance of the differences of cryptosporidium spp. oocyst shedding at the three sampling days in total and within each group of calves. kaplan-meier survival analysis (medcalc software . . ; medcalc, ostend, belgium) was performed for plotting the time needed for the diarrheic calves either to recover after the onset of clinical signs at each group and for the comparison of the obtained curves. a value of p ≤ . was considered significant in all comparisons. the percentage of calves with adequate passive immunity transfer exceeded % (table ) and was not significantly different either between farms (p > . ) or among groups (p > . ). on average, the serum total protein concentration was not significantly different between groups (mean ± se: . ± . and . ± . g/l for groups eco and conts, respectively; p > . ). the infectious agents related with neonatal diarrhea syndrome that were detected at repeated fecal samplings are presented in table . the percentage of animals found positive in rva and cryptosporidium spp. was not significantly different between groups (p > . ) or farms (p > . ). bcov was detected only in farm and the percentage of positive animals was similar among groups (p > . ). fecal shedding of cryptosporidium spp. oocysts was not significantly different between groups at any sampling day (p > . ). friedman repeated measures analysis revealed that fecal shedding of oocyst was significantly reduced on days and compared to day in eco group (mean ranks: . , . and . for days , and , respectively; x = . , p < . ), whereas the reduction was not significant in the control group (mean ranks: . , . and . for days , and , respectively; x = . , p > . ). average ct values obtained from rotavirus real-time rt-pcr were not significantly affected either by oregano oil administration (mean ± se: . ± . and . ± . for groups eco and conts, respectively; p > . ), or by sampling day (p > . ), or their interactions (p > . ). fecal shedding of e. coli was also not significantly affected by group (mean ± se: . ± . and . ± . log cfu/ml for groups eco and conts, respectively; p > . ) or day of sampling (p > . ), or their interactions (p > . ). diarrhea was recorded in out of calves ( . %) that were used in the experiment ( table ). the incidence of diarrhea was significantly lower (p < . ) in calves receiving oregano oil (eco group) compared to the controls (p < . ; table ). within farms, the incidence of diarrhea in farm was also significantly lower in eco group than the conts one (p < . : table ), whereas in farms and no significant difference was recorded among groups (p < . ; table ). thirteen out of ( . %) diarrheic calves had two episodes of diarrhea during the experimental period, without, however, the percentages being significantly different between groups ( % and % for eco and conts groups, respectively; p > . ). the average age of diarrhea onset was not significantly different between groups (mean ± se: . ± . and . ± . days for groups eco and conts, respectively; p > . ). in eco group all diarrhea cases started before day and no new case was recorded between days and of the study. the combinations of infectious agents detected in diarrheic calves are presented in table ; the percentages of positive animals at each agent were not significantly different among groups (p > . ; table ). as it is shown in table , average fecal score recorded in calves throughout the experiment, average number of days that calves had diarrhea and average diarrhea index were significantly affected only by the daily administration of oregano oil and were significantly lower in eco group compared to the controls (p < . ). however, fecal score on days with diarrhea was not significantly affected by the group of animals. the percentage of calves that needed treatment was significantly lower (p < . ) in eco group than the controls (table ) . significantly lower was also the percentage of animals that needed iv fluids and antibiotic treatment in eco group than the conts (p < . ; table ). the survival curves depicting the time until recovery at diarrhea episodes in both groups are presented in fig. . the analysis revealed that the median time until recovery was significantly lower (p < . ) in eco group ( days) compared to the controls ( days). no death was recorded in any experimental group. the objective of the present study was to investigate under field conditions whether daily administration of oregano essential oil could prevent the occurrence and diminish the severity of the diarrhea syndrome in newborn calves. in order to evaluate its possible preventive effect against diarrhea, oregano essential oil was administered to the calves of eco group for the first days of their life, given that the great majority of the diarrhea cases commence during this period, as occurred at the present study. it was selected to continue the administration of the oregano essential oil in calves of eco group that remained diarrheic beyond the age of days in order to be able to estimate its possible treatment effects as well. in the available literature there is not standardized dosage for oregano oil in calves. in a former experiment table incidence of diarrhea, percentages of calves with adequate passive immunity transfer and percentages of positive calves to cryptosporidium spp., rotavirus a (rva), bovine coronavirus (bcov) and enterotoxic escherichia coli k /f (etec) in the three farms of the experiment (farm , and ) and in calves drenched with oregano essential oil (eco) or left untreated as controls (conts). the dosage of mg/kg was used (bampidis et al., ) . according to our experience in practice, diarrheic calves that weight about kg are empirically orally drenched by the farmers with ml of ecodiar liquid %, which corresponds to the dosage rate of . mg/kg that was used at the present experiment. oregano oil, even at the concentration of %, has very strong flavor; in order to avoid any milk consumption refusals it was selected not to mix it with milk but to administer it immediately after feeding as an oral drench, by diluting the drenched quantity with normal saline until ml. the followed practice ensured complete milk consumption and was not associated with discomfort of the calves during administration. the percentage of calves with adequate passive immunity transfer using g/l as the cut-off value for serum total protein concentration exceeded the critical level of % (mcguirk, ) in all farms. these results indicate that the farms selected for the study had successful colostrum management practices (mcguirk, ) and that the distribution of calves with failure and adequate passive transfer is equal between groups. additionally, as expected, the non-significantly different serum total proteins concentrations among groups imply that the administration of oregano oil does not impair the acquirement of passive immunity. the most commonly detected infectious agent at the repeated fecal samplings was rva. the detection of this virus in all three farms of the study and the high percentage of rva positive animals is in accordance to our observations that this virus is prevalent to the great majority of the dairy herds and that, as occurs worldwide (bartels et al., ; cho et al., ; al mawly et al., ) , is a major cause of diarrhea in neonatal calves in our country. the hardly identical percentage of positive animals and the insignificant differences on the virus shedding among groups indicate that oregano essential oil administration does not prevent the infection of calves with rva. this is in accordance to in vitro observations that carvacrol, the main component of oregano oil, does not possess antiviral activity against rotavirus when added before but only when added after the inoculation of cell cultures with the virus (pilau et al., ) . cryptosporidium spp. was the second most commonly detected infectious factor at this study, with similar percentage of positive calves between farms. however, even though non statistically significant, the percentage of positive animals in eco group was lower compared to the controls. this finding was constant in all farms and implies that oregano oil might possibly be associated with reduced infection rates. supporting towards to this point of view is the significant reduction of oocyst shedding from day to day of the experiment in eco group whereas it remained constant in the control group. such an effect warrants further evaluation and cannot be excluded, given that oregano oil is effective against other protozoa in vivo in other animal species (giannenas et al., ; toulah et al., ; mohiti-asli and ghanaatparast-rashti, ) . furthermore, it was recently proved in an in vitro study that at certain concentrations oregano oil as well as carvacrol alone effectively reduced ( %) the infectivity of cryptosporidium parvum on cell cultures (gaur et al., ) . concerning the other pathogens, bcov was detected only in one farm and to a small number of animals and no etec or other enteropathogenic e. coli strain was identified in any calf. however, the possibility that some strains of e. coli were undetected cannot be excluded since fecal e. coli strains are not representative of small intestinal strains (constable, ) . as is commonly observed under field conditions with naturally acquired diarrhea cases (van metre et al., ) , the majority of diarrheic calves at the present study had mixed infections. however, the analysis of the obtained data revealed that the table average (mean ± se) fecal score at the days of the study, number of days with diarrhea, fecal score at the days with diarrhea and diarrhea index in calves receiving oregano oil (eco) and those left untreated (conts). presence of single or mixed infections in the diarrheic calves did not have different impact on the diarrhea course given that the factor "pathogens" did not affect either the duration or the severity of diarrhea. the overall incidence of diarrhea at the present experiment was significantly lower in calves drenched with oregano essential oil compared to the controls. however, this trend was not observed in all farms but only in farm . so, it cannot be supported with certainty that oregano oil administration has preventive effect against neonatal diarrhea syndrome and the hypothesis should be further investigated. however, taking into account that the hygiene management practices in farm were more intensive compared to the other farms, it can be supported that this effect might be possible if oregano oil is drenched to calves that are exposed to low amounts of infectious agents. the significantly lower number of days with diarrhea, the lower diarrhea index, the shorter duration of the diarrhea episodes and the lower number of calves required treatment in eco group compared to the controls, indicate that daily administration of oregano essential oil can effectively diminish the severity of neonatal diarrhea syndrome in calves. this effect could be attributed to the inhibition of coliform bacterial overgrowth in the small intestine of diarrheic calves by oregano essential oil due to its strong antibacterial activity against e. coli (elgayyar et al., ; si et al., ; nazzaro et al., ) . in support to this point of view, bampidis et al. ( ) observed that oregano essential oil drenching had comparable results with neomycin administration per os on the treatment outcome of diarrhea due to e. coli in calves. furthermore, the significantly lower number of calves with systemic illness that needed antimicrobial and supportive therapy in eco group compared to the controls, which is suggestive of lower cases of bacteremia (constable, ) , could also possibly imply that oregano essential oil might inhibit intestinal coliform overgrowth. however, this hypothesis has to be further investigated. the detection of similar number of e. coli in the fecal samples of calves in eco and control group is not contrary to the proposed mode of action of oregano essential oil, given that e. coli numbers in the large intestine do not reflect the numbers in the small intestine (constable, ) . the main conclusion of this study is that daily administration of greek oregano essential oil in calves for the first days of their life effectively diminishes the severity of naturally acquired diarrhea under field conditions. furthermore, it seems that under certain hygiene management practices oregano essential oil administration might possess a preventive effect against neonatal calf diarrhea syndrome. although further research is necessary in order to determine the exact mechanisms of these effects, oregano essential oil appears to be a promising adjunct to antibiotics for the management of diarrhea in neonatal calves. none of the authors has any personal or financial relationships with people or organisations that could inappropriately influence or bias the content of this paper. prevalence of endemic enteropathogens of calves in new zealand dairy farms the effect of scours on calf weaning weight effect of dried oregano leaves versus neomycin in treating newborn calves with colibacillosis calf diarrhoea prevalence, prediction and risk factors of enteropathogens in normal and non-normal faeces of young dutch dairy calves diagnostics of dairy and beef cattle diarrhea case-control study of microbiological etiology associated with calf diarrhea antimicrobial use in the treatment of calf diarrhea cryptosporidium and concurrent infections with other major enterophatogens in to -day-old diarrheic dairy calves in central spain detection of bovine coronavirus using a taq-man-based real-time rt-pcr assay antimicrobial activity of essential oils from plants against selected pathogenic and saprophytic microorganisms multiplex pcr for enterotoxigenic, attaching and effacing, and shiga toxin-producing escherichia coli strains from calves enhancement of detection and quantification of rotavirus in stool using a modified real-time rt-pcr assay oregano essential oil and carvacrol reduce cryptosporidium parvum infectivity of hct- cells effect of dietary supplementation with oregano essential oil on performance of broilers after experimental infection with eimeria tenella enteropathogens and risk factors for diarrhea in norwegian dairy calves staining of cryptosporidia by a modified ziehl-neelsen technique herd-based problem solving: failure of passive transfer. school of veterinary medicine dietary oregano essential oil alleviates experimentally induced coccidiosis in broilers effect of essential oils on pathogenic bacteria the studies on the aetiology of diarrhoea in neonatal calves and determination of virulence gene markers of escherichia coli strains by multiplex pcr development of one-tube real-time qrt-pcr and evaluation of rna extraction methods for the detection of eggplant mottled dwarf virus in different species antiviral activity of the lippiagraveolens (mexican oregano) essential oil and its main compound carvacrol against human and animal viruses comparison of passive transfer of immunity in neonatal dairy calves fed colostrum or bovine serum-based colostrum replacement and colostrum supplement products antibacterial effect of oregano essential oil alone and in combination with antibiotics against extended-spectrum β-lactamase-producing escherichia coli cryptosporidium infection in herds with and without calf diarrhoeal problems the efficacy of origanum vulgare on eimeria tenella evaluation of assays for failure of passive transfer in calves infectious diseases of the gastrointestinal tract this research has been funded by the research committee of aristotle university of thessaloniki (grant number ). key: cord- -da hsuj authors: jamaluddin, a. a.; hird, d. w.; thurmond, m. c.; carpenter, t. e. title: effect of preweaning feeding of pasteurized and nonpasteurized milk on postweaning weight gain of heifer calves on a californian dairy date: - - journal: preventive veterinary medicine doi: . / - ( ) - sha: doc_id: cord_uid: da hsuj abstract the effect of preweaning feeding of pasteurized colostrum and pasteurized waste milk on postweaning performance of dairy heifers on a dairy in the central valley of california was investigated as an extension of a clinical trial examining the effect of preweaning growth and morbidity on performance at weaning. of the heifer calves originally enrolled in the field trial, calves ( treatment, controls) were followed to about months of age, during which time their weights were measured. after weights were adjusted for several potential risk factors and effect modifiers, calves fed pasteurized colostrum and pasteurized waste milk were . kg heavier at days of age than those fed nonpasteurized colostrum and nonpasteurized milk. a previous study indicated that feeding pasteurized colostrum and pasteurized waste milk to calves during the preweaning period resulted in reduced morbidity and improved weight gain compared with those fed nonpasteurized colostrum and nonpasteurized milk (jamaludldin, ) . although several studies had examined the effects of previous exposure to waste milk on postweaning performance, they were focused mainly on examining an association between feeding of waste milk during calfhood and develop-ment of mastitis at first milking (schalm, ) . recent studies have shown no association between feeding waste milk to preweaned heifers and subsequent incidence of subclinical mastitis (caused by srreptococcus agalactiae and staphylococcus uureus) (barto et al., ; roberson et al., ) . studies on children have shown an association between the level of bacterial contamination of food and post weaning growth rates (martorell et al., ; condon-paoloni et al., ; coates, ; bairagi et al., ; cebu study team, ; cebu study team, ; walker et al., ; adair et al., ) . similar studies in animals have indicated that feeding neonates with diets low in bacteria led to a lower incidence of gastrointestinal disorders and improvement in growth compared with animals that received diets with higher levels of bacterial contamination (coates, ; speck, ; black et al., b) . in calves, one possible approach to reduce gastrointestinal disease and perhaps subsequent malabsorption during the preweaning period may be through feeding of pasteurized milk (coates, ; jamaluddin, ) . if diarrhea with accompanying gut inflammation and possible permanent reduction in gut absorption can be reduced during the preweaning period by feeding pasteurized milk, postweaning growth would be expected to be better for calves consuming pasteurized milk than for those consuming raw milk. the objective of this observational study on a californian dairy was to evaluate the effect of feeding pasteurized colostrum and pasteurized waste milk (versus unpasteurized colostrum and waste milk) during the preweaning period on postweaning weight gain of dairy heifers, controlling for demographic and morbidity variables simultaneously. . . source of preweuning morbidity and growth data (previous trial) one hundred and fifty holstein-friesian heifer calves, assessed as clinically healthy (i.e. calves that breathed, walked and nursed normally) had been enrolled in a clinical trial which lasted from birth to weaning at lo- weeks of age on a dairy milking cows in the central valley of california (jamaluddin, ) . the dairy was organized into five smaller dairies, four of which were about . km from each other; the fifth was located about km from the others. calves in this study originated from all five dairies and were raised at one site. seventy-five of the calves in the previous trial were assigned to the treatment group (based on the dam's eartag number) and fed - of pasteurized colostrum (obtained by pooling colostrum from all five dairies) within - h after birth, followed by pasteurized waste milk at day-' until weaning. waste milk originated from cows with mastitis or other disorders, and treated with antibiotics. the other calves were assigned to the control group and were fed the same quantities but of nonpasteurized, pooled colostrum and nonpasteurized waste milk. daily, colostrum and waste milk from all five dairies were taken to a pasteurization site, pooled, and later divided into quantities to be fed either pasteurized or nonpasteurized. calves were housed individually in hutches and identified by an eartag. calves were run concurrently; treated and control calves were placed in separate, adjacent rows of hutches on the same premises. grain feeding consisted of calf-starter pellets (crude protein (cp), % dry matter (dm) basis; crude fat, . % dm basis; crude fiber, % dm basis) and water ad libitum from the third day after birth until weaning. age at occurrence, severity., and duration of each episode of diarrhea and respiratory signs were recorded daily afi:er feeding by a trained worker supervised by the senior author, from birth to weaning. for calves with an episode of diarrhea or respiratory disease signs, recurrence of signs after consecutive sign-free days was considered a new episode. calf weight measured by a trained worker was estimated at birth and once every week until weaning using a heart-girth tape (nasco agricultural science, modesto, ca). the evaluator was not blinded regarding treatment group. four of calves in the control group from the previous study died during that study (crude mortality rate, . %); no deaths occurred in the calves in the treatment group. all calves (n = ) were weaned at about - kg and moved from individual hutches into several common pens where they were mixed with weaned calves from several calf-raising areas on the dairy. fifty-nine of calves in the treatment group continued into the postweaning study; as part of the farm's normal culling procedure, eight freemartin calves and eight small calves were removed from the original study group at weaning, for later sale as beef. of the remaining calves in the control group, were continued to the postweaning study; of the original group four freemartin calves and small calves were removed. all weaned calves were placed in several dirt pens (approxi:mately m x m) that accommodated about calves until about months of age. calves were fed a grain mixture (cp, % dm basis; crude fat, . % dm basis; crude fiber, % dm basis) and alfalfa hay; water was provided twice daily. calves were weighed at , and days after weaning during september-november, . scatterplots of preweaning and postweaning weights versus age were created to visualize how the weights of the two treatment groups differed with time. an orthogonal polynomial function of degree to was fitted to the weight data using bmdpsr (jennrich , ) . the function with the greatest overall fit (that with the least significant f-statistic) was used to provide subjective comparison of calf overall weight gain between treatment and control groups. owing to the time-series nature of the postweaning weight data, autocorrelation was expected and subsequently tested by the durbin-watson statistic using ordinary least square regression (shazam, ) . postweaning weights of calves were compared between the treatment groups, simultaneously controlling for age, birth weight, and number of days calves experienced diarrhea and pneumonia during the preweaning period using a time series cross section regression (proc tscsreg) (statistical analysis systems institute inc., ). diarrhea was defined as watery or loose, unformed feces excreted in voluminous amounts. severity was scored as for no diarrhea, for mild, for moderate and for severe diarrhea. pneumonia was defined as the presence of respiratory signs such as coughing or dyspnea requiring treatment, and was scored in the same manner as diarrhea. evidence of interaction between treatment and postweaning weight versus age was also examined, the presence of which indicated that postweaning weight gain pattern differed between the treatment groups. the da silva model was used to accommodate the structure of random error arising from correlated weight data. this model used a variance component and moving average method that took into account the varying effect of weight between animals for a particular age of weighing. the parameters were estimated using two-stage generalized least square regression (proc tscsreg) (statistical analysis systems institute inc., ). a scatterplot of weight data from birth to the end of the study for treatment and control calves is shown in fig. . a third degree polynomial function fitted to the weight data produced a good fit (unadjusted r = . ) and showed an apparent slight difference in pattern between treatment and control between the polynomial functions of the data were not independent. there was a positive first order autocorrelation in the postweaning weight data as indicated by the durbin-watson statistic, dw, of . (with d = . ) (p < . ). results of the time series cross-section regression analysis using the da silva method of adjustment for autocorrelation indicated the presence of interaction between treatment and postweaning age, suggesting that treatment calves had a better postweaning weight gain pattern than control calves (p < . (table ) . this interaction is illustrated in fig. using the unadjusted data (i.e. not data from the da silva model). at days of age, calves in the treatment group were estimated to be . kg heavier than calves in the control group. calves that experienced several days of diarrhea, pneumonia, or both during the preweaning period tended to be lighter than calves with little or no diarrhea and/or pneumonia, although this difference was not statistically significant (p > . ) ( table ) . results of the present study indicated that calves fed pasteurized colostrum and pasteurized waste milk during the preweaning period continued to perform better after weaning than those fed nonpasteurized colostrum and nonpasteurized milk. we were not able to demonstrate compensatory growth in calves fed nonpasteurized colostrum and nonpasteurized milk before weaning, during the period between weaning and approximately days of age. this was despite the fact that the calves appeared healthy and their diet seemed nutritionally adequate. at about months after weaning, significantly higher postweaning weight was observed for calves fed pasteurized milk compared with those fed nonpasteurized milk, after adjusting for potential confounding factors, effect modifiers, and multiple observations from the same calf. our results provide evidence that the benefit of feeding milk with lower bacterial count to calves persisted for a period of at least months after weaning. several explanations may be offered for the results observed in the present study. in the preweaning trial (jamaluddin, ) , calves fed pasteurized colostrum and milk had onset of diarrhea day later, experienced day less of diarrhea in the preweaning period, and suffered less severe diarrhea than calves fed nonpasteurized milk and colostrum. calves fed pasteurized colostrum and pasteurized waste milk may have had a healthier gut environment during the preweaning period and at weaning and hence experienced reduced morbidity and better weight gain than those fed nonpasteurized colostrum and nonpasteurized milk. this is in line with studies in children which demonstrated that gut flora with predominantly lmtobacillus and s?r-prococcus species were associated with less gastrointestinal disease (coates, ; speck, ) than gut flora with predominantly escherichiu coli (black et al., b) . another explanation is that inflammation of the gastrointestinal mucosa may result in permanent damage to affected tissues, depending on its severity, and hence adversely affect assimilation and absorption (black et al., a) . diarrhea may affect growth by depriving growing cells of nutrition needed for growth (torres et al., ; torres-medina et al., ; hall et al., ) . when a calf has diarrhea, perhaps as a consequence of consuming milk with a high bacterial count, the intestinal epithelium may undergo extensive damage, resulting in conditions such as pseudomembranous ileitis, mucohemorrhagic colitis or proctitis, as observed in e. coli infection (schoonderwoerd et al., ; janke et al., ) . other conditions of the intestinal epithelium that ma:y reduce absorption and assimilation include villous fusion, villous atrophy, or crypt hyperplasia, as observed in rotavirus infection, or villous atrophy of large and small intestines with destruction of crypt epithelium, as in coronavirus infection (radostits et al., ) . damaged epithelium may result in reduced normal nutrient absorptive function of the intestine, while new epithelial cells regenerate, or damage to the epithelium may be too extensive for normal repair to take place, resulting in permanent malnutrition of calves. consequently, calves fed pasteurized milk would be expected to have less compromised intestinal function even after weaning, and would continue to show better growth than calves fed nonpasteurized milk. several studies conducted on humans (black et al., a; barker and osmond, ; ebrahim, ) and in animals (waltner-toews et al., ; correa et al., ; thurmond and pare, ) found a similar relationship in that neonatal morbidity adversely affected subsequent performance. in ou:r study, signs of respiratory disease early in calfhood were associated with poor weight gain after weaning, although the association was not statistically significant. our finding iis consistent with those of previous studies that found a negative association between calfhood pneumonia and weight gain (thurmond and pare, , calving performance (correa et al., , and survivorship (waltner-toews et al., ) . calves that experienced many episodes of pneumonia took longer to reach lb (thurmond and pare, ). children exposed to acute respiratory infection had damaged airways, which caused abnormalities to respiratory function to persist into adulthood (colley et al., ; pullan and hey, ; mock and simpson, ; barker and osmond, ) . even though the negative associations we observed between preweaning exposure to diarrhea and pneumonia episodes and postweaning weight gain were not statistically significant (perhaps attributable to small sample size), there is evidence for similar (statistically significant) effects in human studies. for instance, several large, population-based, longitudinal surveys demonstrated that sick children did not 'grow as well as children ,who were not sick (martorell et al., ; condon-paoloni et al., ; black et al., b; bairagi et al., ; cebu study team, ; cebu study team, ; walker et al., ) . we speculate that findings from this study may be applicable to other dairies, and that similar benefits from feeding calves pasteurized colostrum and pasteurized waste milk would be observed on other dairies. however, it must be emphasized that this study was performed on only one dairy, and therefore the results of this study do not necessarily apply to other dairies. growth dynamics during the first two years of life: a prospective study in the philippines the association between malnutrition and diarrhea in rural bangladesh childhood respiratory infection and adulthood chronic bronchitis in england and wales feeding milk containing staphylococcus aweus to calves malnutrition is a determining factor in diarrhea duration, but not incidence, among young children in a longitudinal study in rural bangladesh effects of diarrhea associated with specific enteropathogens on the growth of children in rural bangladesh underlying proximate determinants of child health: the cebu longitudinal health and nutrition study cebu study team, . a child production function estimated from longitudinal data the gut microflora and growth respiratory disease in young adults: influence of early childhood lower respiratory tract illness, social class, air pollution, and smoking morbidity and growth of infants and young children in a rural mexican village effect of cahhood morbidity on age at first calving in new york holstein herd health in childhood and later performances pathology of calves with diarrhoea in southern britain effects of feeding pasteurized colosttum and pasteurized waste milk on mortality, morbidity, and weight gain of dairy calves: field trial and economic analysis attaching and effacing escherichia coli infection as a cause of diarrhea in young calves polynomial regression acute morbidity and physical growth in rural guatemalan children outcome for acute bronchitis, bronchiolitis and pneumonia in infancy wheezing, asthma and pulmonary dysfunction years after infection with respiratory syncytial virus in infancy diseases caused by virus and chlamydia- . in: veterinary medicine: a textbook of the diseases of cattle, sheep, pigs, goats and horses, th edn staphylococcus aureus intramammary infections (imi): prevalence, sources and modes of transmission in dairy heifers streptococcus agalactiae in the udders of heifers at parturition traced to sucking among calves colitis in calves: natural and experimental infection with a verotoxin-producing strain of escherichia coli oll:nm lactobacilli as dietary supplements and manifestation of their functions in the intestine user's guide, version , nd edn effect of preweaning morbidity on growth of dairy heifers intestinal absorptive defects associated with enteric infections in infants rotaviral and coronaviral diarrhea morbidity and the growth of stunted and nonstunted children, and the effect of supplementation the effect of early calfhood health status on survivorship and age at first calving we gratefully acknowledge the assistance provided by gabe azevedo and ronald haile of modesto, california, and would like to express gratitude to the department of veterinary services, ministry of agriculture, malaysia for sponsoring this study. key: cord- -zsdymkjd authors: gruse, jeannine; kanitz, ellen; weitzel, joachim m.; tuchscherer, armin; stefaniak, tadeusz; jawor, paulina; wolffram, siegfried; hammon, harald m. title: quercetin feeding in newborn dairy calves cannot compensate colostrum deprivation: study on metabolic, antioxidative and inflammatory traits date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: zsdymkjd immaturity of the neonatal immune system is causative for high morbidity in calves and colostrum intake is crucial for acquiring passive immunity. pathogenesis is promoted by reactive oxygen species accumulating at birth if counter-regulation is inadequate. the flavonol quercetin exerts antioxidative and anti-inflammatory effects that may enhance neonatal health. the aim of this work was to study effects of quercetin feeding on metabolic, antioxidative and inflammatory parameters in neonatal calves to investigate whether quercetin could compensate for insufficient colostrum supply. twenty-eight newborn calves were assigned to two dietary groups fed colostrum or milk-based formula on day and and milk replacer thereafter. from day onwards, calves per diet group were additionally fed quercetin aglycone ( mg/(kg body weight × day)). blood samples were taken repeatedly to measure plasma concentrations of flavonols, glucose, lactate, total protein, albumin, urea, non-esterified fatty acids, triglycerides, cholesterol, insulin, glucagon, cortisol, immunoglobulins, fibrinogen, haptoglobin and serum amyloid a. trolox equivalent antioxidative capacity, ferric reducing ability of plasma, thiobarbituric acid reactive species and f -isoprostanes were analyzed to evaluate plasma antioxidative status. expression of tumor necrosis factor, interleukin- α, interleukin- β, serum amyloid a, haptoglobin, fibrinogen, c-reactive protein, catalase, glutathione peroxidase and superoxide dismutase mrna were measured in liver tissue on day . plasma flavonol concentrations were detectable only after quercetin-feeding without differences between colostrum and formula feeding. plasma glucose, lactate, total protein, immunoglobulins, triglycerides, cholesterol, trolox equivalent antioxidative capacity and thiobarbituric acid reactive species were higher after colostrum feeding. body temperature, fecal fluidity and plasma concentrations of cortisol and haptoglobin were higher in formula- than in colostrum-fed groups. hepatic mrna expression of tumor necrosis factor was higher after quercetin feeding and expression of c-reactive protein was higher after formula feeding. data confirm that colostrum improves neonatal health and indicate that quercetin feeding cannot compensate for insufficient colostrum supply. calfhood diseases play a key role in the economy of dairy farms because they increase operating costs and reduce long-term productivity of the animal. incidence of disease is associated with increased mortality rates [ ] , and enteritis is the most common diagnosis in young calves [ ] , which, according to svensson, linder and olsson [ ] , contributes to % of calf losses during the first days of life. neonatal calves are prone to sickness because their immune system is immature. furthermore, the process of birth itself causes an elevated stress level for the newborn and exposure to an oxygen-rich environment leads to an increased generation of reactive oxygen species [ , ] . reactive oxygen species induce peroxidation of lipids and other macromolecules, leading to alteration of cellular components, interaction with signaling cascades and modification of physiological cell functions [ ] . if not properly counterbalanced by antioxidative defenses, excessive production of reactive oxygen species results in oxidative stress, which is a cofactor of disease in humans and farm animals [ , , ] . adequate colostrum supply is vital to calves because colostrum ensures ingestion of nutrients and contains immunoglobulins (ig), peptides, antioxidants and other bioactive factors supporting maturation, antioxidative and immune defense as well as local intestinal immunity [ ] . the ban on antibiotic performance promoters by the european union in increased efforts to establish natural alternatives to enhance health and productivity in breeding. special focus has been directed to phytochemicals because their use can be manifold according to the respective compound [ ] . flavonoids are secondary plant metabolites that are widely distributed in the plant kingdom and are able to modulate inflammation and immune function and exert antioxidative activity [ ] [ ] [ ] . quercetin, which belongs to the subclass of flavonols, is ubiquitous in most plants and is of interest for scientists for its beneficial use in humans and farm animals. its antioxidative capacity can ameliorate the acquisition of passive immunity in neonates, based on the finding that feeding antioxidant-enriched colostrum enhanced igg absorption and antioxidative status in newborn calves and piglets [ , ] . similarly, retskii et al. [ ] showed that correcting the antioxidative balance in newborn calves prior to first colostrum ingestion increases the acquisition of colostral immunity and reduces the incidence of enteric colibacillosis. another beneficial effect of quercetin is its local action in the gastrointestinal tract. in vitro studies of intestinal epithelium demonstrated that quercetin down-regulates the expression of genes related to inflammation in inflamed epithelium [ ] , and lozoya et al. [ ] showed in a clinical study that oral quercetin administration reduced abdominal pain in acute diarrheic disease in humans. in guinea pigs, mice and rats, the inhibitory action of quercetin on prostaglandin e induced ileal contractions and on castor-oil-induced diarrhea has been demonstrated [ , ] . furthermore, quercetin acts as a prebiotic, thus inhibiting adhesion of enteropathogens to caco- cells without affecting the viability of probiotics [ ] , and improves performance in hens by modulating cecal microflora populations [ ] . although a multitude of research on quercetin has been performed in vitro or in animal models for medical conditions, studies of the effects in neonatal farm animals are scarce. the aim of the present work was to investigate the potential health-promoting effects of feeding quercetin to newborn calves during the first week of life and to evaluate whether the healthpromoting effects of quercetin compensate for initial colostrum deprivation in calves. we hypothesized that quercetin improves antioxidative balance and immune function and that local antibacterial and anti-inflammatory effects reduce the incidence of diarrhea and gastrointestinal dysfunctions. experimental procedures were conducted in compliance with the german animal protection regulations with approval of the authorities of the state mecklenburg-western pomerania, germany (landesamt für landwirtschaft, lebensmittelsicherheit und fischerei mecklenburg-vorpommern; lallf m-v/tsd/ . - . - / ). liver biopsies were performed under local lidocaine anesthesia and all calves received metamizole post-operatively for pain relief. twenty-eight male holstein friesian calves were separated from their dams immediately after birth and were housed in single boxes during their first days of life. before the trial started, separate colostrum pools were prepared from the first and third milkings after parturition. according to the colostrums' macronutrient compositions, milk-based formulas with comparable amounts of macronutrients [ ] but without bioactive factors were provided (bergin mat-formula; bergophor futtermittelfabrik, kulmbach, germany). calves were randomly assigned to two dietary groups and were bucket-fed twice daily, receiving either colostrum (col, n = ) or corresponding formula (for, n = ) on days and of life ( % and % of body weight/day, respectively). if appetite was reduced, calves were tube fed to ensure complete ingestion of colostrum or formula. from day until day , all calves were fed commercial milk replacer ( % of body weight/day; g/l; salvalac mirapro ; salvana tiernahrung, klein-offenseth sparrieshoop, germany). on day , the dietary groups were subdivided into control (colq-and forq-; n = per group) and treatment groups (colq+ and forq+; n = per group), the latter receiving quercetin aglycone twice daily with feeding ( mg/(kg body weight × day); quercetin aglycone dihydrate %, carl roth, karlsruhe, germany). the control groups received no quercetin aglycone. treatment. navels were disinfected with % povidone iodine solution (vet-sept; animedica, senden-bösensell, germany). neonatal calves received an oral dose of g iron dextran with their first meal on day (ursoferran; serumwerk bernburg, bernburg, germany). to support immunological defense during the first days of life, all calves received chicken eggderived immunoglobulins with the morning feeding ( . g/kg body weight; globigen life start %, ew nutrition, visbek, germany) [ ] . to prevent cryptosporidiosis, calves were treated with halofuginone ( . mg/kg body weight per os; halocur, intervet, igoville, france) after the evening feeding from day to day . colostrum-deprived calves (forq+, forq-) additionally received b-vitamins ( mg nicotinamide/calf, mg thiamin chloride hydrochloride/calf, s.c.; vitamin-b-komplex, serumwerk bernburg, germany) and bovine colostral immunoglobulins on days (s.c.), and (per os) ( g gammaglobulins/calf with antibodies against escherichia coli, rotavirus, coronavirus; aniserin orinject; animedica, seden-börsensell, germany). furthermore, formula-fed calves were treated metaphylactically with colistin sulfate from day to day ( mg/kg body weight, i.m.; belacol; belapharm, vechta, germany). all calves were weighed immediately after birth and before evening meals on days and . every morning, health status was examined and appetite, general condition, heart rate, respiratory rate, rectal temperature and gut motility were assessed. fecal fluidity was scored according to larson et al. [ ] . calves with reduced vitality after the first days of life were allowed one recovery day before further sample taking. in these cases, the times referred to as days , , and in the results section are days , , and after birth, respectively. due to gastrointestinal imbalances, two calves (one calf of group colq+ and one calf of group forq+) had to be excluded from the study. sample taking. basal blood samples were taken before the morning feeding on days , , and from the jugular vein using evacuated tubes containing either potassium-edta ( . - mg/ml edta) for analyses of plasma metabolites, insulin, glucagon, immunoglobulins and acute-phase proteins or li-heparin ( - iu heparin) for the determination of the cortisol and flavonol concentrations and the antioxidative status in the plasma. for flavonol analysis, additional blood samples were taken before the morning feeding on days and . after centrifugation ( , × g, °c, min), plasma aliquots were stored at - °c until analyses (- °c for analyses of flavonol concentrations and antioxidative status, respectively). plasma flavonols, metabolites and hormones. plasma concentrations of flavonols (quercetin, isorhamnetin, tamarixetin and kaempferol) were measured via hplc as previously described [ ] with a detection limit of nmol/l and a recovery rate of ± %. the intra-and inter-assay coefficients were . and . %, respectively. plasma metabolites were analyzed using an automatic spectrophotometer (abx pentra ; horiba abx, montpellier, france) and respective kits: glucose (#a a ), lactate (#a a ), albumin (#a a ) and triacylglycerides (#a a ) from horiba abx, montpellier, france; total protein (# - ) and cholesterol (# - ) from mti-diagnostics, idstein, germany; urea (#lt-ur ) from labor+technik, e. lehmann, berlin, germany; and non-esterified fatty acids (# - , # - ) from wako chemicals, neuss, germany. plasma concentrations of insulin (#ria- ) and glucagon (#ria- ) were determined by ria using kits from drg instruments, marburg, germany, which were adapted to bovines [ ] . intra-and inter-assay coefficients of variation were . % and . % for insulin and . % and . % for glucagon, respectively. plasma cortisol concentrations were analyzed in duplicate after extraction with diethylether using a commercially available elisa kit (#eia ; drg instruments gmbh, marburg, germany) according to the instructions of the manufacturer. cross reactivities of the antibody to corticosterone and progesterone were % and %, respectively, and < % to any further competing plasma steroids. the assay was validated for use with bovine plasma. the test sensitivity was . μg/l, and intra-and inter-assay coefficients of variation were . % and . %. immunoglobulins (ig) and acute-phase proteins. the concentrations of igg , igg and igm, as well as acute-phase proteins (haptoglobin, serum amyloid a and fibrinogen), were measured in the edta plasma samples taken on days , , and . igg was analyzed by radial immunodiffusion [ ] (modified by gasowska and stefaniak [ ] ) using bovine reference serum (rs - ; bethyl laboratories inc., montgomery, usa) as standard. igg (#e - ) and igm (#e - ) were determined by elisa using kits from bethyl laboratories inc., montgomery, usa. intra-assay coefficients of variation were . % and . % for igg and igm, respectively. the detection limit of igg was . μg/l and that of igm was . μg/l. for detection of serum amyloid a (saa; #tp- ), we used a multispecies elisa kit from tridelta development, maynooth, ireland. the detection limit of saa was . mg/l. the intra-assay coefficient of variation was . %. the haptoglobin concentration was analyzed using the guaiacol method developed by jones and mould [ ] with human haptoglobin hp - (sigma #h ) as a standard. the detection limit of haptoglobin was . g/l. plasma fibrinogen was determined by rapid heat precipitation according to millar, simpson and stalker [ ] . antioxidative status. li-heparinized plasma samples taken on days , and were used to analyze the trolox-equivalent antioxidative capacity (teac, as trolox equivalents (te) in mmol/l) and ferric reducing ability of plasma (frap, as ascorbic acid equivalents, (asce) in μmol/l) as parameters of antioxidative capacity as well as thio-barbituric acid reactive species (tbars, as malondialdehyde equivalents (mdae) in μmol/l) and -iso-pgf α (f -isoprostanes, in ng/l) as markers for oxidative stress. teac was analyzed as described by miller et al. [ ] and modified by re et al. [ ] . frap and tbars were determined according to luehring et al. [ ] . for determination of f -isoprostanes, a commercial elisa kit (#adi- - ; enzo life sciences, lause, switzerland) was used. cross-reactivities of the assay to pgf α and pgf α were . % and . %, respectively, and < % to any further eicosanoids. on day , a liver biopsy was conducted h after the morning meal using a custom-made biopsy trocar [ ] . biopsy tissue was immediately frozen in liquid nitrogen and stored at - °c until further analysis. hippocalcin-like (hpcal ; nm_ ), low-density lipoprotein (lrp ; bc ) and rna polymerase ii (polr a; nm_ . ) were used as reference genes (given accession numbers related to nih genbank). primer sequences, accession numbers and pcr conditions for target genes related to antioxidative status (catalase (cat); glutathione peroxidase (gpx ); superoxide dismutase (sod)) and inflammation (tumor necrosis factor (tnf); interleukin- α and - β (il a, il b); haptoglobin (hp); fibrinogen (fga); serum amyloid a (saa ); and c-reactive protein (crp)) are listed in table . as recently described [ ] , primer products were verified by sequencing using the bigdye terminator v . cycle sequencing kit and an abi genetic analyzer (life technologies, carlsbad, usa). real-time pcr was performed using a lightcycler (roche molecular biochemicals, mannheim, germany); sybr green i was used as the fluorescent dye. melting curve analysis and agarose gel electrophoresis were used to confirm the specificity of the pcr products. quantification cycle values and amplification efficiencies obtained using linregpcr version . [ ] were imported into qbase+ version . . (biogazelle, gent, belgium) for all subsequent calculations and quality controls. the geometric mean of the reference gene abundances was used for normalization. data are presented as the ratio of the copy numbers of genes of interest and the geometric mean of the reference genes' abundances. statistical analyses were conducted using sas software, version . for windows, sas institute inc., cary, usa. descriptive statistics and tests for normality were calculated using the uni-variate procedure of base sas software. body weight, average daily weight gain and data for hepatic gene expression were analyzed by anova with the mixed procedure of sas/ stat taking a model with the fixed factors diet (levels: col vs. for), quercetin (levels: q+ vs. q-) and the interaction diet×quercetin. feed intake, body temperature, heart and respiratory rate and plasma concentrations of metabolites, hormones, flavonols and markers of antioxidative status were analyzed by repeated measurement anova using the mixed procedure of sas/stat software and a model with the fixed factors diet, quercetin and day of life (repeated variable) and all interactions between the fixed factors. repeated measures on the same calf were taken into account using the repeated statement of the mixed procedure and a type for the block diagonal residual covariance matrix chosen in dependence on the levels of day of life. for concentrations of plasma metabolites, hormones and data on antioxidative status in plasma, an unstructured type was used. for the plasma concentrations of flavonols, acutephase proteins and ig, a compound symmetry type was used. for data on heart and respiratory rate, as well as body temperature and feed intake, an autoregressive ( ) type was applied. leastsquares means (lsm) and their standard errors (se) were computed for each fixed effect in the models and all pairwise differences of lsm were tested by the tukey-kramer procedure. the slice statement of the mixed procedure was used to conduct partitioned analyses of the lsm for interactions. fecal score was analyzed by a generalized linear mixed model using the glimmix procedure of sas/stat and a poisson model with the fixed factors diet, quercetin and day of life (repeated variable) and all interactions between these fixed factors. repeated measurements on the same animal were taken into account by the residual option of the random statement of the glimmix procedure using a compound symmetry structure for the block diagonal residual covariance matrix. sick frequencies of calves with respect to diet and quercetin were analyzed with the freq procedure of sas/stat software using two-way tables of diet by sick and quercetin by sick and the exact pearson chi-square test. effects and differences were considered significant at p < . . mean body weight at birth was . ± . kg and increased with age (p < . ) by ± g/ d, without group differences, respectively. all calves received their first meal . ± . h after birth. milk intake related to body weight did not differ among groups on days and but was lower on days and in the colostrum-deprived calves (p < . ; fig a) . on day , appetite was reduced in the formula-fed calves and the amount of tube-fed milk was higher than in colostrum-fed calves (p < . ; fig b) . heart rate decreased with age (p < . ) and respiratory rate tended to decrease (p = . ; fig c and d) . rectal temperature was highest on days and and subsequently decreased (p < . ). rectal temperature and fecal score were higher in formula-fed than in colostrum-fed calves (p < . ; fig e and f ). the number of calves with an allowed recovery day was similar among groups. with the exception of colq +, we treated one calf per group medically for navel infection. four calves in group forq + needed antispasmodic/analgesic treatment during a recovery day because of abdominal pain. thus, col-fed calves tended to be less susceptible to illness (p = . ), whereas quercetin treatment did not affect well-being (p = . ). due to severe disease, we had to remove two calves from the study on day (forq+) and day (colq+). preprandial concentrations of total flavonols in plasma on days and (before first quercetin supplementation) did not reveal significant differences among groups, but very low concentrations were detectable in five colostrum-fed calves. in the control groups forq-and colq-, the plasma concentrations did not change with age. in colq+ and forq+, the plasma flavonol concentrations changed with age (p < . ); they reached the maximum on day and decreased subsequently but tended to differ among groups only on day (p = . ; fig a) . comparing the flavonol fractions in the plasma, the quercetin fraction ( % of total flavonoids) was highest in both dietary groups. the concentrations of isorhamnetin and tamarixetin on day were higher in colq+ than in forq+ (fig b) . plasma glucose concentrations were lowest on day , increased on day in colq-, forq+ and forq-(p < . ) and decreased on day only in forq+ and forq-(p < . ). the mean plasma concentration of glucose was higher in the colostrum-fed than in the formula-fed calves (p < . ; table ). the plasma lactate and non-esterified fatty acid concentrations were highest on day and decreased with age (p < . ) without any group differences ( table ). the concentrations of total protein in the plasma increased only in the colostrum-fed calves (p < . ) and were higher in the colostrum-fed than in the formula-fed calves on day , day and day (p < . ; fig a) . plasma albumin decreased with age (p < . ) in all groups ( fig b) . for both total protein and albumin, the diet×age interaction was significant (p < . ). the plasma urea concentrations increased on day in the formula-fed and on day in the colostrum-fed calves (p < . ; table ). the plasma triglyceride concentrations increased until day only in the colostrum-fed calves (p < . ) and continuously decreased from day to day in the formula-fed calves (p < . ). the plasma cholesterol concentration increased with age (p < . ). the plasma triglyceride (day ) and cholesterol concentrations (day and day ) were higher in the colostrum-fed than in the formula-fed calves (p < . ; table ). the plasma insulin concentrations decreased with age (p < . ; table ). glucagon increased to a maximum plasma concentration on day and subsequently decreased in all calves (p < . ). the glucagon concentration on day and day was higher in the colostrumfed than in the formula-fed calves (p < . ; table ). the cortisol concentrations decreased with age in all groups (p < . ) but decreased earlier in the colostrum-fed calves. the mean cortisol concentrations in plasma during the first week of life were lower in the colostrum-fed than in the formula-fed calves (p = . ; table ). quercetin treatment did not affect the concentrations of metabolites nor hormones in the plasma. the plasma concentrations of igg and igg increased on day only in colostrum-fed calves (p < . ; fig c and d) . the plasma concentrations of igm increased sharply until day in colostrum-fed calves and then slowly decreased (p < . ). in the formula-fed groups, the igm concentration increased until day (p < . ) but was still lower than in the colostrum-fed groups (p < . ) at the end of the trial (fig e) . the concentrations of haptoglobin in blood plasma were below the detection limit on day in all calves and increased only in the formulafed calves (p = . ) and were highest in forq-on day (fig f) . the mean haptoglobin concentration was higher in formula-fed than in the colostrum-fed calves (p = . ). the concentrations of serum amyloid a increased until day and slowly decreased afterwards in all calves (p < . ; fig g) . the concentrations of fibrinogen increased until day and then decreased (p < . ) without differences among groups (fig h) . however, the mean fibrinogen concentration tended to be higher after formula feeding (p = . ). plasma teac increased in all groups until day of life (p < . ) but was higher in the colostrum-fed than in the formula-fed calves (p < . ; fig a) . frap decreased from day to day only in the formula-fed groups (p . ) and was higher (p < . ) on day in the colostrum-fed groups (fig b) . although not statistically significant, the frap decrease was delayed in the quercetin-fed calves compared with the control groups. the mean concentrations of tbars in plasma were higher in the colostrum-fed than in the formula-fed calves (p = . ) and revealed a diet×age interaction (p = . ) with higher concentrations in the colostrum-fed than in the formula-fed calves on day ( fig c) . the mean concentrations of f -isoprostanes decreased from day to day (p < . ) in all calves and tended to increase on day only in forq-(p = . ; fig d) . regarding inflammation markers, the relative mrna abundances of tnf was higher after quercetin feeding (p = . ; table ). the relative mrna abundances of crp was higher (p = . ) and that of saa tended to be higher (p = . ) in the formula-fed than in the colostrum-fed groups (table ) . for the antioxidative enzymes, the mrna abundances of cat tended to be higher in colq-than in colq+ (p = . ) but did not differ between forq-and forq+, revealing a diet×age interaction (p = . ; table ). in this study, we were able to confirm the oral bioavailability of quercetin in colostrum-fed calves and further showed that after colostrum deprivation, quercetin is absorbed in equal amounts. as indicated earlier, oral bioavailability decreases with age due to intestinal maturation and therefore reduced permeability or more effective elimination processes [ ] . the pattern of flavonol fractions in the plasma in this study was different from the patterns in -dayold calves or adult cattle but comparable to observations in -day-old calves [ , ] . this result can be explained by the maturation of intestinal metabolism because it has been shown for rats and pigs [ , ] that orally administered flavonols undergo complete metabolism inside the intestinal mucosa, where glucuronidation is of major importance [ ] . although flavonols were present in the plasma of quercetin-fed calves, we failed to detect any effect on the metabolic parameters or hormone concentrations, which was also the case in previous studies in cattle [ , , ] . however, we could confirm a variety of effects caused by colostrum-deprivation during the first days of life. cortisol is crucial for initiation of parturition and catabolic activity, especially during hypoxia at birth. however, a decrease of the plasma cortisol concentration is delayed in formula-fed calves, as seen in previous studies [ , ] . we assume that the delay was possibly caused by either abdominal pain or by reduced utilization of nutrients, which is proven to increase stress parameters in sheep [ , ] . higher plasma glucose in colostrum-fed groups was most likely caused by enhanced intestinal maturation and therefore enlarged absorptive surface [ , ] . additionally, colostrum feeding seems to accelerate maturation of the pancreas [ ] because glucagon concentrations were also higher in colostrum-fed calves from day onwards. although the basal insulin table . hepatic mrna expression of inflammatory and antioxidative traits in calves. relative mrna expression of genes related to inflammation and antioxidative status in the liver of -d-old calves; data are given in arbitrary units and are presented as the means ± standard errors (se). q+, quercetinsupplemented; q-, control (no quercetin); n = per group. cat, catalase; gpx , glutathione peroxidase; sod, superoxide dismutase; tnf, tumor necrosis factor; il a, interleukin- α; il b, interleukin- β; hp, haptoglobin; fga, fibrinogen; saa , serum amyloid a ; crp, c-reactive protein. concentrations were similar among dietary groups, we showed in a companion paper that the postprandial insulin response is more pronounced in colostrum-fed groups [ ] . higher triglyceride and cholesterol concentrations in colostrum-fed calves are in accordance with observations in other studies [ , ] and are due to the enhanced stimulation of intestinal differentiation by colostral growth factors and hormones. however, it must be considered that diarrhea in formula-fed calves accelerated intestinal transit and therefore reduced absorption time, which might also account for differences in the plasma triglyceride and cholesterol concentrations between dietary groups. similar concentrations of non-esterified fatty acids indicate that the energy balance in the dietary groups is equal and that lipid mobilization seems to be of minor importance to maintaining energy balance; previous studies also failed to demonstrate consistent effects in neonatal calves [ , , ] . as we expected, the concentration of total protein in the plasma increased only in the colostrum-fed groups, although the protein content of the formula and colostrum was similar. the decrease of the albumin concentration from day to day was probably caused by hemodilution after first feed intake and coincides with previous findings [ , ] . because only ig, but not the albumin concentrations in plasma were affected by colostrum feeding, differences in total protein are most likely caused by absorption of colostral immunoglobulins after colostrum intake [ ] . colostrum intake is important not only for maturational processes but also to acquire passive immunity because the bovine placenta is impermeable to antibodies. as reviewed by weaver et al. [ ] , calves with serum igg concentrations below g/l (total protein < g/l) h after birth suffer from failure of passive transfer, which is associated with increased morbidity and mortality as well as reduced performance. in our study, colostrum-fed calves exceeded this threshold; however, failure of passive transfer was a relevant problem in formula-fed calves, although they were parenterally supplemented with bovine immunoglobulins on day . only formula-fed calves showed slight hyperthermia on the first days of life and a higher incidence of diarrhea, probably caused by gastrointestinal inflammation due to missing local immunity [ ] , accompanied by abdominal pain and diminished appetite. obviously, parenteral and oral treatments with immunoglobulins could not prevent local or systemic infections in formula-fed calves, probably because of missing herd-specific immunoglobulins in the formula and the administered drugs. however, we did not perform microbiological analyses in the feces of the calves to determine pathogens that might have caused loose feces. the time course of plasma igm concentration in formula-fed calves indicates that the indigenous production of igm is evident as early as day of life, but the concentrations are too low to effectively protect against infections [ ] . we assume that inflammatory processes also activated the synthesis of acute phase proteins. although the increase of the plasma acute phase proteins in all calves emphasized the immunological burden of the new environment, higher haptoglobin concentrations in formula-fed calves suggest more severe inflammatory processes than in colostrum-fed calves [ , ] , which was underlined by the greater incidence of gastrointestinal infections. because serum amyloid a is more susceptible to stress [ , ] , including physical stress, we suppose that the plasma concentrations were equally high in all groups due to the experimental procedures (e.g., continued venipuncture, restricted feeding, single penning, temporal fixation and dietary changes). on the mrna level, we did not find differences in the acute phase proteins between dietary groups because hepatic transcription precedes translation and protein release into circulation. thus, we obviously missed the time point of elevated mrna abundances of hp. however, mrna abundance of crp, a moderate acute phase protein, was elevated in the formula-fed groups on day , which could indicate the importance of c-reactive protein as a major component of the bovine innate immune system [ ] . hence, increased c-reactive protein production is compensative for the absence of immunoglobulins in colostrum-deprived calves. regarding pro-inflammatory cytokines, we found elevated hepatic mrna abundance of tnf in quercetin-supplemented calves but no differences between dietary groups. under immunocompetent conditions, the impact of various noxae leads to local production of proinflammatory cytokines, e.g., tumor necrosis factor (tnf) or interleukin- , which in turn induce the systemic acute phase response, including synthesis of acute phase proteins [ ] . within signal transduction, reactive oxygen species act as second messengers, thus enhancing tnf-induced gene expression, and oxidative conditions potentiate the activation of respective pathways [ , ] . although high doses of quercetin were repeatedly shown to reduce mrna expression of tnf in vitro [ , ] , application of prophylactic doses increased the ratio of pro-to anti-inflammatory cytokines in murine macrophages [ ] . we assumed that the hepatic quercetin concentration in our experiment did not exceed the prophylactic dose; thus, quercetin might have increased the expression of tnf. unfortunately, we did not measure tnf protein concentration, but we suppose that posttranscriptional processes anticipated tnf signal transduction [ , ] , because quercetin scavenged reactive oxygen species necessary for signal transduction. therefore, the observed quercetin effects on tnf gene expression could not have been forwarded on the expression of target genes, e.g., il b or acute phase proteins, as would have been expected otherwise. concerning the antioxidative status in the plasma, previous findings in neonatal calves are inconsistent. inanami et al. [ ] and stohrer, lutz and stangassinger [ ] concluded from comparisons between calves and dams that the former are highly susceptible to oxidative stress due to immature defense systems, whereas gaál et al. [ ] deduced from high frap values, despite the high reactive oxygen species, that calves are well-prepared to address oxidative stress. although the increase of teac in this and a previous study of our group [ ] indicates rising antioxidative capacity during the first week of life, this result was not supported by determination of frap, another marker of antioxidative capacity. furthermore, the values of tbars, which serve as a proxy to measure the products of lipid peroxidation, were unaffected by age in this study, which contradicts earlier studies [ , , ] . however, the reliability of tbars is criticized for low sensitivity and specificity, and the use of f -isoprostanes is recommended as the most reliable approach to assess oxidative stress in vivo [ , ] . the time courses of f -isoprostanes were significantly decreased in all calves; thus, we support the theory that exposure to an oxygen-rich environment following hypoxia during birth results in severe oxidative stress in the newborn [ ] . although quercetin is known to exert antioxidative effects, we did not find improved antioxidative status in the plasma of calves nor increased hepatic expression of antioxidative enzymes, which is in line with previous findings of our group in research conducted on neonatal calves and lactating dairy cows [ , ] . orally administered quercetin is completely metabolized inside the intestinal mucosa; thus, the gastrointestinal tract is the first site of action for quercetin [ ] , as it is for pathogens and gastrointestinal disorders commonly occurring during the first weeks of life. because we did not examine the intestinal tissue, we cannot exclude local effects of quercetin on antioxidative status. except for f -isoprostanes, the parameters of the antioxidative system in the plasma were higher after colostrum feeding. colostrum is a source of reactive oxygen species, but it also contains a variety of antioxidative factors [ , ] , the latter increasing with time after parturition [ ] . the absorption of proand antioxidants present in colostrum [ ] might have contributed to the rise of the respective parameters in the plasma of colostrum-fed calves and triggered immunological processes, which in turn caused elevated plasma levels. however, the hepatic mrna abundances of antioxidative enzymes seemed not to be affected by diet, but all groups were fed milk replacer from day onwards and potential dietary effects on hepatic antioxidative status might not be longlasting. in conclusion, oral administration of quercetin aglycone at a daily dose of mg/kg body weight to newborn calves during the first week of life is unable to compensate for inadequate colostrum supply. quercetin did not show any positive effect on neonatal antioxidative or antiinflammatory status, whereas colostrum feeding improves neonatal health status by supporting passive immunity and by promoting antioxidative/oxidative status. supporting information s table. complete data set of parameters regarding dry matter intake and health status as shown in fig . (pdf) s table. complete data set of flavonoid measurements in blood plasma as shown in fig . (pdf) s table. complete data set of plasma concentrations of metabolites and hormones as shown in fig and table . table. complete data set of immune and inflammatory status in blood plasma as shown in fig . (pdf) s table. complete data set of antioxidative status in blood plasma as shown in fig . (pdf) s table. complete data set of hepatic mrna expression of inflammatory and antioxidative traits as shown in table . (pdf) calf mortality in norwegian dairy herds calf health from birth to weaning. ii. management of diarrhoea in preweaned calves mortality in swedish dairy calves and replacement heifers xanthine oxidase and the fetal cardiovascular defence to hypoxia in late gestation ovine pregnancy oxidative stress in neonatology. a review oxidative stress, antioxidants, and animal function oxidants and antioxidants in disease: oxidative stress in farm animals impact of oxidative stress on the health and immune function of dairy cattle neonatal immune development in the calf and its impact on vaccine response plant bioactives for ruminant health and productivity the biochemistry and medical significance of the flavonoids the effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer the role of natural plant products in modulating the immune system: an adaptable approach for combating disease in grazing animals selenium addition to colostrum increases immunoglobulin g absorption by newborn calves the effect of ginger extracts on the antioxidant capacity and igg concentrations in the colostrum and plasma of neo-born piglets and sows correcting the antioxidant status of newborn calves for forming higher colostral immunity anti-inflammatory effects of dietary phenolic compounds in an in vitro model of inflamed human intestinal epithelium intestinal anti-spasmodic effect of a phytodrug of psidium guajava folia in the treatment of acute diarrheic disease reduction of agonist-induced contractions of guinea-pig isolated ileum by flavonoids effects of quercetin on the gastrointestinal tract in rats and mice the potential influence of fruit polyphenols on colonic microflora and human gut health effects of dietary supplementation of quercetin on performance, egg quality, cecal microflora populations, and antioxidant status in laying hens the effects of oral quercetin supplementation on splanchnic glucose metabolism in -week-old calves depend on diet after birth bioavailability of the flavonol quercetin in neonatal calves after oral administration of quercetin aglycone or rutin guidelines toward more uniformity in measuring and reporting calf experimental-data bioavailability of the flavonol quercetin in cows after intraruminal application of quercetin aglycone and rutin performance and metabolic and endocrine changes with emphasis on glucose metabolism in high-yielding dairy cows with high and low fat content in liver after calving a single-radial-diffusion method for the immunological quantitation of proteins ocena efektow doustnego podania immunoglobuliny zoltka jaja [igy] cieletom w okresie wchlaniania makromolekul z jelita. folia universitatis agriculturae stetinensis zootechnica adaptation of the guaiacol (peroxidase) test for haptoglobins to a microtitration plate system an evaluation of the heat precipitation method for plasma fibrinogen estimation antioxidant activities of carotenes and xanthophylls. febs letters antioxidant activity applying an improved abts radical cation decolorization assay. free radic vitamin e-sparing and vitamin e-independent antioxidative effects of the flavonol quercetin in growing pigs evaluation of qpcr curve analysis methods for reliable biomarker discovery: bias, resolution, precision, and implications bioavailability of quercetin from its aglycone and its glucorhamnoside rutin in lactating dairy cows after intraduodenal administration the bioavailability of quercetin in pigs depends on the glycoside moiety and on dietary factors the small intestine can both absorb and glucuronidate luminal flavonoids polyphenols: food sources and bioavailability effects of a -wk intraduodenal supplementation with quercetin on energy metabolism and indicators of liver damage in periparturient dairy cows growth performance, metabolic and endocrine traits, and absorptive capacity in neonatal calves fed either colostrum or milk replacer at two levels effects of feeding colostrum and a formula with nutrient contents as colostrum on metabolic and endocrine traits in neonatal calves effect of different dietary energy levels on physio-biochemical, endocrine changes and mrna expression profile of leptin in goat (capra hircus) experimental intestinal stress induced by duodenal distention in sheep feeding colostrum, its composition and feeding duration variably modify proliferation and morphology of the intestine and digestive enzyme activities of neonatal calves intestinal glucose absorption but not endogenous glucose production differs between colostrum-and formula-fed neonatal calves gastrointestinal tract and digestion in the young ruminant: ontogenesis, adaptations, consequences and manipulations metabolic and endocrine traits of neonatal calves are influenced by feeding colostrum for different durations or only milk replacer influence of feeding different amounts of first colostrum on metabolic, endocrine, and health status and on growth performance in neonatal calves maturation of endogenous glucose production in preterm and term calves passive transfer of colostral immunoglobulins in calves transfer of functional immunoglobulin g (igg) antibody into the gastrointestinal tract accounts for igg clearance in calves acute phase proteins as indicators of calf herd health acute phase response in holstein dairy calves affected with diarrhoea influence of physical stress on the plasma concentration of serum amyloid-a (saa) and haptoglobin (hp) in calves c-reactive protein and antibacterial activity in blood plasma of colostrum-fed calves and the effect of lactulose initiation of acute phase response and synthesis of cytokines nf-κb activation by reactive oxygen species: fifteen years later effect of quercetin and its metabolites isorhamnetin and quercetin- -glucuronide on inflammatory gene expression: role of mir- but not its metabolite quercetin- -glucuronide, exerts prophylactic immunostimulatory activity and therapeutic antiinflammatory effects on lipopolysaccharide-treated mouse peritoneal macrophages ex vivo quercetin attenuates tnf-induced inflammation in hepatic cells by inhibiting the nf-κb pathway lipid peroxides and antioxidants in serum of neonatal calves vitamine und zusatzstoffe in der ernährung von mensch und tier symposium free radicals, lipid peroxidation and the antioxidant system in the blood of cows and newborn calves around calving short communication: effects of oral flavonoid supplementation on the metabolic and antioxidative status of newborn dairy calves the comparison of antioxidative/oxidative profile in blood, colostrum and milk of early post-partum cows and their newborns insights into oxidative stress: the isoprostanes measurement of f -isoprostanes as an index of oxidative stress in vivo influence of -week intraduodenal supplementation of quercetin on performance, glucose metabolism, and mrna abundance of genes related to glucose metabolism and antioxidative status in dairy cows antioxidants in bovine colostrum physiological antioxidative/oxidative status in bovine colostrum and mature milk effect of colostrum redox balance on the oxidative status of calves during the first months of life and the relationship with passive immune acquisition we gratefully thank c. fiedler for assistance in conducting the experimental procedures, o. bellmann for veterinary assistance and k.d. witt and b. stabenow for providing experimental facilities. we also thank w. kühl, p. schulz, c. reiko, p. müntzel and u. wiedemuth for excellent laboratory assistance. key: cord- -ixzymz a authors: niskanen, r.; lindberg, a.; tråvén, m. title: failure to spread bovine virus diarrhoea virus infection from primarily infected calves despite concurrent infection with bovine coronavirus date: - - journal: the veterinary journal doi: . /tvjl. . sha: doc_id: cord_uid: ixzymz a abstract previous reports on the spread of bovine virus diarrhoea virus (bvdv) from animals primarily infected with the agent are contradictory. in this study, the possibility of transmission of bvdv from calves simultaneously subjected to acute bvdv and bovine coronavirus (bcv) infection was investigated. ten calves were inoculated intranasally with bvdv type . each of the calves was then randomly allocated to one of two groups. in each group there were four additional calves, resulting in five infected and four susceptible calves per group. virulent bcv was actively introduced in one of the groups by means of a transmitter calf. two calves, susceptible to both bvdv and bcv, were kept in a separate group, as controls. all ten calves actively inoculated with bvdv became infected as shown by seroconversions, and six of them also shed the virus in nasal secretions. however, none of the other eight calves in the two groups (four in each) seroconverted to this agent. in contrast, it proved impossible to prevent the spread of bcv infection between the experimental groups and consequently all study calves became infected with the virus. following infection, bcv was detected in nasal secretions and in faeces of the calves and, after three weeks in the study, all had seroconverted to this virus. all calves, including the controls, showed at least one of the following clinical signs during days – after the trial started: fever (≥ °c), depressed general condition, diarrhoea, and cough. the study showed that bvdv primarily infected cattle, even when co-infected with an enteric and respiratory pathogen, are inefficient transmitters of bvdv. this finding supports the principle of the scandinavian bvdv control programmes that elimination of bvdv infection from cattle populations can be achieved by identifying and removing persistently infected (pi) animals, i.e. that long-term circulation of the virus without the presence of pi animals is highly unlikely. bovine virus diarrhoea virus (bvdv) is endemic in many cattle populations, causing significant disease problems and economic losses. during ± , control programmes aimed at eradicating bvdv without the use of vaccines were initiated in sweden, norway, finland and denmark (husu & kulkas, ; olsson et al., ; bitsch et al., ; waage et al., ; lindberg & alenius, ) . a similar voluntary control programme has also been started in the uk (g. gunn, personal communication). in these schemes, high priority is given to control measures directed towards the livestock trade, in order to control the spread of bvdv between herds. the control measures are focused on excluding the main transmitters of the infection, the persistently infected (pi) animals, from the market, and also on preventing trade with dams that carry pi foetuses. also to be taken into account is the risk of introducing the infection to susceptible herds by other transmission routes without the initial presence of a pi animal. as animals with an acute bvdv infection can have a transient period of viral shedding (kirkland et al., ; bolin & ridpath, ) , direct contact with such animals could be sufficient for transmission to occur. however, reports on the ability of acutely infected animals to serve as a vehicle for the spread of bvdv are contradictory. attempts to transmit the virus by direct contact between primarily infected and susceptible cattle have proved unsuccessful (niskanen et al., ) . furthermore, animals that were experimentally infected by insemination, by parenteral injection or by rectal examination, did not transmit the infection to susceptible in-contact animals (pritchard, ; meyling & jensen, ; lang-ree et al., ) . on the other hand, field studies in large herds have shown different results and seroconversions have occurred during extended timeperiods (up to . years), although the presence of pi animals was not detected and no direct contact with pi cattle was found (barber et al., ; brownlie et al., ; meyling et al., ; moerman et al., ; edwards, ) . these differences in transmission rates may be explained in part by interacting immunosuppressive factors relating to the virus strain, the environment, and the management procedures. furthermore, primarily bvdv infected animals could possibly play a more significant role in the spread of bvdv infection in the presence of co-infections. for example, the rapid virus transmission reported by alenius et al. ( ) may have been due to the fact that a concurrent bovine coronavirus (bcv) infection was present. co-infecting agents may influence the infectivity of bvdv by modifying the immunological response of the host, or increase the amount of virus excreted by aggravating clinical signs such as cough, nasal secretions and diarrhoea. bovine coronavirus is a common bovine pathogen (storz & rott, ; battaglia et al., ; paton et al., ; tra Êve Ân et al., ) and co-infections with this virus and bvdv probably occur frequently. bcv is known mainly as an enteric agent, but this virus also causes respiratory tract infections in calves (mcnulty et al., ; heckert et al., ; storz et al., storz et al., , . the disease is highly contagious, but calves commonly recover from uncomplicated bcv infections within a few days (tra Êve Ân, ) . the experiment described in this paper allowed us to study the transmission of bvdv, from calves simultaneously subjected to acute bvdv and bcv infection, to susceptible bcv-infected calves. twenty-one conventionally reared calves, males and three females, were obtained from two different closed dairy herds. the herds were certified as free from bvdv infection under the swedish control scheme on bvdv (alenius et al., ) . the calves were either swedish red and white breed ( both sexes) or friesian (four males) and aged between and days on arrival at the clinic. they were housed at the experimental unit for one week prior to the experiment, in order to avoid stress effects due to transportation and change of environment. all calves were shown to be free from bvdv, and were antibody-negative to bvdv and to bcv by tests undertaken both in the herd of origin and on the day of arrival. no other cattle were kept on the premises during the trial. foetal calf serum with a non-cytopathogenic bvdv type strain in a titre of  tcid per ml was used to introduce bvdv infection. the inoculum was given intranasally, with . ml sprayed into each nostril. the bcv infection was introduced to the experimental calves by contact with a bcv-inoculated transmitter calf, as previously described (tra Êve Ân et al., ) . briefly, bcv-containing faeces collected from cows during a winter dysentery outbreak were stored at À c with % dmso before use. on day À , ml of a % faecal suspension in phosphatebuffered saline was given through a stomach tube to a two-month-old calf that was kept in solitary in an outdoor pen. in addition, ml of the faecal suspension was instilled into each nostril. two days after inoculation this transmitter calf was introduced to the experimental calves. eighteen calves were matched into pairs according to age, sex, breed and herd of origin. the calves were then randomly allocated to one of two study groups (a and b) until each pair was represented by one calf in each group. two additional calves were kept as uninfected controls (group c). the three groups were housed in two separate units. groups b and c were kept in the same unit but in separate pens divided by a floor-to-roof wall. the units had separate manure-shutters and their own extractor fans for evacuation of stable air. fresh air entered the units via door openings. the units were closed with a sliding door in order to reduce the risk of airborne transmission of bcv between groups b c and a within the building. on the day before the trial started, five calves from group a and b were randomly selected and put in an isolated pen. on day , they were inoculated intranasally with bvdv. the staff that performed all clinical examinations and sampling during the study did not know which calves had been inoculated with bvdv. on day , the calves were returned to their original groups. then all the calves in group a were infected with bcv by placing them in contact with the bcv transmitting calf for h. all calves in group a had nose-to-nose contact with the bcv transmitter twice during the contact period, and they were not allowed to lie down. the calves were repeatedly made to move around in the pen to increase the opportunity for transmission. the bcv transmitter did not defaecate during the h in the pen. directly after removal from group a the bcv transmitter left the experimental unit. all personnel in contact with the calves changed their coats and boots and donned disposable gloves before entering each pen. to minimize the risk of bcv transmission to groups b and c, the staff worked, during any single day, with either group b or group c (always starting with group c) or with group a alone. serum samples ± blood samples were collected from each of the study calves daily from day À upto and including day , and then on days , and . all samples were drawn from the jugular vein using evacuated tubes (becton-dickinson). they were then centrifuged for min at g. the sera were transferred to ellerman tubes and stored at À c until analysis. nasal swabs and faecal samples ± nasal swabs (virocult; medical wire & equipment co.) for bvdv detection were taken from each of the study calves on days , , and , and for bcv detection on days and ( and days after bcv was introduced into group a). faecal samples for bcv detection were taken directly from the rectal ampulla of all calves on day À , days ± , , , and . both nasal swabs and faecal samples were stored at À c until analysis. the study calves were subjected to clinical examinations from day À upto and including day . at examination the following parameters were assessed: body temperature, general condition, faecal consistency, respiratory rate and sounds on auscultation. in addition, the presence of cough or any conjunctival or nasal discharge was recorded. the degrees of diarrhoea, coughing, respiratory sounds on auscultation, nasal and conjunctival discharge were converted to numeric values. the values ranged from to , corresponding to a mild, moderate, and severe degree. similarly, body temperatures between . ± . c, . ± . c and b . c and respiratory rates between ± , ± and b per minute were assigned the numerical values , and . individual scores for general condition, body temperature, diarrhoea and respiratory tract involvement were calculated. for example, for a specific calf, all the daily values for body temperature were summed to produce a cumulative body temperature score for that calf. the respiratory score included scores for respiratory rate, respiratory sounds on auscultation, nasal and conjunctival discharge and coughing. the wilcoxon rank-sum (mann±whitney) test as applied in stata software (stata corp.) was used to test whether the difference in distribution of clinical scores was significant between bvdv-and bcv-infected calves versus calves infected with bcv only. indirect enzyme-linked immunosorbent assays (elisas) (svanova biotech) were used to determinate seroconversions to bvdv and bcv (juntti et al., ; alenius et al., ) . samples generating an absorbance value ! . were regarded as positive for bvdv and bcv antibodies. the presence of bvdv on nasal swabs was detected in an immunoperoxidase test (meyling, ) . bcv antigen on nasal swabs and in faecal samples was detected using a single-coated antigen elisa (svanova biotech). samples generating an absorbance value ! . , were regarded as positive for bcv. the wilcoxon rank-sum (mann±whitney) test was used to see whether there was a significant difference in the number of days with detectable bcv faecal shedding between dually infected calves and calves infected with bcv only. all calves that were intranasally inoculated with bvdv became infected with the virus and seroconverted to bvdv between days and postinfection (p.i.). however, no seroconversion was detected in the other eight calves in groups a and b (fig. ) . the two control calves also remained seronegative to bvdv throughout the observation period. bvdv was detected in nasal swabs on day p.i. from six of the calves intranasally inoculated with the virus, two in group a and four in group b. two of the four positive calves in group b were also positive on day p.i., and one was still positive on days eight and nine p.i. (table i) . bvdv was not isolated from any other nasal swab. all study calves, irrespective of trial group, became infected with bcv. the mean absorbance values in each group on days , , and are shown in table ii . all but three calves had seroconverted to bcv by day or p.i. one more calf in each group (a and b) had seroconverted when tested on day and the remaining calf (in group b) showed seroconversion on day p.i. these three calves had all been inoculated with bvdv. bcv was detected in nasal swabs and/or faecal samples from calves altogether. faecal shedding was detected in six calves in group a, three in group b and in both calves in group c. the calves shed bcv for upto four days in faeces (table i ). the faecal shedding of bcv occurred about two days later in groups b and c than in group a. in total, nine of calves infected with bcv alone shed bcv in faeces, while only three of calves infected with both agents shed bcv in faeces at detectable levels. the number of days of faecal shedding of bcv in faeces was significantly lower (p` . ) in the dually infected calves than in the calves infected with bcv only. four calves in group a and seven in group b shed bcv in nasal secretions on day three and/or day . the control calves both shed bcv in nasal secretions on day (table i) . all calves, including the controls, showed clinical signs following the infections. signs including fever (! c), depressed general condition, diarrhoea and cough were observed between days three and . the median values of the clinical scores for general condition were . and . (p . ) respectively, for calves infected with both bvdv and bcv and for calves infected with bcv only (table iii) . the corresponding values for body temperature were . and . (p . ). in total, calves had diarrhoea. eight of those were dually infected and seven were infected with bcv only. the diarrhoea lasted for ± days, with median clinical scores of . and . (p . ) for calves with dual and single infection, respectively (table iii) . the respiratory tract involvement showed a similar pattern for all calves irrespective of their coinfection status. on days ± and , the calves had an increased respiratory rate and increased respiratory sounds on auscultation. in addition, conjuctival and nasal discharges were present and the calves were coughing. the median values of the clinical scores reflecting respiratory tract involvement were . and . respectively, for dually infected calves and for calves infected with bcv only (table iii) . six calves that had extended periods of fever ( ± days) were treated with antibiotics (benzylpenicillin procaine) for ± days between days and . the two infectious statuses were equally represented among these calves, but four of them belonged to group b (table i) . in this study there was no transmission of bvdv from calves simultaneously subjected to acute bvdv and bcv infection, whereas all study calves became infected with bcv. these bvdv results are in agreement with previously published studies (lang-ree et al., ; niskanen et al., ) and confirm the experiences from ongoing bvdv-control programmes that the virus is not easily transmitted to susceptible cattle by primarily infected animals. on the other hand, the results clearly show that introduction of a bcv-infected animal can result in a rapid spread of bcv to all susceptible animals within a unit. this is consistent with the high morbidity described in diarrhoea outbreaks associated with bcv (durham et al., ; saif, ; alenius et al., ) . all calves inoculated with bvdv became infected, as shown by seroconversion, and six of them were found to shed virus in nasal secretions. susceptible in-contact calves were exposed to the acutely bvdv-infected calves and to their excretions by being housed together in the same pens throughout the experiment. furthermore, bcv was introduced which should, in theory, facilitate the transmission of bvdv by causing increased nasal secretions, cough and diarrhoea, clinical signs that were indeed shown by the dually infected calves. in addition, co-infection as with bcv in this trial may reduce the non-specific resistance and consequently lower the bvdv dose required for infection of the bvdv-susceptible in-contact calves. however, the timing of the co-infection may be crucial. the introduction of bcv one day after the bvdv inoculation was chosen to ensure maximum onset of clinical signs from the bcv infection at the time when bvdv excretion was expected, i.e. ± days after bvdv inoculation (bruschke, ) . also, bcv was introduced before the expected interferon type response to the bvdv infection on days ± (tra Êve Ân et al., ) , in order to minimize the inhibitory effect on bcv replication. by the inadvertent transmission of bcv to group b, we also had an opportunity to study the effect of bcv infection introduced ± days after the bvdv infection, as suggested by bcv shedding in faeces. yet no transmission of bvdv occurred in either group. however, the introduction of bcv before bvdv was not studied in this experiment. it is possible that the susceptibility of the calves to the bvdv challenge could have been increased by a preceding bcv infection. factors contributing to the failure of bvdv transmission may have been low viral excretion by the primarily infected calves, and low virulence of the non-cytopathogenic bvdv strain used. the variation in virulence, as reflected by clinical signs, between bvdv isolates is considerable. for example, animals infected with certain bvdv type strains show serious illness and have high virus titres in serum for a prolonged period of time (corapi et al., ; bruschke, ; carman et al., ) . it is also possible that the spreading of the virus could have been demonstrated if a cytopathogenic biotype of bvdv strain had been used in the experiment. furthermore, the transmission of bvdv might have been more successful if the calves, both transmitters and susceptible in-contact calves, had been exposed to environmental, nutritional and/or transportation stresses. bcv transmission from the transmitter calf to the calves in group a probably occurred by contact with nasal secretions and/or by aerosol, since faecal material was not introduced into the pen of group a. despite the rigorous measures taken to prevent the spread of bcv infection, all calves (including groups b and c) became infected with this virus. bcv infection is highly contagious and infected calves frequently propagate the virus via the enteric and respiratory tracts simultaneously (saif et al., ; heckert et al., ) . we tried to prevent the spread of bcv (via both direct and indirect routes) between groups by housing the calves in two separate units (group a in one unit and groups b and c in separate pens in another unit). the units were located approximately m apart in separate corridors. they also had separate manure-shutters. furthermore, the personnel taking care of and examining the animals only worked with calves in one of the units during any one single day and protective coats and boots were worn when entering each pen. in addition, the units had separate air ducts with extractor fans to evacuate stable air. even so, air may still have been circulating within the building. early bcv-shedding (day ) was detected in nasal secretions, indicating replication of the virus in the upper respiratory tract. this supports our hypothesis that the rapid spread of bcv between units was due to aerosol transmission. all calves infected exclusively with bcv became diseased, which was regarded clinically as a sign of alimentary and respiratory tract involvement, i.e. diarrhoea, nasal and conjunctival discharge and coughing. bcv was found in nasal secretions and in faeces of the calves and the infection was confirmed by seroconversions in all calves. however, the faecal excretion of bcv was significantly lower (p` . ) in the dually infected calves than in calves infected with bcv only. this could indicate a direct or indirect interference of bvdv with bcv replication, possibly mediated by bvdv-induced interferon type (tra Êve Ân et al., ) . a virulent field strain of bcv, originating from an outbreak of winter dysentery in adult cows, was used to introduce bcv infection in the experimental calves. the clinical disease shown by the calves is consistent with previously published experimental studies demonstrating that the outcome of the bcv infection is largely unaffected by both the origin of the virus (cow or calf isolate) and the route of inoculation (nasal or oral) (saif et al., ; el-kanawati et al., ; tra Êve Ân et al., ) . in conclusion, all of the study calves became infected with bcv, whereas none of the calves in close contact with the primarily bvdv-infected calves did. we conclude that spread of bcv infection is very difficult to prevent within a cow-house. to prevent the spread of bcv between herds, all direct and indirect contacts with herds with current or recent outbreaks of diarrhoea among cows and/or calves should be avoided. furthermore, we suggest that spread of bvdv infection is not likely to occur during periods when the only source of virus is primarily infected animals. consequently, bvdv does not seem to be a very contagious agent capable of causing acute epizootic diarrhoea in cattle herds, provided no pi animals are introduced into the herds. therefore, it seems highly likely that the early reports of bvdv in connection with outbreaks of acute epizootic diarrhoea actually described a scenario where other enteric pathogen(s), for example bcv, co-existed with bvdv but escaped detection due to lack of appropriate virus diagnostic methods. bovine coronavirus as the causative agent of winter dysentery: serological evidence a national approach to the control of bovine viral diarrhoea virus disease in a dairy herd associated with the introduction and spread of bovine virus diarrhoea virus serologische u È bersichtsuntersuchung u È ber die verbreitung des bovines coronavirus in der schweiz. schweizer archiv fu Èr tierheilkunde pa Ê vej mot kontrol af bvdv (towards control and eradication of bvdv) differences in virulence between two noncytopathic bovine viral diarrhea viruses in calves pathogenesis and epidemiology of bovine virus diarrhoea virus infection of cattle pathogenesis and vaccinology of bvdv infections. thesis, dlo institute for animal science and health severe acute bovine viral diarrhea in ontario thrombocytopenia and hemorrhages in veal calves infected with bovine viral diarrhea virus coronavirus-associated diarrhea (winter dysentery) in adult cattle observations of bovine viral diarrhoea virus infection in dairy herds, and the response to different control strategies infection and crossprotection studies of winter dysentery and calf diarrhea bovine coronavirus strains in colostrum-deprived and gnotobiotic calves longitudinal study of bovine coronavirus enteric and respiratory infections in dairy calves in two herds in ohio vastusohjelmat naudan tarttuvaa leukoosia ja bvd:ta È vastaa suomen ela Èinla Èa Èka Èrilehti the use of monoclonal antibodies in enzyme linked immunosorbent assays for detection of antibodies to bovine viral diarrhoea virus replication of bovine viral diarrhoea virus in the bovine reproductive tract and excretion of virus in semen during acute and chronic infections principles for eradication of bovine viral diarrhoea virus (bvdv) infections in cattle populations coronavirus infection of the bovine respiratory tract detection of bvd virus in viremic cattle by an indirect immunoperoxidase technique transmission of bovine virus diarrhoea virus (bvdv) by artificial insemination (ai) with semen from a persistently infected bull epidemiology of bovine virus diarrhoea virus. revue scientifique et technique office international des epizooties lack of virus transmission from primarily bovine viral diarrhoea virus (bvdv) infected calves to susceptible peers prevalence of antibodies to bovine virus diarrhoea virus and other viruses in bulk tank milk in england and wales a review of evidence implicating bovine coronavirus in the etiology of winter dysentery: an enigma resolved? u È ber die verbreitung der coronavirusinfektion bei rindern in ausgewa Èhlten gebieten deutschlands: antiko Èrpernachweis durch mikroimmunodiffusion und neutralisation coronavirus isolation from nasal swab samples in cattle with signs of respiratory tract disease after shipping isolation of respiratory bovine coronavirus, other cytocidal viruses, and pasteurella spp from cattle involved in two natural outbreaks of shipping feber winter dysentery caused by bovine coronavirus: no rule without an exception. diagnostics, clinical picture, epidemiology and herd immunity. doctoral thesis, department of ruminant medicine and veterinary epidemiology primary bovine viral diarrhoea virus infection in calves following direct contact with a persistently viraemic calf nationwide survey of antibodies to bovine coronavirus in swedish dairy herd bulk milk the norwegian programme for eradication of bovine viral diarrhoea/mucosal disease the norwegian programme for eradication of bovine viral diarrhoea/mucosal disease world buiatirc congress. th congress of the italian association of buiatrics key: cord- - vraae h authors: gomez, d.e.; arroyo, l.g.; costa, m.c.; viel, l.; weese, j.s. title: characterization of the fecal bacterial microbiota of healthy and diarrheic dairy calves date: - - journal: j vet intern med doi: . /jvim. sha: doc_id: cord_uid: vraae h background: neonatal diarrhea accounts for more than % of total deaths in dairy calves. few population‐based studies of cattle have investigated how the microbiota is impacted during diarrhea. objectives: to characterize the fecal microbiota and predict the functional potential of the microbial communities in healthy and diarrheic calves. methods: fifteen diarrheic calves between the ages of and days and age‐matched healthy control calves were enrolled from dairy farms. the illumina miseq sequencer was used for high‐throughput sequencing of the v region of the s rrna gene (illumina, san diego, ca). results: significant differences in community membership and structure were identified among healthy calves from different farms. differences in community membership and structure also were identified between healthy and diarrheic calves within each farm. based on linear discriminant analysis effect size (lefse), the genera bifidobacterium, megamonas, and a genus of the family bifidobacteriaceae were associated with health at farm , whereas lachnospiraceae incertae sedis, dietzia and an unclassified genus of the family veillonellaceae were significantly associated with health at farm . the phylogenetic investigation of communities reconstruction of unobserved states (picrust) analysis indicated that diarrheic calves had decreased abundances of genes responsible for metabolism of various vitamins, amino acids, and carbohydrate. clinical relevance: the fecal microbiota of healthy dairy calves appeared to be farm specific as were the changes observed during diarrhea. the differences in microbiota structure and membership between healthy and diarrheic calves suggest that dysbiosis can occur in diarrheic calves and it is associated with changes in predictive metagenomic function. n eonatal calf diarrhea accounts for most of the morbidity and mortality in calves, accounting for > % of total deaths, and has a large impact on the economy of dairy farms. although pathogenic bacteria (escherichia coli, salmonella spp), viruses (rotavirus, coronavirus), and protozoa (cryptosporidium spp) can be found in a large percentage of cases, the etiology of disease may be hard to discern because of the common presence of coinfections (> pathogen identified in the same patient) and the fact that most organism also can be found in healthy individuals. these observations suggest that the cause of diarrhea is complex rather than associated with a single pathogen. diagnosis of enteric disease also is likely affected by the complexity of the microbiota (ie, the rich and diverse polymicrobial community present in the gut). the gastrointestinal microbiota plays an important role in maintaining host health by providing vitamins and energy, by facilitating development of gut tissue and the immune system, by modulating inflammatory responses at local and distal organs, and by competing with pathogens for nutrients and attachment sites on the gut epithelial surface. although traditional focus on calf diarrhea has been on individual pathogens, it has become increasingly clear in other species that microbial populations play a key role in health and disease. disruption of this ecosystem, otherwise known as "dysbiosis," can trigger gastrointestinal disorders. to date, the molecular basis of dysbiosis and the key bacterial groups involved remain poorly defined. it is clear however that if the gut microbiota is disrupted (eg, antibiotic treatment, gut inflammation) the risk of disease can increase substantially [ ] [ ] [ ] and re-establishment of the normal microbiota can result in recovery from disease. despite current attention and research on the gut microbiota, few population-based studies have been conducted in cattle to better understand how the microbiota and its functional potential are impacted during neonatal diarrhea. calf diarrhea may be associated with altered microbial diversity and decreased abundance of butyrate-producing microorganisms during the first weeks of life. the gastrointestinal microbiota is a major contributor to the physiological, nutritional, and immunological functions of the gut. therefore, the structure and functional roles of the microbiota in diarrheic calves should be examined in more detail. the objectives of our study were to profile the fecal microbiota and predict the functional potential of microbial communities in healthy calves and calves with diarrhea from large dairy farms with dissimilar management practices. this prospective study used a case-control design. holstein-friesen calves from large dairy farms (f and f ) were enrolled during the spring season of . study farms were selected from a convenience sample of commercial farms within km radius of the university of guelph (guelph, on). on both farms, cows were vaccinated against bovine coronavirus and rotavirus weeks and weeks before calving. a calves were separated from their dams at birth and received bolus containing antibodies against enterotoxigenic e. coli and bovine coronavirus. b farm details and management practices are presented in table . fifteen diarrheic calves between the ages of and days, and age-matched healthy control calves were enrolled from each farm. health status of the calves was assessed with a standardized calf health-scoring chart. this system assisted the assessment of health by evaluating body temperature score ( , = normal; , = fever), fecal score ( , = normal; , = diarrhea), nasal score ( , = normal; , = nasal discharge), eye score ( , = normal; , = eye discharge, crusty eyes), and ear score ( , = normal; , = head tilt, ear or both ears dropped). a diarrheic calf was defined as a calf with fecal and body temperature scores ≥ and score ≤ in the other evaluated systems. once a diarrheic calf was identified, an age-matched (ae days) control calf with health score ≤ in all of the evaluated systems was enrolled. calves were not included in the study if they had a previous episode of diarrhea, other diseases (eg, umbilical abscess, pneumonia), or if they had ever been treated with antimicrobial drugs. healthy calves that developed diarrhea within the days after sampling also were excluded. fecal samples were obtained per rectum from cases and controls, labeled and immediately stored at À °c. the incidence of calf diarrhea, as recorded by farm personnel, for calves < days of age was obtained from farms for the preceding -month period. attributable mortality rates due to calf diarrhea also were calculated for each farm, based on the number of deaths attributed to diarrhea by farm or veterinary personnel divided by the total number of deaths during a period of year. dna extraction, amplification, and sequencing of bacterial s rrna gene total dna was extracted from mg (wet weight) of fecal samples with a commercial kit. c the v region of the s rrna gene was amplified with the forward ( -aytgggydta aagng- ) and reverse ( -tacnvgggtatctaatcc- ) primers the primers were designed with overhanging adapters (forward: tcgtcggcagcgtcagatgtgtataagagacag, reverse: gtctcgtgggctcggagatgtgtataagagac ag) for annealing to illumina universal index sequencing adaptors that were added in a later pcr. the reaction mixture and amplification conditions have been described previously. the pcr products were purified with magnetic beads. d illumina universal adapters (forward: aatgatacgg cgaccaccgagatct acac-index-tcgtcggcagcgtc, reverse: caagcagaag acggcatacgagat-index-gtctcgtgggctcgg) then were added to the purified s rrna gene product by pcr. the pcr products were evaluated by electrophoresis in . % agarose gel and purified as described above. after purification, spectrophotometry e was used to quantify the pcr products. samples were normalized to a final concentration of nm. the library pool was submitted to the genomics facility of the university of guelph and sequenced with an illumina miseq f for cycles from each end. incidence of diarrhea and mortality rates attributed to diarrhea, as well as treatment rates, were compared by a fisher's exact test. the mothur software package g was used for the bioinformatic analysis. paired-end reads were merged to fully overlapping reads and then aligned to the silva s rrna reference database. sequences that were misaligned with the target region were removed. irregular sequences including those with contiguous sequence lengths > bp or < bp and ambiguous base calls also were removed, as were those with runs of homopolymers > base pairs. uchime was utilized to identify chimeras, which then were removed. sequences belonging to nonbacterial domains (chloroplasts, mitochondria, archaea, and eukaryotes) also were removed. the remaining sequences were assigned into operational taxonomic units (otus) by an open otu-picking approach, with a distance limit of . ( % similarity). the otus were classified by the ribosomal database project classifier. h relative abundances of the main phyla, classes, orders, and families (median relative abundance > . %) and the main genera (median relative abundance > . %) were calculated. the shapiro-wilk test was used to evaluate normality of the datasets. the majority of datasets did not meet the assumptions of normal distribution. therefore, comparison of the relative abundances between groups (healthy calves between farms, and healthy and diarrheic calves within farms) was performed by the nonparametric mann-whitney u-test. p-values were adjusted for multiple by a statistical software i to generate q-values. a q < . was considered statistically significant. subsampling was completed to normalize sequence number by random selection of a number of sequences that corresponded to the lowest number of reads for any sample. sampling coverage was assessed by good's coverage value. diversity, evenness, and richness were calculated by the inverse simpson's, shannon's evenness, and chao indexes, respectively, and comparison between groups was made by wilcoxon rank sum test. the core microbiota was investigated by identifying genera with relative abundances of at least % in all samples from a group. the jaccard index (a measure of community membership, which only considers the number of shared genera, but not their abundance) and the yue and clayton index (a measure of community structure, which considers shared genera and their relative abundances) were calculated. unweighted unique fraction metric (unifrac) analysis of molecular variance (amova), homogeneity of molecular variance analysis (homova), and parsimony test were used to compare community membership and structure between groups. the similarity between groups was visualized by dendrograms plotted by figtree v . . . . j clustering of the groups was represented by principal coordinate analysis plotted by a statistical software. k phylogenetic investigation of communities by reconstruction of unobserved states (picrust) was used to predict the functional gene content in the fecal microbiota based on taxonomy obtained from the greengenes reference database (http://greengenes.lbl.gov/cgibin/nph-index.cgi). comparison of the percentages of functional gene content between groups (healthy calves between farms, and healthy and diarrheic calves within farms) was performed by mann-whitney u-test, adjusted for multiple comparisons. linear discriminant analysis effect size (lefse) was used to identify bacterial taxa and predicted functional genes (picrust) that were enriched in feces of healthy and diarrheic calves, based on a p < . and lda score > . . the picrust and lefse were performed online in the galaxy workflow framework (https://hutten hower.sph.harvard.edu/galaxy/). data were made publicly available at the national center for biotechnology information sequence read archive under accession number sub . the incidence of diarrhea in calves < month of age during a -year period was significantly different between farms (f : %, / ; f : %, / ; p = . ). however, the mortality rate due to diarrhea did not differ ( % and %; p = . ). the proportion of calves with diarrhea treated with antimicrobials was high and not different between f ( / , %) and f ( / , %) (p < . ). the age distribution (days) of diarrheic calves was similar across the calves from the farms with mean ages of ae and ae days for f and f , respectively (p = . ). a total of , , reads were obtained with a mean of , reads per calf (standard deviation [sd] , ; median, , ; , range, , to , ). a random subsample of , reads per sample was used to normalize data. subsampling was considered adequate, as evidenced by the coverage of . % obtained for all samples. significant differences in richness, evenness, and diversity of gut microbiota were noted between healthy and diarrheic calves (fig ) . twenty-two different phyla were identified, but firmicutes, actinobacteria, and proteobacteria accounted for > % of sequences (fig ) . fusobacteria, bacteroidetes, and verrucomicrobia were identified in healthy and diarrheic calves at > % of the total number of sequences. comparison between healthy calves from f and f identified a higher relative abundance of fusobacteria (p = . ) and lower relative abundance of proteobacteria (p = . ) in calves from f . firmicutes, actinobacteria, and proteobacteria also dominated the fecal microbiota of diarrheic calves (fig ) . on f , diarrheic calves had a significantly lower relative abundance of actinobacteria than did healthy calves from the same farm (p = . ). there were no differences in phyla between healthy and diarrheic calves on f (table s ) . forty-three different classes, orders, and families were identified, but only , , and accounted for ≥ . % of sequences overall, respectively. the relative abundances of most abundant bacterial taxa identified in feces of healthy and diarrheic calves from f table . in f , there were no statistically significant differences in the relative abundance of any bacterial taxa of healthy versus diarrheic calves. overall, genera were detected. fifty-five of those were present at relative abundance of > . %. the relative abundances of the most abundant genera found in healthy and diarrheic calves from f and f are presented in fig and table . when comparing healthy calves, and bacterial taxa enriched in healthy calves from f and f , respectively, were identified. enriched phylotypes from f were predominantly from the classes bacilli, fusobacteria, alphaproteobacteria, betaproteobacteria, and bacteroidetes, whereas most from f were gammaproteobacteria (fig ) . comparing healthy and diarrheic calves within each farm, genera were enriched in the feces of healthy calves and genera were enriched in the abbreviation "unclass." indicates an unclassified taxonomy within the respective taxonomic group. diarrheic calves from f , whereas in f genera were enriched in healthy calves and in diarrheic calves (fig ) . based on lefse, the genera bifidobacterium, megamonas, and a genus of the family bifidobacteriaceae were associated with health in f , whereas lachnospiraceae incertae sedis, dietzia and an unclassified genus of the family veillonellaceae were significantly associated with health in f (fig ) . as shown in table , there were significant differences in community membership (jaccard index) and community structure (yue & clayton index) between healthy calves from the different farms. healthy and diarrheic calves from f were significantly different in community membership, but there was no difference in community membership between healthy and diarrheic calves from f . community structure was significantly different between healthy and diarrheic calves from f . these differences in community membership and structure were visualized by dendrograms (fig ) and pcoa plots (figs and ). at kyoto encyclopedia of genes and genomes (kegg) level , genes associated with metabolism predominated, accounting for % (n = ) overall. genes encoding genetic information processing ( ; %), environmental information processing ( ; %), cellular information processing ( ; %), and organismal system (immune system, endocrine system, and nervous system; ; %) also were common. at kegg level , % (n = ) of the metabolism genes belonged to xenobiotic biodegradation metabolism, % (n = ) to lipid metabolism, % (n = ) to biosynthesis of secondary metabolites, % (n = ) to carbohydrate metabolism, % (n = ) to amino acid metabolism, % (n = ) to metabolism of terpenoids and polyketides, % (n = ) to metabolism of cofactors and vitamins, % (n = ) to glycan metabolism, % (n = ) metabolism of other amino acids, % (n = ) were unclassified, % (n = ) were related to energy metabolism, and % (n = ) to enzyme families. there were no differences in the percentages of kegg orthologs from either level or (all adjusted p values >. ). the relative abundances of the most abundant kegg orthologs are presented in supplemental fig . when comparing healthy and diarrheic calves, lefse analysis indicated that porphyrin and chlorophyll metabolism pathway genes were enriched in diarrheic calves from f . on f , biosynthesis of vancomycin group antibiotics; valine, leucine, and isoleucine biosynthesis; glycine, serine, and threonine metabolism; folate biosynthesis; pantothenate and coa biosynthesis; and c branched dibasic acid metabolism were enriched in healthy calves, whereas porphyrin and chlorophyll metabolism pathway genes were enriched in diarrheic calves (fig ) . various studies have described the development of the gastrointestinal microbiota of calves , - and the changes associated with different management practices including feeding, housing, and antimicrobial administration during the neonatal period. , however, limited information is available regarding the specific changes occurring in the fecal microbiota and the functional genes in calves with neonatal diarrhea. [ ] [ ] [ ] we identified significant differences in the fecal microbiota and its predicted functional metabolic pathways in healthy and diarrheic calves from large dairy farms with different management practices. the analyses evaluating the similarity of community membership and structure of the fecal microbiota showed that calves from the same farm shared similar microbial communities regardless of the health status when compared to healthy and diarrheic calves from a different farm. the degree of interfarm variation is important to recognize, particularly because earlier studies have been based primarily on animals from single facilities. these data indicate potentially important differences between facilities that must be considered when designing and interpreting microbiota studies. the development of intestinal microbiota in neonatal calves is a dynamic and complex process influenced by external and internal factors that affect intestinal microbial succession. external factors include microbial load in the environment, delivery mode, type of colostrum, type of feeding (raw milk versus pasteurized milk versus milk replacer), housing, and administration of probiotics, prebiotics, or antibiotics. [ ] [ ] [ ] individual factors that can influence gut microbiota include nutritional state, functional immaturity of the immune system, intestinal ph, peristalsis, bile acids, bacterial mucosal receptors, and microbial interactions. therefore, management factors are a likely explanation for the interfarm differences that were noted, but further study of factors that influence both the individual calf and farm-level microbiota is needed. in humans and most other studied species, a small number of phyla tend to account for the majority of the intestinal microbiota. firmicutes tends to be the dominant phylum in most animal species, , , , as was the case here. the predominance of firmicutes, actinobacteria, and proteobacteria noted in these calves is also consistent with previous studies in infants, foals, piglets, and calves. , the predominance of actinobacteria is presumably a reflection of the early colonization of the gut in neonates. bovine milk contains complex nondigestible oligosaccharides that promote proliferation of specific gut microbes, especially bifidobacterium spp. from the actinobacteria phyla. although it is difficult to define normal calf microbiota, general trends can be inferred from previous studies. from a phylum-level perspective, the fecal microbiota of healthy holstein calves during the neonatal period was dominated by firmicutes, with a relative abundance ranging from to %, followed by bacteroidetes ( - %), proteobacteria ( - %), fusobacteria ( - %), and actinobacteria ( - %). at the genus level, several studies identified higher abundance of bacteroides and clostridium spp. in the feces of healthy calves. [ ] [ ] [ ] in addition, in pre-weaned holstein calves relative abundance of faecalibacterium prausnitzii, a microorganism that belongs to the phylum firmicutes, was higher during the first weeks of life and this high abundance was associated with decreased incidence of diarrhea. our results differed with the aforementioned studies in several aspects. the phylum bacteroidetes accounted for < % of the total sequences identified in healthy and diarrheic calves, something that could be accounted for, at least in part, by methodology, because it is well established that differences in bacteroidetes abundance can be found with illumina vs sequencing likely due to the choice of different primers (v -v vs v , respectively). proteobacteria, especially members of the enterobacteriaceae family, were enriched in feces of healthy calves from f , and in feces of diarrheic calves from both f and f . the increase in diarrheic calves is not surprising as this phylum often is associated with intestinal dysbiosis, but the difference between farms was interesting. a recent study of nursing calves from beef farms identified some farms with high relative abundances of proteobacteria, suggesting that higher proteobacteria levels could be a farm-associated effect, perhaps from management practices. this could be of concern because recent studies have identified a mechanistic interrelation among proteobacteria, the gut immune response, and inflammation. , dysregulated innate immune responses can elicit the bloom of proteobacteria that promotes gut inflammation and facilitate inflammation or invasion by pathogens. , additionally, the relative abundance of faecalibacterium spp. was lower in healthy calves, a somewhat surprising result because f. prausnitzii has been associated with anti-inflammatory properties by stimulating the production of anti-inflammatory cytokines, decreasing the secretion of the pro-inflammatory cytokines and by the production of butyrate. , contradictory results regarding the relative abundance of faecalibacterium spp in feces of preweaned calves from different farms also have been reported in dairy , and beef calves. the reasons accounting for these differences are unclear, but differences in methodologies among studies as well as a farm-associated effects (management practices) could explain the differences in gut microbiota. we also identified significant differences in microbiota structure and membership between healthy and diarrheic calves. this observation was not surprising given the differences that have been noted with enteric disease in various animal species and diseases. [ ] [ ] [ ] the differences that were noted with our analyses, along with those identified by other approaches (eg, lefse) highlight the need to look beyond simple comparison of relative abundances when trying to interpret the microbiota, because relative abundance changes were more modest. the relative abundance of actinobacteria was significantly increased in healthy calves. at the genus level, lefse analysis identified enrichment of bifidobacterium and an unclassified genus of the bifidobacteriaceae family in healthy calves. changes in the relative abundance of bifidobacterium have been reported with different dysbiosis-associated intestinal diseases. , bifidobacteria have been reported to prevent gastrointestinal infections by outcompeting pathogenic viruses or bacteria for binding sites on epithelial cells. , bifidobacteria also produce short chain fatty acids that are transformed to butyrate and stimulate growth of butyrate-producing bacteria. butyrate has been shown to have trophic and immunomodulatory effects on the intestinal epithelium. , bifidobacterium also can contribute to the gut health by production of inhibitory substances, , and modulating the gastrointestinal immune system response. , these characteristics can explain the positive impact of bifidobacterium-based probiotics on prevention of gastrointestinal diseases in calves. three-dimensional principal coordinates analyses of the community membership (jaccard index) of the fecal microbiota of healthy and diarrheic calves. coloured points and ellipses indicate groups: healthy calves from farm (green) and farm (red) and diarrheic calves from farm (orange) and farm (blue). three-dimensional principal coordinates analyses of the community structure (yue and clayton index) of the fecal microbiota of healthy and diarrheic calves. coloured points and ellipses indicate groups: healthy calves from farm (green) and farm (red) and diarrheic calves from farm (orange) and farm (blue). the picrust analysis was used to infer functional capabilities of the microbial communities. this approach infers the functional capacity of the microbiota by prediction of functional genes that typically are associated with different taxa. although the biological relevance of this approach is still unclear, lefse analysis identified several functional gene categories that were enriched in healthy and diarrheic calves. diarrheic calves had decreased abundances of genes responsible for metabolism of various vitamins (eg, folate, panthotenate), amino acids (eg, valine, leucine, isoleucine, glycine, serine, threonine) and carbohydrate metabolism. this imbalance might indicate that free vitamin and nutrient availability is altered in diarrheic calves. alterations in amino acid metabolism have been observed in dogs with chronic diarrhea caused by idiopathic inflammatory bowel disease, in cats with acute diarrhea, and in humans with gut inflammation, suggesting that amino acid dysmetabolism may be an important feature of dysbiosis-associated diseases. increased relative abundances of genes associated with porphyrin and chlorophyll metabolism also were present in diarrheic calves. porphyrins are tetrapyrroles that bind covalently to a metal (iron, to form cytochromes, peroxidase, catalase, myoglobin, and hemoglobin; copper or nickel, to form molecules for electron transport in methanogenic bacteria). in nonphotosynthetic eukaryotes such as animals, insects, fungi, and protozoa, as well as in the a-proteobacteria group of bacteria, the committed step for porphyrin biosynthesis is the formation of d-aminolevulinic acid by the reaction of the amino acid glycine with succinyl coa from the citric acid cycle. metabolites such as daminolevulinic acid potentially could be used as a marker of increased abundance of proteobacteria and therefore dysbiosis. however, picrust is only a predictor of metagenomic function, and metabolomic approaches are preferred to identify factual changes in metabolic function of microbiota of diarrheic calves and identify markers of unstable gut microbiota. the intestinal microbiota of healthy dairy calves appeared to be farm-specific as were the changes during diarrhea. significant differences in microbiota structure and membership between healthy and diarrheic calves suggest that dysbiosis occurred in diarrheic calves and was associated with changes in the predictive metagenomic function of the bacterial communities. a metabolomic approach is required however to accurately establish changes in metabolic function. additional supporting information may be found online in the supporting information tab for this article: fig s . median relative abundance of the main metabolic pathways genes identified in feces of healthy and diarrheic calves from different farms (n = per group). table s . relative abundance (median, min and max) of the most abundant phyla, classes, order, and families identified in feces of healthy and diarrheic calves from two different farms. part i: reference of dairy cattle health and management practices in the united states. fort collins co: usda-aphis-vs case-control study of microbiological etiology associated with calf diarrhea host-bacterial mutualism in the human intestine predicted functional composition of metagenomes based on s rrna gene sequencing data. lefse based on the picrust dataset revealed differentially enriched metabolic pathways associated either with healthy (green) or diarrheic calves (red) from farm interactions between commensal intestinal bacteria and the immune system the interplay between the intestinal microbiota and the immune system recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis gut bacteria dysbiosis and necrotising enterocolitis in very low birthweight infants: a prospective case-control study intestinal dysbiosis and depletion of butyrogenic bacteria in clostridium difficile infection and nosocomial diarrhea salmonella enterica serovar typhimurium exploits inflammation to compete with the intestinal microbiota hostmediated inflammation disrupts the intestinal microbiota and promotes the overgrowth of enterobacteriaceae disease management of dairy calves and heifers evaluation of general s ribosomal rna gene pcr primers for classical and next generation sequencing-based diversity studies longitudinal study of the early-life fecal and nasal microbiotas of the domestic pig introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities the silva ribosomal rna gene database project: improved data processing and webbased tools uchime improves sensitivity and speed of chimera detection controlling the false discovery rate: a practical and powerful approach to multiple testing predictive functional profiling of microbial communities using s rrna marker gene sequences greengenes, a chimera-checked s rrna gene database and workbench compatible with arb metagenomic biomarker discovery and explanation characterization of the rumen microbiota of pre-ruminant calves using metagenomic tools postnatal development of bacterial population in the gastrointestinal tract of calves the development of the flora of the alimentary tract in young animals the bacterial content of the small intestine of young calves pyrosequencing reveals diverse fecal microbiota in simmental calves during early development rrna-based analysis to monitor succession of faecal bacterial communities in holstein calves development of colonic microflora as assessed by pyrosequencing in dairy calves fed waste milk the bacteriology of the intestinal tract of young calves with special reference to the early diarrhea ("scours") distribution and virulence of escherichia coli in the small intestines of calves with and without diarrhea changes in small intestinal mor heat-treated colostrum feeding promotes beneficial bacteria colonization in the small intestine of neonatal calves effect of preweaned dairy calf housing system on antimicrobial resistance in commensal escherichia coli ingestion of milk containing very low concentration of antimicrobials: longitudinal effect on fecal microbiota composition in preweaned calves effects of antibiotics (oxytetracycline, florfenicol or tulathromycin) on neonatal calves' faecal microbial diversity developmental microbial ecology of the neonatal gastrointestinal tract new insights in gut microbiota establishment in healthy breast-fed neonates development of the faecal microbiota in foals bovine milk as a source of functional oligosaccharides for improving human health the principal fucosylated oligosaccharides of human milk exhibit prebiotic properties on cultured infant microbiota analysis, optimization and verification of illumina-generated s rrna gene amplicon surveys intestinal microbial communities associated with acute enteric infections and disease recovery assessment of the fecal microbiota in beef calves altered enteric microbiota ecology in interleukin -deficient mice during development and progression of intestinal inflammation dietary-fatinduced taurocholic acid promotes pathobiont expansion and colitis in il -/-mice proteobacteria: microbial signature of dysbiosis in gut microbiota like will to like: abundances of closely related species can predict susceptibility to intestinal colonization by pathogenic and commensal bacteria faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of crohn disease patients alteration of the fecal microbiota and serum metabolite profiles in dogs with idiopathic inflammatory bowel disease the fecal microbiome in cats with diarrhea low counts of faecalibacterium prausnitzii in colitis microbiota effectiveness of bifidobacterium bifidum in mediating the clinical course of murine rotavirus diarrhea changes of the human gut microbiome induced by a fermented milk product lactate-utilizing bacteria, isolated from human feces, that produce butyrate as a major fermentation product commensal microbe-derived butyrate induces the differentiation of colonic regulatory t cells bifidobacterium strains from resident infant human gastrointestinal microflora exert antimicrobial activity proteina effect of bifidobacterium bifidum and lactobacillus acidophilus on gut mucosa and peripheral blood b lymphocytes effect of administration of bifidobacteria and lactic acid bacteria to newborn calves and piglets the treatmentnaive microbiome in new-onset crohn's disease approach to a comparative study of the metabolism of porphyrins and chlorophylls we thank joyce rousseau and william sears for their input and advice during the process of carrying out the experiments and statistical analysis, respectively, described in this manuscript.conflict of interest declaration: authors declare no conflict of interest.off-label antimicrobial declaration: authors declare no off-label use of antimicrobials. key: cord- - cu ly authors: koch, a.; kaske, m. title: clinical efficacy of intravenous hypertonic saline solution or hypertonic bicarbonate solution in the treatment of inappetent calves with neonatal diarrhea date: - - journal: j vet intern med doi: . /j. - . . .x sha: doc_id: cord_uid: cu ly background: the clinical efficacy of iv administered hypertonic saline solution and hypertonic bicarbonate solution (hbs) in the treatment of inappetent diarrheic calves has not been compared yet. hypothesis: hbs is more advantageous than hypertonic saline in the treatment of calves with severe metabolic acidosis. animals: twenty‐eight dehydrated, inappetent calves with neonatal diarrhea. methods: in consecutive clinical studies, calves were initially treated with saline ( . %; ml/kg body weight [bw] over minutes; study i: n = ) or bicarbonate solution ( . %; ml/kg bw over minutes; study ii: n = ), respectively, followed by oral administration of l isotonic electrolyte solution minutes after injection. clinical and laboratory variables were monitored for hours. results: treatment failed in calves of study i and in calf of study ii as indicated by a deterioration of the general condition. all treatment failures had more severe metabolic acidosis compared with successfully treated calves before treatment. in the latter, rehydration was completed within hours after injection; metabolic acidosis was corrected within hours (study i) and hours (study ii) after injection. conclusions and clinical importance: diarrheic calves with slight metabolic acidosis (base excess [be] >− mm) can be treated successfully with hypertonic saline. hbs is appropriate in calves without respiratory problems with more severe metabolic acidosis (be up to − mm). intensive care of the calves is required to ensure a sufficient oral fluid intake after the initial iv treatment. n onspecific neonatal diarrhea represents the most important cause of fatalities and economic losses in calf husbandry. the clinical course of neonatal diarrhea is characterized by severe dehydration and imbalances in the acid-base and electrolyte status. objectives in the treatment of diarrhea are rehydration and correction of the metabolic acidosis. , in diarrheic calves with a failing suckling reflex, the continuous iv infusion of isotonic solution is recommended as the treatment of choice. however, this therapeutic approach is laborious and expensive. as an alternative, the rapid iv injection of hypertonic saline solution (hss) followed by application of oral rehydration solution (ors) has been suggested. in veterinary medicine, hss has been established in the treatment of endotoxic shock in cattle and dogs as well as in the treatment of hemorrhagic shock in sheep, swine, horses, dogs, and cats. hss was also found to be successful for the rehydration of hypovolemic calves, , which were dehydrated experimentally by the administration of sucrose and diuretics. although severely dehydrated, these calves exhibited only a mild acidemia and metabolic acidosis. however, a pronounced metabolic acidosis is typical, in particular for older calves ( days) suffering from neonatal diarrhea. the general condition, the suckling reflex, and the ability to stand are influenced by the extent of the acidosis. , in particular, increases in serum d-lactate concentrations commonly found in diarrheic calves because of extensive microbial fermentation of carbohydrates in the rumen and large intestine were found to have a stronger influence on behavior and posture than metabolic acidosis per se. because parenteral saline induces a mild acidifying effect, , , , the correction of the acidemia in diarrheic calves depends significantly on the consecutive consumption of buffers in ors. the initial application of hypertonic bicarbonate solution (hbs) may be more advantageous in the treatment of calves with metabolic acidosis. however, hbs has been suggested to induce a paradoxical intracellular and cerebrospinal acidosis (pia); the relevance of paradoxical intracellular acidosis in calves after administration of hbs is discussed controversially. , it was the objective of the clinical studies presented here to investigate in inappetent calves suffering from neonatal diarrhea the clinical efficacy of iv administered hss and hbs, respectively. two consecutive studies were performed using an identical experimental protocol. the only differences between them were the solutions for hypertonic treatment and the number of calves. in study i, holstein friesian calves ( females, males) received hypertonic saline as initial treatment. in study ii, hbs was applied to holstein friesian calves ( females, males). all clinical assessments were carried out following a standardized protocol by the same person. both studies were carried out with calves admitted to the clinic for cattle. calves were chosen based on the following selection criteria: age o days, neonatal diarrhea (defined as soupy or watery consistency of the feces), dehydration (distance between the medial canthus and the eyeball at least mm), no suckling reflex, and no clinical symptoms indicating moderate to severe secondary organ diseases. immediately after admission, each calf was weighed. the general condition was assessed using a score system ( agile, physiologic; slight depression, able to stand; moderate depression, sternal recumbency; severe depression, lateral recumbency). a nipple bottle filled with milk was offered to the animal to assess the suckling reflex. the extent of enophthalmus was quantified by estimating the distance between the medial canthus and the eyeball (mm). respiratory rate, heart rate, and rectal temperature were assessed. lung auscultation and navel as well as joints' examination were carried out to exclude secondary diseases. after checking by clinical investigation that all selection criteria were fulfilled, blood was collected from the jugular vein to assess pcv, serum variables, and blood gases. centrifugation of the blood ( ,  g, c, minutes) took place within minutes after withdrawal. finally, a grab sample of feces was taken from the ampulla recti, which was tested for rotavirus, coronavirus, cryptosporidium parvum, and e. coli f a , and for salmonellae by fecal culture. b the initial treatment was conducted after introducing a venous catheter c into the jugular vein. in study i, each calf received ml/kg body weight (bw) saline solution d ( . %) over minutes (study i: nacl) corresponding to mmol nacl/kg bw. in study ii, each calf received ml/kg bw bicarbonate solution e ( . %) over minutes (study ii: nahco ) corresponding to mmol nahco /kg bw. immediately after injection, calves were allowed to suckle l ors. if calves refused to drink, they were administered fluid with a drencher f minutes after injection. ors contained per liter g nacl, . g kcl, . g nahco , and g glucose (calculated osmolarity mosmol/l). procain-penicillin g ( , iu/kg bw sc q h) was administered for days. additional blood samples were withdrawn from randomly chosen calves of each study , , and minutes after injection for assessing the serum concentration of sodium, potassium, and chloride (studies i and ii), and for determination of acid-base status (study ii). clinical examination was carried out , , , , , , , , and hours after the initial treatment. after each of these clinical examinations, blood was collected from the jugular vein to assess pcv, serum variables, and acid-base status. fecal grab samples (approximately ml) were taken after each clinical investigation and kept frozen at À c for subsequent dry matter (dm) determination. milk replacer h (mr; g/l) corresponding to % of bw per day was offered divided into meals ( : am, : am, : pm, : pm). ors was available in nipple buckets from : to : am, : to : pm, : to : pm, and : pm to : am, respectively. recumbent calves were encouraged to drink ors at least minutes at each time. intake of mr and ors was recorded. a treatment failure was defined as a deterioration of the general condition between consecutive clinical investigations during the -hour monitoring period. after the exclusion from the study, calves were treated by an established protocol, ie, a continuous infusion of an isotonic solution in the ear vein ( l of . % saline solution, l of % glucose solution, . l of . % bicarbonate solution). analyses pcv was measured with a hemogram analyzer. i venous blood ph, base excess (be), partial pressure of carbon dioxide (pco ), and standard bicarbonate (shco À ) were determined with a blood gas analyzer. j serum concentrations of total protein (tp), urea, sodium, chloride, and potassium were determined with an automatic analyzing system. k the concentration of l-lactate in plasma was measured by a quantitative enzymatic test. l after thawing of the fecal samples, their dm content was assessed by weighing the sample twice before and after the drying process ( c, hours). anion gap (ag) was calculated as agðmmÞ ¼ ðna þ ½mm þ k þ ½mmÞ À ðcl À ½mm þ shco À ½mmÞ: sigmastat . m was used for statistical analysis of the results. results not differing significantly from a normal distribution as indicated by the kolmogorov-smirnov test are presented as means and standard deviations; otherwise, they are depicted as medians with / quartiles. the significance of differences between the treatment studies i and ii was tested by a t-test and a rank-sum test, respectively. differences between points of time within a study group were checked by one-way repeated-measures analysis of variance (anova) and one-way repeated-measures anova on ranks, respectively. the significance of differences between proportion of treatment failures in studies i and ii was tested by fishers exact test. differences were classified as significant if p o . . infectious agents were found in the fecal smear of all calves investigated. e. coli (f ) were detected in calves ( %); mixed infections with either rotavirus and c. parvum or coronavirus and c. parvum were evident in calves ( %) and in calves ( %), respectively. exclusively, c. parvum were found in calves ( %). salmonellae were found in the feces of none of the calves. patients infected with e. coli (f ) were younger and the metabolic acidosis was not as severe as compared with the calves suffering from neonatal diarrhea caused by viral or cryptosporidium infections, or both (fig ) . the status of the calves before hypertonic rehydration indicated profound diarrhea, moderate to severe dehydration, moderate to severe depression, and a pronounced metabolic acidosis. hypertonic rehydration using saline (study i) was successful in of calves ( %). six calves had to be removed between and hours after injection. these treatment failures were on average days older and exhibited before treatment a more severe acidemia (ph: . ae . ; shco À : ae mm; be: À . mm [À . / À . ]) compared with successfully treated calves (ph: . ae . ; shco À : ae mm; be: À . mm [À . / À . ]); no further differences between successfully treated calves and treatment failures were found (table ) . hypertonic rehydration using bicarbonate solution (study ii) was successful in of calves ( %). the sole calf that was not successfully resuscitated with hypertonic sodium bicarbonate was removed hours after application of the hypertonic solution. this calf had severe metabolic acidosis (ph: . ; be: À mm) before treatment despite only slight dehydration ( % of bw, pcv . l/l; urea . mm). the proportion of treatment failures was , , and hours after injection significantly higher in study i (nacl) compared with study ii (nahco ) (p . ) after injection. no clinically important adverse effects occurred during the administration of hypertonic solutions. roughly % of the calves exhibited signs of discomfort and moderate to severe restlessness during the injection of the hypertonic solutions as indicated by twitching of the limbs, general shivering, and occasionally groaning. no obvious differences with respect to the extent of these clinical signs were observed between calves of study i (nacl) and study ii (nahco ); however, during application of hbs, the respiratory rate increased more profoundly up to roughly breaths per minute compared with the calves treated with hypertonic saline. these signs disappeared gradually within minutes after the injection in all calves. nine calves ( %) of study i (nacl) and calves ( %) of study ii (nahco ) drank voluntarily l ors within minutes after the injection of hypertonic solutions; the remaining calves received the ors by drenching. table . age, body weight (bw), clinical variables, and results of blood analysis before initial iv treatment of diarrheic calves with saline ( . %; ml/kg bw over minutes; study i) and sodium bicarbonate ( . %; ml/ kg bw over minutes; study ii), respectively, followed by administration of l of oral rehydration solution. means and standard deviations or medians and -/ -quartiles are depicted, respectively, for calves successfully treated and those for calves excluded from the study because of treatment failure. within a row, different superscripts indicate significant differences (po. ) between the basal values of successfully treated calves and treatment failures in study i (nacl). tp, total protein; pco , partial pressure of carbon dioxide; po , partial pressure of oxygen; be, base excess; shco , standard bicarbonate; ag, anion gap. the forthcoming comparisons of various variables obtained from the calves of study i (nacl) and study ii (nahco ) are carried out exclusively for only those calves that still remained in the study. thus, the higher percentage of treatment failures in study i (nacl) compared with study ii (nahco ) is not taken into account. the general condition of calves of both studies improved significantly within hours after injection from a mean clinical score of (moderate depression, sternal recumbency) to a mean clinical score of (slight depression, able to stand) ( table ). for successfully treated calves of both studies, the clinical status of the calves improved furthermore, resulting in an almost undisturbed general behavior in the majority of these calves - hours after injection. no significant changes of rectal temperature, heart rate, and respiratory rate were found during the investigation period of hours for calves of both studies (table ) . in both studies, concomitant with the improvement in the general condition, dehydration of the patients could be corrected within - hours after injection. the enophthalmus was significantly reduced already within - hours; a further, but slower improvement was obvious within the subsequent hours ( table ). the pcv decreased in calves of both studies within hours by % from . l/l to roughly . l/l. constant values were reached in both studies hours after injection ( table ). the serum concentration of tp decreased almost linearly in both studies within hours after injection from initially g/l by % to roughly g/l for the remaining investigation period. in both studies, urea concentration decreased from mm (studies i and ii) before administration of hypertonic solutions significantly until hours after injection to - mm (table ) . the metabolic acidosis of calves in study i was equalized gradually within roughly hours as indicated by an increase of blood ph from . ae . before saline application up to . ae . at hours after injection; the be was corrected within hours (À mm before saline application versus À mm hours after injection) ( table ) . a faster and more pronounced increase of plasma ph was observed in calves of study ii (nahco ). the effect of hypertonic bicarbonate application was assessed more in detail by evaluating the short-term effects in a subgroup of the patients ( table ). the results revealed that the calves became seriously alkalemic during the injection period; a ph . ae . and a be of ae mm were measured directly after the -minute injection period. however, this was a transient effect; already minutes later the plasma ph reached the upper limit of table . general condition, rectal temperature, enophthalmus, heart rate, respiratory rate, and fecal dry matter before and after initial iv treatment of diarrheic calves with saline ( . %; ml/kg bw over minutes; study i) and sodium bicarbonate ( . %; ml/kg bw over minutes; study ii), respectively, followed by administration of l of oral rehydration solution. number (table ) . although the plasma ph tended to decrease during the subsequent hours, no serious acidemia developed, as indicated by a ph of . ae . at the end of the investigation period ( hours after injection). the basal serum concentration of l-lactate decreased linearly in both studies within hours after injection by about % from . mm to approximately . ae . mm and remained at this level for the further investigation period (table ) . for calves of study i (nacl), serum sodium concentrations increased significantly from mean basal values of ae mm before saline application to ae mm measured hour after injection. in calves of study ii (nahco ), a massive transient hypernatremia was evident from the results of short-interval collection of samples in calves (table ). however, hours after application of hbs, the mean sodium concentrations were again in the physiologic range ( ae mm). for the subsequent hours, mean concentrations between and mm were found (table ) . serum chloride concentration increased in calves receiving hypertonic saline (study i) within minutes after injection significantly up to ae mm. thereafter, values within the reference range were found. in study ii (nahco ), no significant changes of serum chloride con-centration were observed after hypertonic rehydration (tables and ) . before hypertonic rehydration, hyperkalemia was found in calves of both studies ( mm). a distinct bradyarrhythmia because of hyperkalemia, however, was not detected (table ) . a gradual decrease to a level of about mm was observed within hours in both studies; during the subsequent hours, the serum concentrations remained in this range without significant differences between the calves of both studies ( table ) . intake of mr did not differ between the calves in both studies and tended to increase (p . ) from day to day after hypertonic rehydration (day : . ae . l; day : . ae . l; day : . ae . l [means ae sd]). also, intake of ors did not differ between the calves in both studies and tended to decrease (p . ) from day to day after initial treatment (day : . ae . l; day : . ae . l; day : . ae . l). thus, the amount of total fluid intake remained constant and averaged - l/d. in study i (nacl), the mean percentage of fecal dm remained within the investigation period without significant differences between and %. in study ii (nahco ), a significant difference from calves of study i (nacl) was evident on the rd day after initial treatment when fecal dm ranged between mean values of and % (table ) . table . serum concentrations of electrolytes and ph, partial pressure of carbon dioxide (pco ), base excess (be), and standard bicarbonate (shco ) in venous blood and within minutes after initial iv treatment of diarrheic calves with saline ( . %; ml/kg bw over minutes; study i) and sodium bicarbonate ( . %; ml/kg bw over minutes; study ii), respectively, followed by administration of l of oral rehydration solution. the objective was to evaluate the clinical efficacy of different hypertonic iv rehydration solutions in inappetent diarrheic calves. to prevent adulteration of positive effects of hypertonic rehydration by an insufficiency of other organs, patients with additional secondary infections were disregarded. further health problems can be explained partly by a long history of neonatal diarrhea, but also by a high proportion of hypogammaglobulinemia because of insufficient colostral supply among diarrheic calves. this is confirmed by low serum concentrations of tp after successful rehydration in both studies (table ) ; the catabolic state and intestinal protein losses during diarrhea may have had a further impact. many authors recommend hypertonic saline ( . %) with % dextran for rehydration. , , in the present study, a different concentration of saline had to be used ( . %); this solution is licensed and commercially available in germany, representing prerequisites for application in food animals. nevertheless, because ml/kg bw of this solution were administered, whereas ml/kg are recommended for higher concentrated saline ( . %), almost the same amounts of sodium ( . versus . mmol/kg bw) were given to the patients, ensuring comparability of the results. the hypertonic solutions used in the present studies did not contain dextran (not licensed in germany for food animals). addition of dextran induces a more pronounced expansion of plasma volume and a sustained effect compared with hypertonic saline without dextran because of the predominantly intravascular distribution of dextran with a water-binding capacity of roughly ml water per gram dextran . thus, a shorter effect of the hypertonic saline in study i on plasma volume compared with the studies carried out with . % sodium chloride in % dextran solution had to be expected. , continuous iv infusion of isotonic solution is the most effective and gentle approach in the treatment of severely diarrheic calves. hypertonic rehydration represents an alternative for rehydration in those patients in whom an infusion treatment cannot be carried out for any reason. the primary therapeutic approach is based on a transient replenishment of the intravascular volume from the intracellular and transcellular compartments caused by an increase of plasma osmolarity. thereby, the cardiovascular system is stabilized for - minutes. , , a sustained improvement of the clinical status, however, requires a significant oral intake of fluid containing water, electrolytes, buffer, and energy. in the present study, these principles worked successfully in % ( / ) of the calves of study i (nacl) and % ( / ) of the calves of study ii (nahco ). rehydration, accompanied by a marked improvement of the general condition, and a restoration of the suckling reflex were achieved within - hours; this period is comparable to successful treatments based on continuous infusion of isotonic solutions. the differences in the proportion of treatment failures between study i (nacl) and study ii (nahco ) can be explained predominantly by an advantage of bicarbonate solution in the treatment of diarrheic calves with a severe metabolic acidosis. no differences existed in the status of the calves before hypertonic rehydration between successfully treated calves and treatment failures, except for a more severe acidemia in the latter. calves that failed to respond were on average older than the successfully treated calves, which indirectly confirms the findings that diarrheic calves older than week of age exhibit a more severe acidemia than younger calves , (table ) . as an explanation, infections with rotavirus, coronavirus, or c. parvum induce an osmotic diarrhea, accompanied by microbial fermentation of substrates in the large intestine, leading to excessive production of predominantly d-lactate. calves treated with hbs (study ii) received a double load of sodium compared with calves in study i (nacl). sodium is regarded as the major determinant of rehydration success; however, no obvious differences were found with respect to the velocity of rehydration between calves of studies i and ii (table ) . thus, the concentration of sodium in the hypertonic solution had only a short, transient effect, whereas the final success of the rehydration treatment was determined predominantly by a sufficient intake of ors. unfortunately, the severe metabolic acidosis of recumbent diarrheic calves (be: À to À mm) may be slightly aggravated by application of saline-whether administered as a continuous infusion or as a hypertonic injection. saline creates a strong ion acidosis because the effective sid of calf plasma is meq/l, whereas the effective sid of saline is meq/l. the administration of ml saline ( . %)/kg bw within minutes decreased the plasma ph in swine with hemorrhagic shock and mixed acidosis by . ph units. this decrease has been considered to be clinically inconsequential, predominantly because an improvement of the blood ph is achieved by the intake of buffers from ors. in calves with a pre-existing severe acidemia, however, a further decrease of plasma ph because of hypertonic saline application may be detrimental. in that respect, the shortterm restoration of the suckling reflex is of crucial importance because intake of ors represents a prerequisite for successful rehydration treatment. a negative correlation has been suggested between the degree of the acidosis and the vigorousness of the suckling reflex. also, in our studies, a sustained improvement of oral fluid intake was achieved predominantly in those calves where the acidemia could be corrected quickly. in most calves where hypertonic rehydration failed, the general condition deteriorated, concomitant with a reduction of oral fluid intake and in the face of a prolonged acidemia. the concentrations of d-lactate were most likely massively increased in the diarrheic calves studied compared with healthy calves as indicated by profoundly increased ag (table ) . such increased concentrations of d-lactate contribute significantly to weakness and disturbed consciousness , but they do not seem to influence the suckling reflex. the administration of hbs represents a therapeutic option to correct the metabolic acidosis faster than by hypertonic saline. even calves with a be of À to À mm were treated successfully and plasma ph increased already within hours up to physiologic values. in calves, ml/kg bw bicarbonate solution ( . %) provides a buffer capacity sufficient to equalize a be of about À to À mm. , because of the high effective sid ( meq/l), a strong ion alkalosis is induced and was measured in our calves (table ). double the amount of sodium compared with ml saline ( . %)/ kg bw was administered, leading to the question of whether the application of ml bicarbonate solution ( . %)/kg bw within minutes is more hazardous for the patient than hypertonic saline. three issues have to be taken into account: ( ) the buffering capacity of bicarbonate requires an effective removal of co by the respiratory system. the tachypnea being more pronounced in calves receiving hypertonic bicarbonate than saline is most likely because of the hypercapnia (table ). in study ii (nahco ), several calves with only slight metabolic acidosis were treated, which did not require buffer capacity at all. nevertheless, also these patients had had no problem in coping with the application of hypertonic bicarbonate by enhancing ventilation. even newborn calves with mixed respiratory acidosis because of dystocia (mean blood ph . ) were successfully treated by infusion of on average ml sodium bicarbonate solution ( %) over minutes, combined with doxapram iv and intranasal oxygen insufflation ; no negative effects compared with calves treated with carbicarb were found. also, rapid iv administration of hbs ( . %; ml/kg over minutes) was found to be effective and safe for treating strong ion acidosis in normovolemic calves with experimentally induced respiratory and strong ion acidosis. in these studies, however, lower amounts of bicarbonate were infused than in study ii presented here. many calves, especially with a longer history of diarrhea, also suffer from respiratory infections. it remains unknown whether removal of excessive co after application of hbs by hyperventilation is possible in such patients. reports of practitioners about sudden fatalities in calves after hypertonic bicarbonate infusion may be an indication that such incidents could be related to respiratory insufficiencies. at present, it is recommended to avoid the application of hypertonic bicarbonate in calves suffering from severe respiratory disease, whereas hypertonic saline can also be applied in such patients. ( ) especially in patients with an impaired ability to remove carbon dioxide, a pia after rapid injection of large quantities of bicarbonate has been discussed as a matter of concern. accordingly, bicarbonate ions react with h ions to form co , which readily enters cells and overwhelms the blood-brain barrier because of its high solubility. thus, the intracellular pco increases, which induces a local respiratory acidosis. the clinical consequences are unclear; it has been postulated that an acidification of the cerebrospinal fluid (csf) may exaggerate a depression and increase the respiratory rate. a pia has been demonstrated in in vitro studies after increasing the extracellular pco by approximately mmhg. , however, in vivo the pco rarely increases more than mmhg. it has been suggested that the increase of pco after bicarbonate application can be aggravated by the concomitant release of protons from extracellular nonbicarbonate buffers in severely acidotic patients. berchtold et al induced experimentally a mixed respiratory and metabolic acidosis in calves and found, after subsequent injection of bicarbonate solution ( . %, ml/kg bw over minutes), no ph change in the csf irrespective of a transient increase of arterial pco from to mmhg. a turnover rate of co into the csf of . minute À was calculated. in our patients, a statistical trend (p . ) was found for the relation between plasma ph before injection of hypertonic bicarbonate and the increase of venous pco after injection (table ). however, pco increased only by on average mmhg directly after the initial treatment. thus, pia seems not to be a reason for profound problems after hypertonic bicarbonate application in calves with a healthy respiratory tract. ( ) concerns were expressed with respect to iv treatment of diarrheic calves with bicarbonate solutions because the concomitant hypercapnia and removal of metabolic acidosis may worsen the oxygen extraction in peripheral tissues of such patients. based on the results presented here, these concerns seem to be inappropriate despite an extensive transient metabolic alkalosis immediately after injection of hbs (table ) , which can be explained by incomplete distribution of bicarbonate in the extracellular space. the general condition of most calves that received bicarbonate solution improved rapidly-presumably, the improved blood flow because of increased plasma volume overwhelms the negative impact of a left shift of the oxygen equilibrium curve. because oral fluid intake is of crucial importance for the success of hypertonic rehydration, an intensive fosterage of the calf after initial treatment represents a prerequisite. the recovery of critically ill patients is undoubtedly facilitated by providing many small meals. the variation of milk as well as ors intake during each meal after hypertonic rehydration is high. thus, calves in studies i and ii were fed times per day. moreover, a high feeding frequency reduces the risk of long periods with a low ph in the abomasum, favoring the development of abomasal ulcerations. hypernatremia (serum sodium mm) is a dreaded syndrome in diarrheic calves ; it is often because of oral application of ors with an inadequate composition (sodium mm). the results of the present study indicate that a hypernatremia was not a problem in the hypertonic-rehydrated calves. hypertonic saline ( . %, ml/kg) and hypertonic bicarbonate ( . %, ml/kg) correspond in a kg calf to a total of only and mmol sodium, respectively. serum sodium increased immediately after completing the injection of hbs by mm, triggering an increase of plasma osmolarity of roughly - mosmol/l; an increase in this range has also been reported. the immediate dilution of extracellular compartment by an influx of intracellular and transcellular fluid excludes the risk of a clinically significant hypernatremia. however, hypertonic rehydration is inappropriate in calves that already suffer from hypernatremia before the initial treatment. in conclusion, hypertonic rehydration represented a safe and reliable method to improve the hydration status of inappetent diarrheic calves. hypertonic saline seems to be most appropriate for calves within the st week of life as most of these patients suffer from a slight to moderate metabolic acidosis (be À mm). hbs allows successful treatment of diarrheic calves even with severe metabolic acidosis (be - to - mm) frequently found in diarrheic calves older than week. diarrheic patients also suffering from respiratory disease should not be treated with hbs. a high oral fluid intake is a precondition for successful hypertonic treatment requiring an adequate fosterage. intravenous fluid therapy of calves the treatment of the diarrheic calf: an update hemodynamic response of endotoxemic calves to treatment with smallvolume hypertonic saline solution fluid resuscitation with hypertonic saline in endotoxic shock small-volume resuscitation with hypertonic saline dextran solution resuscitation with . % nacl in % dextran- during hemorrhagic shock in swine: effects on organ blood flow haemodynamic effects of small volume hypertonic saline in experimentally induced haemorrhagic shock hyperosmotic sodium salts reverse severe hemorrhagic shock: other solutes do not small-volume resuscitation with hypertonic saline solution in hypovolemic cats use of hypertonic saline-dextran solution to resuscitate hypovolemic calves with diarrhea comparison of hypertonic saline-dextran solution and lactated ringer's solution for resuscitating severely dehydrated calves with diarrhea severity and nature of acidosis in diarrheic calves over and under one week of age metabolic acidosis in calves untersuchungen zur klinischen einscha¨tzung des azidosegrades von ka¨lbern mit neugeborenendurchfall investigations on the association of d-lactate blood concentrations with the outcome of therapy of acidosis, and with posture and demeanour in young calves with diarrhoea d-lactate production and excretion in diarrheic calves hypertonic saline effect of infusion of hypertonic saline solution of conscious heifers with hypoxemia caused by endotoxin infusion effects of intravenous hyperosmotic sodium bicarbonate on arterial and cerebrospinal fluid acid-base status and cardiovascular function in calves with experimentally induced respiratory and strong ion acidosis clinical and laboratory assessment of hydration status of neonatal calves with diarrhea use of a quantitative strong ion approach to determine the mechanism for acid-base abnormalities in sick calves with or without diarrhea colostrum management in calves: effects of drenching vs. bottle feeding a comparison of several hypertonic solutions for resuscitation of bled sheep fluid and electrolyte therapy in ruminants both l-and d-lactate contribute to metabolic acidosis in diarrheic calves hypertonic saline-dextran resuscitation from hemorrhagic shock induces transient mixed acidosis anion gap correlates with serum d-and dl-lactate concentration in diarrheic neonatal calves investigations of d-lactate metabolism and the clinical signs of d-lactataemia in calves comparison of sodium bicarbonate and carbicarb for the treatment of metabolic acidosis in newborn calves initial effect of sodium bicarbonate on intracellular ph depends on the extracellular nonbicarbonate buffering capacity hydrogen ion homeostasis, tissue oxygenation and their disorders bicarbonate therapy and intracellular acidosis effects of intravenous infusions of sodium bicarbonate on blood oxygen binding in calves with diarrhoea effect of feeding frequency and route of administration on abomasal luminal ph in dairy calves fed milk replacer key: cord- - brqzyn authors: murphy, jacob m.; hagey, jill v.; chigerwe, munashe title: comparison of serum immunoglobulin g half-life in dairy calves fed colostrum, colostrum replacer or administered with intravenous bovine plasma date: - - journal: vet immunol immunopathol doi: . /j.vetimm. . . sha: doc_id: cord_uid: brqzyn in calves, passive immunity of immunoglobulins can be acquired through ingestion of colostrum or colostrum replacers. plasma can been used to supplement immunoglobulins in healthy or sick calves. serum half-life of colostral derived immuglobulin g (igg) is estimated to be days. half-life of igg is important in determining response to antigens and timing of vaccination in calves. to date studies evaluating half-life of colostrum replacer or plasma derived igg are lacking. the objectives of this study were to compare the serum half-life of igg derived from colostrum, colostrum replacer and plasma in dairy calves reared up to days of age. thirty jersey calves were randomly assigned to receive colostrum or colostrum replacer by oroesophageal tubing or plasma by intravenous administration. serum samples were collected at , , , , , , and days. serum igg concentrations were determined by radial immunodiffusion. the results indicated that half-life for igg in colostrum fed ( . days) or plasma transfused calves ( . days) was longer than colostrum replacer fed calves ( . days). further studies are required to evaluate pathogen specific immunoglobulins in order to recommend vaccination timing in calves fed colostrum replacers. the cotyledonary type placenta restricts transfer of immunoglobulins from cow to the fetus during pregnancy (arthur, ) . consequently calves are born hypogammaglobulinemic, thus making it essential for calves to ingest and absorb colostral immunoglobulins to acquire passive immunity. half of all neonatal mortality can be directly attributed to failure to acquire passive immunity at birth (tyler et al., ) . acquiring of passive immunity through ingestion and absorption of colostral immunoglobulins at birth in calves can be achieved by feeding colostrum or colostrum replacers. colostrum replacers are used on dairy farms when sufficient colostrum is not available or to prevent transmission of diseases that can be transmitted through unpasteurized colostrum (foster et al., ) . in veterinary clinical settings, plasma has been used to supplement immunoglobulins in clinically healthy or sick neonatal calves with or without adequate ingestion of colostral immunoglobulins (selim et al., ) . colostral derived immunoglobulins can suppress neonatal immunity by various mechanisms (banks, ) . previous studies demonstrated that colostrum deprived calves produced igg and iga earlier than colostrum fed calves (husband and lascelles, ) . the mechanism of suppression of the immune system by the colostralderived immunoglobulins can be either antigen specific or non-antigen specific (banks, ) . as a result of this immune system suppression, concentrations of colostral derived maternal immunoglobulins must be reduced to a critical low level that does not interfere with response to antigen exposure or vaccination (pastoret, ) . the rate at which this critical low level of immunoglobulins is achieved is dependent on the immunoglobulin concentration and immunoglobulin class absorbed at birth because each class of immunoglobulin is catabolized at a different rate. the half-life of each immunoglobulin can be used to estimate the rate of its catabolism. the serum half-life of colostral-derived immuglobulin g (igg), m (igm), and a (iga) in calves was , . , and . days, respectively (banks, ) . in cattle, the predominant colostral immunoglobulin ( - %) is igg while igm and iga constitute approximately % and % respectively (larson et al., ) . to the best of authors' knowledge, no studies have evaluated the half-life of colostrum replacer or plasma derived igg in dairy calves. we hypothesized that the half-life of maternally derived colostral igg will have a significantly longer half-life compared to colostrum replacer or plasma derived igg. the objective of this study was to compare the serum half-life of igg derived from colostrum, colostrum replacer and plasma in dairy calves reared up to days of age. the results of the study were intended to evaluate if different timing of vaccination should be considered when vaccinating colostrum fed calves as opposed to colostrum replacer fed dairy calves or plasma transfused calves. additionally, information on serum half-life of plasma-derived igg would help predict when to repeat plasma transfusion in clinical settings. the study was approved by the university of california, davis (uc davis) institutional animal care and use protocol (protocol number ). sample size calculation was based on a % mortality rate attributed to calves not ingesting sufficient colostrum (tyler et al., ) , alpha of %, power of % and a mortality rate of up to % during the first weeks of life in calves failing to ingest sufficient colostrum at birth, in california (moore et al., ) . the total sample size required was calves. in order to account for a % dropout due to missed sampling, calves were enrolled. all calves were enrolled from a single jersey dairy farm in hilmar, california (merced county). adult cows on the farm of study were vaccinated annually with a multivalent modified live respiratory vaccine containing infectious bovine rhinotracheitis, bovine viral diarrhea, parainfluenza- and bovine respiratory syncytial viruses and dewormed twice a year with topical ivermectin. additionally, the cows were vaccinated with a multivalent vaccine containing escherichia coli, rotavirus and coronavirus during the dry cow period. thirty jersey bull calves delivered from eutocia and observed births were immediately separated from the dam following parturition. the calves were identified using plastic ear tags and weighed. calves were randomly assigned to receive pooled, pasteurized colostrum from the farm of study or colostrum replacer (the saskatoon colostrum co, saskatoon, canada) or bovine plasma (transfusion services, university of california, davis, ca). colostrum was pasteurized using a batch pasteurizer at • c for min with continuous agitation followed by cooling at • c. all calves received colostrum or colostrum replacer or bovine plasma within h after birth. the bovine plasma was derived from clinically healthy, uc davis veterinary medical teaching hospital blood donor cows. the plasma was evaluated for sterility and considered to be free of transmissible blood borne pathogens. the plasma from the blood donor cows was not pooled. serum samples were collected from all calves prior to any procedure for subsequent serum igg determination. an aliquot ( ml) of colostrum or plasma to be administered was collected prior to administration for subsequent igg concentration determination. no samples were collected from the colostrum replacer because the igg concentration was known according to the manufacturer's label. all calves were enrolled into the study over a -week period. calves in the colostrum (cl, control group) group were fed l of colostrum once by oroesophageal tubing. calves in the colostrum replacer (cr) group were fed . l ( bags) of colostrum replacer, once, according to the manufacturer's recommendations delivering g of igg for absorption by oroesophageal tubing. calves in the plasma (pl) group were administered with bovine plasma at ml/kg (barrington and parish, ) through an intravenous catheter (becton and dickinson co, franklin lakes, nj) aseptically placed in the external jugular vein. infusion of plasma was performed slowly ( ml/kg/h) over the first min. monitoring for transfusion reactions included monitoring heart rate, respiratory rate, color of mucous membranes, and abnormal behavior. in the absence of an immediate transfusion reaction, the remainder of the plasma was transfused over - min. in the presence of a plasma transfusion reaction, transfusion was discontinued for min and resumed at ml/kg/h. the calves were then housed in individual plastic calf hutches at the uc davis beef research facilities. calves were fed l of non-medicated milk replacer (calva products, acampo, ca) and . kg of commercial calf concentrate (department of animal science, university of california, davis, ca) twice daily. at weeks of age, calves were fed l of milk replacer and kg of commercial calf concentrate, twice daily. serum samples were collected at , , , , , , and days of age. all calves were weighed at the end of the study ( weeks of age). samples of serum collected at different time points, colostrum, and plasma administered were stored at − • c until serum igg determination. calves were monitored for health by daily evaluation of appetite, rectal temperature, signs of coughing, diarrhea and lameness. sick calves were treated following recommendations by a uc davis licensed veterinarian as per the uc davis institutional animal care and use protocol. calves that died were submitted for a complete necropsy at the california animal health and food safety laboratory in davis, ca. serum, plasma or colostral igg concentrations were determined using a commercial radial immunodiffusion (rid) kit based on the manufacturer's recommendations (triple j farms, bellingham, wa). briefly, rid plates containing specific anti-bovine igg, agarose gel, . m phosphate buffer ph . , . % sodium azide as a bacteriostatic agent and g/ml amphotericin b as a fungal agent stored in a refrigerator at • c were warmed at room temperature ( - • c). an aliquot ( l) of the provided reference serum at different concentrations were pipetted into each rid wells. an aliquot ( l) of serum (diluted : with phosphate buffer) or colostrum samples (diluted : with phosphate buffer) were pipetted into individual rid plate wells. the plates were incubated at room temperature ( - • c) for h. the diameters of the zones of precipitation were measured using a digital rid plate reader (the binding site inc, san diego, ca) after h. serum or colostral sample igg concentrations were determined by comparing the diameter of the zones of precipitation with a standard curve generated by the reference serum. the regression equation generated in this manner (r = . - . ) accurately predicts inoculum igg concentration. minimum detectable serum igg concentration was mg/dl using the rid. for the purposes of this study, calves determined to have < mg/dl of igg by rid were considered to have mg/dl igg. normality of data was checked using the shapiro-wilk test. in instances where the data was not normally distributed, the median was reported. descriptive statistics were calculated for calf birth weight, -week weight, serum igg concentrations at days, igg concentrations in plasma administered and colostral igg concentrations, morbidity and mortality events among the groups. serum igg concentrations at days were used to compare the presence of failure of passive transfer of immunity among the groups using a kruskal-wallis, -way analysis of variance on ranks. calves with serum igg concentrations of < mg/dl were considered to have failure of passive immunity (besser et al., ) . proportions of calves with failure of passive transfer of colostral immunoglobulins at days or calves that died among the groups were compared using a test or fisher's exact test when a cell had less than counts. in calves that received plasma transfusion, the predicted post-transfusion serum igg concentration was calculated using the following formula (chigerwe and tyler, ) : where weight = mean weight (kg) of the calves in the pl group; . l plasma/kg = plasma volume in jersey calves (quigley et al., ) ; igg = serum igg concentration (mg/dl) prior to administration of plasma; igg in plasma = mean igg concentration (mg/dl) in the plasma administered; and plasma volume = mean volume (l) of plasma administered iv at ml/kg. serum half-life for igg in each of the groups of calves was determined by a non-linear regression analysis using a one-phase exponential decay model with random effects for calf initial serum igg concentrations. the decay of igg over time was assumed to be constant. differences in serum igg half-life among the groups were evaluated by comparing the rate constants using an f-test. in all statistical analyses values of p < . were considered significant. all statistical analyses were performed using a commercial statistical software (prism , graphpad inc, la jolla, ca). mean ± sd birth weight for all calves was . ± . kg. mean ± sd birth weight for the cl, cr and pl group was ± . , . ± . and . ± . kg, respectively. all calves had a serum igg concentration of < mg/dl prior to feeding colostrum or colostrum replacer or administration of plasma. mean colostral igg concentration fed to the cl group was . g/l. mean plasma volume and mean plasma igg concentrations administered to the calves in the pl group were ml and . mg/dl, respectively. none of the calves in the pl group showed immediate transfusion reactions. all calves in the cl and cr group had adequate transfer of immunity (serum igg concentrations > mg/dl at days of age). median (interquartile range) serum igg concentrations at days of age for calves in the cl, cr and pl groups were . ( . ), ( . ), and . ( . ), respectively. only calf in the pl group had serum igg concentrations > mg/dl. there was no difference in median serum igg concentrations at days between calves in the cl and cr groups (p = . ). median serum igg concentrations of calves in the pl group were lower than calves in both cl (p < . ) and cr (p < . ) groups. using eq. ( ) and substituting for a mean weight of . kg, serum igg concentration of mg/dl prior to administration of plasma, mean plasma volume of ml ( . kg × ml/kg) administered, mean plasma igg concentration of . mg/kg, the expected serum igg concentration at days of age in the pl group was . mg/dl. morbidity events were recorded in calves ( % with diarrhea cases and pneumonia case). fourteen of the morbidity cases in the calves required treatment with antibiotics and or oral rehydration electrolyte solutions. six calves died during the -week period. of the calves that died or were euthanized, calves were from the pl group, from the cl group and from the cr group. mortality in the calves occurred during the first days of life. causes of mortality in the calves included enteritis secondary to enterotoxigenic e. coli, rotavirus and cryptosporidium in calves, enterocolitis due to clostridium perfringens in calf and enterocolitis due to e. coli, rotavirus and bovine viral diarrhea virus (bvdv) in calf. given that calf tested positive for bvdv on necropsy, all remaining calves ( calves) calves were tested for bvdv by immunohistochemistry on ear notch skin biopsy. all remaining calves tested negative for bvdv. proportions of calves with failure of birth weight was reported as mean ± sd. mean colostrum igg fed to the cr group was reported as g/l and grams of igg in the cr group. mean igg concentrations transfused to the pl group was reported as mg/dl. median serum igg concentration was reported as median (interquartile range). half-life of igg was reported as days ( % ci). where applicable, values with the same letter superscript within a given row are not different (p > . ). passive transfer between the cl and cr groups were not different (p = ). proportions of calves that died in the pl group were higher compared with the cl (p < . ) and cr (p < . ) groups. mean ± sd weight for the remaining calves at weeks was . ± . kg with a daily average gain of . kg/day. half-life ( % confidence interval) for colostral, plasma and colostrum replacer derived igg was . ( . , . ), . ( . , . ) and . ( . , . ) days, respectively. the half-life for the cl and pl was not different (p = . ). the half-life for the cl (p < . ) and pl (p < . ) groups was longer than that of the cr group. summarized descriptive statistics results are represented in table . the decay curves generated for the groups of calves are represented in fig. . the aim of this study was to compare serum half-life of igg derived from colostrum or colostrum replacer and plasma. the main finding in this study demonstrated that half-life of colostral derived igg was longer than colostrum replacer derived igg. although these study results suggest different timing when vaccinating colostrum or colostrum replacer fed dairy calves, the current studies did not evaluate specific pathogen immunoglobulins to common vaccinations in adult dairy cows and pre-weaned dairy calves. the specific immunoglobulins for pathogens to be considered in future studies include rotavirus, coronavirus, infectious bovine rhinotracheitis, bovine viral diarrhea, parainfluenza- and bovine respiratory syncytial viruses. while the specificity of the colostral-derived immunoglobulins is important to confer specific immunity, it should be noted that at birth calves have greater number of neutrophils compared to adult cattle (rossi et al., ) . primary and secondary lymphoid tissues of newborn calves are populated by immune cells and the circulatory b-lymphocytes can synthesize immunoglobulins following antigenic stimulation (banks, ) . thus passively acquired igg are not the only immunoglobulins involved during conference of immune protection after birth; but cells from lymphoid tissues and neutrophils from circulation are also involved. therefore lack of specificity does not necessarily result in occurrence of a morbidity event. it is important to note that the half-life of colostral derived igg was longer than previously reported in other studies (banks, ; butler, ; besser et al., ) . previous studies reported igg half-life of days (banks, ) , . days (besser et al., ) , . days for igg (butler, ) and . days for igg (butler, ) in calves. while the age of the calves during determination of half-life was not stated in two of the studies (banks, ; butler, ) , serum collection in calves was performed until days of age in the third study (besser et al., ) . we chose to determine serum igg half-life in calves reared up to days of age based on previous studies indicating that antibodies to different bacterial and viral antigens were not produced or did not appear in serum until - days of age (kerr, ; lambert et al., ; thorsen et al., ; husband and lascelles, ) . thus, it can be assumed that the majority of immunologlobulins in the serum are colostral derived in calves aged up to days. the difference in the duration of follow-up is a potential explanation for the difference in the serum igg half-life determinations in the different studies. majority of calves in the pl group were anticipated to achieve adequate transfer of immunoglobulins at days of age based on eq. ( ) and recommended plasma transfusion rate, but only one calf achieved adequate transfer of immunoglobulins. in contrast, the cl and cr group achieved serum igg consistent with adequate transfer of immunity. one explanation for this observation is possible; increased catabolism due to complement activation by igg aggregates from the preparation process of the plasma (lundblad and londeree, ) . in contrast, absorption of orally delivered igg occurs within the first - h of birth is a non-selective passive process through pinocytosis (stott et al., ) . thus, orally derived igg may undergo catabolism through which intravenously derived igg is catabolized. intravenously derived immunoglobulins are resecreted into the gastrointestinal tract and excreted through the feces and urine (besser et al., ) . fecal or urinary igg concentrations were not determined in this study to evaluate rate of excretion among groups. future studies need to determine serum, fecal and urinary igg concentrations in calves intravenously transfused with plasma at shorter intervals (for instance every h) prior to h of age in order to evaluate the catabolism of the immunoglobulins. additionally, evaluation of activity of igg prior to and after plasma transfusion is warranted. the study design in this study differs from previous studies (besser et al., ; jones et al., ) in several ways. in studies by besser and others, calves only received variable volumes of colostrum followed by . ml of labeled sodium iodide intravenously with no group of calves receiving colostrum replacer or plasma. in studies by jones and others fed colostrum was twice to calves while the colostrum replacer was bovine serum derived and the study design did not include a group of calves administered intravenous plasma. it should be noted that in clinical practice, plasma is used to supplement immunoglobulins and thus calves are more likely to have ingested insufficient or sufficient colostrum on presentation. in this study, the pl group only received plasma as the source of immunoglobulins. the results of this study indicate that plasma products with sufficient immunoglobulins may fail to achieve adequate immunity when administered in calves. although the half-life of the pl was longer than the cr group, the % confidence for the half-life estimate is very wide indicating that the estimate is not precise. thus it is difficult to recommend when to repeat plasma transfusions in clinical settings based on the results of this study. the results of this study indicated that colostral derived igg had a longer half-life compared to colostrum replacer derived igg. future studies are required to evaluate pathogen specific immunoglobulins in order to recommend timing of vaccinations in calves fed colostrum replacer at birth. administration of plasma to newborn calves at the recommended dose did not achieve adequate transfer of immunoglobulins. calves intravenously administered with plasma at birth were more likely to experience mortality compared to calves that ingested colostrum or colostrum replacer. the development of the conceptus host defense in the newborn animal ruminant immunodeficient diseases transfer of functional immunoglobulin g (igg) antibody into the gastrointestinal tract accounts for igg clearance in calves comparison of three methods of feeding colostrum to dairy calves bovine immunoglobulins: an augmented review serum igg concentration after intravenous serum transfusion in a randomized clinical trial in dairy calves with inadequate transfer of colostral immunoglobulins serum igg concentrations in dairy calves fed two colostrum replacement products antibody responses to neonatal immunization in calves influence of pooled colostrum or colostrum replacement on igg and evaluation of animal plasma in milk replacer active immunity experiments in very young calves experimental bovine viral diarrhea in neonatal calves immunoglobulin production and transport by the mammary gland the effect of processing methods on intravenous immune globulin preparations influence of arrival weight, season and calf supplier on survival in holstein beef calves on a calf ranch in california challenge and issues of early vaccination in animals and humans estimation of plasma volume in holstein and jersey calves kinetics of detection of blastogenic responses of neonatal responses of neonatal calves inoculated in utero with tetanus toxoid, killed mycobacterium bovis, and killed brucella abortus passive immunotherapy in neonatal calves -ii. the efficacy of a j escherichia coli hyperimmune plasma as immunotherapy in neonatal calves colostral immunoglobulin transfer in calves. i. period of absorption bovine parainfluenza vaccine studies a model partitioning the risk of mortality associated with inadequate passive transfer in dairy calves the authors have no conflict of interest to declare. this research was partly funded by the uc davis student training in advanced research. the authors would like to thank sharon kim, peony kim and dr. elizabeth adams for their assistance. key: cord- -oe yv y authors: dutta, ritaban title: replacement management in cattle: health management date: - - journal: reference module in food science doi: . /b - - - - . - sha: doc_id: cord_uid: oe yv y replacements are the future of the dairy industry. focusing on improving health management of replacements will yield tremendous returns through decreased losses of animals with the greatest genetic potential on the dairy, decreased costs of medication, improved growth rates, improved feed efficiency and earlier entry into the milking herd. health management begins before replacements are born with attention to the nutrition of lactating and dry cows, the vaccination of lactating and dry cows, control of length of the dry period and both control of the disease status of the dams and the cleanliness of the calving environment. greater attention must be paid to animal and environmental biosecurity to prevent introduction of diseases into the herd and to digestive disorders such as diarrhea, internal parasites and appropriate vaccination programs for the calves. health management of replacements is often overlooked because producers do not see the immediate returns for their efforts. common sense management in cattle, historical facts, experience based practice cultural and social aspects, combined with research, would depict that having adequately optimised balanced diets for the replacements, without producing excessive body conditions, could achieve a production of healthy replacements with superior levels of milk production. continual video monitoring of the herd, modern thermal infrared imaging of the dry cows and calves body parts to identify early symptoms, and overall animal health and biosecurity risk analysis could achieve a sustainable and efficient replacement management practice in cattle industry. replacements are the future of the dairy industry. focusing on improving health management of replacements will yield tremendous returns through decreased losses of animals with the greatest genetic potential on the dairy, decreased costs of medication, improved growth rates, improved feed efficiency and earlier entry into the milking herd. health management of dairy replacements begins before the replacements are born. several factors, such as nutrition of lactating and dry cows, vaccinations of lactating and dry cows, length of dry periods, cleanliness of the calving environment and disease status of the dams, will ultimately affect disease resistance and health of replacements. it is important to note, however, that cows that are overfed tend to have difficulty calving because of being excessively over conditioned. cows that are underfed, which results in mineral or vitamin deficiencies or lack of body condition, may produce inferior and low-volume colostrum. they also may experience difficulty calving. protein deficiency in cows during the dry period may lead to low birth weights, low metabolic rates and poor vigor of calves, resulting in poor survivability. some research also indicates that inadequate protein and energy nutrition of the dam results in poor absorption of immunoglobulins from colostrum by the calf. cows that lose condition during the dry period are also at greater risk of experiencing calving difficulty. calves that experience difficult births require more time before being able to stand, experience an increase in the time to voluntary suckling and have a decreased ability to absorb immunoglobulins. all these problems result in decreased transfer of passive immunity from the dam to the calf and increased risk of disease in calves. as the degree of calving difficulty increases, the risk of mortality for calves increases. proper nutrition of dairy cows during lactation and the dry period will help decrease disease risks for replacements. in recent times researcher are employing electronic cattle tags to remotely monitor cows' general health conditions correlated with the movements of the head and other body parts. some farming communities apply more direct video surveillance mechanism to monitor milking herd to prevent any possible disruption in milk production by taking care of early animal health symptoms and unexpected animal behaviours. intelligent processing of video stream, combined with in-situ visual observations by a farmer could provide a better management culture in replacement management. low cost thermal imaging cameras could also be deployed to monitor the cows and calves body temperature, environmental conditions, and any unusual spots in a herd that has significantly higher temperature than normally expected. early prevention of any bacterial and viral disease spread is extremely important for precalving and calving management. this issue has become ever more important in today's world, where market accessibility for a local producer of milk and dairy products, could be global. bigger market access and business proposition comes with a significantly higher level of responsibility. zero tolerance attitudes towards animal and food biosecurity are key to greater market access and business sustainability. greater awareness is needed to achieve a standard biosecurity management in cattle industries. informed decision on planned vaccinations of the dams will also impact disease resistance of dairy replacements and a better preventive management. proper vaccination of the dairy herd will increase the concentration of antibodies (immunoglobulins specific for diseases) in colostrum. dams may be vaccinated during the dry period against pathogens that are common causes of diarrhea in calves, such as escherichia coli, rotavirus and coronavirus. vaccination of the dams increases the concentration of antibodies against these pathogens in colostrum, thus providing increased protection for calves, resulting in decreased incidence or duration of diarrhea. vaccination of the dams during the dry period is more effective for prevention of disease in calves than vaccination of calves at an early age. the immune system of neonatal calves is unable to respond quickly to a vaccination or an infection because the immune system of the newborn is immature at birth. both numbers and effectiveness of antibody-producing cells are lower in calves at birth than in adult cattle. therefore, it is important for calves to obtain antibodies against common diseases of calves by consumption of colostrum rather than from an attempt to vaccinate calves at an early age. vaccination of the dams against pneumonia may also help to decrease the incidence or severity of this disease in replacements. another important factor that may affect the health of replacements is the length of the dry period of the dam. a dry period that is too short, i.e. less than weeks, may not provide enough time for involution of the mammary gland and preparation for the next lactation. cows with shortened dry periods produce small quantities of colostrum that may also have low concentrations of immunoglobulins. it is important for health of replacements, therefore, that cows have at least a -week dry period for production of high-quality colostrum. management of the calving environment has a tremendous impact on the health of replacements. it is important for calves to be born in a clean, dry environment. wet, sloppy stalls provide a perfect environment for growth of bacteria. calving on a grass lot may be the best alternative when the climate is dry and mild. if a maternity barn is used, it is important to clean stalls thoroughly between calvings to prevent transfer of disease. maternity stalls should only be used for calving and never for housing sick cows. maternity pens and sick pens should be kept in separate facilities in order to prevent transfer of disease to highly vulnerable neonates and periparturient dairy cows. it is also important for the cows to be as clean as possible at calving in order to prevent calves from contracting disease organisms when suckling or attempting to suckle their dams. preferably, calves should be separated from dams prior to suckling in order to prevent the calf from ingesting pathogens present on the legs, belly, flanks or udder of the cow as the calf attempts to nurse. separating the calf from the dam and feeding colostrum by bottle also ensures adequate intake of colostrum for transfer of passive immunity from the dam. finally, it is important to know the disease status of cows prior to calving. diseases such as johne's disease, milk fever (hypocalcaemia) close to calving, bovine viral diarrhea (bvd), and bovine leucosis virus (blv) may be passed in utero or through colostrum. calves should only be fed colostrum from cows known to be free of these diseases. it is important, therefore, to maintain a supply of frozen, high-quality colostrum from cows free of such diseases. today's cost could be tomorrow's gain, so conservativeness towards deploying new technologies in replacement management could be addressed by community based farming, to try new practice, to tackle ever growing biosecurity related threats in dairy industries. the importance for baby calves of adequate consumption of immunoglobulins from colostrum has been reviewed elsewhere. mortality resulting from lack of consumption of adequate amounts of immunoglobulins is commonly greater than % and has been reported to be as high as %. others have indicated a -fold increased risk of mortality when calves do not consume colostrum. % of the hypocalcaemia cases occur within day of calving, because extremely high rate of drain calcium drainage (and other substances) from the blood during the milk and colostrum production, which cattle bodies are unable to replace quickly enough, bring severe health risk and subsequent economic loses. along with economic losses from high mortality rates as a result of lack of colostrum consumption, there are also increased costs associated with increased medication and poor feed efficiency. transfer of passive immunity (absorption of immunoglobulins from colostrum) can be determined using commercial kits that measure immunoglobulins in the blood. for adequate protection of calves, blood immunoglobulin concentrations should be at least mg ml À . serum protein concentrations in calves are also highly correlated with the concentration of immunoglobulins in blood and can be used to determine adequate transfer of passive immunity. a hand-held refractometer can be used to measure serum protein; levels greater than . g ml À by h of age indicate adequate consumption of colostrum. the similar technology could be used to measure 'specific gravity of urine' and 'refractive index of serum', as researchers are aiming to combine these factors for better diagnosis. the use of colostrum substitutes and replacers may help improve disease resistance in calves when high-quality colostrum is not available. it's also important to have a better feeding management plan for the dry cows weeks before the calving, as this effects the amount of calcium available to replace blood calcium, and how efficiently body can utilize the available calcium at a very critical period. the most prevalent health problem of calves on most farms in the united states is diarrhea. organisms such as cryptosporidium parvum, rotavirus and coronavirus that cause diarrhea will not respond to antibiotic treatment. for cryptosporidiosis, the only means of prevention is sanitation, which includes controlling flies. for rotavirus and coronavirus, the most effective prevention is vaccination of the dam to increase antibodies in the colostrum against these organisms. other organisms, such as e. coli and salmonella sp., may be resistant to many of the commonly used antibiotics. producers often give antibiotics to calves during episodes of diarrhea in order to prevent secondary infections; however, this practice often does more damage than good, killing beneficial gut microflora and damaging the gut lining. the first step in caring for calves with diarrhea is to provide fluids for hydration and electrolytes for mineral loss, while continuing to provide milk for protein and energy. an electrolyte solution can be fed from min to h after each feeding of milk or milk replacer until feces return to normal. secondly, the organism causing diarrhea should be identified to determine whether antibiotic treatment is needed. pneumonia is the second most prevalent health problem of replacements, especially for replacements raised indoors. research has shown that calves raised in individual hutches (plastic, fiberglass or wooden structures providing individual housing) perform very well and have fewer health problems, especially pneumonia, than calves raised in closed buildings. open-front housing for older heifers should also help prevent pneumonia. adequate, draught-free ventilation is important for prevention of pneumonia. hutches, pastures and open-front housing for replacements provide optimal ventilation. in addition, hutches can be moved from location to location, giving producers the opportunity easily to remove old bedding and to break disease cycles. no matter what type of housing is used for replacements, cleanliness, dry bedding and adequate ventilation are essential to decrease incidence of disease. another important factor for controlling disease in replacements is grouping of heifers. most producers in the united states house young calves individually. in other areas, housing calves in groups and using mob-feeders is an efficient method of rearing calves during the liquid feeding phase. after weaning, calves should be housed in small groups of or fewer until they have successfully made the transition from liquid feed to dry feed and the transition from individual housing to competing for food. the grouping could be done better by multi-sensory historical recording of individual cow behavioural patterns, body temperature, provenance of any previous disease records, long term eating patterns from the young age, and any obvious known symptoms at the time of the grouping. additionally, by housing in small groups (rather than mixing large groups of animals at one time), producers can limit the exposure of calves to disease organisms and match calves more closely by size. as calves age, they can be housed in increasingly larger groups; however, animals should be grouped so there is not more than kg difference in size of animals up to months and not more than kg difference in size for older animals. biosecurity is an outcome of a human systemso people are at its core. all dairy producers must actively institute biosecurity measures to prevent introduction of disease into the herd and to minimize spread of disease within the herd. for replacements, it is extremely important to prevent exposure of younger animals to older animals that may have johne's disease. exposure is not limited merely to animal-to-animal contact, but also includes articles of transmission, such as manure on hands, clothing and boots of workers, manure from older animals on equipment for feeding and handling replacements, or water that has been contaminated by older animals. in addition, flies can transfer diseases from older to younger animals. producers must determine whether to have a closed herd or to allow introduction of new animals to the farm. if new animals are brought to a farm, the producer should work closely with a veterinary surgeon to determine which vaccinations animals should receive prior to coming to the farm. once new animals arrive on the farm, or animals return to the farm from contract-growers or exhibitions, they should be quarantined for at least days. this will allow time to determine if the new animals are likely to become ill and to allow the new animals to be exposed more slowly to any disease organisms currently on the farm. airborne disease spread in cattle farming is rear but when it happens, severity of damage is significantly higher. prions, the proteins that cause mad cow disease and creutzfeldt-jakob disorder could be spread through the air, rather than just through contaminated food, as recent studies have reported. foot-and-mouth disease (fmd) is a severe, highly contagious viral disease of cattle and swine, primarily get spread under favourable weather conditions. the disease may be spread considerable distances by over a route, within a short time period, directed by wind speed, air pressure and temperature. given the fact that airborne spread is probably the one of the most serious issue in animal biosecurity management, a predictive meteorological service for national level biosecurity risk pathway analysis is essential, for prevention of large-scale livestock disease control. in recent time researchers have reported interesting results and applications of a data driven predictive model to track a probable route of an airborne disease, an alerting system to provide better biosecurity decision support for efficient management of cattle health. other potential sources of disease entry into replacements are visitors, vehicles removing dead animals, feed-delivery vehicles, wild and domestic animals, and birds. within the herd of replacements, diseases can be transferred by using needles on more than one animal or using the same glove to palpate more than one animal. producers must identify potential sources for transfer of disease-causing organisms within the herd and from outside the herd and institute a management plan to control them. digestive disorders can occur in dairy replacements, resulting in problems such as acidosis and overeating diarrhea. overeating diarrhea is found in replacements during the liquid feeding phase and may be prevalent in systems using accelerated feeding programs. this form of diarrhea can be treated by decreasing the amount of dry matter offered to calves in the liquid diet until the consistency of the feces returns to normal. care should be taken to determine whether increased fluidity of the feces is caused by overeating or by disease organisms. if caused by disease organisms, treatment should include administration of an electrolyte solution and may require use of antibiotics. acidosis can occur in replacements if they consume large amounts of grains. forages comprise the basis for diets for replacements after months of age. animals that gain access to fields of maize or bags of feed by accident will often suffer acidosis leading to laminitis (founder) or even death. animals that are affected will generally have severe diarrhea. they can be treated by withholding grain until feces return to normal, followed by gradual reintroduction of grain into the diet. several types of internal parasites are found in dairy replacements. perhaps the most common problem is coccidiosis. coccidiosis causes diarrhea, which may be severe, resulting in weight loss, dehydration and anemia. animals can be treated with a coccidiostat, such as amprolium, for severe coccidiosis. coccidiostats such as decoquinate or lasalocid may be included in grain rations or even in milk replacers to help control coccidiosis. another common internal parasite of calves is cryptosporidium parvum. this organism causes diarrhea in young calves at - days of age that lasts approximately a week. there are no cures for cryptosporidiosis and no means of prevention other than sanitation to decrease the pathogen load. treatment involves electrolyte solutions along with continued milk feeding. replacement animals are very vulnerable to internal parasites (especially worms) during their first grazing season. deworming of heifers yields economic returns in growth rates and feed efficiency. producers should consult their veterinary surgeon to determine the most effective method of treating internal parasites both to decrease the parasite load in the animals and to prevent shedding of eggs onto pastures. depending on geographical location, different deworming strategies are needed to control internal parasite populations. producers should be aware that cold temperatures cause larvae to undergo arrest, even when ingested into the host. during this arrested stage, the larvae are resistant to most deworming agents. many external parasites, including various species of flies, affect health and growth of replacements. several species of blood-sucking flies affect replacements. horn-flies, face-flies, stable-flies, ticks, lice and mite can be a major problem for cattle. they can cause substantial blood loss, transmit diseases including mastitis to replacements and decrease growth rates. use of forced back rubs is probably the most effective method of decreasing populations of horn-flies. additionally, removal of manure, which is the major breeding habitat for horn-flies, helps decrease populations. another type of fly, the stable-fly, breeds in wet feed. severe infestations of stable-flies can cause up to a % decrease in milk production. counts of flies per animal cause economically important losses in milk production and growth. removal of waste feed from under feed troughs and other areas to decrease breeding areas is the most important mechanism for control. horse-flies and deer-flies are also blood-sucking flies and may be responsible for spread of several diseases but are impractical to control. common house-flies are not blood-sucking insects but feed on muzzles, eyes and open wounds. they can be contaminated with more than viruses and bacteria, as well as other disease-causing organisms. the main form of control for common houseflies is sanitation and removal of breeding material because many house-flies are resistant to insecticide sprays. cattle grubs are another parasite common in north america. the main damage to cattle is caused by the migration of the grubs through host tissues and production of cysts on the animals' backs. growth rates can be adversely affected with infestations of cattle grubs. appropriate insecticide treatment will kill grubs; however, care must be taken not to administer insecticides when large numbers of grubs may have accumulated in the spinal canal. killing of large numbers of grubs at once can lead to anaphylaxis in cattle. other external parasites that may affect dairy cattle include fleas, lice, ticks and mites. itchiness and formation of scabs should be examined by a veterinary surgeon who can prescribe appropriate forms of treatment. many disease occurrences can be prevented or at least minimized by appropriate vaccination programs. the program that is appropriate, however, will vary from region to region, and even farm to farm. establishment of a vaccination program requires knowledge of diseases prevalent in the area, history of diseases on the farm, history of diseases in the herd, vaccinations used previously in the herd and an assessment of the risk of contracting economically important diseases based on management of the herd (open or closed). producers should, therefore, consult their veterinary surgeon to develop a vaccination program appropriate for their animals, management and location. timing of vaccinations is important for replacement animals. if the colostrum consumed by the calf contained antibodies against the disease organism present in the vaccine, the vaccine will not generate a sufficient immune response in the replacement animal. maternal antibodies obtained from colostrum may be present up to months of age, preventing an adequate response to vaccinations. it may be beneficial to wait until months of age or greater for many initial vaccinations in calves in order to avoid interference from maternal antibodies. additionally, many vaccines are not effective in neonatal calves because their immune system is not sufficiently developed to generate a protective response. common mistakes made in vaccination programs are lack of booster vaccinations at the appropriate time and lack of frequent vaccinations. if the vaccination protocol calls for an initial vaccination followed by a booster vaccination within - weeks, maximum protection will not be achieved without the booster vaccination. essentially, the money spent for the first injection is wasted. the second problem, lack of frequent vaccinations, is seen especially with leptospiral vaccines. leptospiral vaccines should be administered every months to achieve adequate protection. it is also important for heifers to start receiving leptospiral vaccinations at months of age so that they have received two vaccinations by the time they are used for breeding. health management of replacements requires attention to many different areas, ranging from nutrition and management of late lactation and dry cows to vaccinations of replacements. a better-informed management plans for biosecurity risk and analysis is essential for the next generation replacement cattle management. recent development of low cost sensory technology and an introduction to infectious disease control on farms: biosecurity. american feed ingredients association arlington. bovine alliance on management and nutrition large dairy herd management internal parasites of dairy cattle integrated manure management to reduce environmental impact: ii. environmental impact assessment of strategies dynamic cattle behavioural classification using supervised ensemble classifiers vaccines and vaccination programs application of an integrated outbreak management plan for the control of leptospirosis in dairy cattle herds nutrient and immunity transfer from cow to calf pre-and postcalving optimal management of replacement heifers in a beef herd: a model for simultaneous optimization of rearing and breeding decisions replacement management in cattle: health management. encyclopedia of dairy sciences key: cord- - d c zc authors: haschek, b.; klein, d.; benetka, v.; herrera, c.; sommerfeld‐stur, i.; vilcek, Š.; moestl, k.; baumgartner, w. title: detection of bovine torovirus in neonatal calf diarrhoea in lower austria and styria (austria) date: - - journal: j vet med b infect dis vet public health doi: . /j. - . . .x sha: doc_id: cord_uid: d c zc faeces of calves with and without diarrhoea collected during the winter period / in austrian farms (styria and lower austria) were examined for viral, bacterial and parasitic enteropathogens. torovirus‐specific nucleic acid confirmed by reverse transcriptase‐polymerase chain reaction was found in of calves ( . %). ten of these calves were clinically ill, several of them showing signs of dehydration and abnormal faecal consistency at the time of sampling. computer assisted analysis of two nucleotide sequences obtained from austrian bovine samples revealed % similarity to breda strain, but only % or % similarity to equine berne or porcine markelo torovirus strains respectively. phylogenetic analysis grouped austrian torovirus samples into the bovine torovirus cluster indicating the first detection of bovine torovirus in austria. in addition, the following agents were detected in bovine faecal samples: bovine coronavirus, . %; escherichia coli, %; cryptosporidium spp., . %; eimeria spp., . %; rotavirus, . %; clostridium perfringens, . % and giardia spp., . %. salmonella spp. was not detected. neonatal calf diarrhoea is a major problem encountered in calf rearing and leads to losses attributable to growth retardation and death. the most common pathogens are rotaviruses (rv), bovine coronavirus (bcv), escherichia coli k , salmonella spp. and cryptosporidium spp. (koopmans et al., ) . bovine torovirus (botv) was first associated with enteritis of calves by woode et al. ( ) . these enveloped singlestranded rna viruses have a fringe of peplomers on their surface, measure - nm at their largest diameter and contain a tightly coiled nucleocapsid that generally assumes a torus shape in the virion (jamieson et al., ) . by electron microscopy, it is difficult to differentiate between botv and bcv. however, there is no antigenic relationship between these two viruses (pohlenz et al., ) . bovine torovirus fails to grow in cell culture. in order to obtain data about pathology and pathogenesis, gnotobiotic calves were infected orally with botv (woode et al., ; fagerland et al., ) . the inoculated calves developed mild to severe diarrhoea with clinical signs of dehydration and weakness. faecal shedding of botv lasted for - days in these calves. bovine torovirus was described in diarrhoeic calves in various countries (liebler et al., ; duckmanton et al., ; matiz et al., ; smits et al., ) . the faecal prevalence of botv in calf diarrhoea ranges from % in lower saxony, germany (liebler et al., ) to . % in southern ontario, canada . seroepidemiological studies revealed a wide distribution of botv in cattle herds; . % of adult cattle in the netherlands and germany (koopmans et al., ) , . % of cattle in the usa (woode et al., ) and % of cattle in the uk (brown et al., ) were positive for antibodies against botv. to the authorsÕ knowledge, botv has not yet been investigated in austria. there are no data available about sero-or faecal-prevalences. the aim of the study reported here was to determine the faecal prevalence of botv in austrian calves (with and without diarrhoea) and to find out whether there is a significant difference in the occurrence of botv between calves suffering from diarrhoea and healthy calves. furthermore, the faecal prevalences of the most important viral, bacterial and parasitic enteropathogens as co-infecting agents to botv on the one hand and on the other as causative pathogens in neonatal calf diarrhoea were determined. the health status of cattle herds and single animals in relation to a variety of parameters like clinical symptoms, farm management and presence of pathogens were evaluated statistically. during the winter period (october-february) / , a total of farms in styria and lower austria were visited. predominantly milk or milk replacer fed calves from to days of age were included in this study. in of these farms (ÔdiseasedÕ farms), one or more calves were suffering from acute diarrhoea at the time of the visit. other farms (ÔhealthyÕ farms) with a similar structure and geographical distribution had been free from calf diarrhoea for at least weeks and served as a standard of comparison (control group). each calf was examined clinically before collection of a faecal sample. in the ÔdiseasedÕ farms, one to four sick calves and, if available, one to four healthy neighbour calves were selected randomly. in the corresponding ÔhealthyÕ farm, (if available) an equivalent number of calves was included in the study. the health status of each calf was evaluated by clinical examination. healthy calves had to be free from diarrhoea, whereas sick calves showed signs of dehydration and weakness and/or abnormal faecal consistency at the time of sampling or shortly before. farm management characteristics were evaluated for each farm, including parameters like farm size, animal housing, cleaning and disinfection, feeding of the calves and vaccinations of cows etc. faecal samples were stored at ) °c and then tested by reverse transcriptase-polymerase chain reaction (rt-pcr) for specific nucleic acid of botv, bcv and rv. a commercially available kit (qiaampÒ viral rna mini kit; qiagen, hilden, germany) was used for extraction of viral rna from faecal samples. the rt-pcr reaction mixture was prepared following the protocol of a ready-to-use kit (qiagenÒ onestep rt-pcr kit; qiagen). primers were diluted to a final concentration of pmol/ll. to minimize the risk of cross-contamination and dna carry-over from previous reactions, all procedures were strictly separated physically and carried out following good laboratory practice. several negative controls of distilled water as well as one positive control were included in each assay. all working units and laboratory materials were decontaminated with disinfectants and/or ultraviolet light before use. for the detection of specific nucleic acids of botv, bcv and rv, primers designed by tsunemitsu et al. ( ) ; hoet et al. ( ) and schwarz ( ) were used respectively. all details are shown in table . amplified dna was purified using a commercially available kit (nucleospinÒextract; machery-nagel, du¨ren, germany) following the manufacturer's instructions and served as a template for sequencing pcr. two botv-positive samples as well as one randomly chosen sample of bcv and rv each were sequenced in both directions using a commercial sequencing kit (dna sequencing kit; applied biosystems, warrington, uk) and corresponding pcr primers on abi prism genetic analyser (applied biosystems). nucleotide sequences of two botv-specific amplified products and corresponding sequences from genbank were compared using the clustalw program. evolutionary distances were calculated using the program dnadist, employing the kimura two-parameter method (kimura, ) . phylogenetic analysis of bp dna fragments was performed using the neighbor program based on the neighbour-joining method (saitou and nei, ) from phylip inference package programs (felsenstein, ) . statistical parameters of phylogenetic trees were determined by bootstrap analysis carried out on replicates using phylip programs seqboot and consence. the tree was drawn using the drawtree program from phylip. faecal samples were examined for the presence of parasites using a flotation technique with concentrated sugar solution. faecal material was plated on mcconkey and blood agar, and incubated aerobically at °c for - h. for the detection of clostridium spp., samples were plated on blood agar and incubated anaerobically at °c for h. for the detection of salmonella spp., faecal material was incubated in ml of peptone water at °c for h, and then, subcultivated on brilliant green agar at °c for h. data were statistically evaluated using the spss for windows package version . . (spss inc., chicago, il, usa). first of all, possible associations between the health status of the single animal, respectively, of the farms and farm management characteristics like farm size, animal housing, cleaning and disinfection, feeding of the calves, vaccinations of the cows, etc., were investigated by the chi-squared test. whenever an association was hypothesized by an alpha error of p < . , the respective variable was included in the statistical model evaluating the association between the distribution of enteropathogen positive/negative animals and the health status of the single animal. for final evaluation, a logistic regression model was created, which included the infection status of the animal as dependent variable and the health status of the animal and associated variables as independent variables. the resulting odds ratios (or), p-values as well as the % confidence interval (ci) of the respective or are listed in table . furthermore, the distribution of clinical symptoms in infected versus noninfected animals was investigated for different pathogens by descriptive analysis. a total of calves was included in the study. calves originated from ÔdiseasedÕ farms; of these calves were clinically ill at the time of the visit and calves were clinically healthy neighbour calves. on the ÔhealthyÕ farms, calves were examined. reverse transcriptase-pcr with botv-specific primers provided an expected bp dna product originating from s gene with of faecal samples analysed. the specificity of our amplification system was confirmed by sequencing and computer-assisted phylogenetic analysis of two positive samples (aut and aut ). when nucleotide sequence stretches of aut and aut were aligned with torovirus sequences available in the genbank, the analysis revealed % similarity between the two austrian viruses, % similarity to bovine breda virus but only % identities to equine berne virus and % to porcine markelo virus (table ). phylogenetic analysis (fig. ) clearly demonstrated that aut and aut were grouped with botv strain b originating from the netherlands and breda strain isolated in iowa. twelve positive samples for botv rna of faecal samples examinated correspond to a virus prevalence of . %. they were identified on nine different farms, eight of which were ÔdiseasedÕ and one ÔhealthyÕ. ten of the botv-positive calves were clinically ill while the other two showed no clinical symptoms of diarrhoea; one positive calf was a clinically healthy neighbour animal. six botv-positive calves were younger than days, while the age of the other six ranged from to days. the statistical analysis showed a significant difference (p ¼ . ) in the prevalence of botv in healthy and diseased calves. the calculated or-values indicate that calves shedding botv in their faeces are . times more likely to suffer from diarrhoea than calves not shedding botv ( % ci . - . ; p ¼ . ). on clinical examination, several botv-positive calves showed signs of dehydration: skin elasticity was reduced in eight calves, bulbi were sunken and general behaviour was reduced in two calves. three botv-positive calves, two of them diarrhoeic, were suffering from additional lung diseases ranging from bronchitis to bronchopneumonia possibly also due to an additional bcv infection present in all three. one of these calves was further co-infected with eimeria bovis/zuernii and klebsiella spp. faecal consistency was abnormal in seven calves ranging from watery to mushy. admixtures could be found in the faeces of calves: mucus (nine calves) and blood (two calves). single infection with botv was only found in two calves, both suffering from diarrhoea at the time of sampling. the other animals shed one or more other enteropathogens (table ) . the occurrence of other enteropathogens, the p-value and the corresponding or values are listed in table . rotaviruses and cryptosporidia were significantly more frequently detected in calves with diarrhoea with an or of . ( % ci . - . ) and . ( % ci . - . ), respectively, (p < . ). concerning bcv, the or was . ( % ci . - . ) with an alpha error of p ¼ . , and therefore, at least showing a trend towards an association between the detection of bcv and diarrhoea. the following farm management characteristics finally showed significant associations to the infection status with specific enteropathogens (the cited or values are corrected for the influence of the health status of the calves). the or of calves born from cows vaccinated against rotavirus, coronavirus and e. coli to be positive for botv was . ( % ci . - . ; p ¼ . ). the or of calves from farms not having a calving box to be positive for rv was . ( % ci . - . ; p ¼ . ). the or of single kept calves to be positive for rv was . ( % ci . - . ; p ¼ . ). the or of calves from dairy farms to be positive for cryptosporidia was . ( % ci . - . ; p ¼ . ). this is the first study to document the presence of botv in austria extending geographic regions where this virus has already been detected. sequence and phylogenetic analyses of two austrian botv-positive samples (fig. , table ) revealed the highest nucleotide identities with botv strain b (the netherlands) and the north american strain breda of % and % respectively. phylogenetic analysis supported these data as austrian botv clustered clearly within botv belonging to the family coronaviridae. the calculated percentage ( . %) of botv rna shedders in austrian cattle herds is comparable with faecal prevalences to determine the faecal prevalence of botv in austrian calves reported from lower saxony ( %; liebler et al., ) and the netherlands ( %; koopmans et al., ) . in contrast, duckmanton et al. ( ) reported a considerably higher faecal prevalence of . % in southern ontario. compared with other enteropathogens, botv faecal prevalence is rather low. a possible explanation might be the short excretion period of only - days (woode et al., (woode et al., , liebler et al., ) . however, using the very sensitive rt-pcr, hoet et al. ( a) were able to detect botv in faeces as early as h postinfection (p.i.) and up to - days p.i., with the most consistent range between days and p.i. in our study, all animals with different histories and duration of diarrhoea were sampled only once. therefore, it is very likely that not all botv infections have been detected. on the other hand, it has to be considered that the detected rna does not necessarily represent infectious virus and acute infection (hoet et al., a) . according to previous studies (koopmans et al., ; duckmanton et al., ; hoet et al., b) we found botv significantly (p ¼ . ) more frequently in calves with diarrhoea than in asymptomatic calves. calves shedding botv in faeces were . times more likely to have diarrhoea than calves not shedding botv. these results indicate that botv, although not a major cause of disease, is of considerable importance in calf diarrhoea. however, it remains impossible to determine whether botv was the primary cause of diarrhoea in the symptomatic calves, which had concomitant infections with other enteropathogens. even in the calves with botv single infections, the occurrence of other enteropathogens before the time of sampling cannot be excluded. on clinical examination, the majority of botv-positive calves showed signs of dehydration, and faecal consistency was abnormal in seven animals ranging from watery to mushy. three calves were suffering from additional respiratory tract disease (rtd), ranging from bronchitis to bronchopneumonia. in all three calves co-infected with bcv, a possible trigger and cause of rtd, was detected, the aetiological involvement of the two pathogens is impossible to determine. observations by vanopdenbosch et al. ( ) confirmed the pathogenicity of botv for the respiratory tract. respiratory toroviral infections occur mainly during the first month of life and at the age of - months. it is possible that botv might play a role in rtd of young calves (vanopdenbosch et al., ) . as in this study, calves with rtd were not investigated for specific aetiological pathogens, botv might possibly have played a role as a cofactor in those diseases too. the role of botv in rtd has to be clarified by further studies with different sampling techniques. in this study, the most frequently detected enteropathogen was bcv ( . %), followed by various types of e. coli, cryptosporidia, rotavirus and clostridium perfringens. bovine coronavirus is estimated to account for - % of all cases of calf diarrhoea (anonymous, ) , and bcv involvement in calf scours can go up to % (doll, ) . rotavirus was detected with a faecal prevalence of . %, which is rather low compared with investigations from lower saxony (germany), where rv was detected in . % of all collected samples (liebler et al., ) . in particular, for rv and bcv, the respective prevalences might be higher in our calves, as intermittent virus shedding has been reported in both healthy and diseased animals (crouch and acres, ; schwers et al., ; collins et al., ) . cryptosporidia faecal prevalence was . %. faecal prevalences can go up to % (doll, ) , with a peak shedding in calves - weeks old (huetink et al., ) . giardia duodenalis was detected in - % of calves older than weeks (doll, ) , while in our study, it could be found in . % of all calves. eimeria bovis and zuernii and other eimeriae faecal prevalences were . % and . % respectively. depending on animal housing and pasture management, infestation rates can range from % to % (rommel, ) . concerning e. coli subtype, faecal prevalences range from % to % (steiner et al., ; doll, ) , which corresponds to our results ( . %). c. perfringens (faecal prevalence in our study: . %) causes a number of diseases among humans and animals; information in literature goes up to a morbidity of % in single outbreaks (klee, ) . liebler et al. ( ) found rv, bcv and cryptosporidia significantly more often in calves with diarrhoea than in healthy calves. in our study, we found a significant difference only for rv and cryptosporidia (p < . ), but not for bcv. however, or values indicate a tendency that calves shedding bcv are . times ( % ci . - . ) more likely to develop diarrhoea. significant differences between clinically ill and healthy calves could neither be found for bacteria (e. coli, c. perfringens) nor for eimeriae and giardia spp. concerning the significant associations of certain farm management characteristics with specific enteropathogens, a satisfying interpretation cannot be given; further investigations would need to be done. in conclusion, we demonstrated for the first time at the genetic level that toroviruses circulate in austrian cattle populations. seroepidemiological studies are needed to gain more information about the distribution of these agents. additionally, the recently described recombinations between different botv strains, as well as between botv and porcine torovirus, and most interesting with toroviruses of yet unknown origin (smits et al., ) offer a wide field of future research. it remains to be elucidated whether such molecular events are present in the genomes of our torovirus isolates. laboratory methods for detecting calf diarrhea viruses detection of breda virus antigen and antibody in humans and animals by enzyme immunoassay acres, : prevalence of rotavirus and coronavirus antigens in the feces of normal cows innere medizin und chirurgie des rindes, . auflage detection of bovine torovirus in fecal specimens of calves with diarrhea from ontario farms a morphological study of the replication of breda virus (proposed family toroviridae) in bovine intestinal cells phylip (phylogeny inference package), version . c. department of genetics enteric and nasal shedding of bovine torovirus (breda virus) in feedlot cattle comparison of elisa and rt-pcr versus immune electron microscopy for detection of bovine torovirus (breda virus) in calf fecal specimens association of enteric shedding of bovine torovirus (breda virus) and other enteropathogens with diarrhea in veal calves epidemiology of cryptosporidium spp. and giardia duodenalis on a dairy farm human torovirus: a new nosocomial gastrointestinal pathogen a simple method for estimation of evolutionary rates of base substitution through comparative studies of nucleotide sequences clostridiose (enterotoxa¨mie) innere medizin und chirurgie des rindes, . auflage seroepidemiology of breda virus in cattle using elisa association of diarrhea in cattle with torovirus infections on farms zur bedeutung des bredavirus als durchfallerreger in niedersa¨chsischen ka¨lberbesta¨nden torovirus detection in faecal specimens of calves and pigs in hungary: short communication cellular lesions in intestinal mucosa of gnotobiotic calves experimentally infected with a new unclassified bovine virus (breda virus) protozoeninfektionen der wiederka¨uer the neighbor-joining method: a new method for reconstructing phylogentic trees entwicklung einer reversen transkription-polymerase-kettenreaktion (rt-pcr) zum nachweis der persistenz von rotaviren beim schwein. dissertation repeated bovine rotavirus infection and excretion in calves phylogenetic and evolutionary relationships among torovirus field variants: evidence for multiple intertypic recombination events ha¨ufigke-iten und ursachen von ka¨lberverlusten und ka¨lberkrankheiten in mutterkuhbetrieben. ii mikrobiologische und parasitologische diagnosen bei ka¨lbern mit durchfall experimental inoculation of adult dairy cows with bovine coronavirus and detection of coronavirus in feces by rt-pcr breda virus associated with respiratory disease in calves studies with an unclassified virus isolated from diarrheic calves comparative studies on three isolates of breda virus of calves the authors want to thank the organization Ôniederoesterreichischer bauernbundÕ for financial support. s. v. was supported by vega grant no. / / and sp / / . special thanks to ms. michaela stolz for technical support. key: cord- -rp a lj authors: rocha, thaís gomes; silva, fernanda dornelas florentino; gregori, fábio; alfieri, amauri alcindo; buzinaro, maria da glória; fagliari, josé jurandir title: longitudinal study of bovine rotavirus group a in newborn calves from vaccinated and unvaccinated dairy herds date: - - journal: trop anim health prod doi: . /s - - - sha: doc_id: cord_uid: rp a lj reports of rotavirus excretion in calves usually result from cross-sectional studies, and in face of the conflicting results regarding protection of calves born to vaccinated dams against diarrhea, the aim of the present study was to evaluate rotavirus excretion in dairy calves born to vaccinated or unvaccinated dams, to identify the genotypes of bovine rotavirus group a (rva) strains isolated from these animals as well as to investigate characteristics of the disease in naturally occurring circumstances throughout the first month of life. five hundred fifty-two fecal samples were taken from calves, from each farm and, in the vaccinated herd, / samples ( . %) taken from six different calves tested positive for rva while in the unvaccinated herd, / samples ( . %) taken from different calves tested positive. the genotyping of the vp genes showed . % nucleotide sequence identity to g genotype (ncdv strain), and for the vp gene, strains from the vaccinated herd were . % related to b strain, while strains from the unvaccinated herd were % related to p[ ] genotype (uk strain). genotypes found in this study were g p[ ] in the vaccinated herd and g p[ ] in the unvaccinated herd. all calves infected with rotavirus presented an episode of diarrhea in the first month of life, and the discrepancy between the genotypes found in the commercial vaccine (g p[ ] and g p[ ]) and the rotavirus strains circulating in both vaccinated and unvaccinated herds show the importance of keeping constant surveillance in order to avoid potential causes of vaccination failure. neonatal calf diarrhea (ncd) is one of the most important causes of morbidity and mortality in animals aged less than weeks of life, leading to economic losses due to the cost of treatment, prophylaxis, increased susceptibility to other infections, reduced growth rates, and death of calves. colostrum management, size of the herd, type of housing, nutritional status, and the presence of other pathogens are considered risk factors for the development of the infection (lorenz et al. ; cho and yoon ) . ncd is a multifactorial disease, associated with infectious and non-infectious causes. of the bacteria, viruses, and protozoa commonly involved in the etiology of ncd, the most prevalent viral agent is bovine rotavirus group a (rva), as reported in studies conducted in brazil, netherlands, australia, usa, and new zealand (langoni et al. ; bartels et al. ; izzo et al. ; cho et al. ; cho and yoon ; al mawly et al., ) . rotavirus is a genus from the reoviridae family and is divided into groups or species (a-h) based on antigenic properties and nucleotide sequence diversity of the inner viral capsid protein vp (matthijnssens et al. ) . from these, rva is the most prevalent (papp et al. ) . the non-enveloped virion has a triple-layered capsid surrounding dsrna segments that encode structural (vp - , vp , and vp ) and nonstructural (nsp -nsp / ) proteins . since vp and vp induce the production of neutralizing antibodies and protective immunity, these proteins play a central role in strain selection for vaccine production and form the basis of a binary classification system, namely, the g (glycosylated) and p (protease-sensitive) genotypes (estes and greenberg ) . to date, g and p genotypes with different combinations have been described (matthijnssens et al. ; trojnar et al. ) . regarding bovine rotavirus infections, g , g , and g together with p[ ], p[ ], and p[ ], respectively, are the g and p genotypes most commonly found (alkan et al. ) . general recommendations regarding decrease of rva diarrhea include management practices, especially good hygiene and sanitation procedures, as well as pathogen-specific interventions, such as the use of vaccine prophylaxis (alkan et al. ; izzo et al. ) . the vaccination of cows aims an increase of the concentration of specific antibodies in colostrum against targeted pathogens (kaplon et al. ) . although many reports state that vaccination programs of pregnant cows are one of the main strategies for prevention of rva diarrhea in calves, it must be kept in mind that calves that receive a sufficient quantity of specific antibodies through colostrum intake are not completely protected against rotavirus infection, instead, the severity of clinical signs is decreased, reducing deaths from rva infection (parreño et al. ; kaplon et al. ) . the aim of this study was to evaluate rotavirus excretion and to identify the genotypes of rva strains isolated from a vaccinated and an unvaccinated dairy herd from são paulo state, brazil. fifty-six calves were evaluated in a longitudinal design study. these animals were allotted in two experimental groups with animals: group v comprised of animals that belonged to a herd that regularly vaccinated the dams with a commercial vaccine against ncd, and group nv comprised of animals from a herd that did not vaccinate the dams against ncd. the vaccination scheme included administration of a commercial adjuvant vaccine containing inactivated bovine rva strains g p[ ] and g p [ ] , inactivated bovine coronavirus, escherichia coli k colibacterin and type c clostridium perfringens toxoid according to manufacturer's instructionspregnant cows received two intramuscular doses of vaccine, with an interval of approximately weeks, the second dose being administered to weeks pre-partum. cows from group nv did not receive the vaccine in the prepartum period. besides vaccination management, herd v comprised holstein cows in lactation, with a mean production of l of milk/cow/day, and group nv comprised holstein cows in lactation, with a mean production of l of milk/cow/day. colostrum management was similar in both farms, and only calves born to cows that produced a sufficient volume of colostrum to ensure the intake of l in the first meal were included in this study. colostrum management consisted of bottle feeding calves ad libitum with their own mother's colostrum up to h after birth, with a minimum volume of colostrum intake of l per calf. the fecal samples were collected in two municipalities located in the state of são paulo in and . holstein calves were randomly selected and fecal samples were taken directly from the rectum using individual plastic bags before colostrum intake, and at , , , , , and days of age and frozen at − °c until analysis. whenever calves presented diarrhea, samples were taken at h intervals until animals presented two consecutive days of normal feces. fecal samples were classified in scores according to consistency: score (firm feces), score (slightly loose feces), score (loose feces), and score (watery feces) (fernandez et al., ) . scores and were considered normal feces and scores and were considered diarrhea. samples were screened for rva by silver stainedpolyacrylamide gel electrophoresis (page) technique (herring et al. ) modified by pereira et al. ( ) , which is a technique with sensitivity approximately equal to electron microscopy (herring et al. ) . the rt-pcr was employed to characterize the bovine rva genotypes g and p as described previously . fecal samples were prepared as % (v/v) suspensions in depc-treated ultrapure water and centrifuged at ×g for min at °c. total rna was extracted from μl of the supernatant using trizol reagent™ (invitrogen, carlsbad, ca, usa) and cdna was synthesized using random primers (invitrogen, carlsbad, ca, usa) and superscript iii reverse transcriptase (invitrogen, carlsbad, ca, usa) as per manufacturer's protocol. different pcr runs were performed using primers beg , end , end crw , end uk (for g genotypes) or con and con (for p genotypes) (gouvea et al. a; gouvea et al. b; gentsch et al. ; silva et al. ) . therefore, . μl cdna was added into the reaction mix comprising × pcr buffer™ (invitrogen, carlsbad, ca, usa), . mm of each dntp, . μm of each forward and reverse primers, . mm mgcl , and u platinum taq polymerase™ (invitrogen, carlsbad, ca, usa) and nuclease-free water to obtain a final volume of μl. this solution was then heated to °c for min, followed by amplification cycles, with cycle consisting of min at °c, . min at °c, and min at °c; and a final extension step of min at °c. after completion of thermocycles, pcr products were separated by electrophoresis on a . % agarose gel, stained with ethidium bromide, and visualized by uv transillumination. ten samples in which rt-pcr results were satisfactory were used for genotyping. the amplicons were purified using exosap-it® pcr product cleanup (usb products affymetrix, cleveland, usa) and nucleotide sequences of the vp and vp gene segments were determined in both directions using bigdye terminator . ™ cycle sequencing kit (applied biosystems, foster city, usa), according to the manufacturer's protocol and a ™ genetic analyzer system (applied biosystems, foster city, usa). the nucleotide sequences were assembled using bioedit . . software (hall ) and analyzed using the rotac . automated genotyping tool for group a rotavirus to confirm the rva identity of all sequences (maes et al. ). phylogenetic trees were constructed and molecular analyses were performed using the mega® . software by using the neighbor-joining algorithm and the maximum composite likelihood dna substitution model for each viral gene or nucleotide fragment (tamura et al. ) . statistical support was obtained by bootstrap replicates. the identity matrix was constructed using bioedit software version . . . sequences from this study were deposited in the genbank database under the following accession numbers: vp (kx , kx the number of fecal samples taken from calves from the vaccinated and unvaccinated herds; its distribution by scores as well as the number of bovine rva positive samples according to fecal score are shown in table . fecal samples positive for rotavirus detection through page, number and age of calves that tested positive for rva in feces by this technique, number of days of elimination of diarrheic feces throughout the first month of life, as well as results of genotyping are shown in table . the distribution pattern of the dsrna segments was : : : in all the positive samples by page technique. all samples analyzed by rt-pcr for the detection of vp were positive. regarding vp , in four samples from three different animals, it was not possible to obtain amplicons using the primers described, even though samples were tested twice, in different moments, using new rna extractions, and positive and negative controls worked as expected. as sequencing was performed in only ten samples in which rt-pcr results were adequate, including at least one sample from each positive calf, the genotype of the four samples aforementioned could not be determined. genotyping revealed that all rva positive calves from group v were infected with g p[ ] rva, while in group nv, the genotype found was g p [ ] . also, g p[ ] strain was detected in both normal and diarrheic feces, while g p[ ] strain was found only in diarrheic feces. positive calves excreted the virus for to days in the vaccinated herd, and for only day in the unvaccinated herd (table ) . none of the calves that tested positive for rva in feces were aged between and days of life. calves that tested positive for rotavirus in feces were in their second (n = ), third (n = ), or fourth (n = ) weeks of age. evaluation of fecal scores of calves in the first days of life revealed that in group v, . % of fecal samples taken were normal, while . % of feces were diarrheic. in group nv, . % of fecal samples were normal while . % were diarrheic. the nucleotide sequence analysis of the vp genes of brazilian bovine rva strains belonging to both the g p[ ] and g p[ ] genotypes showed the highest nucleotide sequence identity ( . %) to g genotype (ncdv strain) (fig. ) . for the vp gene, two brazilian bovine rva strains (thaf and thaf ) were most closely related to p[ ] genotype (uk strain) ( %). furthermore, the vp gene of the remaining brazilian bovine strains belonging to genotype g p[ ] from this study was closely related to b strain ( . %) (fig. ). although positive samples were found in both normal (n = ) and diarrheic feces (n = ), which agrees with the report by bartels et al. ( ) and freitas et al. ( ) , all calves that tested positive for rotavirus detection in feces presented an episode of diarrhea in the first days of life. this finding is relevant since most of the cross-sectional field research performed investigating rva diarrhea, consider calves in which rva is found in normal feces to be asymptomatic. in the report by oliveira-filho et al. ( ) and cho et al. ( ) , bovine rva was only found in diarrheic feces, however, this sort of information is rare in the literature consulted, which can lead to misinformation. it is quite likely that newborn calves infected with rva will always manifest an episode of diarrhea, and that the virus can be eliminated in all stages of clinical course of the disease, including in moments in which feces are normal. thus, it would be relevant to perform more longitudinal studies in which fecal samples are taken in predetermined moments, as well as whenever the calves manifest diarrhea, in order to evaluate a larger number of animals to confirm these findings and if they are related to the virulence of genotypes of rva. nourmohammadzadeh et al. ( ) reported that the higher prevalence of rotavirus infection occurs in calves aged between and weeks, and lower in animals in the first week of life, which agrees with the results of the present report, in which none of the positive calves were in their first week of life, four calves were in the second week of life, four calves were in the third week of life and one calf was in the fourth week of life. bartels et al. ( ) , evaluating the prevalence and risk factors for the presence of enteropathogens in normal and diarrheic feces of calves aged between and weeks of life reported that the prevalence of normal consistency feces was . %, semiliquid feces was . %, and liquid feces was . %. in the current study, considering the same age groups of calves, percentages found were . and % of normal consistency feces, . and % of semiliquid feces, and . and % of liquid feces, in groups v and nv, respectively. these authors also reported that the percentage of calves with diarrheic feces in the second week of life was higher than in calves with or weeks of age, which was similar to the findings of the present study, in which the percentage of diarrheic calves in their second week of life was . %. this finding is attributable to the immune status of calves, which is higher in the first week of life due to passive immunity provided by colostrum, and gradually decreases through a natural process of catabolism of immunoglobulins, making calves more susceptible to infection in the second and third weeks of life. since most cases of rva diarrhea occur within these age groups, at weeks of age, calves are normally immune to infection through natural resistance (chase et al. ; coura et al. ) . the duration of rotavirus excretion is variable among studies found in literature. a recent report of longitudinal evaluation of diarrhea occurrence in a dairy herd in brazil stated that the excretion of rotavirus in feces lasted for - days, which agrees with the results of the present study (coura et al. ) . in experimental studies, rotavirus excretion has been reported to last up to days (fernandez et al. ) . this difference might be attributable to the method of detection of rva in feces, since both field studies employed page as a screening method for rotavirus in feces, and the limitation of this method lies in the need for a certain concentration of virus particles for the test to be positive, while in the experimental study cited, the method used (elisa) is considered a test able to detect smaller amounts of virus present in feces than page. it is important to point out that there was a difference between the rva strain found between the vaccinated calves (g p [ ] ) and the strains present in the commercial vaccine (g p[ ], and g p[ ]), which might be one of the contributing factors to the lack of efficiency in protecting calves of group v from rotavirus diarrhea. according to alkan et al. ( ) , several factors can contribute to vaccine failures, especially vaccination management. in the present study, there was a special attention with the conservation and protocol of vaccination employed, in order to avoid the risk of vaccine failure due to inappropriate conservation of the vaccine or a longer period of time between the second dose of vaccine and parturition. however, contrary to expectations, incidence of rotavirus diarrhea was higher in group v than in group nv, which might be due to differences in herd size, exposure to virus, and management procedures between herds (meganck et al. ) . the strain found in the vaccinated herd (g p[ ]) was different from the strain of the non-vaccinated herd (g p[ ]), and none of these strains were present in the commercial vaccines available by the time of the experiment. in brazil, g p [ ] has been incriminated as a cause of a diarrhea outbreak in a beef herd (medeiros et al. a ), although there are also reports of its occurrence as the most common genotypic combination in some states of country (silva et al. ) . in ireland, g p[ ] is reported as a low prevalence genotypic combination found especially in dairy herds (collins et al. ) , while in turkey, alkan et al. ( ) reported a high prevalence of this genotype in either unvaccinated herds and in herds immunized against g p[ ]. vaccines containing g p[ ] provide poor heterologous protection against g bovine rva that contain different p genotypes from the vaccine strains (alkan et al. ; medeiros et al. b ). this implies that, considering the latest surveillances performed in brazil and in other regions of the world, strains of rva present in the commercial vaccines should be replaced by genotypic combinations more prevalent in the region of interest to reduce the occurrence of rva diarrhea and all the economic losses associated to this infirmity. in a recent review performed by papp et al. ( ) on the rva strains reported in cattle worldwide, the most prevalent genotypic combinations found in diarrheic calves were g p[ ] (uk strain), g p[ ] (kn strain), and g p[ ] (b strain), comprising . % of all rva strains. also, in argentina, the most prevalent genotypic combinations reported were g p[ ] in dairy herds and g p [ ] in beef herds (badaracco et al. ) . parreño et al. ( ) reported that the protective effect of high titers of serum antibodies against bovine rva significantly reduces the occurrence of diarrhea caused by rotavirus, but does not eliminate the virus from the herd, which reinforces the concept that commercial vaccines currently used are not designed to prevent rotavirus infection, but rather to decrease morbidity, severity of diarrhea, and mortality in field conditions. although this might have impact in some of the economic costs associated with neonatal calf diarrhea, it does not reduce the expenses of treatment aiming to avoid secondary bacterial infections and might still impact on the potential development and performance of calves in their productive life, given that all the animals positive for rva detection in the present study manifested at least one episode of diarrhea throughout the first month of life. the nucleotide sequence analysis of the vp genes of bovine rva strains showed . % nucleotide sequence identity to ncdv strain (g genotype). in the unvaccinated herd, the vp gene was most closely related ( %) to p[ ] genotype (uk strain), while in the vaccinated herd, the vp gene detected was . % related to the b strain. alkan et al. ( ) reported that the g p[ ] lineage found in turkey formed a unique g lineage with only . % amino acid identity to the g strain ncdv found in the vaccines. medeiros et al. ( a) also reported an outbreak of diarrhea in beef calves in brazil caused by a g p[ ] distant from the most common lineages of rva strains g p[ ] and g p [ ] . as the strain found in the vaccinated herd of the present study showed a nucleotide sequence identity of . % to the fig. maximum likelihood phylogenetic tree based on the partial orf nucleotide sequences of bovine rva vp gene. the bovine strains analyzed in this study are marked with a lozange. the scale bar indicates nucleotide substitutions per site. bootstrap values ( replicates) above are shown in the corresponding nodes vaccinal strain ncdv, and the vp antigen associated was different from the vaccinal strain, this could help to explain the lack of protection of the vaccinated herd against rva diarrhea. the small number of calves positive to rva infection found in our study, especially in the unvaccinated herd, as well as the impossibility of comparing herds with similar sizes, production and infection pressure constituted the main limitations of the study, although the results presented here provided important highlights to the characteristics of natural occurring rotavirus diarrhea in calves in the first month of life. more field studies should be performed to evaluate the genotypic profile of rva present in both vaccinated and unvaccinated herds. the results of this study show that all calves infected with rotavirus presented an episode of diarrhea in the first month of life, and that the discrepancy between the genotypes found in the commercial vaccine and the rotavirus strains circulating in both vaccinated and unvaccinated herds, once more, reinforces the importance of constant surveillance in order to avoid potential causes of vaccination failure. prevalence of endemic enteropathogens of calves in new zealand dairy farms distribution of g (vp ) and p (vp ) genotypes of group a rotaviruses from turkish calves with diarrhea family reoviridae-virus taxonomy ninth report of the international committee on taxonomy of viruses phylogenetic analyses of typical bovine rotavirus genotypes g , g , p[ ] and p[ ] circulating in argentinean beef and dairy herds prevalence, prediction and risk factors of enteropathogens in normal and non-normal faeces of young dutch dairy calves neonatal immune development in the calf and its impact on vaccine response an overview of calf diarrhea-infectious etiology, diagnosis, and intervention case-control study of microbiological etiology associated with calf diarrhea detection and characterisation of bovine rotavirus in ireland from longitudinal study of salmonella spp., diarrheagenic escherichia coli, rotavirus, and coronavirus isolated from healthy and diarrheic calves in a brazilian dairy herd rotaviruses passive immunity to bovine rotavirus in newborn calves fed colostrum supplements from cows immunized with recombinant sa rotavirus core-like particle (clp) or virus-like particle (vlp) vaccines, vaccine rotavirus in cattle: risk factors, prevalence and antigenic characterization from dairy calves' samples in são paulo state, brazil, arquivo brasileiro de medicina veterinária e zootecnia identification of group a rotavirus gene types by polymerase chain reaction identification of bovine and porcine rotavirus g types by pcr vp typing of bovine and porcine group a rotaviruses by pcr bioedit: a user-friendly biological sequence alignment editor and analysis program for windows / /nt rapid diagnosis of rotavirus infection by direct detection of viral nucleic acid in silver-stained polyacrylamide gels prevalence of major enteric pathogens in australian dairy calves with diarrhea impact of rotavirus vaccine on rotavirus genotypes and caliciviruses circulatingin french cattle contribution to the study of diarrhea etiology in neonate dairy calves in são paulo state, brazil calf health from birth to weaning: ii. management of diarrhea in pre-weaned calves rotac: a web-based tool for the complete genome classification of group a rotaviruses uniformity of rotavirus strain nomenclature proposed by the rotavirus classification working group (rcwg), archives of virology vp -sequence-based cutoff values as a criterion for rotavirus species demarcation severe diarrhea outbreak in beef calves (bos indicus) caused by g p[ ], an emergent genotype of bovine rotavirus group a, pesquisa veterinária brasileira phylogenetic analysis of a g p[ ] bovine rotavirus strain isolated in a neonatal diarrhea outbreak in a beef cattle herd vaccinates with g p[ ] and g p[ ] genotypes evaluation of a protocol to reduce the incidence of neonatal calf diarrhoea on dairy herds the prevalence of rotavirus in neonatal calf diarrhea, using electron microscopic examination diarrhea in nellore calves: clinical and etiological study review of group a rotavirus strains reported in swine and cattle modulation by colostrum-acquired maternal antibodies of systemic and mucosal antibody responses to rotavirus in calves experimentally challenged with bovine rotavirus electrophoretic study of the genome of human rotaviruses from rio de janeiro molecular characterization of group a bovine rotavirus in southeastern and central-western brazil whole-genomic analysis of porcine group a rotaviruses isolated from symptomatic piglets in brazil during the years of mega : molecular evolutionary genetics analysis version . identification of an avian group a rotavirus containing a novel vp gene with a close relationship to those of mammalian rotaviruses acknowledgements this study was supported by são paulo research foundation (fapesp). statement of animal rights the experimental design was approved by the animal research ethics committee of the school of veterinarian sciences of são paulo state university (unesp), under protocol number / . the authors declare that they have no conflict of interest. key: cord- -wckqscvm authors: maunsell, fiona p.; donovan, g. arthur; risco, carlos; brown, mary b. title: field evaluation of a mycoplasma bovis bacterin in young dairy calves date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: wckqscvm mycoplasma bovis is an important cause of pneumonia, otitis media and arthritis in young dairy calves, and there is a critical need for improved preventative strategies for this pathogen. we conducted a randomized, placebo-controlled, double-blinded field trial to determine the efficacy of a commercial m. bovis vaccine for the prevention of m. bovis-associated disease in calves. calves (n = ) on florida dairies with a history of m. bovis infection received an m. bovis bacterin or a placebo, administered subcutaneously at , and days of age. one of the herds did not experience m. bovis-associated disease; for calves in the remaining herds, the incidence risk for respiratory disease, otitis media and arthritis from to days of age was . , . and . , respectively. vaccination had no effect on the age at first treatment for m. bovis-associated disease, incidence of respiratory disease, mortality, weight gain, or nasal colonization with m. bovis in the first days of life. in one herd, vaccination was associated with an increased risk of otitis media. there was no association between m. bovis-specific serum antibody titers and morbidity in vaccinated calves. under the field conditions in this study, this vaccine was not efficacious for the prevention of m. bovis-associated disease in young dairy calves. mycoplasma bovis is a significant world-wide pathogen of adult dairy cows as well as intensively reared beef and dairy calves [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in the past decade, m. bovis has emerged as an increasingly important cause of respiratory disease, otitis media and arthritis in young calves less than months of age [ , , , , ] . clinical disease caused by m. bovis tends to be chronic, debilitating and unresponsive to antimicrobial therapy [ ] [ ] [ ] [ ] [ ] [ ] [ ] . disease outbreaks with high morbidity rates occur [ , , , , ] and can be economically devastating for the affected farm. the costs of infection are primarily associated with intensive treatment of affected calves coupled with culling of animals that are unresponsive to therapy [ ] . m. bovis-associated disease is also important from an animal welfare perspective as it often results in calves that are subject to severe, chronic illness for which the producer or veterinarian can provide only limited relief. there is therefore a critical need to develop improved preventative and treatment strategies for m. bovis-associated disease. currently, control of m. bovis infection in calves focuses on removal of identified or potential risk factors. colonization of the upper respiratory tract of calves with m. bovis often occurs within the first few weeks of life [ , ] as a result of feeding of milk from cows infected with m. bovis or, probably, by direct or indirect transmission from other calves shedding m. bovis in nasal secretions. removal of infected milk from the diet by pasteurization or feeding of milk replacer has been successfully applied to reduce infection [ , , , , ] . breaks in pasteurization have been associated with subsequent infection outbreaks. management practices to reduce stocking density and improve ventilation are examples of changes that can reduce undifferentiated respiratory disease in housed calves and have been recommended for m. bovis control [ ] [ ] [ ] . similarly, control of other pathogens that are involved in the bovine respiratory disease complex is likely to reduce m. bovis infections. management techniques that improve general immune function, such as improving nutritional status and minimizing environmental stress, have also been suggested as beneficial [ , ] . vaccination is a potential strategy to control m. bovis infection, but efforts to develop efficacious vaccines against m. bovis for use in young calves have been problematic. vaccines against m. bovis have afforded some protection from respiratory disease in european field trials [ ] [ ] [ ] . other vaccines have been efficacious against respiratory disease [ , ] and arthritis [ , , ] in experimental challenge studies. however, in some cases vaccination against m. bovis has significantly exacerbated clinical disease [ , ] . in addition, most experimental challenge studies have been performed in calves that are substantially older than the age at which natural colonization with m. bovis is typically first observed. young calves are often colonized by m. bovis within the first few weeks of life [ , , , , ] , during which time the immune system is undergoing rapid changes associated with maturation [ , ] . therefore, agespecific features of the immune system are likely to be important in determining the susceptibility of the young calf to mycoplasmal disease and the efficacy of particular vaccines. there are several bacterin-based vaccines against m. bovis that are currently marketed in the u.s., as well as a number of companies that manufacture autogenous m. bovis bacterins. however, no commercial vaccines are licensed for use in the very young dairy calf, and, to the best of the author's knowledge, no independent studies have been published on their efficacy. the paucity of studies that critically evaluate currently marketed m. bovis vaccines and autogenous bacterins in well-designed, independent efficacy studies in an appropriate age group is a major gap in understanding the potential of currently available vaccines as a management strategy to control m. bovis infections in young calves. in part to address this gap, we conducted a field trial using a commercial m. bovis bacterin that was approved for use in feeder and stocker calves. the objective of this field trial was to determine the efficacy of this commercially produced m. bovis bacterin for the prevention of m. bovis-associated disease (respiratory disease, otitis media, arthritis) and mortality in dairy calves up to days of age. additional objectives were to compare vaccinated and placebo-treated calves with respect to ( ) weight gain from birth to days of age, ( ) rates of nasal colonization by m. bovis, and ( ) m. bovis-specific serum immunoglobulin (ig) concentrations. we studied holstein heifers in three florida herds using a randomized field trial design. the reference population for this study was heifer calves in florida dairy herds with endemic m. bovis infection. the study unit was a holstein heifer calf clustered in one of the three herds in north-central florida. herds were selected based on their willingness to participate and on a history of mycoplasma-associated disease in calves. according to calf health records, at least % of calves were treated for respiratory disease, otitis media and/or arthritis during each of the years preceding the study. calves were enrolled from march to december, . herd a had approximately lactating cows. calves were bedded on sand in individual hutches placed approximately m apart in an open-sided barn with mechanical ventilation. calves were fed unpasteurized bulk tank milk. calves received a modified live virus (mlv) intranasal vaccine against parainfluenza virus type (pi ) virus and infectious bovine rhinotracheitis virus (ibr) in the first week of life. an intramuscular mlv vaccine against ibr, pi , bovine respiratory syncytial virus (brsv) and bovine viral diarrhea virus (bvdv) types and was administered at , and weeks of age. a -way clostridial vaccine was administered at and weeks of age. calves were weaned at approximately weeks of age and turned out into group pens at approximately weeks of age. herd b had approximately lactating cows. the majority of calves were housed in individual elevated metal crates in a concrete-floored open-sided barn with mechanical ventilation. a small proportion of calves were housed in individual hutches on grass. calves housed in metal crates had nose-to-nose contact with neighboring calves. the feeding protocol varied during the study period and included milk replacer and unpasteurized or pasteurized waste milk. the vaccination protocol was similar to that described for herd a. calves were weaned at - weeks of age and turned out into group pens at - weeks of age. herd c had approximately lactating cows. calves were housed on grass in individual hutches placed at least m apart. calves were primarily fed pasteurized waste milk, supplemented with milk replacer when necessary. several failures of pasteurization were documented during the study period through culture of milk following pasteurization. calves received an oral bolus containing antibodies against bovine coronavirus and escherichia coli at the time of colostrum feeding (first defense, portland, me). the vaccination protocol for mlv intranasal pi /ibr and clostridial vaccines was similar to that described for herd a except that the intramuscular mlv vaccine against pi , ibr, bvdv types and , and brsv was administered at and weeks of age. calves were weaned at - weeks of age and turned out into group pens at - weeks of age. all holstein heifer calves that were born during the study period and were considered healthy by the producer at days of age were enrolled in the study. calves were assigned to one of the two groups based on ear-tag numbers, with odd numbers assigned to one group and even numbers to the other group. assignment of odd and even numbers to groups was decided on a per farm basis by a coin flip. one group received a ml dose of a killed, single strain m. bovis bacterin in proprietary oil-based adjuvant that had a conditional license for the prevention of respiratory disease in u.s. feeder and stocker calves at the time of the study (texas vet. labs, inc., san angelo, tx), while the other group received a placebo (all vaccine components except antigen; control group). vaccine or placebo was administered subcutaneously in the neck at and days of age. a ml dose was administered at days of age. the second or third doses were not administered to calves that were sick; however, if the calf recovered within days the dose was administered week after the due date. calves that failed to recover within days remained in the study but missed that dose; missed doses were recorded. the bacterin and placebo were prepared and blinded by the vaccine manufacturer, and the investigators and farm personnel were blinded throughout data collection and analysis. data recorded for each calf included date of birth, ear-tag number, group allocation, dates of vaccine/placebo administration, any missed doses of vaccine/placebo, and date of weaning. the dates of administration of any preventative treatments or other vaccines were recorded for each calf. the primary outcomes of interest were treatment for respiratory disease, otitis media, and arthritis as well as mortality attributed to these diseases. calves were followed from days until days of age, during which time all treatment for clinical disease was recorded by farm personnel using standardized case definitions. farm personnel were trained by the investigators and followed a standardized protocol for health assessment that was prepared by a veterinarian. respiratory disease was defined as fever (rectal temperature ≥ . • f) plus increased respiratory rate or effort and/or coughing and/or nasal discharge; otitis media/interna was defined as ear droop and/or evidence of ear pain (head shaking, scratching or rubbing ear); arthritis was defined as lameness with painful swelling of any joint. sick calves were treated as per normal farm protocols. for each clinically ill calf, farm personnel recorded the type and dose of antimicrobial, the date(s) of treatment, and the reason for treatment. whenever a calf died, farm personnel recorded the cause of death if this was obvious. in most cases, cause of death was verified by field necropsy performed by the investigators. study personnel visited each of the dairies at least once a week to collect calf health data, monitor compliance, and collect samples. because passive transfer of colostral immunoglobulins can influence the immune response to vaccination or to infectious agents, blood was collected from all calves between and days of age for the measurement of total serum protein concentration. a subset of calves from herds a (n = ) and b (n = ) was studied more intensively. these calves were weighed at birth and approximately days of age. weight gain from birth to days was expressed in kg/day. nasal swabs and blood samples were collected weekly until weeks of age and then at days of age. prior to collecting nasal swabs, gross debris was wiped from the external nares using sterile gauze. a sterile rayon-tipped swab with a polyurethane plastic shaft (bbl tm cultureswab tm liquid stuart medium, bd, franklin lakes, nj) was inserted into the ventral nasal meatus to a depth of approximately in. swabs were cultured to detect nasal colonization with m. bovis. serum was analyzed for m. bovis-specific iga, igm, igg and igg by enzyme-linked immunosorbent assay (elisa). swabs were kept on ice during transport and were processed within h of collection. each swab was used to streak the surface of modified frey's agar. all mycoplasma cultures were performed in modified frey's broth and agar medium containing . % (wt./vol.) mycoplasma broth base (frey) (bd diagnostic systems, sparks, md), . % (wt./vol.) dna from herring sperm, % (vol./vol.) horse serum, % (vol./vol.) fresh yeast extract, . % (wt./vol.) glucose, and supplemented with , u/l each of penicillin g and polymixin b and mg/l of cefoperazone, with the final ph adjusted to . - . . plates were incubated at • c in % co and examined at , , and days for mycoplasmal growth. colonies with typical m. bovis morphology were plugged into broth, incubated at • c for h and stored at − • c until they could be confirmed as m. bovis by polymerase chain reaction (pcr). to prepare samples for pcr, l of broth culture was thawed at room temperature then pelleted by centrifugation at , rpm at • c for h. the pellet was resuspended in l of lysis buffer ( mm tris [hydroxymethyl] aminomethane, ph . with . % [vol./vol.] tween and . mm dithiothreitol). samples were incubated at • c for min then cooled to • c. l of clarified sample was used as the dna template in the pcr. as a positive control, broth was inoculated with the m. bovis type strain pg and processed with nasal isolates. sterile water was used as a negative control template. m. bovis was identified by pcr of the housekeeping gene uvrc, as previously described [ ] . pcr products were analyzed by electrophoresis at v for h in . % agarose gels and visualized by staining with ethidium bromide. blood samples were allowed to clot after collection, and then serum was harvested by centrifugation and stored at − • c. whole-cell lysate antigen [ ] was prepared from a l culture of m. bovis type strain pg grown at • c in modified frey's broth. the protein concentration was determined using a colorimetric assay (bio-rad, hercules, ca) and adjusted to g/ml. the antigen was stored in aliquots at − • c and thawed at room temperature when required. the elisa procedure was optimized using standard methodology. microtiter plates (maxisorb f , nunc, kamstrup, denmark) were coated with g per well of antigen in . m sodium phosphate buffer (ph . ) containing . m nacl and . % (wt./vol.) nan (pbs/a), and incubated overnight at • c. plates were then washed three times with pbs/a containing . % (vol./vol.) tween (pbs/t) using an automated plate washer (elx auto plate washer, biotek instruments, inc., winooski, vt), blocked with l per well of blocking buffer (pbs/t containing % [wt./vol.] egg albumin), and stored at • c for a minimum of h or until needed. sera were diluted ( : for igg assay; : for igm and igg assays; : for iga assay) in blocking buffer and l of the diluted serum was added to duplicate wells; plates were incubated at room temperature for h. plates were washed as described above and l of goat anti-bovine isotype conjugated to alkaline phosphatase (bethyl laboratories inc., montgomery, tx) and diluted to : in blocking buffer was added to each well. plates were incubated at room temperature for h and then washed as described above. l of . % (wt./vol.) p-nitrophenol phosphate was added to each well and plates were incubated in the dark at room temperature for h. the optical density (od) in each well was read at a wavelength of nm using an automated plate reader (elx ultra microplate reader, biotek instruments, inc., winooski, vt). for each microtiter plate, the blank was the mean value for two wells coated with antigen and incubated with the conjugated secondary antibody and substrate only. the blank od value was subtracted from each sample well, and mean values for each pair of duplicate tests calculated. a pool of sera from calves with naturally occurring mycoplasmal disease and high m. bovis-specific titers was included on each plate as a positive control; the negative control was a pool of serum collected from the same calves prior to ingestion of their first colostrum meal. the cutoff for a positive result was the average od value (minus the blank) for the negative control sera plus two standard deviations, established over ten assay runs. all elisa data was reported as the average od value at the standard serum dilution for each isotype. within-batch and between-batch assay variabilities were assessed by using the youden plot graphic method [ ] . the elisa values obtained for the lowest, middle and highest dilution of the control serum included on each plate were used to establish target values and control limits to be used for monitoring the consistency of the assay (ten batches). the values obtained at the beginning of a series of assays were plotted against the values obtained for the same standards at the end of the series. if values for the pooled sera deviated more than % from target values, the assay was repeated. a standard field necropsy was performed by a study veterinarian on most calves that died or had to be euthanized. selected calves were not necropsied when a cause of death was obvious and there had been no clinical evidence of m. bovis infection (e.g. a leg fracture that necessitated euthanasia). calves were examined to determine the cause of death and specifically determine the involvement of m. bovis-associated pathology. cases were defined as m. bovisassociated mortality when there had been a history of respiratory disease, otitis media, and/or arthritis, where m. bovis (with or without other pathogens) was isolated from sites of pathology, and where the observed pathology was consistent with mycoplasmal disease. all necropsies included collection of swabs of the palatine tonsils, tympanic bullae and primary bronchi for mycoplasmal culture. additionally, if the animal had previously been diagnosed with respiratory disease, arthritis or otitis media, or if any macroscopic lung pathology was observed, appropriate samples were collected from the lesion site(s) to determine the involvement of m. bovis as well as other viral and bacterial respiratory pathogens. further samples were collected when deemed necessary to determine the cause of death by the veterinarian performing the necropsy. all swabs and fresh tissue samples were transported on ice to the laboratory as soon as possible and were processed within h after collection. when tissue samples for fixation were collected, they were placed into containers of % buffered formalin and submitted to the diagnostic pathology service, college of veterinary medicine, university of florida. samples then were embedded in paraffin and sections ( m) stained with hematoxylin and eosin. histopathology was read by diagnostic pathologists without knowledge of experimental treatment groups. in addition to culture for mycoplasmas (described under nasal swabs, above), swabs for aerobic microbiological culture were processed and isolates identified using routine clinical bacteriological methods. these methods were focused on identifying bacterial pathogens of the respiratory tract other than mycoplasmas, particularly arcanobacterium pyogenes, histophilus somni, mannheimia haemolytica and pasteurella multocida as well as pathogens that may cause septicemia and associated sequelae in young calves. additional diagnostic testing was performed as requested by the veterinarian who conducted the necropsy based on the presumptive diagnosis and any macroscopic pathology. samples were submitted to the florida state diagnostic laboratory (kissimmee, fl) for detection of bovine respiratory viruses when indicated. morbidity due to m. bovis was the major outcome of interest and was therefore used to calculate sample size. at the time the study was initiated, health records indicated that the incidence of respiratory disease, otitis media and/or arthritis in the study herds was at least %. we hypothesized that a reduction in incidence to % would be biologically and economically significant. using these values together with % confidence and % power, and taking into consideration an attrition rate of approximately %, the calculated sample size was calves per group. for the secondary outcomes of interest from a subset of calves in herds a and b, we hypothesized that a reduction in the nasal colonization rate from % to % would be biologically significant. using these values together with the parameters outlined above, the calculated sample size was calves per group. calves were excluded from analyses if clinical signs referable to other organ systems occurred concurrently with respiratory disease, otitis media or arthritis, with the exception of diarrhea without fever of less than days duration. incidence risk rates were calculated as the number of calves treated for the disease of interest divided by the initial number of calves less half the calves that died during the study for reasons other than the disease of interest. categorical outcome variables (incidence of clinical disease [%], incidence of mortality [%], incidence of missed dose of vaccine/placebo [%], cumulative risk of nasal colonization [%]), were compared among groups using chi-square tests; data were analyzed for effects of herd and passive transfer status by stratified mantel-haenszel analysis. when the expected count in any cell was less than , fisher's exact test was used. quantitative outcome variables (post-colostral total serum protein concentrations [g/dl], age at first treatment for clinical disease [days], average daily weight gain from birth to days of age [kg/day]) were compared among groups using independent sample t-tests, and elisa data were analyzed using repeated measures anova. the relationships between average daily weight gain and serological data (post-colostral elisa data, week elisa data) were examined using pearson correlation analyses. a p value of < . was considered significant. all analyses were performed using commercial statistical software (spss . , spss inc., chicago, il). three hundred and twenty-eight calves from herds b and c ( and calves, respectively) were enrolled and were eligible for inclusion in the study (table ) . despite a history of m. bovis infection, herd a did not experience any m. bovis-associated disease a in herd b, one calf was excluded because of concurrent disease and one calf was excluded because a dose of vaccine/placebo was inadvertently missed; in herd c, one calf was excluded because of concurrent disease and one calf was excluded because it received a dose of vaccine/placebo for the wrong group. during the study and therefore is excluded from some analyses, but data from this herd are included where relevant. the incidence risk for clinical respiratory disease, otitis media, and arthritis was assessed from to days of age (table ) . m. bovis-associated respiratory disease and otitis media were major contributors to calf disease in herds b and c. eight cases of arthritis were observed in herd b, and none were observed in herd c. herd a had a much lower overall mortality risk ( . ) than did herds b ( . ) and c ( . ); m. bovis-associated mortality in herd b accounted for the majority of the mortality risk ( . vs. . overall). vaccinated and control groups had equivalent levels of postcolostral total serum protein. a small percentage of calves did not receive their second vaccine/placebo due to illness, and this was not different between groups. although there were no significant differences for calves that missed the third vaccine/placebo when data from herds b and c were combined ( % [ / ] vaccinate vs. % [ / ] control), no control calves in herd b missed the third placebo as compared with % ( / ) of vaccinated calves that missed the third vaccine (p = . ). vaccination was not efficacious in limiting m. bovis-associated morbidity in the young dairy calves in this field trial. vaccination did not influence the age of first treatment for either otitis media or respiratory disease. the overall age for first treatment of otitis media was ± days for vaccinated calves and ± days for control animals. calves in herd c were treated for otitis at ± days (vaccinated) and ± days (control), whereas treatment was at a later age in herd b ( ± days and ± days for vaccinated and control calves, respectively). a similar pattern was observed for respiratory disease, with no difference in the overall age at first treatment between vaccinated and control calves ( ± days, vaccinated; ± days, control). as for otitis media, calves in herd herd a calves were excluded as no morbidity was observed in these animals. "all respiratory" includes calves that were treated for respiratory disease alone or for respiratory disease in conjunction with either otitis media or arthritis. "all otitis media" includes calves that were treated for otitis media alone or for otitis media in conjunction with either respiratory disease or arthritis. mbad = mycoplasma bovisassociated disease. a one control calf with arthritis also had respiratory disease. b combined (herds b and c) analyses were not performed for data that included otitis media because of the interaction between vaccination and herd for this outcome. c required earlier treatment for respiratory disease ( ± days, vaccinated; ± days, control) than did calves in herd b ( ± days, vaccinated; ± days, control). vaccination did not reduce the overall m. bovis-associated morbidity or the morbidity specifically associated with otitis media or respiratory disease. in fact, vaccination was associated with increased incidence of otitis media (p = . ) in herd b, but no differences in the incidence of otitis media between groups were observed in herd c (table ) . for herds a and b, weight gain was monitored from birth to days of age; no significant difference was observed in average daily gain between groups in herd a. similarly, no significant difference was observed in average daily gain between vaccinated ( . ± . kg/day, n = ) and control ( . ± . kg/day, n = ) calves in herd b, where endemic m. bovis disease was present. neither overall mortality rates (vaccinated, % [ / ] ; control, % [ / ]) nor m. bovis-associated mortality rates (vaccinated, % [ / ]; control, % [ / ] ) differed significantly between vaccinated and control calves. in herd b, m. bovisassociated mortality contributed to % ( / ) and % ( / ) of mortality events in vaccinated and control calves, respectively. although m. bovis-associated mortality also occurred in herd c, it was not the primary cause of calf mortality: only % ( / ) of mortalities in vaccinated and % ( / ) of mortalities in control calves in herd c were attributable to m. bovis. nasal colonization was not affected by vaccination; in herd b where endemic m. bovis disease was present, the mean percentage (±sd) of calves with m. bovis-positive nasal swabs at each sampling time was . ± . % for vaccinated calves and . ± . % in control calves. the average number of sampling times that m. bovis was recovered from each calf was also not associated with vaccination (data not shown). irrespective of vaccination status, however, the temporal pattern of colonization (fig. ) observed in calves from herd a (no mycoplasmal disease) was quite different from that observed in calves in herd b (significant mycoplasmal disease). calves in herd a had minimal to no nasal colonization with m. bovis during the pre-weaning period. calves in herd a were moved out of individual hutches into outdoor housing in group lots after the -week samples were collected; at the next sampling period ( weeks of age), the level of nasal colonization was similar to that in the herd that experienced m. bovis-associated disease. in herd b, calves had m. bovis present in nasal secretions as early as week of age, and by weeks of age over % of calves were colonized in the upper respiratory tract. this level of colonization was maintained throughout the sampling period. the serum antibody subclass response in a subset of calves in herds a and b was assessed by elisa (fig. ) . no significant differences between vaccinated and control calves were found in either herd a or b for iga, igm, or igg . however, a serum igg response to vaccination was detected. significant differences (p < . ) between vaccinated and control groups were first evident at weeks of age in herd a (no endemic disease) and at weeks of age in herd b (endemic disease). we then assessed if there was an association between immunoglobulin subclass response and outcomes in calves from herd b. there was no association of any immunoglobulin subclass response with morbidity or nasal colonization rate (data not shown). the m. bovis-specific serum igg titers at weeks of age were negatively correlated with average daily gain for control calves (r = − . , r = . , p < . ), but not for vaccinated calves (r = − . r = . , p = . ). interestingly, there was no significant association between postcolostral total serum protein concentrations and the incidence or duration of treatment for respiratory disease or otitis media in herds b and c (data not shown). similarly, the incidence of m. bovis-specific calf mortality in herds b and c was not associated with post-colostral total serum protein concentrations (data not shown). there was also no association between post-colostral m. bovis-specific iga, igm, igg or igg serum titers and either . no significant differences were observed between vaccinated and control calves in the iga, igm, or igg subclasses. vaccinated calves in herd a at , and weeks of age and in herd b at weeks of age had significantly higher levels (p < . ) of igg than did control calves at those time points. results are presented as mean optical density ± sd. morbidity or mortality for the intensively studied subset of calves (n = ) in herd b (data not shown). the commercial m. bovis bacterin tested in this trial was not efficacious in the prevention of either m. bovis-associated respiratory disease or otitis media in pre-weaned calves in two florida herds with endemic m. bovis disease. the response to vaccination was herd-dependent, and a higher rate of otitis media was associated with vaccination in one herd. our findings are not unique; although other investigators have reported some protection from m. bovisassociated disease by vaccination of older calves [ , , , ] , adverse outcomes following vaccination against m. bovis have also been reported [ , , ] . protection from mycoplasmal respiratory disease by subcutaneous vaccination of calves with killed whole cell bacterins has been reported [ , , , ] . in a study of an apparently efficacious vaccine in young calves, nicholas et al. [ ] vaccinated -week-old dairy calves with a single dose of inactivated saponin-adjuvanated bacterin. calves received an aerosol challenge with live m. bovis weeks after vaccination. vaccinated calves had fewer numbers of m. bovis at colonized sites, fewer body sites colonized by m. bovis, and reduced severity and incidence of clinical disease and lesions as compared to control calves. there was also a significant decrease in body weight gain in control calves compared with vaccinates. additionally, no vaccinated calves and two of seven control calves developed arthritis. vaccinated calves produced a strong igg response prior to challenge, but igg subtypes were not reported. no adverse events associated with vaccination were reported. a killed vaccine against four bovine respiratory pathogens (brsv, pi , m. bovis, and m. dispar) was evaluated for protection against naturally occurring respiratory disease in beef calves [ , ] . calves were vaccinated subcutaneously and received two boosters at -week intervals. in one study [ ] , three groups of beef calves aged , and weeks at the time of first vaccination were used, and calves were followed for months. respiratory disease occurred in a significantly higher (p < . ) proportion of the control calves ( %) compared with the vaccinates ( . %). in a second study [ ] using the same vaccination protocol, m. bovis and brsv were implicated in outbreaks of respiratory disease during the trial period. morbidity due to respiratory disease was significantly reduced in vaccinated calves ( %) compared with controls ( %), and mortality in the vaccinated group was similarly reduced ( % and % for vaccinates and controls, respectively). no adverse effects of vaccination were noted. there are a number of key differences between the studies reported above and our study that may have influenced vaccine efficacy. firstly, the strain of bacteria, the antigen concentration, the method of bacterial inactivation and the adjuvant used are all factors that influence the efficacy of bacterial vaccines, although there are limited data on how these affect m. bovis vaccines in particular. although some of these data are not reported in the above studies, and some are not available for our vaccine (e.g. the adjuvant used is proprietary), it is likely that all these factors varied among our study and those listed above. secondly, calves in the above studies were first vaccinated at a substantially older age than the calves in our study. immune responses of the newborn calf have unique characteristics and undergo rapid changes during the first few weeks of life [ ] . vaccination at , and days of age (as was performed in our study) may not elicit the same type of immune response as vaccination at weeks of age (as in the nicholas et al. [ ] study, above). our vaccination protocol was chosen based on (a) protocols that were being applied on dairies in florida, and (b) the early age of infection that had been observed in previous studies [ ] . thirdly, calves in endemically infected herds in our study became colonized at a very early age, meaning that infection was likely well established before a vaccine-induced immune response could develop. adaptive immune responses that develop after infection are very inefficient at clearing mycoplasmal infections and often result in detrimental chronic inflammatory responses, and this could contribute to vaccine failure when animals are already infected at the time of vaccination. lastly, the challenge load of m. bovis that calves are exposed to can affect the efficacy of vaccination. given the high incidence of clinical mycoplasmal disease ( % of calves treated for m. bovis-associated disease) and the early age of colonization observed in our endemically infected herds, the level of m. bovis challenge that calves were exposed to may have been significantly greater than that of the calves in other vaccine studies [ , , ] . vaccinated calves in one herd in our study had a greater risk of otitis media than did control calves. the risk of otitis media in control calves in herd b seemed substantially less than that in herd c, but examination of calf health records from previous years in herd b showed that the risk of otitis media observed in control calves was similar to that which had been historically present (data not shown). therefore, vaccination seemed to exacerbate clinical otitis media in this herd. there are other reports of exacerbation of clinical disease following m. bovis vaccination [ , , ] . however, the immune mechanisms associated with adverse outcomes after m. bovis vaccination have not been determined. vaccination of calves did stimulate a systemic humoral immune response, with an increase in serum igg being detectable after the third vaccination. a tendency towards th -biased igg -dominated humoral responses has also been reported after infection of calves with m. bovis [ , ] . as igg is a much more effective opsonin for phagocytosis of m. bovis than is igg , an igg response may be relatively ineffective for control of m. bovis respiratory infections [ ] . it is somewhat puzzling that a humoral response to infection was not obvious in control calves in herd b where there was a high incidence of m. bovis-associated disease. statistical comparison of igg responses in control groups in herds a and b was not conducted. however, it appears that in the control group in herd a, post-colostral igg antibody levels continued to decline throughout the study period (see fig. ), whereas in the control group in herd b, they did not decline after weeks of age. this result may reflect continued stimulation of the immune response as a result of the endemic nature of m. bovis in this herd. additionally, m. bovis infection can result in local mucosal antibody responses without eliciting a substantial systemic humoral response [ ] . other investigators have also noted a poor correlation between serum antibody responses and m. bovis infection in individual calves during the first months of life [ ] . the vaccine used in our study was ineffective at preventing upper respiratory tract colonization with m. bovis in calves, even when colonization occurred after a humoral immune response to vaccination was established. with the exception of one calf, nasal colonization was not detected in calves in herd a until weeks of age, whereas a significant increase in serum igg responses to vaccination was evident by weeks of age. this is consistent with other reports on m. bovis vaccines; even where m. bovis vaccines have been associated with clinical benefits, they typically fail to induce an immune response that prevents upper respiratory tract infection [ , ] . our findings are consistent with the idea that protection from m. bovis infection is better correlated with local mucosal immune responses than with serum antibody titers [ ] . total protein concentrations or m. bovis-specific antibody levels in post-colostral serum were not associated with protection from m. bovis-associated disease in calves in this study. however, as colostrum was not pasteurized on these farms, it is possible that some colostrum containing high antibody concentrations to m. bovis may have come from cows with intramammary infection and therefore may also have contained live m. bovis. this could certainly mask any protective effect of passive transfer when assessed on a herd level. further studies are required to determine the efficacy of passive transfer for prevention of m. bovis-associated disease in a controlled setting. to the best of the authors' knowledge this is the first peerreviewed, controlled, independent efficacy study of any of the m. bovis vaccines available in north america. vaccination was not efficacious in preventing m. bovis-associated disease in pre-weaned calves in two endemically infected florida dairy herds, nor was it effective at preventing colonization of the upper respiratory tract in older calves in a third herd. the vaccine did stimulate a systemic igg response that was detectable after the third vaccination, but most clinical disease occurred prior to this response. calves in these endemically infected herds were colonized with m. bovis at a very young age, and it is likely that this represents the greatest impediment to successful vaccination in this age group. the vaccine used in this field trial was formulated for use in much older cattle (stocker and feeder calves) for the prevention of respiratory disease, and the results of this study should not be extrapolated to infer whether vaccination is efficacious in that age group. our findings highlight the importance of targeting vaccines for use in young calves specifically to this age group, and illustrate some of the challenges to the development of an efficacious vaccine against m. bovis for use in very young calves, if vaccination of this age group is even possible. association of mycoplasma bovis with otitis media in dairy calves use of valnemulin in the control of mycoplasma bovis infection under field conditions isolation of mycoplasma species from the lower respiratory tract of healthy cattle and cattle with respiratory disease in belgium bulk tank milk analysis: factors associated with appearance of mycoplasma sp. in milk mycoplasmal mastitis in dairy herds mycoplasma bovis: disease, diagnosis, and control mycoplasma otitis in california calves naturally occurring mycoplasma bovis-associated pneumonia and polyarthritis in feedlot beef calves clinical study of the disease of calves associated with mycoplasma bovis infection increased severity of calf pneumonia associated with the appearance of mycoplasma bovis in a rearing herd changes in the bacterial flora of the upper and lower respiratory tracts and bronchoalveolar lavage differential cell counts in feedlot calves treated for respiratory diseases immunohistochemical and pathological study of mycoplasma bovisassociated lung abscesses in calves feedlot therapeutics mycoplasma bovis and primary bovine virus diarrhea virus co-association in chronic pneumonia of feedlot cattle: a histopathological and immunohistochemical study ann conf am assoc bov practitioners, rapid city (sd) otitis media in preweaned holstein dairy calves in michigan due to mycoplasma bovis pasteurization of discard mycoplasma mastitic milk used to feed calves: thermal effects on various mycoplasma nasal prevalence of mycoplasma bovis and iha titers in young dairy animals heating of milk to kill mycoplasma bovis destruction of mycobacterium paratuberculosis, salmonella spp., and mycoplasma spp. in raw milk by a commercial on-farm high-temperature, short-time pasteurizer dairy calf pneumonia-the disease and its impact bovine mycoplasmosis mycoplasma infection in cattle. i. pneumonia-arthritis syndrome protection against respiratory disease in calves induced by vaccines containing respiratory syncytial virus, parainfluenza type virus, mycoplasma bovis and m. dispar field trial of a quadrivalent vaccine against calf respiratory disease experiences with herd-specific vaccines against respiratory infections with m. bovis in a large feedlot an experimental vaccine for calf pneumonia caused by mycoplasma bovis: clinical, cultural, serological and pathological findings immunity to mycoplasma bovis infections of the respiratory tract of calves synovial immunoglobulin and antibody in vaccinated and nonvaccinated calves challenged with mycoplasma bovis immunoprophylaxis of experimental mycoplasma bovis arthritis in calves-protective efficacy of live organisms and formalinized vaccines test of an inactivated vaccine against mycoplasma bovis respiratory disease by transthoracic challenge with an abscessing strain pathology of induced mycoplasma bovis calf pneumonia in experimentally vaccinated animals mycoplasmal arthritis and meningitis in calves bovine neonatal immunology effects of age and nutrition on expression of cd , cd , and l-selectin (cd l) on t-cells from neonatal calves species identification of mycoplasma bovis and mycoplasma agalactiae based on the uvrc genes by pcr detection of antibodies to a pathogenic mycoplasma in desert tortoises (gopherus agassizii) with upper respiratory tract disease use of youden plot for internal quality control in the immunoassay laboratory experimental intramammary inoculation with mycoplasma bovis in vaccinated and unvaccinated cows: effect on local and systemic antibody response immune response of cattle to respiratory mycoplasmas characterization of the immune response to mycoplasma bovis lung infection interaction of mycoplasma dispar and mycoplasma agalactiae subsp. bovis with bovine alveolar macrophages and bovine lacteal polymorphonuclear leukocytes association of seroconversion with isolation of agents in transtracheal wash fluids collected from pneumonic calves less than three months of age the authors thank shelly lanhart for her skilled help in sample collection. for help with the laboratory work, we thank janet stevens, barbara crenshaw, dr. marissa curtis and dr. kelly kirk. we are deeply indebted to the herd owners and managers who agreed to participate in the study and to the calf-rearing personnel who recorded data and vaccinated calves. we thank dr. w.h. (bill) wohler at texas vet lab, inc. for providing the vaccine and vaccine vehicle for this study. this work was funded by the florida milk checkoff. key: cord- - m qwl authors: gomez, diego e.; arroyo, luis g.; poljak, zvonimir; viel, laurent; weese, j. scott title: implementation of an algorithm for selection of antimicrobial therapy for diarrhoeic calves: impact on antimicrobial treatment rates, health and faecal microbiota date: - - journal: vet j doi: . /j.tvjl. . . sha: doc_id: cord_uid: m qwl this study evaluated the impact of an algorithm targeting antimicrobial therapy of diarrhoeic calves on the incidence of diarrhoea, antimicrobial treatment rates, overall mortality, mortality of diarrhoeic calves and changes in the faecal microbiota. the algorithm was designed to target antimicrobial therapy in systemically ill calves from on two dairy farms. retrospective (farm : calves; farm : calves) and prospective (farm : calves; farm : calves) cohorts were examined for months before and after implementation of the algorithm. the mantel–haenszel test and kaplan–meier survival curves were used to assess the cumulative incidence risk (cir) and time to development of each outcome before and after implementation of the algorithm. the cir of antimicrobial treatment rates was % lower after implementation of the algorithm on both farms (cir . , % confidence interval . – . ). there was no difference in the cir of overall mortality, but the cri for mortality of diarrhoeic calves was lower in the period after implementation of the algorithm on one farm. the faecal microbiota of healthy calves from both farms at each time period were characterised using a sequencing platform targeting the v region of the s rrna gene. on both farms, there were significant differences in community membership and structure (parsimony p < . ). use of the algorithm for treatment of diarrhoeic calves reduced antimicrobial treatment rates without a negative impact on the health of calves. however, the experimental design did not take into account the potential confounding effects of dietary changes between the study periods. diarrhoea is responsible for more than % of mortality in dairy heifers < month of age in the usa (usda, ) and antimicrobial therapy is commonly recommended regardless of the aetiological agent (walker et al., ) . the reasons for this recommendation are not well established, but include prevention of bacteraemia and elimination of the suspected pathogen from the intestinal tract (constable, ) . however, antimicrobial therapy may not be beneficial in many (or most) cases of calf diarrhoea (e.g. diarrhoea due to viral or parasitic infections), may result in longer recovery times (berge et al., ) , and may contribute to antimicrobial resistance and environmental contamination with antimicrobial compounds (zhao et al., ; sura et al., ) . enteral and parenteral antimicrobial agents and their metabolites can be excreted in significant amounts through faeces and urine (elmund et al., ; feinman and matheson, ; zhao et al., ; sura et al., ) . farm environmental contamination with these antimicrobial residues could reach the gastrointestinal tract of healthy untreated calves, resulting not only in maintenance and development of antimicrobial resistance, but also producing alteration of the normal gut microbial populations (panda et al., ; schokker et al., ) . one approach to reduce and improve the use of antimicrobial agents on dairy farms is the application of algorithms to guide the user towards a more rational course of action (berge et al., ) . simple and cost effective measures such as this could be an asset to the dairy industry to improve calf management and reduce unnecessary usage of antimicrobial agents. therefore, the aims of the present study were to evaluate the impact of an antibiotic use algorithm on calf health (morbidity and mortality) and antimicrobial treatment rates, and to characterise the faecal microbiota of healthy calves before and after implementing the algorithm. impact of an antimicrobial use algorithm on calf health and treatment rates farms two large commercial dairy farms located within a km radius of the university of guelph, ontario, canada, were selected to participate in the study on the basis of convenience, since both farms had a record keeping system for documenting health and disease events, treatment of calves and outcomes. no changes in management in the years before and after enrolment in the study were anticipated (e.g. expansion, new buildings or major changes in disease management practices); however, there was an unexpected change between use of milk replacer and pasteurised milk between study periods on both farms. the characteristics of farms identified in each period are presented in table . the production systems on both farms consisted of free stall housing with an automated milking system. the milking herds consisted of approximately cows on farm and cows on farm . the average milk production was , kg/cow/year on farm and , kg/cow/year on farm . neither farm had a treatment protocol for diarrhoeic calves at the time of enrolment. calves that developed diarrhoea on farm were treated with three antimicrobial agents (trimethoprim-sulphadoxine, spectinomycin and lincomycin), while diarrhoeic calves on farm were treated with one antimicrobial agent orally (sulphamethazine) and one parenterally (trimethoprim-sulphadoxine or sodium ceftiofur). ethical approval for the study was obtained from the university of guelph animal care committee (approval number eaup ). results were compared between a retrospective cohort of calves examined for months before implementation of the algorithm ( calves on farm and calves on farm ) and a prospective cohort of calves examined for months after implementation of the algorithm ( calves on farm and calves on farm ). outcomes assessed before and after implementation of the algorithm were: ( ) incidence of diarrhoea; ( ) antimicrobial treatment rates; ( ) overall mortality; and ( ) mortality of diarrhoeic calves. electronic and paper-based calf health records during the course of the study were reviewed and the following events occurring during the first days of the life of each calf were recorded: ( ) date of birth; ( ) age and date at first diarrhoeic episode; ( ) age and date at time of first treatment for diarrhoea; and ( ) antimicrobial agents used. the outcome (survival or death) at days of life was registered. when the cause of death was not registered, the following decisions were made: ( ) if the calf died while being treated for diarrhoea, the death was attributed to diarrhoea; or ( ) if a calf died suddenly or while being treated for another disease (e.g. pneumonia), the cause of death was not considered to be diarrhoea. in the period following implementation of the algorithm, farm staff registered the cause of death; if the cause of death was not clearly identified, a gross post-mortem examination was performed, but no additional samples were collected for laboratory examination. a multidisciplinary team of large animal internal medicine and infectious disease specialists, an epidemiologist and the veterinary practitioner for each farm collaborated to develop an algorithm for use of antimicrobial agents. the algorithm was designed for farmers to evaluate four main clinical signs: ( ) presence of diarrhoea (defined as loose faeces that stay on top of the bedding, or watery faeces that sifts through the bedding); ( ) fever (rectal temperature > . c); ( ) haematochezia; and ( ) changes in demeanour and milk intake. depending on the table farm characteristics, management practices and antimicrobial treatment protocol on two dairy farms before and after implementation of an algorithm for treatment of diarrhoeic calves. presence of these clinical signs, each calf was assigned to a treatment with or without systemic administration of an antimicrobial agent (fig. ) . a healthy calf was defined as a calf with normal demeanour, faecal consistency and body temperature (rectal temperature < . c), and no major changes in milk intake. farm staff trained in health evaluation and use of the algorithm executed the protocol. regular farm visits were used to communicate with personnel to ensure that there was no misunderstanding or non-compliance. faecal microbiota of healthy calves in the period before and after implementation of the algorithm management practices for both farms during the study period are summarised in table . dietary changes occurred on both farms during the study period. on farm , calves were fed non-antibiotic treated, pasteurised milk during the period before implementation of the algorithm and non-medicated milk replacer in the period after implementation of the algorithm. on farm , calves were fed non-antibiotic treated milk replacer in the period before implementation of the algorithm and non-medicated, pasteurised milk in the period after implementation of the algorithm. faecal samples from healthy calves < days of age were collected from each farm weeks to week before implementation of the algorithm, along with healthy calves, matched for age and farm, months after implementation of the algorithm. calves were excluded if they had experienced a previous episode of diarrhoea, had other diseases (e.g. omphalophlebitis or pneumonia) or had received antimicrobial agents previously. calves that developed diarrhoea within days after sampling were excluded and new calves were enrolled in their places. faecal samples were obtained per rectum and stored at À c. dna extraction, amplification and sequencing of the bacterial s rrna gene dna extraction was performed as described by gomez et al. ( ) . dna was amplified with a set of oligonucleotide primers targeting the v region of the s rrna gene with overhanging adapters for annealing to illumina universal index sequencing adaptors (klindworth et al., ; slifierz et al., ) . the library pool was sequenced with an illumina miseq (illumina rta v . . ; mcs v . ) for cycles from each end. outcomes before and after implementation of the algorithm outcomes considered for epidemiological analysis were: ( ) incidence of diarrhoea; ( ) antimicrobial treatment rates; ( ) overall mortality; and ( ) mortality of diarrhoeic calves. differences in the risk of developing each of these outcomes were evaluated using the periods before and after implementation of the algorithm as the main exposure of interest; the effect of dietary changes on the epidemiological outcomes could not be evaluated. cumulative incidence risk (cir) was evaluated using the mantel-haenszel approach, stratifying on farm as the potential confounder. this approach was used to determine differences in the incidence of each of the four epidemiological outcomes between periods. time to development of each outcome was evaluated using kaplan-meier survival curves. the log rank x test was used to ascertain whether there were differences in the survival experiences of the calves in both periods. the null hypothesis was that the survival curves were similar in both periods. analyses were performed using stata data analysis and statistical software (statacorp lp). mothur software package (v. . . ) was used for bioinformatic analysis (gomez et al., ; weese and jelinski, ) . random subsampling was completed to normalise the sequence count. sampling coverage was assessed using good's coverage value. the inverse simpson's, shannon's evenness and chao- indices were used to calculate a-diversity and comparisons between groups were performed using the steel-dwass test. the community membership and structure were assessed as described previously (gomez et al., ; weese and jelinski, ) . the differences between groups were represented by dendrograms (figtree v . . . ). clustering of the groups was visualised by principle coordinate analysis (jmp , sas institute). relative abundances of the main phyla, classes, orders and families, and the main genera, were calculated and comparisons were performed using the mann-whitney u test (jmp , sas institute). changes in faecal microbiota were evaluated using the period (before and after implementation of the algorithm) as the main exposure of interest. similarly to the epidemiological analysis, the effect of specific management practices, such as the changes in diet between periods of assessment, could not be determined using this approach. benjamini and hochberg's false discovery rate (fdr) (benjamini and hochberg, ) was used to adjust p values for multiple comparisons (r! core team, ). bacterial taxa enriched in faeces in each period were identified using linear discriminant analysis effect size (lefse) (segata et al., ) , based on p < . and a linear discriminant analysis (lda) score > , using the online galaxy workflow framework. the number of different meta-communities (enterotypes) that the data could be clustered into was determined using the dirichlet multinomial mixture model (dmm) (holmes et al., ) . random forests classifier (rfc) (knights et al., ) was also used to determine whether a set of predictive features could be used to accurately identify samples from each period, farms and demographic characteristics, and selected farm practices identified on farms and in each period are presented in table . in the period before implementation of the algorithm, diarrhoeic calves from farm received three different antimicrobial agents concurrently, whereas two antimicrobial agents were used on farm (table ). in the period after implementation of the algorithm, all diarrhoeic calves were treated according to the antimicrobial use algorithm (table ; fig. ). data for antimicrobial treatment rates, incidence of diarrhoea, and overall mortality and mortality of diarrhoeic calves, on both farms for each period are presented in table . on both farms, there was a marked reduction in the cumulative risk of administering antimicrobial treatment following implementation of the algorithm ( table ). the cir of antimicrobial treatment for diarrhoea in the period after implementation of the algorithm was % lower than in the period before implementation (incidence risk ratio, irr, . , % ci . - . ; p < . ) and these estimates were similar between farms. on farm , the cir of diarrhoea was % lower following implementation of the algorithm (irr . ; % ci . - . ; p < . ), but there was no difference in cir before and after implementation of the algorithm on farm (irr . , % ci . - . ; p = . ). the risk of mortality of diarrhoeic calves was lower after implementation of the algorithm on farm (irr . , % ci . - . ; p = . ). table number of calves (and age), calves with diarrhoea, calves treated with antimicrobial agents, overall mortality (and age) and mortality of diarrhoeic calves on two dairy farms before and after implementation of an algorithm for treatment of diarrhoeic calves. age presented as median and range in brackets. difference in the risk of antimicrobial treatment, development of diarrhoea, overall mortality and mortality in diarrhoeic calves before and after implementation of the algorithm. risk ( there was no significant difference in overall mortality before and after implementation of the algorithm (table ) . survival curves indicated that calves raised before implementation of the algorithm were more likely to be treated with antimicrobial agents than those raised after implementation of the algorithm (fig. ) . the time to treatment with antimicrobial agents was different between study periods on both farms and overall (log rank p < . ; fig. ). calves developed diarrhoea at an older age after implementation of the algorithm on both farms (log rank p value < . ; fig. ). there were no significant differences in the time to death (overall mortality and mortality of diarrhoeic calves) between the periods before and after implementation of the algorithm (log rank p > . ; fig. ). the age distribution (in days) of healthy calves included in this study for microbiota assessment was similar within and between farms for both periods before and after implementation of the algorithm. on farm , the mean ages of calves were ae and ae days before and after implementation of the algorithm, respectively; on farm , the mean ages of calves were ae days and ae days, respectively (p values > . for all comparisons). a total of , , reads were obtained, with a mean of , reads per calf (median , ; range , - , ; standard deviation , ). a random subsample of , reads per sample was used to normalise the data. subsampling was considered to be adequate, as evidenced by good's coverage obtained for all samples (median . %; range . - . %). in the period after implementation of the algorithm, there was a significant increase in richness (farms and ) and diversity (farm ) of the faecal microbiota of healthy calves (fig. ) . twenty-eight different phyla were identified; firmicutes, actinobacteria, proteobacteria and bacteroidetes accounted for more than % of sequences (see appendix: supplementary fig. ) . changes in the relative abundances of the main phyla are presented in table and appendix: supplementary fig. . the relative abundance of bacteroidetes was significantly higher in the table relative abundance (median in percentage and ranges) of the main phyla (> %), classes (> . %) and orders (> % of the total of sequences) identified in faeces of healthy calves from farms and before and after implementation of the algorithm. farm period after implementation of the algorithm than in the period before implementation on both farms. on farm , the relative abundance of proteobacteria was significantly lower in the period after implementation of the algorithm. sixty-seven different classes, orders and families were identified; classes, orders and families accounted for ! . % of sequences. the relative abundances of the most abundant bacterial taxa (class, order, family) identified in faeces in the periods before and after implementation of the algorithm are presented in tables and . overall, genera were detected; genera were present at relative abundances of > . %. changes in the relative abundances of the most abundant genera in each period are presented in table and appendix: supplementary fig. . enriched phylotypes in faeces of calves in each period are presented in fig. a (farm ) and appendix: supplementary fig. a (farm ). the genera that were enriched in faecal samples in the period before and after implementation of the algorithm are presented in fig. b (farm ) and appendix: supplementary fig. b (farm ). there were significant differences in community membership (jaccard index) and community structure (yue and clayton index) of faecal microbiota between the periods before and after implementation of the algorithm (see appendix: supplementary table ). these differences can be visualised in the dendrograms (fig. ) and pcoa plots (see appendix: supplementary fig. ) . using the dmm, two meta-communities (enterotypes) were identified; the first group of enterotypes comprised all faecal samples collected before implementation of the algorithm, while the second group of enterotypes contained all samples obtained in the period after implementation of the algorithm. the rfc analysis identified a % error rate for classifying samples (based on the taxa identified in each sample) into the appropriate period (before and after implementation of the algorithm), with a % error rate for classifying samples into the appropriate farm or farm/period. these results indicated that rfc had a stronger ability to separate samples by the appropriate period rather than into the appropriate farm or farm/period. the implementation of an algorithm for treatment of diarrhoea targeting systemically ill calves resulted in a reduction in antimicrobial treatment rates of %, with no identifiable negative impacts on clinical outcome. few clinical trials have investigated the effectiveness of protocols to reduce and refine antimicrobial treatment in pre-weaned calves. a clinical trial investigating the effect of conventional therapy on the health and growth of calves on one farm (four antimicrobial agents administered to any diarrhoeic calf) and targeted therapy (two antimicrobial agents administered to diarrhoeic calves with depression or fever) failed to detect differences in morbidity and mortality rates between groups (berge et al., ) . furthermore, the conventional therapy group had % more days of diarrhoea than the targeted therapy group. similarly, our study demonstrated that targeting antimicrobial therapy to calves that are systemically affected is a feasible approach to decrease the use of antimicrobial agents in diarrhoeic calves, with possible beneficial effects on health (fewer days of diarrhoea) (berge et al., ) . historically, farmers and veterinary practitioners have been concerned that delayed or non-treatment with antimicrobial agents could have a negative impact on calf health and welfare. however, in our study, targeting therapy to systemically ill diarrhoeic calves resulted in lower rates of antimicrobial treatment, without a negative effect on the overall morbidity and mortality attributed to diarrhoea. similar results have been documented in some european countries, in which the use of antimicrobial agents in farm producing animals has decreased by > %, with a minor impact on health and productivity (wierup, ; aarestrup et al., ; speksnijder et al., ) . possible reasons for the lack of adverse effects include improvements in diet, including the quality and quantity of colostrum, water quality, housing and environmental conditions. improving feed quality in pig production can contribute to reduced antimicrobial use and table relative abundance (median in percentage and ranges) of the families (> %) and genera (> . % of the total of sequences) identified in faeces of healthy calves from farms and during the before and after period. farm before farm after adjusted p value farm before farm after adjusted p value maintenance of animal health (postma et al., ) . poor housing conditions are an impediment to decreasing antimicrobial use in pig farms in the united kingdom (coyne et al. ). improvements in housing and environment are important for prevention of disease in dairy cows (leblanc et al., ; vaarst et al., ) . changes in the behaviour of veterinarians and farmers towards the usage of antimicrobials in calves, including focussing efforts on preventative measures (e.g. optimal housing and hygiene practices, climate control, and improved feed and water quality), with the aim to enhance the health of calves and the quality of the environment, may contribute to the reduction in antimicrobial use without a negative impact on the health of calves. differences in bacterial membership and structure of the faecal microbiota of calves in the periods before and after implementation of the algorithm were evident on both farms. diet, pathogen occurrence, environmental factors (e.g. season) and reduction in antimicrobial treatment rates could have influenced the composition of gut microbiota and could play a role in the observed temporal changes (jami et al., ; rey et al., ) . our statistical analyses used the period before and after implementation of the algorithm as the main exposure of interest. this approach meant that we could not differentiate the effects of dietary changes from the reduction in antimicrobial treatment rates on the faecal microbiota of healthy calves. the dmm analysis identified the presence of two groups of enterotypes comprising all samples from the period before and after implementation of the algorithm, respectively, irrespective of the farm of origin, and the rfc analyses had a perfect ability to separate samples into their appropriate period ( % error rate). in addition, the results of the lefse analyses demonstrated that the changes on faecal microbiota were similar in both farms in the period after implementation of the algorithm. these results suggest that a factor common to both farms, i.e. the reduction in antimicrobial treatment rates, may have contributed to changes in the faecal microbiota after implementation of the algorithm. although dietary changes occurred on both farms, the nutritional source was also different between farms (the diet was changed from non-antibiotic treated pasteurised milk to non-medicated milk replacer on farm , while the diet was changed from non-antibiotic treated milk replacer to nonmedicated pasteurised milk on farm ). if the dietary changes had a major role in the observed changes, the dmm would have been expected to identified enterotypes based on diet rather than period (e.g. all calves fed non-antibiotic treated pasteurised milk on farm before implementation of the algorithm and farm after implementation of the algorithm might be expected to be similar, and vice versa) (holmes et al., ) . in addition, rfc would have been expected to assign samples to farm/period rather than to period (knights et al., ) . whilst an impact of antimicrobial agents on calves has been demonstrated previously (smith and crabb, ; grønvold et al., ) , the potential changes in the faecal microbiota of healthy calves from farms having a marked reduction in antimicrobial treatment rates associated with the use of treatment algorithms has not been reported previously. the reduction in antimicrobial treatment rates and dietary changes on both farms after implementation of the algorithm were associated with decreased representation of members of the phylum proteobacteria (family enterobacteraceae and genera escherichia-shigella) in the faecal flora of calves. the higher representation of proteobacteria in healthy calves in period before implementation of the algorithm was unexpected, because enrichment with members of this phylum has been associated with intestinal dysbiosis in other species (costa et al., ; suchodolski et al., ; singh et al., ) , as well as diarrhoea in dairy calves (gomez et al., ) . marked differences in the faecal microbiota of healthy beef calves have been identified among farms, with some farms having firmicutes-dominant microbiota and others proteobacteria-dominant microbiota; in general, higher proteobacteria levels were present on farms with high usage of antimicrobial agents (weese and jelinski, ) . these results are aligned with the hypothesis that antimicrobial agents can have a broader or cumulative impact on farms, where regular use results in the development of a particular microbiota in those calves, regardless of their individual antimicrobial exposure (weese and jelinski, ) . the marked reduction in antimicrobial treatment rates and dietary changes also coincided with a significant increase of the bacteroides and multiple butyrate-producing bacteria (faecalibacterium and unclassified genera from the families lachnospiraceae and ruminococcaceae). these genera have been associated with 'gut health' in different species, including human beings (sokol et al., ) , horses , dogs (suchodolski et al., ) and calves (oikonomou et al., ) . we speculate that a reduction in the use of antimicrobial agents and changes in the dietary source may have had a beneficial effect on the gut microbiota of calves by favouring taxa associated with 'gut health' (bacteroidetes and butyrate-producing bacteria) over those associated with dysbiosis (proteobacteria). the specific changes (especially at the genus level) in microbiota were not consistent between the two farms. one possible explanation is impact of the geographic location and management practices within the farms. differences in the faecal microbiota of healthy calves from different farms have been demonstrated previously (gomez et al., ) . earlier studies based primarily on animals from single farms demonstrated a large degree of fig. . dendrograms representing the similarity of community structure (yue and clayton index, a) and membership (jaccard index, b) in faecal samples collected from healthy calves before (farm , purple; farm , green) and after (farm , blue; farm , red) implementation of an algorithm for antimicrobial treatment. inter-farm variation of faecal microbiota (edrigton et al., ; oikonomou et al., ; klein-jöbstl et al., ) . therefore, the variance in the faecal microbiota of healthy calves from different farms must be considered when designing and interpreting studies of microbiota in calves. differences in the occurrence of pathogens could have also contributed to the specific changes identified on faecal microbiota. in cattle, johne's disease caused by mycobacterium avium subsp. paratuberculosis is associated with increased proteobacteria and reduced firmicutes and bacteroidetes (fecteau et al., ) . similar changes on gut microbiota were identified in calves with undifferentiated neonatal diarrhoea (gomez et al., ) . a limitation of this study is that the two clinical algorithms were not implemented concurrently on the two farms and thus results could have been confounded by other time-dependent variables, such as environment, husbandry and other health management practices. to reduce possibility of confounding, the inclusion criteria aimed to include only herds that had no plan to change treatment and prevention protocols. although a randomised field trial with concurrent treatment arms within the same source population would have been the preferred design, its implementation was not possible because of the cost and effort required from farmers and veterinarians (e.g. farmers were willing to follow only one simple treatment protocol). another major limitation is that the confounding effects of changes in diet between periods on both farms could not be evaluated. however, the results of statistical analyses (i.e. dmm and rfc) suggested that the reduction in antimicrobial treatment rates was the variable with the main effect on the observed changes. the use of an algorithm for treatment of calf diarrhoea decreased the rates of antimicrobial treatment on two dairy farms without an adverse effect on the health of the calves. management practices and reduction in antimicrobial treatment rates at the farm level could have an impact on the development and establishment of faecal microbiota of healthy calves. none of the authors of this paper have a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. changes in the use of antimicrobials and the effects on productivity of swine farms in denmark controlling the false discovery rate: a practical and powerful approach to multiple testing targeting therapy to minimize antimicrobial use in preweaned calves: effects on health, growth, and treatment costs antimicrobial use in the treatment of calf diarrhoea comparison of the fecal microbiota of healthy horses and horses with colitis by high throughput sequencing of the v -v region of the s rrna gene understanding antimicrobial use and prescribing behaviours by pig veterinary surgeons and farmers: a qualitative study role of excreted chlortetracycline in modifying the decomposition process in feedlot waste dysbiosis of the fecal microbiota in cattle infected with mycobacterium avium subsp. paratuberculosis draft environmental impact statement: subtherapeutic antibacterial agents in animal feeds characterization of the fecal bacterial microbiota of healthy and diarrheic dairy calves fecal microbiota of calves in the clinical setting: effect of penicillin treatment dirichlet multinomial mixtures: generative models for microbial metagenomics exploring the bovine rumen bacterial community from birth to adulthood pyrosequencing reveals diverse fecal microbiota in simmental calves during early development evaluation of general s ribosomal rna gene pcr primers for classical and next-generation sequencing-based diversity studies supervised classification of human microbiota major advances in disease prevention in dairy cattle fecal microbial diversity in pre-weaned dairy calves as described by pyrosequencing of metagenomic s rdna. associations of faecalibacterium species with health and growth short-term effect of antibiotics on human gut microbiota alternatives to the use of antimicrobial agents in pig production: a multi-country expert-ranking of perceived effectiveness, feasibility and return on investment establishment of ruminal bacterial community in dairy calves from birth to weaning is sequential long-lasting effects of early-life antibiotic treatment and routine animal handling on gut microbiota composition and immune system in pigs metagenomic biomarker discovery and explanation intestinal microbial communities associated with acute enteric infections and disease recovery longitudinal study of the early-life fecal and nasal microbiotas of the domestic pig the typing of e. coli by bacteriophage, its application to the study of the e. coli population of the intestinal tract of healthy calves and of calves suffering from white scours faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of crohn disease patients reduction of veterinary antimicrobial use in the netherlands. the dutch success model the fecal microbiome in dogs with acute diarrhea and idiopathic inflammatory bowel disease dissipation of three veterinary antimicrobials in beef cattle feedlot manure stockpiled over winter united states department of agriculture animal and plant health inspection service veterinary services (usda-aphis-vs) development and daily management of an explicit strategy of nonuse of antimicrobial drugs in twelve danish organic dairy herds characteristics of dairy calf ranches: morbidity, mortality, antibiotic use practices, and biosecurity and biocontainment practices changes in the faecal microbiota of mares precede the development of post partum colic assessment of the fecal microbiota in beef calves the experience of reducing antibiotics used in animal production in the nordic countries residues of veterinary antibiotics in manures from feedlot livestock in eight provinces of china this work was supported by the ontario minister of agriculture and rural affairs (omafra; grant number ). preliminary results were presented as an abstract at the american college of veterinary internal medicine specialty symposium, denver, co, usa, - june . supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . /j.tvjl. . . . key: cord- -tqc usry authors: godden, sandra title: colostrum management for dairy calves date: - - journal: vet clin north am food anim pract doi: . /j.cvfa. . . sha: doc_id: cord_uid: tqc usry colostrum management is the single most important management factor in determining calf health and survival. unfortunately, a significant proportion of north american dairy calves suffer from failure of passive transfer of antibodies from colostrum, contributing to excessively high preweaning mortality rates and other short- and long-term losses associated with animal health, welfare, and productivity. a successful colostrum management program requires producers to consistently provide calves with a sufficient volume of clean, high-quality colostrum within the first few hours of life. this article reviews the process of colostrogenesis and discusses important components of colostrum. the key components of delivering and monitoring a successful colostrum management program are discussed. the placenta of the cow separates the maternal and fetal blood supplies, preventing in utero transmission of protective immunoglobulins (ig) [ ] . consequently, the calf is born agammaglobulinemic and so depends almost entirely on the absorption of maternal ig from colostrum after birth. the absorption of maternal ig across the small intestine during the first hours after birth, termed passive transfer, helps to protect the calf against common disease organisms until its own immature immune system becomes functional. calves are defined as having failure of passive transfer (fpt) if the calf serum igg concentration is less than mg/ml when sampled between and hours of age [ , ] . achieving early and adequate intake of highquality colostrum is widely recognized as the single most important management factor in determining health and survival of the neonatal calf ( fig. ) [ ] [ ] [ ] [ ] . in addition to reduced risk for preweaning morbidity and mortality, additional long-term benefits associated with successful passive transfer include reduced mortality in the postweaning period, improved rate of gain and feed efficiency, reduced age at first calving, improved first and second lactation milk production, and reduced tendency for culling during the first lactation [ ] [ ] [ ] [ ] . unfortunately, many producers continue to incur significant loss associated with fpt. in the united states mortality rates in preweaned dairy heifers are estimated to range between % and % [ , , ] . poor colostrum management is one of the key factors contributing to these excessive losses. in one study % of calves sampled between and hours of age had fpt (serum igg ! mg/ml) [ ] . it was estimated that approximately % of preweaning mortality events occurring in the first weeks of life were attributed to fpt [ ] . these studies point to the need for producers to adopt practices to improve colostrum management. this article reviews the process of colostrogenesis and discusses important components of colostrum. the key components of developing a successful colostrum management program are discussed. bovine colostrum consists of a mixture of lacteal secretions and constituents of blood serum, most notably ig and other serum proteins, which accumulate in the mammary gland during the prepartum dry period [ ] . this process begins several weeks before calving, under the influence of lactogenic hormones, including prolactin, and ceases abruptly at parturition. important constituents of colostrum include ig, maternal leukocytes, growth factors, hormones, cytokines, nonspecific antimicrobial factors, and nutrients. concentrations of many of these components are greatest in the first secretions harvested after calving (first milking colostrum), then decline steadily over the next six milkings (transition milk) to reach the lower concentrations routinely measured in saleable whole milk (table ) [ ] . igg, iga, and igm account for approximately % to %, %, and %, respectively, of the total ig in colostrum, with igg accounting for % to % of the total igg [ ] . although levels are highly variable among cows and studies, one study reported that mean colostral concentrations of igg, iga, and igm were mg/ml, . mg/ml, and . mg/ml, respectively [ ] . igg, and igg in particular, are transferred from the bloodstream across the mammary barrier into colostrum by a specific transport mechanism: receptors on the mammary alveolar epithelial cells capture igg from the extracellular fluid, and the molecule undergoes endocytosis, transport, and finally release into the luminal secretions [ ] . the alveolar epithelial cells cease expressing this receptor, most likely in response to increasing prolactin concentrations, at the onset of lactation [ ] . smaller amounts of iga and igm are largely derived from local synthesis by plasmacytes in the mammary gland [ ] . although not well understood, colostral transfer of ige also occurs and may be important in providing early protection against intestinal parasites [ ] . normal bovine colostrum contains greater than  cells/ml of immunologically active maternal leukocytes, including macrophages, t and b lymphocytes, and neutrophils [ , ] . at least a portion of colostral leukocytes are absorbed intact across the intestinal barrier [ ] . liebler-tenorio and colleagues [ ] reported that the preferential route of uptake of colostral leukocytes through the intestinal barrier is through the follicle-associated epithelium of peyer patches in the jejunum and ileum. reber and colleagues [ ] proposed that, after entering the neonatal circulation, maternal leukocytes traffic to neonatal nonlymphoid tissues and secondary lymphoid tissues, disappearing from the neonatal circulation by to hours after feeding colostrum. although their functional importance in calves is not routinely measured, early evidence suggests that colostral leukocytes enhance lymphocyte response to nonspecific mitogens, increase phagocytosis and bacterial killing ability, and stimulate humoral immune responses (igg formation) in the calf [ , [ ] [ ] [ ] . presumably these cells would not be viable in pasteurized colostrum or colostrum replacer products. the role and functional significance of colostral leukocytes remains areas of active research. other important components of colostrum include growth factors, hormones, cytokines, and nonspecific antimicrobial factors. bioactive components of colostrum with antimicrobial activity include lactoferrin, lysozyme, and lactoperoxidase [ ] [ ] [ ] . oligosaccharides in colostrum may provide protection against pathogens by acting as competitive inhibitors for the binding sites on the epithelial surfaces of the intestine [ ] . growth factors in bovine colostrum include transforming growth factor beta- (tgf-b ), growth hormone (gh), and insulin, but their function in colostrum is not fully understood (see table ) [ ] . colostral insulinlike growth factor-i (igf-i) may be a key regulator in the development of gastrointestinal tracts of bovine neonates, including stimulation of mucosal growth, brush-border enzymes, intestinal dna synthesis, increased villus size, and glucose uptake increased [ ] [ ] [ ] . trypsin inhibitor, a compound found in colostrum in concentrations nearly times greater than in milk, serves to protect igg and other proteins from proteolytic degradation in the intestine of the neonatal calf. although the immunologic importance of colostrum is frequently discussed, the nutritional significance of the first colostrum meal should not be overlooked. the total solids content (%) in first milking colostrum and whole milk in holstein cows has been reported to average . % and . %, respectively (see table ) [ ] . much of this increase in colostrum solids content is attributed to a more than fourfold increase in protein content of colostrum versus milk, this being because of significant increases in ig and casein content [ ] . the crude fat content of first milking holstein colostrum ( . %) is also significantly higher than for milk ( . %) [ ] . energy from fat and lactose in colostrum is critical for thermogenesis and body temperature regulation. certain vitamins and minerals, including calcium, magnesium, zinc, manganese, iron, cobalt, vitamin a, vitamin e, carotene, riboflavin, vitamin b , folic acid, choline, and selenium are also found in increased concentrations in bovine colostrum versus milk (see table ) [ , ] . to achieve successful passive transfer of igg, the calf must first consume a sufficient mass of ig in colostrum and then be able to successfully absorb a sufficient quantity of these molecules into its circulation. major factors affecting the mass of ig consumed by the calf include the quality and volume of colostrum fed. the major factor affecting the absorption of ig molecules into circulation is the quickness, after birth, with which the first colostrum feeding is provided. the remainder of this article reviews these and other important factors affecting passive transfer, management strategies for preventing bacterial contamination of colostrum, and the use of colostrum supplements and replacers, and provides recommendations for monitoring the colostrum management program. although it is recognized that colostrum contains a wide spectrum of important immune and nutritional components, because the relationship between ig concentrations and calf health is best understood, and because igg composes more than % of total ig in colostrum, the concentration of igg in colostrum has traditionally been considered the hallmark for evaluating colostrum quality. high-quality colostrum has an igg concentration greater than g/l [ ] . the igg concentration in colostrum can vary dramatically among cows. in one recent study, colostrum igg averaged g/l, but ranged from to g/l for individual holstein cows [ ] . some factors affecting colostrum quality, such as breed or age of the dam, may be out of the producer's ability to manipulate. several other important factors affecting colostrum quality, however, including preparturient vaccination, dry period length, and time to colostrum collection, can be managed by producers. this section reviews factors affecting colostrum quality and discusses cow-side testing of colostrum quality. comparative studies have reported that there can be a breed effect on colostrum quality [ , ] . in one study, igg concentration was greater in secretions from beef cows ( . g/l) than from dairy cows ( . g/l) [ ] . in another study, holstein cows produced colostrum with total ig content ( . %) that was numerically lower than for guernsey ( . %) and brown swiss ( . %) cows, and statistically lower than for ayrshire ( . %) and jersey ( . %) cows [ ] . breed differences could be attributed to genetic differences and/or dilutional effects. most, but not all, studies report a tendency for older cows to produce higher quality colostrum, presumably because of older animals have had a greater period of exposure to farm-specific pathogens [ ] [ ] [ ] [ ] . as one example, tyler and colleagues [ ] reported that the mean colostral igg concentration for holstein cows in their first, second, or third and greater lactations was , , and g/l, respectively. in the same study, however, there was reportedly no difference in igg concentration for guernsey cows in their first ( g/l), second ( g/l), and third and greater lactations ( g/l). producers should be discouraged from automatically discarding colostrum from first-calf heifers, because it may be of very good quality. studies generally have shown that ig content of colostrum is not affected by prepartum maternal nutrition [ ] . in a study feeding beef cows either % (co) or % (rs) of national research council (nrc) ( ) [ ] protein and energy requirements, maternal nutrition did not affect either colostrum igg concentration ( . versus . g/l for rs and co, respectively) or the calves' serum igg concentration at hours ( . versus . mg/ml for rs and co, respectively) [ ] . lacetera and colleagues [ ] reported that cows supplemented with injections of selenium and vitamin e in late pregnancy produced a greater volume of colostrum than unsupplemented cows, when all cows were fed a prepartum diet that was deficient in vitamin e and selenium. treatment had no impact on colostrum igg concentration, however. producers should feed dry cows and heifers nonlactating rations balanced according to nrc ( ) guidelines [ ] . some, but not all, studies have reported that exposure to high ambient temperatures during late pregnancy is associated with poorer colostrum composition, including lower mean concentrations of colostral igg and iga, and lower mean percentages of total protein, casein, lactalbumin, fat, and lactose [ , ] . these effects may be attributed to the negative effects of heat stress on dry matter intake resulting in nutritional restriction, reduced mammary blood flow resulting in impaired transfer of igg and nutrients from the blood stream to the udder, or impaired immune reactivity of mammary gland plasmacytes that produce iga [ ] . producers should adopt the similar heat-abatement strategies for prepartum cows and heifers as are routinely used for lactating animals. pritchett and colleagues [ ] observed that cows producing less than . kg of colostrum at first milking were more likely to produce high-quality (o g/l) colostrum than cows producing higher quantities of first milking colostrum (r . kg). this finding was presumed to be attributable to dilutional effects. however, more recent studies report that there is no predictable relationship between colostrum igg concentration and weight of colostrum produced at first milking [ , ] . persistent intramammary infection (imi) during the nonlactating period has not been associated with altered igg concentration. imi is associated with lower colostral volume produced, however [ ] . producers should not feed colostrum from cows with clinical mastitis. pooling of colostrum from multiple dams is generally discouraged because larger volumes of low-quality colostrum may dilute smaller volumes of higher-quality colostrum [ ] . furthermore, pooling raw colostrum may increase the number of calves potentially exposed to colostrum-borne pathogens. although not all studies have shown positive results, a body of research has established that vaccinating the pregnant cow or heifer during the final -to -week period preceding calving results in increased concentrations of protective colostral antibodies, and increased passive antibody titers in calves of vaccinated dams, for some common pathogens including pasteurella haemolytica, salmonella typhimurium, escherichia coli, rotavirus, and coronavirus [ ] [ ] [ ] [ ] [ ] . secretion of ig from the dam's circulation into the mammary gland begins approximately weeks before calving. in one observational study, dry period length (mean ¼ . ae days) was not associated with colostrum igg concentration [ ] . in a controlled study, rastani and colleagues [ ] also reported that colostrum quality was not different for cows with a -or -day dry period, respectively. cows with excessively short dry periods (! days) or no dry period produce colostrum with significantly lower igg concentrations [ , ] . furthermore, dry period length can affect the volume of colostrum produced: in a recent controlled field study cows with a short ( -day) dry period produced . kg less colostrum than did cows with a conventional ( -day) dry period [ ] . the concentration of ig in colostrum is highest immediately after calving, but begins to decrease over time if milking is delayed. in one study, delaying harvest of colostrum for hours, hours, or hours after calving resulted in a %, %, and % decrease in colostral igg concentration, respectively [ ] . to collect the highest quality colostrum, producers should aim to milk the cow within to hours after calving if possible, with a maximum delay of hours. empiric recommendations suggest rejecting colostrum that is visibly watery, bloody, or is from cows that leaked before calving [ ] . it is difficult to predict, based on such factors as dam parity, weight of colostrum produced at first milking, or visual consistency, which colostrum collected will be of high (o g/l igg) versus low quality [ ] . the colostrometer, a hydrometer instrument that estimates igg concentration by measuring colostrum specific gravity, is one rapid and inexpensive cow-side test that may be useful to differentiate high-from low-quality colostrum (specific gravity o . approximates igg concentration o g/l igg). factors such as content of fat and other solids, plus colostrum temperature, affect the hydrometer reading, however. pritchett and colleagues [ ] reported that the sensitivity and specificity of the instrument for detecting low-quality colostrum were . and . , respectively, meaning that the instrument would incorrectly classify two of every three low-quality colostrum samples as acceptable. pritchett and colleagues [ ] suggested that to avoid misclassification error, producers should alter the hydrometer cutoff points to , , or g/l if feeding either . , . , or . l of colostrum at first feeding, respectively. test specificity would be severely compromised by using higher cutpoints, however, resulting in an excessive portion of colostrums being misclassified as deficient [ ] . others have suggested that if a large enough volume (eg, . l) is fed at first feeding, then there may be limited value to using a hydrometer. despite its limitations, the hydrometer may still be useful to differentiate high-from low-quality colostrum used for first versus later feedings, respectively. an alternate tool for differentiating high-from low-quality colostrum may be a commercially available cow-side immunoassay kit (colostrum bovine igg quick test kit, midland bio-products, boone, iowa). chigerwe and colleagues [ ] recently reported that the sensitivity and specificity of this test kit to identify poor-quality colostrum (igg! g/l) were . and . , respectively. with this relatively low specificity, the immunoassay test would incorrectly classify one in every four high-quality colostrum samples as unacceptable. one additional limitation of the immunoassay is that it yields only a positive or negative result, but does not provide an estimate of the actual igg concentration. the immunoassay costs approximately $ (united states dollars [usd]) per sample and takes approximately minutes to run. to achieve successful passive transfer in an average -kg ( lb) holstein calf, experts calculate that producers should feed at least a minimum mass of g of igg in the first colostrum feeding [ ] . so what volume of colostrum should producers feed to meet or exceed this minimum dose? obviously the answer to this question depends on the igg concentration in the colostrum being fed. for example, if colostrum was known to contain g/l igg, then the producer would only need to feed . l ( qt) to achieve the minimum goal of ingesting more than g igg. if the colostrum contained only g/l of igg, however, then the producer would need to feed . l ( qt) to achieve the same ingested mass of igg. besser and colleagues [ ] noted that only % of colostrum samples tested would be of high enough quality to provide greater than g igg if calves were only fed . l. some % of colostrum samples tested would be of high enough quality to provide greater than g igg if calves were fed . l, however. because producers frequently do not know the concentration of igg in the colostrum being fed, it is currently recommended that calves be fed % to % of their body weight of colostrum at first feeding ( . l for a -kg calf). in one study mean serum igg at hours was significantly higher for calves fed l of high-quality colostrum at hours and a further l at hours ( . mg/ml igg) as compared with calves fed only l of high-quality colostrum at hours and a further l at hours ( . mg/ml) (fig. ) [ ] . another study reported that brown swiss calves fed . l (versus . l) of colostrum at first feeding experienced significantly higher rates of average daily gain and greater levels of milk production in both the first and second lactation [ ] . in national surveys, . %, . %, and . % of producers reported feeding or more quarts of colostrum within the first hours in , , and , respectively [ , , ] , indicating that increasing the volume of colostrum fed is still an area of opportunity for most dairy producers. the term ''open gut'' refers to the unique ability of the neonatal enterocyte to nonselectively absorb intact large molecules, such as ig, by pinocytosis [ ] . from there, ig molecules are transported across the cell and released into the lymphatics by exocytosis, after which they enter the circulatory system through the thoracic duct [ ] . in a process referred to as ''closure,'' the efficiency of colostral ig absorption through the intestinal epithelium of the calf decreases linearly with time from birth to completely close at approximately hours [ ] . feeding colostrum after the gut has closed still offers the benefit of local immunity in the gut lumen, but ig absorption into the circulation no longer occurs. the following section discusses factors affecting the efficiency of ig absorption, many of which are under management's control. the major factor affecting efficiency of ig absorption is age of the calf at feeding. the efficiency of ig transfer across the gut epithelium is optimal in the first hours postpartum, but after hours there is a progressive decline in the efficiency of ig absorption over time [ , ] . delaying the first colostrum feeding can only slightly postpone gut closure ( hours) [ ] . producers should aim to feed all calves within to hours after birth and by hours at a maximum. the method of feeding colostrum is worth considering because this can influence the time to first feeding, the volume consumed, and the efficiency of ig absorption. high rates of fpt have been reported in calves left to suckle the dam [ , ] . this finding may be attributable to failure of the calf to voluntarily consume a sufficient volume of colostrum and delays in suckling. edwards and broom [ ] reported that % of calves born to second parity and older cows had failed to suckle within hours after birth. by comparison, % of calves born to first-calf heifers had failed to suckle within hours after birth. these delays could be caused by numerous factors, including weak or injured cow or calf, mastitis or other illness in the cow, low pendulous udders or large teats, or poor mothering ability. it is for this reason that it is currently recommended that the calf be removed from the dam within to hours of birth, and that the calf then be hand-fed a known volume of colostrum using either a nipple bottle or esophageal feeder [ ] . in national surveys, . %, . %, and . % of calves were reportedly fed using a nipple bottle or esophageal tube in , , and , respectively [ , , ] , indicating that progressively fewer producers are relying on suckling the dam for colostrum delivery. producers may have a personal preference for using either a nipple bottle or esophageal feeder for the first colostrum feeding. although the esophageal feeder method is quicker, it is known that when fluid is given with an esophageal feeder, the esophageal groove reflex is not triggered, resulting in fluid being deposited into the forestomachs. this limitation is not significant, however, because outflow of colostrum from the forestomachs to the abomasum and small intestine occurs for the most part within hours [ ] . adams and colleagues [ ] reported that calves fed colostrum using a bottle had only slightly higher serum igg concentrations versus calves fed with an esophageal feeder, but that these differences were numerically small and statistically insignificant. it is generally accepted that either method of feeding achieves acceptable rates of passive transfer provided a sufficient volume of colostrum is consumed [ , ] . veterinarians should train interested producers on how to properly use and clean esophageal feeders. it has been reported that efficiency of ig absorption was improved when calves were housed with the dam [ ] . considering that acceptable levels of serum igg can be achieved without housing the calf with the dam, however, and given that the latter practice may increase the calf's risk for exposure to pathogens from the dam or her environment, it is currently recommended that the calf be removed from the dam within to hours of birth and then hand-fed a known volume of colostrum [ ] . decreased colostral ig absorption in the first hours has been reported in calves with postnatal respiratory acidosis, associated with prolonged parturition [ ] . although hypoxic calves may have delayed igg absorption initially, studies have reported that there is no difference in overall absorptive capacity between hypoxic and normoxic calves and that there is no difference in serum igg concentrations by the time of gut closure [ , ] . weaver and colleagues [ ] suggested that an increased rate of fpt seen in calves with metabolic or respiratory acidosis may be caused by a delay in the animal getting up to nurse, not by reduced absorptive capacity. absorption of ig may be impaired when newborn calves are exposed to extreme cold, possibly because of direct effects on intestinal absorption and transport and indirect effects on the calf's ability to stand and nurse [ ] . bacteria in colostrum may bind free ig in the gut lumen or directly block uptake and transport of ig molecules across intestinal epithelial cells, thus interfering with passive absorption of colostral ig [ ] [ ] [ ] . this effect was demonstrated in a recent controlled study wherein newborn calves were fed either . l of pasteurized ( c  min) colostrum or . l of raw colostrum, with the geometric mean total bacteria counts in the two colostrum treatment groups being cfu/ml or , cfu/ml, respectively [ ] . although the volume, timing, and quality of colostrum fed to the two feeding groups was not different, calves fed pasteurized colostrum had significantly higher mean serum igg levels at hours of age ( . mg/ml) versus calves fed raw colostrum ( . mg/ml). this improvement was attributed to reduced bacterial interference with igg absorption across the gut, resulting in higher efficiency of igg absorption in calves fed pasteurized colostrum ( %) versus calves fed raw colostrum ( %) [ ] . strategies for preventing or minimizing bacterial contamination of colostrum are discussed in the next section. although colostrum is an important source of nutrients and immune factors, it can also represent one of the earliest potential exposures of dairy calves to infectious agents including mycoplasma spp, mycobacterium avium subsp paratuberculosis, fecal coliforms, and salmonella spp [ ] [ ] [ ] . this exposure is a concern because pathogenic bacteria in colostrum could cause diseases such as diarrhea or septicemia. it is also a concern because bacteria in colostrum may interfere with absorption of ig [ ] [ ] [ ] . experts recommend that fresh colostrum fed to calves contain fewer than , cfu/ ml total bacteria count (tpc) and fewer than , cfu/ml total coliform count [ ] . unfortunately, average bacteria counts in colostrum fed on commercial dairies frequently far exceeds this cutpoint [ , ] . in one study of wisconsin dairy herds, % of samples tested exceeded the upper limit of , cfu/ml tpc [ ] . the following section describes management techniques for minimizing bacterial contamination of colostrum. preventing contamination during colostrum harvest, storage, and feeding procedures methods for reducing the risk for pathogen exposure to calves include avoiding feeding colostrum from known infected cows and avoiding pooling of raw colostrum. additionally, all producers should take steps to avoid contamination during colostrum harvest, storage, or feeding processes. in a study of colostrum harvesting and feeding practices on one dairy, total bacteria counts (tpc cfu/ml) were very low or nil in colostrum stripped directly from the gland (geometric mean udder tpc ¼ . cfu/ml). significant bacterial contamination occurred, however, during the process of milking the colostrum into the bucket (geometric mean bucket tpc ¼ , cfu/ ml) [ ] . these results emphasize the importance of minimizing colostrum contamination by properly prepping udders before harvesting colostrum, milking into a clean, sanitized bucket, and handling colostrum using clean, sanitized storage or feeding equipment. bacteria can multiply rapidly if colostrum or milk is stored at warm ambient temperatures [ ] . unless colostrum is to be fed right away, it should be frozen or refrigerated within hour after collection. it is generally accepted that colostrum may be frozen for up to year, provided multiple freeze-thaw cycles do not occur. when thawing frozen colostrum, producers should avoid overheating colostrum (avoid temperatures o c or f) or some denaturation of colostral ig can occur [ ] . options for producers who wish to store fresh colostrum include refrigeration with or without the use of preservatives such as potassium sorbate [ ] . igg in raw refrigerated colostrum is stable for at least week. average bacteria counts in raw refrigerated colostrum may reach unacceptably high concentrations (o , cfu/ml) after days of refrigeration, however. by comparison, average colostrum bacteria counts remained less than , cfu/ml for days of refrigeration when colostrum was preserved with potassium sorbate in a . % final solution [ ] . information on potassium sorbate sources and mixing directions can be found at http://www.atticacows.com/orgmain.asp?orgid¼ &storytypeid¼&sid¼&. an additional tool that may be useful to reduce bacterial contamination of colostrum is pasteurization. early studies tried to pasteurize colostrum using the same conventional methods and high temperatures as are typically used to pasteurize milk ( c [ f] for minutes or c [ f] for seconds). this process yielded unacceptable results, however, including thickening or congealing of colostrum and denaturation of approximately one third of colostral igg [ ] . despite these early setbacks, more recent research has determined that using a lower-temperature, longer-time approach ( c [ f] for minutes) to batch-pasteurize colostrum is sufficient to maintain igg activity and colostrum fluid characteristics, while eliminating or significantly reducing important pathogens including e. coli, salmonella enteritidis, mycoplasma bovis and mycobacterium avium subsp paratuberculosis [ , ] . in one recent on-farm controlled study, calves fed pasteurized colostrum ( c  minutes) experienced a significant reduction in colostrum bacterial exposure and significantly higher serum igg levels at hours of age versus calves fed . l of raw colostrum [ ] . if stored in a clean covered container, the shelf life of pasteurized refrigerated colostrum is at least to days [ ] . the potential short-and longterm health and economic benefits of feeding pasteurized colostrum have not yet been described. farms can occasionally experience periods in which an adequate supply of clean, high-quality, fresh or stored colostrum is not available to feed to all newborn calves. contributing to this problem, some producers may discard colostrum from cows that test positive for m avium subsp paratuberculosis, bovine leukosis virus, or m bovis mastitis. under such circumstances, using colostrum supplements (cs) or colostrum replacement (cr) products may offer producers a convenient way to improve levels of passive immunity in calves while reducing the risk for pathogen exposure through colostrum. powdered commercial cs or cr products contain bovine ig that is typically either lacteal-or plasma-derived. it is recommended that cs or cr products be mixed in water (according to label directions) and fed as a separate meal after any natural colostrum has been fed [ ] . there are important differences between the less expensive cs products ($ -$ per dose) and more expensive cr products ($ -$ per dose). colostrum supplement products typically contain less than g igg per dose, contain no nutrient pack, and are only intended to supplement (not replace) existing colostrum. if given alone, feeding cs products results in significantly lower serum ig and greater risk for fpt in calves as compared with feeding fresh colostrum [ ] . there is no added benefit of feeding cs products if already feeding to l of high-quality bovine colostrum [ , ] . by comparison, cr products contain a minimum of g igg per dose, provide a nutritional source of protein, energy, vitamins, and minerals, and are designed to completely replace (or feed in the absence of) maternal colostrum [ ] . results of cr studies have been mixed, with many products failing to routinely provide the necessary mg/ml igg in serum of calves fed cr [ , [ ] [ ] [ ] . in a controlled study of dairy herds in minnesota and wisconsin, swan and colleagues [ ] reported that commercial dairy calves fed a commercially available cr product (acquire, american protein corporation, inc., ames, iowa) had significantly lower serum igg concentrations ( . mg/ml igg) than calves fed maternal colostrum ( . mg/ ml igg). although a trend was present, the preweaning morbidity and mortality rates were not different for calves fed cr (morbidity ¼ . %; mortality ¼ . %) versus calves fed maternal colostrum (morbidity ¼ . %; mortality ¼ %). other studies have reported better rates of successful passive transfer (mean serum igg o . mg/ml), particularly when calves were fed two doses of cr product [ , ] . in one such study, the average -hour serum igg level for calves fed either one dose ( g igg) or two doses ( g igg) of a lacteal-derived cr, or . l of maternal colostrum, were . , . , and . mg/ml igg, respectively (land o' lakes colostrum replacement, land o' lakes inc., st. paul, minnesota) [ ] . feeding higher doses of cr products may increase the rate of successful passive transfer, but the cost-benefit of this practice has yet to be described. similarly, the effectiveness and cost-benefit of routinely using cr products in johne's or other infectious disease control programs has yet to be described. because of the highly variable performance among different products, veterinarians should review results of peer-reviewed controlled trials when selecting a cr product. veterinarians can help producers develop programs to routinely monitor colostrum management. possible laboratory-based test methods for directly measuring or estimating serum igg concentrations in calves include radial immunodiffusion (rid), turbidimetric immunoassay (tia), enzyme-linked immunosorbent assay (elisa), sodium sulfite turbidity test, zinc sulfate turbidity test, serum gamma glutamyltransferase (ggt) activity, and wholeblood glutaraldehyde coagulation test [ ] [ ] [ ] . in a recent review of these tests, weaver and colleagues [ ] raised concerns about unacceptably high levels of inaccurate results for the sodium sulfite turbidity test when using the % and % sodium sulfite test solutions, the zinc sulfate turbidity test if samples are exposed to co or are hemolyzed, ggt test results, and whole-blood glutaraldehyde coagulation test results. although rid, tia, or elisa would be acceptable tests for use in periodic outbreak investigations, the expense and inconvenience of routinely submitting serum samples to a veterinary diagnostic laboratory would generally discourage their adoption for ongoing monitoring programs. a lateral-flow immunoassay is one tool that could be used for on-farm testing (midland quick test kit -calf igg, midland bioproducts corp., boone, iowa). the manufacturer has reported the sensitivity, specificity, and overall accuracy of this assay to identify calves with serum igg less than . mg/ml as being . , . , and . , respectively [ ] . independent validation of this test is still required. one limitation of the immunoassay is that it yields only a positive or negative result, but does not provide an estimate of the actual serum igg concentration. the assay requires approximately minutes to complete and costs approximately $ . (usd) per sample. measurement of serum total solids (sts) by hand-held refractometer offers a convenient, simple, rapid, and inexpensive on-farm tool by which producers can monitor the colostrum feeding program. the refractometer instrument costs approximately $ (usd). in an early study of calves, sts had a good correlation with serum igg concentration as measured using rid (r ¼ . ) [ ] . calloway and colleagues [ ] , reported that sts concentration test endpoints of . and . g/dl yielded the most accurate results in estimating the adequacy of passive transfer as defined by serum igg . mg/ml or greater (sensitivity o . ; specificity o . ; proportion classified correctly o . ). in that study lower or higher test endpoints misclassified larger numbers of calves. because sts results do result in periodic misclassification of individual calves, the use of sts results as an individual animal diagnostic tool is discouraged. when results are interpreted at the group or herd level, however, sts results accurately reflect the proportion of calves that have fpt, thereby making it a useful on-farm tool for monitoring whether the colostrum management program is succeeding. it is recommended that serum samples be collected from a minimum of clinically normal (not scouring) calves between hours and days of age [ ] . wallace and colleagues [ ] reported that the results of sts refractometry from centrifuge-and noncentrifuge-harvested sources of serum were highly correlated (r ¼ . ), so producers can conduct this test onfarm without need of a centrifuge. mcguirk and collins [ ] suggest that a goal is for % or more of calves tested to meet or exceed a sts cutpoint of . g/dl. tyler suggests that % or more of calves tested should meet or exceed the more accurate sts cutpoint of . g/dl (j tyler, personal communication, ). if it is determined that a disproportionate number of calves have fpt, then the veterinarian and producer must investigate to identify and then correct the root causes of fpt within the colostrum management program. in addition to periodically sampling groups of calves to assess fpt, producers can also periodically submit frozen colostrum samples to a microbiology laboratory for culture. a goal is for a majority of samples submitted to have at total bacteria count of less than , cfu/ml and a total coliform count less than , cfu/ml [ ] . colostrum management is the single most important management factor in determining calf health and survival. unfortunately, a significant proportion of north american dairy calves suffer from failure of passive transfer, contributing to excessively high preweaning mortality. there is considerable opportunity for most dairy producers to improve their colostrum management practices, resulting in improved short-and long-term health and performance of the animal. a successful colostrum management program requires producers to consistently provide calves with a sufficient volume of clean, high-quality colostrum within the first few hours of life. colostrum replacers are useful tools if a sufficient quantity of clean, high-quality maternal colostrum is not available. ongoing monitoring helps producers to more quickly identify and correct problems within the colostrum management program. the development of the conceptus dairy : national dairy health evaluation project. dairy heifer morbidity, mortality, and health management focusing on preweaned heifers. ft. collins (co): usda-aphis veterinary services passive transfer of colostral immunoglobulins in calves national animal health monitoring system. national dairy heifer evaluation project. dairy herd management practices focusing on preweaned heifers. ft. collins (co): usda-aphis veterinary services in: the development, nutrition, and management of the young calf managing the production, storage and delivery of colostrum effects of passive immunity on growth and survival in the dairy heifer effects of passive immunity on subsequent production in dairy heifers factors associated with mortality to days of life in dairy heifers in the united states effects of colostrum ingestion on lactational performance part : reference of dairy health and management in the united states. ft. collins (co): usda-aphis veterinary services availability, storage, treatment, composition, and feeding value of surplus colostrum: a review immunoglobulin production and transport by the mammary gland immunological activities of milk effect of prolactin on in vitro expression of the bovine mammary immunoglobulin g receptor colostral transfer of bovine immunoglobulin e and dynamics of serum ige in calves cellular components of mammary secretions and neonatal immunity: a review intestinal absorption of maternal leucocytes by newborn lambs uptake of colostral leukocytes in the intestinal tract of newborn calves colostrum induced phenotypic and trafficking changes in maternal mononuclear cells in a peripheral blood leukocyte model for study of leukocyte transfer to the neonatal calf the influence of colostral leukocytes on the course of an experimental escherichia coli infection and serum antibodies in neonatal calves effects of the ingestion of whole colostrum or cell-free colostrum on the capacity of leukocytes in newborn calves to stimulate or respond in oneway mixed leukocyte cultures effect of maternal cells transferred with colostrum on cellular response to pathogen antigens in neonatal calves growth factors and antimicrobial factors of bovine colostrum effects of milk-derived bioactives: an overview immunoglobulins, growth factors and growth hormone in bovine colostrum and the effects of processing antioxidants in bovine colostrum effects of dietary insulin-like growth factor i on growth and insulin-like growth factor receptors in neonatal calf intestine peptide regulation of intestinal glucose absorption small intestinal morphology in eight-day-old calves fed colostrum for different durations or only milk replacer and treated with long-r -insulinlike growth factor i and growth hormone passive transfer of immunoglobulin g and preweaning health in holstein calves fed a commercial colostrum replacer regulation of colostrum formation in beef and dairy cows colostral immunoglobulin concentrations among breeds of dairy cattle factors associated with colostral specific gravity in dairy cows management and production factors influencing immunoglobulin g concentration in colostrum from holstein cows colostral immunoglobulin concentrations in holstein and guernsey cows effects of prepartum protein restriction in the beef cow on immunoglobulin content in blood and colostral whey and subsequent immunoglobulin absorption by the neonatal calf nutrient requirements of beef cattle influence of nutritional restriction during late gestation on production measures and passive immunity in beef cattle effects of selenium and vitamin e administration during a late stage of pregnancy on colostrum and milk production in dairy cows, and on passive immunity and growth of their offspring nutrient requirements of dairy cattle composition of colostrum from dairy heifers exposed to high air temperatures during late pregnancy and the early postpartum period use of mammary gland and colostral characteristics for prediction of colostral igg concentration and intramammary infection in holstein cows shortening the dry period from to days does not affect colostrum quality but decreases colostrum yield by holstein cows effects of mastitis on the volume and composition of colostrum produced by holstein cows passive protection of calves against experimental infection with salmonella typhimurium colostral and milk antibody titers in cows vaccinated with a modified live rotavirus-coronavirus vaccine a field trial to evaluate the efficacy of a combined rotavirus-coronavirus escherichia coli vaccine in dairy cattle immune response of pregnant heifers and cows to bovine rotavirus inoculation and passive protection to rotavirus infection in newborn calves fed colostral antibodies or colostral lymphocytes preparturient vaccination to enhance passive immunity to the capsular polysaccharide of pasteurella haemolytica a reducing dry period length to simplify feeding transition cows: milk production, energy balance and metabolic profiles metabolism and mammary secretion of serum protein in the cow effect of delayed colostrum collection on colostral igg concentration in dairy cows alliance on management and nutrition. a guide to colostrum and colostrum management for dairy calves evaluation of the hydrometer for testing immunoglobulin g concentrations in holstein colostrum evaluation of a cow-side immunoassay kit for assessing igg concentration in colostrum comparison of three methods of feeding colostrum to dairy calves effects of quality, quantity, and timing of colostrum feeding and addition of a dried colostrum supplement on immunoglobulin g absorption in holstein bull calves electron microsopic studies of the jejunal epithelium from neonatal pigs fed different diets the ultrastructure of neonatal calf intestine and absorption of heterologous proteins effect of colostral immunoglobulin g and immunoglobulin m concentrations on immunoglobulin absorption in calves intestinal transmission of macromolecules in newborn dairy calves of different ages at first feeding colostral immunoglobulin transfer in calves i. period of absorption effect of suckling followed by bottle feeding colostrum on immunoglobulin absorption and calf survival the period between birth and first suckling in dairy calves the failure of the oesophageal groove reflex, when fluids are given with an oesophageal feeder to newborn and young calves two methods for administering colostrum to newborn calves colostrum management in calves: effects of drenching vs. bottle feeding studies on dairy calves allowed to suckle their dams at fixed times postpartum decreased colostral immunoglobulin absorption in calves with postnatal respiratory acidosis hypoxia in neonatal calves: effect on intestinal transport of immunoglobulins effect of high arterial carbon dioxide tension on efficiency of immunoglobulin g absorption in calves effects of maternal nutritional restriction and cold stress on young calves: absorption of colostral immunoglobulins effect of orally administered duodenal fluid on serum proteins in neonatal calves influence of administered indigenous microorganisms on uptake of [iodine- ] gamma-globulin in vivo by intestinal segments of neonatal calves bacteria in colostrum: impact on calf health [abstract the effect of feeding heat-treated colostrum on passive transfer of cellular and humoral immune parameters in neonatal dairy calves survey of ontario bulk tank raw milk for foodborne pathogens isolation of mycobacterium paratuberculosis from colostrum and milk of subclinically infected cows otitis media in preweaned holstein dairy calves in michigan due to mycoplasma bovis preventing bacterial contamination and proliferation during the harvest, storage and feeding of fresh bovine colostrum heat-treatment of bovine colostrum i: effects of temperature on viscosity and immunoglobulin g effect of on-farm commercial batch pasteurization of colostrum on colostrum and serum immunoglobulin concentrations in commercial dairy calves heat-treatment of bovine colostrum ii: effects of heating duration on pathogen viability and immunoglobulin g improving cleanliness and shelf-life of refrigerated colostrum using heat-treatment and chemical preservatives effects of a colostrum replacement product derived from serum on immunoglobulin g absorption by calves serum immunoglobulin concentrations after feeding maternal colostrum or maternal colostrum plus colostral supplement to dairy calves efficacy of a dried colostrum powder in the prevention of disease in neonatal holstein calves formulation of colostrum supplements, colostrums replacers and acquisition of passive immunity in neonatal calves effect of a why protein concentrate used as a colostrum substitute or supplement on calf immunity, weight gain, and health short communication: absorption of protein and immunoglobulin g in calves fed a colostrum replacer influence of pooled colostrum or colostrum replacement on igg and evaluation of animal plasma in milk replacer serum igg and total protein concentrations in dairy calves fed two colostrum replacement products development of an automated turbidimetric immunoassay for quantification of bovine serum immunoglobulin g evaluation of assays for failure of passive transfer in calves quantitation of bovine immunoglobulins: comparison of single radial immunodiffusion, zinc sulphate turbidity, serum electrophoresis, and refractometer methods evaluation of a lateral-flow immunoassay for use in monitoring passive transfer of immunoglobulins in calves an examination of the influence of husbandry on the plasma immunoglobulin level of the newborn calf, using a rapid refractometer test for assessing immunoglobulin content comparison of refractometers and test endpoints in the measurement of serum protein concentration to assess passive transfer status in calves a comparison of serum harvesting methods and type of refractometer for determining total solids to estimate failure of passive transfer in calves key: cord- -uf eryoy authors: bok, marina; alassia, martín; frank, flavia; vega, celina g.; wigdorovitz, andrés; parreño, viviana title: passive immunity to control bovine coronavirus diarrhea in a dairy herd in argentina date: - - journal: rev argent microbiol doi: . /j.ram. . . sha: doc_id: cord_uid: uf eryoy bovine coronavirus (bcov) is a viral enteric pathogen associated with calf diarrhea worldwide being, in argentina, mostly detected in dairy husbandry systems. the aim of the present work was to study if maternal igg antibodies (abs) to bcov acquired by colostrum intake modulate the development of bcov infection in calves reared in a dairy farm in argentina. thirty holstein calves were monitored during their first days of age. animals were classified into two groups depending on their initial bcov igg ab titers. the “failure of passive transfer” (fpt) group had significantly lower igg abs to bcov than the “acceptable passive transfer” (apt) group of calves (log . vs. . respectively) (p < . ). these differences were also observed when the total protein levels in both groups were compared (p = . ). moreover, % ( / ) of calves from the fpt group showed igg seroconversion to bcov compared to . % ( / ) of animals from the apt group. regarding viral circulation, bcov was detected in % ( / ) of all calves and bcov igg ab seroconversion was detected in % of the total animals showing that almost half of the calves were infected with bcov. in conclusion, calves with high titers of specific bcov igg (≥ ) were mostly protected against viral infection, while animals with low titers of igg (< ) were mostly infected with bcov. igg abs from colostrum origin are critical for prevention of bcov infection. coronavirus; diarrea neonatal del ternero; inmunidad pasiva resumen el coronavirus bovino (bovine coronavirus, bcov) es un enteropatógeno viral asociado a la diarrea neonatal del ternero. el objetivo del presente trabajo fue estudiar si los anticuerpos igg anti-bcov adquiridos pasivamente mediante el calostro modulan la infección por bcov en terneros de un rodeo lechero de argentina. se monitorearon terneros raza holstein durante los primeros días de vida. estos animales fueron clasificados en dos grupos según sus niveles de igg anti-bcov maternales: grupo con transferencia de inmunidad pasiva aceptable (apt) y grupo con fallas en la transferencia pasiva (fpt). este último grupo tenía un título de igg significativamente menor comparado con el primer grupo (log , vs. , , respectivamente; p < , ). la misma diferencia se observó cuando se compararon los niveles de proteínas séricas totales (p = , ). además, el % ( / ) de los terneros del grupo fpt mostró seroconversión de igg , mientras que el , % ( / ) de los terneros del grupo apt la bovine coronavirus (bcov) is a major viral pathogen associated with neonatal calf diarrhea (ncd) , winter dysentery in adult cattle , and respiratory tract disorders in cattle of all ages , . in addition, it causes important economic losses to the beef and dairy industry worldwide , . bcov is comprised of a single stranded, non-segmented positive sense genomic rna, kb long, which associates to the nucleoprotein (n) forming a nucleocapsid having helical symmetry . it belongs to the betacoronavirus genus cluster within the coronavirinae subfamily, coronaviridae family, and the order nidovirales (http://ictvonline.org/virustaxonomy.asp). bcov has several structural proteins which have different functions in the viral cycle. among them, the s protein is responsible for the interaction between the virus and the cellular receptor, also eliciting neutralizing antibodies (abs). bcov is an enteric/respiratory virus that replicates in enterocytes from the gastrointestinal tract as well as in the epithelium of the upper respiratory tract . although it causes severe hemorrhagic diarrhea which is sometimes fatal in young animals, the spiral colon is the hot spot for viral replication in the gastrointestinal epithelium leading to osmotic diarrhea . moreover, bcov is shed both through respiratory and enteric secretions in high amounts ( billion virus particles per ml of feces) for up to days . consequently, bcov infection is transmitted by fecal-oral or respiratory route and generally occurs by horizontal transmission from the mother to the offspring or between calves , . the incidence of bcov varies between % and % in naturally occurring outbreaks worldwide , and lorenzetti reported . % and . % of bcov pcr-detection rates in diarrheic calves from dairy and beef farms in brazil in and respectively. in argentina, the bcov detection rate by elisa was . % in calves with diarrhea, corresponding to . % of the herds analyzed from to . additionally, those argentina-specific strains were distantly related to the mebus reference strain in a phylogenetic analysis , . as previously suggested, bcov outbreaks may occur in calves from beef and dairy herds , . however, in a previous study conducted in argentina, bcov infection was mostly associated with diarrhea in dairy husbandry systems . this difference may be due to the close interaction between calves in dairy farms, since these animals were reared under intensive management systems and fed milk replacers lacking abs, in stark contrast with beef cattle farms, where herds were reared under extensive management systems and calves were fed directly from the dams' milk until they reached months of age . regarding the prevalence of bcov, serological surveys indicate that approximately % of the worldwide cattle population has abs against bcov . however, ohlson et al. observed that bcov ab-positive herds remained persistently high ( --- %) in swedish southern regions compared with northern regions where the percentage of positive herds were lower ( --- %). in argentina, % of the adult cattle population is estimated to be seropositive for abs to covb (dr. parreño, personal communication). colostrum intake is the natural and most useful method to control bcov calf diarrhea . because bcov-associated diarrhea is an early age disease, the continuous presence of neutralizing abs in the intestinal lumen, mostly igg abs, seems to be essential for prevention of bcov diarrhea . protective levels of bcov abs in calves could be achieved by vaccination of the pregnant cows during the last three months of pregnancy. three commercial vaccines are available in argentina, all of them containing the inactivated bcov mebus strain, which confers cross-protection with local circulating strains . however, ab transfer from the colostrum to the calf bloodstream may fail due to deficiencies in the quality and quantity of colostrum produced by the dam, failure of colostrum intake by the calf, or the newborn physical condition . there are few studies reporting the transference of passive maternal abs from the dams to their calves via colostrum intake under field conditions, and its role in the protection against bcov infection . thus, the aim of the present study was to determine if igg passive maternal abs to bcov acquired by colostrum intake modulate the development of bcov natural infection and disease in calves reared in a dairy farm in argentina. thirty holstein calves reared under an educative, intensive dairy management system were monitored during their first days of age. calves were reared tied to individual stakes with no contact of one calf with the other. calves were initially acquired from different farms with varying levels of sanitary status. thus, vaccination and colostrum administration differed depending on the standard operating procedure of each farm. the age of the calves at the beginning of the study (upon arrival to the herd) ranged from one to ten days. all calves were fed with two liters of milk replacer twice a day and progressive amounts of calf starter (aproagro s.a., argentina). neither preventive antibiotics nor anti-parasitic drugs were administered to the calves. in order to evaluate colostrum intake, total serum protein levels for each calf were measured by refractometry at the beginning of the study ( days). additionally, serum samples were collected from each calf every seven days to measure igg ab titers to bcov by elisa. animals were classified into two groups depending on their initial bcov igg ab titers. calves with bcov igg ab titer ≤ (n = ) were assigned to the failure of passive transfer (fpt) group and animals with bcov igg ab titers ≥ (n = ) were assigned to the acceptable passive transfer (apt) group. in order to evaluate the presence of neonatal diarrhea in calves with varying titers of colostrum-derived abs, and to understand the bcov infection dynamics under field conditions, feces were collected daily from each calf and a diarrhea score was recorded ( = normal, = pasty, = semi liquid, = liquid, = hemorrhagic; a fecal score of or higher was considered diarrhea). additionally, we analyzed several parameters associated with the clinical infection of the calves. those parameters were diarrhea onset (days after the beginning of the experiment), age of calves at diarrhea onset, diarrhea duration (in days) and diarrhea severity (measured as the average of the area under the curve of fecal scores of each calve during the period analyzed). furthermore, mortality and bcov infection in calves with different levels of passive immunity was recorded. finally, all calves' weight was registered at days of age and at the end of the study, at days of age. igg ab titers to bcov were measured using a double sandwich elisa. briefly, -well maxisorp nunc plates (thermo scientific ® , usa) were coated with l of guinea pig hyperimmune serum to bcov ( : dilution in carbonate/bicarbonate buffer, ph . ) and incubated overnight (on) at • c. plates were then washed and blocked with % non-fat milk solution diluted in pbs- . % tween . after incubation during hour at • c, bcov mebus strain-infected ( ffu/ml) and mock-infected hrt- supernatants were added as positive and negative antigens, respectively. serial -fold dilutions of the serum samples were tested in duplicate followed by incubation with an hrp-conjugated commercial ab to bovine igg (bethyl laboratories, inc., usa). the reaction was developed using hydrogen peroxide and abts as substrate/chromogen system (sigma aldrich, usa) and read at a wavelength of nm (multiskan ex, labsystems inc.). the titer of each sample was expressed as the reciprocal of the highest serum dilution with a corrected optical density (od c ; od values in the bcov-coated wells minus od values in the hrt- coated wells) greater than the cut-off value of the assay. the cut-off value was established as the average of od c values of four blank wells (pbs- . % tween ) plus three standard deviations (sd). fecal samples from calves were collected daily and stored at - • c until viral detection. the presence of bcov antigens in the calves' feces was detected by an indirect antigencapture elisa that uses monoclonal abs directed to the viral proteins he, n, and s as previously described . virus shedding was confirmed by a ccif assay which detects fluorescent focus forming units of bcov in hrt- cell monolayers inoculated with the fecal samples, and developed using a fluorescent-labeled anti-bcov hyperimmune serum. calves' fecal samples were also tested for group a rotavirus (rva), which is the most common causative agent of neonatal gastroenteritis. the diagnosis was performed using an indirect antigen-capture elisa as previously reported , , , . no bacterial or parasite diagnosis was performed. ab titers to bcov determined by elisa were log transformed prior to the statistical analysis. negative samples at a dilution of : were assigned an arbitrary ab titer of for the calculation of geometric mean titers (gmts). statistical significance was assessed at p < . for all comparisons. statistical analyses were conducted using infostat ® statistical software . group effects on the igg ab titers to bcov were analyzed by a general linear mixed statistical model (glmm). the model included two main fixed factors: group (apt and fpt, as between subjects' factor) and time (with six levels, as within subjects' factor). animals were included in the model as a random factor. the akaike information criterion (aic) was used for choosing the best-fitting model as a minimal adequate one. thus, the model with the lowest aic value was selected. the glmm analysis was conducted by using the glmer function (lme package, r development core team, ). the analysis was performed with r . . (r development core team, ). statistical significance was assessed at p < . for all comparisons. thirty holstein calves reared under an educative intensive dairy management system were monitored for bcov infection during their first days of age. six out of thirty ( / ; %) calves died because of additional causes other than bcov diarrhea. in order to evaluate colostrum intake, total serum protein levels for each calf were measured at the beginning of the study. the mean (±sd) value of total serum proteins in sera was . ± . g/dl. when the kinetics of the igg ab levels to bcov was evaluated by elisa for each individual calf, we observed that the igg ab titers to bcov in the calves' sera (representing colostrum intake) were highly heterogeneous on arrival to the farm (igg ab titers for each calf are shown in table ). calves' passive immunity to bcov was also analyzed in order to understand bcov pathogenesis. animals were classified into the fpt (titers ≤ ) and apt (titers ≥ ) groups depending on their initial bcov igg elisa ab titers (fig. ) . the igg abs to bcov significantly differed between groups depending on the time (glmm group-time f ( , ) = . , p < . ). the fpt group had significantly lower igg ab titers to bcov than the apt calves for up to seven days after the beginning of the experiment. these significant differences were also observed when measuring total protein levels by refractometry (kruskal---wallis rank sum test, p = . ) which is the preferred method used to measure colostrum abs transfer under field conditions. as expected, the higher the initial level of igg abs in the calves' sera, the longest it took for them to decrease to a susceptible level. when bcov igg ab titers decreased to or , bcov infection was evidenced by either virus detection in feces, or by a minimum -fold increase of anti-bcov igg elisa ab titers for around two weeks (defined as seroconversion for the purpose of this study) (table , fig. ). moreover, % ( / ) of calves from the fpt group showed bcov igg seroconversion between and days of the experiment while only . % ( / ) of the animals from the apt group presented sequential seroconversion during the same period (fisher's exact test, p = . ) ( fig. and table ). however, the moments of seroconversion in the different groups of calves are not necessarily correlated with the days of age because, as we mentioned before, the calves' age was between and days at the beginning of the experience. the apt group average age at day of the experience was . days while the fpt group average age was . days (data not shown). considering the presence of neonatal diarrhea in calves with varying titers of colostrum abs, it was observed that all calves were affected by one, two or even three events of severe diarrhea with an average duration of days (table ); bcov shedding of short time duration ( --- days) was detected in % ( / ) of calves (tables and ) and the highest bcov infectious titer was ffu/ml of stool. although viral shedding was detected at a low rate in the calves' stool, bcov igg seroconversion was observed in % of animals, suggesting that almost half of the calves were exposed to bcov (table ) . calves shedding bcov in stools were further analyzed. calf # had a first day-diarrhea episode in which bcov was detected from to days of the study ( , and days of age). this calf had additional diarrhea episodes that were not associated with bcov infection. calf # had diarrhea since the experiment started (ten days of life for this calf) and during days, in which bcov was detected at and days of the experiment ( and days of age). then, this calf had other diarrhea episodes without etiologic diagnosis. finally, calf # had days of severe diarrhea beginning at day two of the study (six days of life), while bcov was detected at and days of the experiment ( and days of age). in these three cases the presence of bcov was associated with severe diarrhea (fecal score ≥ ). two out of three calves showed bcov igg seroconversion at the same time of viral detection (fig. ) . the third bcovpositive calf died with severe diarrhea. calves' fecal samples were also tested for rva as another common causing agent of neonatal gastroenteritis. rva shedding was detected in % ( / ) of the calves, between three to ten days of age. no bacterial or parasite diagnosis was performed; however, taking into consideration that neither antibiotics nor anti-parasitic drugs were administered to the calves, diarrhea caused by either of these groups of agents may not be discarded. as a final important parameter, all the calves' weight was registered. even though all the calves presented severe diarrhea at some point during the study, once the clinical signs of gastroenteritis disappeared, all animals gained weight reaching an average of kg by the end of the experiment. all calves duplicated their weight during the study period ( days) with no significant differences between the groups. in order to better understand bcov infection and prevention in calves reared under artificial management systems, calves were followed up during their first days of life at an educative dairy farm. both, the immunologic status of the calves at birth and the colostrum quality of the dams are the most important factors influencing prevention of neonatal calf diarrhea . the outcome of bcov infection was clearly affected by passively acquired abs from colostrum. igg subtype is the main igg ab isotype actively concentrated during colostrogenesis , and was the isotype analyzed in this study. refractometry is the commonly used method for total protein measurement to evaluate colostrum intake in calves' serum. however, the exact titer of bcov-specific igg transferred to calves from maternal colostrum required to prevent bcov diarrhea in the field remains unclear. total protein level ≥ . g/dl (corresponding with igg ≥ mg/dl) in serum from a healthy calf is considered to be an optimal level of protein transfer . in this study, we observed a mean protein level of . g/dl which is optimal; however, the fpt group had a significantly lower level of total protein in serum than the apt group at day of the experiment (kruskal---wallis non-parametric rank sum test, p = . ). this correlates with the higher detection of bcov igg ab seroconversion in the fpt group compared with the apt animals. this study also aimed to further define bcov pathogenesis, and to better understand bcov-specific ab kinetics. in argentina, we previously reported a significantly higher rate of bcov diarrhea in dairy herds compared to beef herds . we detected viral shedding in a small number of animals but high viral circulation manifested through bcov seroconversion. low viral detection could be due to a lower sensitivity of elisa compared with the rt-pcr assay to detect bcov in feces . however, another country from the southern hemisphere shows a low detection rate using rt-pcr . bcov shedding was detected at high titers during only two or three days differing with other experiments in which bcov shedding was long lasting , . this supports the idea of a lower sensitivity of the elisa assay for bcov detection used. moreover, we showed that calves with high bcov igg ab titers were often protected against infection, while animals with low bcov igg ab titers showed significantly higher bcov infection rates (p < . ). on the one hand, bcov infection dynamics was drastically affected by these ab titers since % of fpt animals showed bcov igg seroconversion during the experiment. the significant difference in ab titers at the beginning of the study was also associated with a significant difference in total protein levels. furthermore, when igg titers decreased over time and reached non-protective levels (bcov igg ab titers of or ), calves seroconverted confirming bcov infection, even when the antigen was not detected in the feces by the methodology used. on the other hand, . % of animals from the apt group maintained elevated igg ab titers over time and only five calves eventually seroconverted. moreover, calves with maternal bcov igg ab titers of or higher seemed to be protected against bcov infection over the course of this study, suggesting that specific igg abs from colostrum are critical for the prevention of bcov infection . although there was a tendency to observe more bcov igg seroconversion in calves from the fpt group compared with calves from the apt group, this difference was not significant (fisher's exact test, p = . ). this could be due to a low number of experimental units in the fpt group. weight gain was evaluated in all calves at the end of the study ( days of age), compared to baseline measurements obtained at the beginning of the study. no significant weight differences between groups were observed. even though we do not have a negative control group ---animals with no signs of diarrhea ---it has been reported that average daily weight gain does not correlate with the occurrence of ncd . however, other studies demonstrated the influence of ncd on weight gain in beef calves . in addition, it has also been established that when bcov is present in diarrhea outbreaks, it causes more aggressive clinical signs compared to other gastrointestinal viruses, which can compromise the animal survival . indeed, one of the animals shedding bcov died due to the severity of the diarrhea during the course of this study. vaccination is the most effective strategy used to prevent ncd. however, vaccination success can be affected by several factors including deficiencies in herd management and varying vaccine quality . as was mentioned previously, we have no information either about the vaccination of dams against neonatal diarrhea or about the quality of commercial covb vaccines. therefore, abs transferred to calves through colostrum intake might be due to previous exposure to bcov or to an active immunization of the dam with this agent. the lack of a good passive immune transference in addition to a diet based on milk replacer might only have contributed to the occurrence of diarrhea in % of the studied animals. all calves suffered a severe and long lasting diarrhea ( to days). the etiology of diarrhea could be diverse since other infection agents were not discarded, and neither antibiotic nor anti-parasitic drugs were used as preventive strategies. bcov infection was evidenced by antigen shedding or seroconversion in % of the calves, while rva infection was evidenced by shedding in % of calves. thus, rva is a major agent associated with ncd, as has been reported elsewhere , , . the present study highlights the critical role of maternal colostrum-derived abs in the development of bcov infection. the systematic immunization of cows with a vaccine of proved efficacy, the monitoring of colostrum quality, and the efficient passive transfer to the calf are key factors to consider when attempting to control bcov-related diarrhea. the results displayed in this study point to useful end-points to measure when evaluating the potency and efficacy of a bcov vaccine under field conditions. prevalence of endemic enteropathogens of calves in new zealand dairy farms bovine rotavirus strains circulating in beef and dairy herds in argentina from phylogenetic analyses of typical bovine rotavirus genotypes g , g , p[ ] and p[ ] circulating in argentinean beef and dairy herds risk factors associated with diarrhoea in newborn calves bovine coronavirus associated syndromes molecular and antigenic characterization of bovine coronavirus circulating in argentinean cattle during crossprotection studies between respiratory and calf diarrhea and winter dysentery coronavirus strains in calves and rt-pcr and nested pcr for their detection evaluation of concurrent shedding of bovine coronavirus via the respiratory tract and enteric route in feedlot cattle bovine coronavirus reduction in morbidity due to diarrhea in nursing beef calves by use of an inactivated oil-adjuvanted rotavirus ---escherichia coli vaccine in the dam enzymelinked immunosorbent assay, immunofluorescent test and electrophoresis analysis of rotaviral rna in the diagnosis and characterization of the bovine rotavirus neonatal immunology longitudinal study of salmonella spp., diarrheagenic escherichia coli, rotavirus, and coronavirus isolated from healthy and diarrheic calves in a brazilian dairy herd prevalence of rotavirus and coronavirus antigens in the feces of normal cows detection of bovine coronavirus using a taqman-based real-time rt-pcr assay molecular characterization of bovine rotavirus circulating in beef and dairy herds in argentina during a -year period a longitudinal study of bovine coronavirus enteric and respiratory infections in dairy calves in two herds in ohio isotype-specific antibody responses to bovine coronavirus structural proteins in serum, feces, and mucosal secretions from experimentally challenge-exposed colostrum-deprived calves mucosal and systemic isotypespecific antibody responses to bovine coronavirus structural proteins in naturally infected dairy calves epidemiologic factors and isotype-specific antibody responses in serum and mucosal secretions of dairy calves with bovine coronavirus respiratory tract and enteric tract infections infostat -sofware estadístico. argentina: universidad nacional de córdoba excretion and persistence of bovine coronavirus in neonatal calves fields virology coronaviral enteritis of young calves: virologic and pathologic findings in naturally occurring infections neonatal diarrhea by bovine coronavirus (bcov) in beef cattle herds diagnosis of viral agents associated with neonatal calf diarrhea surveillance of diarrhea-causing pathogens in dairy and beef cows in yamagata prefecture managing the production, storage, and delivery of colostrum neonatal calf diarrhea: propagation, attenuation, and characteristics of a coronavirus-like agent a longitudinal study of the dynamics of bovine coronavirus and respiratory syncytial virus infections in dairy herds bovine coronavirus in naturally and experimentally exposed calves; viral shedding and the potential for transmission prediction of respiratory disease and diarrhea in veal calves based on immunoglobulin levels and the serostatus for respiratory pathogens measured at arrival failure of passive immune transfer in calves: a meta-analysis on the consequences and assessment of the economic impact winter dysentery in adult dairy cattle: detection of coronavirus in the faeces evaluation of two antigen-capture elisas using polyclonal or monoclonal antibodies for the detection of bovine coronavirus frequency of bcov detection by a semi-nested pcr assay in faeces of calves from brazilian cattle herds biological aspects of the interspecies transmission of selected coronaviruses passive transfer of colostral immunoglobulins in calves protection of human and animal subjects. the authors declare that no experiments were performed on humans or animals for this study.confidentiality of data. the authors declare that no patient data appear in this article. the authors declare that no patient data appear in this article. the authors declare that they have no conflicts of interest. key: cord- -ifwjvvjj authors: beaver, annabelle; meagher, rebecca k.; von keyserlingk, marina a.g.; weary, daniel m. title: invited review: a systematic review of the effects of early separation on dairy cow and calf health date: - - journal: journal of dairy science doi: . /jds. - sha: doc_id: cord_uid: ifwjvvjj abstract concern from the public is growing regarding early cow-calf separation, yet proponents of this practice maintain that artificial rearing is critical for cow and calf health. early separation is assumed to reduce the risk of transfer of pathogens from dam to neonatal calf, but a wide range of health benefits associated with extended cow-calf contact has also been documented. the aim of this systematic review was to report and synthesize conclusions from the literature on dairy cow and calf health in conventional rearing versus cow-calf contact systems. peer-reviewed, published manuscripts, written in english, directly comparing dairy cow or calf health in artificial versus suckling systems, were eligible for inclusion. we conducted targeted searches using web of science to identify key literature on important health conditions. the resulting manuscripts underwent a -step appraisal process, and further manuscripts were sourced from reference lists. this process resulted in a final sample of articles that addressed cow and calf health. sufficient literature was available to assess mastitis in cows, and scours, cryptosporidiosis, johne's disease, pneumonia, immunity, and mortality in calves. the results for cryptosporidiosis, pneumonia, immunity, and mortality were mixed, with some differences between studies likely attributable to flawed comparisons between cohorts. overall, the articles addressing calf scours and mastitis pointed to beneficial or no effects of suckling. the studies addressing johne's disease did not find cow-calf contact to be a significant risk factor. in conclusion, the scientific peer-reviewed literature on cow and calf health provides no consistent evidence in support of early separation. on many commercial dairy farms, it is routine practice to separate the calf from the dam within h of calving (de passillé et al., ; stěhulová et al., ) . proponents of early separation consider it economically beneficial (due to an increase in saleable milk) and ethically preferable (as it is thought to preclude formation of a maternal bond that becomes progressively more difficult to break; flower and weary, ) . one oft-cited rationale for immediate cow-calf separation is the health benefit ostensibly afforded by artificial calf rearing. for example, faubert and litvinsky ( ) claim that this practice reduces the risk of cryptosporidium parvum infections. muskens et al. ( ) make a similar claim for johne's disease, and daugschies and najdrowski ( ) for eimeriosis. however, such literature does not always cite evidence to corroborate these assertions. the concern over disease transmission stems from the agammaglobulinemic state of the neonatal calf and its heightened susceptibility to disease during this time. artificial feeding of calves is thought to allow better control of colostral quality and quantity and thus improve transfer of maternal immunoglobulins to the calf. moreover, the dam's fecal coliform count increases by up to cfu during the periparturient period (pelan-mattocks et al., ) , leading to a concern that calves permitted to remain in the calving area are at an increased risk of exposure to pathogens (mcguirk, ) . despite these concerns, health benefits of prolonged contact have been documented for calves and cows, ranging from increased immunoglobulin absorption from colostrum (stott et al., ) , to decreased mortality rates for calves (alvarez et al., ) , to reduced risk of mastitis for cows (walsh, ) . thus, allowing the cow and calf to remain in contact presents a mosaic of purported health benefits and risks, for which there is a lack of consensus. the aim of the present review is to provide a critical and systematic evaluation of the scientific literature on the health implications of cow-calf contact versus artificial rearing; our compan-invited review: a systematic review of the effects of early separation on dairy cow and calf health ion paper (meagher et al., ) presents the results of a parallel review on the effects of cow-calf contact on measures of behavior, welfare, and productivity. a synthesis of conclusions from the literature is required to offer a measure of resolution to this debate. articles were eligible for inclusion if they were peerreviewed, written in english, complete (e.g., conference abstracts were excluded), available in full-text form, and contained a direct investigation of the effects of cow-calf contact or suckling on dairy cow or calf health. articles were removed if not based upon original data (e.g., review articles or literature-based mathematical models). any manuscript published after the completion of the literature search (may , ) was not included. exclusion and inclusion criteria for the systematic review were developed a priori and agreed upon by all co-authors. systematic searches were conducted using the web of science (wos) database, which allows for integration of boolean operators (i.e., and, or, not) to string together words or phrases, as well as wildcard truncations (denoted as *) to designate a range of possible word forms. the $ symbol was employed to account for alternate spellings (e.g., american versus british english). all searches contained the following fixed set: ("cow-calf" or "cow/calf" or "dam-calf" or "dam/ calf" or "dam rearing" or "reared by the dam" or "reared by cows "or "suckling system*" or "mother rearing" or "reared by the mother" or "contact of calves with adult*" or "leav* calves with dam*" or "stay* with the dam" or "remain* with the dam" or "kept with the dam") and (nurs* or suckl* or separation or contact or "risk factor*") and (calf or calves). seven specific searches were conducted, each containing targeted terms addressing the most relevant calf and cow health conditions: scours (scour* or diarr*), cryptosporidiosis (cryptosporidi*), johne's disease (johne's or paratuberculosis), pneumonia (pneumonia or respiratory), immunity (immunity or "passive transfer"), health and mortality (disease* or infection* or health or morbidity or mortality), and mastitis (mastitis or "intramammary infection*" or "udder health"). several additional search terms were tested but yielded no results meeting the predefined exclusion criteria; these terms included "e. coli," escherichia, salmonella, heidelberg, clostridium, campylobacter, "enteric bacteria," coccidi*, bvd, "bovine viral diarr*," rotavirus, coronavirus, neospor*, bluetongue, diphtheria, "mycoplasma bovis," "mycobacterium bovis," schmallenberg, sarcocystis, anaplasm*, cowdria, blv, "bovine leukemia virus," biv, "bovine immunodeficiency virus," bohv- , bhv- , "bovine herpes virus," piv- , piv , "parainfluenza virus," "mannheimia haemolytica," "pasteurella multocida," "histophilus somni," "arcanobacterium pyogenes," "retained placenta," "placental retention," "f$etal membrane," metritis, "uterine health," "uterine disease," "milk fever," hypocalc$emia, dd, "digital dermatitis," mange, and mites. the selection of these search terms was based upon expert opinion, in addition to several review articles on pathogenesis and health conditions in dairy cows and calves: mulligan et al., ; muktar et al., ; and francoz et al., . results from the unique searches were pooled, and duplicate results were excluded. articles were then selected based upon a -step screening and appraisal process: phase . conference proceedings and articles written in a language other than english were removed. the titles of the remaining articles were scanned to filter out irrelevant results (e.g., literature clearly pertaining to animals other than the dairy cow and calf). phase . abstracts were evaluated to identify and remove additional articles not relevant to the topic of dairy cow or calf health, disease, infection, or mortality (e.g., articles addressing dairy-herd economics). phase . reference lists were mined for additional relevant manuscripts. if full texts were not available online or in the university of british columbia's library system, they were requested via interlibrary loan, re-searchgate, or personal contacts. the reference lists of papers added at this stage were also considered as a source for further manuscripts phase . finally, review articles were removed, and full texts of the remaining papers were read in detail. articles containing experimental research were excluded if the experiment itself did not address the question of cow-calf contact (but rather reviewed literature on the issue as part of a larger discussion). mathematical models were excluded if parameters relating to cow-calf contact were sourced from other literature, or if insufficient information pertaining to real-world data collection was provided to permit recalculation of model parameters. the articles remaining at this stage were included in the systematic review, and in multiple sections if they described more than one relevant effect. to provide a comprehensive overview of the literature, no additional restrictions were placed upon publication year, study type, sample size, or overall quality; however, a quality assessment of the included manuscripts was conducted and is reported in the appendix. from each manuscript, where applicable, we have recorded authorship, publication year, country, breed of cattle under study, dam-calf contact type (e.g., restricted suckling or unrestricted contact), contact duration, sample size, the amount and type of milk provided to artificially reared calves, the author's conclusion, and the direction of this conclusion. inter-observer reliability for data extraction (for all categories except for authorship and publication year) was tested on a random subset of articles, with a result of % agreement. we also present diagnostic test information, relative risks (rr), odds ratios, hazard ratios, and the associated % confidence intervals (ci), where available. these statistics were provided in many of the articles addressing mastitis, johne's disease, and calf cryptosporidiosis. in an attempt to achieve consistency, we calculated the rr (or prevalence ratio in the case of cross-sectional studies) and ci for articles in these categories if not reported by the authors. this was only possible when sufficient information was provided to permit calculation. the following formulae were applied: where a, b, c, and d correspond to number of controls with the disease, number of disease-free controls, number of experimental animals with the disease, and number of disease-free experimental animals, respectively. where se lnrr a c a b a c ( )= + − + − + . results from the -step screening and appraisal process detailed in the materials and methods section are shown in figure . briefly, wos returned unique papers pertaining to cow-calf separation and health. the titles of these papers were scanned, and papers were subsequently excluded ( of these were written in another language, and concerned beef cattle or other species). following an assessment of the abstracts (n = ), an additional papers were excluded. at this stage, articles were sourced from the reference lists of the remaining papers, and from the reference lists of newly included papers. in the final exclusion stage, a further papers were removed because they were review articles (n = ), did not directly compare cow or calf health in relation to cow-calf contact (n = ), or were literature-based mathematical models (n = ). in total, articles (comprised of single-herd studies and multi-herd studies) were selected for inclusion; of these were included in of the specific sections below, and were included in sections. the final manuscripts included in this systematic review were authored between and . the studies originated from continents and countries, most commonly north america (n = : united states = ; canada = ; mexico = ) and europe (n = : central europe = ; scandinavia = ; western europe = ; southern europe = ). additionally, studies originated from south america, from east africa, from asia (middle east = ; south east asia = ), and from oceania (australia = ; new zealand = ). these statistics include only those studies that specified regional information. the single-herd studies included between and relevant groups with a total sample size of between and animals. the multi-herd studies included between and , herds with the total number of animals ranging from to , . scours and enteric pathogens. of the included articles, ( single herd and multi-herd studies) addressed dairy-calf scours of unspecified or multiple etiologies. nine additional articles specifically addressed calf cryptosporidiosis, predominantly infection with c. parvum. one of these studies represented a single-herd evaluation of prevalence, and the other were multi-herd trials (between and herds) that paired questionnaires on management practices with cross-sectional or repeated fecal samples from individual calves. results for scours are reported in table and results pertaining to cryptosporidium are reported in table . two additional articles addressed other enteric pathogens, namely campylobacter, giardia, eimeria, rotavirus, and coronavirus. as shown in table , several studies demonstrated benefits of cow-calf contact on scours (carias and vaccaro, ; nocek et al., ; rajala and castrén, ; weary and chua, ; boonbrahm et al., a; wagenaar and langhout, ) . conversely, studies (svensson et al., ; roth et al., ) reported a greater risk of scours in dam-reared animals. roth et al. ( ) hypothesized that the higher incidence in nurs-ing calves could be explained by the increased quantity of milk consumed by these animals, rather than by infectious causes. indeed, wagenaar and langhout ( ) reported that the type of scouring sometimes observed figure . flowchart depicting the manuscript screening and appraisal process. note that summing the studies included in the qualitative synthesis in each subsection yields studies, rather than . this discrepancy results from the inclusion of studies in specific sections and studies in specific sections. a deletion of the duplicates results in unique studies. listed for each study are country, breed of cattle, type of cow-calf contact, duration of this contact, how milk or colostrum (or both) was fed to calves in the control condition (i.e., in which calves were not allowed to suckle), the amount of milk provided in this condition, the total number of calves followed, the number of herds (and groups within a herd where relevant), and the authors' conclusion and direction of effect (with + signifying a beneficial effect of suckling or cow-calf contact, − signifying a negative effect, and = representing no difference. studies are ordered chronologically within effect direction in suckling calves was characteristically distinct from that of bucket-fed counterparts, primarily because it did not lead to the animals dirtying their backsides. boonbrahm et al. ( a) found that mortality from scours and other conditions was higher in bucket-fed calves ( %) compared with dam-reared calves ( %), again suggesting a different etiology. it is now well known that high volumes of milk may contribute to looser manure in calves (see khan et al., ) , which need not reflect any infection. methods of evaluating scours in calves should therefore take into account differences in fecal consistency associated with level of milk intake. although the balance of the research seems to point to either neutral or positive effects of dam rearing on calf scours, results are mixed with respect to cryptosporidium (see table ). two of the included studies reported a protective effect of the dam's presence (kvac et al., ; duranti et al., ) , reported no difference (maldonado camargo et al., ; mohammed et al., ; delafosse et al., ; garro et al., ) , and the remaining (quigley et al., ; trotz-williams et al., , a indicated that dam-calf contact increases the risk of infection. this lack of consensus may stem from variation in study design and outcome measurements (e.g., herd versus sample-level prevalence), in breed (dairy versus mixed dairy-beef), or in the diagnostic methods chosen. despite the conflicting results, the collective burden of available odds ratios and risk ratios is skewed toward a protective effect of suckling. two papers addressed other specific enteric pathogens in relation to dam-calf contact. klein et al. ( ) found that herds leaving calves with the dam for > h had a . times greater odds of testing positive for campylobacter spp., and quigley et al. ( ) noted an increased risk of giardia in suckling calves, but no differences were found for eimeria, rotavirus, or coronavirus; further investigation of these and other pathogens in suckling systems is needed. johne's disease. of the articles included, directly addressed the relationship between the prevalence of mycobacterium avium ssp. paratuberculosis (map; the causal agent of johne's disease) and duration of cow-calf contact. all studies consisted of management questionnaires paired with serum (n = ) or milk (n = ) elisa testing, unspecified elisa testing (n = ), bulk-milk pcr (n = ), fecal culture (n = ), a combination of fecal culture and serum elisa (n = ), or clinical case reports (n = ). results are shown in table . a limiting factor in many of the cross-sectional studies presented in this review is the absence of a temporal relationship between johne's diagnosis and the imple- listed for each study are country, breed of cattle, study design, groups compared (in reference to length of cow-calf contact), total number of calves sampled, the number of herds included (and groups within herds where applicable), the type of herd, the diagnostic(s) used, and the authors' conclusion and direction of effect (with + signifying a beneficial effect of suckling or cow-calf contact, − signifying a negative effect, and = representing no difference. studies are ordered chronologically within effect direction). hf signifies that breed was reported as holstein, friesian, or holstein-friesian. this designation includes country-specific variants such as danish holstein. pr (ci) = prevalence ratio ( % ci). mentation of management practices. however, none of the included articles were able to identify increased map prevalence among herds permitting cow-calf contact, even in preliminary univariable analyses (with the exception of pillars et al., ) . official johne's disease control programs frequently describe the risks associated with allowing the calf to suckle and remain within the maternity area, or directly recommend immediate separation of cow and calf following parturition; such programs include the three step calf rearing plan of australia (animal health australia, ), the voluntary bovine johne's disease control program of the united states (usda, ), and several regionspecific canadian programs [such as the atlantic veterinary college ( ) johne's disease initiative, which deems calf nursing to be a risky practice, even within test-negative herds]. given the lack of quantitative evidence on the value of immediate cow-calf separation, the persistence and regularity of this recommendation is surprising. a common theme that emerged in our systematic search of this topic was the presence of unsubstantiated claims regarding map prevalence and cow-calf separation. an example of the dissemination of such claims within the literature is shown in figure . the type of citation scheme demonstrates how a largely unsubstantiated claim (in this case, that calves should be immediately separated from the dam to prevent johne's disease) can achieve the status of "common knowledge" in the literature. the perpetuation of these assertions may lead to decisions such as that of norton et al. ( ) to include "duration of dam-calf contact" in multivariable risk assessment modeling due to "biological importance," despite the variable's lack of significance at the univariable screening stage. the hypothesis regarding biological relevance is not unfounded because calves under mo of age are most susceptible to map infection. mycobacterium avium ssp. paratuberculosis may be transmitted from an infected dam to calf in utero or through direct bacterial shedding into colostrum or milk; however, map infection occurs predominantly by means of a contaminated environment, via the fecal-oral route (lombard, ) . we may thus imagine that prompt calf removal could mitigate transmission. several mathematical models (e.g., collins and morgan, ; marcé et al., ) have demonstrated that removing the calf from the calving area, or minimizing the effective number of cow-calf contacts, reduces map prevalence, but such models are often parameterized based upon an assumption that transmission increases for calves contacting adult animals. yet, in some herds, cow-calf separation has supplanted control strategies for which concrete evidence exists to tie the respec-tive strategy to a reduction in map prevalence. for example, wells and wagner ( ) noted that herds in which cows and calves were separated at h (compared with > h) were more than times as likely to have had a previous johne's disease diagnosis. the authors posit that herd managers altered their management practices following the positive herd status report. in contrast, the practice of sourcing > % of animals from outside dairies was associated with a current johne's disease diagnosis in the herd, yet no evidence was provided to suggest that this practice underwent a similar modification (wells and wagner, ) . johnson-ifearulundu and kaneene ( ) reported that the process of cleaning maternity pens was linked to a -fold reduction in the odds of a positive herd-level map status. however, many commercial herds allow multiple animals in the calving area (e.g., % in the united states; usda, ) or permit sick cows to be housed in these areas ( %). of those herds that did use dedicated individual maternity pens, only to % (depending on herd size) cleaned the area after each calving (usda, ). there is evidence for a synergism of infection risk in the calving area, based upon the level of environmental cleanliness, udder hygiene, and presence of other lactating animals (beaver et al., ) . the evidence we have reviewed indicates that prompt calf removal should not be viewed as a substitute for proper hygiene and management in the maternity area. respiratory health. of the included articles, ( single-herd and multi-herd studies) addressed respiratory health in dairy calves. the results of these studies are shown in table . the majority of these papers did not investigate pneumonia as a primary outcome measure; all but one study (gulliksen et al., a) was included in other subsections of this review. five studies failed to find any association between respiratory risk and dam-calf contact or nursing (although of these studies were conducted using the same group of calves: lundborg et al., , and svensson et al., ) . the remaining studies presented opposing conclusions, with gulliksen et al. ( b) reporting a higher risk of pneumonia for calves kept > h with the dam and boonbrahm et al. ( a) noting a lower pneumonia incidence in suckling calves. further research is clearly required before meaningful conclusions can be reached, including studies for which the investigation of specific respiratory pathogens is a primary objective. immunity. of the included articles, described calf immunity or failure of passive transfer (fpt) in suckling compared with artificial systems. the majority (n = ) were single-herd trials, and were multi-herd studies (table ) . the cited studies present conflicting conclusions. for example, stott et al. ( ) and quigley et al. ( ) described a positive association between suckling and immunoglobulin absorption in neonatal calves, and selman et al. ( ) found that the presence of the dam led to improved passive transfer. in contrast, several articles concluded that suckling results in higher levels of fpt (nocek et al., ; besser et al., ; trotz-williams et al., b; beam et al., ). this discrepancy may, in part, be traced back to the source and quantity of colostrum offered to control groups. it is now well established that quantity, quality, and promptness of colostrum administration is pivotal in facilitating immunoglobulin absorption and thus lowering the rate of fpt (see godden, ) . the current recommendation stipulates that calves obtain to % of their bw in colostrum at first feeding, and additional benefits have been shown in calves receiving further doses of colostrum at h postpartum (godden, ) . in several of the older studies (stott et al., ; quigley et al., ) , the volume of colostrum fed to control groups would be considered insufficient by today's standards, and is likely not comparable to the amount a suckling calf would obtain from the dam. moreover, in stott et al. ( ) the colostrum provided to control calves was pooled; pooling has been reported to increase the bacterial burden, leading to inhibition of immunoglobulin absorption (stewart et al., ) . thus, differences in fpt between dam-reared and artificially raised calves may be attributable to colostrum allowance. other studies seeking to compare rates of passive transfer between the groups are often encumbered by different limitations. besser et al. ( ) collected data from separate herds, each with different colostrum management practices (tube feeding, bottle feeding, or suckling). because these herds likely differed in a variety of other dimensions, and successful immunoglobulin absorption is affected by numerous external factors (godden, ) , it is difficult to isolate the effect of suckling. besser et al. ( ) made some effort to measure colostrum quality in all groups, but large volumes were fed to the control animals, and the authors acknowledge that these would far exceed the expected intake of a suckling calf. in nocek et al. ( ) and beam et al. ( ) , calves fed high-quality and promptly administered colostrum were compared with an unmonitored suckling group, with unknown colostral quality and latency to feed. thus, the same concerns regarding insufficient colostrum allowance (stott et al., ; quigley et al., ) are replicated, but in this case, are transferred to the suckling group. several other studies have addressed the question cow-calf removal and fpt without drawing a direct comparison between artificial rearing and suckling systems. for example, mcaloon et al. ( ) found that increased time spent in the calving pen was associated with diminished immunoglobulin absorption, likely because many calves did not suckle in the first few hours after birth. arguably, these conclusions are attributable to latency until farmer intervention rather than time spent with the dam, per se. producers aim to provide prompt administration of high quality colostrum to separated calves and could provide similar interventions for the suckling calf. (goodger et al., ) represents an aggregate of the following management practices: ( ) whether colostrum was harvested from a clean udder (free of manure), ( ) whether the bottles used to store colostrum were clean, ( ) whether colostrum was pooled, and ( ) whether calves were permitted extended contact with the dam. map = mycobacterium avium ssp. paratuberculosis. listed for each study are country, breed of cattle, type of cow-calf contact, duration of this contact, how colostrum was fed to calves in the control condition (i.e., in which calves were not allowed to suckle), the amount of colostrum provided in this condition, the total number of calves followed, the number of herds (and groups within a herd), and the authors' conclusion and direction of effect (with + signifying a beneficial effect of suckling or cow-calf contact, − signifying a negative effect, and = representing no difference. studies are ordered chronologically within effect direction). fpt = failure of passive transfer. it is important to recognize that a large proportion of dairy calves left with the dam fail to nurse within h after parturition ( % of calves born to dams of second or higher parity; edwards and broom, ) . calves may exhibit a higher latency to suckle if they have low vigor or if the dam has experienced a difficult calving (rajala and castrén, ) . thus, the farmer cannot rely upon nature alone but rather should supervise and intervene if necessary to promote adequate passive immune transfer to the calf. calves born to dams with low-hanging udders (ventorp and michanek, ) may also have more difficulty obtaining colostrum by suckling, suggesting that these calves in particular could benefit from active assistance with nursing or administration of colostrum by bottle. together these observations indicate that various types of farmer intervention, including careful observation and supplementary feeding, may be beneficial regardless of whether the calf is separated from the cow. given that bacterial contamination of colostrum can interfere with immunoglobulin absorption (see godden, ) , it is useful to compare bacterial counts in directly stripped colostrum (which a suckling calf would be expected to obtain) and corresponding counts in harvested and stored colostrum sources. one study (stewart et al., ) found that the process of harvesting colostrum into a bucket resulted in dramatically higher bacterial counts than in directly stripped colostrum. additionally, bacteria in colostrum can multiply precipitously when kept at ambient room temperature and may even reach concentrations exceeding , , cfu/ml after h of refrigeration (stewart et al., ) . bacterial growth in colostrum may add variation to how calves perform when fed by bucket or teat; artificial colostrum feeding is therefore likely to work best on farms able to mitigate this risk (e.g., through decontamination of storage containers). thus there may be challenges and benefits associated with both hand feeding and nursing colostrum from the dam. given the conflicting evidence presented, and the flawed comparisons between groups, the common recommendation to separate the cow and calf immediately after parturition to ensure successful immune transfer should not be considered to be evidence based. however, leaving the calf unsupervised with the dam cannot serve as a replacement for careful colostrum management. mortality. of the included articles, addressed the subject of general calf health and directly addressed calf mortality. six were single-herd trials and the remaining incorporated between and herds, with data obtained from questionnaires or surveys on management practices and calf mortality rates (table ). there appears to be little consensus regarding mortality in dam-reared versus conventionally raised calves. the reasons for this variation are likely similar to those previously described for the fpt outcomes. that is, conventionally reared calves may be at risk when study methodology results in inadequate colostrum uptake (as in quigley et al., ) . insufficient colostrum quantity has been definitively linked to fpt, which, in turn, has strong associations with mortality (see godden, ) . similarly, dam-reared calves may experience increased mortality rates when colostrum intake is unmonitored, unaided, or not quality-controlled. the confounding of dam rearing with low-input management may account for the conclusions from large-scale, multi-herd studies such as jenny et al. ( ) and wells et al. ( ) . indeed, jenny et al. ( ) stated that farmers leaving calves with the dam may wrongly assume these calves have obtained sufficient colostrum via suckling. in contrast, farmers separating the calf and dam must undertake the "mothering role" themselves and provide individualized attention. waltner-toews et al. ( ) noted an interesting phenomenon in calves provided assistance at suckling. in this study, calves that suckled colostrum naturally were less likely to require treatment for disease than were calves fed colostrum by bucket; however, calves requiring assistance to suckle also had higher odds of being treated for disease compared with calves suckling naturally. these findings could indicate that farmers were more concerned about the assisted suckling group and therefore were more likely to provide treatment, or that these animals were "weak calves to begin with" (p. ) and thus required more treatment. in any case, these findings do not imply that suckling assistance itself leads to increased disease rates. of the included articles, addressed mastitis in dairy cows. of these, were conducted using a single herd, wherein to groups were evaluated. the cohorts typically consisted of cow-calf pairs in either full or restricted-suckling systems in addition to variants of artificially reared controls; however, one of these articles (wagenaar et al., ) addressed the future udder health of heifer calves raised under contrasting management conditions. of the remaining multi-herd studies, one evaluated cohorts across separate herds, and the other employed a questionnaire on management paired with clinical mastitis assessments for herds. the assessments of udder health and mastitis rates were conducted using the california mastitis test ( ) ( ), electrical conductivity ( ), or unspecified methods ( ). table lists the included studies. consistent with the findings of johnsen et al. ( ) , the studies reviewed here demonstrate a beneficial effect of suckling systems in reducing the risk of mastitis in dairy cows. in addition to the advantages of suckling to remove residual milk from the udder, reduced mastitis rates in suckled cows could be attributed to lysozymes for bacterial inhibition present in calf saliva (mdegela et al., ) . no study in our systematic review demonstrated an increased risk of imi in suckled cows; however, one study did report higher rates of teat damage (thomas et al., ) . this damage was most pronounced in cows suckling calves twice daily for wk compared with machine milking. these authors recommend nursing for shorter durations to prevent the temporary teat damage associated with continuous, long-term suckling. walsh ( ) found that the greatest benefits in mastitis prevention afforded to nursing cows were in early and mid-lactation. together, these results suggest that producers interested in reducing the risk of mastitis should consider the use of suckling, particularly in early lactation (see also kälber and barth, ) . we systematically reviewed literature on the effect of suckling on calf health, with an emphasis on calf enteric health (including general scours, cryptosporidiosis, and johne's disease), respiratory health, immune status, and mortality. in sum, the evidence presented in this review does not support the recommendation of immediate separation to promote calf health. specifically, we found no consistent evidence of increased risk of pneumonia in calves reared with the cow; however, the available literature on this subject was limited and often did not address the subject of respiratory health as a primary research objective. with respect to enteric health, the majority of studies demonstrated that rearing the calf with the dam had no effect on scours, or was associated with a reduced risk. future research should aim to identify differences in etiology (e.g., infectious versus nutritional causes) between scours in dam-reared compared with artificially reared calves. the leading cause of mortality in dairy calves is diarrhea, for which a wide variety of enteric pathogens are responsible (cho and yoon, ) . with the exception of c. parvum (for which the evidence was mixed), common pathogenic causes of scours in the dairy calf have not been evaluated in detail in artificial compared with suckling systems (apart from the work of klein et al., , and quigley et al., ) . johne's disease is commonly considered an important risk associated with cow-calf contact, but we could find little evidence that rearing the calf with the dam increases map prevalence. contact with the dam after birth cannot be considered an appropriate surrogate variable for "contact with adult cow manure," unless aspects of cow hygiene, colostrum management, maternity-pen management, and overall cleanliness of the calving area are poorly governed. as several these variables have been strongly associated with map transmission risk (e.g., see ansari-lari et al., ; tiwari et al., ; donat et al., ) , initiatives to improve these measures should be prioritized over cowcalf separation. more targeted longitudinal research is required to assess whether immediate cow-calf separation provides any substantive benefit across herds with differing cow-level prevalence. for measures of calf immunity and mortality, no consistent pattern was observed, with studies split between those showing benefits versus risks associated with suckling. studies demonstrating benefits associated with suckling typically did not provide sufficient colostrum or milk to the artificially reared calves. similarly, articles detailing the risks of leaving the calf with the dam did not systematically monitor colostrum intake, quality, and suckling latency. interestingly, our systematic review uncovered only a single study addressing immunity and one addressing mortality authored within the last decade (beam et al., , and gulliksen et al., b, respectively) . in nocek et al. ( ) , several experimental groups were completely deprived of colostrum. given our current knowledge of the importance of colostrum quality, quantity, and timeliness of provision, further studies should assess the feasibility of keeping the calf with the dam and providing supplemental high-quality colostrum as soon as possible after parturition. the results of our systematic search into the effects of suckling systems on dairy cow health do not indicate any benefit to early separation. all of the studies included in this review demonstrated a reduced risk of mastitis in nursing dairy cattle or reported no difference. mastitis is one of the most prevalent disorders in dairy systems worldwide, and some evidence suggests that incidence is on the rise in north american herds (see usda, , versus usda, . as mastitis is also, arguably, the most economically significant challenge facing dairy producers (see seegers et al., ; jamali et al., ) , the favorable conclusions with regard to dam-calf suckling should not be taken lightly. our conclusions on mastitis are in agreement with other reviews on this topic (e.g., kamboj and kumar, ; kälber and barth, ; johnsen et al., ) ; the consensus among these authors is that some type of listed for each study are country, breed of cattle, type of cow-calf contact allowed, duration of this contact, how colostrum was fed to calves in the control condition (i.e., in which calves were not allowed to suckle), the amount of colostrum provided in this condition, the age range of calves under study, the total number of calves followed, the number of herds (and groups within a herd), and the authors' conclusion and direction of effect (with + signifying a beneficial effect of suckling or cow-calf contact, − signifying a negative effect, and = representing no difference. studies are ordered chronologically within effect direction restricted suckling system has the potential to reduce mastitis on dairy farms. future research is necessary to determine the exact mechanisms responsible for reducing this risk (e.g., removal of residual milk by the calf, lysozymes present in calf saliva, and so on). surprisingly, there was an absence of literature addressing other common postpartum diseases and conditions such as metritis, ketosis, and retention of the fetal membrane. in their review, flower and weary ( ) address retained fetal membranes and cite the danish work of krohn et al. ( ) , which highlights a beneficial effect of suckling on lowering the risk of placental retention. flower and weary ( ) suggest that this evidence could partially explain the lower incidence of retained fetal membranes in beef compared with dairy cows (noakes, ) . another study demonstrated no effects of immediate calf separation on latency of placental expulsion or placentophagia (lidfors, ) . given the importance of postpartum diseases on the longevity and productivity of dairy cattle (mulligan and doherty, ) , this area of research requires further exploration. the evidence extracted from the included journal articles does not support a recommendation of early dairy cow-calf separation on the basis of calf or cow health. specifically, the body of literature on calf immunity, mortality, scours, and pneumonia does not indicate that early separation is advantageous. moreover, there is an absence of literature to suggest that immediate dam-calf separation confers benefits toward mitigating johne's disease. with respect to cow health, this review indicates that suckling is protective against mastitis. using material sourced from domains and of ro-bis (risk of bias in systematic reviews, whiting et al., ) , we have attempted to identify and address weaknesses in our methodology. we restricted our initial literature screening to a single search engine (wos), as different search engines employ distinct criteria and weightings to generate search results. in addition, the search terms themselves require alteration across different databases; certain search engines automatically account for similar terms whereas others do not. it therefore becomes difficult to integrate reference lists that have been generated based upon unique algorithms. we considered wos to be the best available resource for our search due to its status as a human-curated database with itemized, structured entries. additionally, wos only provides results from journals indexed by clarivate analytics and filters out non-peer-reviewed literature; however, articles from well-known, high-impact-factor journals may be overrepresented in our results. the use of a single search engine may be considered a weakness of the present review. thus, we have attempted to validate this approach by conducting targeted searches in pubmed to ensure no additional articles were discoverable. first, we ran the exact searches as listed in materials and methods in pubmed. results indicated that no exact match was found for the phrases "reared by the dam," "reared by cows," "reared by the mother," and "calf contact with adults." therefore, these phrases were removed to ensure that pubmed would not search within the quoted phrases. our final pubmed search included the following fixed set: ("cowcalf" or "cow/calf" or "dam-calf" or "dam/calf" or "dam rearing" or "suckling system" or "mother rearing") and (nurs* or suckl* or separation or contact or "risk factor*") and (calf or calves). in addition, the following targeted terms were included: (scour* or diarr*) or (cryptosporidi*) or ("johne's" or paratuberculosis) or (pneumonia or respiratory) or (immunity or "passive transfer") or (disease or diseases or diseased or infection or health or morbidity or mortality) or (mastitis or "intramammary infection*" or "udder health"). a total of articles were uncovered in these searches; of these were duplicates from our wos searches. of the unique articles, pertained to beef cattle or other species and were therefore excluded. the titles and abstracts of the remaining articles were scanned, and were considered to be irrelevant to the main research question. two full texts were reviewed and neither study made a direct comparison between cow or calf health in suckling or cow-calf contact systems and conventional rearing. pubmed searches were then conducted for all terms that yielded no results in wos (as listed in materials and methods). no additional relevant results were identified. the "topic" field of wos generates searches of the article title, abstract, and keywords, but not the body of the manuscript. papers assessing a variety of risk factors for a given disease may therefore have been overlooked, particularly if the risk factor of interest was not significantly associated with cow-calf contact. thus, it is conceivable that the results of the wos queries reported here demonstrate a bias away from the null. fortunately, this bias is expected to be present in both directions and is therefore unlikely to affect overall conclusions. to counter this undesired selectivity, we searched within reference lists to uncover additional papers. it must be acknowledged that the authors of these papers may be more inclined to cite papers in agreement with their own conclusions, but this type of selection bias is likely to arise in any systematic review using reference lists as additional resources. similarly, although we placed no restriction on publication year, we did not locate any manuscripts published in or , since very recent papers tend to have fewer citations (from even more recent manuscripts). in the initial scans of the reference lists, we only selected papers with titles that alluded to a potential comparison of cow or calf health in different rearing conditions. for example, a hypothetical paper titled "management decisions linked to heightened prevalence of cryptosporidium oocysts" would have been considered, whereas a paper titled "cryptosporidium parvum in cattle" would not have been investigated further. we may have missed papers that, in spite of a more general title, had in fact addressed the topic in question. this assessment is unavoidably qualitative. it is not possible to measure the number of primary literature articles that may have been overlooked due to our methodology; this highlights the importance of accurate and descriptive key words in manuscripts as emphasized by de boer et al. ( ) . we excluded conference proceedings (both papers and abstracts), as well as book chapters, as we could not be certain that these sources had been peer reviewed. we also excluded literature in languages other than english, as we were unable to critically assess the methods and evaluate the results. we are unable to determine to what extent these exclusions affected the conclusions of this review. to provide an inclusive overview of the topic in question, no exclusion criteria were developed concerning sample size, study quality, or journal quality. here we provide a quality assessment of the included journal articles on the basis of study design, thoroughness of reporting, and potential biases. in the field of human medicine, randomized controlled trials are typically considered the gold standard, and observational research is sometimes excluded from systematic reviews. the number of randomized controlled trials in agriculture and veterinary medicine is comparatively low (sargeant and o'connor, ) , so observational studies are often included out of necessity. with reference to study design, articles in our systematic review ( %) were experimental or quasi-experimental and ( %) were observational. randomization procedures for animals were described or referenced in studies ( % of the experimental studies), but only included some type of blinding (blinding was likely not feasible for some measures, given the obvious differences in management between groups with different levels of maternal contact). random assignment of animals to treatment groups was described in of the single-herd trials ( %), and random selection of herds (or a random selection of animals within herds) was described in ( %) of the multi-herd studies. in tables to , we have reported the authors' conclusions concerning their data, regardless of whether these conclusions were tested using inferential statistics. of the included papers, ( %) either did not confirm their conclusions using statistical testing or failed to report results. in these instances, the reported results often reflected secondary research objectives or descriptive information. as per the inclusion criteria, papers investigating other aspects of cow and calf contact or separation as a primary objective were not excluded, as long as health-related measures were also reported. a further studies ( %) reported conclusions based upon a . α-level threshold. these papers are indicated with footnotes in their respective tables. study limitations, potential biases, and sources of error were formally discussed in manuscripts ( %), although only paper included an assessment of inter-observer reliability. some level of missing data was reported in studies ( %), with explanation for the missing data provided in of these. seven of the single-herd studies ( %) did not contain complete information on the type and duration of cow-calf contact and feeding regimens for control groups. interestingly, ( %) of papers failed to provide information on country or region in which the study was conducted (although the authors' affiliations and the location of laboratories used for sample analysis most often suggested that these studies were conducted in the united states). an additional studies ( %) did not include any information pertaining to cattle breed (of these, were multi-herd trials). for manuscripts addressing johne's disease, cryptosporidiosis, or mastitis, ( %) adequately discussed the diagnostic test used, although only ( %) cited the sensitivity or specificity (or both) of the implemented diagnostic. of the articles in the immunity section that made reference to fpt, only ( %) provided a full definition for the condition and ( %) offered rationales or citations supporting the diagnostic cut points implemented. in the single-herd studies, there was a high variability in what constituted control groups in terms of the level of maternal contact. for example, in of these studies, control calves were given less than h of contact with the dam; in a further studies, control animals were provided with a minimum of d of unrestricted dam-calf contact. the feeding regimens were also different across studies. these discrepancies are likely to have affected conclusions, and thus it is difficult to make direct comparisons between articles. even in studies seemingly describing the same treatment (e.g., "restricted suckling"), the implementation of these treatments was variable. for example, in mejia et al. ( ) calves in the "restricted suckling" group were permitted suckling for min after milking (with one quarter left unmilked), twice daily for mo. in contrast, in fulkerson et al. ( ) , calves were reunited with the dam once per day (for an unspecified amount of time) for approximately mo. further, in several studies (e.g., fallon and harte, ; nocek et al., ) , dam-calf suckling is referenced, but the duration of contact between cows and calves in the control group is not described. several of the older studies had low sample sizes and are more accurately interpreted as case reports. at one extreme, rigby et al. ( ) presented conclusions regarding mastitis based upon a single cow nursing her calf; however, most of the other studies with small sample sizes were able to enroll at least animals. due to the variability in study type, methodology, use of controls, and the amount of information provided, it was not feasible to conduct a meta-analysis. we provided risk ratios, prevalence ratios, and odds ratios where possible, but the variation in study methodology prevented us from generating a meaningful estimate of the overall magnitude of the effects considered. instead, only the direction of association was highlighted, and our discussion is mainly based upon on the number of positive and negative associations. a weakness of this approach is that it weighs equally all studies, regardless of sample size, effect size, and any weaknesses in design and interpretation. it is important to consider that no temporal relationship between management factors (e.g., cow-calf separation) and outcomes (e.g., disease) has been examined in the included cross-sectional studies. thus, management decisions could plausibly have been implemented in response to the disease in question, rather than representing a risk factor for disease. effect on milk production and calf performance of milking crossbred european/zebu cattle in the absence or presence of the calf, and of rearing their calves artificially three step calf rearing plan risk factors for mycobacterium avium ssp. paratuberculosis in fars province (southern iran) dairy herds atlantic johne's disease initiative, risk assessment workbook relationship between frequent milking or suckling in early lactation and milk production of high producing dairy cows prevalence of failure of passive transfer of immunity in newborn heifer calves and associated management practices on us dairy operations comparative risk assessment for new cow-level mycobacterium avium ssp. paratuberculosis infections between dairy production types: organic, conventional, and conventional-grazing systems factor analysis of a johne's disease risk assessment questionnaire with evaluation of factor scores and a subset of original questions as predictors of observed clinical paratuberculosis comparison of three methods of feeding colostrum to dairy calves the influence of calf rearing methods and milking methods on performance traits of crossbred dairy cattle in thailand . calf performance the influence of calf rearing methods and milking methods on performance traits of crossbred dairy cattle in thailand - . milk yield and udder health rearing holstein friesian and brown swiss calves on nurse cows relationships between the presence of johne's disease and farm and management factors in dairy cattle in england prevalence, incidence and geographical distribution of johne's disease in cattle in england and the welsh borders an overview of calf diarrhea-infectious etiology, diagnosis, and intervention epidemiological model of paratuberculosis in dairy cattle herd prevalence and geographic distribution of, and risk factors for, bovine paratuberculosis in wisconsin risk associations to milk elisa result for paratuberculosis in dairy cows in northern portugal using a multilevel regression model path model of individual-calf risk factors for calfhood morbidity and mortality in new york holstein herds eimeriosis in cattle: current understanding invited review: systematic review of diagnostic tests for reproductive-tract infection and inflammation in dairy cows effects of twice-daily nursing on milk ejection and milk yield during nursing and milking in dairy cows cryptosporidium parvum infection and associated risk factors in dairy calves in western france management practices associated with mycobacterium avium ssp. paratuberculosis infection and the effects of the infection on dairy herds management of the calving pen is a crucial factor for paratuberculosis control in large dairy herds risk factors associated with cryptosporidium parvum infection in cattle the period between birth and first suckling in dairy calves methods of feeding milk to young calves natural transmission of cryptosporidium parvum between dams and calves on a dairy farm the effects of early separation on the dairy cow and calf respiratory pathogens in quebec dairy calves and their relationship with clinical status, lung consolidation, and average daily gain health and performance of holstein calves that suckled or were hand-fed colostrum and were fed one of three physical forms of starter effect of restricted suckling on milk yield, milk composition and udder health in cows and behaviour and weight gain in calves, in dual-purpose cattle in the tropics effect of suckling ('restricted suckling') on dairy cows' udder health and milk let-down and their calves' weight gain, feed intake and behaviour improvement in milk production of first calf heifers by multiple suckling prevalence and risk factors for shedding of cryptosporidium spp. oocysts in dairy calves of buenos aires province colostrum management for dairy calves effect of residual calf suckling on clinical and sub-clinical infections of mastitis in dual-purpose cows: epidemiological measurements epidemiologic study of on-farm management practices associated with prevalence of mycobacterium paratuberculosis infections in dairy cattle respiratory infections in norwegian dairy calves calf mortality in norwegian dairy herds calf mortality invited review: incidence, risk factors, and effects of clinical mastitis recurrence in dairy cows management factors associated with calf mortality in south carolina dairy herds is rearing calves with the dam a feasible option for dairy farms? current and future research managementrelated risk factors for m. paratuberculosis infection in michigan, usa, dairy herds practical implications of suckling systems for dairy calves in organic production systems-a review effect of weaning on performance and behaviour of calves and their dams in dairy cows-a review invited review: effects of milk ration on solid feed intake, weaning, and performance in dairy heifers prevalence and risk factors for shedding of thermophilic campylobacter in calves with and without diarrhea in austrian dairy herds comparison of the behaviour of newborn calves housed with the dam and in the calf-house long-term effect of colostrum feeding methods on behaviour in female dairy calves cow-calf relations. : the effect of vs. days suckling on behaviour, milk production and udder health of cows in different stabling age-related and housing-dependence of cryptosporidium infection of calves from dairy and beef herds in south bohemia behavioural effects of separating the dairy calf immediately or days post-partum influence of partial suckling of crossbred dairy cows on milk offtake and calf growth in the ethiopian highlands epidemiology and economics of paratuberculosis herd-level risk factors for infectious diseases in swedish dairy calves aged - days prevalence of and risk factors for shedding of cryptosporidium parvum in holstein friesian dairy calves in central mexico within-herd contact structure and transmission of mycobacterium avium subspecies paratuberculosis in a persistently infected dairy cattle herd restricted suckling of tropical dairy cows by their own calf or other cows' calves relationship between selected perinatal paratuberculosis management interventions and passive transfer of immunity in dairy calves disease management of dairy calves and heifers prevalence and determinants of mastitis and milk-borne zoonoses in smallholder dairy farming sector in kibaha and morogoro districts in eastern tanzania invited review: a systematic review of the effects of prolonged cow-calf contact on behavior, welfare, and productivity effects of restricted suckling versus artificial rearing on milk production, calf performance and reproductive efficiency of dual purpose mpwapwa cattle in a semi-arid climate effect of restricted suckling on milk yield, composition and flow, udder health, and postpartum anoestrus in grazing holstein cows maternal influence on defecation and urination in the newborn calf risk factors associated with cryptosporidium parvum infection in dairy cattle in southeastern new york state a review on major bacterial causes of calf diarrhea and its diagnostic method production diseases of the transition cow a herd health approach to dairy cow nutrition and production diseases of the transition cow herd management practices associated with paratuberculosis seroprevalence in dutch dairy herds effect of management practices on paratuberculosis prevalence in danish dairy herds fertility and obstetrics in cattle, edition . blackwell science influence of neonatal colostrum administration, immunoglobulin, and continued feeding of colostrum on calf gain, health, and serum protein a questionnaire-based cross-sectional study of clinical johne's disease on dairy farms in new zealand fecal shedding of coliform bacteria during the periparturient period in dairy cows management factors related to calf morbidity and mortality rates association between risk-assessment scores and individualcow johne's disease-test status over time on seven michigan, usa dairy herds effects of housing and colostrum feeding on the prevalence of selected infectious organisms in feces of jersey calves effects of housing and colostrum feeding on serum immunoglobulins, growth, and fecal scores of jersey calves serum immunoglobulin concentrations and health of dairy calves in two management systems from birth to weeks of age effect of compliance with recommended calf-rearing practices on control of bovine johne's disease rearing dairy calves by restricted suckling. vii. effect on mastitis development caused by staphylococcus aureus. can influence of artificial vs. mother-bonded rearing on sucking behaviour, health and weight gain in calves effects of restricted suckling versus artificial rearing on performance and fertility of bos taurus and bos indicus cows and calves in tanzania effects of restricted suckling versus artificial rearing on performance and fertility of crossbreed f (holstein friesian x local) cows and calves in vietnam issues of reporting in observational studies in veterinary medicine production effects related to mastitis and mastitis economics in dairy cattle herds absorption of immune lactoglobulin by newborn dairy calves. attempts to produce consistent immune lactoglobulin absorptions in newborn dairy calves using standardised methods of colostrum feeding and management response of dairy cows and calves to early separation: effect of calf age and visual and auditory contact after separation preventing bacterial contamination and proliferation during the harvest, storage, and feeding of fresh bovine colostrum colostral immunoglobulin transfer in calves. iv. effect of suckling morbidity in swedish dairy calves from birth to days of age and individual calf-level risk factors for infectious diseases herd characteristics and management practices associated with seroprevalence of mycobacterium avium ssp. paratuberculosis infection in dairy herds influence of suckling by friesian cows on milk production and anoestrus risk factors associated with mycobacterium avium ssp. paratuberculosis seropositivity in canadian dairy cows and herds passive immunity in ontario dairy calves and investigation of its association with calf management practices association between management practices and within-herd prevalence of cryptosporidium parvum shedding on dairy farms in southern ontario calf-level risk factors for neonatal diarrhea and shedding of cryptosporidium parvum in ontario dairy calves part iv: reference of dairy cattle health and management practices in the united states uniform program standards for the voluntary bovine johne's disease control program dairy cattle management practices in the united states national health monitoring system. dairy : health and management practices on u.s. dairy operations the importance of udder and teat conformation for teat seeking by the newborn calf effect of production system, alternative treatments and calf rearing system on udder health in organic dairy cows practical implications of increasing 'natural living' through suckling systems in organic dairy calf rearing milk secretion in machine-milked and suckled cows dairy calf management, morbidity and mortality in ontario holstein herds. iii. association of management with morbidity effects of early separation on the dairy cow and calf: . separation at h, day and days after birth herd-level risk factors for infection with mycobacterium paratuberculosis in us dairies and association between familiarity of the herd manager with the disease or prior diagnosis of the disease in that herd and use of preventive measures factors associated with mortality to days of life in dairy heifers in the united states robis: a new tool to assess risk of bias in systematic reviews was developed we are grateful to dairy australia (melbourne, australia) for soliciting this review and for providing support to rebecca meagher and annabelle beaver. we also thank angela yu and auguste de pennart (both students at the university of british columbia) for their assistance. key: cord- -yha o l authors: delafosse, a.; chartier, c.; dupuy, m.c.; dumoulin, m.; pors, i.; paraud, c. title: cryptosporidium parvum infection and associated risk factors in dairy calves in western france date: - - journal: prev vet med doi: . /j.prevetmed. . . sha: doc_id: cord_uid: yha o l this study was conducted to determine the prevalence and risk factors for cryptosporidium infection in calf neonates on dairy farms in normandy. fecal samples were randomly collected between july and september from calves ( – days old) on farms. up to calves were selected and sampled per farm, and feces examined for oocysts by microscopy. c. parvum oocyst shedding was scored semi-quantitatively ( – ). a questionnaire about calf-level care and management was completed, and mortality rates were obtained from the french national registration database (bdni). bivariable and multivariable analyses of potential risk factors for c. parvum oocyst shedding were conducted using generalized estimating equation (gee) models (family = binomial).overall, out of calves ( . %) were positive for oocysts, and . % of animals had a shedding score > . seven of the farms ( %) were negative for oocysts in all fecal samples. at the time of collection, calves ( %) had diarrhea, and its prevalence strongly correlated with the score for c. parvum oocyst shedding (p < . ). the mortality rate at days was significantly greater for calves with high combined scores of diarrhea and shedding. factors associated with the shedding of c. parvum were the normande breed (odds ratio = . ; % confidence interval (ci): . – . ), dispensing of colostrum using a bucket (odds ratio = . ; % ci: . – . ), treatment with halofuginone (odds ratio = . ; % ci: . – . ) and feeding with fermented milk (odds ratio = . ; % ci: . – . ). c. parvum is widespread among calves under days old in dairy herds of western france. shedding of c. parvum is associated with a high incidence of diarrhea and increased risk of mortality in young calves. this study identified some associated calf-level factors, although further investigations are necessary to determine appropriate measures that farmers and veterinary practitioners should take to reduce the prevalence of c. parvum. this study was conducted to determine the prevalence and risk factors for cryptosporidium infection in calf neonates on dairy farms in normandy. fecal samples were randomly collected between july and september from calves ( - days old) on farms. up to calves were selected and sampled per farm, and feces examined for oocysts by microscopy. c. parvum oocyst shedding was scored semi-quantitatively ( - ). a questionnaire about calf-level care and management was completed, and mortality rates were obtained from the french national registration database (bdni). bivariable and multivariable analyses of potential risk factors for c. parvum oocyst shedding were conducted using generalized estimating equation (gee) models (family = binomial).overall, out of calves ( . %) were positive for oocysts, and . % of animals had a shedding score > . seven of the farms ( %) were negative for oocysts in all fecal samples. at the time of collection, calves ( %) had diarrhea, and its prevalence strongly correlated with the score for c. parvum oocyst shedding (p < . ). the mortality rate at days was significantly greater for calves with high combined scores of diarrhea and shedding. factors associated with the shedding of c. parvum were the normande breed (odds ratio = . ; % confidence interval (ci): . - . ), dispensing of colostrum using a bucket (odds ratio = . ; % ci: . - . ), treatment with halofuginone (odds ratio = . ; % ci: . - . ) and feeding with fermented milk (odds ratio = . ; % ci: . - . ). c. parvum is widespread among calves under days old in dairy herds of western france. shedding of c. parvum is associated with a high incidence of diarrhea and increased risk of mortality in young calves. this study identified some associated calf-level factors, although further investigations are necessary to determine appropriate measures that farmers and veterinary practitioners should take to reduce the prevalence of c. parvum. © elsevier b.v. all rights reserved. cryptosporidium is one of the most common enteropathogens present in calves during their first two weeks of age (thompson et al., ) . young calves mainly shed cryptosporidium parvum, which has a wide host range and is potentially zoonotic. clinical infection in calves is characterized by non-specific diarrhea, dehydration, depression, anorexia and abdominal pain. in most calves, diarrhea starts - days postinfection and lasts from to days (de graaf et al., ) . oocyst shedding begins at days after birth and peaks at - days, decreasing after weeks (nydam et al., ; trotz-williams et al., ) . higher oocyst shedding is frequently seen during episodes of diarrhea (xiao and herd, ; mccluskey et al., ) . despite much endeavor in the development of effective drugs, halofuginone lactate (hl) is the only drug registered in europe to treat cryptosporidiosis in cattle. hl can reduce, but not completely prevent, oocyst shedding and diarrhea in calves (joachim et al., ) , suggesting that additional hygiene measures must be adopted to provide better control of cryptosporidium infections (de waele et al., ) . other enteric viral and bacterial pathogens such as rotaviruses, coronaviruses and escherichia coli can also cause diarrhea in bovine neonates to the same degree of severity as cryptosporidiosis (de graaf et al., ) . cryptosporidiosis is a multifactorial disease in which many parameters associated with calves, and environment and production practices, may be of significance. previous studies have indicated that cryptosporidiosis may occur more frequently in dairy calves than in beef calves because the former are born throughout the year and are confined to pens or hutches, which can facilitate a high level of yearround transmission (olson et al., ) . other factors linked to passive immunity of the calf and management of large herds may also be relevant (o'handley, ) . several factors have been identified as significant in increasing the risk of shedding cryptosporidium oocysts by dairy calves. most studies have focused on factors associated with management of the herd, and attempted to resolve these issues by presenting a questionnaire to farmers (e.g., garber et al., ; mohammed et al., ; trotz-williams et al., ; silverlås et al., b) . significant risk factors may differ from survey to survey, indicating that different epidemiological practices may need to be adopted on different farms. epidemiological data for dairy calf cryptosporidiosis in france is scarce, and there are no risk factor analysis. the first surveys in france underlined the importance of cryptosporidium in neonatal diarrhea and reported that the prevalence of oocyst shedding ranged from . % to . %, depending on the sampling regime (naciri et al., ; lefay et al., ) . molecular characterization of cryptosporidium isolates shed by dairy calves identified the zoonotic c. parvum as the dominant species, followed by c. bovis and c. ryanae (follet et al., ; rieux et al., ) . the aim of this study was to determine the prevalence and risk factors for cryptosporidium infection in bovine neonates in an area of intensive dairy farming in western france. the sampling pool consisted of herds located in the orne department of normandy (france), from which a computer-generated list of farms was randomly selected (survey toolbox software). farms were visited every two weeks during the calving period between july and september . ten calves that were - days old were sampled on each farm during biweekly visits. calves were randomly selected if there were more than available. individual fecal samples were taken directly from the rectum of each calf using sterile plastic gloves and stored at • c before transfer to the laboratory. at the time of collection, the consistency of each fecal sample and general condition of the calf were scored according to naciri et al. ( ) in the following manner: for diarrhea, absence ( ), mild ( ), heavy ( ); and for general condition, normal vitality ( ), low vitality ( ), very low vitality ( ), recumbency ( ). sampling was done in compliance with animal welfare and did not cause distress according to the french ethics committee for animal experimentation no. . information on the calf mortality rate was obtained from the french national registration database (bdni). mortality was estimated from the number of calves that died before they reached days. calves that were sold before days were excluded from this calculation. screening for oocysts was done by staining fecal smears with ziehl fuchsin and observing at × magnification under a phase-contrast microscope according to heine ( ) . the intensity of shedding was evaluated semiquantitatively according to the average number of oocysts in randomly selected observation fields. six categories were defined as follows: (absence of oocysts), (< oocyst per field), ( - oocysts), ( - oocysts), ( - oocysts) and (> oocysts). one person conducted all tests. considering direct ifat as the gold standard, heine staining of fecal smears has a sensitivity of . % and was used because the scoring ( - ) correlates with the number of oocysts in feces . a questionnaire was used to collect information about the management of each calf sampled. questions were designed to gather information about potential predictors of risk for shedding of c. parvum. data were collected in the field during an interview with the farmer or farm manager. the positive or negative status of each fecal specimen was scored as a dichotomous variable (heine technique score ≥ vs control: score = ). all factors investigated were categorical variables collected at the individual level as follows: age, sex, breed, period of birth, type of housing in which the calf was kept, ease of birth, care given to the calf, vaccines and drugs dispensed to the calf, maternal vaccination against neonatal diarrhea, length of time calf stayed with the dam after birth, calving area hygiene, type of colostrum/milk fed, and equipment used to feed calves (table ) . factors associated (p < . ) with the outcome variable in the bivariable analysis were included in a multivariable analysis. the choice of a screening criterion of . was designed to limit multicollinearity and ensure that potentially important variables were included in the multivariable analysis (dohoo et al., ) . cramer's v coefficient (v) was used to investigate collinearity between categorical predictor variables (cramer, ) . this measure is defined as: v = √ (x /nt) where x is the pearson's chi-squared statistic, n is the grand total of observations, and t is the smaller of the number of rows minus one or the number of columns minus one. when variables were significantly associated (v > . ), only one of the two variables was included in the initial model; the variable was chosen based on the strength of association with the outcome variable and biological plausibility. bivariable and multivariable analyses were conducted using generalized estimating equation (gee) logistic models (liang and zeger, ) to account for within-herd correlation (mcdermott et al., ; kadohira et al., ) . the structure of correlation among observations of the same cluster (herd) was assumed to be constant (option "exchangeable"), which is acceptable when the cluster size is large (> ). all analyses were performed using the statistical software r . . (stats and geepack packages). fecal samples were collected from calves on farms. of the farms originally selected, refused to participate due to time constraints. on one farm, only eight calves could be sampled due to an insufficient number of newborn animals. to sample calves on each farm, several visits were made as follows: visits for farms, for farms, for farms, for farms, for farms and for farm. in total, of the calves ( . %) were positive for oocysts according to heine staining. positive fecal samples were distributed as follows: calves ( . %) with a score of , ( . %) with a score of , ( . %) with a score of or , and ( . %) with a score of . within-herd prevalence of oocyst shedding ranged from % to % (mean, %; first quartile, %; third quartile, %). seven of the farms ( %) were negative for all samples, and, by contrast, farms ( %) had high shedding calves (scores of > ) in more than half of the samples. at the time of sample collection, calves ( %) were diarrheic, and calves ( %) were scored as (heavy diarrhea). within-herd prevalence of diarrhea ranged from % to % (mean, %; first quartile, %; third quartile, %). no calves with diarrhea were found on three ( %) farms, and three ( %) farms had a heavy diarrhea score ( ) in more than half of the samples. calves aged - days were least likely to be shedding c. parvum oocysts (prevalence = % with % having scores > ), while the group of calves aged - days had a prevalence of % (with % having scores > ). the highest prevalence for diarrhea ( %) was detected in calves with heine scores > , and lower percentages for score - ( %), score ( %), score ( %) and score ( %) ( = , p < . ). from the pool of calves with combined high scores of diarrhea (heavy) and shedding (> ), % were in poor general condition (> ) compared to % for calves that had a high score for diarrhea but a low shedding score ( - ) ( = . , p = . ). the mortality rate at days was higher in calves with high combined scores of shedding (> ) and diarrhea (heavy) compared with other classes (table ) . no significant difference was observed in shedding for breeds, sex, season of birth (maximum in august-october, % prevalence; minimum in november-january, % prevalence). candidate factors were age, breed and covariates related to care of the calf, type of milk fed and equipment used to feed calves (table ) . treatment with antibiotics, anti-inflammatory drugs and nutritional products were removed because they were administered as a consequence of diarrhea associated with c. parvum and not as a plausible cause of infection. the final multivariable gee model included five potential explanatory variables (table ). risk factors significantly associated with the shedding of c. parvum were a young age (≤ days) at sampling and the distribution of colostrum using a bucket. a negatively associated factor was feeding the calf fermented milk (table ). the breed (normande vs others, p = . ) and treatment with halofuginone (p = . ) were associated with the shedding of c. parvum (table ) . the aim of this study was to determine the prevalence and risk factors for cryptosporidium infection in bovine neonates in an area of intensive dairy farming in western france. bivariable analysis showed that the prevalence of diarrhea among young calves was correlated with the degree of c. parvum shedding. the general condition of calves was poorer, and the probability of mortality higher, in animals with high scores for diarrhea and shedding compared to those with only a high score for diarrhea. torsein et al. ( ) reported that the prevalence of calves infected by cryptosporidium is different between herds with high mortality (calves between and days) and herds with low mortality (nonsignificant difference, p = . ). these results confirm that c. parvum is a primary enteropathogen. multivariable analysis of our data identified several risk factors. among them, the use of buckets without teats to feed young calves increases the risk of oocyst shedding. a plausible explanation could be that the walls of the buckets are highly contaminated by oocysts, which could occur when farmers stack them for storage. these oocysts are probably not removed by standard washing procedures and may contaminate subsequent food placed in the buckets. an alternative explanation could be the failure to satisfy the calves' need for suckling in those that are quickly separated from their dams and fed using buckets without teats. this separation may lead calves to suckle another calf (cross-suckling), or other points in their housing area or environment, thereby ingesting oocysts that cause early and repeated infection. a study investigating the effect of teat feeding on cross-suckling in dairy calves reported that teat feeding reduces cross-suckling compared to calves fed from buckets (jensen and budde, ) . finally, teat feeding allows a more effective reflex closure of the esophageal groove than feeding from the bucket, which probably prevents fermentation and limits colonization by enteropathogens such as c. parvum (ferran and bouquet, ) . this notion is supported by the findings of a german study of calves (< days old) suffering from acute enteritis, where the esophageal groove did not close in . % of cases (dirr and dirksen, ) . other studies indicate that hygiene measures are important during calf feeding. the use of detergent or soap for cleaning calf feeding utensils was a protective factor against the shedding of cryptosporidium in ontario (trotz-williams et al., ) . similarly, feeding starter grain as well as milk (compared to milk replacers) are factors associated with an increased risk of shedding (maldonado-camargo et al., ; mohammed et al., ) and could be interpreted as practices that increase potential contamination of calf feed. we also identified that normande calves shed more oocysts than other breeds. breed type was previously proposed as a factor associated with infection of c. parvum. in a study of experimentally infected dairy calves, the jersey breed was identified as a risk factor for infection by c. bovis (szonyi et al., ) . in a cross-sectional study of positive fecal samples from dairy cattle in new york state, holstein calves were identified as more likely to be infected with c. parvum (starkey et al., ) . in our study, treatment with hl had a slight protective effect on oocyst shedding. a review of the effects of hl on calf cryptosporidiosis using a meta-analysis (silverlås et al., a) found that for prophylactic treatment, hl has a slight protective effect on infection and diarrhea prevalence on days and post administration. in a recent randomized controlled field trial, the halofuginone group showed a longer prepatent period than the control, although there was no difference in the number of oocysts shed (almawly et al., ) . other studies have reported a significant reduction in the shedding of oocysts and diarrhea by hl-treated newborn calves (klein, ; de waele et al., ; trotz-williams et al., ; keidel and daugschies, ) . supplementation with fermented milk appeared to be a protective factor against the shedding of c. parvum. fermented milk contains more probiotics (e.g., lactobacillus sp.) than unfermented milk, and, through their modifying activities, these beneficial bacteria are able to prevent pathogens from colonizing and multiplying. supplementation of feed with lactobacillus acidophilus and lactobacillus reuteri in mice and rats reduces fecal shedding of c. parvum oocysts (alak et al., ; guitard et al., ) . another study reported that the oral administration of fermented milk supplemented with two probiotic strains significantly increased delayed-type hypersensitivity, plaque-forming cells and half-hemolysis values in healthy adult mice. synbiotic fermented milk may help improve intestinal health and have a positive effect on the humoral and cell-mediated immunity of host animals (wang et al., ) . similarly, lactic acid bacteria isolated from the calf gut inhibit those causing diarrhea in calves (maldonado et al., ) . however, no differences were reported in the incidence of diarrhea and cryptosporidium oocyst shedding in calves receiving probiotic treatment with lactic acid-producing bacteria under field conditions (harp et al., ) . in our study there was no significant association between the risk of shedding and the season of birth, which contrasts with the findings of trotz-williams et al. ( ) who reported that oocyte shedding and the incidence of calf diarrhea was higher during the summer months. contrastingly, others have reported a higher incidence of calf diarrhea during the winter in north america (waltner-toews et al., ; curtis et al., ; frank and kaneene, ) . absence of a seasonal effect in our study may be due to the mild oceanic climate of normandy characterized by low thermal amplitude, allowing oocysts to survive and overwinter in a more conducive environment that allows them to challenge calves all year round. our findings indicated that the type of calf housing used after birth was not a significant risk factor. sischo et al. ( ) observed that contact between calves in the nursery increased the risk of shedding. the lack of association in our study may be explained by the lack of robustness of the small sample size that was unable to detect differences in important cofactors. finally, calves fed with pooled or commercial colostrum did not have a lower risk of shedding. passive immunity does not impart resistance to this parasite in calves (trotz-williams et al., ) , and the lack of association with c. parvum shedding was anticipated. the results of this study show that c. parvum is widespread among calves under days old in dairy herds in the normandy region of france. shedding of c. parvum is associated with a high incidence of diarrhea and increased risk of mortality in young calves. our study identified some risk calf-level factors, such as the use of buckets without teats to feed (causal) or the supplementation with fermented milk (protective). further investigation is necessary to define appropriate measures that farmers and veterinary practitioners can take to control c. parvum infection. none of the authors has any financial or personal relationships that could inappropriately influence the content of this paper. supplementation with lactobacillus reuteri or l. acidophilus reduced intestinal shedding of cryptosporidium parvum oocysts in immunodeficient c bl/ mice utility of halofuginone lactate for the prevention of natural cryptosporidiosis of calves, in the presence of co-infection with rotavirus and salmonella typhimurium. vet. parasitol detection of cryptosporidium oocysts in fresh calf faeces: characteristics of two simple tests and evaluation of a semi-quantitative approach mathematical methods of statistics descriptive epidemiology of calfhood morbidity and mortality in new york holstein herds a review of the importance of cryptosporidiosis in farm animals control of cryptosporidiosis in neonatal calves: use of halofuginone lactate in two different calf rearing systems dysfunction of the esophageal groove (ruminal drinking) as a complication of neonatal diarrhea in the calf an overview of techniques for dealing with large numbers of independent variables in epidemiologic studies how can the closure of the esophageal groove? point vet cryptosporidium infection in a veal calf cohort in france: molecular characterization of species in a longitudinal study management risk factors associated with calf diarrhea in michigan dairy herds potential risk factors for cryptosporidium infection in dairy calves experimental study of the effects of probiotics on cryptosporidium parvum infection in neonatal rats field testing of prophylactic measures against cryptosporidium parvum infection in calves in a california dairy herd eine einfache nachweismethode fűr kryptosporidien im kot the effects of milk feeding method and group size on feeding behavior and cross-sucking in group-housed dairy calves prevalence and control of bovine cryptosporidiosis in german dairy herds assessing infections at multiple levels of aggregation integration of halofuginone lactate treatment and disinfection with p-chloro-m-cresol to control natural cryptosporidiosis in calves preventive and therapeutic efficacy of halofuginone-lactate against cryptosporidium parvum in spontaneously infected calves: a centralised, randomised, double-blind, placebo-controlled study prevalence of cryptosporidium infection in calves in france longitudinal data analysis using generalized linear models lactic acid bacteria isolated from young calves -characterization and potential as probiotics prevalence of and risk factors for shedding of cryptosporidium parvum in holstein freisian dairy calves in central méxico patterns of cryptosporidium oocyst shedding in calves and a comparison of two diagnostic methods study design and analytic methods for data collected from clusters of animals risk factors associated with cryptosporidium parvum infection in dairy cattle in southeastern new york state role of cryptosporidium parvum as a pathogen in neonatal diarrhoea complex in suckling and dairy calves in france number of cryptosporidium parvum oocysts or giardia spp cysts shed by dairy calves after natural infection cryptosporidium parvum infection in cattle: are current perceptions accurate? update on cryptosporidium and giardia infections in cattle molecular characterization of cryptosporidium isolates from high-excreting young dairy calves in dairy cattle herds in western france systematic review and meta-analyses of the effects of halofuginone against calf cryptosporidiosis prevalence and associated management factors of cryptosporidium shedding in swedish dairy herds cryptosporidia on dairy farms and the role these farms may have in contaminating surface water supplies in the northeastern united states factors associated with shedding of cryptosporidium parvum versus cryptosporidium bovis among dairy cattle in new york state evaluation of factors associated with the risk of infection with cryptosporidium parvum in dairy calves risk factors for calf mortality in large swedish dairy herds genotypes and subtypes of cryptosporidium spp. in neonatal calves in northern ireland calf-level risk factors for neonatal diarrhea and shedding of cryptosporidium parvum in ontario dairy calves association between management practices and within-herd prevalence of cryptosporidium parvum shedding on dairy farms in southern ontario efficacy of halofuginone lactate in the prevention of cryptosporidiosis in dairy calves fermented milk supplemented with probiotics and prebiotics can effectively alter the intestinal microbiota and immunity of host animals dairy calf management, morbidity and mortality in ontario holstein herds. ii. age and seasonal patterns infection pattern of cryptosporidium and giardia in calves the authors wish to thank the dairy farmers who participated in this study. key: cord- -z dx ey authors: schaefer, a. l.; cook, n. j.; bench, c.; chabot, j. b.; colyn, j.; liu, t.; okine, e. k.; stewart, m.; webster, j. r. title: the non-invasive and automated detection of bovine respiratory disease onset in receiver calves using infrared thermography date: - - journal: research in veterinary science doi: . /j.rvsc. . . sha: doc_id: cord_uid: z dx ey abstract bovine respiratory disease complex (brd) causes considerable economic loss and biosecurity cost to the beef industry globally and also results in significant degradation to the welfare of affected animals. the successful treatment of this disease depends on the early, timely and cost effective identification of affected animals. the objective of the present study was to investigate the use of an automated, rfid driven, noninvasive infrared thermography technology to determine brd in cattle. sixty-five calves averaging kg were exposed to standard industry practices of transport and auction. the animals were monitored for brd using conventional biometric signs for clinical scores, core temperatures, haematology, serum cortisol and infrared thermal values over weeks. the data collected demonstrated that true positive animals for brd based on a gold standard including core temperature, clinical score, white blood cell number and neutrophil/lymphocyte ratio displayed higher peak infrared thermal values of . ± . °c compared to true negative animals . ± . °c (p < . ). the study also demonstrated that such biometric data can be non-invasively and automatically collected based on a system developed around the animal’s water station. it is concluded that the deployment of such systems in the cattle industry would aid animal managers and practitioners in the identification and management of brd in cattle populations. bovine respiratory disease complex (brd) is common in intensely raised and newly transported calves and refers to the animal displaying an undifferentiated fever in addition to a number of clinical signs, notably respiratory distress (jericho and kozub, ; buckham sporer et al., ) . brd is known to be caused by a number or combination of viruses and microorganisms (jericho and kozub, ; autio et al., ) including mycoplasma bovis (arcangioli et al., ) , coronavirus (decaro et al., ) , bovine para-influenza (pi ) (ellis, ) , bovine respiratory syncytial virus (brsv) (rola and polak, ) , bovine viral diarrhoea virus (bvd) and infectious bovine rhinotracheitis (ibr) (jericho and kozub, ) . brd is a worldwide health problem (griffin, ; horwood and mahony, ) and one of the major and most economically costly diseases affecting the north american beef industry (snowder et al., ; taylor et al., ) . the average incidence of brd in the usa is calculated to be % of receiver calves but can be as high as % (snowder et al., ) . the primary cost is reported to be due to both the cost of treatment as well as a reduction in subsequent animal performance and carcase merit (duff and galyean, ; gay and barnouin, ). in addition to the direct cost of brd, the increasing concern regarding the use of antibiotics and the potential impact on the promotion of resistant microbes is also apparent and is a growing focal point for global health organizations (world health organization (who), ) and the cattle industry generally (jericho and kozub, ; watts and sweeney, ) . this concern has led to the development of antibiotic resistant monitoring systems (wallmann, ) and concern regarding antibiotic resistance has had a significant influence on the national agricultural policy for research and development programs (www.agr.gc.ca). as discussed by cusack et al. ( ) and panousis ( ) the effective management of brd depends on the early recognition and treatment of the onset of the disease. unfortunately, the clinical signs of brd are often not apparent until late in the course of the disease due to the challenge associated with early diagnosis (poulsen and mcguirk, ) . numerous diagnostic approaches have been attempted with varying success including the use of acute phase proteins (humblet et al., ) , pcr techniques (asano et al., ; fulton, ) and elisa (quinting et al., ) , breath analysis (burciaga-robles et al., ), analysis of cough or respiratory sounds (ferrari et al., ) , assessment of feeding and other animal activity (basarab et al., ; hanzlicek et al., ) , analysis of protein profiles (mitchell et al., ) , rumen bolus temperature recording (rose-dye et al., ; timsit et al., ) and various immuno-histochemical techniques (wallmann, ) . the identification of disease in an animal or a population will depend on the information available and on the reliability of that information. currently, the most accurate methods of diagnosis remain analytical techniques such as serum neutralisation, elisa or pcr procedures. however, with cattle at risk of brd the information available is often limited and does not include extensive and expensive serum neutralisation or pcd data. furthermore, if the assessment of an animal's health necessitates the capture and restraint of that animal in order to collect a biological sample then the stress of the process itself will introduce bias. the use of infrared thermography (irt) to diagnose animals with brd has been established (schaefer et al., . these findings are also consistent with those recently reported by hovinen ( ) and polat et al. ( ) for the determination of mastitis in dairy cattle. many of the aforementioned technologies including clinical scores, haematology, acute phase proteins, cytokines, antibody response and core temperature monitoring are useful aids to the diagnosis of brd particularly when its prevalence is high in a population and once the clinical signs of respiratory disease are present. infrared thermography has been shown to demonstrably diagnose brd at an earlier stage of the disease than other conventional technologies . however, the early detection of brd, especially in animal populations where the prevalence of brd and the virulence is low, is more challenging. more difficult still is the development of any technology that can non-invasively and automatically aid in the diagnoses of brd. the objective of the present study was to investigate the use of infrared thermography to non-invasively identify animals with brd in a population with a low prevalence of respiratory disease. the calves used in the present study were exposed to a lower level of stressors for that purpose. furthermore, it was the objective of the current study to examine the feasibility of automating the collection of infrared thermography data. for infrared thermography to be considered as a practical and feasible analytical tool it is necessary that such a system be demonstrated to be compatible with current rfid tags, be automatable and be more user friendly than carrying a hand held camera around cattle pens. sixty-five receiver calves averaging kg were used. the calves were herford x angus from either the agriculture and agri-food canada, lacombe research centre beef herd (n = ) or the animal diseases research centre beef herd (n = ) located at lethbridge, alberta. the calves were weaned and transported approximately h to a commercial auction mart to simulate transport and handling conditions typical of calves received at feedlots in canada (receiver calves). the animals were held overnight without feed or water and then returned to the lacombe research centre beef research unit. this protocol simulated a commercial auction sale and exposure. upon arrival at the lacombe research centre the calves were offloaded, caught in a restraining chute, weighed, blood sampled via a jugular vein venus puncture, sampled for saliva using a cotton swab and placed into outdoor pens measuring approximately  m with one third of the pen having a roof cover. the calves were fed in a conventional bunk feeder a balanced cereal grain silage ration containing % cereal grain which met or exceeded national research council (nrc) recom-mendations (nrc, ) . the calves received straw bedding and free access to fresh water via an automatic system shown in fig. . all management and operating procedures met or exceeded the canadian council of animal care recommendations ( ) and codes of practice for beef cattle guidelines (agriculture canada, ) . in addition, all study protocol was reviewed and approved by the lacombe research centre animal care committee. while contained in their receiving pens the calves were monitored daily by trained personnel for clinical signs of illness using methods described previously (schaefer et al., . briefly, clinical scores were designed to identify bovine respiratory disease (brd) and were based on the appearance of four criteria as follows: respiratory insult: ( - ): = no insult, normal breath sounds (nbs); = very fine crackle (rale) (vfcr) on auscultation and/or a moderate cough; = fine crackle (subcrepitant) (fcr) on auscultation and/or a moderate nasal discharge and moderate cough; = medium crackle (crepitant) (mcr) on auscultation and/or a moderate to severe viscous nasal discharge with cough; = course crackles (ccr), tachypnoea (> % of the norm) and/or a severe discharge with respiratory distress and obtunded lung sounds and = ccr with dyspnoea, tachypnoea, marked respiratory distress and/or lung consolidation. digestive insult: ( - ): = no insult, normal, eating and drinking; = mild or slight diarrhoea with slight dehydration (< %) and reduced eating; = moderate diarrhoea with % dehydration and reduced feed intake (< %); = moderate to severe diarrhoea with % or less of feed intake and more than % dehydration; = severe diarrhoea, and less than % of normal feed intake and = severe diarrhoea and not eating, not drinking and dehydrated. temperature score: core temperature (rectal) ( - ): = < . °c; = . - . °c; = . - . °c; = . - . °c; = . - . °c and = > °c. rectal or core temperatures for the calves were collected at the start and end of the study only since only at these times were the animal restrained. disposition or lethargy score: ( - ): = no lethargy, normal posture; = mild anorexia or listlessness, depressed appearance; = moderate lethargy and depression, slow to rise, anorectic; = recumbent or abnormal posture, largely depressed; = prostrate, recumbent or abnormal posture and = death. with respect to laboratory analysis, salivary and serum cortisol was analysed using an enzymatic assay as described by cook et al. ( ) . haematology analysis and differential counts were conducted on a cell-dyne model haematology analyser (abbott labs, mississauga, ontario). serology assessment was conducted by prairie diagnostic services (saskatoon saskatchewan) and assessment was carried out for bovine viral diarrhoea (bvd) virus types and and infectious bovine rhinotracheitis virus (ibr) via serum neutralisation tests and expressed as a titre or the highest dilution of serum to exert a neutralising effect. additional assessment for coronavirus, bovine para-influenza (pi ), and bovine respiratory syncytial virus (brsv) were conducted by elisa. again, these methods have been referenced previously . the ranking of antibody titre scores was as follows: for bvd and ibr - : = negative, - : = suspicious, - : = low, - : = moderate, > : = high. for brsv, pi and coronavirus < = negative, - = suspicious, - = low, - = moderate, > = high. the determination of an animal true positive or negative for brd was based on the comparison to a set of ''gold standard'' values as per the approach of humblet et al. ( ) and schaefer et al. ( ) . as described by galen and gambino ( ) and thrusfield ( ) this approach is commonly promoted in both veterinary and human medical diagnostic studies. in the current study, the criteria for a true positive animal for brd was defined as an animal displaying three or more of the following signs; a core temperature of > °c, a white blood cell count of less than or greater than  /ll, a clinical score of > or a neutrophil/lymphocyte ratio of < . (leucopaenia) or > . (neutrophilia). a true negative animal was defined as an animal displaying a score of or . these parameters were considered consistent with normal and abnormal ranges suggested by other researchers (kaneko, ; blood et al., ) . for laboratory assessments, all calves were monitored at the beginning of the study and again three weeks later. with respect to infrared thermal measurements, all calves were monitored for radiated temperatures around the orbital area (eye plus one centimetre surrounding the eye) using a flir s broadband camera (flir comp., boston, ma) mounted on a motorised shaft. the scanning camera was interfaced to a control system such that the animal self-collected an infrared image every time it attended the water station. the automatic scanning system used is shown in fig. and schematically in fig. and consisted of the following components: the system was built around a conventional two water bowl float design from ritchie water systems (ritchie cattle fountains. conrad ia, usa). panels were installed along the sides of the water bowls with a partition in the centre to position the calves when they accessed water. extension panels were placed on each side of the water bowls to place the calf's head at the proper focal distance. this system allowed easy access for the calves from two directions. a panel on one side of the water bowls was modified to facilitate a window measuring approximately cm square in order to view the calves while drinking. two inphase loop antennae, specifically designed to read over a defined space where the rfid tags would be located, were mounted on adjacent panels, above and slightly behind each water bowl. each antenna pair was connected to an allflex pnl-oem-modle- control module (allflex eid system. allflex canada inc. st-hyacinthe, p.q.). electromagnetic shielding was placed on the side of the panel exposed to the holding pen to prevent reading tags on calves that were not accessing water. the design of the antenna system provided the identification of the calf at the water station as well as the signal to the control system to rotate the camera if necessary and to initiate capturing images when the calf's head was visible through the viewing window in the panel. the camera/motor assembly, enclosed under a protective cover, was located medially between the two viewing windows at a distance that provided a field of view to cover most head positions of the drinking cattle. rotation of the camera to view a particular window was performed by a geared-head motor, controlled by a set of software commands sent to the motor control circuitry. this design met the good laboratory practice requirements for correct thermography: a fixed focal length and angle with a still image which enabled accurate thermal data collection. software running on a computer located in an instrumentation cabinet read the tag information from the rfid control module, initiated the camera positioning, acquired the infrared image, performed the thermal analysis of the image and stored the acquired information in a database. the cabinet also housed the power supplies and motor control circuitry used in the system. instrument integration, and the hardware and software used in this thermal station was designed and developed at the lacombe research centre. the use of the infrared thermal stations enabled the monitoring of all animals for orbital radiated temperatures every time they attended the water station. the thermal orbital (eye) maximum value for the calves was used in all calculations since this value was found to be most sensitive to stress and disease onset (schaefer et al., . all calves were thus monitored for average daily maximum temperatures and for the mean ratio values (mr). the mean ratio which was calculated as the average of daily radiated maximum temperature for a given animal divided by the average daily maximum value for the group of calves. verified data was entered into microsoft excel (microsoft corporation ). basic statistical calculations for data means, standard deviation and two tailed least squares analysis tests ( tailed t tests) were conducted in microsoft excel. response operant characteristic curves (roc) were used to calculate the relationship between true positive (sensitivity) and true negative ( -specificity) animals for a given biological measurement. these curves were used to calculate the optimal cut off values or values with the greatest efficiency for that parameter. the analysis of data for roc curves, test specificity and sensitivity, optimal cut off value calculations, positive predictive values and negative predictive values were conducted with medcalc ( ) software (medcalc Ò for windows. statistics for biomedical research. version . broekstraat, mariakerke, belgium). infrared thermography data from thermal images were calculated using flir researcher Ò software (version . , boston, ma). the calves used in the present study were exposed to a lower level of stress compared to typical weaned and receiver calves experiencing a multitude of co-mingling, handling and transport stressors. the verified tp incidence in these calves was thus not unexpectedly comparatively low at % ( out of animals show- ( ) side panels, ( ) two bowl water system, ( ) extension panels, ( ) viewing windows, ( ) antennaek ( ) rfid control modules, ( ) electromagnetic, shielding ( ) infrared camera on motor mount within enclosure, ( ) instrumentation cabinet. ing a gold standard value of or / ). of interest, however, was the observation that none of these calves were identified as suspect for brd using subjective clinical signs and none of these animals were deemed to be in need of treatment nor removed for treatment by the animal managers or pen checkers during the study. a total of of the calves displayed tn values (gold standard values of or / ) and a further animals displayed intermediate values (gold score value of / ) for brd incidence. the calves typically would visit the water station between and times per day. the amount of time spent at the water station by any individual varied but again typically twenty or more thermal images were captured per animal per day. the overall average orbital maximum temperature for all calves for the week period was . °c ± . (sd). for this same period, the average for the orbital maximum for the tp calves was . °c ± . and for the tn calves was . °c ± . (p < . ; table ). since daily thermal values for all animals were monitored, it was possible to follow the radiated temperature rise for the tp calves to the point at which a peak temperature occurred (table and fig. ). the peak thermal response for the tp calves as monitored by the thermal station was . °c. the calves displaying intermediate values (a gold standard score of out of ) showed an average orbital irt value of . ± . °c which was not significantly different from the tn calves but was significantly lower than the tp peak values (p < . ). of interest in the present study was the observation that nine of the calves developed brd signs during the study period and for these animals it was possible to automatically and non-invasively follow the thermal radiated response up to the time of peak thermal response and brd onset. these data showed that there was over a °c elevation in temperature for the tp calves over this time. this contrasts to a flat or basically zero average daily change in temperature for the entire group of animals of À . ± . °c/day. the data for the mean ratios (mr. table ) in general paralleled the orbital maximum values. in terms of biological values, compared to the tn animals the tp calves (table ) displayed higher core temperature values, white blood cell counts, clinical scores and serum cortisol values (p < . ) with trends towards a higher value in neutrophil/lymphocyte ratios. these values for the intermediate calves were . ± . °c, . ± .  /ll, . ± . , . ± . and . ± . lmol/l for core temperatures, white blood cell counts, clinical scores, neutrophil/lymphocyte ratio and serum cortisol, respectively. with respect to specific differential counts the tp calves displayed a lower neutrophil count and a higher lymphocyte count, as well as a slight increase in red blood cell numbers (p < . ) ( table ) . salivary cortisol displayed a greater variation among animals and thus there were no statistically significant differences in salivary cortisol values among the groups. the calculation of predictive index values for optimal cut off values, positive predictive values, negative predictive values, test sensitivity and test specificity are shown in table . again, these values were calculated using roc analysis. the collected data may also be used to estimate test efficiency. in the current study, the test efficiency for the irt max values was % which was as high as any test used. in terms of seroprevalence the average and range of titres for ibr and bvd were < : (range : - : ) and : (range < : - : ), respectively. for the pi , brsv and coronavirus these values were (range - ), (range - ) and (range - ). most of the calves displayed a low to moderate titre to one or more viruses. in terms of seroconversion, higher values were seen for the true positive (tp) calves. thirty-three percent of the tp animals seroconverted (> units) to bvd virus, % to ibr, % to brsv, % to pi and % to corona virus. by contrast in the true negative animals a lower extent of seroconversion was seen. twenty-two percent seroconverted to bvd, % to ibr, % to brsv, % to pi and % to coronavirus. the data suggest that the brd viruses measured may have played some significant role in the onset of brd in the tp animals. identifying animals positive for brd is not an easy task, particularly if the cattle have mild signs of brd or the brd prevalence is low. part of the reason for this is that there are few gold standards to identify brd that are absolute (white and renter, ). again, the most accurate determination of a causative agent for brd is still likely to be a serum neutralisation, elisa or pcr technique. however, the gold standards suggested currently in this manuscript have some precedence and support in the literature (humblet et al., ; blood et al., ; kaneko, ; schaefer et al., ) and are reasonable suggested biometric measures for brd. the primary haematological response seen in the tp calves compared to the tn animals was the presence of a leucocytosis characterised by lymphophilia and neutropaenia (tables and ) . considering a lymphocytosis is commonly observed during a fever and a neutropaenia during viraemia (strauss, ) these haematological observations are to be expected in tp animals. in terms of infrared thermography, infrared heat loss is a significant avenue for the dissipation of heat in an animal (kleiber, ) . the technology has been demonstrated to be effective in the non-invasive identification of transport and other environmental stressors that alter heat loss (schaefer et al., ; stewart et al., ) , as well as pain and fear in cattle (stewart et al., ). an earlier comprehensive review of this subject has been published by mccafferty ( ) . using the gold standards criteria in the current study the data suggest that infrared thermography as a biometric measurement also shows utility in identifying the onset of disease in cattle. this is again consistent with previous findings from our own laboratory (schaefer et al., stewart et al., ) and with findings from other laboratories (polat et al., ; hovinen, ; rainwater-lovett et al., ) . indeed, as demonstrated by the roc curve calculations, the efficiency of the irt methods in the current study was equal to or better than any of the other methods ( % efficiency with irt peak values) and in populations with a higher prevalence of the disease has been demonstrated to be superior to other methods in the early identification of disease onset . in the current study the irt maximum values and the mean ratio values appeared to show the greatest utility as single measures. of interest, however, were the observations that the rate of change of an animal's thermal profile as brd onset occurred and even the degree of variation associated with a given measure such as standard deviation did show promise as valuable indicators. further evaluation of such parameters on larger data sets and in data sets with perhaps a greater degree of brd prevalence is merited. in the current study, compared to the industry practice of using clinical signs during pen checking procedures, a lower rate of false negatives was seen. in addition, compared to other procedures using breath analysis (burciaga-robles et al., ) , rumen temperature probes (rose-dye et al., ; timsit et al., ) or any number of biochemical procedures such as pcr, immunohistochemistry, elisa or acute phase proteins (decaro et al., ; fulton, ; quinting et al., ) the non-invasively collected infrared data is likely to be more cost effective, less labour intense and timely as a diagnostic procedure. most calves diagnosed with brd are likely to be treated with one of a few antibiotics regardless of the causative virus. hence, it may make some practical sense to simply provide early diagnostic information to an animal manager earlier. if specific diagnosis is desired then it may still make sense to conduct a first screen with an automated system such as infrared thermography and subsequently test suspected animals with more precise procedures. of interest in the present study was the presentation of a significant number of animals displaying intermediate values for virtually all the biological markers. these animals appeared to be neither tp nor tn. this situation may be a failure of the identification systems to accurately classify animals. or, conversely, these intermediate animals may well be a population of cattle that are either successfully resisting or slowly succumbing to brd. in either case, being able to identify such individuals may still be useful from the position of monitoring potential or suspected or emerging brd cases. . ± . (p < . ). p- equals the day before the peak temperature was evident, p- equals days before, etc. a notable and significant difference of the method used to collect infrared thermal data in the present study to that used in previous studies (schaefer et al., ) is that a multiple animal scanning capability of the system has been developed. mounting the infrared camera on a motor capable of rotating to two different scan windows as signalled from the rfid reader has enabled this system to be placed on two water bowls and hence the capacity to study more animals is significantly increased and can service pens of plus animals. the system was designed to accommodate a second water/thermography station situated parallel to the first station with the camera located centrally between the two stations, hence doubling the animal handling capabilities of the system. also, compared to other prototypes, the present system is automated and non-invasive by using a rfid reader and lap top computer for data storage. these adaptations are significant from an animal behavioural perspective since the calves can attend the water station voluntarily and without restriction or capture. hence, both thermal radiated values from the orbital region, as well as watering frequency, are obtained non-invasively. as a result it can be argued that such data are more representative of the animal's normal or steady state values, compared to values collected during animal capture and restraint. current operation procedures also enable the use of a cell phone communication system to download collected data wirelessly or via the internet. hence, data and animal responses can be monitored remotely to make management and treatment decisions regarding the emergence of brd. furthermore, the use of a non-invasive, automated, remote sensing system such as the infrared scanning station technology, lends itself to easier collection and oversight for early indicators of animal health aberrations. such a system would have clear utility in bio-surveillance and bio-security programs. data collected in the current study demonstrated that the noninvasive, automated collection of infrared thermography data from cattle at risk of brd is effective at identifying true positive and true negative animals. the study further demonstrated that such technology lends itself to the automated, wireless collection of biometric data useful for bio-security and bio-surveillance purposes. recommended code of practice for the care and handling of farm animals. beef cattle, publication /e, communications branch the role of mycoplasma bovis in bovine respiratory disease outbreaks in veal calf feedlots rapid detection of bovine corona virus by a semi-nested, rt-pcr. detecção rápida do coronavírus bovino (bcov) por meio de uma semi-nested rt-pcr etiology of respiratory disease in non-vaccinated, non-medicated calves in rearing herds automatic monitoring of watering behaviour in feedlot steers: potential use in early detection of respiratory disease and in predicting growth performance veterinary medicine transportation of young beef bulls alters circulating physiological parameters that may be effective biomarkers of stress evaluation of breath biomarkers and serum haptoglobin concentration for diagnosis of bovine respiratory disease in heifers newly arrived at a feedlot guide to the care and use of experimental animals dexamethasone reduces transport-induced weight losses in beef calves the medicine and epidemiology of bovine respiratory disease complex in feedlot respiratory disease associated with bovine coronavirus infection in cattle herds in southern italy board-invited review: recent advances in management of highly stressed, newly received feedlot cattle bovine parainfluenza- virus cough sound description in relation to respiratory diseases in dairy calves bovine respiratory disease research a nation-wide epidemiological study of acute bovine respiratory disease in france bovine pasteurellosis and other bacterial infections of the respiratory tract serial evaluation of physiologic, pathological, and behavioral changes related to disease progression of experimentally induced mannheimia haemolytica pneumonia in postweaned calves multiplex real-time rt-pcr detection of three viruses associated with the bovine respiratory disease complex dissertationm, department of production animal medicine acute phase proteins assessment for an early selection of treatments in growing calves suffering from bronchopneumonia under field conditions experimental infectious respiratory disease in groups of calves: lobular distribution, variance, and sample size requirements for vaccine evaluation clinical biochemistry of domestic animals the fire of life the value of infrared thermography for research on mammals: previous applications and future directions statistics for biomedical research. version stress alters the cellular and proteomic compartments of bovine bronchoalveolar lavage fluid dairy calf pneumonia: effective treatment depends on early and accurate diagnosis sensitivity and specificity of infrared thermography in detection of subclinical mastitis in dairy cows respiratory disease of the bovine neonate development of a -step enzyme-linked immunosorbent assay for the rapid diagnosis of bovine respiratory syncytial virus in postmortem specimens detection of foot-and-mouth disease virus infected cattle using infrared thermography aktualne dane na temat zaka _ zeń powodowanych przez wirus syncytialny układu oddechowego bydła rumen temperature change monitored with remote rumen temperature boluses following challenges with bovine viral diarrhea virus and mannheimia haemolytica the effects of fasting and transportation on beef cattle: . acid-base-electrolyte balance and infrared heat loss of beef cattle the early detection of bovine respiratory disease (brd) with infrared (irt) and treatment with nitric oxide infrared detection and nitric oxide treatment of bovine respiratory disease early detection and prediction of infection using infrared thermography the use of infrared thermography as an early indicator of bovine respiratory disease complex in calves early disease detection: implications for industry efficiency, food quality and safety the use of infrared thermography in the non-invasive, automated detection of calves displaying bovine respiratory disease orbital infrared as an early indicator of bovine respiratory disease in sero-negative cattle bovine respiratory disease in feedlot cattle: environmental, genetic, and economic factors infrared thermography as a non-invasive tool to study animal welfare non-invasive measurement of stress in dairy cows using infrared thermography eye temperature and heart rate variability of calves disbudded with or without local anesthetic effects of local anesthetic and a non steroidal anti-inflammatory drup on pain responses of dairy calves to hot iron dehorning effects of an epinephrine infusion on eye temperature and heart rate variability in bull calves white blood cell metabolism and neutropenia and qualitative disorders of neutrophiles the epidemiology of bovine respiratory disease: what is the evidence for predisposing factors? diagnostic testing early detection of bovine respiratory disease in young bulls using reticulo-rumen temperature boluses monitoring of antimicrobial resistance in pathogenic bacteria from livestock animals antimicrobial resistance in bovine respiratory disease pathogens: measures, trends, and impact on efficacy bayesian estimation of the performance of using clinical observations and harvest lung lesions for diagnosing bovine respiratory disease in post-weaned beef calves antibiotic resistance. fact sheet no the authors wish to express their appreciation to agriculture and agri-food canada, growing forward research funding program, the beef cattle research council of the canadian cattleman's association, the national science and engineering research council of canada and the alberta livestock and meat association for funding used in the current study. the authors are also grateful for the valuable technical assistance from p. lepage, l. holt-klemic, d. froehlich and s. marchand. assistance provided by the lacombe beef research unit operational staff is likewise greatly appreciated. key: cord- -ev fzly authors: almawly, j.; prattley, d.; french, n.p.; lopez-villalobos, n.; hedgespeth, b.; grinberg, a. title: utility of halofuginone lactate for the prevention of natural cryptosporidiosis of calves, in the presence of co-infection with rotavirus and salmonella typhimurium date: - - journal: vet parasitol doi: . /j.vetpar. . . sha: doc_id: cord_uid: ev fzly halofuginone lactate (hl) is registered in several countries for the prevention of calf cryptosporidiosis, but the compound's utility in the presence of co-infection with other enteropathogens is not well understood. we performed a randomized controlled field trial of the efficacy of hl for the prevention of natural calf cryptosporidiosis, in the presence of co-infection with rotavirus and salmonella typhimurium. newborn calves on one farm were sequentially enrolled and allocated to a full dose (n = ), half dose (n = ), or a placebo control group (n = ), using a randomized block design. the cryptosporidium oocysts in fecal specimens collected on days , , , and were counted and the severity of the diarrhea was assessed using fecal consistency scores (solid, semisolid, or liquid). the oocyst numbers and fecal consistency scores were statistically compared between the groups. ninety one percent of the calves shed cryptosporidium parvum oocysts during the trial. the full dose group had a longer prepatent period than the control group, but no statistical difference in the number of oocysts was identified between the groups after controlling for the effects of sex and breed. the fecal consistency scores and mortality rates did not differ between the groups. these results indicated that the anti-cryptosporidium activity and clinical benefit of hl were limited. it is concluded that in order to maximize the clinical efficacy of hl in the field, diagnostic efforts should aim to rule out the presence of other enteropathogens. the cosmopolitan protozoan parasite cryptosporidium parvum infects humans and a range of other hosts, and is a common agent of calf diarrhea during the first four weeks of life (gulliksen et al., ; lanz uhde et al., ; millemann, ; trotz-williams et al., ) . the spectrum of severity of cryptosporidiosis in calves varies from subclinical infection to severe diarrhea and dehydration (fayer et al., ; klein et al., ; o'handley et al., ) . typically, natural and experimental infections with c. parvum in calves have a pre-patent period of - days, followed by a patent period characterized by a bell-shaped oocyst excretion curve, with the number of fecal oocysts peaking and then rapidly decreasing to undetectable levels in a matter of - days (fayer et al., ; grinberg et al., ) . oocyst numbers as high as oocysts per gram of feces have been reported at the peak of excretion (fayer et al., ; grinberg et al., ) . whereas infection rates as high as % have been reported in calves in the first month of life, a lower infection prevalence has been consistently observed in weaned animals (bartels et al., ; brook et al., ; fayer and xiao, ; fayer et al., ; garber et al., ) . newborn calves are therefore considered major amplifiers of potentially zoonotic c. parvum in nature, and the prevention of calf cryptosporidiosis is relevant from both animal and human health perspectives (hunter and thompson, ; kiang et al., ; smith et al., ; grinberg et al., ; xiao and feng, ; zhou et al., ) . several features of the c. parvum life cycle make the control of cryptosporidiosis on-farms extremely difficult to be achieved by means of hygienic measures alone. calves can excrete hundreds of millions of oocysts (naciri et al., ; grinberg et al., ) , and considering there is a high probability of infection with a dose as low as oocysts (moore et al., ) , an infected calf could produce enough oocysts to infect thousands of new animals. attempts to interrupt the transmission of c. parvum should therefore include the immediate isolation of every infected animal, but this is rarely feasible on commercial farms. in addition, the c. parvum oocysts are insensitive to the action of numerous disinfectants (chen et al., ; quilez et al., ) and are excreted sporulated and fully infectious (smith et al., ) , requiring daily cleaning for their removal. therefore, in the absence of effective immunizing agents, pharmacological control strategies remain central for the prevention of cryptosporidiosis in calves. a number of compounds, such as halofuginone lactate (hl) (lefay et al., ; jarvie et al., ; klein, ; de waele et al., ; trotz-williams et al., ) , paromomycin sulphate (fayer and ellis, ; grinberg et al., ) , nitazoxanide (ollivett et al., ; schnyder et al., ) and decoquinate (moore et al., ; lallemond et al., ) have been tested for the prevention of cryptosporidiosis in calves, with variable results. hl is a synthetic derivative of a quinazolinone alkaloid with cryptosporidiostatic activity, but a mode of action that is poorly characterized. in naturally and experimentally infected calves, the oral administration of g/kg hl for seven consecutive days from the first day of life delays the onset of oocyst shedding, reduces the number of oocysts excreted, and lowers the severity of diarrhea joachim et al., ; klein, ; lefay et al., ; trotz-williams et al., ; villacorta et al., ) . a formulation containing hl (halocur, intervet ltd., republic of ireland) is currently the only prescription drug registered for the prevention of cryptosporidiosis of calves in several countries. the use of hl has a number of limitations, including a substantial market price and a narrow therapeutic index, with toxicity observable at approximately twice the recommended dose (villacorta et al., ; naciri et al., ; trotz-williams et al., ; http://www.msd-animal-health.co. uk/products public/halocur/ product datasheet.aspx, accessed july ). remarkably, notwithstanding the frequent occurrence of co-infections with cryptosporidium and other enteropathogens in the field (de la fuente et al., ; naciri et al., ; tzipori et al., ) , the utility of hl in the presence of such co-infections is not well understood. indeed, with some exceptions (klein, ; lefay et al., ) , most anti-cryptosporidium efficacy studies of hl did not analyze or took into account the presence of co-infections. co-infections might modify the anti-cryptosporidium effect of hl in different ways. the increased fluid content and intestinal motility determined by the presence of co-infecting pathogens may reduce the activity of hl by dilution, or by reducing the transit time of the drug in the intestinal tract. furthermore, enteric infection with rotavirus or other agents may cause exfoliation of infected cells, altering intestinal cellular function (ramig, ) , potentially increasing the toxicity of hl via systemic absorption. motivated by the need of more data on the utility hl in the presence of co-infections with other pathogens, we performed a randomized controlled field trial of the anti-cryptosporidium preventive efficacy of the compound in calves on a new zealand farm enzootically infected with c. parvum, bovine rotavirus and salmonella typhimurium. the study was performed between july and october , on a farm situated in the taranaki district, new zealand. consent for this study was obtained from the farmer and the use of animals was approved by the animal ethics committee, massey university. the farm managed a seasonal (spring) calving dairy herd of approximately milking cows and a smaller herd of beef cattle. it was recruited following a post-mortem investigation performed in march on two calves conducted by one of the authors (ag), which indicated the presence of cryptosporidium oocysts in the feces of two calves. salmonella was ruled out by culture and rotavirus and coronavirus by antigen elisa. sequence analysis of the cryptosporidium s rrna gene subsequently confirmed c. parvum in both animals. notwithstanding the microbiological results performed in march, bovine rotavirus and salmonella spp. were subsequently identified in multiple enrolled calves during the study (see below). the study was a randomized controlled field trial (see randomization procedure below). a commercial product (halocur, intervet ltd., republic of ireland) registered in new zealand for the prevention and treatment of cryptosporidiosis of calves was used to test the efficacy of hl. the product's recommended preventive dose is mg ( ml) for calves weighing - kg, and mg ( ml) for calves weighing - kg, for seven consecutive days, from birth. the initial aim was to assess the preventive efficacy of half the recommended dose of hl as compared with the full dose and no treatment, and the following treatment groups were established: group (full dose regime; n = ), calves treated orally with ml halocur; group (half dose regime; n = ), calves treated with ml halocur; group (placebo control group; n = ), calves treated with ml water delivered using the product's dispenser. the group sizes were estimated using power analysis for the detection of a difference between means, using pass software (ncss, kaysville, ut). assumptions for the calculations were a log -transformed mean number of oocysts per gram of feces of . for the full dose and . for the untreated-control group at the peak of shedding, and a common standard deviation of . . these means were estimated from a previously published study of efficacy of paromomycin sulphate (grinberg et al., ) . group sizes of calves per group achieved % power to reject the null hypothesis that both group means are . . thus, assuming a conservative infection rate of ∼ %, calves per group were required. management of the enrolled calves followed the same routine procedures used on the farm. briefly, newborn calves were left on the calving paddock for - h after birth, as commonly done in new zealand pasture-based farms to allow for colostrum intake directly from the dam. then the newborns were transferred to a large shed and allocated to a pen for newborn calves, of a capacity of about calves. calves were left in this pen for - days and then transferred to a new pen, in order to create space for new calves in the first pen. subsequently, the calves progressed to new pens (often adjacent) containing - animals of the same age group every - days, until weaning. the allocation of calves to pens was done by caretakers who were not aware of the nature of the treatments given to each calf, effectively creating a commingled pen design. adjacent pens were separated by slatted wooden fences. sawdust was used as bedding material. the feeding regimen included the administration of complete commercial milk replacement for the first two weeks using a tank with multiple nipples, followed by feeding ad libitum using automatic feeders until weaning. roughage was available from the second week of life. no routine vaccinations, preventive treatments or supplementations were administered to the calves during the study. however, inappetent calves could be tube-fed and severely diarrheic calves treated with oral electrolytes and/or other treatments by the caretakers. severely sick calves were transferred to an isolation shed. the study protocol stipulated that the tube fed calves could continue in the study, but animals treated by the farmer other means or transferred to the isolation pen, were removed. newborn calves were sequentially enrolled in the mornings, at arrival from the calving area to the rearing shed. any calf born on the farm was eligible for enrolment, provided no congenital or pathological conditions were identified. at the day of enrolment (day ), calves were identified by the ear or neck tag number, given a sequential number and allocated to one of the three treatment groups. allocation of calves to treatment groups was done using a randomized block design, with blocks of three (one calf per group), using a random list prepared in advance. the fourth calf presented to the investigator after each block of three was systematically left unenrolled and continued its normal life cycle on the farm. treatments were administered in the morning using the commercial product (see above). fecal specimens were collected at the time of treatment from the rectum of each calf using disposable gloves, on days , , , and . at the time of collection, each specimen was scored according to its consistency as , solid (specimen conserved its original shape); , semi-solid (specimen spread across the bottom of the container but was not liquid); or , liquid specimen. specimens were transported on ice to massey university and kept in refrigeration and analyzed between december and april , as described below. the fecal specimens were analyzed by a quantitative method that estimated the number of cryptosporidium oocysts present. briefly, after mixing the specimen with a spatula, g of feces was suspended in ml tap water and strained through a tea sieve. the filtrate was centrifuged at × g for min and the sediment re-suspended in ml of normal saline. a l aliquot of this suspension was deposited as a drop on a slide using a micropipette, airdried and fixed in methanol. fixed drops were stained using a commercial immunofluoresecent anti-giardia and cryptosporidium monoclonal antibody (aqua-glo g/c direct comprehensive kit, waterborne inc., new orleans, usa), according to the manufacturer's instructions. the applegreen fluorescent oocysts present on the entire area of the stained drop were counted using a fluorescent microscope using an excitation wavelength of nm and a × magnification lens. this total number of oocysts (ton) on the slide was used for statistical analysis (see below). samples containing > oocysts were difficult to count and were re-processed by a further dilution of the fecal suspension at − in water, followed by the staining of a l drop as above. the ton present on each rediluted specimen was estimated by multiplying the result by . when the available fecal material was insufficient for counting, a direct fecal smear was stained and a qualitative result (presence/absence of oocysts) was obtained. these qualitative results could not be used for the statistical comparison of the number of oocysts between the treatment groups, but were used for any comparison between proportions of cryptosporidium-positive and negative specimens. in march , dna from three cryptosporidiumpositive specimens from each treatment group was extracted from the stored feces using a dna extraction kit (qiaamp, dna stool mini kit, qiagen, hilden, gmbh), and cryptosporidium parasites were identified by means of pcrsequencing of the s rrna gene. primers were -gtt aaa ctg cga atg gct ca- (forward) and -cca ttt cct tcg aaa cag ga- (reverse) (learmonth et al., ) . amplification was performed in l containing l x pcr buffer, l dntp ( mm), l mgcl ( mm), l nonacetylated bovine serum albumin ( mg/ml) (new england biolabs, usa), picomoles of each primer, and . l of platinum ® taq dna polymerase ( mg/ml) (invitrogen corporation, carlsbad ca, usa). the pcr amplification was carried out in a thermocycler (sensoquest, goettingen, germany) with initial denaturation at • c for min, followed by cycles at • c for s, • c for s and • c for s. pcr products were purified using an in-house ethanol purification protocol, and bidirectional sequencing of an internal segment of the amplicon was performed using primers -ctcgactttatggaagggttg- (forward) and -cct ccaatctctagttg gcata- (reverse). forward and reverse sequences were aligned and edited manually using geneious software version . . (biomatters ltd., http://www.geneious. com/). distal and proximal segments that could not be verified were trimmed and the resulting edited sequences aligned with sequences deposited in genbank using the alignment algorithm blast (http://blast.ncbi.nlm.nih.gov/blast.cgi, accessed on april ). in addition to the analysis for cryptosporidium, fecal specimens taken haphazardly from the three treatment groups were analyzed for the presence of rotavirus, coronavirus, enterotoxigenic k + escherichia coli (k ) and salmonella spp. the analysis for rotavirus, coronavirus and k -positive e. coli was performed by a commercial diagnostic laboratory using antigen-elisa (institut pourquier, montpellier, france). the analysis for salmonella included parallel inoculation of fecal material into tetrathionate and rappaport-vassiliadis soy peptone (rvs) broths, and incubation for h at • c and • c, respectively. this was followed by subculture onto xylose lactose deoxycholate agar plates incubated as above. colonies consistent with salmonella were sub-cultured into triple-sugar iron agar slopes (tsi) and l-lysine decarboxylase broth. lysinepositive bacteria exhibiting tsi patterns consistent with salmonella were subjected to salmonella poly-o slide agglutination using a commercial antiserum (institut für immunpräparate und nährmedien gmbh berlin, berlin, germany), and agglutinating isolates were sent to the salmonella reference laboratory (institute of environmental science and research, porirua, new zealand) for serotyping. the parasitological and clinical effects of the three treatments were statistically compared. the parasitological effects were analyzed by comparing the tons between the groups using an analysis of variance for repeated measurements (rmanova) implemented by the proc mixed procedure of sas (statistical analysis system, , sas institute, cary, nc, usa). tons were log-transformed as log (ton + ) and analyzed using mixed models. the first model (model ) included the fixed effects of treatment (variable 'treatment group'), the day of sampling as repeated factor (variable 'sampling day'), and the interaction of treatment group and sampling day. model included the fixed effects as in model plus the fixed effect of sex of the calves, and model included the same factors as in model plus the effect of breed of the calves as a co-variable (variable 'breed'; friesian calf = ; non-friesian calf = ). fixed effects for variable breed were not included as a class due to unbalanced designs deriving from the uneven distribution of breeds across treatments. finally, model was similar to model , but did not include the data obtained from non-freisian calves, effectively removing any variability due to the breed. all the models included the random effect of calf, to account for the within-calf variability. using the akaike's information criterion, an unstructured error structure was determined as the most appropriate residual covariance structure for repeated measures over time, within animals. finally, the parasitological efficacy was also assessed by comparing the decline of the proportion of parasitologically negative calves as a function of time between the three groups using the nonparametric kaplan-meier method (km) (kaplan and meier, ) . also in this case, the analysis was repeated after omitting all the data from the non-friesian calves. the km analysis was implemented using a code available in "r" (terry therneau, ; a package for survival analysis in r package version . - ). the clinical effects of the treatments were assessed by comparing the fecal consistency scores (considered ordinal data) between the groups on each sampling day, using the non-parametric kruskal-wallis test. these tests were performed using the codes available in "r" (kruskal-wallis: http://stat.ethz.ch/r-manual/r-patched/library/stats/ html/kruskal.test.html; wilcoxon: http://stat. ethz.ch/ r-manual/r-patched/library/stats/html/wilcoxon.test. html). comparisons between proportions of interest were performed using two-tailed fisher's exact tests, which were interpreted using bonferroni-adjusted critical p-values for multiple testing. finally, the existence of association between the fecal consistency and the number of oocysts shed was tested by means of logistic regression, using the log (ton + ) as independent variable and the presence/absence of a liquid specimen (fecal score ) as outcome variable. this analysis was performed using a code available in "r" (http://www.ats.ucla. edu/stat/r/dae/logit.htm; r package version . - ). all the calves were enrolled within h from birth, over a -day period, between july and august . a total of / ( . %) calves were females and there were no statistical differences between the proportion of males and females among the treatment groups. there were ( %) friesian, ( %) angus, ( %) hereford calves and one ( %) jersey × angus crossbred calf (table ) . six calves haphazardly selected at enrolment and weighted using electronic scales had bodyweights between and kg (mean = . , standard deviation = ), indicating under/over-dosing of calves was unlikely to have occurred. one calf that died on the same day of enrolment (calf number ; table ) was substituted by the subsequent calf presented to the investigator (calf number ; table ). all the enrolled calves remained in their pens during the study and no calf was withdrawn due to concomitant treatment. thirty eight calves ( . %) were followed up for the entire observation period, and seven ( . %) died at different stages during the study. the causes of death were defined by the farmer as following: two calves from group died on days and , two from group on days and , and one from group on day , from severe diarrhea. two calves (group and group ) died from improper tube-feeding (possibly, milk inhalation) on day and day . there were deaths in group , three in group and one in group , and these rates were not statistically different (table ) . only / ( . %) fecal specimens could not be retrieved and were not analyzed (table ) . a total of / ( %) calves were cryptosporidiumpositive by immunofluorescence at some stage during the study. two calves belonging to group , that were able to be followed up for the entire observation period, remained parasitologically negative throughout the study (table ) . sequence analysis of the srrna gene of the fecal specimens analyzed by pcr-sequencing indicated the presence of c. parvum in all cases. out of specimens analyzed for other enteropathogens, ( %) were positive for rotavirus and ( %) for salmonella typhimurium. as expected for calves of this age, no e. coli k -positive specimens were identified. although analysis for giardia spp. was not the subject of this study (as the parasite it is not widely considered pathogenic for calves), we note for completeness that giardia cysts were observed in calves by immunofluorescence. the number of cryptosporidium-negative/positive fecal specimens stratified by the treatment groups and sampling days. on the same day, similar superscripts indicate comparisons between proportions resulting in two-tailed fisher's exact test p < . (less than bonferroni-adjusted critical probability). sampling day group (full dose) group (half dose) group (placebo-control) group (full dose) group (half dose) group (placebo-control) day / / / / / / day / * / + / * + / / * / * day / * / / * / / / day / / / / / / day / * / * / / / / the calves in group (control group) showed an oocyst shedding curve which was typical for natural cryptosporidiosis, with the majority of the animals becoming parasitologically positive by day and again negative by day (tables and ) . group started shedding earlier than the other two groups. in this group, the highest mean log (ton + ) was observed on day , and on day oocysts were observed only in two calves (table ). in contrast, in group the peak mean log (ton + ) was lower and occurred later (day ) than in group , and on day , the calves in this group were shedding more oocysts than the other two groups. however, when only friesian calves were considered, groups and showed a very similar oocyst shedding curve, which peaked in both groups on day . comparisons between the crude mean log (ton + ) of the three treatment groups at the various sampling days are reported in fig. . model (which included the effects of treatment group, sampling day and their interaction term), indicated a significant difference between the three treatments (p = . ). post hoc comparisons identified a significant difference between group (full dose) and group (control) (group < log (ton + ) than group ; p = . ), and no significant difference between group and the other two groups. in model , which included also the fixed effect of sex, the significance between the treatment groups was preserved (p = . ), but there was no significant effect of sex on the outcome (p = . ). when variable 'breed' was introduced as a covariate in model , the pvalue of the effect of treatment increased to p = . , and the covariable 'breed' was also significant (friesian < log (ton + ) than non-friesian; p = . ). this increase in the p-value was supported by model (friesian only), which showed p = . for the effect of treatment. all these models produced similar p-values when the interaction of treatment × day was removed (not shown). in order to cross-validate these results we compared the mean log (ton + ) between the treatment groups using bivariate anova for each sampling day separately (not shown), with consistent results: whereas the inclusion of all the animals resulted in statistically significant difference between treatment groups and on days , and (p < . ), all the p-values were > . when only friesian calves were analyzed. the results of the kaplan-meier test indicated a significantly longer prepatent period in group as compared with group . in fact, calves ( %) in group were parasitologically positive on day , whereas only three calves from group ( %) were shedding oocysts on the same day (tables and ). the duration of the prepatent period in group was intermediate, although not statistically different from the other groups. similar results were observed when only data from friesian calves were analyzed (fig. ) . the results of the fisher's exact tests showed that on days and , the proportion of cryptosporidiumpositive calves was significantly greater in group than in group (two-tailed fisher's exact test p < bonferroniadjusted critical value of . ). on the other hand, most calves ( %) in group and only two calves in group and two in group were shedding oocysts on that day (table ) . twenty three calves ( %) passed at least one liquid specimen in the course of the study, and the proportion of liquid specimens on day was relatively high, and very similar in the three treatment groups (table ). the results of kruskal-wallis and wilcoxon tests test did not indicate any significant difference (p > . ) between the fecal consistency scores of the three groups on any sampling day (not shown). except one significant difference between group and group on day , the proportion of liquid specimens did not differ between the groups (table ) , and these result persisted when only friesian calves were included in the analysis (not shown). there was no association between the log (ton + ) and the presence of liquid specimen by logistic regression (p > . ). halofuginone lactate is registered for the prevention of calf cryptosporidiosis in several countries, but the compound has a relatively narrow therapeutic index and a substantial market price. this study was initially designed to assess the efficacy of a reduced dosage regime of hl on a farm infected with c. parvum, with no evidence for the presence of co-infection. however, the identification of rotavirus and salmonella typhimurium in multiple calves provided an opportunity to collect much needed data of the utility of the compound in the presence of such common co-infections. when the study was designed, we predicted a longer prepatent period and a decreased number of oocysts and fecal consistency scores in the full dose group, as reported for hl in the absence of documented co-infections. the coinfections with rotavirus and salmonella typhimurium did not affect the ability of hl (full dose) to delay the onset of shedding. whereas most calves ( / ) in group were still shedding oocysts on day , the majority of the calves in the control group were already parasitologically negative by that day (table ). the prolongation of the prepatent period was not coupled with statistically significant differences between the mean log (ton + ) of the treatment groups after controlling for the repeated measurements, the sex, and the breed of the calves (models and ) . we hypothesize that hl suppressed the parasite's life cycle during the first days of treatment, when the diarrhea caused by the other pathogens was not yet overt, effectively prolonging the prepatent period in group . conversely, about % of the calves in group were passing liquid feces on day , and this early diarrhea (probably caused by the co-infecting pathogens) could have shortened the intestinal transit time of hl in the last days of treatment, effectively abolishing its anti-cryptosporidium effect. some authors suggested that co-infections with multiple agents could cause a more severe diarrhea than mono-infections (de la fuente et al., ; garcia et al., ) . consequently, it could be hypothesized that suppression of one organism could reduce the severity of the table the number of liquid feces (fecal score )/total specimens assessed, stratified by sampling days, treatment groups (the corresponding proportions are in brackets). on the same sampling day, an asterisk indicates a two-tailed fisher's exact test p < . (bonferroni-adjusted critical probability). group (full dose) group (half dose) group (placebo-control) ( ) diarrhea. in this study, there was no significant difference between the fecal consistency scores of the treated and untreated groups, and no association between the ton and the presence of liquid feces was found by logistic regression. furthermore, in agreement with the results of a metaanalysis of the literature (silverlås et al., ), the mortality rates did not differ between the groups. therefore, the results do not allow conclusions to be drawn on a clinical benefit of hl in the presence of co-infections. finally, the statistically significant effect of breed on the intensity of oocyst shedding observed in this study was intriguing. other studies have previously reported lower cryptosporidium infection prevalence in beef calves than in dairy calves (geurden et al., ; kváč et al., ) . the anti-cryptosporidium activity of hl was not fully preserved and the use of the drug was not associated with a clinical benefit in the presence of enzootic co-infection with rotavirus and salmonella typhimurium in calves. diagnostic efforts should therefore aim to rule out the presence of other common enteropathogens in order to maximize the clinical efficacy of hl in the field. one year after the completion of this study, the authors requested and obtained funding from msd animal health new zealand (the new distributors of halocur) to perform an epidemiological study of neonatal calf diarrhea in dairy farms. the current study was performed before such funds were requested and the company was not consulted during the preparation of this manuscript. prevalence, prediction and risk factors of enteropathogens in normal and non-normal feces of young dutch dairy calves prevalence and risk factors for cryptosporidium spp. infection in young calves cryptosporidium and concurrent infections with other major enterophatogens in to -day-old diarrheic dairy calves in central spain control of cryptosporidiosis in neonatal calves: use of halofuginone lactate in two different calf rearing systems cryptosporidium and cryptosporidiosis, second ed paromomycin is effective as prophylaxis for cryptosporidiosis in dairy calves cryptosporidium parvum infection in bovine neonates: dynamic clinical, parasitic and immunologic patterns epidemiology of cryptosporidium: transmission, detection and identification potential risk factors for cryptosporidium infection in dairy calves rotavirus and concurrent infections with other enteropathogens in neonatal diarrheic dairy calves in spain prevalence and genotyping of cryptosporidium in three cattle husbandry systems in zambia controlling the onset of natural cryptosporidiosis in calves with paromomycin sulphate genetic diversity and zoonotic potential of cryptosporidium parvum causing foal diarrhoea enteropathogens and risk factors for diarrhea in norwegian dairy calves the zoonotic transmission of giardia and cryptosporidium effect of halofuginone lactate on the occurrence of cryptosporidium parvum and growth of neonatal dairy calves prevalence and control of bovine cryptosporidiosis in german dairy herds nonparametric estimation from incomplete observations recurrent outbreaks of cryptosporidiosis associated with calves among students at an educational farm programme preventive and therapeutic efficacy of halofuginonelactate against cryptosporidium parvum in spontaneously infected calves: a centralised, randomised, double-blind, placebo-controlled study effect of cryptosporidium parvum infection on the absorptive capacity and paracellular permeability of the small intestine in neonatal calves age-related and housingdependence of cryptosporidium infection of calves from dairy and beef herds in south bohemia field study of the efficacy of halofuginone and decoquinate in the treatment of cryptosporidiosis in veal calves prevalence of four enteropathogens in the feces of young diarrhoeic dairy calves in switzerland genetic characterization and transmission cycles of cryptosporidium species isolated from humans in new zealand efficacy of halofuginone lactate in the prevention of cryptosporidiosis in suckling calves diagnosis of neonatal calf diarrhea prophylactic use of decoquinate for infections with cryptosporidium parvum in experimentally challenged neonatal calves the effect of halofuginone lactate on experimental cryptosporidium parvum infections in calves role of cryptosporidium parvum as a pathogen in neonatal diarrhea complex in suckling and dairy calves in france effect of nitazoxanide on cryptosporidiosis in experimentally infected neonatal dairy calves duration of naturally acquired giardiosis and cryptosporidiosis in dairy calves and their association with diarrhea efficacy of two peroxygen-based disinfectants for inactivation of cryptosporidium parvum oocysts pathogenesis of intestinal and systemic rotavirus infection systematic review and metaanalyses of the effects of halofuginone against calf cryptosporidiosis cryptosporidium and giardia as foodborne zoonoses cryptosporidium excystation and invasion: getting to the guts of the matter prophylactic and therapeutic efficacy of nitazoxanide against cryptosporidium parvum in experimentally challenged neonatal calves prevalence of cryptosporidium parvum infection in southwestern ontario and its association with diarrhea in neonatal dairy calves efficacy of halofuginone lactate in the prevention of cryptosporidiosis in dairy calves an outbreak of calf diarrhea attributed to cryptosporidial infection efficacy of halofuginone lactate against cryptosporidium parvum in calves zoonotic cryptosporidiosis food-borne parasitic zoonoses in china: perspective for control the authors thank the farm owners and staff for their co-operation, and mark stevenson (massey university) for contributing to the statistical analysis. this work was funded by the lewis fitch veterinary research fund, new zealand. the funding body had no active role in this study. bh was funded by a pfizer (currently zoetis) undergraduate summer scholarship. key: cord- -ukn hm x authors: sutherland, m. a.; lowe, g. l.; huddart, f. j.; waas, j. r.; stewart, m. title: measurement of dairy calf behavior prior to onset of clinical disease and in response to disbudding using automated calf feeders and accelerometers date: - - journal: journal of dairy science doi: . /jds. - sha: doc_id: cord_uid: ukn hm x abstract we determined if feeding and lying behavior, recorded by automatic calf feeding systems (acfs) and accelerometers, could be used to detect changes in behavior before onset of neonatal calf diarrhea (ncd) or in response to disbudding pain in dairy calves. at d of age, calves had accelerometers attached to their hind leg and were housed in pens with acfs. calves were examined daily for signs of illness or injury. of the calves monitored, were diagnosed with ncd; activities of calves with ncd were then compared with those of healthy controls (calves that had no symptoms of ncd, other illnesses, or injury). feeding (milk consumption and the number of rewarded and unrewarded visits to the feeder) and lying behavior during the d leading up to calves displaying clinical signs of ncd were analyzed. calves with ncd performed fewer unrewarded visits and consumed less milk than healthy calves during the - and -d periods before diagnosis with ncd, respectively. calves with ncd tended to perform fewer lying bouts than healthy calves over the -d period before diagnosis with ncd. at wk of age, a subset of healthy calves were allocated to of treatment groups: ( ) sham handling (sham, n = ), ( ) cautery disbudding (db, n = ), ( ) administration of local anesthetic (la) and db (la+db, n = ), ) administration of a nonsteroidal anti-inflammatory drug (nsaid) and db (nsaid+db, n = ), and ( ) administration of la, nsaid and db (la+nsaid+db, n = ). feeding and lying behavior were recorded continuously for h pre- and postdisbudding. we found no effect of treatment on the number of rewarded or unrewarded visits to the feeder and milk volume consumed h before administration of treatments. during the -h postdisbudding period, sham calves performed more unrewarded visits than db, la+db, and nsaid+db calves, but the number of unrewarded visits did not differ between sham and la+nsaid+db calves. during the first hour of the posttreatment period we noted a difference in lying times among treatments, with db and nsaid+db calves spending less time lying than sham calves and lying times being similar between sham, la+db, and la+nsaid+db calves. the acfs and accelerometers have the potential to automatically gather valuable information regarding health status and pain in calves. therefore, it may be advantageous to combine both of these measures (acfs and accelerometers) when evaluating ncd on farm or pain in calves in future research. automated techniques for measuring individual calf behavior on-farm are now available; feeding behavior can be detected remotely with automatic calf feeding systems (acfs; svensson and jensen, ; borderas et al., ) and activity can be recorded using accelerometers (e.g., hobo data loggers; bonk et al., ) . automated systems have the advantage of collecting data noninvasively with reduced labor input. feeding and lying behavior have been shown to change in response to sickness (svensson and jensen, ; borderas et al., ; szyszka and kyriazakis, ) and pain (graf and senn, ; heinrich et al., ) in calves. hence, automated measures of feeding and lying behavior could potentially be used to detect early signs of disease and pain in calves. neonatal calf diarrhea (ncd) is an enteric disease that is associated with severe diarrhea. once an animal displays clinical signs of ncd, much of the associated tissue damage to the intestinal submuscosa has already occurred (schroeder et al., ) ; therefore, early detection of ncd would enable calves to be promptly treated and moved to sick pens thereby reducing tissue damage and risk of disease transmission to other calves. previous studies using acfs and accelerometers have reported changes in feeding (svensson and jensen, ; borderas et al., ; lowe et al., ) and lying behavior (swartz et al., ) in response to ncd and respiratory disease. however, those studies predominantly focused on changes in behavior once calves became clinically ill or the time period just prior (svensson and jensen, ; borderas et al., ) , or calves that were diagnosed with respiratory disease (swartz et al., ) . to our knowledge, no studies have measured changes in feeding in conjunction with lying behavior in calves before diagnosis with ncd. it would be valuable to measure changes in feeding and lying behavior concurrently, several days before calves become clinically ill, to assess if information collected from acfs and accelerometers could be used for early detection of ncd on-farm. in the united states, % of dairy cattle producers routinely dehorn their cattle and of these % disbud using cautery (usda, ) . disbudding is often performed without pain relief; however, studies have shown that this procedure causes behavioral changes indicative of pain (reviewed by stafford and mellor, ) . for example, calves spent less time feeding and more time lying after being cautery disbudded than sham-handled controls (graf and senn, ; faulkner and weary, ) . therefore, a need exists to evaluate different pain-mitigation strategies to alleviate the pain caused by disbudding; to do this, however, laborintensive behavioral studies are often needed. it would be useful if automated measures of feeding and lying behavior were sensitive enough to detect behavioral changes in calves in response to a painful procedure such as disbudding. automated measures of feeding and lying behavior could then be used as a research tool to evaluate different pain-mitigation strategies for painful husbandry procedures in calves. disease and pain can negatively affect calf welfare, but these states are often difficult to detect and time-consuming to assess objectively. information on feeding and lying behavior collected from acfs and accelerometers could potentially be measured remotely on-farm and used by producers as a tool to help detect ncd early so that strategies could be promptly implemented to reduce the negative effects of this disease on calf health and production. in addition, automated measures of feeding and lying behavior could provide a useful and less labor-intensive research tool to evaluate different pain-mitigation strategies in calves. therefore, the objectives of our study were to determine if behav-ioral data collected from acfs and accelerometer data loggers could be used to detect changes in behavior before the onset of ncd or in response to disbudding pain in dairy calves. our study was conducted between july and october on agresearch's tokanui dairy research farm in south waikato, new zealand ( ° ′e longitude, − ° ′s latitude). all procedures involving animals were approved by the ruakura animal ethics committee (no. ) under the new zealand animal welfare act (ministry of primary industries, ). seventy-one friesian dairy calves (n = females) and friesian-hereford cross calves (n = females, n = males) were monitored in this study. calves were allocated to of pens (n = , , and calves/ pen respectively) at d of age according to their order of birth. one pen was filled at a time, which ensured pen mates were all of similar size and stage of development. all calves were monitored to obtain a sample of animals displaying clinical signs of ncd (as described below); a subset of calves that never displayed signs of ncd or other health issues or injuries were used in the pain-assessment study. calves were separated from their dams within h of birth and transported to the calf-rearing facility. upon arrival at the facility, all calves were weighed and individually identified using numbered (allflex, irving, tx) and electronic identification ear tags (allflex) placed in the left and right ears, respectively. at this time accelerometers were attached to the lateral side of the hind leg (as described below). the calf-rearing facility had a roof, solid dirt floors, and walls on all sides. calves were housed in indoor pens ( . × . m), located inside the calf rearing facility, with floors covered in wood chip bedding and post and rail fencing. each pen contained an acfs, water troughs, a hay feeder, and meal feeders attached to the side of the pen. water, meal (calf-pro %, seales winslow, morrinsville, new zealand), and hay were provided ad libitum. all calves were trained over a -to -d period to use the acfs from d of the trial. trained staff performed daily health checks to assess the calves' general health and to identify calves with signs of illness. a standard operating procedure developed in conjunction with a veterinarian was used. health checks assessed calves for the presence of diarrhea, high rectal temperatures ( . °c or higher), signs of dehydration (sunken eyes, or poor skin elasticity-assessed using the skin tent test), coat condition (shiny and smooth vs. dry and rough), position of the ears (erect vs. droopy), gut fill (full vs. empty stomachs), signs of navel ill (inflammation, swelling, or discharge from the navel), nasal or ocular discharge, swelling, abscesses, and injury. trained staff were also asked to comment if calves were coughing, had crackly breaths, or were breathing fast. in the present study, calves were diagnosed as having ncd when they presented with diarrhea and high rectal temperatures ( . °c or higher). a calf was considered to have diarrhea if it was observed passing feces with a loose to watery consistency and strong odor. for calves suspected of having diarrhea due to the presence of loose fecal matter on the top of the tail or hind legs, a fecal sample was taken to evaluate fecal consistency and to confirm whether or not that particular calf was diarrheic. only data collected from calves diagnosed as having ncd were considered as sick calves for further analysis. once calves exhibited signs of ncd, blood and fecal samples were collected for analysis to verify etiology of the disease. it is possible that any of the calves could have been experiencing subclinical disease during the course of the study, but it was beyond the scope of this study to test all animals for the presence of subclinical diseases; hence, animals were categorized as sick or healthy based on clinical signs of illness only. calf bw was recorded weekly. of the calves that were monitored, only data from a subset of calves [n = diagnosed with ncd (bw = . ± . kg, mean ± sd) and n = healthy calves (bw = . ± . kg)] were used in this component of the study (n = calves from group , n = calves from group , and n = calves from group ; n = females and n = males). each calf with ncd was matched with a healthy calf that was from the same group, was the same breed, and was approximately the same date of birth and birth weight as the calves with ncd. a calf was considered healthy if during the course of the study it presented no symptoms of ncd or other signs of illness or injury (e.g., coughing, rectal temperatures greater than . °c, navel ill, nasal or ocular discharge, swelling or abscesses, and so on, as defined in the daily health check standard operating procedure above). upon completion of the trial for the first groups, the experimental pens were cleaned in preparation for the final replicates (groups and ), which involved removing and replacing the top layer of woodchip with fresh wood chips. in addition, all surfaces were thoroughly sprayed using a broad-spectrum disinfectant (halamid, axcentive sarl; bouc-bel-air, france) and all water troughs and feed containers were also thoroughly cleaned and refilled. blood chemistry and hematology. blood samples were taken from all calves at , , and d of age and when a calf displayed signs of being clinically ill. blood samples were collected by jugular venipuncture into evacuated tubes that contained sodium fluoride, edta, or no anticoagulant (bd vacutainer, franklin lakes, nj). blood samples were stored at approximately °c until they were delivered to the new zealand veterinary pathology (hamilton, new zealand) laboratory for analysis. blood smear slides were performed on a sysmex xt- iv using veterinary software and sysmex reagents (sysmex corporation, kobe, japan) for estimation of total white blood cell, neutrophil, and lymphocyte counts. white blood cell differentials were performed using a standard cell count and the neutrophil-to-lymphocyte ratio was calculated by dividing the percent of neutrophils by the percent of lymphocytes. serum haptoglobin concentrations were measured using a commercially available colorimetric kit (phase haptoglobin assay cat. no. tp- ; tridelta development limited, maynooth, county kildare) in accordance with the manufacturer's instructions (http:// www .trideltaltd .com/ haptoglobin -main -page .html). the analytical sensitivity of this assay was . mg/ ml. fecal sampling. fecal samples were collected manually via gentle palpation of the rectum from any calves that were considered clinically ill and were tested for the presence of cryptosporidium, rotavirus, coronavirus, and salmonella. cryptosporidium was measured using an acid fast stain to visually detect the presence of cryptosporidium, which could be seen as round bodies measuring to µm in diameter and dark red or pink in color. if more lightly stained, the parasites showed internal bodies that were darker blue or brownish in color. the presence of rotavirus and coronavirus were determined using a commercially available elisa kit (pourquier elisa calves diarrhea; institut pourquier, montpellier, france). the -step, naked eye reading method was conducted in accordance with the manufacturer's instructions (https:// www .idexx .co .uk/ en -gb/ livestock/ livestock -tests/ ruminant -tests/ idexx -rota -corona -k -ag -test/ ). selective enrichment fecal cultures were used for the detection of salmonella. feeding behavior. the acfs (a&d reid, temuka, new zealand) used an electronic identification system to identify individual animals as they entered the acfs. each acfs had a single teat to deliver whole milk (at approximately - °c) on the following schedule: l, times per day with a minimum -min period with no milk access between each complete milk feed (as per normal farm practice in new zealand). if the calf did not consume the whole l during the visit, they could return at any point to consume the remainder of the allowance. the acfs recorded the total number of visits, rewarded visits (calf received part or whole milk allowance), unrewarded visits (calf received no milk), and milk volume consumed per visit. lying behavior. throughout the study period, lying and standing behavior was recorded continuously on all calves using hobo pendant g accelerometer data loggers ( k, onset computer corporation, bourne, ma) set at -min intervals recording the y and z axes. the accelerometers were placed in a durable fabric pouch and strapped onto the lateral side of the hind leg above the metatarsophalangeal joint. accelerometers were placed horizontally on the leg such that the x-axis ran parallel to the ground, pointing in the anterior direction, and the y-axis pointing toward the dorsal plane of the calf. the pouch was held in position using velcro patches, one sewn to the pouch, the other glued (ka-mar, livestock improvement corporation, hamilton, new zealand) to the leg of the calf. the pouch was further held in place by a strap around the leg of the calf. accelerometers were initialized and downloaded using onset hoboware pro software (onset computer corporation, version . . ). the g-force readings were converted into binary values (e.g., lying = , standing = ), and hourly (min/h) and daily (min/d) summaries of lying time and lying bout frequency (number of bouts/d) were calculated in sas . (sas institute inc., cary, nc) using a code designed for this purpose (awp ubc, ) and validated by bonk et al. ( ) for use in calves. at approximately wk of age ( ± . d of age, mean ± sd), female friesian dairy calves (bw = . ± . kg) were selected from the healthy animals and allocated to of treatment groups: ( ) sham handling (sham; n = ), ( ) cautery disbudding (db; n = ), ( ) administration of local anesthetic (la) and db (la+db; n = ), ) administration of a nonsteroidal anti-inflammatory drug (nsaid) and db (nsaid+db; n = ), and ( ) administration of la, nsaid, and db (la+nsaid+db; n = ). the combination of pain relief treatments were included to evaluate whether the automated systems were sensitive enough to measure varying degrees of pain. all calves were restrained in a calf crush with a head bail (front opening calf bail, te pari, oamaru, new zealand) during treatments. for db, la+db, nsaid+db, and la+nsaid+db calves, an electric cautery iron (quality electric debudder, v, w; lister gmbh, lüdenscheid, germany) was used to remove the calves horn buds. for la+db and la+nsaid+db calves, ml of local anesthetic ( % lignocaine hydrochloride; lopaine, ethical agents ltd., auckland, nz) was injected into the cornual nerve ( ml) and injection sites around the base of the horn bud ( . ml/site) min before disbudding. immediately before disbudding, a needle prick test was used to verify the success of the cornual nerve block. for nsaid+db-and la+nsaid+db-treated calves, meloxicam ( . mg/kg of bw; metacam, boehringer ingelheim ltd., auckland, nz) was injected subcutaneously min before disbudding. treatment groups were balanced for age and bw. feeding and lying behavior were recorded continuously for h before and h after administration of treatment, as described above. prior to analysis, all data were assessed for evidence of departures from the assumptions of normality and homogeneity of variance using the univariate procedures of sas version . (sas inst. inc.). the residuals from the log-transformed mixed model analysis showed good consistency with the normality assumptions of the model. for the calf health component of our study, data from calves that were identified with ncd were used (see description above). each calf with ncd was matched with a healthy control from the same pen taking into account sex, breed, birth date, and birth weight. healthy controls never presented with any symptoms of ncd or other signs of illness or injury during the study period. day was the day that calves showed signs of ncd and the equivalent day was used for d for control calves. we were interested in behavioral changes leading up to signs of ncd; therefore, we focused on the d before appearance of ncd. milk consumption, blood chemistry and hematology data were log-transformed to meet the assumptions of the analysis. milk consumption and lying (lying times and frequency of lying bouts) behavior data were analyzed using the mixed model procedure of sas. the model included the fixed effects of treatment (health status: ncd and healthy), day, treatment by day interaction, and the random effects of pen and calf. the model had a repeated structure on time allowing incorporation of heterogeneity of variances across time. for blood chemistry and hematology (cells counts and haptoglobin concentrations) data, only the blood results from d were compared; the model included the fixed effects of treatment (health status: ncd and healthy) and the random effect of pen. feeding behaviors (total number of visits, and number of rewarded and unrewarded visits) were analyzed using the glimmix procedure of sas with a specified poisson distribution. the model included the fixed effects of treatment (health status: ncd and healthy), day, treatment by day interaction, and the random effects of pen and calf. the model had a repeated structure on time allowing incorporation of heterogeneity of variances across time. for the analysis of the pain assessment component of this study, milk consumption was log-transformed to meet the assumptions of the analysis. milk consumption and lying times data were analyzed using the mixed model procedure in sas and the model included the fixed effects of treatment (sham, db, la+db, nsaid+db, la+nsaid+db), day/time, treatment by day/time interaction, and the random effects of pen and calf. the model had a repeated structure on time allowing incorporation of heterogeneity of variances across time. lying times were summarized over the entire -h pre-and posttreatment period and analyzed. in addition, hourly lying times for the first h after administration of treatments were analyzed, as this time period is representative of the cortisol response to cautery disbudding (graf and senn, ; stafford and mellor, ) . feeding behaviors (total number of visits and number of rewarded and unrewarded visits) were analyzed using the glimmix procedure of sas with a specified poisson distribution. the model included the fixed effects of treatment (sham, db, la+db, nsaid+db, la+nsaid+db), day/time, treatment by day/time interaction, and the random effect of pen. the model had a repeated structure on time allowing incorporation of heterogeneity of variances across time. the sample size was determined by a power analysis with % power and % significance level. data displayed in the graphs, tables, and text are actual data summarized by least squares means ± standard error of the means. statistical significance was determined at p ≤ . and . < p ≤ . were considered a tendency. fecal sampling. four calves tested positive for cryptosporidium, with rotavirus, with coronavirus, and with cryptosporidium and rotavirus. no calves tested positive for salmonella. eleven calves identified as having ncd according to the criteria (diarrhea and high rectal temperatures) did not test positive for either cryptosporidium, rotavirus, coronavirus, or salmonella, so the cause of ncd for these calves was unknown. blood chemistry and hematology. total white blood, neutrophil, and monocyte cell counts and the neutrophil-to-lymphocyte ratio were higher (p ≤ . ) and eosinophil cell counts tended (p = . ) to be higher in calves with ncd than healthy calves (table ) . health status of the calves did not affect any other hematology measures or haptoglobin concentrations (table ) . feeding behavior. calves with ncd tended (p = . ) to visit the feeder less than healthy calves over the -d period before signs of ncd (total number of visits to the feeder/d: control: . ± . ; sick: . ± . ). we observed no effect of health status (p = . ) or a health status × day interaction (p = . ) for the number of rewarded visits; however, the number of rewarded visits to the feeder changed (p < . ) over the -d study period (number of rewarded visits to the feeder/d: d − = . ± . ; d − = . ± . ; d − = . ± . ; d − = . ± . ; d − = . ± . ; d = . ± . ). the number of unrewarded visits was affected by health status (p = . ) and day (p < . ; figure ). calves with ncd performed fewer unrewarded visits than healthy calves on d − (p = . ), − (p = . ), and (p = . ). we noted a health status × day interaction for volume of milk consumed (p = . ; figure ); calves with ncd drank less on d − (p = . ), − (p = . ), − (p = . ), and (p < . ) than healthy calves. lying behavior. we found a health status × day interaction for time spent lying (p = . ; figure ). calves with ncd tended to spend more time lying on d − (p = . ) but less time lying on d (p = . ). moreover, calves with ncd tended (p = . ) to perform fewer lying bouts than healthy calves over the entire -d period before diagnosis with ncd (number of lying bouts/d: control = . ± . ; sick = . ± . ). feeding behavior. the total number of visits, rewarded visits, unrewarded visits, and milk volume consumed was similar (p > . ) among all treatments during the -h period before administration of treatments (table ) . during the to h posttreatment period, milk volume consumed was similar (p > . ) among sham and disbudded calves, with or without pain relief (table ) . however, sham calves visited the acfs more during the -h posttreatment period than db (p < . ) and la+db (p < . ) calves, but la+nsaid+db visited the feeder a similar (p = . ) number of times as sham calves (table ) . we observed no differences (p > . ) across treatments in the number of rewarded visits to the acfs (table ) . however, calves experiencing the sham treatment had more unrewarded visits during the -h posttreatment period than db (p = . ), la+db (p < . ), and nsaid+db (p < . ) calves, but the number of unrewarded visits was similar (p = . ) for sham and la+nsaid+db calves (table ) . lying behavior. the proportion of time calves spent lying was similar (p > . ) among all treatment groups over the -h pre-and -h posttreatment period (table ) . however, during the -h posttreatment period, we found a difference among treatments (figure ) . during the first hour, db (p = . ) and nsaid+db (p = . ) calves spent less time lying than sham calves. moreover, lying times were similar (p > . ) among sham, la+db, and la+nsaid+db calves. lying times were similar among all treatments during the -h posttreatment period. feeding and lying behaviors collected using acfs and accelerometers were affected by health status and pain in calves in the present study. the number of unrewarded visits declined before calves showed signs of ncd. differences in the number of unrewarded visits was apparent d before calves showed signs of ncd; however, we found no change in the number of rewarded visits before diagnosis. similarly, svensson and jensen ( ) found that the number of unrewarded visits was a more sensitive measure of sickness in calves than the number of rewarded visits. borderas et al. ( ) suggested that the effect of disease on feeding behavior would vary according to feeding motivation, as milk intake and frequency of visits to the feeder were more dramatically decreased in sick calves being fed a high (ad libitum or l/d) rather than a low ( l/d) milk allowance. in the present study, calves received a milk allowance of l/d, which is closer to the low than the high milk allowance provided in the borderas et al. ( ) study; hence, more dramatic changes in feeding behavior may have been observed if a higher milk allowance was provided to calves in the present study. therefore, milk allowance fed to calves needs to be taken into consideration when using behavioral data from acfs as an indication of ncd in calves. contrary to other studies (svensson and jensen, ; borderas et al., ; lowe et al., ) , milk consumption was reduced in sick calves up to d before displaying signs of ncd in the present study. hence, milk consumption in combination with the number of unrewarded visits maybe a good measure for early detection of ncd in calves. differences in feeding behavior among studies may relate to the pathogenesis of the disease. in other studies, calves were diagnosed with a range of illnesses, including gastrointestinal, respiratory, or a combination of these diseases (borderas et al., ; swartz et al., ) , or other signs of disease such as arthritis, fever, dull calf syndrome, and so on (svensson and jensen, ) . however, in those studies, samples were not taken to verify the pathogenesis of the diseases. in our study, only calves that were diagnosed as having ncd were used; however, even with fecal sampling we could only confirm the cause of ncd in out of calves. therefore, due to the a-c means within each row with different superscripts differ at p < . . sham handling (sham; n = ), cautery disbudding (db; n = ), administration of local anesthetic + db (la+db; n = ), administration of a nonsteroidal anti-inflammatory drug (nsaid) + db (nsaid+db; n = ), and la+nsaid+db (n = ). lying times (min/h; lsm ± sem) of calves during the first h after disbudding or control handling. treatments were sham handling (sham; n = ), cautery disbudding (db; n = ), administration of local anesthetic + db (la+db; n = ), administration of a nonsteroidal anti-inflammatory drug (nsaid) + db (nsaid+db; n = ), and la+nsaid+db (n = ). means within each hour with different letters (a-c) differ at p < . . differences in the pathogenesis affecting calves among our study and previous studies, it is difficult to know the reason for the difference in results. in addition to pathogenesis, age of calves at the time of diagnosis, severity of outbreak, and the time of diagnosis could potentially affect feeding and lying behavior in calves. further research focused on particular pathogens or disease severity would be of interest to assist in our understanding of the relationship between pathogenesis and sickness behavior in calves. calves challenged with a low dose of bacterial endotoxin spent more time lying and performed fewer standing bouts in borderas et al. ( ) , similar to the present study, and sick calves spent more time lying d before displaying clinical signs of ncd and tended to perform fewer lying bouts. a reduction in activity of sick calves may be part of the adaptive strategy adopted by mammals to help combat infections (hart, ) . however, on the day calves were diagnosed as having ncd in the present study their lying times were reduced. at the time calves were diagnosed with ncd, blood and fecal samples were collected and animals were moved to the sick pen and treated with electrolytes; this extra handling may account for the reduction in lying times. in the present study, disbudding treatment affected the total number of visits that calves made to the feeder during the -h posttreatment period. this difference was predominantly due to the number of unrewarded visits as disbudding treatment did not affect the number of rewarded visits. calves experiencing the sham treatment performed more unrewarded visits during the -h posttreatment period than db, la+db, and nsaid+db calves. the greater number of unrewarded visits suggests that even though there was no difference in the amount of milk consumed among treatments, calves that were not disbudded were more motivated to visit the acfs than calves disbudded with la or nsaid or without pain relief. fewer unrewarded visits to the acfs by disbudded calves could reflect a lack of appetite in these animals due to the pain or distress caused by disbudding. indeed, calves spent less time feeding after cautery disbudding (graf and senn, ) and amputation dehorning (sutherland et al., ) . it is also possible that disbudding caused the calves in the present study to become head shy and, hence, more hesitant to place their heads in the acfs due to the potential risk of knocking their head in the confined space. another possibility is that experiencing a painful husbandry procedure caused a change in the emotional state of the animals, which may in turn have affected their motivation to feed (neave et al., ) . interestingly, calves given local anesthetic and an nsaid before disbudding in the present study per-formed a similar number of unrewarded visits as sham calves, which suggests that the combination of la and an nsaid was the most effective pain-mitigation strategy evaluated in the present study. these results are in accordance with previous studies showing that the physiological and behavioral response to disbudding is markedly reduced when calves are given a combination of la and an nsaid before disbudding heinrich et al., ) . total milk consumption was not affected by disbudding, with or without pain relief, in the present study. in contrast, bates et al. ( ) found that when calves were given the same milk allowance as provided in the present study ( l/d), mean cumulative milk consumption over the -d postdisbudding period was greater for calves disbudded with pain relief compared with those without. therefore, it is possible that if milk consumption had been recorded for a longer period of time in the present study, treatment differences may have been detected. furthermore, milk allowance is an important factor to consider when using total milk consumption as an indicator of stress (e.g., pain or disease). borderas et al. ( ) found that sick calves consumed less milk at an acfs when provided with a high milk allowance in comparison to healthy calves, but total milk consumption was not affected when they were provided with a low milk allowance. calves provided only a low milk allowance are more likely to be motivated to feed. in the present study, calves were given access to l/d, whereas calves given ad libitum access to milk will drink up to kg/d (jasper and weary, ) . therefore, providing calves with a higher milk allowance is likely to increase the sensitivity of feeding behavior as a measure of stress (e.g., pain and disease) in calves. the proportion of time disbudded calves spent lying was similar across all treatments groups over the entire -h posttreatment period in the present study; however, during the -h period after disbudding, treatment affected lying behavior. during the first hour after disbudding, calves given local anesthetic showed similar lying patterns to control-handled calves, which is similar to the results reported by mcmeekan et al. ( ) h after amputation dehorning. these findings may suggest that calves given local anesthetic were experiencing less pain initially than calves disbudded without pain relief. moreover, calves given only an nsaid before disbudding showed similar lying times to calves disbudded without pain relief, which corresponds to the mechanism of action of nsaid; giving an nsaid (e.g., ketoprofen) reduces the inflammation-related pain associated with amputation dehorning in cattle, but not the initial pain caused by dehorning (mcmeekan et al., ) .these results suggest that lying behavior mea-sured using accelerometer data loggers maybe sensitive enough to assess pain in calves in response to an acute painful procedure such as cautery disbudding. in conclusion, automated measures of feeding and lying behavior could be used to detect changes in behavior before the onset of clinical signs of ncd or in response to acute pain in calves. the number of unrewarded visits to the acfs appeared to be a more sensitive measure of ncd and pain in calves than automated measures of lying behavior. it would be of interest to confirm these results using animals exposed to different conditions (e.g., different pathogens) or other painful procedures (e.g., castration, lameness). ubc animal welfare program: sop -hobo data loggers, sas code version . university of british columbia effect of analgesia and antiinflammatory treatment on weight gain and milk intake of dairy calves after disbudding technical note: evaluation of data loggers for measuring lying behavior in dairy calves behaviour of dairy calves after a low dose of bacterial endotoxin automated measurement of changes in feeding behaviour of milk fed calves associated with illness reduction in pain response by combined use of local anesthesia and systemic ketoprofen in dairy calves dehorned by heat cauterization reducing pain after dehorning in calves behavioural and physiological responses of calves to dehorning by heat cauterization with or without local anaesthesia biological basis of the behavior of sick animals. neurosci the effect of meloxicam on behavior and pain sensitivity of dairy calves following cautery dehorning with a local anesthetic effects of ad libitum milk intake on dairy calves brief communication: the use of infrared thermography and feeding behaviour for early disease detection in new zealand dairy calves effects of regional analgesia and/or a non-steroidal anti-inflammatory analgesic on the acute cortisol response to dehorning in calves effects of a local anaesthetic and a nonsteroidal anti-inflammatory analgesic on the behavioural responses of calves to dehorning accessed pain and pessimism: dairy calves exhibit negative judgement bias following hot-iron disbudding development and performance evaluation of calf diarrhoea pathogen nucleic acid purification and detection workflow addressing the pain associated with disbudding and dehorning in cattle effect of castration and dehorning singularly or combined on the behavior and physiology of holstein calves identification of diseased calves by use of data from automatic milk feeders short communication: automated detection of behavioral changes from respiratory disease in pre-weaned calves what is the relationship between level of infection and 'sickness behaviour' in cattle usda-animal and plant health inspection service (aphis)-veterinary services (vs)-center for epidemiology and animal health (ceah)-national animal health monitoring system (nahms) the authors thank agresearch staff, in particular suzanne dowling, tokanui farm staff (south waikato, new zealand), theresa topp from waikato university, and alvin reid (a&d reid, temuka, new zealand) for providing the automatic calf feeders. agresearch also gratefully acknowledges that this study was funded by the new zealand ministry of business, innovation and employment (wellington, new zealand). key: cord- -pzjj wxc authors: smith, geof title: antimicrobial decision making for enteric diseases of cattle date: - - journal: vet clin north am food anim pract doi: . /j.cvfa. . . sha: doc_id: cord_uid: pzjj wxc diarrhea in neonatal and adult cattle is common and can be caused by several etiologic agents. as diagnostic testing is not always readily available, practitioners must often decide on a course of treatment based on knowledge of the likely pathogen and their own clinical experience. antimicrobials have long been used to treat diarrhea in adults and neonates; however, there is increased pressure to prevent unnecessary use of antibiotics in food animal species. this article reviews existing data on the use of antibiotics given to cattle with enteric diseases to decide when they are necessary and which antimicrobials should be used. antibiotics to calves to prevent diarrhea cannot be recommended. however, the use of certain antimicrobials to treat select cases of calf diarrhea may be effective in reducing mortality and decreasing the severity and duration of diarrhea. unfortunately, it is unlikely that any of the antibiotics that are currently approved for the treatment of diarrhea in the united states would be effective. instead of mass medicating large numbers of animals, antimicrobial therapy should be targeted to specific animals that are likely to develop septicemia or have systemic signs of disease. there are many causes of diarrhea in adult cattle, and the vast majority of these do not warrant antimicrobial therapy. common enteric diseases of cattle include simple indigestion, rumen acidosis, parasites, coccidiosis, bovine viral diarrhea (bvd), winter dysentery, salmonella, paratuberculosis (johne disease), molybdenosis (copper deficiency), and malignant catarrhal fever (mcf), along with a wide range of toxicities including a host of poisonous plants. the only disease on this list that is likely to truly benefit from antimicrobial therapy is salmonella enteritis; however, an argument could be made for bvd and mcf. both these diseases suppress normal immune function and can lead to an increased occurrence of secondary bacterial infections. it is well understood that bvd is associated with the bovine respiratory disease complex and can lead to higher rates of bacterial pneumonia. however, cases of severe salmonella enteritis have also been reported after bvd infection in cattle causing significant mortality. therefore, it would not be inappropriate to administer a broad-spectrum antimicrobial to cattle suspected of having bvd or mcf, likely one that is labeled for metaphylactic use in cattle at high risk for developing respiratory disease. it is also important to note that many toxic cows with severe mastitis, metritis, or peritonitis often have diarrhea that is a direct result of endotoxemia. the mechanism of endotoxin-induced diarrhea is not completely understood; however, it seems to involve both prostaglandins and nitric oxide. the administration of endotoxin leads to abundant accumulation of fluid inside the small intestines of animals, which is thought to be prostaglandin mediated. endotoxin also increases the enzyme activities of nitric oxide synthase in intestinal smooth muscle, which changes the propagation of jejunal contractions resulting in rapid intestinal transit. the diarrhea observed during endotoxemia in cattle is not profuse but is generally described as low volume. in these cases, choosing to use an antimicrobial would likely not benefit the diarrhea or enteric disease present in the cows but would almost certainly be indicated from the standpoint of treating the primary disease condition. despite the limited number of enteric diseases in adult cattle that would benefit from antimicrobial therapy, surveys indicate that diarrhea is a relatively common reason for the use of antibiotics. in the national animal health monitoring survey (nahms) dairy study, mastitis was the most common reason for antimicrobial use on dairy farms followed by lameness, reproductive diseases (metritis), respiratory disease, and then diarrhea or other enteric disease. results of the survey showed that about % of cattle on dairy farms from the survey population had been treated with an antimicrobial for diarrhea in the preceding -month period and % of farms said they routinely had cows that received antimicrobial drugs because of diarrhea. data from the nahms feedlot study indicated that % of feedlots reported diarrhea or other enteric disease in calves after arrival with . % of calves showing evidence of diarrhea. further data from the study indicated that % of calves with diarrhea received treatment upon arrival. when the survey looked into what specific therapy was administered, % of calves received an injectable antimicrobial, while % of the cattle received an oral antibiotic. when reviewing the data of both the nahms dairy and feedlot studies, it becomes clear that enteric disease is not the primary reason for antimicrobial use in adult cattle. however, it is also apparent that diarrhea is one of the top or reasons cattle receive antimicrobials and that at least half of the cattle diagnosed with diarrhea receive either a parenteral and/or an oral antibiotic. most of the time dairy or beef cattle have diarrhea, it is not clear what the cause is, and therefore they are empirically treated with antibiotics. the assumption in many cases is that the animal has salmonellosis or some other bacterial enteritis. although this is certainly true in some cases, it is very likely that most cases of diarrhea are because of simple indigestion caused by an abrupt diet change, moldy feed, spoiled feed, or perhaps a mild grain overload (rumen acidosis). however, simple indigestion is often difficult or impossible to diagnose definitively and therefore cattle are treated empirically with antimicrobials. if salmonella are the main target of antimicrobial therapy in adult cattle with diarrhea, drug selection should ideally be based on the results of susceptibility testing using bacterial strains recovered from that particular dairy or feedlot. broad-spectrum antimicrobials are usually used pending the availability of susceptibility test results. salmonella show variable resistance patterns to ampicillin, amoxicillin, ceftiofur, florfenicol, neomycin, streptomycin, sulfonamides, tetracycline, and trimethoprim-sulfa and general resistance to penicillin, erythromycin, and tylosin. [ ] [ ] [ ] the most recently published data indicated that salmonella isolates from cattle were most commonly resistant to streptomycin, ampicillin, and sulfonamides, whereas resistance to ceftiofur was extremely low. as salmonella are facultative intracellular pathogens, selecting an antimicrobial with good tissue penetration and the ability to attain intracellular therapeutic drug concentrations within macrophages is desirable. in summary, antimicrobials for the treatment of diarrhea in adult cattle are likely being overused at present in the cattle industry. although diarrhea occurs fairly commonly, most causes are unlikely to respond to antimicrobials. treatment should be primarily supportive care, including fluid therapy, anthelmintics if needed, and provision of good-quality pasture or other forages. mortality rates in most cases of diarrhea in mature cattle are low, and the diarrhea generally resolves within a few days. diseases such as paratuberculosis would have a higher mortality but would still not be likely to respond to antimicrobial therapy. however, when cattle have signs of systemic infection such as pyrexia or bloody diarrhea, it may be rational to begin antimicrobial therapy, particularly on farms that have a history of salmonellosis. when examining an adult ruminant with enteric disease, the practitioner should consider the age of the animal; the onset, severity, and duration of diarrhea (acute vs chronic); the number of cattle affected (is this an individual animal or a herd problem); clinical signs in the animal other than diarrhea (does the animal show systemic signs of disease); nutritional history (especially recent changes in the diet), and whether there has been an introduction of new animals (bvd). all these help to determine a list of possible causes for the diarrhea and may help reduce the use of antimicrobial drugs in cattle that are unlikely to benefit from therapy. prudent use of antimicrobial drugs is recommended with an emphasis on establishing a herd diagnosis and conducting susceptibility testing for the specific salmonella serotype or other bacterial pathogen present and choosing an appropriate antibiotic. calf health should be a priority on both beef and dairy farms. despite this importance, the united states department of agriculture dairy study shows a preweaned antimicrobial decision making heifer calf mortality rate of . % and reports that only % of farms can supply an adequate number of replacements from their own herd. although mortality is slightly less in beef calves, % to % still die before weaning. in both beef and dairy calves, diarrhea represents the most common reason for loss due to death before weaning. therefore, practitioners and producers spend a significant amount of time trying to prevent diarrhea and also making sure good treatment programs are in place when diarrhea does occur. the main principles of diarrhea prevention in both beef and dairy cattle include ( ) using a vaccine in late gestation cattle containing enterotoxigenic escherichia coli, rotavirus, and coronavirus; ( ) making sure a good colostrum program is in place ensuring adequate intake of immunoglobulins by the calf; and ( ) decreasing the load of enteric pathogens in the environment through sanitation, hygiene, housing, and pasture management. historically, many producers (particularly in the dairy and veal industries) have used feeding of oral antibiotics to prevent diarrhea and hopefully decrease mortality in newborn calves. however, the practice of continually feeding antibiotics to calves is now prohibited in many countries, and the efficacy of feeding antibiotics to calves as a method of diarrhea prevention has not proven to be effective in recent studies. almost years ago, a thorough review was published on the efficacy of antibiotics for preventing diarrhea and improving weight gain in dairy calves. the investigator concluded that the addition of chlortetracycline and oxytetracycline to milk replacer in the first weeks of life decreased the incidence and severity of diarrhea. the minimum daily doses necessary for efficacy in this study were . to . mg/lb, which led to the routine inclusion of these antibiotics in milk replacers throughout the united states. unfortunately, this study did not look at critical factors such as mortality rate in calves or incidence of diarrhea. the primary benefits of oral antibiotics were found to be higher weight gain and decreased severity and duration of diarrhea. as discussed in a previous review article, there were several studies done in the s and the s using various antibiotics (including ampicillin, chlortetracycline, furazolidone, neomycin, oxytetracycline, and streptomycin) to prevent diarrhea in calves. although the results of these studies varied, only study documented a decrease in mortality rate from diarrhea due to prophylactic oral administration of chlortetracycline. a few studies did find a decrease in the total number of days of diarrhea associated with antibiotics ; however, other studies (particularly with neomycin) found increased rates of diarrhea in antibiotic-treated calves. , quite a few of these older studies found that oral administration of various antibiotics did not change the incidence of diarrhea in calves when compared with untreated controls. more recent studies have found that either oral antibiotics had no effect on decreasing calf diarrhea or in some cases diarrhea rates actually increased in calves fed antibiotics. for example, a study in california fed group of holstein heifers monensin in the starter ration, whereas another group was fed lasalocid and chlortetracycline (aureomycin) for the first weeks of life (in addition to nonmedicated milk replacer or whole milk). antibiotic-treated calves had no difference in average daily gain, feed efficiency, or the proportion of calves treated for diarrhea. in another study, holstein heifers were fed milk replacer medicated with oxytetracycline and neomycin or an unmedicated milk replacer that contained a probiotic (enteroguard-no longer commercially available). once again, body weight gain, feed efficiency, and the incidence and severity of diarrhea were similar between groups. in a third study, dairy calves were divided into groups: medicated milk replacer (neomycin and tetracycline for the first days of life) plus the administration of trimethoprim-sulfamethoxazole, spectinomycin, penicillin, and bismuth pectin for the treatment of diarrhea (referred to as conventional therapy); medicated milk replacer for the first days of life, bismuth pectin for diarrhea, and other antibiotics only in cases of fever or depressed attitude (targeted therapy); nonmedicated milk replacer with antimicrobial treatment of diarrhea (same treatments as the conventional therapy group above); and nonmedicated milk replacer with targeted therapy. calves fed a medicated milk replacer had % more days with diarrhea when compared with calves fed nonmedicated milk replacer. in a survey, about % of dairy farms in the united states fed medicated milk replacers to preweaned heifer calves, most commonly a combination of oxytetracycline and neomycin. however, a new federal regulation that began in restricts the feeding of medicated milk replacers to a period of to days. thus continuous feeding of antibiotics in the milk from birth to weaning is no longer permitted, and this is meant to transition the use of oral antibiotics in calves from prophylactic to therapeutic. medicated milk replacers should now be reserved for the treatment of bacterial enteritis (diarrhea) and bacterial pneumonia in dairy calves and not for prophylactic prevention. since the late s, the european union has prohibited the sale of milk replacers and other animal feeds containing antibiotics. all the feed and milk replacers for dairy cattle must be sold as nonmedicated, and then antibiotics can be added only for therapeutic use (for example, in calves with diarrhea). australia and new zealand also have strict laws regarding the importation of any animal feed, and these products are generally nonmedicated as well. overall, the conventional practice of adding antibiotics to milk or milk replacers for prophylactic use is being discouraged worldwide. most modern studies fail to find any benefit of using antibiotics as a prevention for diarrhea, and their use in this manner should be discouraged. the use of antibiotics as a treatment in calves with diarrhea is a controversial topic with strong opinions on both sides. several articles have been published indicating that antibiotics are contraindicated in calves with diarrhea or that they serve no beneficial purpose. , in contrast, other studies have indicated that antibiotics are effective in reducing mortality rate and speeding recovery in calves with diarrhea. , to begin the discussion, it is important to establish a reason to use antibiotics in calves with diarrhea. the primary treatment goals of an antibiotic in calves with diarrhea would be ( ) to prevent bacteremia and ( ) to decrease the number of coliform bacteria in the small intestine. several studies have reported that a significant number of calves with diarrhea subsequently develop bacteremia. an initial study in the early s reported that colostrum-deprived calves with diarrhea were frequently bacteremic ( / calves or %). in contrast, none of the diarrheic calves in this study that had received colostrum were bacteremic ( . or %). a study conducted on a large calf-rearing facility in california examined dairy calves with severe diarrhea ; of the calves ( %) had failure of passive transfer and ( %) calves were bacteremic (predominantly e coli). another study done in prince edward island, canada, looked at the prevalence of bacteremia in calves with diarrhea ; of the ( %) calves in this study were bacteremic (predominantly e coli). as noted previously, the percentage of calves with bacteremia was significantly higher in the failure of passive transfer group ( / or %) than in adequate passive transfer group ( / or %). taken together these studies indicate that it can be assumed that one-third of the calves with severe diarrhea are bacteremic and that the percentage is likely significantly higher in calves with failure of passive transfer. although some have argued that antibiotic use in calves with diarrhea is inappropriate and leads to the emergence antimicrobial decision making of resistant bacteria, a case can be made that the use of antibiotics to prevent and/or treat bacteremia in calves with diarrhea and systemic signs of disease is warranted. withholding effective treatment (antibiotics) for a life-threatening disease (such as bacteremia in calves with diarrhea) should not be condoned on animal welfare grounds. another potential reason for antibiotic therapy in calves with diarrhea is coliform overgrowth of the small intestine (fig. ) . research conducted in the s documented increased numbers of e coli in the abomasum, duodenum, and jejunum of calves with diarrhea. , more recent studies have consistently found increased numbers of intestinal e coli in calves with naturally acquired diarrhea regardless of the age of the calf or the cause of the diarrhea. , specifically, the numbers of e coli bacteria increase from -to , -fold in the duodenum, jejunum, and ileum of calves with scours, even when rotavirus or coronavirus is identified as the cause of diarrhea. this small intestinal overgrowth of the intestines with coliform bacteria can persist after the pathogen causing the diarrhea is gone. the increased numbers of coliform bacteria in the small intestine of calves with diarrhea is associated with altered small intestinal function, morphologic damage, and increased susceptibility to bacteremia. therefore there is some logic to the use of antimicrobials in scouring calves to decrease the number of intestinal coliform bacteria. this use could potentially prevent the development of bacteremia, decrease calf mortality, and decrease damage to the small intestine, facilitating digestion and absorption and increasing growth rate. several of which would be illegal to use in the united states (ie, chloramphenicol, furazolidone, or marbofloxacin). the results indicated that specific antibiotics were effective in reducing mortality and increasing growth rate when administered to calves with diarrhea. several studies provided evidence that even calves with simple diarrhea (without systemic signs of disease) seemed to recover faster with antibiotics as opposed to calves that did not receive antibiotics. some veterinarians feel that oral or parenteral administration of antibiotics to calves with diarrhea is contraindicated. the arguments most commonly used to support this approach include: ( ) oral antibiotics alter intestinal flora and thereby induce diarrhea or exacerbate existing diarrhea, ( ) antibiotics harm good intestinal bacteria more than bad bacteria, ( ) antimicrobial use in calves with diarrhea is not effective, and ( ) the use of antibiotics provides a selection pressure on the enteric bacterial population likely leading to increased antimicrobial resistance. there is solid evidence to indicate that the use of antimicrobial drugs can decrease mortality in calves and there is no evidence to support the argument that antimicrobials harm good bacteria more than the bad. however, the emergence of resistant bacteria is certainly serious and is something the veterinarian must take into account before treating calves with diarrhea. table contains a list of antimicrobials currently approved for the treatment or prevention of diarrhea in the united states. at present, oxytetracycline administered parenterally and chlortetracycline, neomycin, oxytetracycline, sulfamethazine, and tetracycline administered orally are the only antimicrobials labeled in the united states for the treatment of calf diarrhea. of these, none have been shown to be consistently efficacious in peer-reviewed studies. as discussed above, when treating calves with diarrhea the primary goals of therapy are to ( ) decrease the number of e coli bacteria in the small intestine and ( ) treat potential e coli bacteremia. with these goals in mind, the target of antimicrobial therapy in calves with diarrhea should be coliform bacteria both in the blood and in the small intestine. as none of the approved drugs for treating diarrhea in the united states are likely to be effective, extralabel use is likely justified. some efficacy has been described for oral amoxicillin in the treatment of calves with experimentally induced diarrhea, , but was not effective in the treatment of naturally acquired diarrhea in beef calves. amoxicillin trihydrate ( mg/kg administered orally every h) or amoxicillin trihydrate-clavulanate ( . mg combined drug/kg administered orally every h) for at least days is one antimicrobial approach that likely has some efficacy for calves with diarrhea. amoxicillin is partially absorbed from the calf small intestine with absorption being similar in both milk-fed and fasted calves. high amoxicillin concentrations are found in bile and intestinal contents after oral administration, with lower concentrations in serum. oral ampicillin could also be used, and its efficacy in one study was shown to be equivalent to that of amoxicillin. although very popular in the united states, oral sulfonamides cannot be recommended for treating calves with diarrhea because of the lack of efficacy studies. most antimicrobial susceptibility studies done in the past years indicate that sulfamethazine (and other sulfonamide drugs) would have poor sensitivity against coliform bacteria in the blood or small intestine. the most logical antimicrobial for parenteral treatment of calf diarrhea in the united states is ceftiofur ( . mg/kg given intramuscularly [im] every h) for at least days. ceftiofur is a broad-spectrum antibiotic that is resistant to b-lactamase. the labeled dose maintains plasma concentrations of ceftiofur above the minimum concentration required to inhibit the growth of % of e coli (mic ) in young calves ( . mg/ml). furthermore, % of the active metabolite (desfuroylceftiofur) is excreted into the intestinal tract of cattle providing activity in both the blood and the small intestine. parenteral ampicillin ( mg/kg im every h) is another antibiotic that would be likely to have efficacy in calves with diarrhea. in europe, parenteral enrofloxacin is labeled for the treatment of calf diarrhea, and several studies have documented efficacy with using fluoroquinolone antibiotics in calves with diarrhea. [ ] [ ] [ ] however, it must be emphasized that the extralabel use of fluoroquinolone antibiotics in the united states is illegal and obviously not recommended. historically, gentamicin was also considered an appropriate treatment for use in calves with diarrhea. however, parenteral administration of aminoglycosides cannot be recommended in calves with diarrhea because of the lack of published efficacy studies, prolonged slaughter withdrawal times ( months), potential for nephrotoxicity in dehydrated calves, and availability of other drugs likely to be equally successful (ceftiofur, amoxicillin, and ampicillin). the issue of whether or not to use antibiotics in a calf with simple diarrhea (without systemic signs of disease) is a little more controversial. although there have been studies to show that these calves gain more weight and recover faster than calves not given antibiotics, there are other studies that indicate no benefit to using antibiotics in these cases. , the clinician must weigh any potential benefit of antimicrobial therapy against the possibility of increasing the population of resistant bacteria on the farm. a fairly recent study demonstrated that individual treatment of sick calves with antibiotics increased the level of resistance to e coli isolates; however, the change in antimicrobial susceptibility was only transient. the next logical question is whether or not antimicrobial susceptibility testing should play a role in determining which drug is used to treat calves with diarrhea. historically, culture and susceptibility results from fecal culture have been routinely used to guide treatment decisions; however, it is not clear whether or not this has any clinical relevance. research validating susceptibility testing as being predictive of treatment outcome for calves with diarrhea is currently not available. part of the problem is that our target is coliform bacteria in the blood and small intestine, which are likely different from fecal bacterial flora. older studies have demonstrated that the predominant strain of e coli in the manure of calves with diarrhea usually changes several times during the course of disease. , these studies also show that about % of calves have different e coli strains isolated from the upper and lower parts of small intestine. so it is logical to conclude that fecal coliform isolates are not representative of what is happening in the intestine. another potential problem with using susceptibility testing to guide antimicrobial selection in cases of diarrhea is that most of the bacterial cultures submitted usually come from dead animals, which represent treatment failures and may have already received antibiotics. preferential growth of resistant bacterial strains can start within a few hours after antibiotic administration, and therefore culture results from dead calves may not be representative of the actual clinical problem. to the author's knowledge, the only study that has tried to assess the predictive ability of fecal antimicrobial susceptibility testing found that it was an inaccurate predictor of clinical outcome. in a large group of experiments evaluating the efficacy of amoxicillin for treating calf diarrhea, calves were divided into groups that either received amoxicillin or did not. diarrhea was experimentally induced using enterotoxigenic e coli and smith rectal swab culture, and susceptibility testing was done. most calves ( %) developed diarrhea after challenge; however, in only about % of cases did calves shed the actual challenge strain of e coli. recovery or treatment success in these studies was defined as normal feces within days after the start of treatment, while treatment failure was defined as death or scouring for more than days. among calves in which the e coli cultured from rectal swabs were susceptible to amoxicillin, % died and % recovered with . as the mean number of days scouring. outcomes were not different in calves that had amoxicillin-resistant strains of e coli cultured from rectal swabs with % death loss, % recovery rates, and . scouring days. in calves given a placebo instead of amoxicillin, mortality was significantly increased ( %), recovery rates were decreased ( %), and the number of scouring days was longer ( . ). the investigators concluded that amoxicillin had a significant effect on disease by decreasing mortality and number of scouring days; however, treatment success could not be predicted by whether the e coli cultured from rectal swabs was susceptible or resistant to the antimicrobial being used. two studies have concluded that there was a good correlation between in vitro antimicrobial susceptibility of fecal e coli isolates and clinical response to treatment; however, neither study had data to statistically analyze this association. , in contrast, other studies reported no correlation between in vitro susceptibility results for coliform isolates and response to antimicrobial treatment. , however, these studies did not differentiate enterotoxigenic and nonenterotoxigenic strains of e coli and also failed to do any statistical analysis of the data. there is a significant need for antimicrobial susceptibility data from e coli and salmonella isolates collected from the small intestine of untreated calves with diarrhea. minimum inhibitory concentrations (mic) could then be compared with free antimicrobial concentrations that are actually achievable in the intestinal tract of calves to determine the best drug to use along with the optimal dosing interval. however, it should be emphasized that antimicrobial concentrations can be altered by multiple variables, such as intestinal ph, which may be quite different between healthy calves and those with diarrhea. therefore even after establishing mic values and setting appropriate breakpoints, these need to be validated through clinical trials examining the use of specific antimicrobial drugs in calves with diarrhea as compared to the pathogen isolated and disease outcome. until then the use of fecal culture and susceptibility testing to guide antimicrobial selection for treating calf diarrhea is probably of little value. drug selection is based on knowledge of the likely pathogen (e coli in the blood and small intestine), pharmacokinetics of the drug (can it achieve therapeutic concentrations at the site of infection), and evaluation of the response to treatment (does the animal get better). on farms in which salmonella or e coli septicemia is a problem, looking at susceptibility results from blood cultures is likely much more appropriate than fecal culture. certainly the overuse of antibiotics is a concern, and the overall philosophy in veterinary medicine is to use antibiotics conservatively to preserve the efficacy of these drugs in both animals and humans. based on the need to minimize the use of antibiotics and because of the lack of any demonstrated recent efficacy, the feeding of antimicrobials to calves as a method of diarrhea prevention is not recommended. however, calves with diarrhea and systemic signs of illness should receive antibiotics targeted toward coliform bacteria in the blood (because of likelihood of bacteremia) and the small intestine (because of bacterial overgrowth). a clinical sepsis scoring system to predict bacteremia based on physical examination does not seem to be sufficiently accurate to guide antimicrobial decision making, and therefore the clinician should assume that calves are bacteremic when they exhibit inappetence, coinfection with bovine viral diarrhea virus and mycoplasma bovis in feedlot cattle with chronic pneumonia outbreak of salmonella enterica serotype newport in a beef cow-calf herd associated with exposure to bovine viral diarrhea virus supportive therapy of the toxic cow effect of lipopolysaccharide on diarrhea and gastrointestinal transit in mice: roles of nitric oxide and prostaglandin e effects of endotoxin on regulation of intestinal smooth muscle nitric oxide synthase and intestinal transit united states department of agriculture animal plant health inspection service (usda aphis) feedlot part iv: health and health management on u.s. feedlots with a capacity of , or more head salmonella in calves antimicrobial susceptibility of salmonella isolates recovered from calves with diarrhea in australia prevalence of antimicrobial resistance among salmonella on midwest and northeast usa dairy farms antibiotics as growth stimulants for dairy cattle: a review use of antibiotics to prevent calf diarrhea and septicemia antibiotics and calf diarrhea diarrhea and malabsorption in calves associated with therapeutic doses of antibiotics: absorptive and clinical changes adverse effect of oral antibacterial prophylaxis and therapy on incidence of neonatal calf diarrhea evaluation of an oral glucose-glycine-electrolyte formulation and amoxicillin for the treatment of diarrhea in calves a field trial comparing the effects of supplementation with aureomycin plus lasalocid or monensin on the health and production performance of dairy calves growth and health of holstein calves fed milk replacers supplemented with antibiotics or enteroguard targeting therapy to minimize antimicrobial use in preweaned calves: effects on health, growth, and treatment costs no effect of a homeopathic preparation on neonatal calf diarrhea in a randomized double-blind, placebo-controlled clinical trial a rational approach to treatment of calf diarrhea antimicrobial use in the treatment of calf diarrhea treatment of calf diarrhea: antimicrobial and ancillary treatments observations on the etiology of neonatal diarrhea (scours) in calves bacteriological culture of blood from critically ill neonatal calves model to predict septicemia in diarrheic calves the distribution of the colon-aerogenes group of bacteria in the alimentary tract of calves the bacteriology of the intestinal tract of young calves with special reference to early diarrhea distribution and virulence of escherichia coli in the small intestine of calves with and without diarrhea changes in small intestinal morphology and flora associated with decreased energy digestibility in calves with naturally occurring diarrhea pathogenesis and prevention of infectious diarrhea (scours) of newborn calves amoxycillin: distribution and clinical efficacy in calves a clinical evaluation of antimicrobial agents and temporary starvation in the treatment of acute undifferentiated diarrhea in newborn calves oral absorption and bioavailability of ampicillin derivatives in calves clinical efficacy of amoxicillin in calves with colibacillosis field evaluation of efficacy of marbofloxacin bolus in the treatment of naturally occurring diarrhea in the new born calf comparison of danofloxacin with baquiloprim/sulphadimidine for the treatment of experimentally induced escherichia coli diarrhea in calves efficacy of danofloxacin % injectable solution in the treatment of escherichia coli diarrhoea in young calves in europe field trial evaluating the influence of prophylactic and therapeutic antimicrobial administration on antimicrobial resistance of fecal escherichia coli in dairy calves the typing of e. coli by bacteriophage, its application to the study of e. coli populations of the intestinal tract of healthy calves and of calves suffering from white scours passage of antibiotics through the digestive tract of normal and scouring calves and their effect upon the bacterial flora discrepancy between antibiotic (amoxycillin) resistance in vitro and efficacy in calf diarrhea further observations on the effect of chemotherapy on the presence of drug-resistant bacterium coli in the intestinal tract of calves the sensitivity to chemotherapeutic agents of a further series of strains of bacterium coli from cases of white scours: the relationship between sensitivity tests and response to treatment neonatal diarrhea in calves escherichia coli and salmonella newport in calves: efficacy of prophylactic and therapeutic treatment dehydration, lethargy, or fever. in calves with diarrhea and no systemic signs of illness (normal appetite for milk, no fever), evidence suggests that the clinician continue to monitor the health of the calf and not administer antibiotics unless the calf's condition deteriorates. key: cord- -ygd xv x authors: björkman, camilla; von brömssen, claudia; troell, karin; svensson, catarina title: disinfection with hydrated lime may help manage cryptosporidiosis in calves date: - - journal: vet parasitol doi: . /j.vetpar. . . sha: doc_id: cord_uid: ygd xv x diarrhea is common in young calves and is often caused by cryptosporidium parvum infection. the aim of this study was to investigate if disinfection of calf pens with hydrated lime would reduce contamination of c. parvum oocysts and improve calf health in herds with c. parvum associated diarrhea problems. four dairy herds with ongoing c. parvum associated calf diarrhea problems each participated in the study over six to seven months. during the study period, all pens/huts for young calves were cleaned according to the usual farm routine before a new calf entered. hydrated lime was then used to disinfect half of the pens/huts. diarrhea incidence was recorded by the farmers and by veterinarians, who clinically examined the calves every second month. in total, calves participated in the study. the farmers detected diarrhea in ( %) calves, with similar proportions in calves kept in lime disinfected and control pens. age at diarrhea onset was significantly higher in lime disinfected pens than in control pens, . days and . days, respectively. there was no difference between the groups regarding duration or severity index recorded by the farmers. the body condition score in – week old calves was significantly higher in calves that had been kept in lime disinfected pens during their first weeks of life, indicating that calves in disinfected pens/huts were less affected by their infections. faecal samples from to day old calves, were collected on four occasions at each farm (n = ). cryptosporidium positive samples were found at all samplings in all four herds. cryptosporidium spp. was detected in ( %) samples with no difference between lime disinfected and control pens. c. parvum was the dominant species. two different c. parvum subtypes were found; iiaa g r b in three herds and iiaa g r b_variant in one herd. only one subtype was found in each herd. disinfection of calf pens with slaked lime delayed onset of diarrhea and improved the body condition in the calves, but did not affect diarrhea incidence or duration. although lime disinfection alone will not be sufficient to control cryptosporidium associated diarrhea in herds with extensive calf diarrhea problems, these results suggest that it can be a valuable complement to other measures. diarrhea is a significant health problem in young calves, and the protozoan parasite cryptosporidium parvum is one of the major infectious causes of calf diarrhea worldwide (blanchard, ; thomson et al., ) . clinical cryptosporidiosis is mostly seen in calves up to six weeks of age and the most prominent clinical symptoms are watery diarrhea, inappetence, depression, and sometimes death (robertson et al., ) . co-infection with other pathogens or presence of noninfectious diarrheal causes can lead to more severe disease. oocysts, the infective stage of the parasite, are excreted with the faeces and an infected individual can shed millions of oocysts per gram faeces (uga completely prevent or cure disease (silverlås et al., a; almawly et al., ; meganck et al., ) . thus, sanitation is still the most important tool in disease prevention. however, the parasite is resistant to all commonly used chemical disinfectants, making it particularly difficult to control (fayer, ) . hydrated lime, or calcium hydroxide, can be used to sanitise animal facilities from bacteria and viruses (kristula et al., ; anonymous, ) . it has been shown that hydrated lime decreases the viability of c. parvum in the laboratory (zintl et al., ) , and when hydrated lime disinfection of the calf pens was used as a complement to the standard cleaning procedures in two swedish herds with long-lasting cryptosporidiosis problems, the farmers reported a lower incidence and less severe diarrhea in the calves (c. axén personal communication). recently, matsuura et al. ( ) reported about a successful sanitation of a herd that for about years had experienced severe calf diarrhea problems due to mixed c. parvum and giardia intestinalis infections. they emptied the calf stable, removed bedding and faecal matter, let the pens dry for several days and then washed them with boiling water, coated them with milk of lime, and exchanged all wooden boards dividing the pens. during the two-year period following the sanitation no calves died and no c. parvum oocysts or g. intestinalis cysts were found (matsuura et al., ) . this indicates that hydrated lime disinfection might be an effective control measure in cattle herds. the aim of this study was to investigate if disinfection of calf pens with hydrated lime would decrease contamination of c. parvum oocysts and improve calf health in herds with c. parvum associated diarrhea problems. four herds were recruited to this cohort study. inclusion criteria were that i) the herd had a prevailing calf diarrhea problem for at least two months, ii) more than % of the calves developed diarrhea before three weeks of age, and iii) c. parvum, but not rotavirus or coronavirus, had been detected in calves from the herd. furthermore, the herd should not use hydrated lime or other chemicals for disinfection of the calf pens, and the design of the calf facilities should allow arrangement of calf pens into a treatment and a control section. preferably, the calves should be housed individually until they reached four weeks, but at least until two weeks, of age. potential experimental herds were identified by field veterinarians at växa sverige, an advisory and ai service company with knowledge about calf health in dairy herds in the different regions of sweden. based on clinical findings and laboratory diagnostic results, the field veterinarians considered c. parvum to be the main cause of the diarrhea problem in these herds. only herds that fulfilled all requirements were included in the study. several herds had to be excluded because they already used hydrated lime, and few herds kept their calves in individual pens longer than one week. owners of potential experimental herds were interviewed by one of the authors (cs) about management and health of the pre-weaned calves using a predefined questionnaire. before it was finally decided if a herd could be included, faecal samples were collected from five calves under three weeks of age and analysed for presence of cryptosporidium spp., rotavirus and coronavirus. only herds in which cryptosporidium spp., but no rotavirus or coronavirus, was found were accepted. each of the four herds participated in the study over six to seven months and was visited by a project veterinarian every second month, i.e. four times, during this period. at the first visit the magnitude of the calf diarrhea problem and the feeding and cleaning routines were recorded. the veterinarian and the farmer together decided how the pens/huts used for newborn calves should be divided into experimental and control areas, and how the calves should be distributed between the pens. in herds one to three, cleaning routines meant that all pens/huts forming one row in the stable were cleaned at the same time. in these herds, three to nine consecutively born calves were therefore allocated to experimental and control pens/huts, respectively. in herd four, every other calf born was put in an experimental and a control pen, respectively. the farmers themselves decided which area was used as experimental and control. this was, as far as possible, kept unknown to the veterinarian. the farmers were instructed to clean all pens/huts according to their usual farm routine before a new calf was entered. in the experimental pens/huts a thin layer of hydrated lime should then be spread onto the walls and floor. the recommendation was to use no more than g hydrated lime to coat both floor and walls of a . m pen. farmers were instructed to apply the lime while the surface was still damp after washing in order to allow the lime to attach to the walls. if the farm routine did not include cleaning using water, a small volume of water was to be sprinkled onto the walls and floor before the hydrated lime was applied. the lime was allowed to work for a minimum of h and farmers were instructed to brush the walls before the pen/ hut was bedded and a new calf introduced. the control pens were to be left empty for at least h after cleaning before a new calf was entered. ethics approval for this study was granted by the regional ethical review board in uppsala (reference number c / ). for each calf born during the study period, farmers were requested to record the identity number, date of birth, breed, sex, date when the calf was separated from the dam and moved to an individual pen, whether it was kept in an experimental or control pen, and the date the calf was moved from the individual pen to a group pen. if a calf developed diarrhea they were requested to record the date when the diarrhea was first observed, the length of the disease period, if the diarrhea was accompanied by decreased general condition and/or decreased appetite, if the calf received any treatment, and if it was euthanised or died. at each of the four visits, the project veterinarian performed clinical examinations, scored the body condition and collected faecal samples. all calves up to four weeks of age were clinically examined. general condition was graded from to ( = normal behavior, alert, gets up when approached, interested in the surroundings; = depressed, must be stimulated to get up; = gets up with help; = unable to stand even with help). dehydration was graded from to ( = none; = mildly dehydrated; = moderately to severely dehydrated). faecal consistency was graded from to ( = sausage-like; = porridgelike; = gruel-like; = watery). as a measure of disease severity, body condition was scored on a . scale from to for all two to four and six to eight week old calves based on shape of the lower back ( = pointed; = rounded, = flat; = inverted). faecal samples were collected for analysis of cryptosporidium spp., rotavirus and coronavirus from up to five - day old calves in the experimental and control group, respectively. samples were taken directly from the rectum. at the first and fourth visits, blood samples were also collected from five - day old calves for analysis of serum protein as an estimate of the colostrum management in the herd. one gram of each faecal sample was cleaned and concentrated by a saturated sodium chloride flotation method and analysed for cryptosporidium oocysts by epifluorescence microscopy as described by (silverlås et al., b) ). the entire wells were examined by epifluorescence microscopy, and oocysts enumerated at x magnification. an animal was considered cryptosporidium positive if at least one oocyst was detected in the microscope. the lower detection limit of this method is oocysts per gram of faeces (opg). all cryptosporidium positive samples collected at the first and fourth visits were analysed further to determine species and c. parvum subtype. dna was extracted using the powerlyzer ® powersoil ® dna isolation kit (mo bio laboratories, cat no. -s) according to the manufacturer's recommendations but with the following modifications: up to ml of each cleaned sample was spun down at , rpm for three minutes in an eppendorf centrifuge and the pellet dissolved in μl bead solution. the solution was transferred to the glass bead tube and μl solution c was added. the tubes were briefly vortexed and incubated at °c in a thermal block for min, followed by bead beating using a mp fast prep bead beater set at . m/s for one minute. finally, the dna was eluted in μl solution c . a nested pcr protocol for partial amplification of a ∼ bp fragment of the s rrna gene was set up using kapa g robust hotstart pcr kit (kapa biosystems, cat no kk ). the reaction mixture for all reactions consisted of μl kapa buffer a, . μl of a mm nucleotide mixture, . μl of forward and reverse primer ( μm stock), . μl of kapa g polymerase and μl template in a total volume of μl. the reactions were run in a bio-rad s thermal cycler. primers for the s amplification were as described in (santín et al., ) ). samples containing c. parvum were subtyped through partial amplification (∼ bp) of the -kda glycoprotein (gp ) gene using the same pcr mixture as for s but with primers described in (alves et al., ) . s reaction conditions were °c for three minutes followed by cycles of °c for s, °c for s and °c for s, with a two minutes extension at °c after the last cycle. two μl were used as templates for the second reaction using the same reaction conditions, except that the annealing temperature was raised to °c. the gp annealing temperature was °c in the first pcr and °c in the second. achieved sequences were compared with sequences deposited in genbank using basic local alignment search tool (blast, ncbi http://www.ncbi.nlm.nih.gov/blast/blast.cgi). analyses for rotavirus and coronavirus were performed at the diagnostic laboratory at the swedish national veterinary institute. up to five samples are pooled at the laboratory and then rotavirus is detected by antigen-elisa and coronavirus by pcr. for each sampling, faecal samples from the experimental group and the control group, respectively, were pooled before analyses. serum protein was analysed by refractometer. data were entered and edited in microsoft office excel spreadsheets (© microsoft corporation). results presented in the text are original, not modeled, data. statistical analyses were performed with generalised linear mixed models using sas (version . , sas institute inc., cary, nc, usa) with the binary variable of whether or not a calf was kept in a hydrated lime disinfected pen as the main predictor. 'herd' was included as a random variable in all models. 'sex' was initially included as a fixed variable but had a negligible effect and is therefore not presented in the final results. 'breed' and 'housing' (i.e. pen or hut) were excluded from the models as they were associated with 'herd'. results were considered significant when p ≤ . . for farmer recorded data, the association between whether or not a calf was kept in a hydrated lime disinfected pen and the binary outcome variable 'presence of diarrhea' was investigated by using a generalised linear mixed model with a binomial distribution for the response, i.e. a logistic regression model. the continuous outcome variables 'age at diarrhea onset' and 'days with diarrhea' were investigated assuming a normal distribution for the error term. as the residuals for the analysis of 'days with diarrhea' did not follow a normal distribution, data were log-transformed before analysis. the assumption of normality and equal variances was checked by appropriate residual plots. a variable 'severity' was created by combining the farmers´records of presence of diarrhea and whether the diarrhea had been accompanied by decreased general condition and/or decreased appetite. the 'severity' variable had three categories; = diarrhea, but general condition or appetite not affected, = diarrhea and decreased general condition or decreased appetite, and = diarrhea and decreased general condition and decreased appetite. therefore, a proportional odds model was used to model the probabilities to fall in any of these categories when the pen was hydrated lime disinfected or not. for veterinary recorded data, 'general condition' data were categorised into (normal) and (affected, including all records > ), 'dehydration' into (normal) and (dehydrated, including all records > ), and 'faecal consistency' into (normal, including records = sausage-like and = porridge-like), and (diarrhea, including records = gruel-like and = watery). these variables were investigated by logistic regression models. body condition score was investigated by general linear models (glm), i. e. assuming normally distributed errors. the variables 'oocyst concentration', i.e. opg, and 'oocyst level > , opg' were investigated by glm on log-transformed data and a logistic regression model, respectively. the second variable was created because it has been suggested that , opg might be a suitable cut off to distinguish clinical cryptosporidiosis and subclinical infection (operario et al., ) . differences between serum protein values in first and fourth visits and between herds, respectively, were tested in a two-way anova without random factors. information obtained by interviewing the owners of the four herds before the study started is presented in table . the calves were usually kept with their dam for up to h and then moved to individual pens or huts (two herds had pens, one had huts, and one had both pens and huts) where they were kept for two to four weeks until moved and mixed with other calves in group pens or huts. in all four herds the calf diarrhea problems had been ongoing for several years and the farmers estimated the morbidity among - day old calves to be - % over the last six months. caretakers gave colostrum manually in all herds except in herd four in which some of the calves suckled their first colostrum. in total, calves were born during the study period, of which were excluded either because the farmers' records were not complete or because the pen had not been kept empty for h before the calf was introduced. thus calves were finally included in the study. of these, and were kept in hydrated lime disinfected pens and control pens, respectively. the farmers' recordings are presented in table . overall, the farmers detected diarrhea in ( %) of the calves, with similar proportions in experimental and control pens. the mean age when diarrhea was first seen was . days (range - days) and it lasted for . days on average (median days). 'age at diarrhea onset' was significantly (p < . ) higher in experimental pens than in control pens, . days and . days, respectively,. there was no significant difference between calves kept in experimental and control pens for the variables 'presence of diarrhea', 'days with diarrhea', or 'severity' (table ; appendix table i) . veterinarians examined calves, i.e. all calves that were up to four weeks old at the time of the visits. there was no significant difference in the veterinary registrations regarding either 'general appearance', 'dehydration', or 'faecal consistency' between calves kept in experimental pens or control pens (appendix table ii ). body condition was scored for calves at two to four weeks of age and from calves at six to eight weeks of age. mean scores for calves from experimental and control pens respectively were . (range: . - . ) and . (range: . - . ) at two to four weeks and . (range: . - . ) and . (range: . - . ) at six to eight weeks. the six to eight week old calves kept in experimental pens had a significantly higher body condition score (p = . ) than calves in control pens, but the difference between the groups in the younger calves was not significant (appendix table iii ). faecal samples were collected from calves. cryptosporidium positive samples were found at all samplings in all four herds. cryptosporidium spp. was detected in ( %) samples and oocyst counts were - × opg, with a median of , opg in the positive samples. there was no difference between experimental or control pens in either the variables 'oocyst concentration' or 'oocyst level > , opg' (table ; appendix table iv) . when the cryptosporidium positive samples from the first and fourth samplings (n = ) were analysed by molecular biology methods, the species could be determined in samples. c. parvum was the dominant species, found in samples. c. bovis was found in four samples. two different c. parvum subtypes were found; iiaa g r b in three herds, and iiaa g r b_variant in one herd (table ). only one subtype was found in each herd. rotavirus was found in all the herds and in pools from both experimental and control calves. in total, pools ( from each herd) were analysed and rotavirus was found in ( %) of them. coronavirus was not found in any pool. the mean serum protein value was . g/l (range - g/l), with no significant differences between the first and fourth visits ( . and . , p = . ) or between herds (p = . ). in this study, performed in dairy herds with pervasive c. parvum-associated calf diarrhea problems, calves kept in the hydrated lime disinfected pens were older when diarrhea was first recorded and had a higher body condition score at six to eight weeks of age. no other significant differences between calves kept in the experimental and table background information collected by interviewing four dairy herd owners before the herds were enrolled in a study on the effect of hydrated lime on cryptosporidiosis. table i ). experimental = calves kept in individual pens that were cleaned, coated with hydrated lime and left empty for at least h; control = calves kept in individual pens that were cleaned and left empty for at least h. control pens were observed by farmers or veterinarians. in disinfected and control pens the mean age at diarrhea onset was . and . days, respectively. although the difference between the groups is small, it is clinically relevant and represents a significant improvement in herds with pervasive diarrhea problems. body condition score in calves is an easily accessible but rough estimate of body condition and was used as a measure of disease severity. the significantly higher body condition score in the calves in disinfected pens at - weeks of age indicates that the disease was less severe in these calves. that no difference was recorded at - weeks of age may be due to the fact that several calves got diarrhea at a late stage; the diarrhea onset varied from and days of age. c. parvum and rotavirus are the two predominate infectious causes of diarrhea in young calves in sweden, whereas coronavirus is found sporadically and e. coli f + is an uncommon pathogen (björkman et al., ; torsein et al., ) . all herds were suggested to the study by field veterinarians who had identified cryptosporidium spp. as the main pathogen associated with the diarrhea problems. when we sampled the four herds before including them in the study, we detected no rotavirus or coronavirus in any of them. all four herds did, however, show the presence of cryptosporidium spp. during the study, cryptosporidium spp. oocysts were found in a high proportion of the samples ( %) at all four sampling points for all the herds. rotavirus was also found, but only a one-third of the sample pools were positive. together, this suggest that c. parvum was the major pathogen, but that coinfection with rotavirus might have contributed to the diarrhea. lime has been shown to decrease the viability of oocysts of c. parvum (zintl et al., ) . however, we found no difference in oocyst shedding between calves kept in the experimental and control pens. this might be due to the study design, in which faecal samples were taken at veterinary visits scheduled every two months. this sampling schedule was therefore not optimised in relation to either diarrhea occurrence or potential oocyst excretion patterns. we also note that for practical reasons, the lime was mostly left to react for only h, whereas the shortest time used by zintl et al. ( ) was h. disinfection might have been more effective if the lime had been allowed to react for h in the study pens. the infection dose of c. parvum is very low; oocysts have been reported to induce clinical infections in neonatal calves (zambriski et al., ) . in problem herds we can suspect a particularly high infection load, which is a challenge to any disinfectants. furthermore, zintl et al. ( ) concluded that in order to avoid reinfection, disinfection would have to be repeated at times when the pens were unoccupied. in the present study, disinfection was repeated, and each pen was unoccupied when coated. however, the barns were not emptied and cleaned before the start of lime disinfection. lime disinfection was only added as a complement to the ordinary cleaning routines and was started during the housing season, when the infection load was already high. cleaning routines and the quality of the equipment in the calf barns varied among the experimental herds. for example, in one herd the pens were not cleaned with water between each calf, and in another herd the boards dividing the pens were worn and difficult to clean. it has been suggested that components in cattle faeces enhance the oocysts' resistance to environmental stress (robertson et al., ) , and remnants of faecal material might have impaired the effect of the lime. we could speculate that the disinfection might have been more effective if the barn had been vacated, thoroughly cleaned, and allowed to dry for several days before the start of lime disinfection, as described by matsuura et al. ( ) . lime has also been found to be effective in reducing rotavirus contamination (hansen et al., ) . in the present study, an inclusion criterion was that no rotavirus had been detected on the farm. however, the samplings taken during the course of the study revealed presence of this virus in all herds. the study design did not permit determination of the significance of this infection in the herds. the passive protection obtained by a high uptake of antibodies from maternal colostrum is essential to combating disease in young calves. total protein content in blood serum can be used to estimate colostrum uptake; concentrations below g/l are considered to indicate failure of passive transfer (tyler et al., ) . in contrast, higher concentrations are associated with improved calf health (waldner and rosengren, ; furman-fratczak et al., ) . although the mean serum protein value was above g/l at both serum samplings, all herds had individual calves with lower total protein values, and only of calves had igg values above g/l, indicating suboptimal colostrum feeding that may have rendered the calves particularly susceptible to cryptosporidial infections. in agreement with a previous study on calves from swedish herds with ongoing cryptosporidium-associated calf diarrhea problems , c. parvum was the dominant species, and c. bovis was only found in a few samples. however, in swedish herds without diarrhea problems, c. bovis is the major species seen in young calves (silverlås et al., ; silverlås and blanco-penedo, ). the c. parvum subtype iiaa g r b that was found in three of the herds is the most common subtype in sweden, both in cattle and in humans. it was, for example, the subtype involved in a recent outbreak among veterinary students in sweden (genbank: eu ) (kinross et al., ) . the iiaa g r b_variant is not as common and is slightly different from iiaa g r b: the tcg repeat is in a different position in the repeat region. it was first seen in sweden in when it was identified in children who had fallen sick after visiting dairy herds at public events held on the occasion of letting the cows out on pasture for the first time in spring (anonymous, ) . the sequence is published (genbank: kt . ). disinfection of calf pens with slaked lime delayed the onset of table presence of cryptosporidium spp. oocysts in faecal samples collected from to day old calves in four dairy herds participating in a study on the effect of hydrated lime on cryptosporidiosis. oocyst concentration > , opg . × ( . × - . × ) ( %) . × ( - . × ) ( %) . × ( . × - . × ) ( %) . × ( - . × ) ( %) . × ( . × - . × ) ( %) . × ( . × - . × ) ( %) ( - . × ) ( %) . × ( . × - . × ) ( %) . × a ( . × - . × ) ( %) a . × a ( - . × ) ( %) a experimental = calves kept in individual pens that were cleaned, coated with hydrated lime and left empty for at least h; control = calves kept in individual pens that were cleaned and left empty for at least h. oocysts/gram feces (opg): median (interquartile range). a = within each row, values are not significantly different (p = . ) in statistical analysis performed with generalised linear models (appendix table iv ). diarrhea and improved the body condition of the calves but did not affect diarrhea incidence or duration. although lime disinfection alone will not be sufficient to control cryptosporidium-associated diarrhea in herds with extensive calf diarrhea problems, these results suggest that it can be a valuable complement to other measures. declarations of interest: none utility of halofuginone lactate for the prevention of natural cryptosporidiosis of calves, in the presence of co-infection with rotavirus and salmonella typhimurium subgenotype analysis of cryptosporidium isolates from humans, cattle, and zoo ruminants in portugal practical guidelines on the use of lime for the prevention and control of avian influenza, foot and mouth disease and other infectious diseases cryptosporidiuminfektion under cryptosporidium parvum and giardia intestinalis in calf diarrhoea in sweden diagnostics of dairy and beef cattle diarrhea general biology the influence of colostral immunoglobulin concentration in heifer calves' serum on their health and growth retrospective cohort study of an outbreak of cryptosporidiosis caused by a rare cryptosporidium parvum subgenotype inactivation of adenovirus type , rotavirus wa and male specific coliphage (ms ) in biosolids by lime stabilization cryptosporidium parvum infections in a cohort of veterinary students in sweden evaluation of free-stall mattress bedding treatments to reduce mastitis bacterial growth report of fatal mixed infection with cryptosporidium parvum and giardia intestinalis in neonatal calves evaluation of a protocol to reduce the incidence of neonatal calf diarrhoea on dairy herds duration of naturally acquired giardiosis and cryptosporidiosis in dairy calves and their association with diarrhea correlation between diarrhea severity and oocyst count via quantitative pcr or fluorescence microscopy in experimental cryptosporidiosis in calves molecular characterization of cryptosporidium isolates from pre-weaned calves in western france in relation to age a small outbreak of human cryptosporidiosis associated with calves at a dairy farm in norway. scand cryptosporidiosis in farmed animals survival of cryptosporidium parvum oocysts under various environmental pressures prevalence and age-related variation of cryptosporidium species and genotypes in dairy calves systematic review and meta-analyses of the effects of halofuginone against calf cryptosporidiosis cryptosporidium spp. in calves and cows from organic and conventional dairy herds is there a need for improved cryptosporidium diagnostics in swedish calves? prevalence and associated management factors of cryptosporidium shedding in swedish dairy herds molecular characterisation of cryptosporidium isolates from swedish dairy cattle in relation to age, diarrhoea and region bovine cryptosporidiosis: impact, host-parasite interaction and control strategies risk factors for calf mortality in large swedish dairy herds detection of low serum immunoglobulin concentrations in clinically ill calves prevalence of cryptosporidium parvum infection and pattern of oocyst shedding in calves in japan factors associated with serum immunoglobulin levels in beef calves from alberta and saskatchewan and association between passive transfer and health outcomes characteristics of cryptosporidium transmission in preweaned dairy cattle in henan cryptosporidium parvum: determination of id and the dose-response relationship in experimentally challenged dairy calves survival of cryptosporidium parvum oocysts in the presence of hydrated lime the study was funded by swedish farmers´foundation for agricultural research (grant number v ). we thank helena bosaeus-reineck and harri ahola for valuable help in the laboratory. we are grateful to the owners and staff of participating herds. veterinarians gunilla blomqvist and ylva ståhl at växa sverige are acknowledged for their contribution in collecting samples and examining calves. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.vetpar. . . . key: cord- -s xmdmpf authors: lorenz, ingrid; fagan, john; more, simon j title: calf health from birth to weaning. ii. management of diarrhoea in pre-weaned calves date: - - journal: ir vet j doi: . / - - - sha: doc_id: cord_uid: s xmdmpf calfhood diseases have a major impact on the economic viability of cattle operations. the second of this three part review series considers the management of diarrhoeic diseases in pre-weaned calves. in neonatal calf diarrhoea, oral rehydration therapy is the single most important therapeutic measure to be carried out by the farmer and is usually successful if instigated immediately after diarrhoea has developed. continued feeding of milk or milk replacer to diarrhoeic calves is important, to prevent malnourishment and weight loss in affected calves. indiscriminative antibiotic treatment of uncomplicated diarrhoea is discouraged, whereas systemically ill calves can benefit from systemic antibiotic treatment for the prevention of septicaemia or concurrent diseases. ancillary treatments and specific preventive measures are discussed. eimeriosis has a high economic impact on the farming industries due to direct cost of treatment and calf losses, but especially due to decreased performance of clinically as well as sub-clinically affected animals. emphasis lies on prophylactic or metaphylactic treatment, since the degree of damage to the intestinal mucosa once diarrhoea has developed, makes therapeutic intervention unrewarding. calfhood diseases have a major impact on the economic viability of cattle operations, due to the direct costs of calf losses and treatment and the long term effects on performance [ ] . further, calf health was prioritised as one of the most important animal health issues facing the irish livestock industry in a recent expert policy delphi study conducted on behalf of animal health ireland (ahi) [ ] . as part of ongoing ahi work, a group of experts was commissioned to provide evidence-based advice on calf health and disease management to irish farmers, agricultural advisers and veterinary practitioners. as an initial step, a three-part review series on calf health from birth to weaning has been generated, specifically to provide a scientific evidence base for the development of advisory tools on calf health, and to identify gaps in current knowledge to be filled with targeted research. even though the envisaged output will be specific for irish husbandry systems, the scope of the reviews should make them useful for the same purpose elsewhere. the reviews cover both suckler and dairy calf management. however, due to the differences in the nature of these systems, some topics will deal mainly or exclusively with either dairy or suckler calves. neonatal calf diarrhoea is recognised worldwide as one of the biggest challenges for both the beef and dairy industries. about one third of us beef cow-calf owners agree that it has an economic impact on their operations [ ] and it has constantly accounted for more than % of unweaned dairy heifer deaths since [ ] . in ireland, diarrhoea is the most common cause of death in calves from birth to one month of age submitted for post mortem examination (regional veterinary laboratories -surveillance report ). disease prevention, though preferable, is not always possible in intensive calf rearing systems. appropriate calf management, once diarrhoea has developed, is crucial to avoid further economic losses, animal welfare impact and farmer distress. the second part of this three part review series concentrates on the management of diarrhoea in pre-weaned calves. the first and third parts focus on general aspects of disease prevention in pre-weaned calves [ ] and disease prevention and management with particular reference to calf pneumonia [ ] , respectively. enterotoxic escherichia coli, cryptosporidium parvum, rotavirus and coronavirus are usually seen as the most common infectious causes of neonatal calf diarrhoea [ ] . these infectious agents can also be found in faecal samples from healthy calves and in calves from farms without diarrhoea problem [ ] [ ] [ ] [ ] . clinical disease develops due to an unfavourable relation between the resistance of the calf and the infectious pressure. the main management factors with impact on the resistance of the calf are calving management to prevent dystocia, timely provision of adequate amounts of colostrum and appropriate diet thereafter, as previously discussed [ ] . the infectious pressure can be lowered through general hygiene in the areas of calving, feeding, housing and in general calf handling. enterotoxic e. coli usually only cause secretory diarrhoea in the first four days of life. the other common infectious agents involved in neonatal calf diarrhoea cause damage to the intestinal mucosa resulting in mixed malabsorptive and secretory diarrhoea. even if therapy against the causal pathogens was available this pathophysiological mechanism would make it unlikely that the duration of diarrhoea could be significantly influenced [ ] . for this reason replacement of fluid and electrolyte losses remains the single most important treatment measure in uncomplicated calf diarrhoea. oral rehydration therapy, originally developed in human medicine for the treatment of cholera, is generally recognised as one of the most significant medical advances of the th century [ ] . the general requirements for an efficient oral rehydration solution (ors) are that it should be efficiently absorbed, normalise the extracellular fluid volume and correct acidosis [ ] . there are several factors to consider while choosing an appropriate ors. since sodium is the osmotic skeleton of the extracellular fluid, it must be present in adequate concentration in ors. a study comparing three ors with different sodium concentrations in calves found that the solution with a sodium content of mmol/l corrected dehydration, whereas solutions with much lower concentrations did not [ ] . even though there is little evidence that solutions with sodium concentrations > mmol/l are harmful, it is generally suggested that the sodium concentration for ors should be between and mmol/l [ ] . the critical scientific step facilitating the development of oral rehydration therapy was the discovery of the coupled transport of sodium and glucose [ ] . besides glucose, neutral amino acids (e.g. glycine or glutamine) and volatile fatty acids (e.g. acetate or propionate) have been shown to enhance sodium absorption in the intestines [ , ] . glucose-tosodium ratios of : to : have been recommended [ , ] . metabolic acidosis is known as a frequent and potentially severe complication of neonatal calf diarrhoea. diarrhoea leads to loss of bicarbonate via the faeces, decrease of glomerular filtration of hydrogen ions and accumulation of l-lactate in case of severe dehydration. in addition, the production and absorption of d-lactic acid plays a major role in diarrhoeic calves [ ] . this pathophysiological abnormality appears to be more common in ruminants than in other domestic species or in infants [ ] [ ] [ ] [ ] [ ] , which suggests that newborn ruminants are more prone to developing severe metabolic acidosis than infants during diarrhoea. this is most probably the reason why the current recommendation on the alkalinising capacity for ors used in calves ( - mmol/l [ ] ) is considerably higher than that of the current who-ors formula ( mmol/l [ ] . alkalinising agents commonly used in commercial ors are bicarbonate and bicarbonate precursors, mainly acetate and propionate. bicarbonate alkalinises the abomasum to a higher degree than propionate and acetate, thus lowering the non-specific resistance of the calves against bacterial infection [ , ] . the impact of bicarbonate in ors on milk clotting and calf performance is controversial [ ] [ ] [ ] [ ] [ ] . however, in the absence of contrary evidence, it seems reasonable to avoid the use of bicarbonate-containing ors less than to hours following milk feeding. as in humans, the purpose of ors in diarrhoeic calves is to replace electrolytes and fluids that are lost via the intestines. therefore, ors should be given to calves as an extra feed (that is, in addition to each normal milk meal) as soon as diarrhoea is observed [ ] . the efficiency of this measure relies on early detection, through thorough observation, of diarrhoeic calves. oesophageal intubation of ors produces similar, albeit slightly delayed, resuscitative effects compared to ors that is suckled. therefore, oesophageal intubation is recommended for calves that are either anorexic or otherwise not likely to drink from an artificial teat [ ] . milk should not be force-fed to calves that are depressed and not interested in drinking. force-feeding always leads to dysfunction of the oesophageal groove, so that milk fermented in the reticulorumen can further contribute to metabolic acidosis [ ] . even though there is little experimental evidence about limits for oral rehydration therapy, it is generally accepted that intravenous fluid therapy is indicated in severely depressed, recumbent, severely dehydrated (> %) and prolonged anorexic (> h) calves. the principles and techniques of intravenous fluid therapy in calves have recently been reviewed [ ] . it has traditionally been recommended that milk feeding is withdrawn from diarrhoeic calves, either for a defined period of time or for as long as diarrhoea persists [ ] . however, no scientific evidence is available to suggest that starvation of diarrhoeic calves leads to improved clinical outcomes. indeed, it is now recognised that milk feeding does not worsen or prolong the course of diarrhoea, despite a somewhat lowered digestive capacity. rather, withdrawal of milk rapidly results in malnourishment and weight loss [ , ] . continued milk feeding not only provides the energy required for weight gain and growth throughout the period of diarrhoea, but also provides the nutrients that are necessary for the recovery of the intestinal mucosa [ ] . a similar scientific evolution in human medicine led to the inclusion of continued feeding into the standard management protocols for diarrhoea by the who in [ ] . the reluctance of veterinarians and farmers to adopt the principle of continued feeding of the diarrhoeic calf led to diverging developments in osmolality of ors for calves and humans. commercially available ors for calves range from isotonic to highly hypertonic, whereas who recently changed their recommendation towards a hypotonic formula [ ] . higher osmolality ors for calves is a reflection of a higher concentration of glucose which is added to provide additional nutritional support. nonetheless, the provision of high-energy ors cannot prevent negative energy balance in calves [ ] and hypertonic solutions are known to slow abomasal emptying rates compared with isotonic solutions, thereby delaying plasma volume expansion [ ] . there is increasing pressure on the veterinary profession to promote prudent use of antibiotics, noting that indiscriminate use of antibiotics promotes the selection and subsequent proliferation of antibiotic-resistant strains of bacteria [ ] . in this context, the benefit of antimicrobial treatment in neonatal calf diarrhoea has been reviewed by constable [ ] , who came to the following conclusions: • routine use of oral or injectable antibiotics cannot be recommended in calves without systemic illness. • in calves with diarrhoea and systemic involvement (marked depression, anorexia, fever), the risk of bacteraemia or septicaemia as well as bacterial overgrowth of the small intestine is increased. in such circumstances, administration of broad-spectrum beta-lactam antimicrobials (ceftiofur, amoxicillin or ampicillin), potentiated sulphonamides, or fluoroquinolones (where permitted) is recommended. • susceptibility tests of bacteria cultured from faecal samples do not reliably predict treatment outcomes in diarrhoeic calves [ ] . non-steroidal anti-inflammatory drug therapy with meloxicam has proven effective in improving food intake and weight gain in diarrhoeic calves [ , ] . flunixin meglumine showed some beneficial effect in experimental calves orally challenged with heat-stable escherichia coli enterotoxin [ ] and in calves with naturally acquired bloody diarrhoea, but not in calves without faecal blood [ ] . additional ancillary treatment has been either suggested or used to treat diarrhoeic calves in the past, but is now either contraindicated (glucocorticoids, motility modifiers) or cannot be supported due to insufficient evidence of efficacy (intestinal protectants, probiotics) [ ] . for the management of the individual diarrhoeic calf, the knowledge of infectious agents involved is of little value. if specific preventive measures are considered, faecal samples from untreated calves early in the course of clinical disease can be submitted for laboratory analyses [ ] . care has to be taken with the interpretation of results, since the enteropathogens most commonly implicated in calf diarrhoea outbreaks (rotavirus, coronavirus, pathogenic strains of escherichia coli, cryptosporidia, salmonella spp.) can also be found in faecal samples from healthy calves and in calves from farms without diarrhoea problem [ ] [ ] [ ] [ ] . postmortem examination of calves dying or euthanased in the acute stage of disease can be beneficial especially for the diagnosis of outbreaks of salmonellosis [ ] . rotavirus and cryptosporidia are the most frequently identified infectious agents in faecal samples from diarrhoeic calves in ireland [ ] and elsewhere [ ] . vaccination is the only prophylactic measure available against rotavirus infection. vaccination of the dam before calving has been used to enhance the content of rotavirus specific antibodies in colostrum [ ] . commercially available vaccines usually also contain coronavirus and e. coli f antigen. there is no doubt that modern vaccines are able to increase the level of specific antibodies in serum and milk of vaccinated cows as well as in serum of calves that have ingested colostrum from vaccinated dams [ ] [ ] [ ] . however, evidence as to clinical efficacy in naturally acquired diarrhoea is either not available for all vaccines or is conflicting. using the same vaccine, lerousic et al. [ ] found a reduction in severity of diarrhoea in calves born from vaccinated cows, whereas kohara et al. [ ] failed to find any clinical effect. in either case, available data are not sufficient to assess the economic benefit associated with vaccine usage. oral vaccination of newborn calves is not effective [ ] . in herds endemically infected with salmonella spp., vaccination of the dams prior to calving can be considered [ ] . halofuginone has a demonstrated cryptosporidiostatic effect and is licensed for prevention and treatment of cryptosporidiosis in calves. in a recent review, this substance was found to be beneficial for prophylactic use in cases with severe cryptosporidium-associated diarrhoea. however, data were insufficient to evaluate therapeutic efficacy [ ] . in a study on an irish farm, halofuginone was effective in reducing clinical signs of cryptosporidiosis and environmental contamination [ ] . however, it did not delay the onset of diarrhoea or reduce the risk of infection in group-housed calves. the use of halofuginone should be combined with hygienic measures and improvement of the husbandry management system. infection with eimeria spp. has a high prevalence in cattle, especially calves and yearlings [ ] . clinical coccidiosis is most often caused by infection with e. zuernii or e. bovis, generally linked with conditions of high infectious pressure [ , ] . in contrast, infection with other eimeria spp., as well as more pathogenic species but under conditions of low infectious pressure, lead to subclinical coccidiosis [ ] . e. alabamensis has been reported in outbreaks of watery diarrhoea in calves on pasture in northern europe [ , ] . the economic impact of clinical, but also subclinical, coccidiosis on the farming industry is considerable, due to both the cost of treatment and impaired performance of affected animals [ ] . the general conditions of animal husbandry discussed previously (e.g. housing hygiene, ventilation, immunocompromising factors) each contribute to infection risk and should be critically assessed during coccidiosis outbreaks [ ] . individual animal testing is of limited value because eimeria spp. are frequently found in the faeces of healthy calves [ , ] . to relate clinical observations of diarrhoea to infection with eimeria spp., it is recommended that faecal samples are collected and examined from several animals in a clinically affected group, followed by differentiation of oocysts to the species level [ ] . in clinical coccidiosis, the development of diarrhoea is caused by the late stages of the life cycle (second merogony and especially gamogony [ ] . therapeutic intervention at this stage is of limited value and therefore emphasis should be given to metaphylactic treatment in outbreak situations or prophylactic treatment of at-risk groups [ , ] . diarrhoea is generally the most common cause of morbidity and mortality in pre-weaned calves. a range of measures are critical to disease prevention, including colostrum management and subsequent nutrition (lorenz [ ] et al., b). oral rehydration therapy, continued milk feeding and prudent use of antibiotics are each important in the successful management of neonatal calf diarrhoea. vaccines for neonatal calf diarrhoea are available, however, efficacy reports are variable and data on economic benefit are lacking. none of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. the technical working group includes employees of pfizer inc. (cc) and volac ireland (lg); these companies played no role in the design, development or journal submission of this review series. calf and disease factors affecting growth in female holstein calves in florida, usa setting priorities for non-regulatory animal health in ireland: results from an expert policy delphi study and a farmer priority identification survey usda: beef - , part iv: reference of beef cow-calf management practices in the united states usda: dairy calf health from birth to weaning. i. general aspects of disease prevention calf health from birth to weaning. iii. disease prevention and management with particular reference to calf pneumonia pathophysiology of diarrhea in calves microbiology of calf diarrhoea in southern britain enteropathogens and risk factors for diarrhea in norwegian dairy calves prevalence, prediction and risk factors of enteropathogens in normal and nonnormal faeces of young dutch dairy calves cryptosporidium infection in herds with and without calf diarrhoeal problems reducing deaths from diarrhoea through oral rehydration therapy why has oral rehydration for calves and children diverged: direct vs. indirect criteria of efficacy the comparative effectiveness of three commercial oral solutions in correcting fluid, electrolyte and acid-base disturbances caused by calf diarrhoea treatment of calf diarrhea: oral fluid therapy oral therapy for acute diarrhea. the underused simple solution effects of sugars and amino acids on sodium movement across small intestine effect of acetate or chloride anions on intestinal absorption of water and solutes in the calf who: oral rehydration salts. production of the new ors. who (world health organization) document production services influence of d-lactate on metabolic acidosis and on prognosis in neonatal calves with diarrhoea d-lactic acidosis in lambs d-lactic acidosis secondary to exocrine pancreatic insufficiency in a cat serum d-lactic concentrations in cats with gastrointestinal disease floppy kid syndrome caused by d-lactic acidosis in goat kids effect of suckling an isotonic solution of sodium acetate, sodium bicarbonate, or sodium chloride on abomasal emptying rate and luminal ph in calves influence of different oral rehydration solutions on abomasal conditions and the acid-base status of suckling calves the effects of feeding milk to diarrheic calves supplemented with oral electrolytes skilnick p: a comparison of three oral electrolyte solutions in the treatment of diarrheic calves effects of electrolyte solutions for oral administration on clotting of milk comparative effects of two oral rehydration solutions on milk clotting, abomasal luminal ph, and abomasal emptying rate in suckling calves comparison of two oral electrolyte solutions and route of administration on the abomasal emptying rate of holstein-friesian calves treatment of calf diarrhea: intravenous fluid therapy a clinical evaluation of antimicrobial agents and temporary starvation in the treatment of acute undifferentiated diarrhea in newborn calves whole milk and oral rehydration solution for calves with diarrhea of spontaneous origin comparison of two oral electrolyte solutions for the treatment of dehydrated calves with experimentallyinduced diarrhoea why is anti-microbial resistance a veterinary problem as well? antimicrobial use in the treatment of calf diarrhea efficacy of meloxicam (metacam) as adjunct to a basic therapy for the treatment of diarrhoea in calves nonsteroidal anti-inflammatory drug therapy for neonatal calf diarrhea complex: effects on calf performance effect of flunixin meglumine on escherichia coli heat-stable enterotoxin-induced diarrhea in calves evaluation of flunixin meglumine as an adjunct treatment for diarrhea in dairy calves treatment of calf diarrhea: antimicrobial and ancillary treatments factors that may affect the occurrence of enteropathogens in the faeces of diarrhoeic calves in ireland salmonella in calves prevalence of four enteropathogens in the faeces of young diarrhoeic dairy calves in switzerland group a rotavirus veterinary vaccines enhancement of passive immunity with maternal vaccine against newborn calf diarrhea serological, colostral and milk responses of cows vaccinated with a single dose of a combined vaccine against rotavirus, coronavirus and escherichia coli f (k ) comparative lactogenic antibody responses of cattle from european field trials with a new enteric disease vaccine use of colostrum from rotavirus-immunised cows as a single feed to prevent rotavirus-induced diarrhoea in calves field trial evaluation of a reocoronavirus calf diarrhea vaccine calf salmonellosis: prophylaxis by maternal immunization systematic review and meta-analyses of the effects of halofuginone against calf cryptosporidiosis control of cryptosporidiosis in neonatal calves: use of halofuginone lactate in two different calf rearing systems an observational study of eimeria species in housed cattle on dutch dairy farms some pathophysiological changes associated with infection of eimeria zuernii in calves experimentelle eimeria bovis infektionen beim kalb: . parasitologische und klinische befunde zur epizootologie der eimeria-alabamensis-kokzidiose bei jungrindern. monatsh veterinärmed eimeria alabamensis infection as a cause of diarrhoea in calves at pasture the economic impact of coccidiosis in domestic animals eimeriosis in cattle: current understanding experimental eimeria bovis infection in calves: a histopathological study pathology and treatment of eimeria zuernii coccidiosis in calves: investigations in an infection model the effect of a metaphylactic treatment with diclazuril (vecoxan) on the oocyst excretion and growth performance of calves exposed to a natural eimeria infection submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution the authors from animal health ireland's technical working group (twg) on calf health would like to thank the remaining colleagues of the twg for their valuable input authors' contributions il drafted the manuscript and compiled the literature. all authors made substantial inputs to the review, critically discussed the progressing manuscript and approved the final manuscript. key: cord- -cyul w authors: mcguirk, sheila m. title: disease management of dairy calves and heifers date: - - journal: veterinary clinics of north america: food animal practice doi: . /j.cvfa. . . sha: doc_id: cord_uid: cyul w this article focuses on the most important diseases of dairy calves and heifers and presents clinical approaches that can improve detection, diagnosis, and treatment of herd-based problems. a systematic herd investigation strategy is pivotal to define the problems, understand important risk factors, develop a plan, and make recommendations for disease management accurately. a review of records, colostrum and feeding routines, housing and bedding management, routine procedures, vaccination, and treatment protocols begins the investigation and determines which diagnostic procedures and testing strategies are most useful. disease management is most effective when the problem source is well defined and the exposure can be limited, calf immunity can be enhanced, or a combination of both. screening examinations performed regularly or done at strategic time points improves detection of disease, can be used to monitor treatment outcomes, and can avoid disease outbreaks. analysis are used to define problems, sources of infection, opportunities for improving resistance, disease detection, and prevention. the investigation of herd-based calf diarrhea begins with an accurate understanding of the age of onset, morbidity, and mortality data. for an endemic herd problem, it is optimal to review months of retrospective data. the minimum database includes the total number of calves born; the number of heifer calves alive at or hours (depending on when they leave the calving pen); the number affected; primary age-group affected; treatment history; and the mortality rate. for calf enteritis outbreaks, it is useful to see at least months of similar data. calf records may not be kept or may provide minimal information but a review of the adult cow records can provide enough information to calculate calf mortality rate. prospective record keeping may be necessary and forms that are simple and useful (table ) can be provided to the dairy before the investigation. a verbal clinical history is necessary and important but the scope, which is frequently dominated by the most recent cases, requires some validation from a minimum of months of records. other records of potential importance to the investigation are laboratory results from calf fecal specimens, blood cultures, tissue specimens, or postmortem examinations. most calf diarrhea problems are caused by a combination of factors, not all of which are infectious. the purpose of the herd investigation is to elucidate the potential enteric pathogens and to focus on the environment, calf immune status, nutrition, and management to define other contributing factors. colostral immunity is an essential part of enteric disease management and is discussed elsewhere in this issue. most calf diarrhea herd problems are caused by mixed infections [ ] and the agents may change over time, depending on season of the year and population dynamics within the environmental site of exposure. by analyzing the fecal shedding patterns of calves in the affected age groups, potential pathogens can be identified. knowledge of the agents can better define sources or sites of exposure, can result in the development of more effective treatment plans, and can result in more specific preventive recommendations. to determine the potential enteric pathogens to which calves have been exposed, fecal specimens are obtained from untreated calves within the affected age group. a good clinical history and calf health records provide the initial evidence for the age group of calves from which fecal specimens are obtained. the population at-risk can be confirmed by identifying the age of calves being treated for diarrhea on the day of the farm investigation. unless, the age-of-onset of diarrhea is in calves less than days of age, diagnostic tests for enterotoxigenic escherichia coli are not performed. for calf diarrhea problems with age-of-onset between and days, the age group most commonly affected in most calf diarrhea investigations, samples are submitted for rotavirus, coronavirus, salmonella spp, and cryptosporidium parvum. diarrhea problems in calves older that days or in weaned heifers may include diagnostic tests for attaching and effacing e coli, salmonella spp, eimeria spp, and giardia spp. fecal samples are obtained from a minimum of six calves by inserting a gloved finger into the rectum carefully to extract feces that are present or by gently massaging the rectal lining. most calves defecate with the stimulation and the feces can be collected into a -oz specimen cup. one should remove gloves before sampling the next calf, clean the outside surface, and seal the cap of the specimen cup. four cotton swabs can be used to obtain a rectal smear from calves that do not produce manure. the calf identification, age or birth date, and fecal consistency score are recorded as shown in table . sample handling should follow the directions of the diagnostic laboratory receiving the samples, but within minutes of collection it is best to place feces for salmonella culture into transport or selective salmonella media like tetrathionate, selenite, or both. we bring media to the farm and inoculate each with where fecal score is ¼ normal consistency; ¼ semiformed or pasty; ¼ loose but enough consistency to remain on bedding; ¼ watery feces that sift through bedding material; b ¼ blood is present. a -to -g (pea-size) portion of fresh feces. c parvum and viral samples can be prepared according to the laboratory's specifications. for acid-fast stained smears, a dry cotton swab is dipped into the fecal specimen and then used to make a thin fecal smear on each of two glass slides that are appropriately labeled and submitted for c parvum. the remainder of the fecal sample is submitted for rotavirus and coronavirus testing. from expected level of exposure to potential fecal pathogens in the environment [ ] [ ] [ ] , it is anticipated that up to % of the calves sampled may be shedding rotavirus, coronavirus, or c parvum. fecal shedding of virus in orally vaccinated calves is uncommon [ ] , so when two or more of six calves sampled are positive for the enteric viruses or c parvum or if any calf is salmonella spp positive, the exposure is considered abnormally high. enteric pathogens revealed in fecal shedding profiles can be validated as the cause of herd diarrhea problems when intestinal microscopic lesions described in postmortem specimens are consistent with the pathogens present in the feces. as shown in table , fecal shedding results show evidence of increased exposure to c parvum and salmonella newport in a herd with calf diarrhea problems in -day-old calves. previous postmortem examinations from the herd had demonstrated some intestinal villus blunting and clumping, consistent with c parvum infection, but the fibrino-purulent and necrotizing enteritis from three recently examined dead calves had not been linked to a salmonella spp isolate. with abnormal fecal shedding patterns present, locating the source of infection is important and provides incentive to find solutions that bypass, dilute, or distance calves from that site. finding the sources of infection for a dairy herd with calf diarrhea must take into account the health status of the dam, routes of infection of the potential pathogens, the traffic pattern of the calves, incubation period of the potential pathogens, and the behavior of calves. sick and bacteremic calving cows are more likely to have septicemic calves than calves with enteritis. salmonella dublin carrier cows, whether or not they have clinical manifestations, are a high risk for colostrum transmission of the disease to their calves, in which clinical disease is most common between weeks and months [ ] but can also occur at an earlier age. most enteric pathogen transmission between the dam and calf, however, occurs by fecal-oral spread by colostrum or the environment. even healthy cows have a large increase in fecal coliform bacterial counts during the periparturient period [ ] , putting the calves that commingle with cows in the calving environment at much higher risk for enteric infection. fecal-oral transmission of enteric pathogens to calves can occur by contaminated bedding; commingled animals; pets; pests; colostrums; feeds; feeding utensils; esophageal feeders; or the hands, boots, or clothing of calf caregivers. salivary secretions from sick calves that reach the mouth of susceptible calves can transmit salmonella spp and other enteric pathogens, making the disposal of refused milk, water, and feed away from the calf environment an essential aspect of disease management. esophageal feeders, balling guns, clothing, and hands can facilitate salivary-oral transmission in a dairy herd experiencing calf diarrhea problems and must be cleaned and disinfected between successive calf uses. as the newborn calf moves from the birthing pen to its final preweaning home, every place of short-term occupancy is regarded as a potential source of infection. from the maternity pen to the warming or drying area, to the transport vehicle or temporary hutch, all of the environments should be evaluated for enteric pathogens. considering the percentage of time that calves most at risk for enteric infections spend lying [ ] , the most likely environmental source of an enteric infection is the bedding. qualitative assessments of bedding cleanliness are subjective, unreliable, difficult to communicate, and easy to dismiss. objective data that can be reported and compared with appropriate benchmarks provide motivation for change. the concept of quantifying bedding bacteria as a risk assessment tool for mastitis [ , ] is well established, whereas the interest in identifying specific bacteria like multidrug-resistant salmonella in bedding material is more recent [ ] but is useful for locating an environmental site of infection. an evaluation of different calf bedding materials [ ] also demonstrates the use of this approach to assessing disease risks in the environment where calves spend most of their time. for herd problems of enteric disease in calves, bedding materials from each environment that has housed calves are submitted both for bacterial counts (university of minnesota laboratory for udder health, minnesota veterinary diagnostic laboratory, st. paul, mn) and salmonella spp culture. samples are taken with gloved hands from the perimeter of the pen in each of the four quadrants and from the center of the pen, specifically avoiding sampling fecal material. samples for bacterial count are collected in a sealable storage bag and stored in the refrigerator overnight before shipment to the laboratory. bedding samples for salmonella culture are placed in room temperature buffered peptone water pre-enrichment media, which is sealed tightly and shipped overnight to the laboratory in biohazard bag containers. bedding sample results from a dairy with diarrhea that starts in -day old calves is shown in table . on that dairy, calves leave the maternity pen, move to a currently unoccupied maternity pen hutch, from which location they are taken by truck to a second farm, where they are placed in a clean hutch. because of a -to -day incubation period for the fecal pathogens identified in the calves, bedding from a -day occupied hutch is also sampled. from the data in table , the bedding from the unoccupied hutch in the maternity pen and the truck are the most likely sources of infection for the calves on that dairy. for the enteric pathogens of most concern to calves, the incubation periods range from hours to days. when a herd diarrhea problem affects calves within the first days of life, the source of infection is usually encountered before the calf reaches its final preweaning pen. alternatively, the source of infection for diarrhea that begins after days of age is usually found in the calf housing area. the optimal bedding material for calves depends on age of the calf, temperature, cost, bedding, season, and management. when granite fines, sand, rice hulls, long wheat straw, and wood shavings were compared [ ] , performance indices were similar under the moderate temperatures of the study period but calves on sand and granite fines had more scours. adding clean, dry bedding to maintain a minimum of in between the calf and the base of the pen and the removal of all feed refusals from the calf housing area are two very effective ways to dilute and distance calves from potential enteric pathogens. continuous occupancy of calf raising facilities is a major risk factor that increases both the number and survival time of enteric pathogens in the environment. a goal of having % more calf pens than calves at maximum occupancy [ ] provides time for cleaning, sanitizing, and resting pens between successive occupants. strategic filling of calf raising facilities to empty large areas of the barn, rather than a single row at a time, can reduce endemic enteric disease of calves. disinfection protocols are useful if prior cleaning of facilities and pens has removed all organic debris; if the disinfectant is effective for the agents encountered in that facility; and if contact application, time, and surface are as specified. safe, broad-spectrum disinfectants that can be used in housing facilities, have penetration into soil or porous surfaces, can be cross-protective for boots, and can be applied in novel ways lead to greater compliance and improved calf disease management [ ] [ ] [ ] . in addition to bedding contamination, other sources of enteric disease pathogens for calves are feeds, feeding equipment, pathogens on the skin, and the pen itself. colostrum bacterial contamination is discussed elsewhere in this issue. milk replacer and pasteurized milk have a low risk for bacterial contamination, especially fecal coliform bacteria, when there is proper mixing, storage, delivery, and feeding with clean equipment. unpasteurized whole milk can present a high risk for enteric infection when it is nonsaleable milk that, if not fed immediately, has not chilled. to determine the level of risk coming from the liquid feed, a bulk tank milk culture can be performed. of most interest in reviewing the culture results is the total bacterial count and the lactose-positive (fecal) coliform count. table shows goals and the ranges in milk and milk replacer bacterial numbers from bucket . culture swabs are used to assess cleanliness of feeding equipment, esophageal feeders, nipples, feeding bottles, and buckets for potential enteric pathogens. only lactosepositive coliform growth is reported as evidence of inadequate sanitizing procedures. self-grooming, a normal behavior of calves, can introduce enteric pathogens from the skin of calves. although this is not considered a major risk factor for transmission of enteric disease, commingled calves, calves with contact across open pen dividers, or calves housed in barns that are power-washed while still occupied may be at risk. aerosolized bacterial spread of enteric pathogens, although possible [ ] , is rarely the primary source of enteric disease in calves. a review of current vaccination, routine health management, and treatment protocols is an important part of enteric disease management in calves. colostrum management, as discussed elsewhere in this issue, is the most effective way to transfer immunity to the specific enteric pathogens, enterotoxigenic e coli, coronavirus, rotavirus, and clostridium perfringens types c and d from vaccinated cows to newborn calves. similarly, vaccinated cows may transfer the benefit of gram-negative core antigen vaccine and siderophore receptor porin s newport vaccine immunity to calves. because most calf diarrhea problems occur within the first weeks of life, immune colostrum may be the only way effectively to protect young calves. the vaccines labeled for administration to the young calf to aid in preventing diarrheal diseases are limited and, with one exception (entervene-d, fort dodge, fort dodge, iowa), are administered at birth. although many extralabel protocols attempt to improve the immunity of colostrum-deprived or susceptible calves against diarrheal diseases, there is little scientific basis for safety, efficacy, or disease protection. where the veterinarian investigating calf diarrhea problems can be influential is in eliminating practices that have potential to do harm or that make calves more susceptible to disease. avoid gram-negative bacterial vaccines not labeled for young calves. do not routine medications, feed additives, and well calf treatments should be reviewed closely in calf herds with enteric disease problems. individually, feed additives like immunoglobulins, mannan oligosaccharides, coccidostats, antibiotics, direct-fed microbials, immune modulators, charcoal, amino acids, and other ingredients may benefit calves, but unrestrained combinations, concentrations, and packaged remedies may change intestinal flora, transport time, digestibility, absorption, and intestinal health of calves. simplicity and consistency is a good starting point for most calf health programs. the treatment protocol for calf diarrhea is based on early and effective detection followed by appropriate intervention. calves with diarrhea (fecal score or , with or without blood as described in table ) should be identified and currently on a treatment protocol. as part of a calf diarrhea investigation, determine the disease detection rate by dividing the number of calves currently being treated for diarrhea by the number of calves with fecal scores (loose but enough consistency to remain on bedding) and (watery feces that sift through bedding material). the goal of an % or greater detection rate can be achieved by twice weekly fecal scoring of all calves weeks of age or less. in table , the detection of one of four calves with diarrhea is below expectations. diarrhea treatment protocols for farm use must be straightforward and trainable. it should penalize neither the calf nor the person administering treatments. for compliance, it must be effective, frequently monitored, and updated. the most important component of the treatment protocol is rehydration, and intravenous and oral fluid and electrolyte therapy of calves have been reviewed [ , ] . feeding calves through the course of diarrhea maintains caloric intake and adds fluid volume and electrolytes to supplemental fluid administration. continued feeding may facilitate the induction of digestive enzymes but may not be beneficial if force-feeding is required [ ] . therapeutic antibiotics are recommended for calves with diarrhea that have signs of systemic illness [ , ] . for the herd calf diarrhea protocol, criteria for treatment is clearly established as any calf with a fecal score or as described in table . if the examination of the calf reveals blood in the feces, a temperature greater than or equal to f, or the calf is dull, depressed, or off feed a -day course of antibiotics is started. if the diarrheic calf has no signs of systemic illness, fluid therapy is the basis of the treatment protocol. to encourage voluntary suckling, dividing the normal feeding volume into three or four smaller volume feedings may be better tolerated by sick calves. milk or milk replacer is not given to diarrheic calves with a distended abdomen or to one that is down and cannot be assisted to maintain sternal recumbency. either a veterinarian is called or the recumbent calf is given intravenous fluid therapy. in addition to feeding milk, calves with diarrhea need (diarrhea score ) or qt (diarrhea score ) of warm electrolyte solution each day. the electrolyte solution can be fed immediately after (but not mixed in) the milk replacer or it can be fed at a time different than the milk replacer feeding. the approach i prefer is feeding four times per day: qt milk followed by qt oral electrolyte solution at the regular morning feeding, at noon, again at the regular afternoon feeding, and last thing in evening. alternatively, the four-time-a-day feeding schedule can provide two feedings for milk and two feedings for oral electrolyte solution for calves with a fecal score of . oral electrolyte solution (always mixed in water, not milk replacer) is fed until the diarrhea score returns to or . as the fecal consistency improves, the -qt electrolyte solution feeding can be placed between the two milk feedings. fresh warm water should be available to all calves but especially to diarrheic calves. water is either fed at pleasure or within to minutes of a milk feeding so that calves drink before they leave the buckets to lie down. the selection of a therapeutic antibiotic is based on the fecal culture results or its gram-negative bacterial spectrum [ , ] . once started, an antibiotic protocol is not changed before the -day treatment is completed. the antibiotic recommendation may look like one of the three extralabel protocols shown next. dose: mg/kg ¼ tablet ( -mg size) for a -lb calf twice daily. give two pills on the first dose. route: oral, crushed and added to milk, crushed and dissolved in water-karo syrup combination, or bolus administered by balling gun used slowly and gently. frequency: . calves ! weeks: two times daily for days . calves - weeks: three times per day . naxcel/excenel (ceftiofur) dose: mg/kg ¼ . ml for a -lb calf. this dose is . times the dose for respiratory disease and is specific for salmonella. route: in the muscle frequency: two times daily for days . nuflor (florfenicol) dose: mg/kg ¼ ml per lb. unlike the protocol for respiratory disease, calves with diarrhea receive a daily dose for days. route: subcutaneously frequency: one dose daily for days treatment is successful if the calf is aggressively eating and has a bright attitude, even if the feces stay somewhat loose (score or ). it may take to days for return to normal intestinal function and fecal consistency. pneumonia is responsible for . % and . % of preweaned and weaned heifer deaths, respectively, at an estimated cost of almost $ per calf year [ , ] . despite the importance of the disease, early detection is hampered by use of diagnostic criteria that are poor predictors of pneumonia in the preweaned calf age group. delayed diagnosis results in prolonged use of antibiotics, a high recurrence rate, the development of refractory sequelae, such as pulmonary abscessation, ear infections, and endemic herd problems. dairy calf and heifer pneumonia has serious economic consequences because subclinical, clinical, and chronic pneumonia of calves has a negative impact on growth, reproductive performance, milk production, and longevity [ ] [ ] [ ] . pneumonia is typically viewed as a postweaning problem of dairy heifers but preweaned calves are frequently affected [ , ] and are the source of pneumonia outbreaks in group pens. early detection of pneumonia is a significant problem in dairy calves, however, because typical signs of illness, such as depressed appetite, dull attitude, or an infrequent cough, are not exhibited. in investigating a dairy calf or heifer pneumonia problem, the review of records to determine morbidity and mortality data, seasonal patterns, health, management, housing, number of calves at maximum occupancy, nutrition, vaccinations, procedures, case definition, and treatment protocols is important. the site for disposal of liquid and solid feed refusals and pen management between successive calf occupants is also important. calf housing, with the number of calf pens or hutches, barn and pen construction, layout and dimensions, type, amount and condition of the bedding, calf traffic patterns, and stocking density have an impact on respiratory disease that is described elsewhere in this issue. weaning parameters, age of weaning, and routine health management procedures are additional data of importance. tests for colostral immunity, infectious disease testing, and laboratory or postmortem data are assembled and reviewed. the true age of onset of respiratory disease and prevalence is determined on the day of the herd investigation using a respiratory disease screening tool [ , ] (http://www.vetmed.wisc.edu/dms/fapm/fapmtools/calves. htm). individual calves in pens are examined and assigned a clinical score of (normal), (variation of or slightly abnormal), (abnormal), and (severely abnormal) for temperature, nasal discharge, cough and eye discharge, and ear position. calves with a total respiratory score over are considered to have respiratory disease based on bronchoalveolar fluid cytology and culture validation (mcguirk, unpublished data, ) . all of the calves are scored if there are less than calves. for larger groups, a representative sample up to calves are screened by the scoring system to determine the earliest age of onset and barn, pens, or location of most of the affected calves. in group pens, respiratory disease evaluations are similar but based on the percentage of calves in the pen with abnormal ocular or nasal discharge, abnormal ears, or coughing as shown in table . with completion of the scoring examinations, a detection rate is calculated by dividing the number of calves currently on treatment for respiratory disease by the total number of calves with a total respiratory score greater than . as with enteric disease, the goal is detection of at least % of the calves that require treatment, but this goal is rarely met until the farm is trained to use the respiratory screening procedure. with digital thermometers that have a -second reading time, an individual examination can be completed in less than minutes per calf. calves are easiest to examine between milk feedings when they are resting. the nasal discharge, eye, and ear scores can be assigned without entering the calf pen. spontaneous coughing can also be noted from outside the pen, giving the calf points in that category and obviating the need to use tracheal compression for cough induction. proactive use of the respiratory scoring system improves early detection, provides more reliable information on case rate, monitors for treatment efficacy, and determines which calves can move into the postweaning group pen. having identified the age of onset of respiratory disease through scoring, the youngest age group of affected calves is used for further diagnostic testing. if the goal of the investigation is simply to improve early detection and initiate a more effective treatment protocol, nasal swabs are obtained from six untreated calves with respiratory disease. from each calf, two deep nasal swabs are taken using flexible culturettes that contain a transport system for aerobic and anaerobic bacteria (bbl culture swab plus, benton dickenson, sparks, maryland). one of the swabs is submitted for bacterial culture and the second is submitted for mycoplasma bovis culture. from the nasal pasteurella multocida, mannheimia haemolytica, and histophilus somnus isolates, the antibiotics to which all isolates are susceptible are used to predict the susceptibility of bacterial isolates from the lung [ ] . if more than one of six calves cultures m bovis from the nose [ ] , b-lactam antibiotics are not recommended for the routine treatment protocol. from the nasal swab antibiotic susceptibility patterns shown in table , ceftiofur, florfenicol, trimethoprim-sulfonamide combination, and tulathromycin are considered suitable for respiratory disease treatment if fewer than two of six calves cultures m bovis from the nasal swab. prioritization of the antibiotic protocols is based on the farm, age of calves being treated, compliance, acceptance, and cost of the drugs. bronchoalveolar fluid collection from preweaned calves in a herd with respiratory disease is a relatively efficient way to confirm and characterize the type and severity of respiratory inflammation and provide fluid for bacterial culture. the bronchoalveolar fluid is collected from sedated calves using a sterilized, flexible  -in french catheter with a -ml balloon cuff (mila international, medical instrumentation for animals, florence, kentucky). five to minutes after administration of . mg/kg xylazine intramuscularly, the sedated calf is restrained and the nostrils are cleaned with a dry  -in gauze sponge. the head and neck of the calf are extended to facilitate passage of the sterile bronchoalveolar catheter by a person wearing surgical gloves. before catheter introduction into the nostril, sterile saline is dripped into the catheter to lubricate the guidewire stylette. the bronchoalveolar catheter is introduced into the ventral meatus of the nose through which it is advanced until it encounters resistance in the caudal pharynx. at that point, the restrainer pushes the poll of the calf's head ventrally while simultaneously elevating the ventral mandible and the catheter is advanced down the trachea during the inspiratory phase of the respiratory cycle. repeated coughing is induced with proper catheter placement and it is rapidly advanced until resistance is met as it wedges in a cranial lung lobe bronchus. a failure to induce spontaneous coughing subsequent to passage beyond the pharynx usually implies passage into the esophagus. in the wedged position, the catheter is held firmly in place while the guidewire stylette is removed. the balloon cuff is then inflated with ml of air and ml of sterile saline is infused using -ml syringes with a stopcock and catheter-tipped adapter attached. immediately after the -ml infusion, negative pressure is applied to aspirate fluid, a process that usually yields to ml of clear to mildly turbid, foamy fluid. the returned fluid sample is placed into a sterile -oz specimen cup. a second -ml infusion is introduced and aspirated as described and the pooled fluid is sealed in the specimen cup and preserved in a cooler until it can be processed. the fresh bronchoalveolar fluid sample is processed within hours of collection or refrigerated until it can be analyzed. a -ml aliquot of the pooled sample is used for bacterial and mycoplasma cultures. the remaining fluid is submitted for cytologic interpretation, which is based on routine staining of cytospin and direct smear preparations. bronchoalveolar fluid that yields homogenous (o cfu/ml) bacterial or positive m bovis culture is considered abnormal. a disproportionate lowering of macrophages (! %) or elevation of neutrophils (o %) provides evidence of an inflammatory response with or without a positive culture (mcguirk, unpublished data, ) . the ability to troubleshoot respiratory problems in calves is hampered if the problem is not respiratory disease; the methods for and detection of the problem are neither sensitive nor specific; or the treatments use inappropriate drugs, routes of administration, dose administered, duration of therapy, or storage. the perceived problem of high morbidity or poor cure rates may be a problem of poor disease definition, inaccurate diagnosis, overwhelming exposure, unusual susceptibility, ineffective treatments, or a combination of these factors. respiratory disease management of calves and heifers is not complete without a thorough review of the vaccination protocols for the herd. with preweaned respiratory disease problems, the emphasis is placed on vaccination of the adult cows and an effective colostrum feeding program. as more and more is learned about the effectiveness of vaccinating calves for respiratory pathogens in the face of maternal immunity, vaccination of preweaned calves may become more common [ ] . where colostral immunity is consistently good, most dairy heifers have the first modified live virus vaccines at or months of age. in the absence of adequate colostral immunity, earlier vaccination schedules have been instituted and, at least for viral respiratory pathogens, are relatively safe. respiratory disease investigations present three opportunities to reduce endemic problems in calves and heifers. regular implementation of a screening examination can find calves at an early age when treatment is extremely effective. scoring calves after a -or -day treatment protocol can determine which calves are cured and which require additional treatment. calves scored before moving into a group pen can result in fewer uncured pneumonia cases causing a respiratory disease outbreak in the weaning pen. nasal swab results can guide the implementation of effective treatment protocols and bronchoalveolar fluid can more specifically characterize respiratory inflammatory changes. finally, characterizing and resolving calf housing risk factors for respiratory disease can reduce the exposure to aerosolized bacteria and lower the prevalence of respiratory disease. descriptive epidemiology of morbidity and mortality in minnesota dairy heifer calves partitioning the mortality risk associated with inadequate passive transfer of colostral immunoglobulins in dairy calves part i: reference of dairy health and management in the united states mortality in swedish dairy calves and replacement heifers abortion and calf mortality in danish cattle herds morbidity from nonrespiratory diseases and mortality in dairy heifers during the first three months of life dairy replacement programs: cost and analysis what affects the costs of raising replacement dairy heifers: a multiple component analysis custom dairy heifer grower industry characteristics and contract terms pathology of calves with diarrhoea in southern britain potential risk factors for cryptosporidium infection in dairy calves prevalence of bovine group a rotavirus shedding among dairy calves in ohio association of enteric shedding of bovine torovirus (breda virus) and other enteropathogens with diarrhea in veal calves rotavirus shedding in feces of gnotobiotic calves orally inoculated with a commercial rotavirus-coronavirus vaccine evaluation of an indirect serum elisa and a bacteriological faecal culture test for diagnosis of salmonella serotype dublin in cattle using latent class models fecal shedding of coliform bacteria during the peripartum period in dairy cows growth performance and health of dairy calves bedded with different types of materials growth of environmental mastitis pathogens in various bedding materials bacterial counts associated with recycled newspaper bedding long-term persistence of multi-drug resistant salmonella enterica serovar newport in two dairy herds neonatal diarrhea in calves: investigation of herd management practices efficacy of two peroxygen-based disinfectants for inactivation of cryptosporidium parvum oocysts broad-spectrum microbicidal activity, toxicologic assessment, and materials compatibility of a new generation of accelerated hydrogen peroxide-based environmental surface disinfectant efficacy of directed misting application of a peroxygen disinfectant for environmental decontamination of a veterinary hospital transmission of salmonellae among calves penned individually oral electrolyte therapy intravenous fluid therapy of calves effects of additional milk replacer feeding on calf health, growth and selected blood metabolites in calves antimicrobial use in the treatment of calf diarrhea a clinical trial evaluating prophylactic and therapeutic antibiotic use on health and performance of preweaned calves efficacy of ceftiofur for treatment of experimental salmonellosis in neonatal calves veterinary and nonveterinary costs of disease in california dairies participating in the national animal health monitoring system from to calf and disease factors affecting growth in female holstein calves in florida, usa the effect of early calfhood health status on survivorship and age at first calving the relationship of calfhood morbidity with survival after calving in new york holstein herds epidemiologic and pathologic characteristics of respiratory tract disease in dairy heifers during the first three months of life calf respiratory disease and pen microenvironments in naturally ventilated calf barns in winter troubleshooting dairy calf pneumonia problems comparison of pasteurella spp. simultaneously isolated from nasal and transtracheal swabs from cattle with clinical signs of bovine respiratory disease the nasal mycoplasmal flora of healthy calves and cows evaluation of protection against virulent bovine viral diarrhea virus type in calves that had maternal antibodies and were vaccinated with a modified-live vaccine key: cord- -c w pf t authors: gebregiorgis, ashenafi; tessema, tesfaye sisay title: characterization of escherichia coli isolated from calf diarrhea in and around kombolcha, south wollo, amhara region, ethiopia date: - - journal: trop anim health prod doi: . /s - - - sha: doc_id: cord_uid: c w pf t this study was carried out from october to end of february in and around kombolcha, amhara regional state, ethiopia, using a total of neonatal calves aged day to months and suffering from diarrhea. the objectives of the study were to isolate escherichia coli from diarrheic calves, and to determine e. coli biotypes and risk factors associated with its isolation. the fecal samples were collected, transported, and processed following standard microbiological procedures. seventy-four isolates of e. coli were identified. yellowish diarrhea, younger age, and low-colostrum feeding were significantly associated with rate of e. coli isolation (p < . ). then the isolates of e. coli were biotyped using fermentation of sugars and grouped into biotypes; the most dominant was biotype iii ( . %). finally, by comparing with studies elsewhere, from the isolated biotypes, of them were suggested to be enteropathogenic e. coli (epec), entherotoxigenic e. coli (etec), and adhesion and effacing e. coli (aeec) pathogenic strains. the present study showed that e. coli accounted for % of calf diarrhea, with very diverse biotypes. newly born calves represent an important source of animal production for either meat or breeding worldwide. diarrhea is one of the very common disease syndromes in neonatal calves in different countries, and this can have severe impacts both economically and in terms of animal welfare (africa union ) . calves are at greatest risk of developing diarrhea within the first month of life, and the incidence of diarrhea decreases with age (meganck et al. ) . neonatal calf diarrhea is a multifactorial disease which despite decades of research in the topic remains the most common cause of neonatal calf mortalities (uhde et al. ). there is a multitude of interactions of the noninfectious causes (predisposing factors) (flaws or gaps in management-inadequate nutrition, exposure to severe environment, insufficient attention to the newborn calf, or a combination of these) and infectious causes (blanchard ; cho and yoon ) . several enteropathogens are implicated in neonatal calf diarrhea, and their relative prevalence varies geographically but the most common prevalent infections in most areas are escherichia coli, rotavirus, and corona virus, clostridium perfringens, salmonella, and cryptosporidium. causes of neonatal calf diarrhea are commonly associated with more than one of these agents, and the causes of most outbreaks are usually multifactorial (foster and smith ; blanchard ; cho and yoon ) . e. coli strains are the most common primary agents causing calf diarrhea and septicemia (kolenda et al. ) . escherichia coli is a motile gram-negative bacilli that falls within the family enterobacteriaceae. it colonizes the infant gastrointestinal tract within hours of life, and thereafter e. coli and the host derive mutual benefit for decades (welch ; foster and smith ) . many strains of the bacterium are harmless to the calf, but certain strains that acquire virulence genes can cause moderate to severe scours and even death (wani et al. ) . two of the more prominent virulence factors identified for enterotoxigenic e. coli strains are (i) expression of fimbrial (pilli) antigens that enable the bacteria to adhere to and to colonize the luminal surface of the small bowel and (ii) elaboration of one or more enterotoxins that influence intestinal secretion of fluids (chen and franke ; welch ). severity of e. coli symptoms varies from mild transient diarrhea to severe sustained diarrhea resulting in death. typical presentation is in calves less than days of age and as early as h of life. e. coli typically produces a secretory diarrhea switching the intestinal epithelial cells from an absorption mode to a secretion mode. diarrhea is typically whitish to yellowish in color (ata et al. ; cho and yoon ) . e. coli are able to ferment a variety of carbohydrate substrates, generally by converting them to glucose or to a substrate on the fermentative chain of the breakdown of glucose. the ability to ferment a given sugar of the types described above by a strain of e. coli is dependent on the strain having the requisite enzymes to convert it to glucose or to a substance on the degradative chain from glucose (aklilu et al. ) . this is the basis of biotyping of e. coli. these tests are also easy to perform, by determining, whether a strain of e. coli will produce acid following growth in the presence of the carbohydrate. of the various typing systems available, study of the organisms' biological properties (bbiotyping^) appears to be a useful method of identification (aklilu et al. ) . in this regard, chattopadhyay et al. ( ) classified six different biotypes of verotoxin-positive e. coli on the basis of sugar fermentation reactions of three sugars, viz., sorbitol, raffinose, dulcitol, and decarboxylase test with lysine, arginine, and ornithine. in addition, murinda et al. ( ) reported diagnostic significance of rhamnose fermentation test; they recorded rhamnose non-fermenters belonging to e. coli o were % stec producers. there were no previous studies conducted on calf diarrhea as well as on diversity of e. coli biotypes in the study area. however, there are frequent cases of calf diarrhea in the study area leading to morbidity and mortality and often seeking professional intervention. therefore, this work was conducted to determine the diversity of e. coli using standard sugar fermentation tests and identify risk factors associated with its isolation from diarrheic calf feces in the area. we believe the data in this work contributes to diagnosis and control of calf diarrhea in ethiopia, in particular, and worldwide, in general. this work was carried out from october to end of february using a total of neonatal calves of different ages ( day- months old) examined clinically for diarrhea on dairy farms in and around kombolcha, south wollo, amhara region, ethiopia. kombolcha is located km northeast of addis ababa at a latitude of ° ′ n ° ′ e, longitude of . °n . °e, and elevation between and m above sea level (fig. ) . the woreda has an annual mean temperature of . - . °c (csa ) and receives the shortest rainfall from march to may and the longest rainfall from june to september ( - mm). there are many small-scale and large-scale dairy farms in this area that supply milk and milk products to consumers of the town and surrounding urban areas. these dairy farms contain either local or exotic breeds depending on the scale of production. animals included in this study were calves under months of age that were clinically affected with diarrhea and exhibiting signs of systemic disease (e.g., poor appetite, fever, dehydration, decreased mentation, and reduced suckle reflex) and had pasty-watery feces with different colors. all of these calves were found suffering from different degrees of diarrhea, dehydration, emaciation, and weakness. the calves were born in different farms located in the area, dairy farms in kombolcha, and dairy farms around kombolcha; in all of the dairy farms, the management system was similar, i.e., animals were kept under intensive system. the calves were kept in isolated pens for the whole day except for few hours when the calves were released in the compound for exercise. in some farms, the calves were released for suckling their dam. in all of the farms, the floors were concrete and cleaning of the calves' pens was practiced daily when calves were out. in addition, in all of the farms, calves were fed on colostrum three times per day from their respective dams with different amount and for different periods of time. the weight of each calf was determined by the heart girth system. the study was purposive type, i.e., samples were collected from calves that show clinical signs of diarrhea. approximately ml of fecal samples were collected from non-treated diarrheic calves directly from the rectum by using disposable plastic gloves and transferred immediately to sterile universal bottles. the samples were then transported in ice box to the kombolcha veterinary diagnostic laboratory, kombolcha, for processing. feces were stored at °c until the time of processing. fecal samples were inoculated onto blood and macconkey agar and incubated at °c overnight. from each macconkey agar plate, lactose-fermenting colonies were inoculated on eosin methylene blue (emb) agar medium, which selectively grows members of the enterobacteriaceae and permit differentiation of enteric bacteria on the basis of morphology. colonies showing characteristic metallic sheen on emb agar were then picked up and considered as presumptive e. coli. the purified cultures of e. coli were stored temporarily as nutrient broth cultures for further identification by biochemical tests. all the isolates were stained by gram stain to determine the cell morphology, gram reaction and purity of the isolates under the oil immersion lens (× ). identification of suspected e. coli colonies was conducted following standard bacteriological procedures described in quinn et al. ( ) . thus, e. coli isolates were preliminarily characterized by imvic tests, viz., indole, methyl red, voges-proskauer, and citrate utilization. the isolates which exhibited the imvic pattern of +, +, −, and −, respectively, were presumed as e. coli isolates. the identified isolates were then further characterized for their sugar fermentation reactions on nine sugars, viz., dulcitol, raffinose, rhamnose, salicin, sucrose, inositol, lactose, maltose, and xylose according to aklilu et al. ( ) . the isolates grown in phenol red broth were inoculated into % of each sugar medium. tubes were incubated at °c for days, and readings were then recorded after every h. production of yellow color was considered as positive reaction and proper controls were kept for each of the sugar tests performed. furthermore, literature was searched for previous studies that utilized similar sugars like ours in biotyping of e. coli and conducted strain identification in parallel. comparison was made between our biotype data and those published works; whenever a pathotype strain fermenting similar sugars with any of our biotypes was encountered, this strain was suggested as a possible pathotype for our biotype. data describing the diarrhogenic conditions suggestive of e. coli infection observed on calves along with the amount of colostrum given, sex, weight, diarrhea type, and age were classified, filtered, and coded using microsoft excel® . the data were then exported to spss windows version . (spss inc. chicago, il) for appropriate statistical analysis. the detection rate of e. coli and the abundance of identified biotypes were determined by using descriptive statistics. chisquare (χ ) was used to measure associations between the detection rates of e. coli and their biotypes with relevant factors. associations were reported as statistically significant if p value is less than %. during the present study, ( . %) out of the diarrheic calves that showed calf diarrhea were e. coli positive. calf diarrhea and e. coli isolation associated with host and management factors among the diarrheic fecal samples, six types of diarrhea, namely, yellowish, blood-tinged, pure bloody, greenish, watery, and mucoid, were observed at different proportions. there was a statistically significant association between rates of e. coli isolation and color type of diarrhea (p= . ), isolation rates being highest in blood-tinged diarrhea and lowest in greenish diarrhea. in contrast, there was no e. coli isolation in calves with mucoid type of diarrhea (table ). the majority of the isolates were originated from calves with yellowish diarrhea due to the large number of calves with yellowish diarrhea in the area. the diarrheic calves examined were classified into four age groups which yield different proportions of e. coli isolates. there is a high occurrence of diarrhea on week old calves, followed by - days old group, and then by - days old group. there was no direct association between age of calves and occurrence of diarrhea; however, the isolation rate of e. coli was highest at the earlier age groups, decreasing as the age of diarrheic calf increases. the e. coli isolates that were recovered from the age groups of - days and - days constitute % of the total isolates. this difference in isolation rate of e. coli was statistically significant (p= . ) ( table ) . it was also shown that isolation rate of e. coli was not statistically associated with either sex (p= . ) or body weight (p = . ) of the calves studied. similarly, higher amount of colostrum feeding does not reduce significantly isolation rate of e. coli (p= . ) ( table ) . bloody diarrhea was observed only in the youngest and oldest age groups; whereas, the other types of diarrhea were observed in all age groups. distribution of the diarrheal types among the different age categories of calves showed no significant association (table ) . types and relative abundance of e. coli biotypes in calves all the e. coli isolates were studied for their fermentation activities on sugars; ( . %) isolates showed the ability to utilize one or more sugars while ( . %) isolates failed to utilize any of the sugars tested. the most abundant e. coli biotypes from calf diarrhea, namely, biotypes iii, viii, v, iv, and i, constituted the majority of the isolates ( . %). furthermore, literature search showed that gargan et al. ( ) , levine et al. ( ) and johan et al. ( ) have used similar sugars like this study for biotyping; they also reported the pathotype strains of their biotypes. accordingly, by comparison with their data, the biotypes iii, ivand vii, and viii of the current study could possibly be entherotoxigenic e. coli (etec), enteropathogenic e. coli (epec), and adhesion and effacing e. coli (aeec) strains (table ) . the present study showed variation on the pattern of distribution of biotypes among calf age groups; the predominant biotypes in the age groups of -days, - days, - days, - days, and - days were iii, iv, iv, v, and viii, respectively. there was a statistically significant association between the different age groups and occurrence of the different biotypes (p< . ). overall, biotype iii was the most predominant biotype. it was also observed that as age increased, the diversity of biotypes observed decreased, the highest numbers of biotypes observed were in the youngest age ( - days); only one and two biotype variants were detected in the age groups of - days and - days, respectively (table ) . furthermore, associations on the distribution of the different biotypes among diarrhea types were assessed; the predominant biotypes in the diarrhea types of yellowish, bloodtinged, pure bloody, and greenish were iii; iii, iv, and v; iv, and viii, respectively (table ). neonatal diarrhea is a major health problem in dairy farms, leading to high mortality and is hindering sustainable development of the dairy sector; e. coli is the most important cause of bacterial scours in calves (charles et al. ; razzaque et al. ; kolenda et al. ) . the involvement of e. coli in calf diarrhea has not been well studied in ethiopia, with the exception of a recent study by dawit ( ) . in the present study, e. coli was isolated from . % of calf diarrhea cases which is in agreement with the report by razzaque et al. ( ) and bekele et al. ( ) who reported and %, respectively. this finding is also consistent with the work of amoki ( ) in the central high land of ethiopia. the higher calf diarrhea due to e. coli in the present study might be attributed to the variations in age groups examined as well as environmental and management conditions of the farms such as insufficient and/or poor-quality colostrum intake by the calves as stated by charles et al. ( ) . in addition to that poor hygiene often allows buildup of pathogenic strains in the young animal's environment. besides, a large dose of pathogenic e. coli may overcome colostral immunity (quinn et al. ). the present study shows much lower isolation rate than achá et al. ( ) ( ) ( %). on the other hand, lower ( . %) percentages of calf diarrhea caused by e. coli were reported by darsema ( ) , . %, and uhde et al. ( ) , . %. the reason why the result of the current study varies from the reports in other areas might be due to variations in farm management conditions. as documented in charles et al. ( ) and radostits et al. ( ) , gaps in management specifically calf handling practices including inadequate nutrition, exposure to severe environment, insufficient attention to the newborn calf, or a combination of these, qualitative and , , , , , , , , , , , ( ( ) a the sugar inositol has not been used by johan et al. quantitative conditions of the colostrum, are often involved in scours outbreaks. the color and consistency of feces in e. coli scours have their own value in making a diagnosis of any type of diarrhea (ata et al. ; cho and yoon ) . in the present study, the color of diarrhea in relation to involvement of e. coli in calf diarrhea was also considered. accordingly, four types of diarrheal color were identified, the most dominant being the yellowish one ( . %) from which . % were positive for e. coli. in comparison, bartels et al. ( ) reported that among the . % of the yellowish-colored calves' diarrhea, . % was caused by e. coli. ata et al. ( ) also reviewed that in calf scours due to e. coli, the small intestine may be filled with fluid and the large intestine may contain yellowish feces. factors that may contribute to these differences between studies could be related to presence or absence of mixed infections, variations on management conditions of the farms, and the age of the calf as also supported by the present study. in support of this, charles et al. ( ) stated that onset of e. coli diarrhea varies in color and consistency that could be consistent with intestinal overload possibly due to high-volume colostrum feeding. there were agreements and contradictions on isolation rates of e. coli from the other diarrhea types in the present study and previous studies conducted elsewhere. thus, the isolation rate of e. coli from blood-tinged diarrhea in the present study ( . %) was inconsistent with that of james and james ( ) , who isolated e. coli from % of bloodtinged calf diarrhea. in support of this, naylor and smith ( ) reported that diarrhea due to e. coli is characterized by profuse, pasty to watery, foul smelling, and occasionally flecked with blood (blood-tinged). the isolation rate of e. coli from pure bloody diarrhea in the present study ( . %) was consistent with the % reported by james and james ( ) . the overall variations in color of feces may be as a result of differences on the basis of pathogenic features and mechanisms of the disease. exudative diarrhea is observed in inflammatory diseases when mucosal inflammation and ulceration cause outpouring of plasma, mucus, and blood into the stool. diarrhea of the large intestine is associated with frequent small-volume stools with the presence of blood (naylor and smith ; foster and smith ) . involvement of mixed infections could also complicate the appearance of feces (ata et al. ; cho and yoon ) . when we look at sex variation, female calves ( . %) yielded higher e. coli isolation rate compared to male calves ( . %). in contrary, male animals do generally get less attention and management care as their role in the farms is considered irrelevant especially as the replacement stock. furthermore, the association between the different age groups with the occurrence of diarrhea due to e. coli was studied. the highest percentage ( . %) of e. coli-positive ( ) samples were identified in those calves found in the - days age group; this result is inconsistent with reports of achá et al. ( ) ( %). the causes of variation might be as a result of variations in management of the farms and immunity of the animals. most newborn calves are exposed to e. coli from the environment, particularly when sanitation is marginal (charles et al. ) . the author stated that young neonates under week of age are particularly susceptible because the normal flora of the intestine is not fully established. in addition to that, they have a naive immune system and also receptors for the adhesions of e. coli are present on the first week of life of the calves (villarroel ). the result of this study is in agreement with the above idea. in addition, other authors reported that this is the age group most commonly affected clinically (maddox-hyttel et al. ; santin et al. ) . the isolation rate of e. coli from the age group of - days ( . %) was consistent with the literature (villarroel ) where it is said that e. coli affects calves within the first - days of age. the isolation rates of the bacteria in the subsequent age groups were shown to decrease as supported by the reports of villarroel ( ) . in support of this, calf diarrhea was noted to decrease as the age of the calves increased. this could be due to the poorly developed immune system of the days old calves as compared to older ones to fight against the disease-causing agents (darsema ; bekele et al. ). scott et al. ( ) and radostits et al. ( ) also documented that neonatal infection is higher during their early stage of life because of the stress and infection pressure encountered shortly after birth. the cause of high isolation rate in the calves receiving less amount of colostrum could be for various reasons. an inadequate, in quality and quantity, supply of colostrum and delay in first colostrum feeding, which leads to failure of transfer of passive immunity (fpt) is an important reason. calves with inadequate colostral immunoglobulin concentration within h of birth were at greater risk of neonatal morbidity and mortality. in addition, fpt could be due to bacterial contamination of the fed colostrum (keith et al. ) . colostrum feeding practices also have effect in that allowing calves to nurse their dam may predispose them to fpt since they consume late and small amount (meganck et al. ) . the present study also investigated the identity of different types of e. coli biotypes in calf diarrhea. biotyping based on sugar fermentation tests has been utilized by various workers for identifying diversity within a bacterial spp. the most dominant biotype was biotype iii ( . %) which fermented dulcitol, rhamnose, and inositol. this result was consistent with a report by gargan et al. ( ) who determined e. coli biotyping using the same type of sugars. they assigned biotype i as an isolate fermenting the above three sugars, which accounted . % of the isolates, and based on the api e system, they concluded that the identified biotype i confirmed with serotyping was etec containing o-antigen. so according to their data, biotype iii of the present study could be etec (entherotoxigenic e. coli). chattopadhyay et al. ( ) classified six different biotypes verotoxin-positive e. coli on the basis of sugar fermentation reactions in three sugars, viz., sorbitol, raffinose, dulcitol, and decarboxylase test with lysine, arginine, and ornithine. in addition, murinda et al. ( ) reported diagnostic significance of rhamnose fermentation test; they recorded rhamnose non-fermenters belonging to e. coli o were % stec producers. the current study identified different biotypes. dawit ( ) reported biotypes based on sugars (dulcitol, raffinose, rhamnose, salicin, starch, and sucrose) in the central part of ethiopia. their data showed that the biotype that fermented dulcitol was the most dominant one ( %), followed by the biotypes that ferment dulcitol, raffinose, and rhamnose ( . %). one of the limitations of this study was the lack of pathotype strain identification of the e. coli isolates due to lack of resource. literature was searched for similarity in sugars used and relation of biotypes and strains; then comparison was made with the biotypes of this study. johan et al. ( ) also reported different biotypes, using four carbohydrates, from healthy and diarrheic rabbits; biotype ( . %) (dulcitol, rhamnose, and sucrose fermenters) and biotype ( . %) (dulcitol and rhamnose fermenters) induced lesions characteristic of attaching and effacing e. coli (aeec). serotyping showed a close relationship between biotype and serotype of the aeec examined. even though the proportions of biotype of the above result was dissimilar to the present study of biotype iv ( . %) (fermented dulcitol, inositol (not used by the above author), rhamnose, and sucrose), their fermentation activity was identical. but the result and fermentation activity of the present study of biotype vii ( . %) (dulcitol and rhamnose fermenter) was consistent with the result of biotype of johan et al. ( ) . in reference to their work, biotypes iv and vii of the present study could be aeec. variation in results of biotype iv of the present study and biotype of johan et al. ( ) might be the result of differences in sample size and animal species studied. these results were also in agreement with levine et al. ( ) who stated that a given strain from diarrheic rabbit (that fermented dulcitol, rhamnose and xylose and assigned as biotype ) isolated at the rate of . % was able to induce watery diarrhea and high mortality after experimental infection. this was in consistent with biotype viii ( . %) of the present study. similar observations have been made with other strains in england (varga and pesti ) , belgium and the netherlands (peeters et al. ) , and france (camguilhem et al. ). none of these strains produced heat-labile or heatstable enterotoxins, nor were they enteroinvasive. histology and electron microscopy has also showed their tight adherence to the brush border of intestinal epithelial cells. so they are considered to be enteropathogenic e. coli (epec). so, biotype viii of the present study could be suggested to be epec. in the present study, associations between age groups and the different biotypes were also assessed. hence, young calves in the first age group ( - days) and in the second age group ( - days) were at a significantly high risk of being affected with diarrhea (p< . ). this finding is also comparable with the result obtained from the biochemical test results which indicated these groups of calves were at a significantly high risk of being affected with e. coli-caused diarrhea than the older age groups (p< . ). in conclusion, during the present study, calf diarrhea was investigated at kombolcha town in order to isolate e. coli and determine the biotypes of the isolates as well as identify risk factors associated with calf diarrhea. using sugars, biotypes were identified, among which, biotype iii was the most dominant one affecting calves in the first week of age. finally, by comparing with studies elsewhere, from the isolated biotypes, of them were enteropathogenic e. coli, enterotoxigenic e. coli, and adhesion and effacing e. coli. biotyping is relatively cheaper and can be conducted by less-experienced personnel. compared to molecular and serotyping techniques, the technique would be valuable for resource poor countries. however, further studies involving comparison of biotyping with pcr and serotyping should be conducted in order to study the applicability of the method for strain identification among pathogenic e. coli strains. studies on calf diarrhea in mozambique: prevalence of bacterial pathogens bulletin of animal health and production in africa identification and biotyping of escherichia coli from diarrheic lambs in and around debre birhan town management of dairy calves in holleta area, central high land of ethiopia calf scours: definition and causes prevalence, prediction and risk factors of enteropathogens in normal and non-normal faces of young dutch dairy calves prevalence and incidence rate of calves morbidity and mortality and associated risk factors in small holder dairy farms in hawassa diagnostics of dairy and beef cattle diarrhea, veterinary clinics of north america food animal practice reproduction experimental chez le lapin en engraissement d'une diarrh e provoquee par une souche d' escherichia coli de sdrogroupe calf scour: cause, prevention and treatment, (extension service search for shiga toxin producing escherichia coli (stec) including o :h strains in and around kolkata enteropathogenic escherichia coli: unravelling pathogenesis an overview of calf diarrhea -infectious etiology, diagnosis, and intervention agriculture sample enumeration statistical abstract major causes of calf mortality in dairy farms and two cattle ranches in western amhara region, north western ethiopia isolation and identification of enterotoxigenic escherichia coli strengthen from diarrheic calf faeces in addiss ababa and debre zeit pathophysiology of diarrhea in calves, veterinary clinics of north american food animal practitioner a concise biotyping system for differentiating strains of escherichia coli advances in prevention and therapy of neonatal dairy calf diarrhoea: a systematical review with emphasis on colostrum management and fluid therapy diarrheagenic escherichia coli biotype, serotype, and pathogenicity of attaching and effacing enteropathogenic escherichia coli strains isolated from diarrheic commercial rabbits comparison of passive transfer of immunity in neonatal dairy calves fed colostrum or bovine serum-based colostrum replacement and colostrum supplement products a systematic review and meta-analysis of the epidemiology of pathogenic escherichia coli of calves and the role of calves as reservoirs for human pathogenic e. coli new knowledge on pathogenesis of bacterial enteric infections as applied to vaccine development cryptosporidium and giardia in different age groups of danish cattle and pigs: occurrence and management associated risk factors food borne pathogens and disease neonatal ruminant diarrhea infectious agents associated with diarrhea in commercial rabbits: a field study infectious causes of neonatal diarrhea in cattle in kuwait with special reference to cryptosporidiosis clinical veterinary microbiology veterinary microbiology and microbial disease diseases associated with bacteria iii dairy calf rearing in hot arid environment of kuwait, paper ii: impact of interventions on health performance of pre-weaned calves prevalence and age-related variation of cryptosporidium species and genotypes in dairy calves infectious diseases of the gastrointestinal tract prevalence of four enteropathogens in the faces of young diarrheic dairy calves in switzerland serological and some pathological characteristics of escherichia coli strains isolated from rabbits scours in beef calves: causes and treatments escherichia coli o associated with an outbreak of calf diarrhea the genus escherichia, prokaryotes acknowledgments we are grateful to the personnel working at the microbiology laboratory of kombolcha regional veterinary laboratory, south wollo, ethiopia. in addition, we thank the owners of the dairy farms at kombolcha town for their cooperation during the study. conflict of interest the authors declare that they have no competing interests. key: cord- -crjjhpl authors: graef, geneva; hurst, natalie j.; kidder, lance; sy, tracy l.; goodman, laura b.; preston, whitney d.; arnold, samuel l. m.; zambriski, jennifer a. title: impact of confinement housing on study end-points in the calf model of cryptosporidiosis date: - - journal: plos negl trop dis doi: . /journal.pntd. sha: doc_id: cord_uid: crjjhpl background: diarrhea is the second leading cause of death in children < years globally and the parasite genus cryptosporidium is a leading cause of that diarrhea. the global disease burden attributable to cryptosporidiosis is substantial and the only approved chemotherapeutic, nitazoxanide, has poor efficacy in hiv positive children. chemotherapeutic development is dependent on the calf model of cryptosporidiosis, which is the best approximation of human disease. however, the model is not consistently applied across research studies. data collection commonly occurs using two different methods: complete fecal collection (cfc), which requires use of confinement housing, and interval collection (ic), which permits use of box stalls. cfc mimics human challenge model methodology but it is unknown if confinement housing impacts study end-points and if data gathered via this method is suitable for generalization to human populations. methods: using a modified crossover study design we compared cfc and ic and evaluated the impact of housing on study end-points. at birth, calves were randomly assigned to confinement (n = ) or box stall housing (n = ), or were challenged with x ( ) c. parvum oocysts, and followed for days. study end-points included fecal oocyst shedding, severity of diarrhea, degree of dehydration, and plasma cortisol. findings: calves in confinement had no significant differences in mean log oocysts enumerated per gram of fecal dry matter between cfc and ic samples (p = . ), nor were there diurnal variations in oocyst shedding (p = . ). confinement housed calves shed significantly more oocysts (p = . ), had higher plasma cortisol (p = . ), and required more supportive care (p = . ) than calves in box stalls. conclusion: housing method confounds study end-points in the calf model of cryptosporidiosis. due to increased stress data collected from calves in confinement housing may not accurately estimate the efficacy of chemotherapeutics targeting c. parvum. a a a a a cryptosporidium is a genus of protozoal parasites that infect a wide range of hosts including wild and domestic mammals, amphibians, and reptiles [ ] . the most pathogenic species are c. hominis and c. parvum, which primarily affect people and cattle, respectively. c. parvum, which is zoonotic, represents an important public health threat. in resource-poor countries this is of particular concern for children who live in close proximity to cattle, and in resourcerich countries the zoonosis impacts agricultural laborers on commercial dairy farms, where herd prevalence can be as high as % [ , ] . in both human and cattle hosts, disease is characterized by villus atrophy, intestinal crypt inflammation, and malabsorptive, maldigestive diarrhea that may be secretory in nature [ , ] . in addition, large numbers of infective oocysts are shed in the stool, which impacts environmental parasite loading and propagates fecal-oral transmission [ ] . diarrhea has been identified as a leading cause of death in children < yrs globally [ , ] . in resource-poor countries, cryptosporidiosis is the second leading cause of diarrhea in children (after rotavirus) and is associated with an increased risk of death in the first yrs of life [ ] . individuals that are immunocompromised due to other etiologies, such as hiv or severe malnutrition, are more likely to develop life-threatening diarrhea [ , , ] . pediatric cryptosporidiosis has also been associated with cognitive impairment and stunting, regardless of the development of diarrhea [ ] [ ] [ ] . despite the adverse impact on human lives there are no treatments or vaccines that are consistently effective against cryptosporidium [ ] [ ] [ ] [ ] [ ] . nitazoxanide is the only therapy currently approved for the treatment of cryptosporidiosis in people. it has been demonstrated to have modest efficacy in hiv-seronegative children, but not hivseropositive children [ ] . similarly, in veterinary patients there is a dearth of treatment options. halofuginone is the only drug licensed for use in cattle to control cryptosporidiosis, but it is not an ideal treatment option. it is registered in a few countries, excluding the united states, is only effective when used prophylactically and not therapeutically, has a very narrow margin of safety, and is not cost-effective [ ] [ ] [ ] . given the substantial impact of cryptosporidiosis on child health in resource-poor settings and the lack of options to prevent and treat disease in human and animal populations, there is an urgent need to identify and test new chemotherapeutic agents. to develop effective chemotherapeutics, repeatable and reliable in vivo and in vitro models are essential. cryptosporidium is notoriously difficult to culture and study in vitro, though advances are being made using hollow fiber technology, and in vivo models either fail to achieve comparable severity of clinical illness or are cumbersome to execute [ , ] . murine models of cryptosporidiosis are commonly used because they permit induction of infection with c. parvum. although c. parvum is not host-adapted to mice, this model is widely applied because it is tractable. cryptosporidium species do not induce severe clinical illness in mice. this includes c. muris, which is host-adapted to mice [ ] . studies in the mouse model report weight loss, listlessness, and occasional pasty stool, but do not report fulminant diarrhea [ , ] . the inability to induce measureable clinical illness is a major limitation of mouse models, because we must be able to measure a response to treatment in order to evaluate chemotherapeutic efficacy. the calf model of cryptosporidiosis is the best approximation of human disease because calves experience natural infection and clinical illness that mirrors human symptomology. however, the model is not consistently applied across research studies, which may impact evaluation of study end-points such as fecal oocyst shedding, severity of diarrhea, and degree of dehydration. the type of housing system used in calf model studies of cryptosporidiosis is dictated by the desired method of fecal sampling. the two methods most commonly used to collect and evaluate stool from calves experimentally infected with c. parvum are the complete fecal collection (cfc) method and the interval collection (ic) method. for cfc the total amount of stool produced by a calf is collected every hours, blended for homogenization, measured either by weight or by volume, and an aliquot (~ . g) is removed for evaluation [ , ] . in the interval collection method, a small fecal sample (~ g) is collected directly from the rectum of the calf every or every hours [ , [ ] [ ] [ ] . historically, cryptosporidium challenge studies conducted in human volunteers have used cfc [ , ] . for human participants cfc is considered to be the gold-standard for evaluating reduction of diarrhea in response to treatment. another potential advantage to cfc is that homogenization of the stool sample prior to oocyst enumeration mitigates the risk of variation in oocyst counts associated with intermittent oocyst shedding or diurnal fluctuations. though there is a report of possible diurnal variation in oocyst excretion in confinement housing, it is not clear whether this would be an issue in box stall housing or in all situations of confinement housing [ ] . a major disadvantage to cfc is that it is cumbersome and difficult to execute in animal models. with human volunteers, a variety of sample collection methods exist to permit simple, non-invasive collection of cfc. with neonatal calves collection of cfc is a formidable challenge requiring use of confinement housing that severely restricts calf movement and their ability to engage in natural behaviors, such as grooming. calves may rise or lay down in sternal or lateral recumbancy, but they cannot turn around or ambulate. the use of this degree of confinement is necessary in order to direct the fecal matter expelled by the calf directly into collection pans. enlarging the confinement system to let the calf ambulate or to allow sufficient space for the calf to turn around would not permit collection of the entire stool sample, especially at the peak of illness when calves may pass stool - times per hour. this housing system, originally developed for use in the veal industry, is not ideal for calf welfare and may not be acceptable to institutional animal care and use committees, particularly those at european institutions. in contrast to cfc, the use of the ic method allows calves to be housed in box stalls where they can move freely. it also has the added advantage of reduced labor requirements for sample collection and calf husbandry. in addition to required use of confinement housing, implementation of cfc is expensive to maintain, labor intensive, and age restricted. calf enrollment cannot exceed days as calves older than days are too large for confinement housing. the "guide for the care and use of agricultural animals in research and teaching" specifically states that calves can not be placed in confinement after day of life [ ] . thus the window of data collection in confinement housing is limited to days. in addition, this limits study efficiency, as it is difficult to manage large numbers of calves, which typically restricts enrollment to - animals. there is also a risk that data collected during this period may not be representative. it has been demonstrated that calves are more likely to have higher levels of cortisol in the first days of life, indicating higher susceptibility to stress [ , ] . however, the impacts of confinement housing on cortisol levels have not been evaluated and the impacts of elevated cortisol on study end-points have also not been assessed. no studies have been conducted comparing cfc and ic, nor are there studies comparing confinement housing and box stalls in the calf model of cryptosporidiosis, which is a substantial knowledge gap considering how integral this model is to cryptosporidium drug development. it is unknown if the ic method is an acceptable alternative to cfc with respect to relevant study end-points such as diarrheal severity and magnitude of fecal oocyst shedding. it is also unknown if these end-points are impacted by calf confinement. lastly, there are no studies that compare cortisol levels and other negative health outcomes, such as risk of calf injury and need for supportive care, in association with housing type. therefore, the objective of this study was to determine if data collected via ic is comparable to data collected via cfc, and if confinement housing impacts the study end-points used to evaluate chemotherapeutic agents targeting c. parvum in the calf model. this study was reviewed and approved by the washington state university institutional animal care and use committee (asaf ). the study adhered to the guidelines put forth by the animal welfare act and specifically the animal welfare act & regulations blue book for usda animals. a total of holstein bull calves were enrolled contemporaneously on a commercial dairy farm located in pasco, wa. calves were enrolled for a period of days. to ensure that calves were not exposed to cryptosporidium and to limit exposure to other microorganisms, all calvings were attended and assisted. the perineum of the dam was cleaned thoroughly with a povidone-iodine scrub, and each calf was delivered using a single-use plastic sheet or a designated wheelbarrow that was thoroughly cleaned and disinfected between each calving. a physical exam was conducted and each calf was weighed. calves with normal physical exam findings that weighed more than . kg were enrolled. upon enrollment, the umbilicus of each calf was dipped in an iodine tincture, and ml of vitamin e and selenium (bose, intervet/ merck animal health, germany) was administered subcutaneously. calves received l of ! g igg/l commercial colostrum replacer within hours of birth (bovine igg, colostrum replacement, land o'lakes inc., st. paul, mn) via an esophageal tube feeder. calves were transported to washington state university in a dedicated trailer bedded with sterile straw. animals were randomly assigned to box stall (n = ), confinement housing (n = ), box stall negative control (n = ), or confinement housing negative control (n = ) treatments and housed in a bsl isolation facility. negative control calves served as sentinels to aid in monitoring for inadvertent transmission of parasite between calves. inadvertent transmission would result in some calves receiving a larger parasite inoculum than others and could impact severity of clinical illness. calves in box stalls were housed individually in approximately . m ( ft ) of space, bedded in sterile wood shavings, and had a mirror placed at eye-level for environmental enrichment. calves in the confinement housing group were placed inside of commercially manufactured elevated calf stalls (wenke manufacturing, pender, nebraska) [ , ] . to provide environmental enrichment, stalls were placed in each bsl room and calves were faced toward each other at a distance of . m ( ft) to prevent cross-contamination. the surface area of each stall was . m ( . ft ) ( . cm long by . cm wide), and each stall was raised . cm off of the ground (s fig) . stalls were constructed from . cm ( . in) galvanized square framed tubing with an open-grate rubber coated floor to permit feces to fall below the stall into collection bins. in compliance with the "guide for the care and use of agricultural animals in research and teaching," on day of life, calves randomized to confinement housing were removed from confinement and placed in box stalls [ ] . all calves were followed for days post-infection. over the course of enrollment, calves were fed every h via nipple buckets with a commercial, non-medicated milk replacer containing % crude protein and % fat (maxicare, land o'lakes, shoreview, mn). water was provided ad libitum. calves were randomly assigned to inoculation (n = ) or negative control groups (n = ). calves in the inoculation group were challenged with a commercially available iowa laboratory isolate of cryptosporidium parvum within - h from birth at a dose of × oocysts (bunch grass farms, deary, id). oocysts were administered within month of original isolation and were cleaned for one minute in . % sodium hypochlorite to inactivate possible viruses and bacteria co-purified with the oocysts, then washed four times with phosphate buffered saline to remove the sodium hypochlorite. the oocysts were delivered orally in a ml suspension via the rigid oral portion of an esophageal feeder, followed by approximately ml of water to ensure that the entire oocyst suspension was given to the calf. negative control calves were sham challenged to maintain blinding. all calves in confinement housing underwent cfc every h and had an ic sample collected every h. calves in box stalls had an ic sample collected every h. for ic sampling, up to g of stool was collected directly from the rectum of the calf via digital manipulation with a gloved hand. cfc methods previously described for stool collection were used [ ] . briefly, a fecal collection pan was placed beneath each calf, and urine was diverted to a h adult diaper to prevent contamination of the fecal pans. all stool from the fecal pan was collected every h and blended for homogenization to ensure uniform oocyst distribution. the contents of the blender were then weighed (kg). after homogenization, a . g aliquot of feces was removed from all ic and cfc samples for enumeration via real-time quantitative polymerase chain reaction (qpcr). oocysts counts were interpolated by qpcr using serial dilutions of commercially purified c. parvum oocysts (waterborne, inc., new orleans, la). total nucleic acid was extracted from supernatants of mg of fecal sample, oocyst suspension, or negative control homogenized in μl of pbs using a magnetic bead based automated procedure (am , applied biosytems, foster city, ca). an exogenous control (ms phage) was added to the lysis buffer to control for extraction efficiency [ ] . qpcr for cryptosporidium spp. s rrna was performed using a previously described assay on the applied biosystems -fast placfcrm using an inhibitor-optimized master mix (part , quantabio, beverly, ma), nm forward primer, nm reverse primer and nm of probe (labeled with fam) [ ] . for both cfc and ic samples, the count was standardized by the fecal dry weight percentage. dry weight analysis of fecal samples was obtained by taking a - g portion of each original fecal sample, drying it at ˚c for a minimum of h (squaroid vaccuum oven, labline, kochi, india), then weighing it directly (scout pro sp , ohaus corporation, pine brook, nj) [ ] . the calves were examined and evaluated every - h. a complete physical exam was conducted every h at feeding time. at the time of physical exam any new injuries or clinical signs were documented, this included abrasions and wounds, as well as swellings, areas of localized pain, and fever. in order to minimize inter-observer bias, standardized and validated rubrics were used as previously described [ , , , ] . in addition, all research personnel underwent training to maximize inter-observer agreement. appetite, fecal consistency, mentation (state of mental activity and responsiveness), and hydration status were each individually evaluated on an ordinal scale of - , where a score of represented normal clinical findings and a score of was consistent with severe clinical illness in the specified category (s table) . qualitative measures of hydration such as skin turgor and degree of enophthalmos were used. monitoring for enophthalmos was necessary for identifying calves at risk for development of corneal ulcers. as severity of dehydration worsens in calves and the globes of the eyes recede further into the orbit, the eyelids roll inward causing the eyelashes to rest directly on the cornea, abrading the surface. hydration status was also assessed quantitatively via measurement of urine production every h. to determine urine production without the use of urinary catheters, an adult diaper was weighed, secured over the calf's penis, and re-weighed to determine the difference (kg). blood samples were collected via jugular venipuncture on days , , , and post-infection. all samples were collected at : a.m. samples were obtained by individuals trained in blood collection using a vacutainer system. for all enrolled calves, to reduce the risk of falsely elevating cortisol levels, efforts were made not to create excessive stress prior to blood collection, e.g., loud noises, changes in lighting, use of excessive restraint, more than venipuncture attempts. calves in confinement housing were already restrained within the housing system. additional restraint of these animals was not required and could have resulted in additional stress. blood was collected from calves in confinement housing by reaching directly through the bars of the housing mechanism. calves in box stalls were restrained in sternal recumbancy to visualize the jugular vein. restraint was for less than sec and attempts were made to avoid chasing calves to capture them in the stalls. however, calves in box stalls were generally more energetic and therefore ambulated more frequently prior to blood collection. cortisol was extracted from calf plasma by solid phase extraction and quantified using ultra performance liquid chromatography in tandem with mass spectrometry (uplc-ms/ms). cortisol and cortisol-d were purchased from cerilliant (round rock, tx) and stock solutions were separately prepared in % methanol at concentrations of μg/ml and ng/ ml, respectively. a calibration curve was generated in calf plasma at nominal concentrations of , , , , , , , , , and ng/ml. quality control (qc) samples were prepared at nominal concentrations of , , and ng/ml. μl of calibration and qc samples were added to a -well plate along with the study samples. next, μl of the cortisol-d solution was added to each well followed by addition of μl methanol. samples were aspirated and then centrifuged at rcf for minutes. following centrifugation, μl of the resulting supernatant was removed and placed in a well plate. μl of % phosphoric acid was added to each μl aliquot, and the samples were mixed thoroughly. samples were then placed in an oasis prime hlb μelution plate (waters, milford, ma) and washed with aliquots of μl % methanol. next, aliquots of / acetonitrile:methanol were added to each sample, and the resulting eluate was diluted with μl water. . μl of each sample was injected onto an acquity uplc in tandem with a waters xevo tq-s. a zorbax eclipse plus c column (agilent, santa clara, ca) heated to ˚c and mobile phases a: water with . % formic acid and b: acetonitrile with . % formic acid were used with the following gradient: %- % a over the first minute, %- % a from to . minutes, a further decrease to % a until minutes, followed by an increase back to % a until minutes. the flow rate of the solvents was . ml/min. the transitions (m/z) . > . and . > . were used to quantify cortisol and cortisol-d , respectively. endogenous cortisol observed in the calf serum was used to generate the calibration curve, and the amount of naturally occurring cortisol was subtracted from each calibration curve and qc sample. all qc samples were within % of their expected values, and the coefficients of variation (cv%) were < %. descriptive and inferential methods were used. the shapiro-wilk test was used to determine if the data was non-gaussian. hypothesis tests for normally distributed continuous data were analyzed using the student's t test or analysis of variance. categorical variables were tested via chi-square or fisher's exact. non-normal continuous variables were either log-transformed or analyzed via the wilcoxon rank sum test. the wilcoxon rank sum was used to assess differences in fecal oocyst counts and fecal dry matter percentage between ic and cfc samples for confinement housing calves and between ic samples in confinement housing and box stall calves. the wilcoxon rank sum test was also used to evaluate the differences in plasma cortisol, daily weight gain, daily milk replacer consumption, volume of fluid therapy, and frequency of non-fluid therapy treatments in confinement housing and box stall calves. chi-square or fisher's exact tests were used to describe the association between frequency of injury and housing type. the frequency of injury was counted once at the time of diagnosis. the frequency of non-fluid therapy treatment was counted with every intervention following diagnosis. the frequency of injury and non-fluid therapy treatments were recorded in this manner to enable the ability to track the occurrence of new injuries as well as to assess the amount of researcher effort required to subsequently treat and manage those injuries. bivariate analysis was used to evaluate the relationship between log oocysts per gram of fecal dry matter and plasma cortisol. in order to identify the best predictors of total fecal output, simple backward stepwise regression was carried out to achieve the most parsimonious model. individual explanatory variables with a p-value . in bivariate analysis were retained. data were analyzed using jmp pro . (sas institute inc., cary, nc). enrolled calves weighed an average of . ± . kg at birth (range . - . kg). the mean serum total protein measurement at h of life was . ± . (g/dl) (range . - . g/dl). there were no differences in serum total protein across groups (p = . ), and no calves experienced failure of passive transfer. all calves challenged with c. parvum oocysts developed fecal oocyst shedding within days post-infection (pi) ( % at day pi, % at days pi, and % at days pi). the median duration from parasite challenge to onset of fulminant diarrhea and clinical illness consistent with cryptosporidiosis was days post-infection ( . ± . ). on day pi, all calves were still shedding oocysts in their stool. stool was tested via qpcr for coinfection. all enrolled calves were negative for e. coli k , salmonella spp., rotavirus, and corona virus. the two negative control calves did not develop cryptosporidiosis. in order to prevent calves from turning around in the stall and urinating in the fecal collection bin, we applied the head-catch to animals. both calves died after they became cast in the stall and were unable to rise. these animals were removed from the study. final analysis included confinement housed calves. for calves housed in confinement housing (n = ), the mean total fecal output was . ± . kg/day (range . - . kg/day) and a mean total urine output was . ± . kg/day (range . - . kg/day) during the day study period ( table ). the uninfected control calf in confinement housing had a mean total fecal output of . ± . kg/day (range . - . kg/day) and a total mean urine output of . ± . kg/day (range . - . kg/day) ( table ) . over the day period of confinement, there were no significant differences in mean log oocysts enumerated per gram of fecal dry matter between cfc ( . ± . ) and ic samples ( . ± . ) (p = . ), nor were there any diurnal variations in oocyst shedding for morning and evening ic fecal samples (p = . ) (fig ) . the fecal dry matter percent was significantly lower in cfc samples (p = . ) ( table ) . table . descriptive summary of daily and stool and urine production for calves (n = ) in confinement housing from days to post-infection. stool production/day (kg) mean ± sd . ± . -- urine production/day (kg) mean ± sd . ± . --iqr . - . fecal dry matter percentage/day mean ± sd . ± . à . when comparing across calves in confinement housing (n = ) and calves in box stalls (n = ), calves in confinement housing shed significantly more oocysts in their stool (p = . ) (fig ) . the mean peak in oocyst shedding for confinement housing calves was log . oocysts/ gram fecal dry matter, which is nearly orders of magnitude greater than the mean peak in box stall calves. fecal dry matter percentage was significantly lower in cfc samples from confinement housing calves as compared to ic samples from box stall calves (p = . ), however, there was no significant difference (p = . ) in fecal dry matter percentage for ic samples collected from confinement housing and box stall calves (tables and ) . for days - post-infection, calves in confinement housing experienced significantly more severe clinical outcomes in comparison to box stall calves across all study end-points except for average daily weight gain, even though calves in confinement housing consumed less milk replacer (p = . ) ( table ) . calves in confinement housing required significantly more fluid therapy to maintain their hydration status both during the day period of confinement (p = . ) and after being removed from confinement and placed in box stalls (p = . ) ( table ) (s fig). on average, for days - post-infection, confinement housing calves required . l/day of parenteral or oral fluid therapy while box stall calves required . l/day (table ) . calves in confinement housing were also more likely to become injured. the frequency of abrasions or lacerations was significantly greater (p = . ) in confinement housing calves as was the frequency of pressure sores (p = . ) ( table ). confinement impact of confinement housing in the calf model of cryptosporidiosis housing calves received significantly more non-fluid therapy supportive care (p = . ) in the form of administered pain medication, topical eye and skin treatments, wound care, and bandage changes. plasma cortisol samples from infected confinement housing calves (n = ) and box stall calves (n = ) were evaluated on days , , , and post-infection. the distributions of plasma cortisol and log oocysts per gram of fecal dry matter were plotted and outliers in the confinement housing group were identified and removed from analysis. the first measured plasma cortisol value for each of these calves was greater than ng/ml. testing was limited to calves in confinement and calves in box stalls due to sample loss. mean plasma cortisol levels were significantly higher in confinement housing calves ( . ± ng/ml) than in box stall calves ( . ± ng/ml) (p = . ) (fig ) . as the mean plasma cortisol level increased, the mean log oocysts per gram of fecal dry matter significantly increased (p < . ) (fig ) ( table ). for every point increase in plasma cortisol there was a . log increase in oocyst shedding (fig ) . both housing groups initially had similar plasma cortisol levels, but the table . descriptive summary of study end-points for calves in confinement housing (n = ) and box stalls (n = ) from days to post-infection. table . descriptive summary of supportive care administered to calves in confinement housing (n = ) and box stalls (n = ). daily to determine the best predictors of total fecal output, backward stepwise regression was carried out. the following explanatory variables were entered into the model: plasma cortisol, percent of milk replacer consumed, log oocysts per gram of fecal dry matter, and fecal dry matter percentage. also included were the interactions for log oocysts per gram of fecal dry matter and plasma cortisol, and for log oocysts per gram of fecal dry matter and fecal dry matter percentage. the interaction terms were found to be non-significant and were removed from the model. the remaining variables were retained. log oocysts per gram of fecal dry matter was found to be non-significant (p = . ) and was removed. the best predictors of total fecal output were plasma cortisol (p = . ), percent of milk replacer consumed (p = . ), and fecal dry matter percentage (p = . ) ( table ). recent large multicenter studies investigating etiologies of diarrhea in children have demonstrated that cryptosporidium spp. is one of the main pathogens contributing to the global burden of pediatric diarrhea, yet there are no vaccines to prevent infection and the only licensed drug for treatment has poor efficacy [ , ] . efforts to accelerate identification of a lead drug candidate have generated increased demand on the calf model of cryptosporidiosis. this surge in demand has underscored the need to critically evaluate and refine the calf model of cryptosporidiosis in order to garner the best data possible for application in veterinary medicine and translation to human patients. alarmingly, as the model has been more frequently applied, inconsistencies in model execution have come to light. one of the most pronounced inconsistencies pertains to use of confinement housing for research calves. the desire to mimic human challenge studies and collect the total fecal output from research calves has necessitated the use of confinement housing [ , , , ] . use of confinement housing conflicts with the five freedoms of animals, which are commonly used to define parameters for animal welfare and to determine if animal housing, environment, and handling are adequate [ ] [ ] [ ] . in the case of confinement housing, the freedom to express natural behaviors is not observed. in research settings, the five freedoms may be compromised if there is scientific justification or if no other acceptable alternative exists. to date, no one has evaluated confinement housing side-by-side with box stall housing to determine if box stall housing is an acceptable alternative in the calf model of cryptosporidiosis. our study demonstrates that use of confinement housing is unnecessary to measure study end-points such as total oocyst excretion and diarrhea quantity and quality. moreover, the use of confinement housing appears to stress calves and lead to increased oocyst excretion that may confound data and underestimate the efficacy of the chemotherapeutics being studied. impact of confinement housing in the calf model of cryptosporidiosis our findings challenge the assumption that cfc is required to get accurate measurements of oocyst excretion or the quantity and quality of diarrhea. first, we did not find a difference in log fecal oocysts enumerated per gram of fecal dry matter in cfc or ic samples among calves in confinement. previously, it was thought that homogenization of the cfc sample would result in more accurate oocyst enumeration [ ] , but this was not supported by our data. to the contrary, there was greater variation in oocyst enumeration for cfc samples, as is evidenced by the larger standard error bars (fig ) . since these counts are standardized to the fecal dry matter percentage, this would indicate that these differences are attributable to increased water content of the stool sample. it has also been suggested that cfc must be collected and homogenized due to possible diurnal variations in fecal oocyst shedding [ , ] , but we observed no significant difference in the number of oocysts enumerated in samples collected in the morning or evening. it is important to note that while cfc permits capture of the total fecal output, the entire cfc sample is not analyzed. for both cfc and ic a mg aliquot of stool is homogenized with equal amounts of pbs. after qpcr is performed, oocyst counts are standardized by fecal dry matter percentage. therefore, our findings indicate that a representative stool sample can be attained via ic and collection of the total fecal output is not necessary in order to attain a representative stool sample for oocyst enumeration via qpcr. the fecal dry matter percentage was significantly lower in cfc samples as compared to ic collected from calves in confinement. this was controlled for in oocyst enumeration by standardizing to the fecal dry matter percentage. the increased water content in cfc samples could be from more severe diarrhea or improved ability to measure water content due to sample homogenization. alternately, it could be due to urine contamination of the fecal bin which has been previously described or to increased urine production secondary to increased frequency of fluid therapy [ ] . although adult diapers were secured to the calves using duct tape ( m, st. paul, mn) and elastikon (johnson & johnson, new brunswick, nj) diapers did slide out of place with calf movement and exposed the penis. in addition, during the first - days of confinement, calves were small enough to turn around in the stall, thus there were instances when they defecated in the urine bin, which would falsely lower the fecal dry matter percentage of stool collected from the fecal bin and falsely elevate the volume of urine collected (s fig). if the lower fecal dry matter percentage detected in confinement housing calves is a true finding, indicating that cfc better detects increased water content in the stool, this would indicate that confinement exacerbates severity of diarrhea. this is a possibility, given that calves in confinement housing required significantly more supportive care than calves in box stalls. calves in confinement housing received an average of . l of fluid therapy each day, as compared to box stall calves, which only received . l/day. thus, confinement housing calves may experience greater volume of fluid elimination due to greater volume of fluid support. however, when comparing ic samples from confinement housing calves to ic samples from box stall calves, there is no significant difference in fecal dry matter percentage. thus, it is probable that the difference in fecal dry matter percentage between cfc and ic samples for confinement housing calves is at least partially attributable to urine contamination of the fecal bin. calves in confinement housing shed significantly more oocysts in their stool. at peak shedding, this difference was orders of magnitude greater in confinement housing calves. since there was no difference in shedding detected in ic or cfc samples from confinement housing calves, it stands to reason that the difference in shedding detected between box stall and confinement housing calves is a true effect. moreover, since all qpcr data was standardized to the fecal dry matter percentage, this data is not susceptible to dilutional effects of increased water content in the stool sample. it is possible that this difference in fecal oocyst shedding can be explained by the significant difference in plasma cortisol concentration for confinement housing and box stall calves. in healthy calves, plasma cortisol concentration is reported to be high at birth ( - ng/ml) and gradually decreases over the first days of life ( - ng/ml) [ , ] , which is consistent with our findings for box stall calves. calves in confinement housing impact of confinement housing in the calf model of cryptosporidiosis did not follow this pattern. plasma cortisol was elevated at - days post-infection ( ng/ml) in confinement housing calves, coinciding with the observed peak in fecal oocyst shedding. thus, it is feasible that increased stress due to confinement exacerbated the degree of fecal oocyst shedding in confinement housing calves. this conclusion is supported in the bivariate analysis in which plasma cortisol level and log oocysts per gram fecal dry matter were significantly associated. it could be argued that confinement housing effectively creates an immunosuppressed calf model of cryptosporidiosis. this may be deemed desirable by cryptosporidium researchers, as many children infected with cryptosporidium are immunocompromised due to other comorbidities. however, unlike immune suppressed mouse models, where diminished immune function is intentionally induced, the presumed reduction in immune function due to hypercortisolemia for calves in confinement housing is a pathological state. while chemotherapeutic efficacy in the face of immunocompromise is desirable, when testing chemotherapeutics in animal models, the degree of immune suppression induced must be predictable and repeatable. our findings also indicate that calves in confinement housing were substantially more physically and metabolically decompensated than calves in box stalls. confinement housing calves required more fluid therapy and ate significantly less milk replacer. reduced milk replacer consumption is an indication of reduced calf wellbeing. in the calf model of cryptosporidiosis this has additional relevance, as failure to meet daily fluid requirements through consumption of milk replacer puts calves at greater risk for severe dehydration and development of metabolic acidosis, which can result in calf death or removal from the study. confinement housing calves also received significantly more non-fluid treatments. not only does this indicate reduced calf welfare, it also indicates increased time demands on study personnel for provision of care to sick calves. box stall calves experienced no abrasions, lacerations, or pressure sores. these types of injuries only occurred in confinement housing calves, as they were more likely to injure themselves on the metal framework of the calf stall, and the lack of shock absorption when lying down predisposed calves to repeated injury over their joints and bony prominences. the severity and duration of these injuries may have been impacted by elevated plasma cortisol levels. mice placed in confinement to induce repeated restraint stress experienced impaired wound healing, partially through stress-induced glucocorticoid release [ ] . given these findings, we wanted to determine if variables that could be collected without the use of confinement housing would accurately predict the total fecal output produced by a calf experimentally infected with c. parvum. in our regression model, holding other variables constant, plasma cortisol, the percent of milk replacer consumed, and the fecal dry matter percentage from ic samples were the best predictors of the total fecal output measured via cfc. these variables accounted for the greatest amount of inter-calf variation in total fecal output. this indicates that when used in combination, these study end-points are better measures of a treatment effect than total fecal output alone. the regression model also suggests that the total fecal output is sensitive to hypercortisolemia. interestingly, the log oocysts per gram of fecal dry matter was not a good predictor of total fecal output. a previous study has shown that the magnitude of oocyst shedding tracks with the ordinal measures of severity of diarrhea [ ] . when considered alongside our data, this indicates that ordinal measures of severity of diarrhea and total fecal output are not synonymous, and that fecal dry matter percentage is a better proxy measure of total fecal output. the findings of this study suggest that confinement housing of calves may affect study endpoints that are important for the evaluation of novel chemotherapeutics targeting cryptosporidium. fecal oocyst shedding, appetite, and frequency of fluid therapy are study end-points that were all significantly impacted by the use of confinement housing. our results indicate that representative data can be captured via ic from calves housed in box stalls, and that this data is less likely to be impacted by the immunomodulatory effects of stress hypercortisolemia. while collection of total fecal output mirrors methodologies used in human challenge studies, use of confinement to capture total fecal output confounds data used to assess other study end-points and reduces overall comparability to the human challenge model. given that confinement housing calves experience increased severity of illness, higher plasma cortisol levels, greater risk of injury, and higher requirements for fluid therapy, use of confinement housing to evaluate novel chemotherapeutics targeting cryptosporidium spp. may result in reduced ability to detect a treatment effect. that said, our study is limited by the fact that we did not compare outcomes in the two housing systems during a trial of an actual cryptosporidium therapeutic. however, box stall housing has recently been used successfully in a trial evaluating a novel drug targeting cryptosporidium [ ] . our study was also subject to potential bias due to the inability to blind observers to housing system. for this reason, we did not report and evaluate subjective observer assessments in this study, such as mentation. these assessments were restricted to use in determining need for supportive care. to reduce risk of bias in evaluation of injuries, severity was not assessed. instead assessments of injuries, such as abrasion or pressure sore, were dichotomous: present or not present. blinding was maintained for laboratory analysis of stool and blood samples. while we were able to report an association between plasma cortisol and oocyst shedding, this study was not designed to assess stress as a primary end-point. cortisol is just one marker of stress. a more complete assessment of stress, including behavioral assessment, and other markers such as substance p, would aid in the interpretation of the data. another concern is the loss of calves in the confinement group due to death, which could result in imprecise estimates. however, we feel that imprecise estimates are the minor concern relative to the major concern of compromised research animal welfare. when considered together, the risk of imprecise measurements due to loss as well as reduced welfare resulting in death and injury, further substantiates the need to revise the methods used for studying cryptosporidiosis in the calf model. lastly, while the study authors have extensive experience using box stalls, this was our first study using confinement housing. it could be argued that in more experienced hands the study findings may have been different. to address this, we consulted with researchers currently executing these studies in confinement and followed protocols identical to those previously published [ ] . in conclusion, it is our opinion that box stall housing should be preferentially used in the calf model of cryptosporidiosis, as confinement housing compromises calf welfare, alters important outcomes, appears to stress calves, and leads to sicker and more heavily infected calves. supporting information s table. rubric for the evaluation of appetite, mentation, fecal consistency, and hydration. cryptosporidium: a water-borne zoonotic parasite association between management practices and within-herd prevalence of cryptosporidium parvum shedding on dairy farms in southern ontario prevalence of cryptosporidium parvum infection in southwestern ontario and its association with diarrhea in neonatal dairy calves cryptosporidium spp. and cryptosporidiosis. microbiol rev impact of confinement housing in the calf model of cryptosporidiosis plos neglected tropical diseases quantitative risk assessment of cryptosporidium species infection in dairy calves burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the global enteric multicenter study, gems): a prospective, case-control study pathogen-specific burdens of community diarrhoea in developing countries: a multisite birth cohort study (mal-ed). lancet glob health asymptomatic and symptomatic cryptosporidiosis: their acute effect on weight gain in peruvian children parasite prevalence and the worldwide distribution of cognitive ability effects of cryptosporidium parvum infection in peruvian children: growth faltering and subsequent catch-up growth prophylactic use of decoquinate for infections with cryptosporidium parvum in experimentally challenged neonatal calves paromomycin is effective as prophylaxis for cryptosporidiosis in dairy calves effect of nitazoxanide on cryptosporidiosis in experimentally infected neonatal dairy calves prophylactic and therapeutic efficacy of nitazoxanide against cryptosporidium parvum in experimentally challenged neonatal calves effect of nitazoxanide on morbidity and mortality in zambian children with cryptosporidiosis: a randomised controlled trial efficacy of halofuginone lactate in the prevention of cryptosporidiosis in suckling calves effect of halofuginone lactate on the occurrence of cryptosporidium parvum and growth of neonatal dairy calves european public assessment report: halocur (halofuginone) continuous culture of cryptosporidium parvum using hollow fiber technology novel bioengineered three-dimensional human intestinal model for long-term infection of cryptosporidium parvum. infect immun infectivity of cryptosporidium muris (strain rn ) in various laboratory animals immune responses to cryptosporidium muris and cryptosporidium parvum in adult immunocompetent or immunocompromised (nude and scid) mice protein malnutrition impairs intestinal epithelial cell turnover, a potential mechanism of increased cryptosporidiosis in a murine model antibody fusions reduce onset of experimental cryptosporidium parvum infection in calves novel bumped kinase inhibitors are safe and effective therapeutics in the calf clinical model for cryptosporidiosis cryptosporidium parvum: determination of id and the dose-response relationship in experimentally challenged dairy calves description of fecal shedding of cryptosporidium parvum oocysts in experimentally challenged dairy calves effect of nutritional plane on health and performance in dairy calves after experimental infection with cryptosporidium parvum the infectivity of cryptosporidium parvum in healthy volunteers infectivity of cryptosporidium parvum in healthy adults with pre-existing anti-c. parvum serum immunoglobulin g federation of animal science societies. guide for the care and use of agricultural animals in research and teaching comparison of four methods of calf confinement. i. physiology neonatal changes in the plasma levels of cortisol, cortisone and aldosterone in the calf use of bacteriophage ms as an internal control in viral reverse transcription-pcr assays correlation between diarrhea severity and oocyst count via quantitative pcr or fluorescence microscopy in experimental cryptosporidiosis in calves a comparison of fecal percent dry matter and number of cryptosporidium parvum oocysts shed to observational fecal consistency scoring in dairy calves cryptosporidium parvum: intensity of infection and oocyst excretion patterns in healthy volunteers cryptosporidium hominis: experimental challenge of healthy adults report of the technical committee to enquire into the welfare of animals kept under intensive livestock husbandry systems moving beyond the "five freedoms" by updating the "five provisions" and introducing aligned updating animal welfare thinking: moving beyond the "five freedoms" towards "a life worth living hemato-biochemical and cortisol profile of holstein growing-calves supplemented with vitamin c during summer season restraint stress impairs early wound healing in mice via alpha-adrenergic but not beta-adrenergic receptors a cryptosporidium pi( )k inhibitor is a drug candidate for cryptosporidiosis the authors would like to thank the undergraduate and veterinary students who tirelessly cared for the calves and collected data points, amber adams-progar for essential inputs on housing and calf behavior, renee anderson for qpcr technical support, mike kluzik for operational and logistical assistance, the office of the campus veterinarian, in particular, gay lynn clyde, nina woodford, and gwen anderson for providing care to the calves, the animal resource unit, and specifically jan luft and josh steele, who insured excellent husbandry and facility management, and wesley van voorhis and douglas call for their thoughtful review and editing of the manuscript. we also wish to express our gratitude to jaime andrade and d dairy farm for their unwavering support of this research, and to lazaro and the maternity pen team for their patience, flexibility, and good humor. lastly, we want to thank the calves and to acknowledge their invaluable contribution to the advancement of science and medicine. conceptualization: geneva graef, tracy l. sy, jennifer a. zambriski. key: cord- -ipoelk h authors: crouch, c. f. title: vaccination against enteric rota and coronaviruses in cattle and pigs: enhancement of lactogenic immunity date: - - journal: vaccine doi: . /s - x( ) - sha: doc_id: cord_uid: ipoelk h passive immunity against enteric viral infections is dependent upon the continual presence in the gut lumen of a protective level of specific antibodies. this article examines methods currently used to enhance the titre and duration of specific antibody in the mammary secretions of cows and pigs with particular reference to rotavirus and coronavirus infections. in addition, some of the potential problems to be found in attempting to produce vaccines against these viral infections are outlined neonatal diarrhoea is a complex disease associated with a number of infectious agents occurring either singly or in combination ~- . in domestic animals economic losses are suffered, as a result of mortality (ranging between and %), and also veterinary costs and decreased productivity of the survivors. the viral agents most commonly associated with this syndrome are rotavirus and coronavirus, both of which have been found to be primary pathogens in calves ,~ and piglets - . these viruses are most frequently isolated during the period from birth to weaning, and animals of this age have been the most intensively studied because of the frequency and severity of these infections. animals of all ages are, however, susceptible, with subclinical infections apparently common in both adult cows and pigs ,'°. in neonatal calves the incidence of rotavirus and coronavirus associated diarrhoea is similar varying between and % '~'-~ . the situation in neonatal piglets is less clear, rotavirus infections are apparently common .t -tt, w.hilst transmissible gastroenteritis virus (tgev), the prototype enteric coronavirus in swine, is an example of a seasonal cold-weather disease, probably related to both the thermal sensitivity of the virus ~ and the effect of cold-stress on converting subclinical to clinical infections ~ . the pathogenesis of enteric rotavirus and coronavirus diseases of swine and cattle are similar. in contrast to tgev, however, rotaviruses appear to be confined to the alimentary tract, predominantly the small intestine, although there is some evidence in both lambs and piglets for infection of the large intestinal , °. the infections are characterized by diarrhoea and dehydration caused by the functional and anatomical loss of the absorbtive cells of the intestine. the principal site of virus replication has been shown to be the intestinal villus epithelium. the infected cells are lost from the tips of the villi and are replaced with immature crypt cells. generally, there is a dimunition in the number and size of the villi and a progressive replacement of the epithelium with squamous and cuboidal cells which lack a brush bordep - .~. °- . such immature cells have been shown to possess reduced levels of disaccharidases =~. . the loss of the absorptive cells of the intestine is assumed to result in the observed malabsorption syndrome. this is further exacerbated by the decreased ability to utilize dietary lactose, resulting in its accumulation in the large intestine, thereby preventing further absorption of water by exerting an osmotic effect. as a result of the severity of these enteric viral infections during the first few weeks of life, passively acquired antibody is the major source of protection. in calves and pigs there is no selective transfer of immunogiobulins from the maternal to the foetal circulation during the last third of the pregnancy. instead, during the period immediately following birth, maternal immunoglobulin is acquired from the colostrum ofthe dam z ' °. absorption of colostral immunoglobulins by the intestinal epithelial cells is a non-selective process t- lasting - h , s. factors present in colostrum may influence the absorption of immunoglobulins , or help prevent their proteolytic degredation . in addition to immunogiobulins, colostrum and milk have recently been shown to contain functional immunocompetent cells including macrophages and t and b lymphocytes '~°. in contrast to colostral absorption, highly specific mechanisms operate in the colostrum-forming m a m m a r y glands of cattle and pigs causing large amounts of igg (relative to iga and igm) to concentrate in the coiostrum ' ' q ' . lgg passively acquired by the neonate from colostrum persists in the serum for several weeks protecting against systemic infection. in tgev infection of pigs ' and rotavirus infection of calves 'g circulating antibody has been found to be of little value. resistance to these infections appears to be mediated instead by local immunity at the epithelial surface of the intestine. in cattle the selective transfer of igg~ from serum to milk continues throughout lactation, although at a reduced level when compared with colostrum formation. the concentration of all three classes ofimmunoglobulin is significantly reduced ( to -fold) in milk and in consequence igg~ remains the primary immunoglobulin in bovine milk ~. in contrasl in pigs the concentration of lgg~ decreases about -fold during the first week of lactation, whilst that of secretory lga declines only about three-fold, leaving it to become the predominant class of immunoglobulin in swine milld t,' . most adult cattle are seropositive for both rotavirus ,s° and coronavirus s~ antibodies. there is a dramatic decline in these colostral antibody titres during the transition to milld ' "s~- , reflecting this reduction in concentration of immunoglobulins. this partially explains the high incidence of rotavirus and coronavirus infections in calves older than five days, as the titres of passively derived protective antibody decline. despite the presence of one or more common antigens it has been demonstrated that rotaviruses isolated from different species can differ antigenically from each other s - . more recently it has been shown that different serotypes exist within isolates obtained from single species. the existence of at least two different serotypes of porcine rotavirus ° and at least three distinct bovine serotypes ~ have been described. bridger et al have suggested the occurrence ofintermediate bovine rotavirus types , although more work is essential to clarify this situation. some recent isolates possessing the distinctive morphology of rotaviruses have been found to lack the common group antigen. to date, these atypical rotaviruses have been isolated from humans, birds, calves, lambs and pigs ~ . in pigs, preliminary results using two previously characterized atypical isolates , ° have indicated that these are distinct and do not share a common group antigen g ,~'. these observations have been extended by snodgrass et al ga who suggest the occurrence of at least four distinct groups ofrotaviruses based on their group antigen. the significance of the serotypic differences observed between rotaviruses in vitro still needs to be fully assessed in vivo. orbiviruses (also members of the reoviridae) possess many serotypes and require the use of multivalent vaccines . many of the cross-protection studies carried out using different rotavirus serotypes are contradictory and the data inconclusive. for example, in utero vaccination of calves with a bovine rotavirus was found to protect against diarrhoea caused by challenge with human rotavirus serotype , although challenge virus was still shed v . in contrast, one out of three calves was protected against a bovine rotavirus challenge after vaccination with a human serotype or an equine rotavirus tm and this animal shed no detectable virus. furthermore, piglets vaccinated with human rotavirus and challenged with porcine rotavirus were protected against the clinical disease but enhancement of lactogenic immunity:. c f. crouch shed virus ~s. using a more defined challenge system. evidence has been obtained indicating that rotavirus isolates from different animal species and of different serotypes show poor cross-protective properties in vivo tm. this observation has been confirmed and extended by studies in gnotobiotic calves and piglets showing that cross-protection only occurred between rotaviruses of the same serotype, and that even a minor serotype difference could be sufficient to affect cross-protection °,g~. further evidence for a lack of cross-protection between rotavirus serotypes can be obtained from studies of sequential infections, where subsequent rotavirus infections were found to be associated with different serotypes ~ . the situation with coronaviruses is simpler. to date, the coronaviruses isolated from mammals and birds have been grouped into four antigenic classes, where little or no cross-reactivity can be demonstrated between classes tm. tgev is antigenically distinct from bovine enteric coronavirus tm as well as from another as yet unclassified coronavirus causing diarrhoea in pigs (cv ) °. two approaches have been used in an attempt to provide calves with protection against rotavirus and coronavirus infections. the first approach involves oral vaccination with live attenuated virus in order to stimulate active immunity in the calf(scourvax ii, norden laboratories). the incidence of diarrhoea in neonatal calves orally vaccinated with attenuated rotavirus was found to be reduced ~- , but the vaccine was not proven to be effective in blind field trials s - . there are a number of limitations associated with this approach. these include the potential of the vaccine to regain virulence: a high incidence of seropositive adult animals, leading to the possibility of interference with vaccine virus replication by maternally derived (milk) antibodies: and the relative immaturity of the neonate's immune system, the second approach utilizes passive protection produced through lactogenic immunity, stimulated by maternal vaccination. attempts to vaccinate dams using an attenuated live vaccine (calf guard, norden laboratories) have failed to significantly enhance milk antibody titres ", ~ (table ) , whilst in- activated, adjuvanted rotavirus preparations have been found to enhance levels of specific antibody in coiostrum and milk (tables and ) . a number of parameters need to be considered in attempting to optimize the enhancement of antibody production in mammary secretions. dose and form of vaccine. in considering inactivated vaccines, it ~s to be expected that relatively large amounts are necessary to achieve a satisfactory response. further. the process of inactivation may decrease the immunogenicity of some viral polypeptides. table shows that no significant differences in milk antibody titres were obtained following vaccination of cows with rotavirus preparations containing either or elisa units (after inactivation) emulsified in freund's incomplete adjuvant. in contrast, if the same preparations were used. but adjuvanted with aluminium phosphate, the higher dose resulted in a greater antibody response. a similar result using an oil adjuvanted rotavirus vaccine has been previously reported ss. formaldehyde inactivated rotavirus vaccines have been used to successfully enhance milk antibody titres as compared with controls s "- °. other workers have reported increased antibody responses using/~-propriolactone as the inactivating agentol although saif et al found that antibody titres in mammary secretions were at least tenfold greater from cows vaccinated with binary ethylenimine inactivated rotavirus compared with those vaccinated with , -propriolactone inactivated rotavirus . adjuvant snodgrass et a found that oil-based adjuvants were more effective than alhydrogel for the enhancement of rotavirus antibody titres in mammary secretions. this concurs with the data presented in table . most workers have demonstrated a satisfactory immune response following vaccination using oil-based adjuvants. generally freunds incomplete adjuvant (table ). route and timing of vaccination. to some extent the route and timing of vaccination are dependent upon the type ofcattle being farmed. thus the intramammary route used successfully by saifetal , whilst applicable to dairy cattle, may not be practical in beef cows. similarly. from an administrative viewpoint a single vaccination would be preferable to a regime utilizing several doses. the majority of studies have reported a significant increase in rotavirus antibody titres in mammary secretions using either subcutaneous or intramuscular injection of oiladjuvanted vaccines. all such vaccines have also proved to be effective when administered as either single or double doses injected prior to or at parturition . - ( table ) . the efficacy of immune milk as a mechanism for providing passive immunity against rotavirus challenge has been examined by a number of workers(table ). the alactogenic antibody originated from either vaccinated (vacc), control (cont) or normal cows (normal). bcalves were either suckled naturally (suckled) or fed a supplemented diet containing antibody (supp). ccalves were either challenged experimentally (exp) or naturally exposed under field conditions (field). d days after challenge edays after birth, r days after start of experiment. ~cows vaccinated with commercial vaccine nr, not reported results, however, are difficult to compare, due to variations in the feeding regime used for the immune milk and also the challenge systems used. the amount and the timing of the feeding oflactogenic antibody and the dose, virulence and serotype of the virus challenge strain used will all affect the apparent susceptibility of the calf to infection. further, in situations where a field challenge has been used. failure of protection may be due to infection by rotavirus serotypes other than those used in the vaccine, or possibly by other agents capable of causing diarrhoea. generally, these investigators reported either a reduced incidence of rotavirus shedding or diarrhoea or both. in only one study did the feeding oflactogenic antibody fail to significantly affect the incidence or onset of diarrhoea. the majority of animals receiving passive immunity appear to be capable of developing active immunity during this period " , consequently vaccination should lead to elimination of clinical disease rather than a delay in its onset investigation of the immunoglobulin isotypes associated with this protective antibody induced by vaccination in bovine milk and colostrum suggests that igg, plays the major role , . these observations are in agreement with those discussed earlier concerning passive immunity in the bovine. in contrast to the bovine system, evidence suggests that milk or colostral immunoglobulin of the iga isotype is more effective than those of the igg isotypes at protecting piglets against infection by tgev -~°°. high persisting levels of lgg may, however, provide some degree of vaccine, vol. , s e p t e m b e r enhancement of lactogenic immunity:. c e crouch protection against virus challeng~ ~. as a result of these observations` most studies have examined methods for optimising the stimulation of secretory iga antibodies in milk. the origin of tgev-specific iga found in mammary secretions remains somewhat obscure, although there is a good correlation with the presence of an infection in the intestinal tract ~, , °°,' . secretory iga in porcine milk is almost certainly locally produced in the mammary glan& °=-~° . in order to explain this phenomenon, it has been suggested that specificallysensitized iga-secreting lymphocytes may migrate to the mammary gland following initial sensitization in the intestine s~-~°°. such an inter-relationship between the intestinal and the mammary immune systems has also been proposed in rabbits =° and humans ~° . direct evidence for such migration, under the influence of pregnancy-associated hormones, has been obtained in micd ° . a summary of various investigations into the antibody response and efficacy of lactogenic immunity following different vaccination protocols is given in table . reduced immunogenicity in pigs of cell culture attenuated tgev has been described ~° . oral vaccination with a live, attenuated tge vaccine, whilst producing neutralizing antibody, did not stimulate good lactogenic immunity in suckling pigs ~°°,~° .~'°. intramuscular vaccination of sows with live, attenuated tgev leads to the enhancement of specific igg levels in colostrum and milld~,"l higher titres oftgev-specific igg have been achieved using intramammary injection, with an associated increase in the protection provided to suckling pigs ~. these results are supported by the observations of other workers "=-" . feline infectious peritonitis virus (fipv) is a member of the same antigenic class as tgev and consequently the two viruses are serologically related. good levels of cross-protection, associated with high titres of tgev-specific neutralizing antibody have been reported in sows vaccinated orally with fipw ~. in contrast, the results of a more recent study have shown that whilst tgev neutralizing antibodies of the lgg subclass are stimulated in milk and colostrum, the survival rate for suckling pigs was low i". it may be possible to boost the level oflga in mammary secretions. preliminary investigations have revealed that specific secretory lga levels in milk can be enhanced by the parenteral injection, at parturition, of tgev or rotavirus into naturally infected (orally primed) animals "s." . a similar approach also combining oral with parenteral antigen administration has been proposed as a means of providing lactogenic immunity against colibacillosis in pigs t=°. lgg can be induced readily in the mammary secretions of cattle, by intramuscular or subcutaneous injection of adjuvanted immunogen. in pigs however, whilst live, virulent virus is capable of inducing high levels in iga iri milk. it is apparent that the ideal candidate vaccine virus must be sufficiently attenuated to produce only mild or no disease in neonatal pigs, whilst retaining sufficient virulence to infect the intestinal tract of adult swine. more work is essential in the possible use of inactivated vaccines for the boosting of existing iga levels in mammary secretions. these may require prior natural infection of the sow, the incidence of which will vary between herds, with an associated affect upon the efficacy of such a vaccine. further investigation into the variety of strains and serotypes of rotaviruses is of obvious importance, as is the response to vaccination of cattle and swine by rotaviruses or coronaviruses. current data suggests that crossprotection between rotavirus serotypes is limited, although there is little information concerning the specificities of the antibodies induced by vaccination of previously infected animals. such animals naturally exposed to a variety of serotypes may produce a heterogeneous antibody response, capable of reacting with a broad spectrum of rotavirus serotypes. it is apparent that the enhancement of lactogenic immunity through the vaccination of the dam provides a suitable mechanism by which neonatal pigs and calves can be protected against rotavirus and coronavirus infections. the production of truly effective vaccines, however, awaits further work in some of the areas outlined above. acute undifferentiated neonatal diarrhoea in beef calves . occurrence and distribution of infectious agents a-l pathogenic relationships of rotavirus, escherichia colz and other agents in mixed infections in calves pathological and microbiological observations made on spontaneous cases of acute neonatal calf diarrhoea pathology of neonatal calf diarrhoea induced by a reo-like virus pathology of neonatal calf diarrhoea induced by a corona-like agent vet_ path the isolation of reovirus-like agents (rotaviruses). from acute gastoenteritis of piglets a transmissible gastroenteritis in pigs reovirus-like agent associated with fatal diarrhoea in pigs shedding of rotavirus in feces of sows before and after farrowing prevalence of rotavirus and coronavirus antigens in the faeces of normal cows monoclonal antibody capture-enzyme-linked immunosorbent assay for the detection of bovine enteric coronavirus coronaviral enteritis of young calves: virologic and pathologic findings in naturally occurring infections diagnosis of viral agents associated with neonatal calf diarrhoea rotaviral diarrhoea in pigs: brief review j. am serologic detection of rotavirus in swine from the region of brittany rearing regimen producing piglet diarrhoea (rotavirus) and its relevance to acute infantile diarrhoea transmissible gastroenteritis of swine effects of ambient temperatures on clinical and immune responses of pigs infected with transmissible gastroenteritis virus rotavirus infection in lambs: pathogenesis and pathology pathogenesis of porcine rotaviral infection in experimentally inoculated gnotobiotic piglets virusintestinal cell interactions, ) immunofluorescence, histopathology and virus production in the small intestine through the course of infection el scanning electron, light and immunofluorescent microscopy of intestine of gnotobiotic calf infected with calf diarrhoeal coronavirus calf diarrhoea (scours) reproduced with a virus from a field outbreak pathological changes in the small intestine of neonatal calves naturally infected with reo-like virus (rotavirus) pathological changes in the small intestine of neonatal pigs infected with a pig reovirus o like agent (rotavirus) serial studies of virus multiplication and intestinal damage in gnotobiotic piglets infected with rotavirus virus particles in epithelial cells of duodenal mucosa from children with acute nonbacterial gastroenteritis changes in intestinal structure and function of neonatal calves infected with reovirus-like agent and escherichia coil austr the transmission of passive immunity from mother to young c the transfer of antibodies by neonates and adults relative efficiency of absorption of igg , igg , iga and igm in the newborn calf the specificity of the intestinal absorption of large molecules by the newborn calf effect of dietary regimen on cessation of intestinal absorption of large molecules (closure) in the neonatal pig and lamb porcine colostral iga and igm antibodies to escherichia coil and their intestinal absorption by the neonatal piglet electrophoretic and immunological studies on sera from calves from birth to weaning acceleration of the absorption of unchanged globulin in the newborn calf by factors in colostrum effect of altered serum glucocorticoid concentrations on the ability of the newborn calf to absorb colostral immunoglobulin trypsin inhibitor from bovine colostrum e colostral cell-mediated immunity and the concept of a common secretory immune system mitogen and antigen response in milk lymphocytes symposium on the young farm animal: the immunoglobulin system of the suckling pig localised humoral immunity with particular reference to ruminants biophysical and immunological studies on bovine immunoglobulins with evidence for selective transport within the mammary gland from maternal plasma to colostrum secretory iga and antibodies to escherichia coil in porcine milk and their significance in the alimentary tract of the young pig transmissible gastroenteritis of swine as a model for the study of enteric d=sease~ gastroenterology levels of colostral antibodies against neonatal calf diarrhoea virus synthesis and distribution of immunoglobulins classes of immunoglobulins related to immunity in the pig: a review studies on rotaviral antibody in bovine serum and lacteal secretions using radioimmunoassay the rotaviruses radioimmunological (ria) and enzymimmunological (elisa) detection of coronavirus antibodies in bovine serum and lacteal secretions vaccination of cows with a combined rotavirus/ enterotoxigenic escherichia coil k vaccine to protect newborn calves against diarrhoea immune responses of pregnant cows to bovine rotavirus =mmunization passive immunity in calf rotavirus infections. maternal vaccination increases and prolongs immunoglobulin g antibody secretion in milk comparison of different antigen preparat=ons as substrates for use in passive haemagglutin~tion and enzyme-linked immunosorbent assays for the detection of antibody against bovine enteric coronavirus` j. c/in. microbiol relation between viruses from acute gastroenteritis of children and newborn calves antigenic relat=onship between human and simian rotaviruses serological relationships between rotaviruses from different species as studied by complement fixation and neutralization morphological and antigenic relationships between viruses (rotaviruses) from acute gastroenteritis of children, calves, piglets, mice, and foals ljo isolation and serotyping of porcine rotaviruses and antigenic comparison with other rotaviruses antigenic relationships among some bovine rotaviruses: serum neutralization and crossprotection in gnotobiotic calves antigenic variation in porcine and bovine rotaviruses detection of a rotavirus-like agent associated with diarrhoea in an infant isolation of human pararotavirus isolation of rotaviruses from turkeys and chickens: demonstration of distinct serotypes and rna electrophoresis detection by electron microscopy of caliciviruses, astroviruses and rotavirus-like particles in the faeces of piglets with diarrhoea rotavirus-like, calicivirus-like and nm virus-like particles associated with diarrhoea in young pigs compar=son of atypical rotaviruses from calves, piglets lambs and man characterisation of an antigenically distinct porcine rotavirus. infec~ immun porcine pararotavirus: detection, differentiation from rotavirus, and pathogenesis in gnotobiotic pigs molecular characterisatlon of rotaviruses with distinct group antigens viruses of vertebrates rotaviral immunity in gnotobiotic calves: heterologous resistance to human virus induced by bovine virus studies on crossprotection induced in calves by rotaviruses of calves, children and foals the clin,cal response of gnotobiotic calves, pigs and lambs to inoculation with human, calf, pig and foal rotavirus isolates antigenic relationships among some animal rotaviruses: virus neutralization in vitro and cross-protection in piglets epidemiologyof human rotavirus type and type as studied by enzyme-linked immunosorbent assay the biology and pathogenesis of coronaviruses comparative structure of coronav=ruses a new coronavirus-like particle associated with diarrhoea in swine neonatal calf diarrhoea: results of a field trial using a reo-like virus vaccine immunity to neonatal calf diarrhoea virus survey of the field efficacy of reoviral calf diarrhoea vaccine licensing and use of the calf scours vaccine. part ii an analysis of scourvax-reo the efficacy of a modified live reo-like virus vaccine and an escherichia coli bacterin for prevention of acute undifferentiated neonatal diarrhoea of beef calves rotavirus infections in calves: efficacy of oral vaccination in endemically infected herds colostral and milk antibody titres in cows vaccinated with a modified live rotaviruscoronavirus vaccine l passive immunity in calf diarrhoea: vaccination with k antigen of enterotoxigenic escherichia coli and rotavirus passive immunity in calf rotavirus infections: maternal vaccination increases and prolongs immunoglobulin g antibody secretion in milk evolution des anticorps anti rota dans le lait de vaches traitees en fin de gestation soit par le vaccine ant=-rota complet, soit par i'adjuvant seul passive protection of newborn calves against rotavirus by vaccination of their dams die wirksamkeitsprufung eimer inaktiv=erten rotavirus-vakzine in einem milchviehbestand passive immunity to bovine rotavirus in newborn calves fed colostrum supplements from immunized or non-immunized cows rotavirus infection in lambs: studies on passive protection development of passive immunity to porcine rotavirus in pigs protected from disease by bovine colostrum diarrhoea in dairy calves reduced by feeding colostrum from cows vaccinated with rotavirus. res. vet-scl antibody responses in serum, colostrum and milk of swine after infection or vaccination with transmissible gastroenteritis virus isolation of porcine immunoglobulins and determination of the immunoglobulin classes of transmissible gastroenteritis viral antibodies passive immunity in transmissible gastroenteritis of swine: immunoglobulin characterisation of antibodies in milk after inoculating virus by different routes passive immunity in transmissible gastroenteritis of swine: immunoglobulin classes of milk after oral/intranasal inoculation of sows with a live low cell culture-passaged virus immunology of transmissible gastroenteritis transfer of immunoglobulins igg, iga and igm to lacteal secretions in the parturient sow and their absorption by the neonatal piglet secretory iga and antibodies to escherichia coli in porcine colostrum and milk and their significance in the alimentary tract of the young pig the transfer of immunoglobulins igg, iga and igm from serum to colostrum and milk in the sow the induction and characteristics of secretory iga antibodies lidinjanson, ( . and sohi-akerlund, a. antibodyforming cells in human colostrum after oral immunization e hormonal induction of the secretory immune system in the mammary gland immunogenicity and distribution of transmissible gastroenteritis in pigs transmissible gastroenteritis in: diseases of swine immunity in tge of swine after infection and vaccination passive immunity in transmissible gastroenteritis of swine: intramuscular injection of pregnant swine with a modified live-virus vaccine experimental immun,zation of sows against transmissible gastroenteritis experimental immunization of sows with cell-cultured tge virus experimental immumzation of sows with inactivated transmissible gastro'enteritis (tge) virus. car~ j present status of products for use against transmissible gastroenteritis cross-protection studies between feline infectious peritonitis and porcine transmissible gastroenteritis viruses efficacy of vaccination of sows with serologically related coronaviruses for control of transmissible gastroenteritis in nursing pigs passive immunity against enteric viral infections passiveimmunityagainstentericviral infections of piglets intestinal defence of the neonatal pig:-inter-relationships of gut and mammary function providing surface immunity against colibacillosis i would like to thank dr s.d. acres for his permission to include some ofthe data obtained during my employment at vido. key: cord- -a l e z authors: selim, s. a.; aziz, k. m. s.; sarker, a. j.; rahman, h. title: rotavirus infection in calves in bangladesh date: journal: vet res commun doi: . /bf sha: doc_id: cord_uid: a l e z faecal samples from calves under year of age ( diarrhoeal and normal) were collected from three dairy farms and one village in selected areas of bangladesh. the samples were tested by an enzyme-linked immunosorbent assay (elisa) to detect the presence of rotavirus antigen. of dairy calves tested, ( . %) were positive, of which ( . %) were from diarrhoeic calves and ( . %) from non-diarrhoeic calves. rotavirus infection varied from farm to farm ( . – . %) and there was no positive response from any of the village calves. rotavirus was most commonly found in calves of week of age or less (up to . % in one group) but was not found in any calves later than months of age. more than % of rotavirus-positive samples from diarrhoeic calves exhibited a titre of or more (geometric mean ± . ), whereas non-diarrhoeal calves had titres less than or equal to (geometric mean= ± . ), suggesting that rotavirus infection in calves in bangladesh was mostly associated with diarrhoea. rotavirus infection in calves is very common, with a worldwide distribution (mcnulty, ; kurstak et al., ) . the role of the virus in causing diarrhoea, especially in young subjects, is well established (mebus et al., % ; woode and crouch, ; castrucci et al., ) . although rotavirus infection in diarrhoeic calves usually involves multiple enteropathogens including escherichiu cob, corona virus, and/or cryptosporidia (morin et al, ) , single infections are not uncommon (tzipori, ) . in calves, the infection is mostly associated with diarrhoea, sometimes as the primary agent, in naturally infected and experimentaily produced cases (mebus et al., % ; woode and crouch, ) and the infection varies widely depending on various factors (mcnulty, ; tzipori, ) . calf diarrhoea (gastroenteritis syndrome) remains the most often reported clinical problem in calf management and in rural conventional cattle rearing systems in bangladesh (debnath et al., ) . this study was undertaken to determine the prevalence of rotavirus infection in selected dairy farms and in conventionally reared village calves in bangladesh, and to study the prevalence of rotavirus in diarrhoeic and age matched non-diarrhoeic calves. farms a and b are close to each other, km apart, in dhaka district. farm c and the selected village are km away in mymensingh district. the populations of calves under year of age on the farms were , and respectively at the beginning of the study. the equivalent calf population in the village could not be ascertained but was estimated to be around so. calves on farms a and b were holstein crossed (fl) with either sindi, sahiwal (tropical breeds) or local improved nondescript zebu. farm c calves were cross breeds between sindi and sahiwal, while most calves of the village were improved zebu crossed with sindi or sahiwal. all the calves were grouped as diarrhoeic (d), having clinical diarrhoea with liquid or semi-liquid faeces, or non-diarrhoeic (n), without any abnormal fluidity of the faeces, regardless of any previous history of illness. all the calves were further categorized into four groups on the basis of age: group i, calves from birth up to week of age; group ii, from over week to month old calves; group iii, calves aged over month to months; and group iv, calves from over months to year old. calves were reared in individual metal calf pens until they were month of age. they were then moved to pens containing lo- calves where they were housed up to year of age. on farm c, the newborn calves were reared in groups of or and transferred to a common calf shed at months of age where they remained up to year of age with occasional moving to similar sheds. each farm calf was bottle-fed colostrum and natural milk from their dams soon after birth, usually within hours. sick calves were usually transferred to a separate calf shed in all the dairies. the village calves were reared in the conventional way for backyard rearing systems in bangladesh. most farmers in the village had - cattle, the cows being used for dual purposes, i.e. draught power and milk. their calves receive milk naturally by sucking from their respective dams. these calves usually received sufficient colostrum and milk, and occasionally roamed freely in the small grazing fields. sampling procedures and processing of samples faecal samples were collected from calves during weekly visits to each location. the diarrhoeic samples were collected first, followed by the collection of up to an equal number of normal faeces from age-matched non-diarrhoeic calves. a total of samples, diarrhoeic and non-diarrhoeic, were collected over a lo-month period (table i) . faecal samples (ca. g) were collected directly from the rectum, kept in a sterile screw-capped, labelled container, and transported to the laboratory as soon as possible in a thermostable box. each sample was diluted : in phosphate-buffered saline (pbs, ph . ), mixed, and centrifuged at oog for min ( °c). the supernatants were separated, labelled and kept at - °c until the samples were used for elisa. a commercially available kit (dakopatts a/s, code k , denmark) was used to analyse the samples for the presence of rotavirus in the faecal materials. the test was done as described elsewhere (ellens and de leeuw, ; ellens, ) and following instructions provided in the kit. each sample was tested in duplicate. the readings were taken spectrophotometrically at nm wavelength using automated equipment. positive results were assessed by comparing the light absorbance of the test samples with that of the known standard positive and negative controls. if a test sample had an absorbance value of times that of corresponding negative control, it was taken as positive. the p/n ratio was . titration of the positive samples was done with a twofold serial dilution of each sample using duplicate rows of elisa plates and adopting the above elisa methods. a : dilution of a positive sample was used as the initial dilution, being considered to have a titre of . titre was defined as the reciprocal of the highest dilution of a sample at which it was still positive by the above elisa interpretation. the distribution of samples positive for rotavirus as between locations and ages of calves is shown in tables i and ii respectively. a higher prevalence was observed in young diarrhoeic calves than in the older groups, whereas in non-diarrhoeic samples the prevalence was higher in older calves (table ii and figure ). no rotavirus was detected in calves over months of age. the titres in diarrhoeic calves with positive samples ranged from to (median ). the highest and the lowest titres were and respectively (median ) in samples from non-diarrhoeic calves. figure shows the distribution of the titres of the positive samples from both diarrhoeic and non-diarrhocic calves. age groups shown as i, ii and iii represent calves of not more than week, from over week to month, and from over month to months of age respectively little was known about the status and viral aetiology of calf diarrhoea in bangladesh under field conditions, although rotavirus-associated human infantile diarrhoea is prevalent (sack et al., ; huq et al., ) . a . % infection of diarrhoeic calves as detected in the present study appears low in comparison with fmdings elsewhere (de leeuw et az., ; moerman et az., , bellinzoni et uz., . rotavirus infection may vary widely from place to place or even from farm to farm (tzipori, ) . our findings, however, show similarity with limited observations (debnath et al., ) in the area of dhaka in diarrhoeic calves up to month old. the variation in the rate of infection on different farms (p=o.o ) needs more investigation before reaching any firm conclusions. hygiene measurement is one of the important factors, along with other interacting variables (tzipori, ) . farm b, which had the lowest prevalence ( . %) of the virus differed significantly (p=o.o ) from farm a and was noted to have a better cleaning and disinfecting procedure than the other two farms. a higher infection rate (table ii) in the first week of life suggests widespread rotavirus in this group of younger calves. other studies (acres and babiuk, ; de leeuw et al., ) showed similar results. in farm c, infection in the non-diarrhoeic calves was relatively high (table i) . subclinical infection is not uncommon on premises where clinical infection occurs in calves (de leeuw et al., , snodgrass and sherwood, ) . the overall infection rate in non-diarrhoeic calves in this study corresponds with some other reports (bellinzoni et al., ) . as numerous factors (tzipori, ) interplay in precipitating clinical diarrhoea, it is difficult to make absolute conclusions based on the limited information we have. however, rotavirus infection has been shown to be more important than other agents in diarrhoea in young calves of around week of age ( - days) (de leeuw et al., ) . although our findings did not reveal a significant difference in rotavirus infection between diarrhoeic and non-diarrhoeic calves, rotavirus was mostly associated (p =o.l) with young diarrhoeic calves. the age-related (r=o. ) prevalence in diarrhoeic calves (figure ) tends to agree with the report by tzipori ( ) . the absence of detectable rotavirus antigen in non-diarrhoeic calves up to week old and the high prevalence rate in diarrhoeic calves in the same age group (table ii) emphasizes the association between rotavirus and diarrhoea in such calves. the higher viral antigen titres (figure ) in samples from diarrhoeic calves, for which the geometric mean (gm) of titre was . as against those from non-diarrhoeic calves (gm = . ) confirms an association of the virus with diarrhoea in these young dairy calves. the small numbers of conventionally reared village calves makes it difficult to draw firm conclusions from the absence of rotavirus in these animals. the study, however, suggests that the rotavirus infection in young calves also exists in different locations of the country outside the dhaka region and is the first report of the existence of the virus in high concentration in association with diarrhoea in young dairy calves in that country. studies on rotaviral antibody in bovine serum and lacteal secretions calf diarrhea (scours): reproduced with a virus from a field outbreak prevalence and significance of viral enteritis in dutch dairy caives neonatal calf diarrhea: pathology and microbiology of spontaneous cases in dairy herds and incidence of enteropathogens implicated as etiologic agents sero-epidemiology of rotavirus infection in rural bangladesh aetiology of diarrhoea in young calves the aetiology and diagnosis of calf diarrhea. veterinary record, lot? naturally occurring and experimentally induced rotaviral infections of domestic and laboratory animals the study was sponsored by the icddr,b, mohakhali, dhaka , bangladesh (project no. - (p)). national science and technology division (bangladesh) supported the work by offering a ncst fellowship to the senior author. suggestions by drs ca. mebus, p.w. de leeuw and s. tzipori at the start of the study are acknowledged. journal of the american veterinary medical association, , - bellinzoni, rc., mattion, n.n., la torre, j.l. and scodeller, ea., key: cord- - wx v b authors: tautenhahn, a.; merle, r.; müller, k. e. title: factors associated with calf mortality and poor growth of dairy heifer calves in northeast germany date: - - journal: prev vet med doi: . /j.prevetmed. . sha: doc_id: cord_uid: wx v b a cross-sectional study was conducted on fifty german dairy farms to identify risk factors for high mortality and poor growth in calves. between and , farm visits took place and a questionnaire on farm management practices was completed by in-person interview. in addition, heart girth measurements of calves around weaning were performed to estimate average daily weight gain. furthermore, blood and faecel samples of calves were collected and a score addressing calf welfare was determined. the epidemiological associations between risk factors and high mortality and poor growth were estimated using two regression models. the factors significantly associated with high calf mortality (> %) were a high rate of calves with failure of passive transfer (> %) and the metaphylactic use of halofuginone lactate. a small amount of concentrates consumed around weaning, relocating calves more than twice until weaning and a low incidence risk of milk fever (< %) were found to be significantly associated with poor growth (median: grams). although the fifty farms cannot be considered as a representative sample for north-east germany, the results indicate that the farm management has a big impact on growth and survival of dairy calves and needs to be addressed more thoroughly when raising the future dairy cow. defence, by improvement of colostrum management and nutrition as well as minimizing stress by professional stockman ship in animal handling and by reducing the number of pathogens in the calf's environment (lago et al., ; windeyer et al., ; al mawly et al., ; urie et al., b) . previous studies reported average calf mortality rates between . % and . % for norway, canada, switzerland, united states and the netherlands (gulliksen et al., ; bleul, ; windeyer et al., ; urie et al., a; santman-berends et al., ) . few current data is available on calf mortality rates in germany. zitzmann et al. ( ) reported an average calf mortality rate of . % for thuringia in , hoedemaker ( . % for lower saxony in and sanftleben ( ) , % for mecklenburg-western pomerania in . no data is available on growth rates of preweaned dairy calves and calf rearing practices on german dairy farms. identifying risk factors for high dairy calf mortality and poor growth, will contribute to improve housing, feeding and management strategies on german dairies. therefore, the objective of the present study was to identify factors associated with high mortality (> %) in dairy calves during the first six months of life and with poor growth in calves in the first three months of life. the study was designed as an observational study and consisted of two models. in both models, the herd was the unit of concern. in the model "risk factors associated with high calf mortality", a cross-sectional study was j o u r n a l p r e -p r o o f conducted with calf mortality, defined as mortality of calves between the second day and sixth month of life, as the dependent variable. at herd level calf mortality was not normally distributed and was therefore dichotomized in herds with high risk of calf mortality (> %) and herds with a low risk (≤ %). the classification of herds was based on the median calf mortality in the study population in , which is in agreement with the target value for calf mortality of % proposed by roy ( ) . in the model "risk factors associated with median adg from birth to weaning", average daily weight gain (adg) of calves from birth until twelve weeks of life was the dependent variable at herd level. due to the limited study period and resources, we decided to enrol a total of dairy herds in the study. for logistical reasons farms had to be located in northeastern germany (brandenburg, saxony, lower saxony and mecklenburg-western pomerania). inclusion criteria for the study were an average herd size of ≥ dairy cows and in-house rearing of calves and heifers. most farms were enrolled in the projects "brandenburger testherden" of the cattle breeders' association brandenburg (visited/contacted: n= / ) and "veredelungsland sachsen " of the state control association of saxony (visited/contacted: n= / ) and were invited to participate in the study by letter (müller et al., ) . additional farms belonged to the clientele of the clinic for ruminants and swine of the some of these farms have been working together with the clinic for several years; others contacted the clinic due to problems with calf rearing. we offered them to participate in the study as a first diagnostic step. during the study visits no other investigations than on the purpose of this study were made. in october , members of the project "brandenburger testherden" and of the educational and experimental institution for animal husbandry echem were asked for help with farm acquisition. between october and october the remaining farms were visited. overall, farms were invited to participate in the study. in detail, farms were contacted by letter and farms by phone with a response rate of % and %, respectively. the overall response rate was %, which can be considered a high response rate. all farms entered the study voluntarily. each herd was visited on a single occasion by the same veterinarian (corresponding author) between september and october . a questionnaire on farm management practices, morbidity and calf mortality was completed by in-person interview. in addition, blood und faecel samples were obtained to evaluate for failure of passive transfer (fpt), selected metabolic parameters (serum albumin, globulin) and enteropathogens often observed in calves with ncd. heart girth measurements were conducted around weaning ( th week) to estimate adg from birth to weaning. additionally, a score addressing calf welfare (animal needs index by winckler et al. ( ) ) was determined. all variables were collected and retrieved on farm by a single investigator at a single farm-visit. this made a separate data collection of exposure and outcome variables impossible and therefore rules out blinding of the study. this could lead to biased data what should be in mind when interpreting the results of this study. the questionnaire was created hypothesis driven and corresponding to literature (taffe et al., ; schäffer et al., ; bundesministerium, ; lorenz et al., a; lorenz et al., b) in this study we applied the centimetre scale. as described by heinrichs et al. ( ) , heart girth measurements were carried out and the described formula was used to estimate life weight in kilograms for the breed german holstein. one herd kept mainly the breed jersey and was excluded from the model "risk factors associated with median adg from birth to weaning", because the calculation of adg is not suitable for this breed. colostral antibody transfer was estimated on basis of serum total protein levels the test was carried out following the operating instructions. for evaluation of husbandry conditions with respect to calf welfare a modified version of the animal needs index for calves (tiergerechtheitsindex für kälber) described by winckler et al. ( ) was applied. the animal needs index (ani) for calves covers seven areas of influence: movement behavior, feeding behavior, social behavior, resting behavior, comfort behavior, hygiene and supervision. in each area of influence, several parameters are graded using a score. a farm can achieve calf mortality risk was calculated as follows: the average birth weight of kg refers to trilk and münch ( ) , who determined the birth weights for the breed german holsten in northern germany. data at individual calf level from blood and faecel testing as well as from heart girth measurements were transformed into data at herd level using the herd median or percentage of calves. the cut-off value for fpt and successful antibody transfer was set at < g/l and ≥ g/l, respectively, as recommended by vandeputte et al. ( ) when the biuret-test for serum total protein examination is applied. at herd level, the percentage of calves with fpt was used for further analyses. serum albumin levels in calves around the twelfth week of life was used to evaluate appropriate nutrition and health status before weaning. the authors assumed a threshold value of < g/l serum albumin to be an indicator for malnourishment (and chronic disease) of preweaned calves. at herd level, the percentage of calves with low serum albumin was used for further analyses. after execution of the ani, the results were transferred into score values for each area of influence. at herd level results were expressed as percentage of the achieved score from maximum possible score value in each area of influence. for example: a farm with points in the area of influence "supervision" (maximum possible score value: points) achieved % of all possible points in this area of influence. due to possible information bias, we excluded the incidence risks of ncd, brd and navel-ill as outcome variables. diseases were treated at very different degrees of severity and most farmers estimated all incidence risks freely. others counted the cases out of the treatment register (herd management program or stable book) and calculated incidence risks by dividing counted cases by the number born alive calves for ncd, brd and navel-ill and by the number of calvings for milk fever and ketosis in the past j o u r n a l p r e -p r o o f twelve month before farm visit. because of the importance of these variables for the outcome variables calf mortality and adg we kept them in these models. due to the convenience sampling strategy of farms selection bias cannot be ruled out. confounding was controlled by including potential confounders in the multivariable regression models. before statistical analyses the data set was revised by using data cleaning techniques as removing duplicate data and correcting or marking obviously incorrect data. missing or incorrect data was marked as missing values. all statistical analyses were performed using the software spss (version . , spss inc. , usa). for the investigation of associations between potential risk factors with calf mortality and adg from birth to weaning a multivariable model each was built. at first, the large data set was reduced. therefore, descriptive analyses of all data at herd level were performed. continuous not normally distributed data was dichotomized or categorized. potential risk factors were eliminated from further analyses if they were lacking variance (< % observations per category in binary outcomes), were missing biological causality or had too many missing values (> %, except for "amount of concentrates consumed around weaning in kg" in the model for poor growth). then all variables were tested for correlation using the spearman's rank correlation coefficient. if the correlation coefficient was above . , only the factor with the better biological plausibility was selected for further analyses. the remaining potential risk factors as well as categories and observed j o u r n a l p r e -p r o o f frequencies are summarized in table . table is displayed in the supplements of this article. the associations of calf mortality (binary: ≤ %, > %) with potential risk factors (n = ) were tested in univariable analyses. candidate variables with binary outcome were tested using chi-square-test and those with categorical or continuous outcome using logistic regression. factors with a p-value ≤ . (n = ) qualified for multivariable analysis. due to a limited number of herds (n = ) in the study the number of candidate variables was reduced according to the p-value to a maximum of seven candidate variables. in the first step, the multivariable logistic regression model was reduced by using a manual stepwise backward procedure, with p < . as criterion for retention. in the second step, excluded variables were reentered one by one. the final model only included variables that were significant at a significance level of p < . . confounding and interaction effects were considered in the model. in the end, no confounders were discovered in the model and none of the two-way interactions were significant in the model and therefore, all interaction terms were removed. variables with binary outcome were tested using student's t-test, those with categorical outcome using analysis of variance (anova) and with continuous outcome using linear regression. factors with a p-value ≤ . (n = ) qualified for multivariable analysis. as described in the previously model a maximum of seven candidate variables were included in a multivariable linear regression model. the potential risk factor "amount of concentrates consumed around weaning" was used in the model with an adjusting factor to weight the observations, to account for heteroscedasticity of the data (weighting was done using "weighted least squares"). further analysis was performed as described above. in the model "risk factors associated with median adg from birth to weaning", the fit of the model was evaluated with the adjusted r-squared and by assessing the residuals. plots of the standardized residuals against the predicted values were observed for their pattern and distribution and for outliers. herd size ranged from to cows (median: cows) with a median milk yield of kg per cow and year (range: - kg). median calf mortality risk in was . % (range: . % - . %). herd characteristics of low and high mortality herds are presented in table . median herd level incidence risk for ncd, brd and navel-ill in calves (treatment) was . % (range: . % - . %, q %: . %; q %: . %), . % (range: . % - . %, q %: . %; q %: . %) and . % (range: . % - . %, q %: . %; q %: . %), respectively. median herd level incidence risk for milk fever and ketosis treatment of dairy cows j o u r n a l p r e -p r o o f was . % (range: . % - . %, q %: . %; q %: . %) and . % (range: . % - . %, q %: . %; q %: . %), respectively. with fpt compared to farms visited in winter (p = , ). serum albumin, total protein and globulin levels were determined in calves around weaning with a herd median of . g/l (range: . g/l - . g/l), . g/l (range: . g/l - . g/l) and . g/l (range: . g/l - . g/l), respectively. . % of the herds had at least % calves per herd with a serum albumin concentration < . g/l. the authors assume a threshold value of ≥ g/l serum albumin to be an indicator for malnourishment of preweaned calves. respectively. relevant descriptive data of the questionnaire and the ani is presented in a summarized form in table . results from univariable analyses are presented in table . seven of these variables were introduced to the model "risk factors associated with high results from univariable analyses are presented in table . seven of these variables were introduced into the model "risk factors associated with median adg from birth to weaning" selected by the lowest p-value in univariable analyses. after manual backward selection, the variables "amount of concentrates consumed around weaning" (p < . ), "frequency of relocation of calves between birth and weaning" (p < . ) and "incidence risk of milk fever" (p = . ) remained in the final multivariable linear regression model (table ) and explained . % of the variance between herds (adjusted r-squared). the amount of consumed concentrates around weaning was associated with adg until weaning. median adg increased by grams ( % confidence interval - ) at herd level. frequent relocation of calves turned out to be a risk factor for poor growth until weaning. calves that were relocated on the same farm more than twice from birth until weaning gained grams per day less until weaning than calves that were relocated not more than twice ( % confidence interval - -- ). farms with a high incidence risk of milk fever treatment in periparturient cows (> %) had grams more adg until weaning than farms with a lower incidence risk ( % confidence interval - ). fpt of more than % of the neonatal calves in a herd was associated with high calf mortality. this finding is in accordance with results of previous studies demonstrating that total serum protein levels below g/l in j o u r n a l p r e -p r o o f neonatal calves are a risk factor for lethal outcome (rea et al., ; tyler et al., ; torsein et al., ) . in the present study, cryptosporidium parvum was detected in faecel samples obtained from calves on ( %) farms with a median percentage of . % of calves between and days of age. halofuginone lactate was administered to all neonatal calves on nearly half ( %) of the farms visited. farms on which routine administration of halofuginone lactate was performed had . times higher odds for calf mortality risk above %. similar observations were made by fourichon et al. ( ) , who observed a higher mortality in neonatal calves on farms applying medical prevention for digestive disorders. one explanation could be that farms with high risk of calf mortality due to ncd use halofuginone lactate more often than farms with less problems with ncd. in contrast to these observations, other researchers demonstrated the beneficial effect of metaphylactic halofuginone treatment (keidel and daugschies, ) . a toxic effect of halofuginone lactate as reason for increased calf mortality is unlikely as villacorta et al. ( ) observed toxic side effects at much higher dosages ( µg/kg bw) than those recommended by producers ( - µg/kg). risk factors that did not remain in the multivariable model for high calf mortality, but showed a significant (p < . ) association with calf mortality, are discussed in this section. the risk factor "age at first offer of hay" did not remain in the model although no offer of hay in the first days of life compared to early supply was associated with higher calf mortality in the univariable analysis (p = . ). j o u r n a l p r e -p r o o f torsein et al. ( ) and perez et al. ( ) observed lower calf mortality rates on farms that offered roughage to calves from the first week of life onwards compared to those farms that offered roughage shortly before weaning. perez et al. ( ) assumed that the supply of roughage to neonatal calves enhances early rumen development. in practice, feeding concentrates to young calves is preferred to offering hay as concentrates were proven to enhance rumen development more efficiently than hay (noci, ). in addition, hay feeding was shown to reduce the amount of concentrates consumed (Žitnan et al., ) . early supply of high amounts of concentrates without any roughage was shown to cause hyperkeratosis and clumping of rumen papillae. to this end, the hay provided is thought to prevent hyperkeratosis of rumen papillae by evoking a scratching effect on the rumen epithelium (suárez et al., ; noci, ) . promoting solid feed intake of calves during the pre-weaning period has a positive effect on rumen development and facilitates the transition from milk to solid feed at weaning (khan et al., b) . the intake of high amounts of concentrates in the pre-weaning period was associated with high adg until weaning (+ adg per kg concentrates). this is in line with the results of relocating calves more than twice from birth until weaning was related to lower adg. corresponding observations were made by mormède et al. ( ) and siegel and latimer ( ) in rats and chickens. as a result of repeated regrouping and relocation, animals showed signs of chronic stress such as aggression against herd mates and insufficient adg. veissier et al. ( ) did not observe adverse effects on adg due to regrouping and relocating of calves but measured an increased stress response compared to controls. they suspected a negative effect of regrouping and relocation on adg and calf health under field conditions when more than two calves form a group. more research is needed to confirm their assumptions. the association between incidence risk of milk fever in dairy cows and adg in calves was an unexpected result. calves showed higher adg on farms with a higher milk fever incidence risk. this result is in contrast to former publications that describe significant health implications for both dams and calves on farms with a high milk fever incidence risk, among these, dystocia and the weak calf syndrome (gröhn et al., ; houe et al., ; wilhelm et al., ) . weak calves consume less first colostrum, fall ill more often and have reduced adg (besser et al., ; wittum and perino, ; weaver et al., ) . one explanation for our findings could be a higher percentage of cows with ≥ parities on farms with higher incidence risk of milk fever treatment. milk fever is usually a disease of dairy cows in their third lactation or beyond (correa et al., ) . at the same time multipara j o u r n a l p r e -p r o o f have richer first colostrum (tyler et al., ; scholz et al., ) and therefore their offspring has a decreased risk of fpt, sickness and reduced adg (robison et al., ; wittum and perino, ; windeyer et al., ) . risk factors that did not remain in the multivariable model for median adg, but showed a significant (p < . ) association with adg, are discussed in this section. as expected the risk factors "time per day without staff at the farm" (p = . ) and "maximal time interval between calving and harvesting first colostrum of the dam" (p = . ) were associated with adg. a precondition for early first colostrum harvesting is that staff members are present to take over the latter task. in our study, collecting first colostrum within two hours after calving at maximum versus a more extended period was associated with better growth in neonatal calves. several studies proved before, that a delay in first colostrum harvesting leads to reduced immunoglobulin g concentrations due to the diluting effect of milk synthesis in the freshly calved cow (moore et al., ; morin et al., ; scholz et al., ) . calves that consume first colostrum with low concentrations of immunoglobulin g have a higher risk of fpt (besser et al., ) and thus are at risk for diseases and poor growth (robison et al., ; wittum and perino, ; windeyer et al., ) . feeding at least three litres of first colostrum at first meal was associated with higher weight gains before weaning (p = . ). this finding is in accordance with observations by others (faber et al., ; trotz-williams j o u r n a l p r e -p r o o f beam et al., ) . early and sufficient colostrum supply is one of the main protective factors against fpt, which is connected to poor growth, as described above. farms that started offering concentrates to calves beyond two weeks of life had lower adg from birth to weaning than farms that fed concentrates earlier (p = . ). most of the study farms reared their calves with a restricted milk feeding routine und tried to wean calves as early as possible to reduce feeding costs. in particular, calves fed restricted depend on early rumen development for sufficient growth. therefore, concentrates that are supposed to be the most effective feed for early rumen development need to be offered to calves as early as possible (tamate et al., ; forbes, ; noci, ) . farms that abandoned hay from the diet of preweaned calves had higher adg in this study than farms that fed hay unchopped and separately (p = . ). this result is in line with studies (warner et al., ; jahn et al., ; kertz et al., ) in which calves were exposed to a restricted milk feeding routine. pre-ruminant calves consumed less concentrates when they were fed hay separately. the authors concluded that limited rumen capacity in young calves determines the amount of solid feed intake. to this end, the simultaneous offer of hay and concentrates would reduce adg due to the lower energy density of hay compared to concentrates. in further studies, pre-ruminant calves were fed chopped hay either separately, or mixed with concentrates (coverdale et al., ; suárez et al., ; khan et al., a) . none of these authors could show a negative effect of hay on adg. taking the protective effect of hay on calf mortality into account hay feeding to calves seems to be recommendable. evaluating the data from the animal needs index, we observed higher adg on those farms that fulfilled the requirements of calves more efficiently than farms with insufficient adg. farms with more than % of points in the area of influence "supervision" in the animal needs index had higher adg than farms with less points (p = . ). differences between farms relied mainly on differences in documentation of symptoms and treatment of diseases. this corresponds to the results of lundborg et al. ( ) . they found a positive correlation between proper documentation and good calf health and draw the conclusion that thoroughness in documentation reflects a good quality of calf raising management. in our study, we found a strong positive correlation of median serum albumin concentrations and adg until weaning (p < . ). at herd level, each onegram increase in serum albumin concentration was associated with a rise in adg by grams. low serum albumin levels are a constant finding in malnourished and underweight children (clarke et al., ; cripps et al., ) . in piglets and calves, the predictive capacity of serum or plasma albumin concentrations has been examined in the context of different rearing intensities (restricted vs. ad libitum feeding) in the first month of life (hammon et al., ; liu et al., ; maccari et al., ) . a positive association between body weight and plasma albumin concentration was detected in mature humans by jeejeebhoy et al. ( ) . although in individual animals low serum albumin concentrations can be the j o u r n a l p r e -p r o o f consequence of either malnutrition or a variety of disorders (protein loosing enteropathy, liver degeneration), on herd level, we consider low serum albumin (< g/l) as predicting indicator for malnourishment or for an elevated proportion of calves with chronic diseases leading to insufficient adg from birth to weaning. its suitability as indicator, however, has to be investigated in subsequent studies. our statistical analyses included dealing with data challenges such as multicollinearity, finding associations 'due to chance alone', confounding and interaction effects (dohoo et al., ) . we used techniques to reduce the number of independent variables and to select for variables with the highest explanatory power before inserting them into multivariable models. furthermore, confounding and interaction effects were considered for in the models. due to the limited number of farms in this study, the number of independent variables per final multivariable regression model had to be reduced to a maximum of seven variables to reduce the possibility to find associations just due to chance alone. since more than seven risk factors may effect multifactorial diseases in calves, we had to select the variables carefully. the results of the present field study indicate that high calf mortality (> %), fpt in the neonate (serum protein < g/l) and low adg from birth to weaning (< g/day) are still a problem on dairy farms under livestock production conditions in northeast germany. as the welfare of food producing animals and in particular of young stock comes more and more j o u r n a l p r e -p r o o f into the focus of society, the management of calf rearing should be systematically evaluated and improved in the light of recent insights into nutrition, husbandry conditions, animal handling and disease prevention. especially colostrum and feeding management routines on most farms need to be revised and checked more frequently. feeding routines should focus on high rates of adg until weaning. offering water and concentrates right from the start enables calves to consume sufficient amounts of concentrates at weaning and leaves them prepared for the milk deprivation at weaning. animal handling and husbandry conditions should be reviewed from an animal perspective to reduce unnecessary stressful routines as frequent relocating of calves until weaning. connected with the funding associations, helped with the farm acquisition and supervised the corresponding author in this study. the author roswitha merle supported the study with the development of the assessment strategy and by performing data analysis. all examinations and sample collections were performed in line with legal regulations on the performance of experiments on animals (bundesministerium, ) . in conduct with this research quality standards like standard operating procedures for peripheral venous blood sampling and faecel sampling of the clinic for ruminants and swine of the freie universität berlin and ‚safeguarding good scientific practice' of the german research foundation (dfg) were taken into account. j o u r n a l p r e -p r o o f risk factors for neonatal calf diarrhoea and enteropathogen shedding in new zealand dairy farms tiergerechtheit bewerten. kuratorium für technik und bauwesen in der landwirtschaft e meta-analysis of the effect of initial serum protein concentration and empirical prediction model for growth of neonatal holstein calves through weeks of age comparison of three methods of feeding colostrum to dairy calves decreased colostral immunoglobulin absorption in calves with postnatal respiratory acidosis risk factors and rates of perinatal and postnatal mortality in cattle in switzerland investigation about quantitative benchmarking of animal health. dissertation an examination of the influence of marketing organisations on animal health and meat quality of fattening pigs on the basis of collected slaughter check results, ph-values and meat temperature of the ham verordnung zum schutz landwirtschaftlicher nutztiere und anderer zur erzeugung tierischer produkte gehaltener tiere bei ihrer haltung: tierschnutzv. in: bundesgesetzblatt tierschutzgesetz: tierschg the influence of various degrees of confinement and supervision on the incidence of dystokia and stillbirths in hereford heifers new common animal health strategy case study: effects of colostrum ingestion on lactational performance effects of increased dietary protein and energy on composition and functional capacities of blood mononuclear cells from vaccinated, neonatal calves physiological changes affecting voluntary food intake in ruminants critical points related to housing and management in control programmes for calf morbidity and mortality in french dairy herds. th international congress in animal hygiene fuel feeds function: energy metabolism and the t-cell response management risk factors associated with calf diarrhea in michigan dairy herds potential risk factors for cryptosporidium infection in dairy calves epidemiology of metabolic disorders in dairy cattle: association among host characteristics, disease, and production calf mortality in norwegian dairy herds feed intake patterns, growth performance, and metabolic and endocrine traits in calves fed unlimited amounts of colostrum and milk by automate, starting in the neonatal period predicting body weight and wither height in holstein heifers using body measurements verminderung von aufzuchtverlusten in niedersächsischen milchviehbetrieben milk fever and subclinical hypocalcaemia--an evaluation of parameters on incidence risk, diagnosis, risk factors and biological effects as input for a decision support system for disease control nutritional assessment: comparison of clinical assessment and objective variables for the prediction of length of hospital stay and readmission integration of halofuginone lactate treatment and disinfection with p-chloro-m-cresol to control natural cryptosporidiosis in calves an early weaning calf program: summarization and review hay intake improves performance and rumen development of calves fed higher quantities of milk invited review: effects of milk ration on solid feed intake, weaning, and performance in dairy heifers farm characteristics and calf management practices on dairy farms with and without diarrhea: a case-control study to investigate risk factors for calf diarrhea calf respiratory disease and pen microenvironments in naturally ventilated calf barns in winter effects of dietary protein/energy ratio on growth performance, carcass trait, meat quality, and plasma metabolites in pigs of different genotypes calf health from birth to weaning. iii. housing and management of calf pneumonia calf health from birth to weaning. i. general aspects of disease prevention effects of two different rearing protocols for holstein bull calves in the first weeks of life on health status, metabolism and subsequent performance multikausale infektionskrankheiten effect of delayed colostrum collection on colostral igg concentrationin dairy cows effect of colostral volume, interval between calving and first milking, and photoperiod on colostral igg concentrations in dairy cows multiple neuroendocrine responses to chronic social stress: interaction between individual characteristics and situational factors bewertung von hygiene effects of different rations on growth and rumen development of calves. dissertation effects of chronic environmental cold on growth, health, and select metabolic and immunologic responses of preruminant calves composition and functional capacity of blood mononuclear leukocyte populations from neonatal calves on standard and intensified milk replacer diets management factors related to calf morbidity and mortality-rates the effects of disease, management, and nutrition on average daily gain of dairy heifers from birth to four months herd health and production management in dairy practice prediction of calf mortality by use of tests for passive transfer of colostral immunoglobulin relationship of various incoming cattle traits with feedlot performance and carcass traits effects of passive-immunity on growth and survival in the dairy heifer invited review: influence of climatic conditions on the development, performance, and health of calves influence of weaning method on health status and rumen development in dairy calves the calf: volume management of health vorbeuge von kälberkrankheiten durch richtige aufzucht und fütterung in der milchviehhaltung. . fachtagung lkv/rgd quantifying calf mortality on dairy farms: challenges and solutions critical control points (ccp) for the housing and management of calves factors of influence on the quality of colostrum from dairy cows preweaned heifer management on us dairy operations: part vi. factors associated with average daily gain in preweaned dairy heifer calves social interactions and antibody titres in young male chickens (gallus domesticus) the effect of respiratory disease and a preventative antibiotic treatment on growth, survival, age at first calving, and milk production of dairy heifers effect of roughage source and roughage to concentrate ratio on animal performance and rumen development in veal calves morbidity in swedish dairy calves from birth to days of age and individual calf-level risk factors for infectious diseases kälbersterblichkeit senken, aufzuchtverluste minimieren effect of various dietaries on the anatomical development of the stomach in the calf risk factors for calf mortality in large swedish dairy herds connections between health of calves, growth and later yields of dairy cattle use of serum protein concentration to predict mortality in mixedsource dairy replacement heifers colostral immunoglobulin concentrations in holstein and guernsey cows preweaned heifer management on us dairy operations: part i. descriptive characteristics of preweaned heifer raising practices preweaned heifer management on us dairy operations: part v. factors associated with morbidity and mortality in preweaned dairy heifer calves comparison of four refractometers for the investigation of the passive transfer in beef calves calves' responses to repeated social regrouping and relocation efficacy of halofuginone lactate against cryptosporidium parvum in calves ruminant nutrition, dietary factors influencing development of ruminant stomach passive transfer of colostral immunoglobulins in calves effect of serum calcium status at calving on survival, health, and performance of postpartum holstein cows and calves under certified organic management tiergerechtheitsindex für kälber factors associated with morbidity, mortality, and growth of dairy heifer calves up to months of age antimicrobial resistance global report on surveillance: summary. world health organization influence of dietary concentrate to forage ratio on the development of rumen mucosa in calves risk factors for calf mortality influence the occurrence of antibodies against the pathogens of enzootic bronchopneumonia the study was supported by the cattle breeders' association brandenburg "brandenburger testherden", the managers of the project "veredelungsland sachsen " of the state control association of saxony and the educational and experimental institution for animal husbandry echem by helping with the farm acquisition. the authors thank the participating farmers for their interest and support. declarations of interest: none. key: cord- -a h irel authors: santman-berends, i.m.g.a.; de bont-smolenaars, a.j.g.; roos, l.; velthuis, a.g.j.; van schaik, g. title: using routinely collected data to evaluate risk factors for mortality of veal calves date: - - journal: prev vet med doi: . /j.prevetmed. . . sha: doc_id: cord_uid: a h irel from to a gradual increase in on-farm mortality of dutch veal calves was observed. in , the cattle industry decided that more information was needed on risk factors for mortality in both veal herds and herds of origin to enable implementation of risk mitigating measures. routinely collected data were available from seven different data sources and contained information from . million white veal calves that were fattened in the period between january and june . survival analysis techniques (kaplan-meier), multilevel poisson and multilevel logistic regression models were applied to analyse the data. two different models were assembled in which risk factors for veal calf mortality in respectively veal herds and herds of origin were identified. univariable and multivariable regression techniques were used to detect risk factors significantly associated with mortality of veal calves during the fattening period. during the study period, the mean mortality was . % per production cycle. the probability to die was highest during the first weeks after arrival in the veal herds and declined thereafter. important risk factors included a veal herds with a higher use of antimicrobials, hair colour as proxy for breed, certain countries of origin, veal herd management with a limited amount of supplied feed and a not having an all-in / all-out system. a higher body weight at arrival in the veal herd was associated with lower mortality as well as veal calves that were fed an above median amount of milk, roughage and concentrates. from the calves that were fattened during the study period, observations of . million calves originated from the netherlands and were available to study risk factors for veal calf mortality associated with the herd of origin. important risk factors included purchase, herds with high mortality rates in the quarter in which the calf was born, fast growth in herd size, high cattle replacement rates and a higher antibiotic use in the quarter of birth. calves that originated from herds that were certified bvd-free, salmonella-unsuspected or paratuberculosis-unsuspected, had a lower odds to die during the subsequent fattening period in a veal herd. veal calf mortality was influenced by risk factors at the herd of origin as well as at veal herds. adequate collaboration between the different industries is necessary to optimize veal calf management leading to a reduction in veal calf mortality during the fattening period. annually, . million veal calves are fattened in europe (sans and de fontguyon, ). with . million fattened veal calves per year, the netherlands has one of the largest veal industries in europe (brscic et al., ; sans and de fontguyon, ) . in this industry, surplus calves mostly born in dairy herds throughout europe, are collected, fattened and eventually slaughtered. given the fact that veal calves account for more than one-third of the total cattle population in the netherlands, it is important to monitor their health. on population level, cattle health is monitored on a quarterly basis through the national cattle health surveillance system (chss) (santman- berends et al., ) . one of the key-monitoring indicators in this system is mortality rate. between and the end of a gradual increase in mortality rate of veal calves was observed in the chss. in the corresponding period, the policy with regard to antimicrobial use was altered, resulting in reduced possibilities to apply antibiotics in livestock followed by a tremendous decrease (> %) in antimicrobial usage (sda, ) . it was hypothesized that this decrease in antibiotic use was associated with the increased mortality rate. nevertheless, other potential causes were identified such as increased imports of calves persistently infected with bvdv, increased herd sizes and factors related to the herd from which the calves originated. yet, even with all these potential conditions that might have led to an increased mortality in veal herds, the large between herd variation in veal calf mortality provided potential for improvement in herds with high mortality rates. to enable the implementation of risk mitigating measures for veal calf mortality, more information was needed on risk factors associated with veal calf mortality. although it is well known that diarrhoea and respiratory infections are the main causes of mortality among veal calves (pardon et al., ; hoet et al., ; webster, ) , the risk factors associated with occurrence of these disorders are ambiguous. various studies describe pathogens as the cause of these problems such as rotavirus, coronavirus, bvdv and cryptosporidium (hoet et al., ; bendali et al., ) , but lack to describe management factors on which the farmer can intervene. there have been studies that described factors associated to either diarrhoea, respiratory syndromes or both. these factors include colostrum intake (lorenz, ; perez et al., ) , bvdv (pardon et al., and pardon et al., ) , weight at arrival in the veal herd (brscic et al., ) , housing (brscic et al., ; lorenz, ) and type and amount of feed (brscic et al., ) . nevertheless, it is unknown to which extent these risk factors for diarrhoea and respiratory syndromes are associated to veal calf mortality and whether these results are applicable to the white veal industry in the netherlands. additionally, this information does not provide insight in the combination of factors related to both the veal herd and the herd of origin that are associated to mortality. therefore, the aim of this study was to evaluate risk factors in veal herds and in the herd in which the calves were born that are associated with mortality of veal calves. a retrospective cohort study was carried out exclusively focusing on white veal calves in the netherlands. white veal calves mainly consists of superfluous dairy calves (sometimes calves from suckler herds) that are transported to veal herds at an age of on average eighteen days. first, the calves are housed individually until an age of eight weeks after which they are held in group housing. milk is the most important nutrition throughout the calves' life and at the age of approximately six months the white veal calves are slaughtered. data were available from all white veal calves that were fattened by four veal cooperations from january to june . cooperations were selected on the inclusion criteria that they had to digitally register data of their white veal herds and on an individual animal level during the complete study period. additionally, they had to be willing to share their data for the study. eventually, the delivered data consisted of records of white veal herds, which represented around % of the total white veal herd population in the netherlands. at onset, an inventory was conducted to evaluate which parameters were hypothesized to be of interest for inclusion in this study and were available in routinely collected datasets. data were available on either individual level, level of the production cycle or at herd level. for this study, animal movement data (identification and registration database) and data concerning the body weight at arrival in the veal herd were available on calf level. on the level of the production cycle, the amount of feed supplied, the veal herd system (all-in / all-out) and data about the amount of antibiotics that were supplied were provided. the production cycles were based on registrations of the veal industry that provided the production cycle number for each individual calf. the same production cycle number was given to a group of calves housed in either the same herd (all-in/ all-out) or in a barn in which all calves had approximately the same age. at the herd, level information about the compliance to critical control points set by the veal branch organisation (ikb, ) were accessed (table ) . additionally, information about the herd of origin was obtained for calves born in the netherlands. this information consisted of the herd health status, mortality rates in the quarter of the year in which the calf was born and animal movement data in the herd of origin. for the calves that were imported, only the country of origin was available (table ) . additionally, information about the geographical location of the veal herds and meteorological records were available for the study period. to each data supplier anonymity was guaranteed and the delivered data were send to and encrypted by an external firm (intofocus data transformation services (idts), deventer). this firm encrypted all variables in the data that might link the data back to the original source, such as the unique herd identification number (uhi) of the farm, number of the production cycle and the unique identification code of individual animals (animal id). the same encryption code was used for all datasets to ensure that data of the different data sources could be combined for analysis. thereafter the data were send to the researchers for validation and analysis. the level of detail of the data and the number of observations varied depending on the source of the data and the level of aggregation (table ) . software scripts in sas . ® (sas institute inc, ) were developed to combine the different datasets and to validate the data. observations with non-existing animal identification numbers and records with unlikely values (such as starting weights of kg or negative amounts of feeds) were set to removed. the body weight of the calf at arrival was either estimated based on the difference in weight between the empty and full truck divided by the number of calves in that truck ( %) or known based on weighing each individual calf at arrival in the veal herd ( %). the amount of feed was provided as total amount of concentrates, roughage (sometimes further specified to, for example, amount of corn silage and straw) and milk powder in kilograms per production cycle. based on this information the amount of feed per calf per day in that specific production cycle was calculated by dividing the total amount per type of feed by the number of calf-days in the herd. the latter was defined as the number of calves in the herd multiplied by the number of days that each calf was present. daily regional measures of humidity and temperature were obtained from the royal netherlands meteorological institute (knmi, zeist the netherlands) during the study period. for each veal calf, the daily meteorological results from the month of birth from the nearest weather station (there are weather stations in total) were downloaded from the national database (knmi, ) and were averaged to a monthly measure that was used as an explanatory variable in the models. for the analysis, stata ® version (stata, ) was used. descriptive statistics were applied to present the data. an attempt was made to apply survival analysis techniques on the data. survival time was defined as the time between entry in the veal herd and either death or the end of the follow up period i.e. the date of slaughter. white veal calves are slaughtered between and days of age thus the maximum study duration was set at days. observations of calves that outlived this period, were censored at days. the kaplan-meier survival function in stata (stel et al., ; kaplan and meier, ) was used to determine the daily hazard rate, conditional on having survived until that specific day. parameters such as mortality rates, probability of survival and the cumulative probability of dying were presented using life tables. univariable associations between mortality rate and potential confounders were described using kaplan-meier survival curves. cox proportional hazard frailty models were evaluated to test the association between potential risk factors and the time to survive, correcting for the hierarchical structure of the data. the attempt to correct for within herd clustering combined with the large number of observations lead to non-converging models and thus survival analyses seemed unfeasible for multivariable analysis of our data. therefore multivariable regression techniques were applied for analysis of the data and it was decided to build two different models. the first model evaluated risk factors for veal calf mortality in veal herds and was conducted at the level of production cycle. the second model assessed risk factors for veal calf mortality associated with the herd of origin and was conducted on animal level. the latter was exclusively conducted on observations from veal calves originating from the netherlands because information about the herd of origin was only available for dutch calves. multilevel poisson regression models were used to determine the associations between veal herd risk factors and mortality at the level of production cycle. the number of deaths within each production cycle were included as dependent variable and the number of calves that entered the production cycle was incorporated as exposure. the uhi was included as random variable to correct for clustering within herds. first, all potential risk factors were univariably pre-screened and parameters which were significantly associated with the mortality rate (p-value < . ) entered the multivariable model. the final multivariable model was selected using a forward selection and elimination method in which every round the variable with the lowest p-value was added until all variables in the model were significantly (p-value < . ) associated with the outcome and adding additional variables did not result in a significant improvement of the model (non-significant in the likelihood ratio test). significance of relevant two-and three-way interaction terms and the amount of variance that was explained by the final multivariable model were assessed. additionally, confounding was checked by evaluating the change in model coefficients after introduction of possible confounders (e.g. age at arrival as possible confounder for body weight at arrival). if the introduction of the confounder resulted in a substantial (> %) change in coefficients of significantly associated variables, the variable was retained. otherwise the variable was removed from the model. in the final model, the different classes of categorical variables were presented relative to the average of the complete study population. the risk factors in the herd of origin were evaluated on animal level because they were available for each calf individually. again, the number of observations was too large for a cox proportional hazard frailty model. therefore, it was decided to use a logistic regression model in stata ® in which mortality (yes/no) was included as dependent variable and potential risk factors as independent variables. in this model, we included the uhi from the veal herd as random variable to account for clustering of calves and for variability in mortality that was associated with the veal herd. univariable pre-screening at pvalue < . was conducted. the remaining variables were used to construct the final model using a forward selection procedure. after combing and validating the data, records of approximately . million veal calves that were fattened during the study period from production cycles fattened in different white veal herds where available for analysis. these calves represented more than % of the total white veal population in the netherlands. from the population of . million calves that entered the veal herds from january to july , , calves died before slaughter (table ) . corrected for censoring of calves that were removed from the herd before the moment they were slaughtered, resulted in a mean mortality rate of . % per production cycle ( table ). the median mortality rate was . % per production cycle with an interquartile range ( th - th percentile) ranging from . % to . %. veal calves that originated from dutch cattle herds ( % of the study population) arrived in the veal herds at an average age of days. imported calves ( % of the study population) were on average days old at arrival. the probability to die was highest during the first weeks after arrival in the veal herds, with a peak mortality rate between the first and second week and a declining rate thereafter (table , fig. ) . the probability to survive ( -mortality rate) the fattening period was highest in with . % and lowest in with %. after , the probability to survive the fattening period increased again (fig. ) . the difference in survival rate between and the following years arose in the first days after arrival and remained stable during the remaining period. the higher survival rate in and compared to was observed in the second half of the fatting period ( - days) (fig. ) . in total, potential risk factors for veal calf mortality that were related to the veal herd were evaluated. out of these, were significantly associated with calf mortality of which variables were related to compliance (yes, no or not applicable) with critical control points defined by the branch organisation. eleven variables were excluded from the multivariable model because of an insufficient number of observations (n < ) in one of the categories. additionally, seven variables were highly correlated (r > . ) with other variables and of those only the variables that were highest associated with the dependent variable were retained for inclusion in the initial multivariable model. from the variables that entered the multivariable multilevel model selection process, variables were significantly associated with the mortality rate. additionally, the random herd-effect was significant. the final model included production cycle observations from veal herds and had a pseudo r of . . the risk factors that were significantly associated with a higher mortality rate during the fattening period were a higher use of antimicrobials, hair colour as proxy for breed, country of origin, not having an all-in/all-out system, and the answer 'not applicable' at the critical control point "medicines are stored in a closed cabinet or room separated from animals and feed" (table ). veal calves that had a limited supply of feed had a higher mortality rate whereas being fed more than the median amount of milk and roughage was associated with a significantly lower mortality rate. additionally, being born in the summer or autumn appeared to be associated with a higher mortality ratio (irr = . and . , respectively), whereas born in winter and spring were associated with a lower risk (irr = . and . , respectively) when corrected for temperature and humidity. further, a higher body weight at arrival in the veal herd was associated with a lower mortality rate. per kilogram increase in initial body weight, the incidence rate refers to the number of days each calf is treated during a specific period (such as fattening period). b the average of the complete study population is included as reference. c < median: the calves were fed less than the median amount of the specific type of feed per day, > median: the calves were fed more than the median amount of the specific type of feed per day. median amount of product in kg per calf per day throughout the whole production cycle: concentrates , ; milk powder , ; roughage , . d the answer category "yes" is included as reference category. * median and interquartile range. santman-berends et al. preventive veterinary medicine ( ) - ratio was . (corresponds to . times lower mortality). thus veal calves from a certain production cycle with an average weight at arrival of kg had a . times lower mortality rate during the fattening period than calves from a cycle with an average weight at arrival of kg. finally, veal herds in which the predominant breed was belgian blue (based on the hair color), had a mortality rate of . compared to the population average. observations of . million calves that were fattened during the study period and were born in the netherlands were available for inclusion in the model. the majority of these calves originated from dairy herds ( %), followed by suckler cow herds ( %) and other herds ( . %). in total parameters were evaluated for their potential association with calf mortality. twelve parameters and the random herd effect correcting for clustering within veal herds remained in the final model (table ). the final model included thousand records from calves originating from more than thousand different herds that were eventually fattened in different veal herds. the association between the size of the herd of origin and mortality was inconsistent. purchase of cattle into the herd of origin on or prior to the date the calf was born was associated with a higher mortality odds during the subsequent fattening period (table ). herds with high mortality rates in the quarter in which the calf was born (both in calves and older cattle), fast growth in herd size, high cattle replacement rates and a higher antibiotic use in the quarter of birth were also associated with a higher odds for mortality during the fattening period (table ). calves that originated from herds certified bvd-free, salmonellaunsuspected or paratuberculosis-unsuspected, had a lower odds to die during fattening compared to calves that originated from herds with an unknown status for these diseases. originating from a herd that was certified ibr-free was associated with a higher odds to die during fattening (table ) . in this study, many risk factors were identified for veal calf mortality during the fattening period, both related to the veal herd and the herd of origin. the mortality per production cycle varied between . % and . % during the study period and was in the same range as previous studies on mortality in veal calves. in an italian study conducted by brscic et al ( ) , mortality rates per production cycle varied between . and . %, and in switzerland a mortality rate of . % was found (bähler et al., ) . furthermore, pardon et al ( ) found that mortality rates were on average . % per production cycle in belgium veal herds. additionally, we found that the gradual increase in mortality rate that was observed in the period before the initiation of this study, stabilized during the study period. the highest mortality rates in veal calves were observed in and declined thereafter. the decreased probability of mortality appeared to occur in the second half of the fattening period, indicating that the reduction was associated with veal calf management rather than management in the herd of origin. our study confirmed some risk factors already determined in previous studies, but also detected new risk factors. one of the most important factors in our model was the body weight of calves at arrival, with a higher starting weight being associated with a lower mortality. we checked whether the association between mortality and body weight would change when we corrected for age at arrival as confounder, but no change in association was observed. our results were in agreement with the results of brscic et al. ( ) , who found that a lower weight at arrival was associated with an increased risk to develop respiratory infections, which is an important cause for mortality. additionally, both winder et al. ( ) and brickell et al. ( ) found that a lower body weight at arrival in the veal herds was associated with an increased risk to die in the first six months. veal calves of the belgian blue breed had a lower mortality risk than other breeds of calves in our study. even though, these calves might have a higher body weight at arrival, the lower mortality risk was also observed when we table results of the multivariable multilevel logistic regression model of the association between veal calf mortality during the fattening period and risk factors associated with the herd of origin based on , dutch veal calves that were fattened in the period between january and july . corrected for weight at arrival as confounding factor. it was hypothesised that the lower mortality rate was due to the fact that belgian blue calves are very valuable resulting in application of additional care to maximise the probability of survival. this hypothesis was further supported by the fact that the veal cooperations stated that they only select specific farmers for fattening belgian blue calves. another known risk factor that was found in our study was purchase of cattle. both in the herd of origin as in the subsequent veal herd (herds without an all-in/all-out system), purchase was a risk factor for a higher mortality rate. this finding was in accordance with previous studies of pardon et al. ( ) and gorden and plummer ( ) , who found that purchase resulted in a higher risk for respiratory infections during the fattening period, which consequentially may result in a higher mortality risk. the kaplan-meijer survival curve showed that the detrimental effect of purchase i.e. not having an all-in/all-out system, occurred from day after arrival in the veal herd and onwards (results not presented). generally, after this moment a new production round of calves enter the veal farm without all-in/all-out system resulting in an increased risk of introduction of infections and increasing the mortality risk. the significant association between veal calf mortality and number of calves in the specific production cycle that was found in this study was incoherent as both the smallest and largest veal herds were protective factors, whereas the size groups in between had a higher risk. moreover, the association between veal calf mortality and the information from the critical control points was ambiguous. the answers could either be yes the farm complies with the demand, no the farm does not comply or not applicable. for most points, the answer "no" did not occur and the answer "not applicable" was significantly associated with the mortality rate. nevertheless, information about the rationale behind this answer was not provided and might differ between farms. in our study, a higher use of antibiotics in either the veal herd or the herd of origin was associated with a higher mortality rate. both results indicate that health problems, either during the first weeks after birth or during the subsequent fattening period are associated with a higher probability to die during the fattening period. this finding is supported by earlier research of jarrige et al. ( ) and bähler et al. ( ) , who both found that antibiotic treatment was associated with higher mortality. in the netherlands, prophylactic use of antibiotics is forbidden and a higher use of antibiotics is thus an indication for increased health problems that result in a higher mortality rate. moreover, a higher use of antibiotics might result in antibiotic resistance in time, which can deteriorate the possibility to treat infections and thus increase mortality (hoet et al., ; bos et al., ) . furthermore, we also observed that calves originating from herds with a higher health status for endemic diseases (i.e. bvdv, salmonella, paratbc), had a lower probability to die during their subsequent fattening period. providing a higher than median amount of either milk and roughage or all three types of feed (milk, roughage and concentrates), was associated with a lower mortality rate. additionally, application of a lower than median amount of milk and roughage or all three types of feed was associated with higher mortality. previous study already stressed the importance to provide sufficient amount of feed, especially when diarrhoea problems occur (lorenz, ) . in our study we were only able to distinguish concentrates and roughage and were unable to specify the feed on a more detailed level. based on previous studies, not only the amount of feed but also the specific feed that is administered is indicated to be associated to health problems and mortality (webb et al., ; berends et al., ; brscic et al., ) . although no comprehensive conclusion about the risk of certain types of feed can be drawn, it seems that a certain amount of roughage supports the development of the rumen and have a positive effect on calf health. unexpectedly, born in winter was not associated with an increased mortality while earlier studies found this birth season to be a risk (bendali et al., ; brscic et al., ; winder et al., ) . when we excluded the humidity and temperature from our model, we did find winter to be a risk factor. we therefore argue that it is not the season itself, but the weather conditions in that specific season that are associated with mortality. to our knowledge, this study was the first that studied risk factors for veal calf mortality during fattening related to both the veal herd and the herd of origin. this approach offered additional insight compared to research that only evaluated risk factors during the fattening period. nevertheless, there was still one factor missing in our study of which it is known that it influences health of veal calves, namely transport to the veal herd. we evaluated the possibilities to include such information, but at the time of the study, data of the transporter was not yet centrally registered. nevertheless, given the strict regulations regarding transport on both european and national level (ec, ; ikb, ) , we assumed that the variation in quality of transport was limited and thus that transported played a minor role compared to factors related to management in the herd of origin and the subsequent veal herd. ideally, both risk factors related to herds of origin and risk factors related to veal herds would be included in the same model. however, this was impossible due to a lack of background information from calves that were imported (in this study % of the veal calves). additionally, while most of the information of the veal calves during the fattening period were available at the level of the production cycle, the information of the herd of origin (if born in the netherlands) was available on individual level. this enabled more detailed evaluation of risk factors for veal calf mortality associated with the origin of the calves. for selection of the most appropriate model, both multilevel poisson regression and negative binomial regression models were evaluated. both regression models resulted in the same final model and because the model with the log-linear distribution showed the best fit to the data, it was decided to present those results in this paper. for this analysis, only routinely collected data was available. the advantage of this type of data is the possibility to include a large number of observations resulting in a high analytical power. however, a drawback is that we could not include factors that are hypothesized to be important but that were not available from central databases. such factors include for example quality and amount of colostrum received by the calves (perez et al., ; lorenz, ; osorio and drackley, ) , housing (lorenz, ; brscic et al., ) and climate in the barn (lorenz et al., ; bähler et al., ) . it is therefore commendable to collect additional management data to evaluate the importance of the factors that were observed in this study compared to management factors that are not present in generic big datasets. the large dataset allowed detection of many risk factors associated with mortality of veal calves during the fattening period. both factors in the herd of origin and the veal herds were found to influence mortality during the fattening period. adequate collaboration between the different industries is necessary to optimize veal calf management leading to a maximal reduction in veal calf mortality during the fattening period. risk factors for death and unwanted early slaughter in swiss veal calves kept at a specific animal welfare standard pattern of diarrhoea in newborn beef calves in south-west france effects of early rumen development and solid feed composition on growth performance and abomasal health in veal calves livestockassociated mrsa prevalence in veal calf production is associated with farm hygiene, use of antimicrobials, and age of the calves mortality in holstein-friesian calves and replacement heifers, in relation to body weight and igf-i concentration, on farms in england veal calves' clinical/ health status in large groups fed with automatic feeding devices prevalence of gastrointestinal disorders recorded at postmortem inspection in white veal calves and associated risk factors prevalence of respiratory disorders in veal calves and potential risk factors on the prot ection of animals during transport and related operations and amending dir ectives / /e ec and / / ec and regulation control, management, and prevention of bovine respiratory disease in dairy calves and cows association of enteric shedding of bovine torovirus (breda virus) and other enteropathogens with diarrhea in veal calves supply chain regulations for transport of veal calves in the netherlands: voorschriften transporteurs rules and regulations for veal farmers, veal hauliers and veal assembly centres quantitative and qualitative analysis of antimicrobial usage in white veal calves in france nonparametric estimation from incomplete observations freely accessible data base of daily meteorological records of the netherlands diarrhoea of the young calf: an update inadequate colostrum intake decreases growth of calves on intensified feeding programs longitudinal study on morbidity and mortality in white veal calves in belgium management factors related to calf morbidity and mortality rates the use of antimicrobials in farm animals in veal calf industry economics surveillance of cattle health in the netherlands: monitoring trends and developments using routinely collected cattle census data data analysis and statistical software. statacorp lp monitoring of antimicrobial susceptibility of streptococcus suis in the netherlands effects of roughage source, amount, and particle size on behavior and gastrointestinal health of veal calves control of infectious diseases in housed veal calves mortality risk factors for calves entering a multi-location white veal farm in ontario. can this study was funded by the organisation of the dutch dairy supply chain (zuivelnl/dairynl, the hague the netherlands) and the dutch calf industry association (stichting brancheorganisatie kalversector (sbk), nieuwegein). the authors would like to thank the data suppliers that delivered the data for this study and e. damen for her participation in this study. key: cord- -ajj qy c authors: maier, g. u.; love, w. j.; karle, b. m.; dubrovsky, s. a.; williams, d. r.; champagne, j. d.; anderson, r. j.; rowe, j. d.; lehenbauer, t. w.; van eenennaam, a. l.; aly, s. s. title: management factors associated with bovine respiratory disease in preweaned calves on california dairies: the brd study date: - - journal: journal of dairy science doi: . /jds. - sha: doc_id: cord_uid: ajj qy c abstract the objective of this cross-sectional study was to determine how management practices on california dairies may be associated with bovine respiratory disease (brd) in preweaned calves. a convenience sample of dairies throughout california, providing a study population of , calves, were visited between may and april . during each farm visit, in-person interviews with the herd manager or calf caretaker were conducted to collect information about herd demographics, maternity pen, colostrum and calf management, herd vaccinations, and dust abatement. a random sample of preweaned calves was identified and evaluated for the presence of brd using a standardized tool. a survey-adjusted generalized linear mixed model with a logit link function was fitted with calf as the unit of analysis and dairy as the random effect. mean study herd size (±se) was , (± . ) cows. survey-adjusted estimates of breed types in the sample were . % (± . ) holstein, . % (± . ) jersey, and . % (± . ) crossbred or other purebred breeds, and calf sex proportions were . % (± . ) female and . % (± . ) male. overall survey-adjusted brd prevalence in the study herds was . % (± . ). housing factors positively associated with brd were metal hutches compared with wood hutches [odds ratio (or) = . ; % confidence interval (ci) = . – . ], calf-to-calf contact in calves > d of age (or = . , % ci = . – . ), feeding holstein calves < . l of milk or replacer per day (or = . , % ci = . – . ), and lagoon water used for flushing manure under hutches compared with no flush (or = . , % ci = . – . ). providing extra shade over hutches (or = . ; % ci = . – . ), feeding calves at least % saleable milk (or = . , % ci = . – . ) or pasteurized milk (or = . ; % ci = . – . ), and feeding > . l of milk or replacer per day to jersey calves (or = . ; % ci = . – . ) were negatively associated with brd. our study identified management practices on california dairies with variability and that may contribute to differences in brd prevalence, which will be incorporated into a risk-assessment tool to control and prevent brd in preweaned dairy calves. bovine respiratory disease (brd) complex is one of the most common causes of death in dairy calves and poses a significant welfare and economic burden on the industry (panciera and confer, ) . reported morbidities for calves in the nahms study evaluating the health of preweaning heifers on dairies was only . % in calves up to weeks of age (usda, ) . in , respiratory disease in dairy heifers was reported as the cause in . % of deaths before and . % of deaths after weaning (usda, ) . in addition, . % of preweaning heifers on dairy heifer-raising operations were reportedly affected by pneumonia, making it the second most common calf illness after diarrhea (usda, ) . hence, over the last few decades, no improvement has been reported in morbidity from brd in dairy calves. given the lack of improvement in brd incidence in us dairy cattle, despite the availability of numerous vaccines and antimicrobial drugs labeled for brd, novel approaches that target prevention in addition to control management factors associated with bovine respiratory disease in preweaned calves on california dairies: the brd study should be evaluated. the complexity of etiologic agents and predisposing factors for brd combined with the difficulty of accurate diagnosis pose challenges in the prevention and control of this disease on the farm that may be addressed using a risk-assessment approach in combination with a disease-scoring system. a multitude of tests can be used to identify pneumonia calves in a herd; however, scoring systems require minimal training, are low cost, and can be reasonably accurate, making them a viable tool to estimate the disease burden in the herd (love et al., (love et al., , b . repeated use of a risk-assessment tool to target preventive management practices combined with a scoring system to benchmark the burden of brd in a calf herd over time may offer a low-cost, rapid, and comprehensive control program for brd. in contrast to a chronic disease, such as johne's disease, where changes implemented to control the disease may not result in a reduction in incidence for many years, brd primarily presents as an acute disease. with the daily influx of newborn calves, changes in incidence may be observed within months of implementation of the prevention or control program. dairies in california differ in many ways from dairies in other parts of the united states. for example, the average dairy herd size for california in was , milking cows, with herds up to , milking cows, whereas the latest report on dairy cattle management practices published by usda categorizes herd sizes with more than cows as large (california department of food and agriculture, ; usda, a). other differences include california's climate, which allows for year-round outdoor housing of calves and cattle, which is impractical in many parts of the country (lago et al., ) . climate and dairy management practices in california are not homogeneous across the state, however. as an example, in , california held . % of the nation's organic dairy cows, most of which were found in the northern part of the state . it is clear, that the development of a risk-assessment tool for california dairies cannot be based on national averages but should be based on data collected from within this unique industry. the objective of our study was to identify factors associated with brd in preweaned calves on california dairies by correlating management practices on california dairy farms to prevalence of brd in calves exposed to these practices. our hypothesis was that brd is associated with management practices related to risk factors for brd, such as the amount of colostrum and milk fed, housing type, and vaccinations. the research described here is a further step in the development of a risk-assessment tool for brd in preweaned calves on california dairies following the survey of management practices related to brd in preweaned calves on california dairies (love et al., a) . a cross-sectional study was designed to collect data on management practices and estimate brd prevalence in preweaned dairy calves on a convenience sample of california dairies. in , managers of , california dairy herds were surveyed using mail questionnaires followed by a reminder card after each mailing. the survey aimed to identify the variety of management practices known to be associated with the incidence of brd in preweaned calves with the purpose of characterizing these practices, comparing them across geographic regions of the state, and determining the principal components that explain the variability in management between herds (love et al., a) . respondents to the survey were given the option of providing contact information if they were interested in participating in a follow-up study. in addition to respondents who showed willingness for a follow-up herd visit, we sought local dairies, veterinarians, consultants, farm advisors, and dairy owners through personal contacts to enroll farms. emphasis was placed on inclusion of a wide range of herd sizes, geographical locations, and management styles into the sample. the study protocol was approved by the university of california at davis animal care and use committee (approval # ) . a sample of dairies was targeted, which is the number of groups required to obtain unbiased estimates for fixed effects if group sizes are or higher (moineddin et al., ) . a sample size calculation for the number of dairies required to estimate the state prevalence of brd in california yielded n = based on a total of , dairies (n = , ) in the state and assuming a conservative prevalence estimate of . , to maximize n. the sample size to estimate the state prevalence was calculated using the formula where n = number of dairies in the state, p = . , and b = % for the bound on the error of estimation of p = . = var p ( ) (scheaffer et al., ) . within dair-ies, a simple random sample of calves was drawn to estimate the prevalence of brd on each of the participating dairies based on equation , but where n = number of calves on the farm, p = . for the estimate of herd prevalence (love et al., b) , and b = % for the bound on the error of estimation of p = . = var p ( ) (scheaffer et al., ) . the principal components identified in the survey formed the basis for the on-farm questionnaire (love et al., a) . briefly, the questionnaire collected information about demographics (location, herd size, and organic or conventional production), maternity pen management (density and individual or group pens), colostrum management (source, heat treatment, storage, quality, volume fed, and testing of calves for failure of transfer of passive immunity), calf management (housing, time of weaning, treatment of sick calves, criteria for treatment, and type and volume of milk fed), vaccinations (type, timing, and frequency of administration to adult cattle and calves), and dust abatement (whether perceived as a problem and type of abatement). study outcome was the presence of brd at the time of the visit in a simple random sample of calves at the dairy that were either housed individually or housed in groups provided they were fed milk as part or all of their diet. calves were scored using the standardized and validated california scoring system for brd in preweaned calves based on a point system (love et al., ) . in the california scoring system, points are assigned for the presence of each of these clinical signs: points for cough, ocular discharge, dyspnea, or a rectal temperature of ≥ . °c ( . °f); points for nasal discharge; and points for a head tilt or ear droop. a calf that scores points or more is scored as positive for brd. dairies were visited between may and april and in-person interviews with owners or herd managers were conducted. the individuals conducting the interviews consisted of veterinarians, graduate students, research assistants, animal scientists, extension and outreach specialists, and farm advisors who were all familiar with the california dairy industry. all interviewers were trained on the use of the california brd scoring system and were accompanied by a more experienced team member during their first farm visit. interviewees were instructed to answer questions based on management practices in place only as far back as when the oldest calf eligible to be enrolled was born. the number of calves on the premises was determined as part of the questionnaire. once the questionnaire was completed, study personnel proceeded to score a random number of calves based on the total number of calves on the farm to estimate the calf herd's brd prevalence. the random sample was drawn from all individually housed calves and calves in groups on a milk diet and included calves that were recently weaned if still housed in a hutch. sample sizes of calves to be scored for n = (n = ), (n = ), (n = ), (n = ), , (n = ), and , (n = ) calves on the farm were calculated per equation and sheets with random numbers between and n prepared in a spreadsheet (excel . the sheet with the next larger n was used for selecting the sample of calves. scoring started typically with the youngest calf followed by counting of hutches according to the random number sheet until the next calf on the list was reached and scored and so on until the oldest eligible calf was reached. apart from the clinical signs that are part of the brd score, the calf's ear tag number, sex, and date of birth were recorded. breed was recorded as holstein, jersey, or other, the latter including other purebred breeds as well as crossbred animals. hutch type for each calf was described in terms of floor and wall materials, whether a roof was partial, full, or absent, whether extra shade was provided, and the dimensions of the hutch or pen. in addition, the elevation of the hutch floor from the ground, the presence and type of a flush system for manure, and the number of calves in the enclosure were recorded as well as whether calf-to-calf contact with other calves was possible. further, the distance from the hutch to the nearest dust area was measured. maternity pens were visited, dimensions of pens were measured, and hygiene scores for cows were assigned according to a -point system. a score of indicated no manure soiling above the pastern; score indicated manure soiling on the lower leg; score indicated manure soiling on the upper leg, udder, and abdomen; and score indicated manure soiling on udder or abdomen and toward spine (farmers assuring responsible management, ) . if fewer than cows were present in the maternity pen, all cows present were scored. the dimensions of hutches (width and depth) as well as maternity pens and group pens (width and depth) were measured in feet and inches and transformed to metric measurements for analysis. of , calves that were originally enrolled, calves on dairies were removed from analysis due to missing information on their date of birth. several dairies kept records with approximate ages of calves (e.g., birth month), but these calves were also excluded from the final model once age was confirmed as an important confounder. calves that had missing data for any of the predictors in the final multivariable model were excluded from analysis as well, resulting in , calves in the final model ( figure ). no imputation of missing data was performed. survey-weighted proportions of calves missing from the analysis were compared with calves in the final model in terms of prevalence with pearson's chi-squared test. all statistical analyses were performed using a commercial software package (stata . , stata corp., college station, tx). calf-level explanatory variables included age, breed, and sex; housing-related variables, such as the type of hutch the calf is housed in, including the wall and floor materials; whether extra shade was available; whether the hutch was elevated or not; whether or not calf-to-calf contact was possible; whether the calf was individually housed or in groups; if and what type of flush system for manure was in place; and what type of surfaces were close to the calves. in addition, milk-feeding related variables were assigned at the calf level by recording each calf's age and relating it to the source and amount of milk being fed, and whether or not the milk was pasteurized or anything was added to the milk. vaccine-related variables were at the calf level by determining whether or not a calf had received a certain vaccine based on the age of the calf and the vaccination protocol discussed while filling in the questionnaire. all other variables, such as all variables relating to maternity pen management, colostrum management, vaccinations of cows and heifers, or herd demographics, were recorded at the herd level. all analyses were performed at the calf level with the unit of analysis being the calf and values of p < . considered significant. relational database design. individual calves scored during visits were matched to questionnaire answers by dairy identification in a database (access . calf records were linked to survey responses with a query in access, matching the age, sex, and breed demographics of the calf to the respective management based on the survey. most differences in management were related to the amount, source, or number of milk feedings by age, but also included different timing of movement to group housing by breed or management differences concerning diet or colostrum by breed or sex. all records were checked for completeness, plausibility, and accuracy. descriptive statistics. characteristics of dairies and calves in the sample were described using proportions for categorical and means for continuous variables as well as standard errors and % confidence intervals of these estimates. estimates at the calf level, including prevalence estimates, were survey adjusted, where each observation was weighted by the inverse of the sampling fraction ( /f j ), with f j being the number of calves scored divided by the number of calves on the jth premises. comparisons between levels of categorical variables were performed with chi-squared tests and bonferroni-dunn adjustments for multiple comparisons. strata with single sampling units were omitted from proportion estimates because they prevented calculation of a standard error. multivariable logistic regression model. the dependent variable for all analyses was the presence or absence of brd in study calves, as assessed via the california brd scoring system. continuous variables were assessed for linearity using the box tidwell test (box and tidwell, ) . variables that did not meet this criterion were categorized using biologically sensible cut points. two-by-two tables of categorical variables with case status were inspected for cell frequencies and categories were collapsed as necessary to achieve cell sizes of or greater. survey-adjusted univariate generalized linear mixed models with a logit link function with a random effect for dairy were fitted to each variable with the melogit command in stata. variables were then added one at a time to a survey-adjusted multivariable generalized linear mixed model with a logit link function with dairy as a random effect. variables were assessed in the multivariable model in the groups colostrum management, housing, precalving management and immunity, and feed. variables were added in order of increasing p-value obtained in univariate analysis. the variables age, breed, sex, season, herd size, and region were considered possible confounders and model fit was assessed frequently during model building by adding these covariates as confounders. biologically relevant interaction terms for colostrum, feeding, and housing management with the variables breed, herd size, sex, and age were tested during model building. the variables for herd size, breed, and region were forced into the final model to adjust for their potential confounding effects. variables that either had a significant association with the outcome in the univariate analysis or that had a significant association in the multivariable model but were eliminated after adjusting for another variable were tested again in the final multivariable model. akaike information criterion was used to assess model fit with models producing lower values considered better fit. age was divided into categories of < , to , and > d. season was categorized as spring [april-june; reference (r)], summer (july-september), fall (october-december), and winter (january-march) based on distinct climate patterns that may be relevant to brd, such as precipitation and temperature fluctuations. milking herd size was categorized as < (r), - , , - , , and > , milking cows. regions were categorized based on differences in climate and types of dairies as northern california (nca; r), the northern san joaquin valley (nsjv), and greater southern california (gsca), where the southern san joaquin valley and southern california are combined into a single region due to low recruitment success for southern california dairies (love et al., a ; figure ). questions that could be answered with yes or no resulted in dichotomous variables regarding colostrum, including supplementation with a colostrum replacer, heat-treatment, bacterial content testing (either amount or type of bacteria before or after heat treatment), storage for greater than h between milking and feeding calves, and use of a preservative. colostrum source was categorized as % individual cow or nursing from dam (r), % pooled from multiple cows, or a mix of those sources including colostrum replacer. colostrum from individual cows and colostrum nursed from the dam were combined into a single category because the quality of the colostrum from these sources was regarded as equal. for those dairies that heat-treated their colostrum, the time between colostrum harvest and heat-treatment was categorized into either less than (r) or more than h. the distribution of storage time for colostrum before feeding in hours underwent a square root transformation to achieve normality. if colostrum was stored for less than h, zero hours were recorded for colostrum storage time. storage temperature for colostrum was recorded as either room temperature (r), refrigerated, frozen, or first refrigerated then frozen. a variable for colostrum storage temperature had indicator variables for each of the storage temperatures. the type of storage container for colostrum was dichotomized as solid (r) or bags, as thawing frozen colostrum stored in bags may achieve a more uniform increase in colostrum temperature compared with colostrum stored in plastic bottles and may result in less heat damage to immunoglobulins. the variable for percent of colostrum fed that is from firstcalf heifers was dichotomized into the categories any or no (r) colostrum from first calf heifers. the amount of colostrum fed in the first h was dichotomized into < . l (r) or ≥ . l ( quarts), as feeding l of colostrum was considered a relevant biological cutpoint (godden et al., ) . four dairies let calves nurse from their dams to meet the calves' colostrum needs; hence, estimates for the amount of colostrum fed in the first h on these dairies could not be made. in addition, a dichotomous variable for whether or not (r) colostrum was tested for immunoglobulin content and a dichotomous variable for whether or not (r) calves were assessed for failure of transfer of passive immunity based on serum total protein were explored in the model. data to create variables concerning housing of calves were drawn from the questionnaire sections that captured herd-level data as well as from data collected at the individual calf level. questionnaire data. proportions of calves housed individually or in groups, average group size of grouphoused calves, housing types, weaning age, age when moved from individual hutches to group housing, percent sick preweaned calves moved to a hospital pen, clinical signs to detect brd, and treatment choices for brd were used only for descriptive statistics purposes. this was done because these variables provide information about the whole cohort of preweaned calves and are not suitable for analyzing the association of management practices and brd in individual calves. data on the percent of calves raised on the premises from other dairies was dichotomized into any or no (r) calves from other dairies on the premises. the variable feeding order by age was created with indicator variables for the answer choices youngest typically fed first (r), calves fed in no particular order, and oldest typically fed first. only dairy in the sample used a brd scoring system; hence, the variable describing use of such systems was dropped from analysis. as some dairies gave multiple answers to the question about what surfaces, driven on daily, cover the roads adjacent to the calf-raising area, dichotomized variables for paved, gravel, dirt, or other (hay, fly ash, and shavings) surfaces were created and analyzed as separate variables. similarly, dichotomized variables for dust-abatement procedures were created describing whether or not water, magnesium chloride, or other dust-abatement measures (limiting speed, no driving through the area, restricting entry, oiling of driveway) were implemented (amato et al., ) . for the question on how often dust was a problem in the area where preweaned calves were housed, a categorical variable was created with never as the reference and the indicator variables rarely (weeks pass without dust), no week passes by without dust, daily during the nonrainy season, and daily all year round. calf-level data. the number of calves in the enclosure was dichotomized into group or individual housing type (r). whether calves had calf-to-calf contact or not (r) was recorded as a dichotomous variable. hutch space was defined as the product of the width and depth, as measured during the visit. a categorical variable was created, categorizing the data as less than (r), greater than, or within standard deviation around the mean space for wooden hutches in the sample population, and a separate category for group pens. hutch elevation from the ground was dichotomized into elevated or not (r). dichotomous variables for each of the reported floor materials were specified where absence of the specific floor material was the r [concrete, rubber, sand, tenderfoot (tandem products, inc., minneapolis), dirt, grass, wood, and bedding]. in addition, dichotomous variables for slatted floors (wooden floors with slats) and floors that allow airflow (slatted floors and tenderfoot) were specified. wall materials were categorized with indicator variables for only wood (r), only metal, only plastic, and a category for a mix of materials or group pens. fourteen dairies had at least some hutches that were made of a combination of materials, such as wood and metal or metal and plastic. calves housed in groups were added to the latter category to be able to assess the association of a single hutch material on an individually housed calf without losing group-housed calves or calves in hutches made from a mix of materials in the analysis. in addition, a dichotomous variable for solid walls (r) or nonsolid walls (wire metal or metal bars as material) was specified to explore an association with air flow in hutches and brd. a categorical variable describing the presence and type of flush system under the calf hutches was specified with levels: no flush (r), fresh water flush, or flush with lagoon water. a categorical variable describing the presence and type of extra shade structures above calf hutches was specified with levels no cover (r), partial roof, complete roof without side walls, complete roof with to side walls, and fully indoors. the calving space in square meters per cow was calculated by dividing the average number of cows per pen by the dimensions of the maternity pen. calving space was normalized using the natural log transformation. the percent of calving area that is pasture was dichotomized into any or no calving on pasture (r). the number of times the bedding is changed in the calving pens was dichotomized into less than or equal to times (r) and more than times per month. typical maternity pen bedding type was summarized into categories of plant fiber (almond shells, rice hulls, straw, wood shavings, sawdust, wood chips, cotton stock, recycled wood product), recycled manure (r), gypsum or lime, dirt (which included sand), and pasture. those dairies that answered no maternity pen exists or no bedding in maternity pen were assigned to the category sand or dirt. dichotomous variables were created for the presence or absence (r) of each type of bedding, as multiple dairies used more than type of bedding. the data for the percent of calves removed in h from the dam was dichotomized into ≥ and < % (r). vaccine timing and frequency for cows and calves was dichotomized into whether a type of vaccine was given or not (r). the time since vaccination for each calf was estimated as the time between the reported age when calves receive a certain type of vaccine as stated by the interviewee in the questionnaire and the age of the calf at the time of visit. dichotomous variables were created describing whether a certain type of vaccine was given at least d before the visit to account for the fact that there is a variable lag time between vaccine administration and an immune reaction. hygiene scores for up to cows present in the maternity pen during the dairy visit were transformed into cumulative hygiene scores, which facilitated comparisons of percent of cows scoring , and , or and and versus the percent of cows scoring the respective remaining scores. questions that could be answered with yes or no resulted in dichotomous variables including if milk was ever tested for bacterial content (either amount or type of bacteria) both before pasteurization and after pasteurization and if any medication was added to milk or milk replacer (r = no testing or no medication). the source of milk for each diet fed on the study dairies was recorded as a percentage of the total diet during the farm visit. for the analysis, these percentages were converted into dichotomous variables per milk source describing whether a calf's diet consisted of > % of one of the sources saleable milk, waste or hospital milk, unmedicated milk replacer, or medicated milk replacer for at least d before the farm visit or not (r). no distinction was made between waste milk from fresh cows and milk from cows in a milk-withdrawal period due to medication. one dairy fed waste from a yogurt producer, which consisted of a water-yogurt mixture created during flushing of the production lines. a separate dichotomous variable for feeding of the yogurt waste product was created. the variable describing whether milk fed had been pasteurized (r = not pasteurized) contained a category for weaned calves to avoid exclusion of weaned calves from the model. for the number of times calves were fed milk per day, a categorical variable was created with twice feeding as the reference and levels of once daily as well as a level including or times or free choice feeding. two calves in the study were suckling from the dam based on their age and management of neonatal calves described by the producer and had no amount of milk fed or number of times fed assigned. the total volume of milk fed per day, which was calculated from the number of times calves were fed milk and the volume offered at each feeding, was categorized into ≤ . l ( quarts), between . l and up to . l ( quarts; r), and > . l. finally, dichotomous variables for whether or not (r) antimicrobial drugs, vitamins, or electrolytes were added to the milk were explored. the type of medication used to treat brd was used for descriptive statistics only. for this cross-sectional study, a convenience sample of grade-a dairies across california was drawn, resulting in an initial study population of , calves. of dairies visited during the study period, were eliminated from analysis because calf ages were not available ( calves, figure ). characteristics of the remaining dairies and calves in the study population included in the analysis are available in supplemental table s (https: / / doi .org/ . / jds . - ). mean (±se) and median reported herd size were , . (± . ) and , (interquartile range = - , ) milking cows, respectively, with herd size ranging from to , cows. average reported breed composition on the study dairies was . ± . % holsteins, . ± . % jerseys, . ± . % other purebred, and . ± . % crossbred (due to rounding, these percentages add up to . ). about in dairies ( . ± . %) in the study was certified organic, with the majority of organic dairies located in the nca region ( . ± . %). calves were reported to be predominantly housed in individual housing units ( . ± . %). the most commonly reported type for individually housed calves was the california hutch system ( . ± . % of dairies)-a wooden hutch with separate compartments housing calf each-followed by a type described as hutch with adjacent enclosed individual exercise area ( . ± . % of dairies), and other types ( . ± . % of dairies). multiple dairies used more than hutch type ( ± . % of dairies) the mean age in days when calves were moved to group housing ( . ± . ) was similar to the mean age in days when weaning occurred ( . ± . ). nasal signs ( . ± . %), such as dry nose or nasal discharge, abnormal breathing ( . ± . %), droopy ears or head tilt ( . ± . %), and cough ( . ± . %), were the clinical signs most commonly used to diagnose brd in calves on study dairies. most dairies used more than clinical sign to diagnose brd ( ± . %). only of the study dairies used a clinical scoring system for detection of brd, a hybrid of the wisconsin and california systems (mcguirk and peek, ; love et al., ) . phenicols ( . ± . %, which was represented exclusively by florfenicol) and macrolides ( . ± . %) were the antibiotic classes most frequently named for the treatment of brd in calves among the dairies where data on drug choices were available. of the dairies in the study where drug choices for brd treatment in calves were known, ± . % used more than drug. almost half the respondents ( . ± . %) indicated they were using a nonsteroidal anti-inflammatory drug in the treatment of brd. the sample of , randomly selected calves from the study dairies across california that were either preweaning or days to a few weeks after weaning but still housed individually in hutches consisted of . ± . % holstein, . ± . % jersey, and . ± . % crossbred or other breeds. of the calves in the study, . ± . % were female and . ± . % were male. of the , calves with known exact ages, . ± . % were less than d old, . ± . % were between and d old, and . ± . % were to d old (due to rounding, these percentages add up to . ; supplemental table s ; https: / / doi .org/ . / jds . - ). survey-weighted prevalence estimates for brd in the sampled calves are shown in table . overall prevalence of brd was . ± . % with a range of to . %. we found no significant differences in breed-specific prevalence between holstein ( . ± . %), jersey ( . ± . %), and other purebred or crossbred calves ( . ± . %; p = . ). the prevalence in female calves ( . ± . %) was numerically smaller than in male calves ( . ± . %) but not statistically significant (p = . ). the age group between and d of age had higher prevalence with . ± . % compared with the youngest calves under d of age, with . ± . % affected (p < . ). in conventionally managed dairies, prevalence ( . ± . %) was not different from that in certified organic dairies ( . ± . %; p = . ). regionally, the nsjv region had lower prevalence ( . ± . %) than either nca ( . ± . %) or the gsca region ( . ± . %; p = . ). table shows the associations between predictors and the presence of brd in calves in the study population after testing all main effects, as well as biologically relevant confounders or effect modifiers. the proportion of variance at the dairy level was ± . %. demographics and seasons. herd size, breed, and region were forced into the model and were not significantly associated with brd. fall compared with spring was significantly associated with brd [odds ratio (or) = . , % ci = . - . , p = . ]. predictors concerning colostrum management. none of the predictors describing colostrum management was associated with brd in our study population. interactions between heat treatment of colostrum and the source of the colostrum, as well as between the volume of colostrum fed and breed or sex, were explored and none were significant. additionally, confounding of colostrum related variables by season, herd size, breed, sex, and age was investigated and no change in estimates found after adjusting for possible confounders. predictors concerning housing. the association of calf-to-calf contact on brd depended on age, where increased odds of brd were observed in calves > d of age that had calf-to-calf contact. (or = . , % ci = . - . , p = . ) versus those that did not. however, the latter association was not observed in younger calves. when contrasting age groups among those calves with calf-to-calf contact, calves > d old had almost times higher odds of brd (or = . , % ci = . - . , p = . ) than those < d of age. calves between and d old had over times higher odds of brd compared with calves < d old (or = . , % ci = . - . , p = . ; table ). the use of fresh water for flushing under hutches was not associated with brd; however, the use of a flush lane the presence of an extra shade cover over the hutches in the form of a full roof with no sidewalls was negatively associated with brd (or = . , % ci = . - . , p = . ). hutch space was not significantly associated with brd. predictors concerning precalving management or immunity. none of the variables characterizing whether calves or dams had been vaccinated with either a killed, modified live, or intranasal vaccine for the prevention of respiratory disease showed a significant association with brd. likewise, none of the other variables in this group was significantly associated with brd in our study population. predictors concerning milk feeding. feeding pasteurized milk was negatively associated with brd compared with calves drinking unpasteurized milk (or = . , % ci = . - . , p = . ). we compared calves that had been consuming at least % saleable milk for at least d before the farm visit, according to milk diets described in the survey, with those that had been on a different diet (i.e., waste milk, milk replacer, or less than % saleable milk in combination with waste milk or milk replacer) and found saleable milk feeding was negatively associated with brd (or = . , % ci = . - . , p < . ). we found a significant interaction between the total volume of milk or replacer fed per day and the breed of the calf. holstein calves compared with jersey calves fed < . l of milk or replacer had higher odds of brd (or = . , % ci = . - . , p = . ). feeding holstein calves between . and up to . l of milk or replacer compared with < . l resulted in lower odds or brd (or = . , % ci = . - . , p = . ). feeding jersey calves > . l was associated with lower odds of brd compared with feeding between . and . l (or = . . % ci = . - . , p = . ) or < . l (or = . , % ci = . - . , p = . ). in addition, feeding jersey calves > . l compared with feeding holstein calves > . l was also associated with lower odds of brd (or = . , % ci = . - . , p = . ; table ). calves omitted from analysis. there was no difference between calves in the final model (n = , ) and those eliminated from analysis (n = ) in terms of prevalence of brd (p = . ). the current study is the first to evaluate brd in preweaned calves housed on dairies throughout california and to associate brd in these calves with management practices. our results suggest that management relative to both housing and calf feeding practices may be the most important areas associated with the prevalence of brd in young dairy calves on california dairies. using lagoon water for flushing under hutches, the use of metal hutches, as well as calf-to-calf contact in older calves, and feeding holstein calves ≤ . l of milk or replacer per day were all positively associated with brd. pasteurizing milk, feeding saleable milk, feeding jersey calves more than . l of milk or replacer per day, and providing extra shelter were all negatively associated with brd. interestingly, no associations were found between vaccinating dams or calves or the amount or quality of colostrum fed and brd. management factors significantly associated with brd in our study may reflect some of the areas where the california dairy industry is less uniform and where changes may have the biggest effect on brd prevalence. current knowledge based on numerous studies underscore colostrum management and vaccinations as important components of brd prevention; however, the current study's dairies may have exhibited uniform colostrum and vaccination practices. the principal component analysis of a california survey on management practices related to bovine respiratory disease in preweaned dairy calves performed in identified calf housing as the component construct with the largest variation proportion (love et al., a) . the component construct contained the variables hutch style, calf contact, flush used, and waste milk fed to heifers. three of these variables (hutch style, calf-to-calf contact, and flush) were significantly associated with brd in our study, supporting the idea that these are areas of highly variable management on california dairies. although we did not observe an association between the fourth variable in the construct (waste milk fed to heifers) and brd in our study, we did observe a negative association between brd and feeding saleable milk for at least d, which is one of the alternatives to feeding waste milk. the state-average dairy herd size in california in was , milking cows (california department of food and agriculture, ), which is less than the average herd size of , milking cows but close to the median herd size of , milking cows in our study. herd sizes are expected to increase (usda, a), so the calves enrolled in our study likely represent a growing proportion of dairy calves raised in california. nationally, holsteins make up . % (± . se), jerseys . % (± . se), and other breeds . % of us dairy cows (usda, a), compared with . % holsteins, (± . se), . % jerseys (± . se), and . % crossbred (± . se) calves in our study. jersey cows have increased in popularity over the last years in california due to their higher milk fat content and potential for higher feed-to-milk conversion ratios among other health related reasons (mackle et al., ; washburn et al., ; white et al., ) . although no official statistics for california dairy breeds are available, it may not be unreasonable to assume that jersey cows are more common in california than nationally. organic farms make up . % of california dairies and are overrepresented in our sample ( . %; usda, b); however, this overrepresentation has the advantage of providing more accurate estimates for management practices from this sector of california's dairy industry. the mean percentage ( . %) of calves housed individually on dairies in our study of california dairies table . estimated odds ratios (or) for joint effects for total volume of milk or milk replacer fed per day and breed on bovine respiratory disease (brd) in , calves on california dairies was similar to the proportion of dairies that reported housing calves individually ( . %) in the survey of california dairies by love et al. ( a) , but larger than the mean nationally ( . %; usda, a). given the similar order of magnitude in breed distribution and other factors, such as organic status and individual calf housing, results of our study could be extrapolated to dairies in california and elsewhere that fall within the study herds' description and climate. case definitions for brd vary widely between studies and, therefore, any comparisons of results should be interpreted with caution. some studies rely on treatment records by dairy staff, whereas others use certain clinical signs for diagnosis, which may also differ between studies. most epidemiological studies on brd in dairy calves report incidence rates rather than prevalence. lago et al. ( ) found a prevalence of . % in dairy calves housed in barns in the winter in wisconsin and diagnosed with the wisconsin brd scoring system for preweaned calves. buczinski et al. ( ) found a median prevalence of % in the summer and % in the winter for lung consolidation diagnosed by ultrasound, consistent with brd in preweaned calves in dairy herds in québec. indoor housing and poor ventilation are associated with brd, so the higher prevalence in the wisconsin and québec studies in the winter is not unexpected (callan and garry, ; gorden and plummer, ) . both buczinski et al. ( ) and lago et al. ( ) reported a gradual increase in prevalence from birth to the sixth and seventh week of life, respectively; we observed a similar rise in prevalence, which continued until d of age (supplemental figure s ; https: / / doi .org/ . / jds . - ). the fact that most of the calves in the current study were housed outdoors may explain the lower prevalence observed of . %, comparable to what buczinski et al. ( ) observed in the summer. distributions of the percent of cases by age stratified by breed or region are shown in supplemental figures s and s (https: / / doi .org/ . / jds . - ), respectively. even though we observed lower prevalence in the nsjv region compared with the other regions of the state, region was not significantly associated with brd when adjusting for other factors in the multivariable model. we observed lower prevalence in herds between , and , milking cows compared with herds with fewer than or more than , milking cows. likewise, we found no association between herd size and brd in the final model. little evidence exists in the literature to support associations of breed or herd size with brd. a previous study found a higher risk of preweaning calf pneumonia in ottawa for increasing number of calvings per farm per year (waltner-toews, ) . no interpretation for this result was offered by the authors and it may not be relevant to the large california dairy farms of today. in the current study, we observed positive associations of calves housed in hutches made from metal components and brd. in a study based on the same data set, the prevalence of brd observed in calves housed in wooden hutches ( . ± . %) was similar to the prevalence of calves housed in metal hutches ( . ± . %). however, in nca, brd prevalence for calves housed in hutches or pens made of metal (including wire, pipe, or sheet metal; . ± . %) was significantly higher compared with those housed in wooden hutches ( . ± . %, p < . ; karle et al., ) . furthermore, a longitudinal study of , preweaned calves in california showed that calves housed in hutches made of a combination of wood and metal were at higher risk of brd compared with calves housed in hutches made of wood only . calf-to-calf contact in the age group over d was also significantly associated with brd. results of past studies on the effects of hutch type on calf health were mainly focused on the effect of plastic type hutches or available space. calvo-lorenzo et al. ( ) housed calves in wooden hutches of sizes between april and july in california to assess the effect of hutch space on health, performance, and respiratory immunity. calves were raised in conventional california-style wooden hutches and allowed either . , . , or . m /head of space. those authors concluded that increased space may improve pulmonary immunity and health, although it was not apparent which component of the increased space allowance environment influenced the finding. we did not observe an association of hutch space with brd in the present study, possibly because hutch material may be the more important component associated with brd. the temperature in plastic hutches can average to °c higher than in wooden hutches and ventilation is comparatively poor, leading to accumulation of heat, carbon dioxide, and humidity (lamb, ; lammers et al., ) . several studies have compared the effect of plastic hutch types on performance and health outcomes in dairy calves, and although agreement exists that plastic hutches increase heat stress, none have found significant associations with adverse health outcomes (macaulay et al., ; higginbotham and stull, ; peña et al., ) . no references regarding the effect of metal style hutches were found in the literature, but heat stress and possibly cold stress are likely associated with metal hutches and may explain the current study findings. specifically, % of metal hutches housing individual calves on dairies in the current study also had metal roofs, whereas % on dairies had no roof but were under a shed or other shade structure. the difference in health outcomes between hutch types should be researched further, ideally using an experimental study design comparing a limited number of hutch designs, including hutches made from metal components. the large number of different hutch designs in our study made it difficult to elucidate the association between hutch material and brd and has resulted in large confidence intervals around estimates. the current study identified a positive association between calf-to-calf contact and brd in calves older than d. callan and garry ( ) recommend spacing hutches at least . m ( feet) apart to prevent calf-to-calf contact and transmission of respiratory pathogens. a distance of . m between hutches was rarely, if at all, the case on the california dairies visited for our study, and the feasibility of this strategy and its cost in terms of land needed to raise the same number of calves may be a challenging constraint. in calves that are close to weaning age (in this study those > d of age), housing-or nutrition-related factors may be less important; thus, calf-to-calf contact with animals shedding pathogens may become a more important factor for their brd status. lagoon flush water could be a source of noxious gases (ammonia, hydrogen sulfide, and methane) that may irritate respiratory mucous membranes, making calves susceptible for opportunistic infections with commensals. the role of lagoon water on a dairy as a source of infective agents has mainly been studied with respect to enteric pathogens, including mycobacterium avium ssp. paratuberculosis, salmonella spp. and escherichia coli and its effect on antimicrobial resistance in some of the pathogens (aly and thurmond, ; grewal et al., ; li et al., ; ravva and sarreal, ) . alhamlan et al. ( ) found members of the flaviviridae in lagoon water samples, a family of viruses that contains bovine viral diarrhea virus (bvdv). although the viruses were not categorized to the species level in alhamlan et al. ( ) , it is conceivable that lagoon water could be a potential source for exposure to respiratory pathogens if used as flush water, especially if aerosolized particles are created, potentially resulting in in-halation of pathogens. if flush water is close enough to hutch floors, calves could also be able to ingest or have direct exposure of oral or nasal mucosae to pathogens. in addition to bvdv, bovine coronavirus is shed via feces (oma et al., ) and could cause respiratory disease in calves. the significance of lagoon water as a source of pathogenicity with respect to brd should be further evaluated. we also observed a negative association between the presence of an additional shade structure over the hutch area and brd. shade could reduce heat stress as well as protect from rain or frost, resulting in lower odds of brd. the association between shade and brd was not observed for shade structures with to sidewalls, which could be due to an offset of the benefits due to a lack of ventilation. coleman et al. ( ) studied the effect of supplemental shade structures over polyethylene calf hutches in alabama during the summer on feed consumption, growth, calf stress, and bedding contamination. their study found no differences between groups in terms of health-related outcomes, but humidity and coliform counts in bedding were higher in shaded hutches, which may be due to the difference in climate between the states, specifically the higher humidity in alabama during the summer months. the current study findings included higher prevalence in the fall compared with spring. in a comprehensive review on the effects of temperature and humidity on animal diseases, dennis ( ) concluded that sudden changes of weather might be more important than steady extremes of temperature and humidity to which an animal may adapt. callan and garry ( ) also mentioned rapidly changing environmental temperatures as a predisposing factor for brd. feeding pasteurized milk versus nonpasteurized milk, including milk replacer, was associated with reduced brd prevalence. numerous studies have underlined the importance of pasteurizing milk fed to dairy calves for the prevention of enteric disease as well as exposure to potential respiratory pathogens (e.g., mycoplasma spp. or bvdv; pfützner and sachse, ; callan and garry, ) . mycoplasma spp. are mastitis pathogens that can be shed by clinically or subclinically infected cows and can colonize the nasopharynx of calves fed the milk resulting in otitis and respiratory disease (pfützner and sachse, ; walz et al., ) . mycoplasma and other bacterial pathogens ingested in the milk can also spread hematogenously to the lungs, where they cause respiratory disease (callan and garry, ) . a study from spain found decreased morbidity and mortality in calves with adequate transfer of passive immunity (≥ . g/dl serum total protein) during the first wk of life that were fed heat-treated colostrum and pasteurized milk versus those that received nonheat-treated colostrum and nonpasteurized milk (armengol and fraile, ) . a separate study by godden et al. ( ) found a higher risk of pneumonia in calves fed milk replacer than for calves fed pasteurized waste milk. those authors argued that the improved immune function was attributable to higher energy and protein intake or the presence of medium-chain fatty acids in the whole milk, which have shown antimicrobial effects in pigs. we also observed a negative association of feeding a diet consisting of at least % saleable milk for at least d at time of farm visit with brd versus any other milk diet, including waste milk or milk replacer. several studies have evaluated the effect of milk intake volume on calf performance (jasper and weary, ; johnson et al., ) . studies evaluating the amounts of milk fed to calves on health outcomes have found mixed effects. medrano-galarza et al. ( ) found no associations between within-pen prevalence of brd and peak milk allowance in a study of majority holstein group-housed dairy calves fed with automated milk feeders. however, calves in that study were fed at least l per calf per day before and at least l per calf per day after introduction to the group pen. a different study, similarly found no difference in incidence of brd between holstein calves fed a maximum of or l of milk replacer per day in a randomized trial comparing performance and health response of dairy calves offered different milk replacer allowances (bach et al., ) . higher amount and quality of milk replacer fed was found to be associated with superior immune function in jersey calves (ballou, ) . the group of holstein calves fed ≤ . l of milk or replacer per day in our study was fed less than the lowest amounts supplied to calves in the above-mentioned studies and were likely not able to consume enough starter to meet their nutrient requirements for growth and development if they were under mo old (jasper and weary, ) . results in our study showed increased odds of brd for holstein calves fed ≤ . l and decreased odds of brd in jersey calves fed > . l of milk or replacer, which may reflect the effects of nutrition on immune function but should be interpreted with caution due to the relatively small samples in those categories, resulting in large confidence intervals for estimates. restricting the analysis to calves ≤ d of age did not change the sign or statistical significance of contrasts except for the comparison of > . to ≤ . l of milk or replacer in jersey calves, which became nonsignificant (p = . ). future research should quantify the benefits of saleable milk compared with waste milk or milk replacer further, as well as how the association with brd is modified by pasteurization and amount of milk fed in the major dairy breeds. the current study found no significant associations between calf or dam vaccination status for respiratory pathogens and brd in the study population. due to the cross-sectional nature, it was not possible to ascertain the vaccination status of calves in the study with certainty, instead we relied on the in-person surveyreported vaccination protocols, although respondents were asked to answer the questions with respect to the current cohort of calves on their premises during the visit. if calves were older than the age of vaccination stated in the questionnaire they were considered vaccinated. however, the true vaccination status and time since vaccination may have been different for an unknown number of calves. no associations between reported vaccination status and brd were found when calves at least d older than age of vaccination were considered vaccinated to account for time to initiate an immune response. our findings correlated with a randomized clinical trial by windeyer et al. ( ) of , heifer calves on commercials dairy farms in minnesota and ontario, in which they found no difference in risk of brd between calves vaccinated at or wk of age or both with a commercial multivalent modified live vaccine against the common respiratory viruses compared with a placebo control group. those authors cited interference by maternal antibodies, unresponsiveness of the neonatal immune system, timing of immunity relative to pathogen exposure, disease caused by pathogens other than the viruses in the vaccine, or herd immunity as possible explanations for their findings. the vaccination of dams may only be effective in preventing calfhood brd in combination with adequate transfer of passive immunity. many factors can affect the efficiency of transfer of passive immunity, from the amount, quality, and timing of colostrum fed to the storage time and temperature (pithua et al., , williams et al., , lago et al., . it is possible that any positive effect of dam vaccination may have been diluted by other variables affecting transfer of passive immunity. in a longitudinal study following over , preweaned calves on dairies in california and associating management factors to brd in the calves, vaccinating dams with either a killed or a modified live vaccine reduced the risk of disease in calves. . the longitudinal study design combined with close follow-up of management practices by verifying changes every to mo in a few dairies may have been the reason that study found these associations, whereas in the present study they were not apparent. the complexity of factors involving vaccination of neonatal calves combined with the present study design make it difficult to extend recommendations based on our findings. although the benefits of colostrum on calf health have been previously reported (denise et al., ; weaver et al., ) , we found no association between volume, source, quality, or storage conditions of colostrum and brd in the study herds. windeyer et al. ( ) reported serum total protein levels ≥ . g/dl during the first week of life as well as supplementation with an antibody product at birth to be negatively associated with brd, but the predictive value positive was low ( %) when using serum total protein levels at the cutpoint of < . g/dl to predict brd. in a study by virtala et al. ( ) , low postcolostral total serum igg concentrations ≤ , mg/dl was associated with increased risk of brd in a prospective cohort study of preweaned heifer calves. diagnostic accuracy of prediction in that study was %. both groups of authors (windeyer et al., ; virtala et al., ) stated that additional factors could play a role in the development of brd. the current study did not include data on failure of transfer of passive immunity for the study calves. the data on volume of colostrum fed as well as whether colostrum from first-calf heifers was used and if the quality of colostrum was assessed served as proxies for adequate passive transfer. the fact that all colostrum-management variables had to be assessed on a herd-level basis may have masked the effect of colostrum quality fed to individual calves in the study, as it was unknown which calves received colostrum from first-calf heifers or colostrum stored at a certain temperature or for a certain amount of time if there was variability within a dairy. the cross-sectional nature of the study prohibits drawing of causal inferences. although a longitudinal study design would have allowed us to draw causal inferences, the large number and wide geographic distribution of dairies enrolled did not allow such a study. however, findings reported here provide the basis for hypothesizing potentially causal factors and promote the design of further studies with longitudinal design to directly examine housing and management factors found to be associated with brd. participating dairies were not chosen randomly from all dairies in california, and it could be argued that those willing to invite researchers onto their dairy might differ from those who do not. however, considering the wide geographic distribution of dairies, the inclusion of both organic and conventional operations, and a wide range of herd sizes, as well as representation of holstein and jersey breeds, in the sample further ensures that the spectrum of california dairy operations was captured in the current study population. the california dairy industry contains both very large herds with several thousand cows in the central and southern parts of the state, as well as many organic dairies in the northern part of the state, and hence may collectively represent a large number of us dairies despite the state's unique dairy industry. the current study evaluated which management practices on california dairies were associated with the prevalence of brd in preweaned calves. housing-related factors positively associated with brd included using recycled flush water, housing calves in metal hutches, and calfto-calf contact in calves over d of age. feeding holstein calves less ≤ . l of milk or replacer per day was also positively associate with brd. negatively associated with brd were an additional shade structure over hutches, feeding pasteurized milk and feeding saleable milk, as well as feeding more than . l of milk or replacer to jersey calves. the fact that no associations between colostrum management and vaccinationrelated factors were found in the current study may indicate the lack of variability regarding these factors in the california dairy industry, rather than diminished importance in prevention of brd. metagenomics-based analysis of viral communities in dairy lagoon wastewater evaluation of mycobacterium avium subsp paratuberculosis infection of dairy cows attributable to infection status of the dam effects of road dust suppressants on pm levels in a mediterranean urban area colostrum and milk pasteurization improve health status and decrease mortality in neonatal calves receiving appropriate colostrum ingestion performance and health responses of dairy calves offered different milk replacer allowances immune responses of holstein and jersey calves during the preweaning and immediate postweaned periods when fed varying planes of milk replacer transformation of the independent variables herd-level prevalence of the ultrasonographic lung lesions associated with bovine respiratory disease and related environmental risk factors pages - in california dairy statistics annual. division of marketing services, dairy marketing branch, sacramento pages - in california dairy statistics annual. division of marketing services, dairy marketing branch, sacramento biosecurity and bovine respiratory disease wooden hutch space allowance influences male holstein calf health, performance, daily lying time, and respiratory immunity supplemental shade for dairy calves reared in commercial calf hutches in a southern climate effects of passive immunity on subsequent production in dairy heifers the effects of temperature and humidity on some animal diseases-a review epidemiology of bovine respiratory disease (brd) in preweaned calves on california dairies: the brd k study farmers assuring responsible management economic analysis of feeding pasteurized nonsaleable milk versus conventional milk replacer to dairy calves improving passive transfer of immunoglobulins in calves. ii: interaction between feeding method and volume of colostrum fed control, management, and prevention of bovine respiratory disease in dairy calves and cows persistence of mycobacterium avium ssp. paratuberculosis and other zoonotic pathogens during simulated composting, manure packing, and liquid storage of dairy manure performance and health of dairy calves using different types of commercial housing effects of ad libitum milk intake on dairy calves analysis of pre-weaning feeding policies and other risk factors influencing growth rates in calves on commercial dairy farms regional management practices and prevalence of bovine respiratory disease in california's preweaned dairy calves calf respiratory disease and pen microenvironments in naturally ventilated calf barns in winter efficacy of colostrum replacer versus maternal colostrum on immunological status, health, and growth of preweaned dairy calves comparison of plastic domes with wooden hutches for housing of dairy calves the effect of plywood and polyethylene calf hutches on heat stress antibiotic-resistant e. coli in surface water and groundwater in dairy operations in northern california survey of management practices related to bovine respiratory disease in preweaned calves on california dairies development of a novel clinical scoring system for on-farm diagnosis of bovine respiratory disease in pre-weaned dairy calves sensitivity and specificity of on-farm scoring systems and nasal culture to detect bovine respiratory disease complex in preweaned dairy calves comparison of calf housing types and tympanic temperature rhythms in holstein calves feed conversion efficiency, daily pasture intake, and milk production of primiparous friesian and jersey cows calved at two different liveweights timely diagnosis of dairy calf respiratory disease using a standardized scoring system associations between management practices and within-pen prevalence of calf diarrhea and respiratory disease on dairy farms using automated milk feeders a simulation study of sample size for multilevel logistic regression models bovine coronarvirus in naturally and experimentally exposed calves; viral shedding and the potential for transmission pathogenesis and pathology of bovine pneumonia effect of housing type on health and performance of preweaned dairy calves during summer in florida mycoplasma bovis as an agent of mastitis, pneumonia, arthritis and genital disorders in cattle efficacy of feeding a lacteal-derived colostrum replacer or pooled maternal colostrum with a low igg concentration for prevention of failure of passive transfer in dairy calves survival of salmonella enterica in aerated and nonaerated wastewaters from dairy lagoons survey sampling national dairy heifer project - , dairy heifer morbidity, mortality, and health management focusing on preweaned heifers heifer calf health and management practices on u.s. dairy operations usda-aphis-vs, ceah, national animal health monitoring system (nahms) the effect of maternally derived immunoglobulin g on the risk of respiratory disease in heifers during the first months of life dairy calf management, morbidity and mortality in ontario holstein herds. iii. association of management with morbidity otitis media in preweaned holstein dairy calves in michigan due to mycoplasma bovis reproduction, mastitis, and body condition of seasonally calved holstein and jersey cows in confinement or pasture systems passive transfer of colostral immunoglobulins in calves milk production and economic measures in confinement or pasture systems using seasonally calved holstein and jersey cows effect of three colostrum diets on passive transfer of immunity and preweaning health in calves on a california dairy following colostrum management training the effects of viral vaccination of dairy heifer calves on the incidence of respiratory disease, mortality, and growth factors associated with morbidity, mortality, and growth of dairy heifer calves up to months of age the authors thank the dairy owners, their staff, and herd veterinarians for their participation and jeffery stackhouse (cooperative extension, division of agriculture and natural resources, university of california, eureka), nyles peterson (cooperative extension, division of agriculture and natural resources, university of california, san bernardino), and paul rossito, courtney faria, and arianna villarreal (veterinary medicine teaching and research center, school of veterinary medicine, university of california-davis, tulare) for technical assistance. funding for this project was provided by the university of california's division of agriculture and natural resources, competitive grants key: cord- - fse h f authors: gow, sheryl p.; waldner, cheryl l. title: antimicrobial drug use and reason for treatment in western canadian cow–calf herds during calving season date: - - journal: preventive veterinary medicine doi: . /j.prevetmed. . . sha: doc_id: cord_uid: fse h f abstract investigators examined antimicrobial drug use practices and reason for treatment as part of a large on-farm observational study of cow–calf herds from western canada. reason for treatment and antimicrobial drug use (amu) were described using data collected during the calving season (january to june , ). the study included , calves and , cows and heifers from beef herds. all herds had more than cows. individual animal treatment records and a herd-level standardized questionnaire were collected from every herd. during the period of january to june , at least one treatment was reported in % ( % ci, – ) of calves and . % ( % ci, . – . ) of cows and heifers from these herds. the median percent of calves reported as treated per farm was . % (range – %) while the median percent of cows and heifers reported as treated was . % (range – %). antimicrobial drugs used during the calving season were primarily for disease treatment rather than prevention or growth promotion. diarrhea was the primary reason for treating calves and metritis was the primary reason for treating cows. parenteral antimicrobial drugs were the most common formulation used in both calves and cows. the most commonly used antimicrobial drugs in these herds were tetracyclines, sulphonamides, and florfenicol. this study provides baseline estimates of the frequency of antimicrobial drug exposure, the types of drugs used, and diseases treated in these cow–calf herds. the challenges identified in collecting these data can be used to improve the design of future on-farm studies. the treatment of infectious disease in food producing animals is an essential component of veterinary medicine. antimicrobial drug therapy is an important tool available to producers and veterinarians and is necessary to ensure that animal health and welfare are maintained. in addition to therapeutic use, antimicrobial drugs are also used to prevent disease, for growth promotion, and to increase production efficiency. while a few studies have provided some insight to more intensive livestock production units such as hog farms (dunlop et al., ; rajic et al., ) , there is no information about antimicrobial drug use (amu) in western canadian cow-calf herds. based on farm cash receipts the beef industry is the largest livestock commodity in canada (anon., a) . the provinces of alberta and saskatchewan contain more than % of the beef cow, breeding heifer, and calf populations in canada (anon., b) . a better understanding of amu patterns in this population is essential to develop a baseline and determine investigators examined antimicrobial drug use practices and reason for treatment as part of a large on-farm observational study of cow-calf herds from western canada. reason for treatment and antimicrobial drug use (amu) were described using data collected during the calving season (january to june , ) . the study included , calves and , cows and heifers from beef herds. all herds had more than cows. individual animal treatment records and a herd-level standardized questionnaire were collected from every herd. during the period of january to june , at least one treatment was reported in % ( % ci, - ) of calves and . % ( % ci, . - . ) of cows and heifers from these herds. the median percent of calves reported as treated per farm was . % (range - %) while the median percent of cows and heifers reported as treated was . % (range - %). antimicrobial drugs used during the calving season were primarily for disease treatment rather than prevention or growth promotion. diarrhea was the primary reason for treating calves and metritis was the primary reason for treating cows. parenteral antimicrobial drugs were the most common formulation used in both calves and cows. the most commonly used antimicrobial drugs in these herds were tetracyclines, sulphonamides, and florfenicol. this study provides baseline estimates of the frequency of antimicrobial drug exposure, the types of drugs used, and diseases treated in these cowcalf herds. the challenges identified in collecting these data can be used to improve the design of future on-farm studies. ß elsevier b.v. all rights reserved. the need for future monitoring in the canadian cow-calf industry. as part of a large on-farm observational study of factors affecting the productivity of cow-calf herds, an opportunity was available to examine amu during calving season. the objectives of this study were to describe the frequency of treatment with any antimicrobial drug in beef calves and cows, common reasons for amu, the types of antimicrobial drugs used, and risk factors for treatment in western canadian cow-calf herds during the calving season. the herds included in this study were concurrently enrolled in a comprehensive investigation of risk factors affecting the productivity and health of cow-calf herds in western canada. the baseline study collected data from the spring of through the end of calving in . the investigation of antimicrobial drug use practices was undertaken to maximize the industry benefit from this unique opportunity for on-farm access to individual animal data from a large number of cow-calf herds. the design of the baseline health and productivity study, including sample size calculations and herd selection criteria, have been described in detail elsewhere (waldner, ) . a brief overview of the herd selection criteria is provided here. mixed and large animal practitioners from the western provinces of saskatchewan, alberta and british columbia were asked to participate in the study. sixty-one veterinary clinics referred producers who indicated some level of interest in learning more about the study (waldner, ) . study personnel initially contacted this group by phone to provide background information about the project. if, after the call, the producer was still interested, study personnel visited the farm to collect some baseline information on herd size and management to ensure that selection criteria were met. herds were eligible for enrollment based on completeness of animal identification, existing calving records, presence of animal handling facilities, use of pregnancy diagnosis and bull evaluation, and a relationship with a local veterinary clinic. herds of less than animals, greater than animals, and herds not using a winter/spring calving season were excluded. of those initially contacted herd owners provided basic demographic data. from this group, herds that best fit the selection criteria and that agreed to participate after learning more about the study were enrolled. characteristics of participating and non-participating herds were compared (waldner, ) . producers that were not enrolled in the study were more likely to have < or > cows, less likely to use pregnancy diagnosis and bull breeding soundness exams, and were less likely to have complete calving records (waldner, ) . participants were more likely than non-participants to be - years old and less likely to be older than (waldner, ) . five producers changed their minds before the start of the study and data collection began in herds in the spring of (waldner, ) . two herds then withdrew form the study in the fall of , and two more were lost in the winter of leaving herds providing data for the calving season (waldner, ) . participating herds were visited regularly by one of six veterinarians contracted by the health and productivity study to collect samples and data and to monitor the quality and consistency of on-farm records. the individual animal treatment records from january to june , were summarized for study herds. treatment data for spring-born calves were investigated separately from that of cows and replacement heifers in the breeding herd. in the first step of the analysis only calf treatment data were considered. the analysis was restricted to information collected for calves born alive between january and may , . potential risk factors for treatment were summarized for calves and their dams meeting these inclusion criteria (table ). the second step of the analysis included treatment data reported for all cows, bred heifers, and yearling heifers present in the herd on january , . cows and bred heifers with stillborn calves, non-pregnant cows and heifers were included in the total number of animals available for investigation. any cows or heifers purchased after january , were not included. variables considered as potential risk factors for treatment were summarized for cows and heifers (table ) . available calving records for each animal included dam identification, calf identification, date of calving, single or twin birth, sex of the calf, the type of assistance provided to the dam, any post-calving problems, and calving outcome (born alive, stillbirth and died later). if the calf died, the date of death was reported. other data recorded for each herd included the ecological region in which the herd was located, the veterinary clinic servicing the herd, vaccination status for infectious bovine rhinotrachietis (ibr) and bovine viral diarrhea virus (bvdv), and vaccination status for neonatal diarrhea (coronavirus, rotavirus and escherichia coli). dam body condition (bcs) was scored ( -point scale) at the time of pregnancy diagnosis and again before or during the early part of the calving season. records from herds across alberta ( ), saskatchewan ( ), and northern british columbia ( ) were included in this study. on january , , herd size ranged from to mature, breeding females, with a median herd size of . of the included in the study, herds ( %) had between and mature, breeding females. the , calves born alive from january and may , were included in the study. this period captured % of all calves born alive in these herds and provided a minimum one-month observation period for the youngest calves included in these data. the majority of calves were born in march and april ( %). most calves born alive were unassisted; . % of live births were twins (table ) . about half of the dams were vaccinated for bvdv and ibr prior to breeding in , while about one third of the dams received some type of vaccination to prevent neonatal calf bacterial or viral diarrhea (table ) . most dams had a bcs of or higher on a -point scale at pregnancy testing and again at calving (table ) . there were , cows and heifers reported in study herds on january , ( yearling replacement heifers and , cows and bred heifers expected to calve in the spring of ). this number included all cows and heifers that had calves born alive from january to may , (dams of calves described above), cows and heifers that had abortions or stillborn calves during this period, and non-pregnant cows and heifers. the majority of cows were between and years of age (table ) . dystocia was reported in < % of cows/heifers, and < % of cows/heifers had post-partum complications such as retained placentas, prolapses, or metritis (table ) . data on amu were collected using individual treatment records as well as a herd level questionnaire and these data were summarized for the period of january to june , . the data used in this study were limited to this time frame for two reasons. first, in western canadian herds that have cows calving in the winter and early springmonths cows are often confined for ease of monitoring and feeding. confinement also simplifies identification and treatment of sick animals and the recording of these treatments. in contrast, during the summer and fall months cattle are generally turned out into large pastures where routine observation and identification of sick animals is more difficult and treatment records are more sporadic. the second reason for collecting data during this period is that the majority of treatments in cow-calf herds occur during the calving season. calving during the winter and spring often exposes young animals to severe weather extremes. close confinement during this period increases the risk of disease transmission. by restricting the analysis to the period between january and june the investigators captured the most accurate treatment records available for these herds during the time frame when the majority of treatment episodes were expected to occur. producers reported individual animal treatments in a record book originally designed for the primary health and productivity study. treatment type was reported as a coded pick list: injectable antimicrobial, oral antimicrobial, oral and injectable antimicrobial, fluids, and other. coded lists were also provided to standardize the responses for class of animal, reason for treatment, and type of treatment. a notes section allowed producers to write in any comments. where completed by the producers, the notes section was used by investigators to help further classify diagnoses or treatments. producers could report more than one reason for, or type of treatment for, each treatment episode. the producer was asked to record each treatment occurrence; however, animals reported as treated more than once for the same diagnosis within a -day period were classified as having one treatment event for the purpose of analysis. treatment data were summarized in two ways. first, to provide an estimate of treatment intensity per herd for the period of january to june , , a count of total treatment events per herd was determined. this was calculated separately for the calves and then for the cows and heifers. the treatment intensity was reported as the number of treatment events per every animals at risk during the -month period. second, risk of treatment was described using the cumulative incidence for the period of january to june , for calves and then for the cows and heifers. this was calculated as the number of animals that were reported as ever having been treated as a percentage of the number of animals per herd at risk of treatment at any time during the study period. a herd-level questionnaire was developed to identify the types of antimicrobial drugs most commonly used on each cow-calf farm. this was done because the individual animal records did not consistently include information on the type of antimicrobial drug used for treatment. herd owners were asked about the frequency of use for sulphonamides, tetracylcines/oxytetracyclines, trimethoprim/sulphadiazine, and penicillins. antimicrobials that did not fall into these broad categories were classified as ''other''. producers were asked to report separately the number of treatments for both cows and calves for each drug category listed above. the number of treatments for each category was coded as follows: l- animals treated, - animals treated, and greater than animals treated. the quality of the treatment records were assessed at the end of the study. the veterinarians responsible for data collection and entry were asked for a subjective, comparative assessment of the quality of the data. they classified the data for each herd into one of the following categories: excellent, good or satisfactory, and less than satisfactory. herd owner compliance in completing treatment records was also evaluated by considering the relative frequency of calf mortality in the herds that did not report any treatments. inventory and post-mortem examination data that were available from the baseline productivity study were used to verify calf mortality records. the plausibility of no reported treatments was assessed when compared to the percent calf death loss in each herd. all data were entered into a customized database (microsoft office access , microsoft corporation). descriptive analyses were completed and variables were recoded as necessary for statistical modeling using commercially available software (spss . for windows, spss inc., chicago, illinois). factors affecting the occurrence of treatment, a class variable with two levels (treated or not treated), were examined in calves as well as cows and heifers using mixed models with a binomial distribution and logit link function. the calculations were performed using penalized quasilikelihood estimates ( nd order pql) (mlwin version . , centre for multilevel modelling, institute of education, london, uk). the strength of the association between outcome and exposure was reported as an odds ratio (or) with % confidence intervals. a null model (intercept only) was created for each outcome variable. random intercepts were examined to assess degree of clustering for treatments reported within herd, veterinary clinic, and ecological region (ecoregion). ecoregion is a geographical delineation characterized by regional ecological factors such as vegetation, soil, climate, water and fauna (wiken, ) . within-herd clustering was accounted for as a random intercept in all models. veterinary clinic and ecoregion where not included as random intercepts in any model as neither explained a substantial part of the variation. models were checked for the presence of extra-binomial variation, but extrabinomial parameters in the range of . - . were reset at . (binomial variation). data from the null models were used to estimate the intra-class (i.e., intra-herd) correlation coefficient (icc or r) to measure clustering of each outcome within herd (dohoo et al., ) . the null models were also used to generate population-average estimates of the risk of calf treatment and cow and heifer treatment (dohoo et al., ) . the unconditional associations between each of the potential risk factors (table for calves and table for cows and heifers) and the odds of treatment were examined. all potentially important risk factors (p . ) were identified and a final model was then developed using backwards stepwise elimination. any potential risk factors where p < . or that acted as important confounders (removal of the potential risk factor from the model changes the effect estimate for the exposure by %) were retained in the final model. after establishing the main effects model, biologically reasonable (dohoo et al., ) first order interaction terms were tested if two or more variables (p < . ) were retained in the final model. the adequacy of all models was evaluated using plots of residuals to check that all assumptions had been met as appropriate. associations between calf treatment and mortality in each herd were investigated using generalized estimating equations (sas v. . for windows (proc genmod); sas institute, cary, north carolina, usa). the number of calves with any treatment (numerator) as a proportion of the total number of calves in the herd (denominator) was the outcome of interest. the predictor variable, percent calf mortality for the herd, was categorized into quartiles (< %, - . %, - . % and > % mortality). the model specifications included a binomial distribution, logit link function, repeated statement with subject equal to herd, and an exchangeable correlation structure. herd owners reported treating % ( % ci, - %) of calves with an antimicrobial drug at least once between january and june , . the median age of calves at the time of their first treatment was (range - ) days of age and % ( / ) of calves were first treated between birth and days of age. the median percentage of calves treated at least once on each farm was . % (range - %). the median number of treatment events per farm was . (range - ) for every calves at risk from january to june , . the most commonly recorded antimicrobial drug formulation used in calves was injection (table ) . parenteral antimicrobial drugs were used in ( %) herds on < % of the calves, in ( %) herds on - % of the calves, in ( %) herds on - % of the calves, and in ( %) herds on > % of the calves. four herd owners treated - % of the calves and three herd owners treated all of their calves at least once. the maximum number of times a calf was treated with parenteral antimicrobial drugs was (median, ; range - ). the second most commonly reported protocol for calves included treatment with both oral and parenteral antimicrobial drugs (table ) . oral and parenteral antimicrobial drugs were used together on < % of the calves in ( %) herds, on - % of the calves in ( %) herds, on - % of the calves in ( %) herds, and on > % of the calves in ( . %) herds (maximum, %). oral formulations were used alone as an antimicrobial treatment in < % of calves (table ) . one hundred and forty-four ( %) herd owners treated < % of their calves with oral antimicrobial drugs, ( %) treated - % of the calves, ( . %) treated - % of the calves, and ( . %) treated between and % of their calves with oral antimicrobial drugs. diarrhea was the most commonly reported reason for treatment in calves (table ). the percentage of calves treated for diarrhea per farm ranged from to % (median, . %). within these herds, ( %) producers treated to % of their calves for diarrhea, ( . %) treated - %, ( %) treated - %, and ( . %) producers treated > % of their calves for diarrhea. the next most common reason for treatment was pneumonia (table ). the frequency of calf treatments for pneumonia per farm ranged from to % (median, . ). one hundred and twenty-six ( %) producers treated % of their calves for pneumonia, ( %) treated - % of their calves, ( . %) treated - % of their calves, and ( . %) treated > % of their calves for pneumonia. treatment and prevention of omphalitis (navel infection) made up the third most common recorded reason for treatment (table ). four producers treated between and % of their calves prophylactically for omphalitis. between january and june , , . % ( % ci, . - . %) of the cows and heifers were treated at least once. the median percent of cows and heifers ever treated per farm was . % (range - %). since almost all cows and heifers that were treated were only treated once during the period of january st to june th, the number of treatment table administration routes for antimicrobial drugs used in calves a and in cows/heifers b at the animal and herd c level between january and june , d . percent ( events per cows and heifers at risk was also . (range - ). the most commonly reported treatments in cows and replacement heifers were with parenteral antimicrobial drugs (table ). very few cows or heifers were reported to receive either oral antimicrobial drugs or oral and parenteral antimicrobial drugs together (table ) . oral treatments were only given in ( . %) herds and to < % of the cows in these herds; whereas, oral and parenteral treatments were given in ( . %) herds to < % of cows. one hundred and eleven ( %) producers treated < % of their cows with parenteral antimicrobial drugs, ( %) treated - % of their cows, and ( %) producers treated > % of their cows with parenteral antimicrobial drugs. treatments other than antimicrobial drugs were more commonly reported for cows and heifers than for calves (table ) . treatments categorized as ''other'' were given on ( %) herds to . - % of the cows. other treatments included non-antimicrobial treatments such as mineral oil or other products for gastrointestinal disorders. table summarizes the primary reasons for treatment of cows and heifers. metritis was diagnosed in - % of the animals with calving records in ( . %) herds, and in > % of the animals in ( . %) herds. one hundred seventyfour ( %) herd owners treated < % of their cows and heifers and ( %) treated - % of their cows and heifers for interdigital necrobacillosis (footrot). retained placentas were diagnosed and treated in < % of the animals with calving records in ( %) herds, in - % of the animals in ( . %) herds, and in > % of the animals in ( . %) herds. the most commonly reported antimicrobial drugs used are summarized for calves in table , for cows and heifers in table , and for all herds in table . ionophores were used in the feed supplied to cows or heifers in % ( / ) of herds. reason for use was not specified. enrofloxacin was reported as used on calves in herds. one herd reported treating of ( %) calves with a combination of enrofloxacin and sulbactamampicillin. for all calves treated with enrofloxacin, the recorded reason for treatment was diarrhea. florfenicol was listed specifically in the notes section for the treatment records of calves on farms. of these calves, ( %) were treated for diarrhea, ( %) for omphalitis, ( . %) for pneumonia, ( . %) for arthritis, and ( %) for other reasons. neither the location of the herd by ecoregion or differences in recommendations among referring veterinary clinics explained a substantial part of the variation in reported treatment practices for either calves or cows. however, treatment practices were clustered within herd (calf treatment, r = . ; cow treatment, r = . ). calf gender, assistance at parturition, and the percent of cows and heifers treated in the herd were unconditionally associated with the odds of a calf having been reported as treated by the herd owner. after accounting for other variables in the model, female calves were less likely to be treated than male calves (or, . ; % ci, . - . ). calves for which manipulation or traction was applied during calving were . ( % ci, . - . ) times more likely to be reported as treated than calves that were not assisted. the odds of calf treatment also increased . ( % ci, . - . ) times for every percent increase in cow treatment reported for the herd. yearling heifers were one fifth (or, . ; % ci, . - . ) as likely to be treated as all other breeding females. other risk factors, including breed, vaccination status and body condition score, did not significantly contribute to the odds of a cow or heifer being treated. ( ( ) . ( ) . ( ) . ( ) . ( ) calf sulbactam-ampicillin injectable ( ) . ( ) . ( ) . ( ) . ( ) calf ceftiofur ( ) . ( ) . ( ) . ( ) . ( ) calf amprolium hydrochloride oral ( calving-related factors were examined only for mature females with calving records in . after accounting for other risk factors, cows or bred heifers were more likely to be treated if they were assisted at the time of calving or if they experienced post-calving problems such as prolapse, retained fetal membranes or metritis. cows or bred heifers for which manipulation/traction or caesarian sections were reported at calving were respectively . ( % ci, . - . ) and ( % ci, . - ) times more likely to be treated than cows and bred heifers that did not require assistance at calving. cows or bred heifers with postcalving problems such a prolapse, retained fetal membranes or metritis were respectively ( % ci, - ), ( % ci, - ) and ( % ci, - ) times more likely to be treated than cows or bred heifers that did not have these conditions. the study-employed veterinarians responsible for collection and entry of all herd data subjectively rated % of the herd treatment records as excellent, % as good or satisfactory, and % as less than satisfactory. herd owner compliance in recording these data was also investigated by comparing herd calf mortality and treatment rates in the ( %) herds that had no reported treatments. of these herds, had no calf mortality, had < % calf mortality, had - % calf mortality, had - % calf mortality and had > % calf mortality. the risk of calf mortality was not associated with the proportion of calves treated in all study herds (p = . ). the extent of under reporting of antimicrobial drug use was difficult to estimate using the data available from this study. based on the crude subjective and comparative assessment of the quality of the data, at least % of the herds had less than satisfactory treatment records. when combined with information on the calf mortality and the proportion of herds reporting no treatments there is further evidence that there was under reporting of amu by some study herds. for example, it is unlikely that herds with greater than % calf death losses would have no calf treatments. while under reporting was a potential limitation of this study, complete and accurate farm-based antimicrobial drug use records are difficult to obtain from extensively managed livestock operations. complete recording of use information is demanding for producers especially during busy times where farm labor resources are limited. another limitation of the study was the lack of consistent reporting for individual animals detailing which antimicrobial drugs were used to treat specific conditions and the dose used. while investigators attempted to collect information on the types and relative amounts of drugs used in each herd, these data were potentially subject to recall bias since the questionnaire was administered at the end of the calving season and relied on producer accounts of the number of animals treated with each class of antimicrobial. future studies should focus on working with producers to prospectively report the amount and type of each antimicrobial used to more accurately assess antimicrobial drug exposure. while the baseline health and productivity study provided a unique opportunity to collect information about on-farm treatment practices, the data collection tools were not specifically designed to measure the amount and frequency of individual antimicrobial drugs used in these herds. a producer-friendly data collection instrument designed for the purpose of capturing detailed information on both the type and amount of antimicrobial drug used and the class of animal treated would be necessary to more exactly measure antimicrobial drug exposure. despite the limitations, this study does provide the first available documentation of the proportion of calves and proportion of cows and heifers treated during the calving season and the types of conditions most often treated in western canadian herds. the study also provides some initial information about antimicrobial drug use practices in these herds which can be used to design future studies of extra-label drug use, prophylactic treatment, and the importance of managing dystocia in reducing treatment of cows or calves. the relatively small proportion of treated animals is consistent with the finding that the primary reasons reported for antimicrobial drug use in cow-calf operations were for individual therapeutic use rather than prophylaxis, metaphylaxis, or growth promotion. in contrast, between and % of all dairy cattle receive prophylactic antimicrobial drugs to prevent mastitis (sischo et al., ; usda, ) . depending upon the size of the feedlot, the type of cattle placed and bovine respiratory disease risk designation, anywhere between - % of feedlot cattle in the united states (usda, ) and - % of feedlot animals in canada receive prophylactic injectable antimicrobial drugs on arrival for the control of bovine respiratory disease (radostits, ; personal communication with calvin booker, feedlot health management services ltd., february , ) . there are few available antimicrobial drug use studies for cow-calf herds (bair and mcewen, ; powell and powell, ; sayah et al., ) . powell and powell ( ) and sayah et al. ( ) both gathered information via questionnaires while bair and mcewen ( ) had access to treatment records. the previously reported studies had substantially fewer cattle and herds enrolled than the present study. also, the reporting structure of the previous three studies did not differentiate between drug use in cow-calf herds and drug use in feedlots limiting direct comparisons to the present study. extra-label drug use was reported in some cow-calf herds in the current study. the most commonly reported antimicrobial drugs used in these herds were tetracyclines, sulphonamides, and florfenicol. in canada, florfenicol is labeled for bovine respiratory disease and for the treatment of interdigital phlegmon (compendium of veterinary products, ), but the individual animal treatment notes indicate that it was also used in an extra-label manner in calves for diarrhea and omphalitis. extra-label use of fluoroquinolones and cephalosporins were also reported. powell and powell ( ) also reported off label use of enrofloxacin in their survey of ontario beef producers. at the time of these studies, enrofloxacin was not readily obtainable by cattle producers because in there was only a small animal formulation available in canada. since the time of this study, a cattle formulation has been approved for use in canada for the treatment of bovine respiratory disease (compendium of veterinary products, ) . follow up studies are needed to see how antimicrobial drug use patterns might change following the release of new products. in addition to describing use patterns, this study identified factors associated with the reported frequency of calf treatment. calves that were assisted during birth were more likely to be reported as treated. sanderson and dargatz ( ) also reported that increasing incidence of dystocia in a herd was associated with increased morbidity. another potentially related finding was that female calves were less likely to be treated than male calves. this could be because male calves are often larger than females (bellows et al., ) . larger calves are more likely to experience delayed parturition, increased fetal stress, and reduced vigor; potentially negatively affecting passive transfer and calf health. calves were also more likely to be treated in herds where more cows or heifers were treated. this finding might reflect an increased likelihood of exposure to disease on these farms because of management or other factors, or it may reflect an increased tendency of these producers to administer and report treatment. yearling heifers were less likely to be treated than cows. risk factors for cow or bred heifer treatment included assistance at calving and post-calving problems such as a prolapse, retained fetal membranes, and metritis. because cows and bred heifers were more likely to be treated if there was assistance at calving, further investigation is needed into whether producers are providing treatment prophylactically because of the intervention or it they are treating an observed morbidity, either related to parturition or for some other reason. this distinction is not clear from the available data. calves were more likely to be reported as treated than cows or heifers in this study and the primary reason reported for calf treatment was diarrhea. diarrhea was also the most commonly reported illness in beef calves in the united states (usda, ) and in a survey of beef producers in ontario (powell and powell, ). in the current study the primary reported reason for treatment of cows or bred heifers was metritis followed closely by interdigital necrobacillosis; whereas, in the united states infectious keratoconjunctivitis and interdigital necrobacillosis were listed as the two primary disease conditions reported among breeding females (usda, ) . the current study only looked at treatment from january to june; whereas, the national animal health monitor system (nahms) study questions spanned the entire year. infectious bovine keratoconjunctivitis (ikc) is more common in the summer months. although there was a difference in the primary reasons for cow and heifer treatment between western canada and the united states, both studies did report a relatively low occurrence of disease and treatment in breeding females. because herds were enrolled in the larger productivity study based on their ability to provide the required data, these herds probably represent some of the more progressive, commercially viable, and intensively managed herds in western canada. antimicrobial drug use may be different in very large cow-calf herds or the small herds present on some mixed or hobby farms. data from this study can be applied to typical commercially viable cowcalf farms in canada with to about cows which would represent the core of the industry in western canada. these data demonstrate that antimicrobial drug use on cow-calf farms, during the calving season, is primarily for treatment of disease and that on most farms few animals are exposed to antimicrobial drug therapy. researchers can use data collected in this study for baseline estimates of antimicrobial drug exposure and the types of diseases treated in this population. they can also learn from the challenges identified in collecting these data to improve future study designs. the risk factors identified by this work can be used by industry to develop strategies to optimize the use of antimicrobial drugs in these herds. census of agriculture cattle numbers by province antimicrobial use in the ontario beef industry occurrence of neonatal and postnatal mortality in range beef cattle. ii. factors contributing to calf death compendium of veterinary products veterinary epidemiologic research individual and group antimicrobial usage rates on farrow to finish swine farms in ontario a mail survey of ontario beef producers' attitudes about antibiotics herd health in food animal production medicine reported antimicrobial use in swine farms in alberta risk factors for high herd level calf morbidity rate from birth to weaning in usa beef herds patterns of antimicrobial resistance observed in escherichia coli isolates obtained from domestic and wild animal fecal samples, human septage and surface water prevalence of the contagious pathogens of mastitis and use of mastitis control practices united states department of agriculture (usda), . part i: baseline reference of feedlot management practices part iii. reference of dairy cattle health and health management practices in the united states western canada study of animal health effects associated with exposure to emissions from oil and natural gas field facilities. study design and data collection. . herd performance records and management ecological land classification series no the authors would like to thank the western interprovincial scientific studies association (wissa) for support of the beef productivity study that provided much of the data for this analysis. we would also thank the producers and veterinarians who provided data and the project veterinarians who collected it. key: cord- -lh ixof authors: van weeren-keverling buisman, a.; mouwen, j. m. v. m.; wensing, t.; breukink, h. j. title: intraruminal administration of milk in the calf as a model for ruminal drinking: morphological and enzymatical changes in the jejunal mucosa date: journal: vet res commun doi: . /bf sha: doc_id: cord_uid: lh ixof in order to develop a calf model for studying the syndrome of ruminal drinking (rd) in veal calves, three dual-fistulated calves were used to test the effect of intraruminal administration of milk replacer on the jejunal mucosa. biopsies of the proximal jejunal mucosa were taken through a jejunal fistula and the mucosal morphology and the activities of two brush border enzymes, lactase and alkaline phosphatase, were determined. means of villus length and brush border enzyme activities decreased during the period of intraruminal administration of milk. the hyperplastic villus atrophy in this model was similar to that found in chronic rd patients in previous studies. this could not be associated with isolation of pathogenic micro-organisms from the faeces and is probably the consequence of the intraruminal milk feeding procedure itself. clinical recovery from the signs of rd occurred rapidly after intraruminal administration of milk ceased and was followed by restoration of villus length and brush border enzyme activities – weeks later. ruminal drinking (rd) calves are known to have an insufficient reticular groove reflex while drinking milk. this causes deposition of a substantial amount of milk into the rumen (breukink et al., ) . during the retention of this milk in the rumen, fermentation takes place and volatile fatty acids and lactate are produced. a distinct reduction in ph is also noticed (van weeren-keverling buisman et al., ) . at necropsy of chronic rd calves, hyperkeratosis of the rumen is found (van bruinessen-kapsenberg et al., ; breukink et al., ) . chronic rd calves usually have a reduced growth rate and are small for their age (van bruinessen-kapsenberg et al., ) , apparently the result of disturbed energy and protein balance (breukink et al., ) . the latter may be partially due to inappetence but may also be affected by jejunal villus atrophy (de visser and breukink, &q, breukink et al., ; van weeren-keverling buisman et al., a) and brush border enzyme deficiency (de visser and breukink, ) . the purpose of this study was to determine the influence of intraruminal administration of milk on the morphology of the villi of the proximal jejunum. information about the functional capacity of the small intestinal epithelium was obtained by determining the activities of the brush border enzymes lactase and alkaline phosphatase (ai'). three clinically normal male friesian holstein calves (e, h and s) were used. the housing and feeding regimens were as described by van weeren-keverling buisman et al. ( ) . they were fed commercial milk replacer twice a day and no additional solid food was given. in week of the fattening period, a cannula was placed in the proximal jejunum under general anaesthesia (van weeren-keverling buisman et al., b) . procain penicillin iv/kg and dihydrostreptomycin mg/kg were administered intramuscularly twice daily for four days postoperatively. a rumen cannula was placed under general anaesthesia during week (van weeren-keverling buisman et al., ) and this was followed by mg/kg intravenous sodium ampicillin every h for h. this second cannula was used for the intraruminal administration of milk. the experiment was divided into three periods, the pre-induction period, the induction period and the recovery period. in the pre-induction period (weeks l- ), during which both operations took place, a commercial milk replacer (tentofeed i, tentego bv, mijdrecht, the netherlands) containing . % protein (mainly skim milk powder), . % fat, . % ash and . % lactose was administered by an open bucket. in the induction period, from week onwards, the milk replacer was administered intraruminally in increasing proportions until the faeces became clay-like. in week , % of the ration was infused into the rumen twice daily, the remaining portion being fed from an open bucket. in the recovery period the entire milk replacer diet was again consumed from an open bucket. in calves h and s the induction period was five weeks. in calf e, the induction period was prolonged by two weeks, during which time the percentage of intraruminally administered milk replacer was raised to %. consequently, the recovery period in this calf started two weeks later. during all three periods, milk replacer from the same batch of powder was fed. the fast sample of the proximal jejunal mucosa was taken in week of the fattening period during the insertion of the cannula. from week onwards, jejunal biopsies were taken twice a week throughout all three experimental periods to cm caudally from the intestinal fistula, using a crosby capsule (lam&is, utrecht, the netherlands) (van ssb) . data obtained in the different experimental periods were compared within each calf. the jejunal biopsies were fmed in % buffered formalin. stereomicroscopic examination of all biopsies was carried out with a zeiss stereomicroscope at a magnification of - x in order to determine the shape of the villi (mouwen, ) . thin slices of the biopsies perpendicular to the mucosal surface were then embedded in paraffin and serial sections, pm in thickness, were stained with haematoxylin and eosin. the lengths of full-length villi and crypts were measured with an ocular micrometer (mouwen, ) . the same crypts were used to determine the index of mitosis, so as to indicate the proliferative status of the crypt epithelium. the number of mitotic cells in metaphase and anaphase was counted and the total number, multiplied by , was divided by the total crypt depth in pm. the other jejunal biopsies (one biopsy from each week) were immediately frozen and stored in liquid nitrogen at -l%"c during the experimental period. the biopsy was homogenized (ika-thyristor speed controller tr , janke and kunkel, ika werk, stauben, frg) for min in ml of buffer solution ( mmol/l mannitol and mmol/l tris at a ph of . ), in melting ice. the homogenate was centrifuged at , rpm for min at °c (eppendorf s, merck bv, amsterdam, the netherlands) and the supernatant was stored at - °c for up to two days prior to analysis. determination of the protein content in the supematant was carried out by the method of lowry et al. ( ) . the method for determining the lactase activity of the biopsies was first assessed using a commercially available lactase (ec . . . ). a mixture containing . ml of the -fold diluted supernatant in the same mannitol-buffer and . ml of lactose solution ( mg/ ml) was incubated at °c for min. lactose hydrolysis was then ended by putting the mixture in ice. the amount of glucose was measured by the gluco-quant method (boehringer mannheim, frg). lactase activity was expressed in mm glucose/g protein. alkaline phosphatase activity (ar, ec . . . ) in the supernatant was determined calorimetrically by the optimized standard method (boehringer mannheim, frg) on the multistat iii (instrumentation laboratory (benelux) bv) and expressed in u/g protein. throughout the experimental period a weekly sample of the faeces was taken directly from the rectum of each calf. bacteriological examination for salmonella and pseudomonas was performed by direct isolation on modified brilliant green agar or after selective enrichment in sodium selenite-brilliant green-mannitol broth (oxoid, hampshire, uk), followed by an antimicrobial sensitivity test (neo-sensitabs, rosco, taastrub, denmark). the samples were examined for cryptosporidia by the modified ziehl neelsen technique. the presence of rotavirus was tested for by latex agglutination (wellcome diagnostics, dartford, uk) (microbiologic diagnostic centre, state university of utrecht). virological examination for the presence of coronavirus in the faeccs was carried out using a double antibody-sandwich (das) elisa test from week onwards (central veterinary institute, lelystad, the netherlands). serological examination for antibodies against rotavirus, coronavirus and bovine diarrhoea virus (bdv) was performed by blocking elisa in weeks and . a tube agglutination test with h and antigen was done to detect antibodies against salmonella dublin and salmonella typhimurium (central veterinary institute, lelystad, the netherlands). in the induction period, the faeces developed a clay-like consistency with greyish-white colour. faeces with these characteristics, typical for chronic rd calves, were produced in week by calves h and s and in week by calf e. during the recovery period, the faeces of calves e and s regained their normal character within a few days. in calf h, recovery from the clinical signs of rd w&delayed for about three weeks after intraruminal administration of milk stopped. in this calf, clinical recovery was only achieved after modification of the feeding regimen by increasing the feeding frequency, giving smaller meals and using a floating nipple. the results of the microbiological examination of the weekly samples of the faeces are shown in table i . no sahnonellae were isolated during the entire experiment. in weeks and , other pathogens (pseudomonas, cryptosporidia, rotavirus) were isolated in all three calves. in most cases no diarrhoea was observed. from weeks to , all the faecal samples were negative for potential pathogens and no diarrhoea was observed. from weeks to , pseudomonas, rotavirus and coronavirus were again isolated. diarrhoea was observed sporadically on a few days during the experiment. only calf s experienced diarrhoea during the larger part of the recovery period. in no case was there any need for treatment or adjustment of the feeding regimen. pseudomonas species were found in large numbers in calf e from weeks to but were never associated with diarrhoea. three of the six diarrhoeic samples from calf s contained pseudomonas. all pseudomonas isolated were resistant to most of the antibiotics tested. the antibody titre against both types of salmonella and bdv was negative in all three calves. although the titre against rotavirus (calves e and h) and coronovirus (calf s) was unchanged or slightly elevated in week compared to the titre in week , no seroconversion occurred in any calf. the jejunal biopsies of the three experimental calves showed a mixture of finger-shaped and long tongue-shaped villi during the pre-induction period and the first part of the induction period. in calves h and s, from week onwards, these villus forms were replaced by predominantly long to short tongue-shaped villi with a few finger-and leaf-shaped ones. in calf e, this change was observed in week only. there was no essential change in villus form in the recovery period in calves h and s compared to that in the induction period. the mean villus length and the individual values for calves e, h and s respectively are given in figure . individual values are given because each calf serves as its own control. the measurements are compared with the initial villus length in week , at the time of the frost operation. in the pre-induction period, all three calves exhibited a temporary decrease in villus length to % of the initial length, which resolved in week , shortly after ruminal infusion of milk was initiated. from weeks to the villus length was again reduced compared to the initial values. the shortest villus length ( - % of initial length) were observed in all three calves during week . in week , during the recovery period, all values had almost returned to their initial values. crypt depth did not alter after intraruminal milk administration; it ranged from to pm in the experimental calves. the index of mitosis for each calf is given in table ii . peak values ( . ) were obtained in weeks lo- in calf e, weeks - in calf h and weeks & in calf s. the mean and individual lactase activities for the three experimental calves during the three experimental periods are presented in figure . the first measurements are from week , when the first biopsies were taken. a substantial variation in lactase activity was observed during the experimental period, although the activity in all three calves fell in weeks and , to % and % respectively of their values in week . figure the mean and individual ap activities for the three experimental calves are presented. variation is less than with lactase activity but the low points in activity for both lactase and ap in weeks and were essentially the same. ai' activity was reduced to % in week and to % in week . the use of an intestinal fistula, by which repeated biopsies of the jejunal mucosa can be taken, made it possible to compare within each calf the data obtained in the different periods. this compensates for the individual morphological and functional variations. the clinical signs of rd in this calf model were less obvious than in rd patients. the typical clay-like faeces were only observed for a short period in the three experimental calves. this may be the consequence of more rapid clearance of rumen contents in this calf model than in chronic rd patients (van weeren-keverling buisman et al., ) . when considering the detection of pathogenic micro-organisms in the faeces, two periods of excretion can be distinguished. the first period (weeks - ) was directly post-operative, when pathogenic micro-organisms were found in out of samples of the faeces, but only one calf (s) was diarrhoeic in week . this may be attributed to increased susceptibility in the surgically stressed animals, post-operative antibiotic treatment, or both. ampicillin, for instance, is known to reduce colonization resistance maintained by the normal gut flora (van der waay, ), as a result of which pathogenic micro-organisms may start to colonize the intestine. the second period of shedding of micro-organisms was from weeks - , during the recovery period; pseudomonas aentginosa, rotavirus and coronavirus were isolated. calf s suffered from slight diarrhoea during the larger part of the recovery period; pseudomonas was present in of the faeces samples. this is similar to observations in naturally occurring rd calves, in which a period of diarrhoea is observed after initiation of reconditioning therapy. in more than half of these cases, pseudomonas were found in the faeces (van weeren-keverling buisman et al., a) . pseudomonas usually shows a multiresistant pattern towards antibiotics (batra and garg, ) . pseudomonas ueruginosu is able to cause a haemorrhagic gastroenteritis in calves (nilsson and thiirne, % ) but clinically healthy intestinal carriers have also been reported in dairy calves and cows (matthews and fitzsimmons, % hoadley and mccoy, ; batra and garg, ) . the incidence of pseudomonas intestinal carriage in veal calves is probably even higher, due to frequent antibiotic treatment or to the subtherapeutic doses of antibiotics which are frequently added to the milk replacer (hamstra and van haeringen, ) . no information is available about the pathological changes in the intestine in cases of subclinical pseudomonas infection. rotavirus was detected in calf h in weeks , and by the latex agglutination test but was not accompanied by diarrhoea. coronavirus was detected only in the recovery period, in calf e in week and in calf h in week . rotavirus and coronavirus can both cause diarrhoea and villus atrophy in the jejunum of young calves under three weeks of age (torres medina et al., ) . however, in this experiment no pathogenic micro-organisms were shed during the period of intraruminal milk administration, while the vilhrs length was decreasing gradually. thus, the observed vihus atrophy during the induction period is probably caused by another (non-infectious) factor, resulting from the intraruminal administration of the milk. the transitory decline in villus length shortly after the operations may be associated with the well-known relationship between surgery, stress and the shedding of pathogens. the observed vilhrs forms in the pre-induction period, a mixture of finger-shaped and long tongue-shaped villi, is the normal stereomicroscopic appearance for veal calves (van weeren et az., ) . in the induction period, these villus forms are replaced by long to short tongue-shaped villi with few finger-and leaf-shaped ones. the index of mitosis was increased at the end of the induction period and the beginning of the recovery period, although the crypt depth in the experimental calves did not change. this indicates that the villus atrophy induced by intraruminal feeding is one of the hyperplastic type. viius atrophy with hyper-regenerative crypt epithelium in chronic rd calves was also found in previous studies (breukink et az., ; van weeren-keverling b&man et al., a) . a reduced lactase activity compared to the initial values was found in all three experimental calves in week , at the end of the induction period. a similar decrease in lactase activity was found in week , shortly post-operatively. these changes in enzyme activity were accompanied by parallel changes in villas length and indicate a reduction in functional intestinal epithelium, caused by immaturity or damage to the epithelial cells (landsverk, ) . lactase is localized mainly in the brush border of the epithelium of the villi and the apical portions of the crypts in the proximal small intestine (landsverk, ) . its function is the hydrolysis of lactose into glucose and galactose, which are absorbed in the small intestine (coombe and smith, ) . normally, lactase activity is highest in very young calves (huber et al., l%l) and decreases with age. toofanian et al. ( ) observed a gradual decrease in activity during the first four weeks of life; after that the lactase activity remained constant in calves up to months old. lactase activity is also influenced by the type of diet offered: continuation of a liquid lactose-containing diet and prevention of the functional development of the rumen prevented the normal post-weaning decline in lactase activity (toofanian et az., ) . supplementation of milk diets with extra lactose resulted in an even higher lactase activity (huber et al., ) . the lactase activity in our study was therefore probably not only influenced by the observed villus atrophy but was also partly the result of a reduced amount of substrate. in the rumen, an unknown amount of lactose is converted into lactate and volatile fatty acids (van weeren-keverling buisman et al., ) . comparison of the absolute values of lactase activity in our study with those found in the literature is difficult because of the differences in determination of enzyme activity. a reduced lactase activity was reported in diarrhoeic calves between and days of age, which also showed villus atrophy and crypt hyperplasia in the proximal jejunum (landsverk, ) . youanes and herdt ( ) reported similar results. dargel and hartmann ( ) also found that lactase activity in diarrhoeic calves from to days of age was about half that of healthy calves of the same age. a reduction in ap activity was observed at the same times as the reduction in lactase activity, i.e. during weeks and . restoration of the depressed activity of this enzyme after the induction period was somewhat delayed when compared with the lactase activity. at the end of the recovery period, in week , the ap activities of the experimental calves had almost completely regained their initial values. this suggests that ap activity may also be influenced by villus atrophy. alkaline phosphatase (ap) is a brush border enzyme with slight additional activity in the golgi apparatus (landsverk, ) . it is mainly localized in the duodenum and proximal and mid jejunum (landsverk, ) , where it is involved in energyconsuming sugar and lipid absorption (stighnair-herb ef az., ). it is not known if diet or age influence ap activity. however, in diarrhoeic calves up to days of age with experimental escherichia coli and rotavirus infections, ap activity is reduced (stighnair-herb et al., ) . this reduction was also found in k&week-old calves with parasitic gastroenteritis (benz and ernst, ) . in dogs with small intestinal bacterial overgrowth, caused by aerobic flora such as enterococci and escherichiu coli, ap activity was reduced, while lactase activity and the activities of other brush border enzymes were not altered significantly. this suggests that bacterial secretions or metabolites also specifically act on ap activity (batt and hall, ) . it can be concluded that intraruminal administration of half the milk ration to normal veal calves results in clinical symptoms of rd and villus atrophy in the proximal jejunum. this hyperplastic villas atrophy is similar to that found in chronic rd patients and is accompanied by a reduction in brush border enzyme activities. clinical recovery is fast after returning to the normal feeding regimen, with restoration of villus length and brush border enzyme activities in three to four weeks. occurrence of yersnia, serr&z and pseudomonas in health and gastrointestinal disturbances of men and cattle along with the detection of pigs and rats as their natural carriers chronic enteropathies in the dog alkaline phosphatase activities in intestinal mucosa from calves infected with cooperia punctata and eimeria bovis consequences of failure of the reticular groove reflex in veal calves fed milk replacer. the veterinary quarter indigestionen der mastkllber infolge fehlenden schlundrinnenreflexes. tier&ztliche umschau, i absorption of glucose and galactose and digestion and absorption of lactose by the preruminant calf die activitlt der fl-galaktosidase im darmkanal von klinisch gesunden und durchfallkranken k;ilbem sowie ferkeln some observations on the ecology of pseudomonas aerugiraosa and its occurrence in the intestinal tract of animals. the cornell veferinarian digestive enzyme activities in the young calf effect of level of lactose upon lactase concentrations in the small intestines of young calves histochemical distribution of enzymes in the small intestine of young milk-fed calves an enzyme histochemical investigation of the intestinal mucosa in diarrheic calves protein measurement with the folin phenol reagent the incidence and distribution of pseudomonas aerugiinosa in the intestinal tract of calves white scours in piglets. i. stereomicroscopy of the mucosa of the small intestine white scours in piglets at three weeks of age pseudomonas aeruginosa -en mikro-organism med iikad aktualitet. en i+ersikt i&r pseudomonasinfectioner enzyme histochemistry of the small intestinal muccxa in experimental infections of calves with rotavirus and enterotoxigenic escherichia coli the postnatal development of intestinal disaccharidases in the calf rotaviral and coronaviral diarrhea. veterbzaty clinics of north america, symposium on calf diarrhea pensdrinkers en kleischijters. tijdrctifi voor diergeneeskumfe colonization resistance of the digestive tract as a major lead in the selection of antibiotics for therapy % villus atrophy in ruminal drinkers villus atrophy in ruminal drinking calves and mucosal restoration after reconditioning. the veterinary quarterly a simple and effective biopsy technique of the small intestine in the calf intraruminal administration of milk in the calf as a model for ruminal drinking: clinical aspects and biochemical and morphological changes in the rumen changes in small intestinal morphology and flora associated with decreased energy digestibility in calves with naturally occurring diarrhea the authors thank dr ma. van der velden (department of large animal surgery) for performing the operations. h.c. den hartog, j. van dasselaar and a.c. westeneng are acknowledged for technical assistance. dr th. h. herdt is gratefully acknowledged for his critical reading of the manuscript. key: cord- -igmb yfe authors: oma, veslemøy sunniva; tråvén, madeleine; alenius, stefan; myrmel, mette; stokstad, maria title: bovine coronavirus in naturally and experimentally exposed calves; viral shedding and the potential for transmission date: - - journal: virol j doi: . /s - - -x sha: doc_id: cord_uid: igmb yfe background: bovine coronavirus (bcov) is a widely distributed pathogen, causing disease and economic losses in the cattle industry worldwide. prevention of virus spread is impeded by a lack of basic knowledge concerning viral shedding and transmission potential in individual animals. the aims of the study were to investigate the duration and quantity of bcov shedding in feces and nasal secretions related to clinical signs, the presence of virus in blood and tissues and to test the hypothesis that seropositive calves are not infectious to naïve in-contact calves three weeks after bcov infection. methods: a live animal experiment was conducted, with direct contact between animal groups for h as challenge procedure. four naïve calves were commingled with a group of six naturally infected calves and sequentially euthanized. two naïve sentinel calves were commingled with the experimentally exposed group three weeks after exposure. nasal swabs, feces, blood and tissue samples were analyzed for viral rna by rt-qpcr, and virus isolation was performed on nasal swabs. serum was analyzed for bcov antibodies. results: the calves showed mild general signs, and the most prominent signs were from the respiratory system. the overall clinical score corresponded well with the shedding of viral rna the first three weeks after challenge. general depression and cough were the signs that correlated best with shedding of bcov rna, while peak respiratory rate and peak rectal temperature appeared more than a week later than the peak shedding. nasal shedding preceded fecal shedding, and the calves had detectable amounts of viral rna intermittently in feces through day and in nasal secretions through day , however virus isolation was unsuccessful from day six and day from the two calves investigated. viral rna was not detected in blood, but was found in lymphatic tissue through day after challenge. although the calves were shedding bcov rna days after infection the sentinel animals were not infected. conclusions: prolonged shedding of bcov rna can occur, but detection of viral rna does not necessarily indicate a transmission potential. the study provides valuable information with regard to producing scientifically based biosecurity advices. electronic supplementary material: the online version of this article (doi: . /s - - -x) contains supplementary material, which is available to authorized users. bovine coronavirus (bcov) is an important livestock pathogen with a high prevalence worldwide. the virus causes respiratory disease and diarrhea in calves and winter dysentery in adult cattle. these diseases result in substantial economic losses and reduced animal welfare [ ] . one way of reducing the negative consequences of this virus is to prevent virus transmission between herds. inter-herd transmission is possible either directly via transfer of live animals [ , ] , or indirectly via contaminated personnel or equipment [ ] . measures to prevent virus spread between herds must be based upon knowledge of viral shedding, the potential for transmission to susceptible animals and the role of protective immunity. several observational studies have been published on bcov shedding in feces of diarrheic calves and after transportation to feedlots [ , [ ] [ ] [ ] [ ] [ ] [ ] . however, relatively few studies on bcov pathogenesis with emphasis on transmission potential under controlled conditions have been published. bcov belongs to the genus betacoronavirus within the family coronaviridae, also including the closely related hcov-oc , which causes respiratory infections in humans, and the human pathogens sars-cov and mers-cov [ ] [ ] [ ] . bcov consists of one serotype with some antigenic variation between different strains [ , ] . acutely infected animals develop antibodies that persist for a long period, possibly for several years [ ] [ ] [ ] . however, the protective immunity is shorter and incomplete. in two experimental studies, infected calves were not protected against reinfection with a different bcov strain three weeks after the first challenge, but did not develop clinical signs [ , ] . bcov is transmitted via the fecal-oral or respiratory route [ ] . it infects epithelial cells in the respiratory tract and the intestines; the nasal turbinates, trachea and lungs and the villi and crypts of the small and large intestine, respectively [ , ] . replication leads to shedding of virus in nasal secretions and in feces. important factors for the pathogenesis are still not fully explored, such as how the virus infects enterocytes shortly after introduction to an animal. viremia has been detected in one study by park et al. [ ] . clinical signs range from none to severe, and include fever, respiratory signs and diarrhea with or without blood [ , ] . as the time of infection is usually unknown and laboratory diagnostics are usually not performed, occurrence of clinical signs is the most relevant parameter to relate to viral shedding. the majority of experimental studies have used bcov inoculation as challenge procedure, which may influence clinical signs and viral shedding, and thereby the transmission potential compared to natural infection. it has been hypothesized that bcov can cause chronic subclinical infections which could be an important virus source [ ] . kapil et al. documented viral antigen in the small and large intestines of infected calves three weeks post inoculation [ ] . crouch et al. found that ten cows were shedding bcov-immune complexes in the feces for weeks [ ] . it is, however, difficult to establish whether there is true persistence of virus, or reinfection of partially immune animals and whether these animals represent a risk to other animals. there is a lack of experimental studies investigating viral shedding pattern for longer periods than two weeks, with sensitive detection methods. viral load and infectivity also needs to be determined. this is of high practical relevance, since the farmers need guidance on biosecurity in trade and transport of live animals. the current study was conducted to fill prevailing gaps in the knowledge on fundamental aspects of bcov infection. the specific aims were to: a live animal experiment with the natural host was conducted. the experimental units were groups of calves and the intervention consisted of direct contact with bcov-infected animals. the primary outcome was clinical signs, and the secondary outcome was presence of bcov rna and bcov antibodies. three experimental groups were included; the field group (fg, n = ) that was naturally infected with bcov, the naïve exposed group (eg, n = ) and the naïve sentinel group (sg, n = ). an overview of the study design is shown in fig. . twelve bcov seronegative weaned bull calves between six and twelve weeks of age were included, seven were swedish red and white, four were swedish holstein and one swedish mountain breed. they originated from two dairy herds, initially negative for antibodies to bcov in milk from primiparous cows. the calves were allocated to groups according to herd of origin and day of arrival. the sequence of euthanasia of the eg and sg calves was random, determined by drawing of lots. natural outbreak of winter dysentery fg originated from a herd that was in an early phase of a winter dysentery outbreak. when fg was transported to the research facility, the calves showed mild signs of respiratory disease. two days later, a severe outbreak confirmed by rt-pcr and serology to be caused by bcov with bloody diarrhea and reduction in milk production, took place in the herd. the experiment was conducted at the stationary clinic at the department of clinical sciences at the swedish university of agricultural sciences. the facility was closed for other animals during the experiment, and had restricted admission for people. personnel used designated clothing, and had no contact with other cattle the same day. each group was housed in separate pens within the same room. due to the type of facility and design of the study, acclimatization period was not possible for any of the groups. clinical examinations and sampling were consistently done in the order sg, eg and fg. to mimic standard managerial conditions, direct contact was chosen as challenge procedure for both eg and sg. the commingling was done by moving eg into the other two groups' pens for h. efforts were made to minimize the stress and discomfort for the animals involved. the calves were kept groupwise in pens with straw bedding, were fed a commercial calf concentrate twice daily and had access to haylage ad libitum. the animals were monitored by a trained animal technician and a veterinarian at least three times a day. indications for antibiotic treatment ( iu procaine benzyl penicillin/kg bodyweight/day i.m. for five consecutive days) were abnormal sounds on lung auscultation or prolonged high temperature. indication for treatment with a non-steroidal anti-inflammatory drug (metacam vet, boehringer ingelheim vetmedica, germany) was severe depression, and oral fluid with electrolytes was to be given to moderately dehydrated animals. euthanasia was achieved by i.v. injection of pentobarbital (euthasol vet., le vet, netherlands). daily clinical examinations were performed by a veterinarian and clinical signs were scored as presented in fig. timeline of the experiment. the solid lines symbolize the timespan when the calves participated in the experiment. the dashed lines symbolize commingling of the indicated animals for h; e.g. the field group arrived at the research facility on day − , commingled with the exposed group day and left the research facility day . the calves in the exposed group were sequentially euthanized from day to day . the sentinel group arrived at the research facility day and commingled with the exposed group the following hours the score from each category was added to give a daily clinical score for each of the calves in the experiment score above six on three consecutive days as moderate disease and a score above eleven was categorized as severe clinical disease. nasal swab specimens and fecal samples from fg were collected approximately every third day from day − (d- ) to d . from eg, nasal swabs and fecal samples were collected every day from d to d and then every third day until d . nasal swabs from sg were collected d , d and d . the nasal specimens were collected by rotating a flocked eswab™ (copan, brescia, italy) approximately five cm inside one of the calf's nostrils. the specimens were frozen and stored at − °c before further processing. blood was drawn from the jugular vein upon arrival and d , d , d , d , d , d , d , d , d , d and d using sterile evacuated tubes with and without edta-anticoagulant. the edta-blood was centrifuged and the cell fractions were stored separately at − °c before further processing. sera were stored at − °c until analyzed. tissue samples from lung, medial retropharyngeal and mesenteric lymph nodes, ileum, and colon were stored in rna-later at − °c. serum samples were analyzed for anti bcov igg by svanovir bcv-ab (boehringer ingelheim svanova, uppsala, sweden) according to the manufacturer's instructions. samples from sg were also tested for antibodies to bovine respiratory syncytial virus (brsv) by svanovir brsv-ab (boehringer ingelheim). the optical density (od) at nm was measured and corrected by subtracting the od for the negative control. percent positivity (pp) was calculated as (sample od/positive control od) × , and a pp-value of < was regarded as negative. fecal samples (diluted : in pbs) and nasal swab specimens were centrifuged at x g for min. rna was extracted from μl supernatant and μl plasma by qiaamp viral rna mini qiacube kit (qiagen, hilden, germany), eluted in μl and frozen at − °c. rna from blood cell fractions from calf e on d and calf e on d was extracted with qiazol (qiagen) and chloroform phase separation mixed with % ethanol ( : ) and purified using rneasy mini kit column (qiagen), while rna was extracted from - mg tissue samples, using rneasy plus universal mini kit (qiagen). rt-qpcr was performed using rna ultrasense™ one-step quantitative rt-pcr system (invitrogen, ma, usa). two microliters of rna was added to a μl reaction volume containing nm each of forward and reverse primers and nm taqman probe [ ] . the thermal profile included an rt step with min at °c followed by °c for min. thereafter, cycles with s at °c and s at °c were conducted. the rt-qpcr was performed on a stratagene mx p™ (agilent technologies, ca, usa) and a positive and a negative control were included in each run. in order to evaluate inhibition of the rt-qpcr, rna extract from some fecal samples were diluted : and compared to undiluted rna. the ct-values in these samples suggested negligible levels of inhibitors. inhibitors in plasma and cell extracts were evaluated by spiking with mengovirus rna. comparison of ct-values showed that plasma had no negative effect, while the cell fractions had an inhibitory effect, giving an increase of one ct-value. in order to estimate the number of bcov viral rna copies (vrc) in the clinical samples, a standard curve was prepared using tenfold dilutions of a plasmid containing the bcov target sequence. aliquoted bcov rna was used as a calibrator and included in every rt-qpcr plate to adjust for inter plate variation. the number of vrc in the clinical samples was calculated using the formula: where q s = viral rna copies in sample, q c = viral rna copies of calibrator, ct s = ct value of sample, ct c = ct value of calibrator and m = slope of the standard curve. the standard curve covered the range from . to . × plasmid copies, and showed a strong linear relationship with a high coefficient of determination (r = . ) and a high amplification efficiency ( . %). the limit of quantification (loq) for the plasmid was . copies which represented . log bcov vrc per nasal swab and ml plasma, . log vrc/g feces and . log vrc/g tissue. virus infectivity was tested by virus isolation from nasal swabs from e and e between d and d (d , d , d , d , d , d , d , d and d ). the swab supernatants were diluted : in dulbecco's modified eagle medium (dmem, thermo fisher scientific, paisley, scotland), filtered through a . μm filter (sartorius stedim biotech, goettingen, germany) and added to a monolayer of -days-old human rectal tumor cells (hrt- g, attcc crl- ) in a -well plate. in addition, infective virus was titrated from one nasal swab supernatant using two-fold endpoint dilutions in a well plate. after h incubation at °c, the inoculum was replaced with dmem with % fetal calf serum and antibiotics ( iu penicillin and mg streptocillin/ ml). after two days at °c and % co , the cells were fixed with intracellular fixation buffer (ebiosience, ca, usa) and stained with : dilution of monoclonal mouse anti-coronavirus antibody labelled with fluorescein isothiocynate (biox diagnostics, rochefort, belgium) and dapi nuclear counterstain (thermo fischer scientific). the wells were observed under a fluorescent microscope for antigen positive cells. an overview of clinical signs in all groups is presented in table . five out of six fg calves showed mild clinical disease. eg's daily clinical scores are shown in fig. . three out of four eg calves showed mild disease, and one calf moderate clinical disease. sg did not develop clinical signs that were categorized as disease in the clinical scoring system. however, both calves had some days with intermittent nasal discharge and sporadic cough and s had a few days with intermittently runny feces. blood-tinged diarrhea or nasal discharge was not observed in any of the groups. all calves tested negative for antibodies to bcov at the beginning of the trial. at d all calves in fg and eg had seroconverted (additional file : table s ). the sg was still seronegative to bcov d and did not show an increase in titer for antibodies to brsv. bcov rna was not detected in any of the blood samples analyzed. the nasal shedding of bcov rna from fg and eg is presented in fig. a , and fig. shows eg calves' individual shedding. briefly, fg was shedding bcov rna d- through d , and in eg all swabs were positive from d through d , and at least one out of four calves was positive through d (fig. a) . two calves were positive in nasal swabs with a concentration of . log and . log vrc/swab the day of commingling with sg. none of the nasal swabs from sg were positive. fecal shedding of bcov rna in fg and eg is shown in fig. b , and the individual shedding from eg in fig. . viral rna was detected in fecal samples from fg between d- and d . fecal samples from eg were negative d and d . at least two out of four calves were positive every day from d through d and bcov rna was intermittently detected through d . after d , three calves had a period of four to six days with negative results, before they again started shedding bcov rna for three to five days (fig. ) . the association between bcov pcr results and selected clinical signs is shown in figs. and . the overall clinical score showed good correlation with detection of bcov rna. general depression and cough were the individual scores that showed the best association with bcov rna shedding. the highest mean respiratory rate and rectal temperature appeared more than a week later than the peak shedding. the calves were exposed to bcov in the field (f - ), were exposed to f-animals (e - ) or exposed to e-animals (s - ). a peak rectal temperature (rt) b runny to watery stools were considered diarrheic. c mucopurulent or purulent nasal discharge (nasal discharge score = ). d five days of i.m. treatment with iu procaine benzylpenicillin was initiated on indicated day. e calf f was treated for six days viral rna was detected in lymph nodes from the eg calves euthanized three, four, five and six weeks after infection (table ). viral rna was also detected in ileum and colon from the animals euthanized five and six weeks after infection, but not in lung tissue. virus was isolated from nasal swabs from calf e on d and from e in the period d to d . a photograph of infected cells is shown in fig. . the titer of infective bcov in the nasal swab was per μl swab medium ( ml in total) corresponding to . log infective particles in a swab containing . log vrc, giving a total to infective particles ratio (t/i) of log . the present study showed that calves infected with bcov shed viral rna for five weeks, and harbored viral rna in intestinal tissues and lymph nodes even longer. interestingly, contact with these calves three weeks after challenge, when the clinical condition had improved and the calves had seroconverted, did not lead to infection in sentinel calves and virus isolation was not possible from calves shedding viral rna at this time point. in concordance with other studies [ , ] , all eg calves became bcov positive shortly after contact with infected calves and shed viral rna continuously for two weeks. this supports that introduction of bcov into a naïve population leads to a high basic reproduction number (r ). r depends on the duration of the infectious period, the number of exposed susceptible individuals and the probability of a susceptible individual to be infected. in herds and transportation systems where cattle from different herds are commingled, the risk of virus transmission is high. the detection of bcov rna in nasal swabs from naïve calves in eg shortly after exposure might be due to passive inhalation of virus excreted by the fg, or to virus replication in the respiratory tract. since the viral load in the nasal swabs from eg exceeded that of fg at d , the study confirms that bcov replicated massively in the airways of eg calves already at d . fecal shedding started later than nasal shedding which is in concurrence with other studies [ ] . saif and colleagues found that when inoculating calves intranasally, bcov was first detected in nasal epithelial cells and secondly in feces. in contrast, in calves inoculated orally, fecal detection of bcov preceded detection in nasal swab specimens. they concluded that the infection route could determine the sequence of infection of the respiratory and intestinal tract [ ] . the present study supports that the respiratory route is the most common infection route when calves are naturally infected by direct contact. with indirect virus spread, the fecal-oral route could be more common. nasal swabs were more often positive for bcov than fecal samples in this trial, most likely due to a higher limit of detection for bcov in feces than in nasal swabs. for diagnostic purposes, nasal swab specimens therefore seem advantageous to fecal samples for virus detection in calves with suspected bcov related disease. moving and commingling are associated with stress, which has been found to affect the intestinal immune system [ ] . it is possible that stress increased the bcov rna shedding observed in the eg calves after introduction of the sentinel calves. buying and selling of calves often involve extended transportation and commingling with susceptible cattle. the stress response, and a possible increased fecal shedding of virus, would probably be higher under field conditions. shedding of virus in conventionally reared piglets, only in gnotobiotic piglets [ ] . this indicates that secondary pathogens and changes in microbiota are important for disease development and clinical signs. the present study supports that after the acute stage of disease other factors than virus replication are important for clinical signs; for instance secondary bacterial infections. although the sentinel calves did not get infected with bcov, they showed sporadic unspecific signs during the trial, but below the mildest category "mild disease" in the clinical scoring system. since acclimatization was not possible, the calves changed environment including feeding routines when enrolled in the experiment, which could cause the signs observed. other infectious agents could also have been present, and if so, most likely less virulent pathogens. bovine virus diarrhea virus and bovine herpesvirus are not present in sweden [ ] , and the sentinel calves showed no serologic response to brsv. co-infection between bcov and other agents is likewise possible in fg and eg, as is the case under field conditions. unlike most enteric viruses, bcov is enveloped and therefore susceptible to environmental inactivation [ ] . one might expect that the conditions in the forestomaches and abomasum would inactivate bcov and one possibility is that bcov is transported from the oronasal cavity to the small intestines through the bloodstream. however, viremia was not detected in the present study, and transport of the virus to the intestines appears to have been through the digestive tract. park and colleagues [ ] detected bcov rna in serum samples from calves infected with a winter dysentery strain between day three and eight post inoculation. they used nested pcr for detection, which is generally a more sensitive method than rt-qpcr, but also more vulnerable for contamination [ ] . short viremic period or intake of a lower virus dose in naturally infected calves could also explain the negative results in the present study. inhibition of the rt-qpcr by plasma components was tested and ruled out. despite the absence of detectable viremia in the present study, bcov rna was found in mesenteric lymph nodes at late stages of the infection. viral rna must have been transferred in low concentrations in blood or lymph to the draining lymph node, by antigen presenting cells or as free virus particles. the finding of bcov rna in lymph nodes, ileum and colon six weeks after infection indicates coronavirus persistence in calves, however, the importance of this persistence for virus transmission is uncertain. other coronaviruses are known to create persistent or chronic infections in mice and cats [ , ] . mers-cov is shown to be excreted for more than a month in humans [ ] and human coronavirus e creates persistent infections in vitro [ ] . although fecal shedding of bcov rna was detected five weeks post infection in the present study, the transmission potential at this stage is most likely negligible, as at three weeks post infection. bcov vrc were quantified by rt-qpcr, which does not give information on the number of infective particles. the ratio of total to infective particles (t/i) is challenging to establish for bcov due to difficulties in cultivating virus from clinical samples. in the present study, virus titration showed a t/i ratio of approximately tissue samples from lymph nodes, lung, ileum and colon were harvested from exposed group calves euthanized at the indicated number of days after exposure to field group calves. the number of viral rna copies (vrc) of bcov was quantified with rt-qpcr and the limit of quantification was . log vrc/g tissue log . with this high t/i ratio it is not surprising that virus isolation was unsuccessful after d , when the vrc numbers are decreasing. it also agrees with the sentinel calves not getting infected d . in contrast, roughly . log vrc were detected per nasal swab and gram feces from the seronegative fg calves that infected the eg calves. with a t/i ratio of log , each nasal swab and gram of feces contained more than . log infective virus particles. the high t/i ratio and the failure of virus isolation after d could be due to either few infective particles or low sensitivity of the isolation method. low levels of infective particles could be caused either by high production of defective particles or by neutralizing effect of antibodies. low sensitivity could be caused by suboptimal conditions in cell culture compared to in vivo (particularly for virus from clinical samples not adapted to cell culture growth), dilution of viral content in the swab, and freezing and thawing of the material. for feline enteric coronavirus, the t/i increased from - log during the first week after infection, to up to log days post infection [ ] , the increase possibly caused by the antibody response. few methods are available for studying transmission potential apart from live animal experiments, although ethically challenging and resource demanding. existing literature is based on experimental studies examining bcov shedding for [ , , ] to [ , ] days. to the authors' knowledge, the present study is the first to study the shedding for as long as six weeks under experimental conditions. in addition, it is also the first to study the impact of this shedding using sentinel calves. although a low number of calves were used, the results indicate that calves are not infectious three weeks after exposure to bcov. this information is important and relevant in order to produce scientific based advices on how to avoid introduction of bcov into herds. further investigation of calves at different stages of disease is recommended to verify and corroborate these findings. the effect of stress related to transport on viral shedding and infectivity should also be considered. in the present study, the virus that caused winter dysentery in adult cattle primarily gave respiratory disease in calves. niskanen et al. also found that bcov derived from an outbreak of winter dysentery caused mainly respiratory disease in weaned calves [ ] , supporting that bcov is an important cause of respiratory disease in calves [ , ] and winter dysentery in adults [ ] . the economic and welfare consequences of bcov therefore include the combined effects of neonatal enteritis, respiratory disease in young cattle and winter dysentery in adults. also considering the high prevalence worldwide, bcov is an important loss-inflicting factor in the cattle industry. the current study shows that calves infected with bcov are rt-qpcr positive in nasal and fecal specimens for a longer period than earlier recognized. however, contact with naïve calves three weeks after exposure did not lead to infection. a low level of infective particles could be due to either production of a high level of defective particles and/or production of neutralizing antibodies. the study provides highly relevant information when designing biosecurity advice regarding animal trade and coronaviral disease in cattle. additional file : table s . antibodies to bcov. (docx kb) bovine coronavirus associated syndromes severe outbreak of bovine coronavirus infection in dairy cattle during the warmer season bovine coronavirus (bcv) infections in transported commingled beef cattle and sole-source ranch calves bioexclusion of diseases from dairy and beef farms: risks of introducing infectious agents and risk reduction strategies evaluation of concurrent shedding of bovine coronavirus via the respiratory tract and enteric route in feedlot cattle sensitivity comparison for detection of respiratory bovine coronaviruses in nasal samples from feedlot cattle by elisa and isolation with the g clone of hrt- cells isolation of bovine respiratory coronaviruses from feedlot cattle and comparison of their biological and antigenic properties with bovine enteric coronaviruses antibody titers against bovine coronavirus and shedding of the virus via the respiratory tract in feedlot cattle antibody responses to respiratory coronavirus infections of cattle during shipping fever pathogenesis detection of respiratory and enteric shedding of bovine coronaviruses in cattle in an ohio feedlot evolutionary history of the closely related group coronaviruses: porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, and human coronavirus oc coronaviruses post-sars: update on replication and pathogenesis the structure and functions of coronavirus genomic ′ and ′ ends a serological comparison of bovine coronavirus strains bovine coronavirus antibody responses of cattle with respiratory coronavirus infections during pathogenesis of shipping fever pneumonia are lower with antigens of enteric strains than with those of a respiratory strain bovine coronavirus as the causative agent of winter dysentery: serological evidence experimental reproduction of winter dysentery in lactating cows using bcv -comparison with bcv infection in milk-fed calves crossprotection studies between respiratory and calf diarrhea and winter dysentery coronavirus strains in calves and rt-pcr and nested pcr for their detection infection and cross-protection studies of winter dysentery and calf diarrhea bovine coronavirus strains in colostrum-deprived and gnotobiotic calves dual enteric and respiratory tropisms of winter dysentery bovine coronavirus in calves experimentally induced coronavirus infections in calves: viral replication in the respiratory and intestinal tracts excretion and persistence of bovine coronavirus in neonatal calves chronic shedding of bovine enteric coronavirus antigen-antibody complexes by clinically normal cows bovine respiratory syncytial virus iscoms-immunity, protection and safety in young conventional calves bovine respiratory syncytial virus iscomsprotection in the presence of maternal antibodies molecular characterisation of cryptosporidium isolates from swedish dairy cattle in relation to age, diarrhoea and region detection of bovine coronavirus using a taqman-based real-time rt-pcr assay failure to spread bovine virus diarrhoea virus infection from primarily infected calves despite concurrent infection with bovine coronavirus transmission of bovine coronavirus and serologic responses in feedlot calves under field conditions stress modulates intestinal secretory immunoglobulin a origin, evolution, and virulence of porcine deltacoronaviruses in the united states surveillance of infectious diseases in animals and humans in sweden identification of a -to -fold repetitive bp dna fragment in toxoplasma gondii, and its use for diagnostic and quantitative pcr the biology of coronaviruses persistence and transmission of natural type i feline coronavirus infection kinetics and pattern of viral excretion in biological specimens of two mers-cov cases persistent infection of human oligodendrocytic and neuroglial cell lines by human coronavirus e experimental feline enteric coronavirus infection reveals an aberrant infection pattern and shedding of mutants with impaired infectivity in enterocyte cultures development of nasal, fecal and serum isotype-specific antibodies in calves challenged with bovine coronavirus or rotavirus experimental inoculation of adult dairy cows with bovine coronavirus and detection of coronavirus in feces by rt-pcr bovine respiratory coronavirus respiratory infections in norwegian dairy calves we thank kerstin mover berglund, dennis larsson and bengt norén for taking good care of the calves during the trial, mamata khatri for performing the virus isolation and hanne haatveit for preparing the picture of the stained cells. authors' contributions ms was the project leader; vso, mt, sa, mm and ms conceived and designed the experiment; vso, sa and mt performed the experiment; vso and mm planned and performed the lab analyses. all authors wrote, read and approved the manuscript. the authors declare that they have no competing interests. the trial was conducted in line with national and international guidelines for the care and use of animals and approval was given by the ethics committee for animal experiments, uppsala, sweden [protocol number c / ].• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- - iy l f authors: sharon, k. p.; hulbert, l. e.; davis, e. m.; ballou, m. a. title: effects of plane of milk-replacer nutrition on the health, behavior, and performance of high-risk holstein bull calves from a commercial calf ranch date: - - journal: applied animal science doi: . /aas. - sha: doc_id: cord_uid: iy l f abstract objective this research study aimed to determine whether preweaning plane of milk-replacer nutrition influences health, standing and oral behaviors, and performance of high-risk calves. materials and methods thirty-six holstein bull calves ( d of age) from a commercial calf ranch were assigned to either a high plane of milk-replacer nutrition (hpn; n = ; and % dm lipid and protein, respectively, cow’s match, land o’lakes animal milk protein co., shoreview, mn) or a low plane of milk-replacer nutrition (lpn; n = ; % dm lipid and protein, herd maker, land o’lakes animal milk protein co.) through weaning. total serum protein concentrations confirmed that and % of calves had failure of passive transfer (< . g/dl) in hpn and lpn, respectively. all calves were bottle fed twice daily. the lpn were fed g of dm/d of milk replacer (mr) until weaning, and the hpn calves were fed g of dm/d of milk replacer during the first d and , g from d until weaning. calf starter and water were offered ad libitum. calves were step-down weaned beginning at d and completely weaned after d when consuming kg of calf starter (as-fed basis). data are reported as hpn versus lpn throughout, respectively. results and discussion risk for bloat and scouring were greater for calves fed the hpn, but there was no difference in antibiotic treatment or mortality. the hpn calves had greater performance over the preweaning period including final bw, adg, and feed:gain (total kg of milk replacer + calf starter, kg of dm/kg of bw gain), but weaning was more stressful for hpn calves. implications and applications in conclusion, the high-risk calves fed a hpn had increased preweaning adg, but there was also an increased incidence of scours. there may be differences in the health status of calves that may affect the ability to consume and use high mr levels. when feeding high-risk calves there may be a need for alternate strategies when determining the quality and quantity of mr being fed when compared with low-risk calves. differences in gut integrity may influence the ability of high-risk calves to handle high levels of mr early in life. potential issues with high-risk calves being fed a hpn might be reduced with increased feeding times per day, a gradual step-up method, or other nutritional strategies that improve gastrointestinal development and function. determining the effects of an increased plane of nutrition (pon) on the health, behavior, and performance of high-risk dairy calves can help establish recommendations for feeding calves more milk solids to increase measures of gain and animal health without compromising normal calf behavior. feeding calves more milk earlier in life has the potential to increase bw and age at first weaning and may have implications into the entire lifespan and productivity of the calf. a common practice in the dairy industry is to restrict the quantity of milk solids fed to calves to accelerate starter consumption and decrease the age of weaning. however, an increasing number of operations are beginning to feed greater quantities of milk solids (nahms, ) . a higher pon increases preweaning growth performance as well as future lactation performance (davis rincker et al., ; ollivett et al., ; soberon et al., ) . the influence that pon has on the risk for enteric disease during the preweaning period continues to be debated. a study completed by quigley et al. ( ) reported that calves force fed greater quantities of milk solids had increased incidence of scours and antimicrobial treatments, whereas ollivett et al. ( ) reported improved recovery and better hydration following a cryptosporidium challenge in calves that were free fed. it is generally accepted that when calves are fed greater quantities of milk solids, their fecal consistency appears looser or they tend to have more diarrhea. liang et al. ( ) reported that fecal consistency may not be the best indicator of enteric health because even though the feces look more loose, there was no difference in the dm percentage of the feces from healthy calves fed lesser versus greater planes of milk-replacer nutrition. another concern with feeding a reduced pon is that a lower pon does not satiate calves and possibly can cause prolonged expression of extra nonnutritive oral behaviors. high-risk calves experiencing failure of passive transfer (fpt) of colostrum require particular care and management, and increased rates of sickness may impede nonnutritive oral behavior development (hulbert and moisá, ) . increased health risks, including gastrointestinal ailments, are common in colostrum-deprived calves. high-risk calves are calves that may have fpt, increased exposure to pathogens, or increased stressors such as transport in the first few days of life. thus, nutritional planes may especially influence these high-risk calves. therefore, the objective of this study was to determine whether preweaning pon influences performance, behavior, and health in high-risk neonatal calves. all experimental procedures were in compliance with the guide for the care and use of agricultural animals in research and teaching and approved by the institutional animal care and use committee of texas tech university. thirty-six holstein bull calves ( d of age) were acquired from a local calf ranch and consisted of calves from commercial dairies within a -km radius of lubbock, texas. calves were randomly assigned to of dietary treatments (n = calves per treatment): a low plane (lpn) of milk-replacer nutrition or a high plane (hpn) of milkreplacer nutrition. calves fed the lpn were fed g of dm/d of a % cp, % lipid milk replacer (mr) at % solids dm (herd maker, land o'lakes animal milk protein co., shoreview, mn; table ). calves fed the hpn were fed g of dm/d from d of age to d of age and , g of dm/d from d until weaning of a % cp, % lipid mr at . and . % solids dm (cow's match, land o'lakes animal milk protein co., table ). calves were fed mr from bottles twice daily at and h. all calves were offered ad libitum access to a texturized calf starter (table ) and water. peripheral blood samples were collected from the jugular vein into evacuated tubes without any additive at enrollment and analyzed for total serum protein using a hand-held refractometer. a total serum protein concentration of . g/dl was used as the threshold to determine the proportion of calves that had fpt of immunoglobulins, based on information from tyler et al. ( ) , who developed a regression equation where a total serum protein concentration of . g/dl was equivalent to , mg/dl of serum igg . total serum protein concentrations confirmed that % of hpn and % of lpn calves had fpt (< . g/dl). all calves were housed outdoors in commercially available calf hutches ( . × . m, agri-plastics, cortland, ny) with an attached pen ( . × . m) bedded with sand, and bottle holder, water-bucket, and grain-bucket located inside the hutch. calf starter and water intakes were measured daily. calves were vaccinated with a -way (infectious bovine rhinotracheitis, bovine viral diarrhea virus i and ii, bovine respiratory syncytial virus, parainfluenza- ; triangle ; boehringer ingelheim, ridgefield, ct) vaccine at d and a booster at d. weaning was initiated at d by removal of the afternoon feeding. calves were completely weaned from mr beginning after d once they consumed kg as fed/d of calf starter. individual calf bw was recorded at enrollment and at , , and d of the study period. voluntary mr refusals were recorded min after each feeding. peripheral blood samples were collected into -ml heparin vacutainers (bd vacutainers, fisher scientific, waltham, ma) at h on , , , , and d relative to enrollment via jugular venipuncture. blood samples were centrifuged at room temperature ( , × g for min) within min of collection, and plasma was stored at − °c until subsequent analyses. plasma glucose and urea nitrogen were analyzed using commercially available enzymatic kits (stanbio laboratory, boerne, tx). all procedures were followed per the manufacturer's instructions for the manual method; however, sample and reagent volumes were scaled for use in -well microplates. all colorimetric data were measured on a spectramax pc (molecular devices, sunnyvale, ca). control serum (randox laboratories, oceanside, ca) was used to calculate the inter-assay cv of . and . % for plasma glucose and urea nitrogen, respectively. the mean intra-assay cv for plasma glucose and urea nitrogen were . and . %, respectively. fecal scores were classified twice daily around feeding according to the guidelines outlined by larson et al. ( ) , by independent trained observers that were not blinded to treatment: = firm, well-formed (not hard); = soft, pudding like; = runny, pancake batter; and = liquid, splatters, pulpy orange juice. data for each calf were averaged over -d periods before statistical analysis, and a scouring event was reported as consecutive fecal scores of . the independent evaluations were compared and any disagreement between the evaluations on a calf were reconciled by both observers looking at the calf together and agreeing on a fecal score. bloat was assessed before and within h after each feeding. disposition scores for both the body and head were scored twice daily imme-diately before each mr feeding. calves were scored on a -to- scoring system. the body disposition scores were = standing; = lying sternal; and = lying lateral. the head scores were = alert, ears up; = depressed, head distended or ears droopy; and = head extended. calves with either a body disposition score of or head score of or had a rectal temperature taken before the a.m. feeding. any calf with a rectal temperature greater than . °c or less than . °c was treated with resflor gold (merck animal health, kenilworth, nj). ex vivo leukocyte responses were performed as described by hulbert et al. ( ) with minor modifications. whole blood was stimulated with lipopolysaccharide from escherichia coli o :b to estimate the ability of whole blood cells to secrete tumor necrosis factor-α (tnf-α). for the cultures, μl of whole heparinized blood was added to μl of roswell park memorial institute (rpmi) medium (# - , invitrogen life technologies, grand island, ny) containing, at a final concentration, % antibiotic and antimycotic (# - , invitrogen life technologies) and μg/ml lipopolysaccharide from e. coli o :b , and incubated in a °c water-jacketed incubator at % co for h. following incubation, cultures were centrifuged at , × g for min at room temperature, and resulting supernatant was collected and stored at − °c until analysis for tnf-α concentration via commercially available bovine-specific elisa kits (#dy for tnf-α, randd systems, minneapolis, mn) using -step serially diluted recombinant bovine tnf-α standard. at d of age, accelerometers (ua- - ; onset computer corp., bourne, ma) were placed on each calf's right or left hind leg with vetwrap cohesive bandage ( m products, st. paul, mn) as previously described by calvo-lo- ) . at midnight at d of age, the accelerometers and event loggers began recording every s each time the calf manipulated the dummy nipple (at a rate of hz), respectively. every to wk, all logging devices were removed, data were downloaded, and loggers were refixed to the calves and hutches. automated logger data were downloaded and processed using methods previously described (ledgerwood et al., ; hill et al., ; hulbert et al., ) ; standing duration and pacifier use (min/d) were the main variables of interest. the observed dummy nipple was correlated (r = . ; p ≤ . ) with the sensor-activated event-recording logger in a previous study (ruiz, ) . the sample size was based on a -sided t-test with expected proportions of calves with scours of % for the hpn and % for the lpn with a and % protection against type and error, respectively. all repeated, continuous data were analyzed by restricted maximum likelihood anova for a completely randomized design using the mixed procedure of sas (sas version . , sas institute inc., cary, nc). a linear mixed model with the fixed effects of treatment, time, and the interaction of treatment by time were fitted. calf nested within treatment was the subject of the repeated statement. the mean model was run with all available covariance structures for the withinsubject measurements. the appropriate covariance structure was chosen for each analysis based on the schwarz's bayesian information criterion. initial bw and total serum protein at enrollment were included in the model as covariates and remained in the model even if they were not significant. total serum protein was a significant covariant (p = . ) for calf starter intake. pairwise differences were performed at each time interval for significant treatment × time interactions using a sliced effect multiple comparison approach using a tukey-kramer adjustment. fecal scores were averaged by consecutive days, and the residuals were normally distributed and therefore analyzed as a continuous repeated measure. head and body score as well as incidence of scours, bloat, mortality, and antibiotic use were analyzed using a fisher's exact test with the freq procedure of sas (sas version . , sas institute inc.). before analysis, repeated data were tested for normality of the residuals by evaluating the shapiro-wilk statistic, normal probability plots of the residuals, and histograms of the residuals using the univariate procedure of sas (sas version . , sas institute inc.). in addition, all nonrepeated data were evaluated for homogeneity of variance. least squares means (±sem) are reported throughout. treatment differences with p ≤ . were considered significant, and . < p ≤ . were considered tendencies. two of the calves died in both treatments, which is within the expected mortality rate for high-risk calves. nutrient contents of both diets are reported in table . there was a treatment × time interaction (p ≤ . ) in adg, where hpn calves had greater (p ≤ . ) adg during the preweaning period but were not different (p = . ) from lpn calves after weaning (table ). there was also a treatment × time interaction (p ≤ . ) in feed: gain ratio, where hpn had greater (p = . ) efficiency from to d but were not different (p ≥ . ) from to d or to d (table ) . feed-to-gain ratio was calculated as [(total kg of mr + calf starter, kg of dm)/kg of bw gain]. there were treatment × time interactions (p ≤ . , . , and . ) for milk, starter, and total intakes, respectively ( figure ). the hpn calves consumed more milk solids, whereas the lpn consumed more calf starter. the hpn consumed more total intake during the preweaning periods, whereas the lpn calves consumed more total intake during the weaning and immediate postweaning period. there was a treatment × time interaction (p = . ) in water intake; however, a slice effect at each week revealed lpn had greater (p ≤ . ) water intake throughout the study ( . vs. . ± . l; table ), which is consistent with the increased calf starter intake. plasma glucose and urea n concentrations are reported in table . calves fed a hpn refused more (p ≤ . ) milk than lpn calves (table ) . no differences (p ≥ . ) in the distribution of frequencies of head or body scores were observed between treatments (data not shown). there was a tendency (p = . ) for more hpn calves to bloat than lpn calves during the study ( . vs. . %; table ). further, the hpn calves had greater (p = . ) incidence of scouring ( . vs. . %; table ) as well as greater (p = . ) fecal scores (table ) compared with lpn calves. however, there was no difference (p = . ) in the percentage of calves that received systemic antibiotics ( vs. %; table ). calves fed a hpn tended to have greater tnf-α concentrations in whole blood cultures compared with lpn calves (table ). there were no treatment × time (p = . ) or treatment (p = . ) differences for daily standing duration; however, standing duration was influenced by time (p ≤ . ). treatment × time differences were observed (p = . ; figure ) in dummy nipple use after the second mr feeding was removed at d of age. the hpn calves increased their use of the dummy nipple after complete weaning, whereas lpn calves did not change their dummy nipple use. feeding a higher pon positively affected calf performance including final bw, adg, and the feed-to-gain ratio. however, the improved adg was more moderate than reported in other studies where a comparable mr was fed (bartlett et al., ; ballou, ) . studies using calves with fpt reported reduced adg compared with calves with appropriate passive transfer (pithua and aly, ) . calves fed hpn had greater adg in every period, with the exception of to d. at this time, calves were undergoing weaning. a probable contributor to the reduced adg among hpn calves was likely due to the lower calf starter intake as well as digestibility of that calf starter at that time. calves fed a hpn ate less starter throughout the study. similarly, huber et al. ( ) reported calves fed greater mr amounts consumed less calf starter. however, once weaned, previous studies observed that the hpn rapidly increase calf starter intake and can exceed lpn calves a few weeks after weaning (ballou, ; obeidat et al., ; ballou et al., ) . in agreement with previous studies, hpn calves had greater fecal scores throughout the study (nonnecke et al., ; bascom et al., ; liang et al., ) . greater fecal scores in calves fed a hpn are routinely considered a consequence of greater milk solids consumed. more importantly, the hpn calves had a greater incidence of scouring than lpn calves. scours were considered if a calf had consecutive fecal scores of . previously, ballou ( ) reported that anything greater than or equal to a fecal score of was considered scours. however, liang et al. ( ) reported the dm of feces from calves with a fecal score and concluded it would be unlikely for most calves to become dehydrated with a fecal score of or less. the moderate increase in fecal scores reported by others when feeding hpn would not increase the risk for dehydration. in contrast, when a calf has a fecal score of , the calf is at an increased risk to become dehydrated or develop metabolic acidosis. scours can be infectious or noninfectious; however, the consequences are similar. a study completed by quigley et al. ( ) reported findings that high-risk calves force fed a variable hpn program had greater incidence of scours and were treated with more antimicrobials than calves fed a restricted quantity of milk solids when figure . intakes of milk solids, calf starter, and total (milk replacer + calf starter) during the preweaning period among calves fed either a low (lpn) or high (hpn) plane of nutrition. the lpn calves were fed g of dm/d of milk replacer ( % dm lipid and protein) until weaning, and the hpn calves were fed g of dm/d of milk replacer ( and % dm lipid and protein) during the first d and , g from d until weaning. intakes were recorded daily. treatment × time interactions existed (p ≤ . ) for milk replacer intake and total intake, and a tendency existed (p = . ) for starter intake. treatment differences (p ≤ . ) are reported as milk, starter, and total for milk replacer, calf starter, and total intakes. data are presented as least squares means. largest sem estimates were . , . , and . for milk replacer, calf starter, and total intakes, respectively. raised on bedding contaminated with coronavirus. force feeding high-risk calves has been reported to increase scours and is not a recommended nutrition strategy for sick calves and, therefore, was not included in this study design. liang et al. ( ) reported that healthy -wk-old calves fed either a low or high pon had no differences in the de or me efficiencies, concluding healthy neonatal calves are capable of digesting greater mr concentrations during the first week of life. in fact, healthy neonatal calves fed a high pon may be more capable of absorbing and utilizing those additional nutrients than calves fed a low pon. further, liang et al. ( ) reported increased digestible cp and nitrogen retention as a percentage of intake when fed the high pon. they concluded that healthy calves are able to digest and use the extra nutrients very efficiently during the first week of life; however, they constrained the inferences to healthy calves. healthy calves may be more capable of absorbing nutrients than high-risk calves; therefore, a hpn may not be as advantageous among high-risk calves as it is in healthier low-risk calves. calves failing to receive colostrum were reported to have reduced gastrointestinal integrity, as reflected in decreased mucosal thickness, as well as decreased villus length and width compared with calves that received colostrum (yang et al., ) . further, increased colostrum intake improved intestinal epithelial cell proliferation and maturation as well as enzymatic activity (blättler et al., ) . colostrum not only influences gut integrity but also contains immune components and nutrients that are considered a major factor in preweaning health. failure of passive transfer is associated with poorer preweaning performance and health (naylor et al., ; donovan et al., ; pithua and aly, ) . these highrisk calves receiving the hpn may have had reduced gut integrity and local immune protection and, as a result, were less tolerant to the additional milk solids, which increased the risk for scours. calves fed the hpn also had a tendency for increased incidence of abomasal bloat. although the incidence of bloat is often sporadic and the etiology not completely understood, a reduced abomasal emptying rate and increased fermentable substrates in the abomasum are risk factors (burgstaller et al., ) . increasing the solids content or osmolality of mr fed can delay abomasal emptying, and feeding a high pon also may increase the nutrients available for rapid fermentation by clostridium perfringens, a microorganism often suspected in abomasal bloat. more research is needed to understand how pon influences abomasal dynamics and health. the expression of nonnutritive oral behaviors early in life may be important to establish mature feeding behaviors and reduce the incidences of unwanted behaviors (e.g., cross sucking) after calves are weaned and transitioned from individual housing to group housing (veissier et al., ; horvath and miller-cushon, ) . before weaning, pon did not influence dummy-nipple use, except at d, when lpn calves tended to use the nipple more than hpn calves. the day before this ( d), daily standing duration was increased among all calves, which was likely caused by research personnel spending most of the day setting up video recording equipment. nonetheless, after weaning was completed, the hpn calves increased dummy-nipple use to over times their preweaning frequencies until d of age. this is an indicator that the stress of weaning was likely greater among hpn calves. the hpn calves had mr as the main component of that diet, and the hpn calves were only motivated to use the dummy nipple when that source of satiety was removed. the hpn calves returned to their preweaning dummy-nipple use by d, and by this age the calves may have been consuming enough starter to become satiated. this is in contrast to data reported by bortoluzzi ( ) , where step-down weaning calves fed a hpn at d did not increase their use of the dummy nipple, whereas hpn calves either stepdown weaned at d or gradually weaned by decreasing the quantity of milk offered from to d had relatively greater use of the dummy nipple. the authors concluded it may have been less stressful to wean those calves from hpn at the older age, d in that study, because they were consuming more starter. even though the calves in the present study were weaned at approximately the same age and in the same manner as the calves in the study by bortoluzzi ( ) , the high-risk nature of the calves resulted in reduced calf starter intake and, therefore, required either a later or possibly an even more gradual weaning. benefits of feeding programs that increase preweaning adg are established. improvements in calf performance were reported in stressed calves fed times per day versus times a day (schingoethe et al., ) . starter intake was improved with increased feeding frequency (kmicikewycz et al., ) . further, it is possible the issues observed with hpn in high-risk calves could be reduced with increased feeding frequencies and should be considered in future research. no difference in either serum glucose or urea nitrogen was observed between treatments. similarly, obeidat et al. ( ) and smith et al. ( ) reported no difference in these metabolites in calves fed varying levels of mr. conversely, calves fed greater levels of mr had greater plasma glucose concentrations in previous studies (smith et al., ; quigley et al., ; ballou et al., ) . others have reported lower urea nitrogen concentrations in calves fed greater mr levels (ballou et al., ) . calves in the present study did not appear to have any differences in protein or glucose metabolism. calves fed a hpn had greater bw compared with lpn calves; however, they also had increased risk for preweaning scours and abomasal bloat as well as increased dummynipple activity during weaning. differences in gut integrity may influence the ability of high-risk calves to handle high levels of mr. therefore, when feeding high-risk calves, al-ternative strategies may need to be considered in comparison with more healthy or low-risk calves. potential issues with high-risk calves being fed a hpn may be reduced with increased feeding times per day, a gradual step-up method, or other nutritional strategies that improve gastrointestinal development and function. one intern was funded through with the science without borders program-brazil. affiliates of kansas state university and behavioral supplies were partially funded through kansas state university ha (hatch act of ) distributions representing the usda-national institute of food and agriculture multistate projects: w- (impacts of stress factors on performance, health, and well-being of farm animals) and nc case study: effects of a blend of prebiotics, probiotics, and hyperimmune dried egg protein on the performance, health, and innate immune responses of holstein calves immune responses of holstein and jersey calves during the preweaning and immediate postweaned periods when fed varying planes of milk replacer plane of nutrition influences the performance, innate leukocyte responses, and resistance to an oral salmonella enterica serotype typhimurium challenge in jersey calves growth and body composition of dairy calves fed milk replacers containing different amounts of protein at two feeding rates influence of dietary fat and protein on body composition of jersey bull calves feeding colostrum, its composition and feeding duration variably modify proliferation and morphology of the intestine and digestive enzyme activities of neonatal calves applied ethology management methods for resilient calves invited review: abomasal emptying in calves and its potential influence on gastrointestinal disease space allowance influences individually housed holstein bull calf innate immune measures and standing behaviors after castration at weeks of age effect of intensified feeding of heifer calves on growth, pubertal age, calving age, milk yield, and economics associations between passive immunity and morbidity and mortality in dairy heifers in florida, usa short communication: intensive measurement of standing time of dairy calves housed in individual pens within a naturally ventilated, unheated nursery over different periods of the year the effect of milkfeeding method and hay provision on the development of feeding behavior and non-nutritive oral behavior of dairy calves influence of feeding different amounts of milk on performance, health, and absorption capability of baby calves effects of changing milk replacer feedings from twice to once daily on holstein calf innate immune responses before and after weaning stress, immunity, and the management of calves milk replacer plane of nutrition influences calf nutritive and non-nutritive oral behaviors effects of a modified intensive milk replacer program fed two or four times daily on nutrient intake, calf growth and health guidelines toward more uniformity in measuring and reporting calf experimental data evaluation of data loggers, sampling intervals, and editing techniques for measuring the lying behavior of dairy cattle the digestive system of -week-old jersey calves is well suited to digest, absorb, and incorporate protein and energy into tissue growth even when calves are fed a high plane of milk replacer dairy cattle management practices in the united states bartholomew. . plasma total protein measurement for prediction of disease and mortality in calves composition and functional capacity of blood mononuclear leukocyte populations from neonatal calves on standard and intensified milk replacer diets nutrient requirements of dairy cattle. th rev plane of nutrition during the preweaning period but not the grower phase influences the neutrophil activity of holstein calves effect of nutritional plane on health and performance in dairy calves after experimental infection with cryptosporidium parvum a cohort study of the association between serum immunoglobulin g concentration and preweaning health, growth, and survival in holstein calves effects of additional milk replacer feeding on calf health, growth, and selected blood metabolites in calves behavioral biomarkers for calf health increased solids intake and feeding frequency for calves in hutches during cold weather effect of nutrient intake on the development of the somatotropic axis and its responsiveness to gh in holstein bull calves preweaning milk replacer intake and effects on long-term productivity of dairy calves evaluation of assays for failure of passive transfer in calves does nutritive and nonnutritive suckling reduce other oral behaviors and stimulate rest in calves? colostrum quality affects immune system establishment and intestinal development of neonatal calves the authors thank kansas state university research assistants for behavior data management and supply prepa- key: cord- -bt kaix authors: quigley, j. d.; martin, k. r.; bemis, d. a.; potgieter, l.n.d.; reinemeyer, c. r.; rohrbach, b. w.; dowlen, h. h.; lamar, k. c. title: effects of housing and colostrum feeding on serum immunoglobulins, growth, and fecal scores of jersey calves date: - - journal: journal of dairy science doi: . /jds.s - ( ) - sha: doc_id: cord_uid: bt kaix abstract ninety-six jersey calves were used to evaluate the effects of housing and method of colostrum feeding on serum ig concentrations, incidence and severity of scours, intake, and bw changes from birth to d of age. calves were separated from the dam and fed l of colostrum in nipple-bottles or allowed to nurse the dam for d. calves were housed in individual hutches or wooden pens in a barn. intake of colostrum by calves allowed to nurse the dam was not controlled. serum igg and igm concentrations at h of age were greater for calves that nursed the dam. scours were less severe when calves were housed in hutches, but number of days scouring was unaffected by treatment. calves fed colostrum in nipple-bottles and housed in the barn consumed more starter than did other calves from to wk of age. the bw were greater for calves allowed to nurse the dam and housed in hutches. feed efficiency over the -d study was improved when calves nursed the dam. optimal transfer of passive immunity and housing in hutches appeared to maximize health and growth in this study. a large proportion of dairy calves in the us develop diarrhea during the first mo of life ( ) . mean mortality of preweaned calves is . % in the us (se =. ); > % of fatalities are associated with scours ( ) . therefore, management of calves often involves attempts to minimize the spread of infectious organisms and initiation of treatment when scours develop. two factors that influence the incidence and severity of scours are colostrum feeding and housing. the importance of consumption of an adequate amount of colostrum on acquisition of passive immunity is widely recognized ( ) . however, less clear are effects of the method of feeding colostrum on serum ig concentration and subsequent health. failure of transfer of passive immunity is greater when calves are allowed to suckle ( , , ) , although efficiency of ig absorption is improved ( ) . calves left with the dam for h may be exposed to greater numbers of infectious organisms associated with the dam and environment, thereby increasing the risk of disease. housing also affects incidence and severity of scours ( , , ) . calves raised in hutches are less likely to be treated for scours than are calves raised in individual pens in a calf barn ( ) . however, other reports ( , ) indicate that overall calf management influences morbidity more than housing system. methods of housing and feeding colostrum may alter the exposure to infectious organisms, which may predispose calves to scours, thereby reducing intake and bw gain. therefore, our objectives were to determine the effects of housing and method of colostrum feeding on the incidence and severity of scours, intake, bw gain, and feed efficiency for neonatal jersey calves. jersey cows (n = ) were housed in a drylot until approximately d prior to parturition, when they were moved to an individual calving pen bedded with straw. at birth, calves were blocked by sex ( heifers and bull calves) and date of birth and assigned to one of four treatments in a randomized complete block design. calves were allowed to nurse the dam for d or were separated from the dam and fed l of colostrum in nipple-bottles at and h after birth and housed in individual fiberglass hutches or individual pens in an unheated calf barn. stillborn calves (n = ) and calf that died at d of age were replaced, and other calves that died during the study were not replaced; therefore, a total of calves completed the study. calves fed colostrum from nipple-bottles were moved to the hutch or barn before nursing. the unheated calf barn contained individual wooden pens ( . x . m) and housed calves prior to the initiation of the study. mechanical ventilation was provided in the barn throughout the study. calf hutches were located in an area not previously exposed to calves. pens and hutches were thoroughly washed, disinfected, and allowed to dry prior to use. hutches were moved to a new location after each use. calf pens and hutches were bedded with a minimum amount of sawdust and straw to facilitate estimation of fecal scores. journal of dairy science vol. , no. . calves fed by nipple-bottle were fed first milking colostrum from the dam. cows were milked by hand, and a -ml sample of colostrum was obtained and stored (_ o q prior to compositional analysis ( ) . calves were fed i l of colostrum as soon as possible after birth and again h later. following the second feeding, calves were fed l of colostrum twice daily to d of age using second and third milking colostrum from the dam. calves that nursed the dam were observed for the first h after birth and were assisted if nursing did not commence by h. thereafter, calves were left with the dam until d of age. no attempt was made to measure quantitatively the amount of colostrum consumed by nursed calves. commercial calf starter (tennessee farmers cooperative, lavergne, tn) was offered for ad libitum consumption from d . starter was formulated to contain a coccidiostat (deccox®; rhone-poulenc, atlanta, ga) at mg/kg. commercial milk replacer (land o'lakes, inc., ft. dodge, ia) was reconstituted to % dm and fed twice daily (. l per feeding) from to d of age. milk replacer was formulated to contain oxytetracycline ( mglkg) and neomycin base ( mglkg). refused milk replacer and calf starter were weighed and reported daily. water was available at all times. second-cutting alfalfa hay was offered for ad libitum consumption after d. although hay orts were not measured, few calves consumed significant quantities of hay. calves were weighed at birth and every d thereafter to d. milk replacer, calf starter, and hay were sampled monthly and analyzed for dm, cp, ash ( ), ndf ( ), and minerals. incidence and severity of scours were estimated at the a.m. feeding by the method of larson et a!. ( ) . when fecal score exceeded or when a calf exhibited other signs of disease (fever, cough, or congestion), antibiotic therapy was initiated. scours were also treated with electrolyte therapy (enterim- ™; shering-plough animal health, kenilworth, nj); milk replacer was not discontinued during electrolyte therapy. fecal grab samples were collected from all calves every tuesday and friday during the study to detennine the prevalence of rotavirus, coronavirus, k + escherichia coli, cryptosporidium, giardia, and eimeria ( ) . approximately ml of jugular blood were taken at h after birth and allowed to clot. serum was separated by centrifugation ( x g) and frozen (- "c) until analysis in duplicate for igg and igm by radial immunodiffusion (vmrd, inc., pullman, wa). weekly mean fecal scores, number of days of scouring per week, bw gain, feed efficiency, and days x fecal score were analyzed as a randomized complete block design in a repeated measures analysis of covariance using a general linear mixed models algorithm ( ) . tenns in the model were block, treatment, block x treatment, week of age, week x treatment, and error. block x treatment was used as the error tenn to test the effects of treatment, and error was used to test week and week x treatment. intake and bw data could not be measured by repeated measures analysis of covariance because of heterogeneity of error variance. therefore, data were analyzed by pooling of data across weeks and analysis as a randomized complete block design. weekly intake and bw data were analyzed by the method of allen et al. ( ) using cubic regression to obtain coefficients for each calf, which were subjected to multivariate analysis of covariance. serum ig concentrations at h of age were analyzed as a randomized complete block design. all data were covariately adjusted for bw at birth. data from calves that died during the experiment were removed prior to analysis. significance at p < . was used unless otherwise noted. mean mortality was . % for all calves during the experiment and . % for calves born alive. serum igg and igm concentrations at h were generally low for calves that died, although serum igg exceeded gil in calves. although the amount of protection provided by serum ig is dependent on environment and exposure to pathogens, serum igg i ( ) or igg ( ) concentrations < gil have been used to denote the failure of transfer of passive immunity. under the assumption that to % of igg is igg i • serum igg concentrations < to gil indicate failure of transfer of passive immunity. using this criterion, of calves ( %) that died failed to achieve acceptable transfer of passive immunity. serum igg and igm concentrations at h were greater when calves were allowed to nurse the dam (table ) . serum igg concentrations in this study were greater than those in reports on holstein calves ( , , ) . tennant et al. ( ) also reported that serum ig was higher for jersey than for holstein calves and attributed differences in serum ig to improved efficiency of ig absorption. absorption of ig improved when calves were allowed to nurse the dam ( ), although the biological basis for improved absorption has not been determined. calves that were allowed to suckle may have consumed more colostrum than bottle-fed calves and, therefore, had higher serum ig concentrations. however, others have indicated that calves allowed that were to nurse consumed smaller amounts of colostrum ( , ) , began consuming colostrum at a later age than calves fed colostrum by bottle ( ) , or failed to suckle altogether ( ) . brignole and stott ( ) reported that to % of calves left with the dam failed to obtain sufficient passive immunity. apparent efficiency of igg absorption in calves fed by nipple-bottle (calculated as serum igg concentration x bw x . /g of igg intake) was negatively correlated with colostral igg concentration (r =-. ; p < . ), which supports the hypothesis that ig absorption from colostrum is linearly related to colostral ig concentration ( ) . mean consumption of igg and igm for calves fed by nipple-bottle was . and . g over h, respectively, and ranged from . to . and . to . g, respectively. (table ) , indicating that environment affected the severity of scouring, regardless of method of colostrum feeding. total weekly fecal scores ( figure ) were affected by period and by an interaction of period x treatment (p < . ). single degree of freedom contrasts were used to determine the effects of treatment within week. weekly scores (days of scouring x fecal score) markedly increased for all treatments during wk and then declined to the end of the study. incidence of scours generally increases during the first to wk of life when rotavirus, coronavirus, and cryptosporidium are primary infectious organisms ( , , ) . fecal scores during the first wk of age were greater for calves fed colostrum by nipple-bottle (p < . ) than for those nursing the dam. however, fecal scores were greater at wk of age and lower at wk of age for calves fed colostrum by nipple-bottle and housed in the barn. consequently, severity and frequency of scours for these calves occurred at an earlier age than for calves on other treatments (p < . ). because the calf barn used in this study housed calves previously, calves placed in the barn may have standard error = . . been exposed to a greater number and variety of potentially pathogenic organisms. calves fed via nipple-bottle had lower serum ig concentrations (table ) and probably were less well protected against the greater infectious challenge, which increased fecal scores. fecal scores for those calves at wk may have been lower because calves were exposed to one or ore infectious agents during the first wk of age and had subsequently recovered. others ( , , , ) have recommended housing calves in individual hutches as an effective method to minimize contact among calves and, consequently, prevalence of disease. reduction of fecal scores by . units ( . %) supports this hypothesis, but reductions in morbidity were not as marked as those in other studies ( ) . although contact between calves is minimized, pathogens can be transferred to isolated calves by farm workers and other vectors. stepwise multiple regression was used to determine variables affecting the incidence and severity of scours in calves during the study. dependent variables were weekly incidence of scours ( = fecal score~ at least once per week; = no scours occurred during the week), severity of scours (mean of fecal scores during the week), numbers of days of scouring per week, and mean fecal score x number of days of scouring. independent variables included prevalence of rotavirus, coronavirus, eimeria, giardia, and cryptosporidium, week of age, serum igg and igm concentrations, colostral igg and igm concentrations, sex, housing (barn vs. hutch), method of colostrum feeding, interaction of housing x method of colostrum feeding, date of birth, mean daily bw gain, dmi, and intakes of starter and milk replacer. squared and logarithmic terms of quantitative independent variables were also included in the model. variables entered the model and were retained in the model if significance was p :::; . . results of all regressions indicated that the prevalence of cryptosporidium was the most significant factor influencing the incidence of scours, severity of scours, and the number of days of scouring per week in this herd ( table ) . prevalence of rotavirus also contributed to the incidence and severity of scours, but to a lesser extent than cryptosporidium. other organisms measured did not significantly contribute to incidence or severity of scours. the coefficient for mean daily bw gain was negative in all regressions, indicating that increasing incidence or severity of scours reduced bw gain. the highly negative coefficient for mean daily bw gain in regression of days x severity (table ) indicated that bw gain was markedly reduced when scouring was severe or lasted for an extended period. serum igg and igm concentrations and colostral igg concentration were significant in one or more regression equations, reemphasizing the importance of transfer of passive immunity to minimize the incidence and severity of scours. serum igm was related to incidence of scours, number of days of scouring, and interaction of days x severity; serum igg was related to incidence and severity of scours. milk replacer intake was negatively related (p < . ) to incidence and severity of scours because milk replacer was refused only when calves scoured severely. the regression coefficient for sex was significant for number of days of scouring, indicating that heifers scoured fewer days than bulls. mean number of days of scouring for bulls and heifers was . and . d/wk (se = . ), respectively. the cp in calf starter (table ) was higher than nrc recommendations ( ) for starters, and protein and ndf in hay were typical of medium quality forage. calves left with the dam were not offered milk replacer until d of age. from d to the end of the study, intake of milk replacer did not vary by treatment. milk replacer refusals were minimal and occurred only when the calf exhibited severe scours; consequently, differences in dmi were caused by differences in journal of dairy science vol. , no. , starter intake. intakes of calf starter, dm, and cp over the -d study were greater for calves fed colostrum in nipple-bottles (fable ). an interaction of colostrum feeding x housing tended toward significance (p < . ), indicating that calves fed colostrum in nipple-bottles and housed in the barn consumed more calf starter, dm, and cp than other calves. multivariate anova of starter intake regression coefficients (fable ) indicated a significant effect of housing on the change in starter intake during the study. calves housed in the barn, but particularly those fed colostrum in nipple-bottles, consumed more starter than other calves, primarily during the last wk of the study. also, during wk , starter intake did not increase as rapidly when calves were housed in the barn, as indicated by the more highly negative cubic regression coefficients (- vs. - ; p < . ). decreasing rate of increase in starter consumption during wk may have been due to greater prevalence of eimeria in feces of calves housed in the barn during wk and . although weekly fecal scores were not markedly increased by the prevalence of eimeria, damage to intestinal epithelium may influence intake and digestibility ( ) . mean daily intake of decoquinate was . , . , . , . , and . mglkg of bw from wk to , respectively, which was lower than the amount (. mglkg of bw) required to minimize incidence of coccidiosis in dairy calves, although smaller amounts may partially suppress fecal oocyst discharge and diarrhea ( ) . calf starter dmi averaged g at d, which was slightly lower than those amounts recommended for weaning ( ) . calf bw at d and bw gain from to d were affected by an interaction of colostrum feeding x housing. calves that were allowed to nurse the dam and housed in hutches were heavier at the end of the study than other calves (table ). regression coefficients of bw over time (table ) also tended (p < . ) to be affected by colostrum feeding, indicating that growth patterns differed by treatment; most differences in bw occurred during the last wk of the study. mean bw change was - and gld during the first wk, respectively. prevalence of cryptosporidium was particularly high during the first wk of life, which probably contributed to increased scours and bw changes. thereafter, bw gain increased with age, but most rapidly in calves allowed to nurse the dam and housed in hutches. prevalence of cryptosporidium and eimeria oocysts was lower, and prevalence of rotavirus tended to be lower, in feces of calves housed in hutches, which may have contributed to differences in bw gain. the combination of improved overall health of calves nursing the dam and reduced exposure to cryptosporidium, eimeria, and rotavirus by housing in hutches may have contributed to increased rate of bw gain, particularly in the last wk of the study. stepwise multiple regression indicated that weekly bw gain increased as age and cp intake increased. the number of days of scouring and the prevalence of rotavirus and cryptosporidium reduced bw gain, suggesting that management related to minimizing prevalence of these organisms would have been most effective in improving the growth of the calves used in this study. efficiency of feed and cp use pooled across all weeks of the study were improved when calves were allowed to nurse the dam (table ). improved efficiency of feed use was not due to the lower intake of starter because starter intake was not significant when included as a covariable in the analysis of dm or cp efficiency. heinrichs et al. ( ) proposed that calf housing and management interact to influence health and growth of calves. our data support this hypothesis, particularly in relation to the method of colostrum feeding and the acquisition of passive immunity. in our study, assisted nursing (ensuring that all calves nursed the contrasts: =effect of colostrum feeding, =effect of housing, and =interaction of colostrum feeding x housing. p> . . does not include hay intake, which was not measured. tp < . . *p < . . "p < . . dam by h of age) promoted increased serum igg and igm concentrations, decreased prevalence and severity of scours, number of days of scouring, and improved feed efficiency. although allowing calves to nurse the dam can increase the potential for failure of transfer of passive immunity, assisting calves to nurse the dam may alleviate some problems associated with failure to suckle or delayed suckling. however, the amount of time and management required to assist calves to suckle may preclude widespread use of this practice. a recent survey of dairy producers in the us indicated that . % of producers assisted calves in nursing the dam compared with % who fed first colostrum via bucket or botde, . % that allowed unassisted nursing, and . % that fed colostrum via esophageal feeder ( ). methods of housing and colostrum feeding affected the incidence and severity of scours, growth, and intake of young calves in this study. allowing the calf to nurse the dam increased serum igg and igm concentrations at h of age. most scours occurred during the first wk of age and were a result of the prevalence of cryptosporidium and rotavirus. housing in the barn increased fecal scores, journal of dairy science vol. , no. , probably as a result of increased exposure to eimeria and rotavirus. isolation of calves by housing in hutches and maximizing transfer of passive immunity by allowing calves to nurse the dam increased bw gain and improved health of calves in this study. analysis of repeated measurements from animal experiments using polynomial regression association of official analytical chemists effect of colostral immunoglobulin g and immunoglobulin m concentrations on immunoglobulin absorption in calves comparison of three methods of feeding colostrum to dairy calves general linear mixed models (glmm) user's manual effect of suckling followed by bottle feeding on immunoglobulin absorption and calf survival dairy herd management practices focusing on preweaned heifers outdoor individual portable pens compared with conventional housing for raising dairy calves intestinal coccidiosis in cattle. the veterinary clinics of north america. parasites: epidemiology and control failure of passive transfer of colostral immunoglobulins and neonatal disease in calves: a review. page in epidemiologic factors and isotypespecific antibody responses in serum and mucosal secretions of dairy calves with bovine coronavirus respiratory and enteric tract infections survey of calf and heifer housing on pennsylvania dairy farms management of dairy heifers calf mortality in virginia dairy herd improvement herds indoor versus outdoor calf rearing at three weaning ages guidelines toward more uniformity in measuring and reporting calf experimental data prevalence of bovine group a rotavirus shedding among dairy calves in ohio dairy calf mortality rate: influence of management and housing factors on calf mortality rate in tulare county. california decoquinate in the control of experimentally induced coccidiosis of calves nutrient requirements of dairy cattle. th rev prevalence of cryplosporidium infection in dairy calves in western washington effects of housing and colostrum feeding on the prevalence of selected infectious organisms in feces of jersey calves immunoglobulin concentration. specific gravity. and nitrogen fractions of colostrum from jersey cattle studies on natural suckling in cattle during the first eight hours post partum. ii. behavioral studies (calves) colostral immunoglobulin absorption linearly related to concentration for calves colostrum immunoglobulin transfer in calves. iv. effect of suckling neonatal alterations in serum gamma globulin levels of jersey and holstein-friesian calves dairy calf management. morbidity and mortality in ontario holstein herds. iii. association of management with morbidity the authors thank l. wallis, d. johnson, and the farm crew at the dairy experiment station in lewisburg, tennessee for their capable assistance in care, feeding, and sampling calves, the american jersey cattle club for partial financial support, and a. m. saxton for statistical advice. key: cord- -gvlkc r authors: foster, d.m.; smith, geof w. title: pathophysiology of diarrhea in calves date: - - journal: vet clin north am food anim pract doi: . /j.cvfa. . . sha: doc_id: cord_uid: gvlkc r infectious diarrhea in calves is most commonly associated with enterotoxigenic escherichia coli, cryptosporidium parvum, rotavirus, coronavirus, or some combination of these pathogens. each of these agents leads to diarrhea through either secretion or malabsorption/maldigestion, though the specific mechanisms and pathways may differ. specific pharmacologic control and treatment are dependent on gaining a greater understanding of the pathophysiology of these organisms. etec diarrhea in calves is k , which is more appropriately referred to as f . , the f and p antigens can also be found in calf etec isolates, often in conjunction with f . , because the k antigen is only expressed at an environmental ph level of less than . , the distal small intestine is the initial site of colonization. this is because the ph level of the intestinal fluid increases as it moves caudally, and it only reaches this threshold at the ileum. [ ] [ ] [ ] [ ] [ ] the ability of k etec to bind to the small intestinal epithelium is age dependent and gradually decreases from hours of age to weeks of age. however, there is not a precipitous drop in the binding ability that would explain the age resistance to etec. the attachment of etec allows the bacteria to colonize the ileum, proliferate, and spread proximally through the small intestine. , , , once established in the gut, etec produces heat stable toxin leading to secretory diarrhea. classically, mechanistic discussions of enterotoxin-mediated secretory diarrhea have focused on the cholera toxin of vibrio cholerae and the heat labile toxin (lt) of e coli. these are both significant causes of diarrhea in humans, and have a similar mechanism of action involving increases in intracellular cyclic adenosine monophospate (camp), which activates the cystic fibrosis transmembrane conductance regulator (cftr) and ultimately causes secretion of chloride. this movement of chloride ions osmotically draws water into the lumen of the intestine, leading to diarrhea. , these models of human diarrhea have less bearing on toxin-mediated secretory diarrhea in the calf because the heat stabile toxin (sta) of etec is the primary mediator. , , sta is an -or -amino-acid peptide that is secreted by many strains of etec; however, the production can vary up to , fold between strains when cultured under identical conditions. , after being secreted by e coli, sta binds to guanylyl cyclase-c (gcc), a brush border membrane enzyme that is present throughout the villi and crypts. , the concentration of gcc appears to be highest in the lower villous, but this may vary by species, and its precise location on the villous has not yet been determined in the calf. in contrast to rodents and humans, in which concentrations of gcc decrease in the distal small intestine, gcc is present throughout the gastrointestinal tract of calves and is concentrated in the ileum. , in both mice and humans, the density of this receptor declines after birth, , and it remains present in pigs until up to weeks of age. no specific research has been done detailing the expression of gcc at various ages of calves, however inoculation with sta induces diarrhea in animals up to days of age. this indicates that gcc is present until at least weeks of age and down-regulation of the receptor is not the reason for age-dependent resistance to etec diarrhea. binding of sta to gcc leads to the production of intracellular cyclic guanylyl monophospate (cgmp), which acts as a second messenger to activate cgmp-dependent protein kinase ii (cgkii). this kinase phosphorylates cftr, inducing movement of the protein to the cell surface and activation, which in turn leads to chloride secretion. this up-regulation of chloride secretion osmotically pulls water into the intestinal lumen, which outweighs the absorptive ability of the villous (fig. ) . blocking the cftr dramatically decreases intestinal fluid secretion, indicating the importance of this protein in the pathogenesis of etec diarrhea. however, secretion is not completely prevented, indicating that sta may have additional effects in the small intestine. further research has shown that sta can induce bicarbonate secretion through a tyrosine kinase that is independent of the gcc/cgmp/cftr pathway, and this secreted bicarbonate can act as an osmotic agent to pull water into the lumen of the intestine. , the receptor and other messengers in this pathway have not been elucidated. an opposing model of sta-induced diarrhea has also been proposed that is not based on fluid secretion caused by movement of chloride or bicarbonate, but which instead is caused by decreased fluid absorption. in addition to activating the cftr, sta inhibits the apical na-h exchanger, leading to failure of sodium absorption. this failure of sodium absorption decreases fluid movement from the intestinal lumen to the interstitial space. the importance of this mechanism of etec diarrhea in calves is unknown because this has been most conclusively shown in the duodenum and proximal jejunum of rodent models. , similar to expression of k , production of sta is ph dependent. when the environmental ph is less than . , toxin production is severely limited. , therefore, toxin production is maximized in the distal small intestine because the ph level is greatest in this segment. although it has not been directly investigated, it can be theorized that sta-mediated secretion of bicarbonate and inhibition of the na-h exchanger would have the net effect of alkalinizing the proximal small intestine. this would create an environment more hospitable to etec promoting its spread to the proximal small intestine. the autonomic and enteric nervous systems are known to be involved in the secretory response to cholera toxin through the actions of prostaglandin e (pge ), -hydroxytriptamine ( -ht), and vasoactive intestinal polypeptide (vip). [ ] [ ] [ ] sta-mediated secretion may also involve local reflex arcs in the enteric nervous system (ens); however, it does not involve the autonomic nervous system. most of the support for this idea comes from studies that inhibit the ens and subsequently reduce the secretory effect of sta. [ ] [ ] [ ] the neurotransmitters critical in these responses are nitric oxide (no) and vip, , whereas pge and -ht are not involved. furthermore, a well-defined example of the influence of the ens in etec is its role in exacerbating k etec binds to an intestinal epithelial cell, and heat stable toxin (sta) is secreted, which binds to the receptor gcc. the enteric nervous system becomes activated by the secretion of sta, but the mechanism of this activation is unclear. at this point, cftr is not active. frame : sta binds to gcc, which converts guanylyl triphosphate (gtp) to cgmp. cgmp activates cgkii to phosphorylate the cftr, and the cftr moves to the luminal surface and is activated, leading to chloride (cl) secretion. frame : secreted sta activates tyrosine kinase through an unknown pathway, which leads to bicarbonate (hco ) secretion. sta also directly inhibits the sodium-hydrogen exchanger, decreasing the movement of sodium (na) and hydrogen (h) across the membrane. sta-mediated secretory diarrhea in states of malnutrition, [ ] [ ] [ ] however the importance of these mechanisms in the calf is unknown because this was found in a rodent model of human disease. ultimately, the pathophysiology of etec is dependent on several factors. first is the exposure to and ingestion of the organism. once ingested, etec must survive the acidic ph of the abomasum. this is facilitated in neonatal calves because the ph level of their abomasum ranges from approximately to , which enables survival of etec. the ph of the abomasum decreases to less than by days of age, which is low enough to kill etec strains. , once etec reaches the ileum, both the k antigen and sta are expressed as a result of the increased ph level, yet this may occur sooner because the ph level can be higher in the proximal gi tract of neonatal calves. k allows attachment of the organism, leading to colonization of the ileum. production of sta induces secretion and may increase the luminal ph level, which would make the normally acidic proximal small intestine more hospitable for the organism. etec bacteria then move proximally toward the duodenum, and secretion dramatically increases, leading to diarrhea and dehydration. , treatment of escherichia coli diarrhea the focus of treatment for etec diarrhea should be to remove the organism from the gastrointestinal tract and combat dehydration until normal absorption is restored. based on an extensive review on the topic by constable in , the only antibiotic with documented efficacy and legal use in food animals in the united states is amoxicillin trihydrate, which is recommended at a dose of mg/kg, orally every hours. ideally, this would only be used in calves with signs of systemic illness caused by diarrhea. oral electrolyte solutions remain the mainstay of on-farm fluid replacement therapy for most calves with etec diarrhea. based on the pathophysiology of the organism, two characteristics of oral replacement fluids are critical. the first is to maximize sodium absorption through means other than the na-h exchanger, because this may be inhibited by sta. most oral electrolyte solutions take advantage of the sodium-glucose cotransporters to improve sodium absorption, which bypasses the inhibited na-h exchanger. although this will not reduce the secretory response (and diarrhea), it will improve the hydration status of the calf. second, increasing the ph of the abomasum and proximal small intestine favors the survival of etec, as discussed above. hence, oral replacement fluids with bicarbonate as the alkalinizing agent may favor the proliferation of etec, expression of the k antigen, and secretion of sta. if secretion of bicarbonate caused by sta is a significant component of the disease in calves, as it appears to be in some models, the additional bicarbonate load from an oral electrolyte solution could even exacerbate the secretory response. because of the potential harm of bicarbonate, oral electrolyte solutions containing acetate are recommended for treatment of etec diarrhea. additional approaches for increasing the abomasal ph are discussed in the article by marshall found elsewhere in this issue. c parvum is one of the most commonly isolated gastrointestinal pathogens from dairy calves and immunosuppressed humans and is a significant cause of waterborne diarrhea outbreaks. infection occurs when oocysts are ingested from the environment. once in the host, the organism goes through a complicated life cycle that involves multiple stages. the cycle starts with exposure to gastric acid and bile salts, leading to excystation of the oocyst to the first life stage, the sporozoite. the sporozoites invade the intestinal epithelial cells of the ileum, where the infection is typically concentrated, but they can infect the gastrointestinal tract anywhere from the abomasum to the colon. the sprorozoites create an invagination of the luminal membrane, allowing them to maintain an extracytoplasmic but intracellular location known as a parasitophorous vacuole. from this location, the sporozoites transform into trophozoites. at this stage, asexual reproduction occurs and type i meronts are formed. merozoites are then released into the lumen. these organisms can form additional type i meronts or type ii meronts, which form micro-and macrogamonts. micro-and macrogamonts reproduce sexually to create thin-and thick-walled oocysts. the thin-walled oocysts lead to autoinfection, whereas the thick-walled oocysts pass out with feces to contaminate the environment. these oocysts are infective immediately, and remain viable in the environment for extended periods of time. [ ] [ ] [ ] c parvum oocyst shedding occurs as early as days of age, peaks at weeks of age, and can continue to occur in adult cattle. however, diarrhea caused by c parvum rarely occurs after months of age. [ ] [ ] [ ] [ ] [ ] [ ] [ ] after infection, clinical signs peak at to days and last to days. , some studies have shown that up to % of dairy calves become infected with c parvum, , and become the major source of environmental contamination because calves shed up to oocysts per gram of feces. shedding in beef calves is much less frequent and occurs in less than % of calves. , calves appear to be resistant to subsequent infection after the initial episode of c parvum diarrhea. severity of diarrhea and incidence of clinical signs in calves shedding oocysts can be variable within and between farms, leading some to question the true importance of c parvum as a primary pathogen; however, it has been repeatedly isolated independent of other known pathogens in clinical cases. infection with c parvum has been shown to induce severe villous atrophy ( fig. ) in calves and other food animal species. , , this atrophy is caused by the loss of villous enterocytes and the subsequent retraction of the villous to maintain a continuous epithelial barrier. crypt hyperplasia also occurs in an effort to replace the lost epithelial cells, however in severe infections, disruption of the epithelial barrier can occur despite these efforts. , , furthermore, both cell culture and animal models have shown an increase in epithelial permeability after c parvum infection when the loss of epithelial surface area is taken into account. , in spite of this well recognized consequence of c parvum infection, the precise mechanism of cell loss remains elusive. it is still not understood whether the cell loss is an effect of the pathogen or is part of the host response in an effort to resolve the infection. there are two potential mechanisms for the increased loss of epithelial cells in c parvum infections. the first is a direct cytotoxic effect of the organism on the intestinal epithelium, but this is not well supported by the current literature. in a few cell culture models of c parvum infection, the cytosolic enzyme, lactate dehydrogenase, has been shown to leak into the cell media. [ ] [ ] [ ] however, this may simply be caused by the deformation of the apical membrane by the organism as it attaches to and is enveloped by the membrane. the second and more likely mechanism for cell loss is apoptosis because apoptotic cells are consistently found in both in vitro and in vivo models of infection. [ ] [ ] [ ] [ ] [ ] [ ] [ ] yet there is evidence in cell culture models that the loss of epithelial cells is minimized by the inhibition of apoptosis during the infection, , and many infected cells are not apoptotic. specifically, research has shown that the activation and inhibition of apoptosis is calf diarrhea related to the life stage of c parvum, and that apoptosis is inhibited during the trophozoite stage when the organism is most dependent on the host, but then increases later during the infection. furthermore, the incidence of apoptosis will vary over time between infected cells and uninfected neighboring cells. this may be beneficial to the host to limit spread of the organism, limit the severity of cell loss, and/or speed clearance of the organism. pharmacologically induced apoptosis in infected cell cultures is also prevented, indicating that apoptosis mechanisms are actively inhibited, , which has been shown to be mediated by nf-kb. additional research is needed to elucidate the ultimate beneficiary of this apoptotic regulation: the organism, to maintain its intracellular habitat, or the host, to limit cell loss and spread of infection. irrespective of how or why epithelial cell loss and villous atrophy occurs, this leads to a malabsorptive diarrhea. the net absorption of fluid is caused by the movement of sodium coupled with either chloride or other nutrients in the villous tip versus the secretion of anions in the crypts. therefore, absorption is impaired because of the loss of the mature villous epithelial cells and their associated transporters as well as a decrease in total surface area. , , , absorption of sodium and water can still occur to some degree in the crypts when coupled with glucose or neutral amino acids (eg, glutamine), which can be used to improve absorption of oral rehydration solutions, , but overall absorption of carbohydrates, lipids, and amino acids is reduced. [ ] [ ] [ ] [ ] this malabsoption leads to diarrhea that can range from very mild to life threatening, depending on the dose of organism and coinfection with other pathogens. however c parvum has not been shown to decrease overall growth in calves once the infection has resolved. epithelial cell loss, villous atrophy, and malabsorption cannot account for all the fluid loss seen in c parvum infections, and studies have documented a prostaglandin-mediated anion secretion (cl À or hco À ) and inhibition of neutral nacl absorption (fig. ) . the prostaglandins pge and prostaglandin i (pgi ) are found at higher concentrations in infected tissue, and blocking the secretion of these prostaglandins reverses the anion secretion and inhibition of nacl absorption. , however, in vivo, inhibition of prostaglandins exacerbated the villous atrophy, indicating that this approach is unlikely to be useful therapeutically. the source of prostaglandins in the infected tissue is unknown, but is believed to be leukocytes that infiltrate the lamina propria in the infection. macrophages appear to be the most likely source because they invade the lamina propria after infection and can induce prostaglandin secretion from mesenchymal cells, , , , whereas inhibition of neutrophil migration into infected tissue had no effect on prostaglandin synthesis. furthermore, no, which has been shown to be important in defense against c parvum infections, stimulates prostaglandin-mediated secretion when no production is augmented by arginine supplementation. fig. . infection of intestinal epithelial cells with c parvum induces the epithelial cell to secrete pge and leads to activation of macrophages (m ) in the lamina propria. this leads to secretion of pge and pgi from the mesenchymal cells. pgi activates the enteric nervous system to secrete acetylcholine (ach) and vip. the secretion of ach, vip, and pge leads to an increase in intracellular calcium and camp, which activates anion secretion (cl and hco ) and inhibits neutral sodium and chloride absorption (nacl). the mechanism of action of the two prostaglandins differs because pge acts on the enterocyte directly, whereas pgi exerts its effect through the ens. pgi causes % of the secretion in c parvum infection by stimulating the nicotinic ganglia and the vip-ergic and cholinergic motor neurons that innervate the intestinal mucosa. prostaglandin secretion ultimately leads to increases in calcium and camp that increase anion secretion and decrease sodium absorption. , , inhibiting the effects of prostaglandins on the ens is a potential method of decreasing the diarrhea associated with c parvum without exacerbating the villous atrophy. specifically, peptide yy, which is naturally found in the intestinal epithelium, is a potent inhibitor of vip and can abolish the secretory response to pgi . furthermore, if the inhibition caused by prostaglandins can be blocked, the intestine is capable of absorbing nacl and water despite the villous atrophy, indicating that the transporters are fully functional, even in the immature enterocytes. [ ] [ ] [ ] treatment and prevention of cryptospordium parvum diarrhea because of the privileged location of c parvum, drug delivery can be difficult. drugs in the lumen of the intestine may pass through without actually reaching the organism, whereas drugs that penetrate the intestinal epithelial cell would concentrate in the cytoplasm instead of the extracytoplasmic parasitophorous vacuole. despite these challenges, multiple drugs have been studied as potential treatments with varying degrees of success, and none are currently licensed for calves in north america. , halofuginone is licensed for prevention of c parvum infection in europe when administered during the first days of life. unfortunately, clinical trials have not shown it be consistently effective. when used as directed in a study of bull calves, there was no difference in the incidence of or treatment rates for diarrhea between the treated and control calves. there was a significant delay in shedding of oocysts, but upon withdrawal of the drug, the treated calves began to shed a similar number of organisms. there was no difference in milk intake, weight gain, or age at weaning between the two groups. another study in which halofuginone treatment was initiated at to days of age saw no difference in the number of calves shedding oocysts or in the incidence of diarrhea. however, the total number of oocysts shed was reduced for the days during treatment as well as the following days. oocyst shedding then rebounded and was greater than in control animals days after the start of treatment. other studies have shown more favorable results in which the incidence of diarrhea and excretion of oocysts was reduced. re-excretion of oocysts after stopping treatment continued to be a problem, but was less if a lower dose was used. [ ] [ ] [ ] in an experimental infection, the only difference noted with treatment was a decrease in the number of calves dying. however, calves began excreting oocysts after treatment was discontinued, even though they had been housed individually and reinfection was unlikely. this indicates that halofuginone is cryptosporidiostatic but is unlikely to effectively kill oocysts. halofuginone appears to be effective at decreasing oocyst shedding only when it is being administered. it may or may not reduce clinical signs in the calf, and has not been consistently shown to be effective as a treatment for c parvum diarrhea. paromomycin was shown to be effective in one trial of experimental infection as a prophylaxis for c parvum infection. the drug was administered day prior to infection and continued for a total of days. oocyst shedding and diarrhea were decreased, but calves began to shed organisms at the end of the treatment period, and the shedding continued after treatment was stopped. decoquinate has also been used to control c parvum in calves, but trials have not consistently shown it to reduce diarrhea or oocyst shedding in calves. , , in a study from turkey, azithromycin was shown to be effective as a treatment of calves that were know to be shedding c parvum when it was administered at a dose of , mg/calf/day for days. treatment decreased oocyst shedding and diarrhea, and improved weight gain. however, the cost of azithromycin in the united states would likely prevent its use to treat cryptosporidiosis at this time. in a small study, activated charcoal with wood vinegar liquid was effective in stopping diarrhea and oocyst shedding when administered after the start of experimentally induced c parvum diarrhea. this effect was noted hours after addition of this product to the milk replacer. it remains to be seen if this effect can be duplicated in a large-scale field trial. similar to chemotherapeutic agents, both active and passive vaccination have not been consistently successful in preventing c parvum infection, diarrhea, and oocyst shedding. vaccination of dry cows with whole organisms or a recombinant protein both reduced oocyst shedding and clinical signs, but neither have been validated under field conditions. an oral vaccine to be given to calves at birth prior to colostrum administration showed promise initially, but was ineffective in a field trial. , because of the questionable benefit of mass medication or vaccination for control, prevention should be focused on decreasing exposure to the organism by appropriate hygiene and husbandry. because c parvum is a zoonotic agent, appropriate personal hygiene is also important for public health and farm employee safety. specific treatment for c parvum-infected calves also cannot be recommended in the united states at this time, although the extralabel use of azithromycin or activated charcoal with wood vinegar appears promising. in general, treatment should be focused on appropriate fluid therapy and supportive care because most calves will recover from cryptosporidiosis if there is not an overwhelming infection or coinfection with another pathogen. rotavirus was one of the first identified viral causes of diarrhea, and was initially known as neonatal calf diarrhea virus. subsequently, it has been found throughout the world and has been identified as a significant pathogen of children and most other mammals. , antibodies to rotavirus can be found in more than % of unvaccinated cattle, and the virus was isolated from % of dairy calves at a large dairy and calf ranch during the first weeks of life. it has also been isolated from approximately % of calf diarrhea samples, , and from at least one calf on % of farms. calves become infected after ingesting the virus from fecal contamination of the environment, because the virus remains quite stable if the temperature does not get near freezing. the virus typically affects calves less than weeks old, with a peak incidence at days of age. after ingestion of the virus, the incubation period is approximately hours, with resolution of diarrhea in uncomplicated cases in days. classically, rotavirus diarrhea is thought to be primarily a malabsorptive diarrhea, but recent evidence indicates that there is also a toxin-mediated secretory component as well. similar to c parvum, rotavirus preferentially targets the mature villous enterocytes and spares the crypts, generally causing moderate villous damage. the virus attaches to these cells via specific receptors and invades through an unknown mechanism. the virus replicates within the cells, leading to enterocyte death. malabsorption will then occur because of the loss of surface area, and unabsorbed glucose and other carbohydrates create an osmotic load pulling fluid into the lumen. furthermore, fluid secretion from the crypts increases the amount of fluid in the intestinal lumen relative to villous absorption, which leads to diarrhea. , , however, the severity of clinical signs does not always correlate with histologic damage to the villi. this has led to speculation that there may be another mechanism contributing to the diarrhea seen with rotaviral infections, and that enterocyte damage is less critical than previously believed. in the mid- s, a viral enterotoxin was demonstrated to be crucial to the pathogenesis of rotaviral diarrhea. this was the first time an enterotoxin could be identified in a viral diarrheal pathogen, and this changed our fundamental understanding of rotavirus diarrhea. the rotavirus protein, nonstructural glycoprotein (nsp ), was found to induce a dose-and age-dependent diarrhea that is clinically similar to rotavirus diarrhea. unlike the bacterial enterotoxins, diarrhea due to nsp is unrelated to camp, cgmp, or cftr. , the protein is initially produced during intracellular viral replication and acts on the infected cell. it is secreted or released upon cell death, and acts in a paracrine manner. exogenous exposure of intestinal epithelial cells to nsp allows binding to caveolae, special lipid rafts within the endoplasmic reticulum (er) and cell membrane. it specifically binds to caveolin- , a transmembrane, hairpin protein unique to these rafts. , binding to caveolin- leads to an increase in intracellular calcium concentrations by causing the release of calcium from er stores and increasing movement across the plasma membrane. this is mediated by phospholipase c (plc), which increases the level of intracellular inositol , , -triphosphate (ip ), , - however intracellular nsp causes the release of calcium independent of the plc pathway. extracellular and intracellular exposure to nsp causes several changes in the movement of nutrients and water across the epithelium (fig. ) . increases in intracellular calcium inhibit the translocation of disaccharidases from the intracellular vesicles to the luminal surface, decreasing the ability to digest carbohydrates and leading to maldigestion and exacerbation of the diarrhea. - nsp also directly inhibits sodium glucose cotransporter sglt , the primary sodium and glucose cotransporter that is critical for effective water absorption, significantly contributing to the pathogenesis of rotaviral diarrhea. the actions of nsp better account for the maldigestion and malabsorption that are seen in rotavirus diarrhea, and is are likely more important to the pathogenesis than is histologic damage to the epithelium. nsp has also been implicated in causing secretion of chloride through the increase in intracellular calcium, but the importance of this finding is being increasingly questioned because the increase is relatively mild and only occurs early in the course of diarrhea. , as previously mentioned, the actions of nsp are independent of cftr, so the ion channel that is important for this chloride movement is unknown, and has been hypothesized to be created by nsp . , an alternate mechanism that may explain the chloride secretion occurring in rotaviral diarrhea is activation of the ens. pharmacologically inhibiting the ens dramatically reduces the diarrhea seen with rotavirus infections, although the mechanism by which the virus activates ens-dependent secretion is unknown. , , prostaglandins and other inflammatory mediators may also play a role in causing secretion by affecting the ens, similar to other intestinal pathogens such as c parvum. , , the ens appears to play a critical role in rotavirus-induced secretion, but the mechanism responsible for this effect is unclear. treatment of rotavirus diarrhea is focused solely on rehydration because there are no currently available pharmacologic methods of controlling the infection in calves. whereas the inhibition of sglt and subsequent decrease in sodium and glucose absorption mediated by nsp would theoretically make sodium-and glucose-containing oral rehydration solutions less effective, this has not been shown clinically or experimentally. currently, the centers for disease control and prevention recommend a low-osmolality rehydration solution that contains sodium and glucose for children with acute gastroenteritis. this recommendation is irrespective of the cause, yet rotavirus is likely involved in a majority of the cases. similar fluids would be expected to be effective in rehydrating calves with rotaviral diarrhea as well. enhancing colostral antibody transfer to the calf from the dam appears to be the most effective method of control for rotaviral diarrhea in calves. first, proper once rotavirus replicates in an intestinal epithelial cell, the enterotoxin nsp is produced. it has autocrine effects by causing calcium (ca) release from the endoplasmic reticulum. nsp has paracrine effects by being secreted and binding to caveolin- . this activates plc, which increases cytoplasmic ip . ip increases intracellular calcium by increasing release from the endoplasmic reticulum and increasing calcium movement across the luminal membrane. the increased intracellular calcium inhibits movement of disaccharidases to the luminal surface. nsp directly inhibits the sglt which decreases the absorption of sodium (na) and glucose, and increases chloride (cl) secretion by an unknown mechanism, but may involve a channel created by nsp . nsp also activates the enteric nervous system by an unknown mechanism. colostrum management is critical to ensure that an adequate igg mass is delivered to each calf. this can be enhanced by administering vaccines to cows in the dry period to increase the amount of rotavirus-specific antibodies in the colostrum. experimental evidence has shown these vaccines to be effective at decreasing clinical signs; [ ] [ ] [ ] [ ] however, some authors feel that the commercial vaccines are not as effective clinically. there appears to be protection initially that is caused by the presence of specific antibodies in the intestinal lumen, but immunity in subsequent weeks is dependent on the resecretion of igg into the intestine. oral vaccination of the calves at birth has been found to be less successful, and is not recommended. similar to other gastrointestinal pathogens, reducing exposure is also critical for control of rotaviral diarrhea in calves. therefore, appropriate housing, stocking density, and hygiene cannot be ignored. the epidemiology and pathophysiology of coronavirus diarrhea in calves overlaps significantly with that caused by rotavirus. antibodies to coronavirus are ubiquitous in cattle, and the virus is frequently found in both normal and diarrheic feces of calves. coronavirus typically affects calves with the first weeks of life, and peak incidence occurs between days and . the virus is ingested from the environment, which is contaminated by other calves or adult cattle. , clinical signs begin approximately days later and continue for to days. , diarrhea secondary to coronavirus is mainly caused by intestinal epithelial cell loss and malabsorption. this virus has also been implicated in respiratory disease outbreaks in older calves as well as a diarrheal disease of adult cattle (winter dysentery), but discussion of these syndromes is beyond the scope of this report. coronavirus infection begins in the proximal small intestine, but then usually spreads throughout the jejunum, ileum, and colon. initially, the virus attaches to the enterocyte via the spike and hemagglutinin glycoproteins, which also allow fusion of the viral envelope with the cell membrane or endocytotic vesicles. , [ ] [ ] [ ] once in the cell, the virus replicates and is released using normal secretory mechanisms and upon cell death. , diarrhea begins at the time of virus entry into the cell (before cell death occurs), but it is unknown if this is the result of secretion, malabsorption, or both. infected cell loss is significant by days after onset of diarrhea, and villous blunting occurs. the mature villous epithelial cells are the primary target for the virus, but crypt enterocytes are also affected. , as in rotavirus and c parvum infections, maldigestion and malabsorption lead to diarrhea. because the crypt enterocytes and the colonocytes can be affected by coronavirus, the clinical signs often have a longer duration. , treatment and control of coronavirus diarrhea like rotavirus diarrhea, there are no specific treatment methods for coronavirus infections, and little research has been done to confirm specific control measures for coronavirus in calves. oral electrolyte solutions should be provided to prevent dehydration and treat acidosis. methods aimed at controlling rotavirus infections (proper housing and hygiene, good colostrum management, and dry cow vaccination) are believed to be the best measures for control of coronavirus as well. furthermore, salmonella is the other major infectious cause of diarrhea in calves, but it is discussed in a separate article in this issue. a few other minor causes of diarrhea in calves are worth brief discussion. in addition to etec, there are several other types of e coli that are potential pathogens in calves, and these fall into the broad category of attaching and effacing e coli. these bacteria are characterized by the presence of the eae gene, which encodes the protein intimin, a key component of the outer membrane that mediates attachment to the intestinal epithelium. if these bacteria do not secrete any enterotoxins, they are classified as enterpathogenic e coli (epec). epec organisms attach to the epithelium, disrupt the microvilli, and cause malabsorption. they also use a type iii secretory protein to inject effector proteins into the host cell, inducing a secretory response by an undefined mechanism. furthermore, they disrupt tight junctions between epithelial cells and lead to inflammation; all of which contribute to diarrhea. the importance of epec as a pathogen of calves is debatable. it can be found in abnormal fecal samples, but is also frequently found in healthy calves or not found at all. enterohemorrhagic e coli (ehec) are typically defined as expressing the eae gene and shiga toxin. strains that lack eae but secrete shiga toxin are designated stec. shiga toxin mediates many of the systemic effects that are seen in humans with e coli o :h infections, and may cause some intestinal damage in some species. many epidemiological studies have shown that ehec and stec are commonly found in calves with normal and abnormal feces, and there is significant interest in these bacteria from the public health standpoint. , , [ ] [ ] [ ] [ ] however, shiga toxin receptors cannot be found in the intestine of calves or adult cattle, and no diarrhea was seen following experimental infection, calling into question claims that these bacteria are pathogens of calves. diarrhea caused by clostridium difficile appears to be an emerging problem in both humans and veterinary patients. diarrhea caused by c difficile is mediated by bacterial toxins that lead to epithelial cell death, damage to epithelial cell tight junctions, inflammation of the mucosa and submucosa, and activation of the ens. c difficile and its toxins can be found in the feces of both normal and diarrheic calves, - but its role as a pathogen has not been clearly established. purified toxins will cause epithelial damage and an increase in luminal fluid in a calf intestinal loop model, however experimental infection has not been successful. giardia giardia organisms can be found in the feces of calves with diarrhea throughout the world, but is also commonly found in the feces of normal calves. , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] some of these studies also found other pathogens along with giardia, and none were controlled experiments. only a single study has documented attempted experimental infection of calves with giardia. in that study, histologic changes were found in only of calves, clinical signs were simply described as not severe, and calf diarrhea the incidence of diarrhea was not reported. giardia has been documented to cause villous atrophy in naturally infected calves, and is known to cause a malabsorptive diarrhea in other species. , therefore, some have proposed that it may not be a significant cause of disease, but could still negatively impact calf growth. this has also not been experimentally proven. although giardia is commonly found in the feces of both dairy and beef calves, it is unknown if it is truly a pathogen. in the early s, an infectious agent similar to coronavirus was identified in a herd of beef cattle in iowa. it was initially named breda virus, but has subsequently been renamed torovirus. since that time, it has been identified in both beef and dairy calves throughout the world, and % of adult cattle are seropositive. torovirus is found in calves with normal and abnormal feces, but is isolated more frequently in diarrheic feces. the incidence in calves with diarrhea ranged from % to %, while it was never isolated from more than % of normal calves. other pathogens were commonly, but not always, found in conjunction with torovirus, but none appeared to be consistently associated with torovirus infection. [ ] [ ] [ ] [ ] [ ] after ingestion, the virus infects the epithelium of the distal half of the jejunum, the ileum, and colon. histopathologic lesions in experimental infections include necrosis of the crypt and villous enterocytes and villous atrophy, but infection does not consistently lead to clinical signs or histologic damage. , , although it has not been conclusively shown, these lesions would be expected to lead to a malabsorptive diarrhea. there is no specific information on control of torovirus, but as with other viruses, proper housing, decreasing exposure to adult cattle, and good hygiene are likely important to prevent its spread. pathophysiologic mechanisms of infectious diarrhea in calves can be generally divided into malabsorptive/maldigestive, secretory, or both, and research into these mechanisms at the cellular level may ultimately lead to more specific control and treatment methods. currently, most information must be extrapolated from other research models because calves are not commonly used. further elucidation of the mechanisms by which these pathogens affect calves is critical because diarrhea is a significant cause of morbidity and mortality in both dairy and beef cattle. reference of beef cow-calf production management and disease control dairy heifer morbidity, mortality, and health management focusing on preweaned heifers. usda survey enterotoxigenic escherichia coli infections in newborn calves: a review acute undifferentiated neonatal diarrhea of beef calves: the prevalence of enterotoxigenic e. coli, reo-like (rota) virus and other enteropathogens in cow-calf herds prevalence of enterotoxigenic escherichia coli in calves in scotland and northern england occurrence and characteristics of enterotoxigenic escherichia coli isolated from calves with diarrhea the development of the flora of the alimentary tract in young animals observations on the flora of the alimentary tract of animals and factors affecting its composition the role of k antigens of enteropathogenic escherichia coli in colonization of the small intestine of calves scanning and transmission electron microscopic study of the small intestine of colostrum-fed calves infected with selected strains of escherichia coli some infectious causes of diarrhea in young farm animals prevalence of four enterotoxin (stap, stah, stb, and lt) and four adhesin subunit (k , k , p, and f ) genes among escherichia coli isolates from cattle hybridization of clinical escherichia coli isolates from calves and piglets in new york state with gene probes for enterotoxins (stap, stb, lt), shiga-like toxins (slt- , slt-ii) and adhesion factors (k , k , f , p) influence of bovine intestinal fluid on the expression of k pili by escherichia coli functioning of the digestive tract of young fasted calves gastro-intestinal disorders and the functioning of the digestive tract of young calves observations on the aetiology of neonatal diarrhoea (scours) in calves pathophysiology of calf diarrhea: new concepts. presented at the acvim forum proceedings development of resistance with host age to adhesion of k escherichia coli to isolated intestinal epithelial cells the pathogenesis of enteric colibacillosis in neonatal unsuckled calves pathophysiology of neonatal calf diarrhea toxigenic diarrheas, congenital diarrheas, and cystic fibrosis: disorders of intestinal ion transport hybridization of bovine escherichia coli isolates with gene probes for four enterotoxins (stap, stah, stb, lt) and one adhesion factor (k ) escherichia coli heat-stable enterotoxin in feces and intestines of calves with diarrhea characterization of heat-stable enterotoxin from a hypertoxigenic escherichia coli strain that is pathogenic for cattle coli heat-stable enterotoxin and guanylyl cyclase c: new functions and unsuspected actions solubilization and partial characterization of the intestinal receptor for escherichia coli heat-stable enterotoxin distribution of heat-stable enterotoxin/guanylin receptors in the intestinal tract of man and other mammals studies on the mechanism of diarrhoea induced by escherichia coli heat-stable enterotoxin (sta) in newborn calves characterization of the interaction of escherichia coli heat-stable enterotoxin (sta) with its putative receptor on the intestinal tract of newborn calves age-dependent variation in the density and affinity of escherichia coli heat-stable enterotoxin receptors in mice effect of age on activation of porcine intestinal guanylate cyclase and binding of escherichia coli heat-stable enterotoxin (sta) to porcine intestinal cells and brush border membranes sta and cgmp stimulate translocation to the surface of villus enterocytes in rat jejunum and is regulated by protein kinase g prevention of toxin-induced intestinal ion and fluid secretion by a small-molecule cftr inhibitor heat-stable enterotoxin of escherichia coli stimulates a non-cftr-mediated duodenal bicarbonate secretory pathway effects of heat-stable escherichia coli enterotoxin on intestinal alkaline secretion and transepithelial potential difference in the rat intestines in vivo escherichia coli heat stable (sta) enterotoxin and the upper small intestine: lack of evidence in vivo for net fluid secretion a reconsideration of the evidence for escherichia coli sta (heat stable) enterotoxin-driven fluid secretion: a new view of sta action and a new paradigm for fluid absorption conditions for the production of eschericha coli enterotoxin in a defined medium effect of vasoactive intestinal polypeptide (vip) antagonism on rat jejunal fluid and electrolyte secretion induced by cholera and escherichia coli enterotoxins comparison of the mechanisms of action of cholera toxin and the heat-stable enterotoxins of escherichia coli role of the enteric nervous system in the pathophysiology of secretory diarrhea lack of evidence in vivo for a remote effect of escherichia coli heat stable enterotoxin on jejunal fluid absorption the net fluid secretion caused by cyclic -guanosine monophosphate in the rat jejunum in vivo is mediated by a local nervous reflex the enteric nervous system participates in the secretory response to the heat stable enterotoxins of escherichia coli in rats and cats enterotoxin escherichia coli sta activates a nitric oxide-dependent myenteric plexus secretory reflex in the rat ileum role of the enteric nervous system in the maintained hypersecretion induced by enterotoxin sta in the nutritionally deprived intestine fluid hypersecretion induced by enterotoxin sta in nutritionally deprived rats: jejunal and ileal dynamics in vivo neurally maintained hypersecretion in undernourished rat intestine activated by e. coli sta enterotoxin and cyclic nucleotides in vitro effect of feeding frequency and route of administration on abomasal luminal ph in dairy calves fed milk replacer observations by the ligated intestinal segment and oral inoculation methods on escherichia coli infections in pigs, calves, lambs and rabbits antimicrobial use in the treatment of calf diarrhea a global challenge massive outbreak of waterborne cryptosporidium infection in milwaukee, wisconsin: recurrence of illness and risk of secondary transmission giardiasis and cryptosporidiosis in ruminants cryptosporidiosis: biology, pathogenesis and disease a review of the importance of cryptosporidiosis in farm animals duration of naturally acquired giardiosis and cryptosporidiosis in dairy calves and their association with diarrhea prevalence and infection pattern of naturally acquired giardiasis and cryptosporidiosis in range beef calves and their dams cryptosporidium parvum infection in bovine neonates: dynamic clinical, parasitic and immunologic patterns prevalence and age-related variation of cryptosporidium species and genotypes in dairy calves molecular and phylogenetic characterization of cryptosporidium and giardia from pigs and cattle in denmark resistance of calves to cryptosporidium parvum: effects of age and previous exposure villous atrophy, crypt hyperplasia, cellular infiltration, and impaired glucose-na absorption in enteric cryptosporidiosis of pigs infection pattern of cryptosporidium and giardia in calves an examination of the prevalence of and risk factors for shedding of cryptosporidium spp. and giardia spp. in cows and calves from western canadian cow-calf herds cryptosporidiosis in bovine and human health experimental infection of lambs with cryptosporidium isolated from a human patient with diarrhoea enteric lesions and diarrhea in gnotobiotic calves monoinfected with cryptosporidium species host responses to cryptosporidium infection temporal changes in permeability and structure of piglet ileum after site-specific infection by cryptosporidium parvum cryptosporidium parvum is cytopathic for cultured human biliary epithelia via an apoptotic mechanism cryptosporidium parvum infection of intestinal epithelium: morphologic and functional studies in an in vitro model cryptosporidium parvum infection of human intestinal epithelial cells induces the polarized secretion of c-x-c chemokines cryptosporidium parvum infection of caco- cell monolayers induces an apical monolayer defect, selectively increases transmonolayer permeability, and causes epithelial cell death infection of human and bovine epithelial cells with cryptosporidium andersoni induces apoptosis and disrupts tight junctional zo- : effects of epidermal growth factor cryptosporidium parvum induces apoptosis in biliary epithelia by a fas/fas ligand-dependent mechanism cryptosporidium parvum activates nuclear factor kappab in biliary epithelia preventing epithelial cell apoptosis intestinal epithelial cell apoptosis following cryptosporidium parvum infection caspase-dependent apoptosis during infection with cryptosporidium parvum absence of weight loss during cryptosporidium infection in susceptible mice deficient in fas-mediated apoptosis host cell apoptosis impairs cryptosporidium parvum development in vitro cryptosporidium parvum at different developmental stages modulates host cell apoptosis in vitro prostanoids inhibit intestinal nacl absorption in experimental porcine cryptosporidiosis glutamine stimulates prostaglandin-sensitive na( )-h exchange in experimental porcine cryptosporidiosis glutamine transporter in crypts compensates for loss of villus absorption in bovine cryptosporidiosis cyclooxygenase blockade and exogenous glutamine enhance sodium absorption in infected bovine ileum evidence for the absence of an intestinal adaptive mechanism to compensate for c. parvum-induced amino acid malabsorption in suckling rats impairment of amino-acid absorption in suckling rats infected with cryptosporidium parvum effect of cryptosporidium parvum infection on the absorptive capacity and paracellular permeability of the small intestine in neonatal calves pulmonary excretion of h in calves with cryptosporidium-induced malabsorption role of the enteric nervous system in piglet cryptosporidiosis tumour necrosis factor alpha changes porcine intestinal ion transport through a paracrine mechanism involving prostaglandins neutrophils do not mediate the pathophysiological sequelae of cryptosporidium parvum infection in neonatal piglets nf-kappab-mediated expression of inos promotes epithelial defense against infection by cryptosporidium parvum in neonatal piglets oral delivery of l-arginine stimulates prostaglandin-dependent secretory diarrhea in cryptosporidium parvum-infected neonatal piglets peptide yy inhibits intestinal cl-secretion in experimental porcine cryptosporidiosis through a prostaglandin-activated neural pathway effect of halofuginone lactate on the occurrence of cryptosporidium parvum and growth of neonatal dairy calves field study of the efficacy of halofuginone and decoquinate in the treatment of cryptosporidiosis in veal calves efficacy of halofuginone lactate against cryptosporidium parvum in calves specific serum and local antibody responses against cryptosporidium parvum during medication of calves with halofuginone lactate efficacy of halofuginone lactate in the prevention of cryptosporidiosis in suckling calves the effect of halofuginone lactate on experimental cryptosporidium parvum infections in calves paromomycin is effective as prophylaxis for cryptosporidiosis in dairy calves prophylactic use of decoquinate for infections with cryptosporidium parvum in experimentally challenged neonatal calves efficacy of azithromycin dihydrate in treatment of cryptosporidiosis in naturally infected dairy calves feeding activated charcoal from bark containing wood vinegar liquid (nekka-rich) is effective as treatment for cryptosporidiosis in calves speculation on whether a vaccine against cryptosporidiosis is a reality or fantasy efficacy of hyperimmune bovine colostrum for prophylaxis of cryptosporidiosis in neonatal calves protection of calves against cryptosporidiosis with immune bovine colostrum induced by a cryptosporidium parvum recombinant protein protection of calves with a vaccine against cryptosporidium parvum strategies for the control of cryptosporidium parvum infection in calves field testing of prophylactic measures against cryptosporidium parvum infection in calves in a california dairy herd pathogenesis of intestinal and systemic rotavirus infection rotaviral and coronaviral diarrhea prevalence of neutralizing antibody to the calf rotavirus in new york cattle prevalence of group a and group b rotaviruses in the feces of neonatal dairy calves from california molecular epidemiology and subgroup determination of bovine group a rotaviruses associated with diarrhea in dairy and beef calves frequency of group a rotavirus in diarrhoeic calves in brazilian cattle herds prevalence of bovine group a rotavirus shedding among dairy calves in ohio age-dependent diarrhea induced by a rotaviral nonstructural glycoprotein interactions between rotavirus and gastrointestinal cells microbes and microbial toxins: paradigms for microbialmucosal interactions. viii. pathological consequences of rotavirus infection and its enterotoxin nsp elicits age-dependent diarrhea and ca( )mediated i(-) influx into intestinal crypts of cf mice a functional nsp enterotoxin peptide secreted from rotavirus-infected cells the rotavirus enterotoxin nsp directly interacts with the caveolar structural protein caveolin- full-length, glycosylated nsp is localized to plasma membrane caveolae by a novel raft isolation technique the rotavirus enterotoxin nsp mobilizes intracellular calcium in human intestinal cells by stimulating phospholipase c-mediated inositol , , -trisphosphate production rotavirus infection induces an increase in intracellular calcium concentration in human intestinal epithelial cells: role in microvillar actin alteration rotavirus infection induces cytoskeleton disorganization in human intestinal epithelial cells: implication of an increase in intracellular calcium concentration cytoplasmic calcium measurement in rotavirus enterotoxin-enhanced green fluorescent protein (nsp -egfp) expressing cells loaded with fura- disaccharidase activities in small intestine of rotavirus-infected suckling mice: a histochemical study rotavirus infection reduces sucrase-isomaltase expression in human intestinal epithelial cells by perturbing protein targeting and organization of microvillar cytoskeleton a cyclic amp protein kinase a-dependent mechanism by which rotavirus impairs the expression and enzyme activity of brush border-associated sucrase-isomaltase in differentiated intestinal caco- cells direct inhibitory effect of rotavirus nsp ( - ) peptide on the na( )-d-glucose symporter of rabbit intestinal brush border membrane mechanisms of net chloride secretion during rotavirus diarrhea in young rabbits: do intestinal villi secrete chloride? how do the rotavirus nsp and bacterial enterotoxins lead differently to diarrhea? role of the enteric nervous system in the fluid and electrolyte secretion of rotavirus diarrhea managing acute gastroenteritis among children: oral rehydration, maintenance, and nutritional therapy enhancement of passive immunity with maternal vaccine against newborn calf diarrhea passive immunity to bovine rotavirus in newborn calves fed colostrum supplements from cows immunized with recombinant sa rotavirus core-like particle (clp) or virus-like particle (vlp) vaccines modulation by colostrum-acquired maternal antibodies of systemic and mucosal antibody responses to rotavirus in calves experimentally challenged with bovine rotavirus enteric viral infections of calves and passive immunity reduction in morbidity due to diarrhea in nursing beef calves by use of an inactivated oil-adjuvanted rotavirus-escherichia coli vaccine in the dam viral diarrhea in calves, lambs, kids, piglets, and foals. in: veterinary medicine: a textbook of the diseases of cattle, sheep, pigs, goats, and horses detection by radioimmunoassay and enzymelinked immunosorbent assay of coronavirus antibodies in bovine serum and lacteal secretions aetiology of diarrhoea in young calves bovine coronavirus pathophysiologic changes due to coronavirus-induced diarrhea in the calf the s protein of bovine coronavirus is a hemagglutinin recognizing -o-acetylated sialic acid as a receptor determinant isolated he-protein from hemagglutinating encephalomyelitis virus and bovine coronavirus has receptor-destroying and receptor-binding activity initial events in bovine coronavirus infection: analysis through immunogold probes and lysosomotropic inhibitors scanning electron microscopic characterization of bovine coronavirus plaques in hrt cells coronaviral morphogenesis and ultrastructural changes in intestinal infections of calves dual enteric and respiratory tropisms of winter dysentery bovine coronavirus in calves attaching and effacing activities of rabbit and human enteropathogenic escherichia coli in pig and rabbit intestines enteropathogenic escherichia coli diarrheagenic escherichia coli enteropathogenic escherichia coli: physiological alterations from an extracellular position characterization of eae escherichia coli isolated from healthy and diarrheic calves longitudinal prevalence study of diarrheagenic escherichia coli in dairy calves escherichia coli that cause diarrhea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorrhagic, and enteroadherent frequency of shiga toxin-producing escherichia coli (stec) isolates among diarrheic and non-diarrheic calves in brazil occurrence and characteristics of enterohemorrhagic escherichia coli o and o in calves associated with diarrhea occurrence and characteristics of enterohemorrhagic escherichia coli o in calves associated with diarrhoea characterization of shiga toxin-producing escherichia coli strains isolated from calves in poland cattle lack vascular receptors for escherichia coli o :h shiga toxins the comparative pathology of clostridium difficile-associated disease a possible role for clostridium difficile in the etiology of calf enteritis prevalence of pcr ribotypes among clostridium difficile isolates from pigs, calves, and other species clostridium difficile pcr ribotypes in calves natural and experimental infection of neonatal calves with clostridium difficile cryptosporidium parvum and giardia intestinalis in calf diarrhoea in sweden epidemiology of cryptosporidium spp. and giardia duodenalis on a dairy farm concurrent infections of giardia and cryptosporidium on two ohio farms with calf diarrhea diagnosis of giardia infection in calves mixed giardia duodenalis assemblage a and e infections in calves molecular characterization of cryptosporidium and giardia isolates from cattle from portugal molecular epidemiology of giardia and cryptosporidium infections in dairy calves originating from three sources in western australia cryptosporidium and giardia in different age groups of danish cattle and pigs-occurrence and management associated risk factors identification of genotypes of giardia intestinalis isolates from a human and calf in japan outbreaks of enteric infections caused by multiple pathogens associated with calves at a farm day camp efficacy of vaccination in preventing giardiasis in calves morphological changes in the jejunum of calves naturally infected with giardia spp. and cryptosporidium spp pathophysiology of small intestinal malabsorption in gerbils infected with giardia lamblia effects of murine giardiasis on growth, intestinal morphology, and disaccharidase activity studies with an unclassified virus isolated from diarrheic calves seroepidemiology of breda virus in cattle using elisa detection of bovine torovirus in fecal specimens of calves with diarrhea from ontario farms detection of bovine torovirus and other enteric pathogens in feces from diarrhea cases in cattle association of enteric shedding of bovine torovirus (breda virus) and other enteropathogens with diarrhea in veal calves detection of bovine torovirus in fecal specimens of calves with diarrhea in japan detection of bovine torovirus in neonatal calf diarrhoea in lower austria and styria (austria) a morphological study of the replication of breda virus (proposed family toroviridae) in bovine intestinal cells cellular lesions in intestinal mucosa of gnotobiotic calves experimentally infected with a new unclassified bovine virus (breda virus) key: cord- - n knt authors: elad, d.; brenner, j.; markovics, a.; yakobson, b.; shlomovitz, s.; basan, j. title: yeasts in the gastrointestinal tract of preweaned calves and possible involvement of candida glabrata in neonatal calf diarrhea date: journal: mycopathologia doi: . /a: sha: doc_id: cord_uid: n knt to examine the possibility of a mycotic involvement in neonatal calf diarrhea (ncd) the presence of fungi was assessed in (a) the intestinal contents of dead calves and fecal samples submitted for routine laboratory examination, (b) fecal specimens, sampled once in winter and once in summer, of calves raised on farms with different management systems, and (c) mucosal scrapings of various segments of the digestive tract of a diarrheic calf, massively shedding candida glabrata. c. glabrata was the most prevalent fungal species isolated from the routine samples. it was the only fungus which was shed by the calves on the farms, for continuous, more or less prolonged periods, but exclusively in the winter months. diarrhea and c. glabrata shedding seemed to be associated. c. glabrata colonized the abomasum (the functional equivalent of the monogastric stomach) but not the other segments of the digestive tract of the euthanized calf based on the findings of this study it seems that while some yeast species may be considered as commensals of the digestive tract of calves, and consequently their isolation from intestinal contents or fecal samples has no clinical significance, others, such as c. glabrata may be involved in enteric pathogenic processes. moreover, characteristics of the culture, previous chemotherapeutic treatments, the animal's age and possibly climatic conditions should be taken into account before deciding on the fungal isolate's clinical relevance. determination of mycotic involvement in ncd by routine mycological examination of intestinal contents and fecal samples of diarrheic calves may be useful to avoid unnecessary and potentially harmful antibacterial therapy. neonatal calf diarrhea (ncd) is arguably the most important pathological entity in causing economic losses due to morbidity and mortality. ncd may be caused by a highly virulent primary pathogen, such as enterotoxigenic escherichia coli, or, more frequently, by mixed infections of various microorganisms [ ] . while the role of viruses, bacteria and protozoa in causing ncd has been investigated [ ] , the possible role of fungi in enteric pathology has received only limited attention. this is probably the result of the assumption that fungi are part of the normal microbial flora of the digestive system [ ] and consequently of no clinical relevance, unless they are isolated in conjunction with pathological changes. this assumption, until recently prevailing in human medicine, has been subject to reconsideration. a number of reports published during the last years [ ] [ ] [ ] indicate the possibility of connection between fungi, especially yeasts, and cases of diarrhea in human patients, especially children, with no recognizable underlying conditions to justify infection by opportunistic pathogens. association between yeasts and disorders of the digestive tract in these cases was made by exclusion (no other pathological microorganisms found), the presence of an elevated number of yeasts in the feces, or fol-lowing failure of antibacterial therapy but successful treatment of the patient with antimycotic drugs. this study was devised to appraise the possibility of a similar involvement of fungi in ncd. to assess the possibility of mycotic involvement in ncd a study consisting of parts was initiated: . assessment of the prevalence of fungi in the gastrointestinal tract (git) of calves: during a period of months, the intestinal contents of calves that died after showing clinical signs of ncd and fecal samples of diarrheic calves, up to month of age, which were submitted for routine microbiological examination, were examined mycologically. . study of the clinical significance of fungi in the git: a field trial in which the presence of mycotic agents in feces of calves of various ages and under different management and climatic conditions was investigated. . examination of the anatomic sites in the git to be colonized by fungi and the interaction between these microorganisms and the mucosa: one day old diarrheic calf, massively shedding c. glabrata, was euthanized and mucosal scrapings of various segments of the digestive tract (abomasum, rumen, duodenum, jejunum, ileum, cecum, colon and rectum) and their contents were examined microscopically and microbiologically. samples of the segments were examined histopathologically. mycology. approximately . g of intestinal contents or fecal specimens material were suspended in ml sterile saline. from this suspension, . ml was inoculated onto chromagar (chromagar, paris, france) plates which were incubated at • c [ ] . thus a semiquantitative indication of the number of yeasts present in the samples, as well as the ratio between different yeast species, in case of mixed growth, was obtained. two yeast colonies (of each species in mixed culture) were subcultured on sabouraud dextrose agar and identified by the id c system (bio-meneux sa, marcy l'etoile, france). chromagar plates were kept for days before being discarded as negative. feed was examined for the presence of mycotic contamination as previously described [ ] . bacteriology. enterotoxigenic escherichia coli: one loopfull of the above mentioned suspension was inoculated onto e-agar [ ] plates, incubated overnight at • c and the resulting cultures tested by the slide agglutination test with anti k- monoclonal antibodies (central veterinary laboratory, weybridge, uk). for salmonella enrichment, tetrathionate broth was added to the rest of the suspension, the tubes were incubated at • c overnight, following which they were inoculated onto mcconkey and brilliant green agar plates. these plates were reincubated for hours under the same conditions and colonies suspected to be salmonella spp. were tested biochemically and serologically (welcome diagnostics, dartford, england). virology. the fecal samples were examined for the presence of coronavirus and rotavirus (serogroup a) by elisa with the pathasure bovine enteritis system (cambridge veterinary sciences ltd., uk). were examined for the presence of cryptosporidia by sugar flotation and modified ziehl-neelsen staining [ ] , as previously described. experimental design. to assess environmental influence on the epidemiology of yeasts in the digestive tract of calves, dairy farms with different management systems were included in the survey. in addition, based on previous findings that ncd is influenced by seasonal factors [ ] , samples were taken during the hot summer months (july-august) and the cooler, rainy, winter months (december-january). animals and sampling. twenty four female calves, divided into groups were included in the survey. the groups consisted of: daily rectal fecal samples were taken during the first days of life and once every days up to days of age. after that age samples were taken as long as diarrhea and/or yeast shedding persisted. shedding was assumed to have stopped after consecutive fecal samples negative for yeasts. one week after the last sampling an additional fecal sample was taken to assess whether the absence of yeast from the feces was definitive or not. specimens were processed within minutes after the sampling. management. feeding practices on both farms included in the survey were similar. each calf was fed immediately after it's birth liters of first colostrum, followed by additional liters hours later. the colostrum was obtained from the dam on farm a and from a colostrum pool on farm b. on days and the calves were fed second and third day colostrum, respectively, by the same scheme. from the fourth day on, calves were fed liters of commercial milk substitutes ( g/l), twice daily. dry food, composed of corn and cotton seed, commercial concentrates, hay and dry milk substitute were available. water was accessible ad libitum. farm a was located in an agricultural research facility. each calf was housed in an individual pen, placed on the ground. consequently no direct contact between the animals was possible. highly experienced personnel were in charge of the calves and individual care was given to each calf. the dry food made available to the calves was not medicated. farm b was located in a large collective (kibbutz) holding. calves were kept in raised individual pens up to the age of - days and then transferred to yards, where - animals of homologous age groups were reared. personnel were not as experienced as on farm a (due to frequent substitutions) and individual supervision of each calf was limited. the commercial concentrate incorporated in the dry food, made available to the calves, was oxytetracycline medicated. samples of the dry food and milk replacer fed to the calves on each farm were examined for mycotic contamination as previously described [ ] . one calf, intensely shedding c. glabrata was euthanized. slides of mucosal scrapings of the various segments of the euthanized calf's digestive system were giemsa stained and examined microscopically. scrapings as well as contents of each segment were examined microbiologically. pas stained samples of each segment were examined histopathologically. to assess the capability of the c. glabrata strain isolated from the euthanized calf's abomasum (the functional equivalent of the monogastric stomach) and of the fecal isolates ( from each farm) to grow under the highly acidic conditions present in this organ -ph - . - [ ] , the strain was suspended in distilled water to a turbidity equaling on the mcfarland standard. of this suspension, . ml was inoculated into sabouraud dextrose broth, acidified with hcl to ph values as low as . . the inoculated broth tubes were incubated at • c for days. growth was examined daily. yeasts were isolated from . % ( / ) of the intestinal contents of dead calves and . % ( / ) of the fecal samples. twenty six of the isolates ( . %) from dead calves and ( . %) of the fecal isolates were identified as candida glabrata (fig. ). other fungal species were isolated on rare occasions: c. catenulata (n = ), c. rugosa (n = ), c. krusei (n = ), geotrichium candidum (n = ), trichosporon cutaneum (n = ), c. tropicalis (n = ), c. kefyr (n = ), c. lypolytica (n = ), c. colliculosa (n = ) and c. parapsilosis (n = ). unlike c. glabrata, however, cultures of these species consisted of only a few ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) colonies. twenty three ( . %) of c. glabrata positive calves were in their first week of life, ( . %) in their second week and ( . %) were between and days old (fig. ) . summer months: on farm a, all the calves had diarrhea, starting at the end of their first week of life, for to days. on farm b, only one calf had diarrhea, on days to . microbial findings on both farms were similar: bacterial pathogens and coronavirus were not found in the calves during this period, rotavirus and cryptosporidia, the latter in very low numbers, were found sporadically during the whole week period but were not associated with the clinical signs. candida krusei, c. rugosa and trichosporon cutaneum were isolated from some fecal samples but neither the number of colonies ( ) ( ) ( ) ( ) ( ) nor the duration of the yeasts' presence ( - days) indicated colonization by these microorganisms. a few colonies of c. glabrata were isolated from the feces of one calf from farm a on day , following a week of diarrhea. two days later this calf died of incarcerated diaphragmatic hernia and its intestinal contents yielded rich cultures of c. glabrata. winter months: the results of the survey during this period are more complex then those of the summer months and are presented in fig. . all calves on farm a had diarrhea which started between day and of their lives. c. glabrata was isolated from all but one of the calves. shedding of this yeast started usually - days after the onset of diarrhea. the periods of c. glabrata shedding and intermittent diarrhea were generally overlapping. this phenomenon is especially evident in calf no. , which had diarrhea for an exceptionally long period (until days of age) and shed c. glabrata for an equally prolonged time span (until days of age). based on the semi quantitative character of the culture method, an initial increase and a final decrease in c. glabrata colony counts was observed. during most of the shedding period, however, yeast cultures were extremely rich and consisted of confluent or semiconfluent growth of the c. glabrata. c. krusei, c. catenulata, rotavirus and cryptosporidia were found sporadically (fig. ) in low numbers and for not more than a few consecutive days. one exception was calf no. (fig. ) , from which mixed cultures of c. glabrata, c. krusei and c. catenulata were isolated (the latter two in numbers higher than in other cases) for days, during which it was treated with antibiotics (penicillin/streptomycin) against a mouth infection caused by actinomyces pyogenes. on farm b, out of the calves had diarrhea but its duration was generally shorter than on farm a, and did not exceed the age of days. the animals which had diarrhea were the same animals that shed c. glabrata. unlike farm a, the shedding of the yeast started in calves before or concomitantly with the diarrhea. a few colonies ( - ) of t cutaneum, geotrichium can-didum or c. rugosa were isolated once from calves. rotavirus was found sporadically but cryptosporidia were absent. enterotoxigenic e. coli and salmonella spp. were not isolated during the survey. c. glabrata was not found in samples of the milk replacer or dry feed fed to the calves. no pathological changes were observed at the necropsy of the calf. cultures of abomasal and intestinal contents (all the segments) yielded rich cultures of c. glabrata. a few colonies of c. krusei and c. catenulata were also present but c. glabrata was clearly the dominant yeast species. microscopic examination of the mucosal scrapings of the various segments showed that the abomasal mucosa was heavily colonized by yeasts. no hyphae, pseudohyphae or elongated yeasts (typical of c. krusei) were observed. only a few yeast cells, were present on the intestinal segments. the histopathological examination of the various segments confirmed these findings: the abomasum was the only segment to be colonized by yeasts which seemed to interact with the mucosa (fig. ) . no macroscopic or microscopic pathological changes of the git were observed. all the c. glabrata strains examined for acid resistance grew at ph = . after overnight incubation, and at ph = . after days. enterotoxigenic e. coli and salmonella spp. were not isolated from the various segments, rotavirus was not found and cryptosporidia were found in very low numbers ( /cc). during the last years fungal diarrhea, especially in children, has received much attention [ ] . c. albicans, c. tropicalis, c. krusei, c. glabrata and other yeast species have been associated with the syndrome [ ] . various diagnostic criteria have been suggested to differentiate between normal individuals shedding yeasts and patients afflicted with noninvasive fungal diarrhea. the presence of hyphae in the stools [ ] is not sufficient as cases of diarrhea in which only budding yeast cells were observed in the feces and which were successfully treated with antimycotic drugs [ ] have been reported. moreover, one of the salient characteristics (and the cause of many taxonomic controversies) of c. glabrata is that it does not form either pseudo-or true hyphae. heavy to confluent growth, such as cultured from the calves in this study, may be considered an indication of non-invasive fungal diarrhea [ ] . in our epidemiologic study, yeast were found in the git of . % of the examined samples, with c. glabrata emerging as the most prevalent species ( . %) in preweaned calves suffering from ncd. although it was not possible to obtain sufficient data regarding antibacterial treatment of the dead and diarrheic calves which were examined in the first part of the study, it may be, however, safely assumed that most (if not all) of the former and many of the latter received such drugs. c. glabrata has been reported to be the most prevalent yeast species in young calves, especially if fed with milk replacers, but was not found to be connected with diarrhea [ ] . in addition, c. glabrata is one of the most prevalent species isolated from human sources, often second only to c. albicans [ ] . the field trial confirmed the findings of the epidemiologic study as to the prevalence of c. glabrata. in addition, it showed that c. glabrata was the only yeast present in very high numbers in the feces of young calves for periods longer than days. other species were present only sporadically and for brief periods. this indicates that the ability of c. glabrata to colonize the git of young calves under the conditions prevailing during the trial exceed that of other yeast species. the comparison between diarrhea and presence of c. glabrata in the feces of young calves during the winter months indicates a connection between these two observations (fig. ) . this connection is underscored by the case of calf on farm a, in which both diarrhea and excretion of the fungus were exceptionally prolonged. the nature of this connection is not clear and several possibilities must be considered: although no pathological changes were present in the euthanized calf, other mechanisms, such as fungal metabolites, might be involved. this mode of action would be analogous to the one observed in enterotoxemic colibacillosis, where no or only mild pathologic changes are observed in the git during diarrhea [ ] . (b) conditions produced in the digestive tract by the diarrhea favor the proliferation of the yeast. the appearance of c. glabrata in the feces of the calf days before its death following an incarcerated diaphragmatic hernia seems to support this possibility. (c) a common underlying condition, such as interference with the process of establishing a natural microflora, causes both diarrhea and colonization by c. glabrata. further studies are necessary to clarify the nature of this connection. the examination of the euthanized calf showed that the abomasum was the only segment of the git in which an interaction between the mucosa and the yeasts was observed. the clear predominance of c. glabrata in cultures made from these segments indicates that the yeasts observed microscopically are c. glabrata as well. this assumption is further substantiated by the morphology (absence of hyphae and pseudohyphae) of the yeast cells. colonization of the human stomach by yeasts, including c. glabrata, has been reported [ , ] , often without the induction of pathological changes [ ] . due to the low ph, the only yeast species able to grow in this organ have to be able to do so under stringent acidic conditions. the c. glabrata isolates examined in this study were found comparable to human isolates [ , ] , to be highly acid resistant, and thus capable of growth in the abomasum. higher ph values and a competitive intestinal microflora, established earlier than in the abomasum, are likely causes for the limited colonization of the intestine by c. glabrata. a number of reports dealing with fungal infections of the digestive tract of preweaned ruminants have been published, most indicating the involvement of various yeast species in cases of necrotic abomasitis [ ] [ ] [ ] . a common factor to these cases was the treatment of the animals with antibacterial drugs, considered predisposing to fungal infections. animals included in the field trial, (with the exception of calf on farm a), were not subjected to antibacterial treatment. the medicated dry food made available to the calves on farm b is unlikely to have been ingested in relevant quantities given the young age of the animals. it is noteworthy that the only period during which yeast species other than c. glabrata (c. krusei and c. catenulata) were isolated for consecutive days was parallel to the period of antibacterial treatment of calf on farm a. this observation, in addition to the self-limiting characteristic of c. glabrata colonization in untreated calves seems to support other reports [ ] [ ] [ ] [ ] which indicate that antibacterial treatment may induce pathological processes in which yeasts are involved. the source of the c. glabrata strains isolated during our study is not clear. it has not been found in the feed samples and it does not seem to remain in the digestive tract of calves for more than a few weeks. the examination of additional potential environmental sources might clarify this point. oro-fecal transmission of c. glabrata between calves is likely. this hypothesis is supported by the earlier onset of c. glabrata shedding on farm b than on farm a (see in next paragraph). based on the findings of this study it seems that the assumption that yeasts in general are part of the normal flora of the digestive tract of ruminants, and consequently that their isolation from intestinal contents or fecal samples has no clinical significance, should be reconsidered. some species, such as c. catenulata, c. rugosa or g. candidum, appeared in our study to be harmless, others, such as c. glabrata may be involved in enteric pathogenic processes. consequently, the yeast species, qualitative (pureness) and quantitative (cfu number) characteristics of the culture, previous chemotherapeutic treatments, the animal's age and possibly climatic conditions should be taken into account before deciding on the fungal isolate's clinical relevance. we suggest that at least some yeast species should be considered with other potentially pathogenic microorganisms which may cause diarrhea under certain conditions but are not considered primary enteric pathogens. diarrhea in which yeasts are involved is mild and self limiting. antibacterial treatment of such cases, might be not only unnecessary but counterproductive, resulting in the intensification of the microbial imbalance in favor of the mycotic flora in the git. massive necrosis of the forestomach mucosa caused by c. glabrata infection exacerbated by antibacterial treatment has been reported [ ] . determination of mycotic involvement in ncd by routine mycological examination of intestinal contents and fecal samples of diarrheic calves may be useful to avoid inappropriate antibacterial therapy. epidemiological study of neonatal calf diarrhoea in israel -a one year survey of faecal samples microbiological and pathological findings in young calves suffering from neonatal diseases the alimentary system prevalence of candida species in nigerian children with diarrhoea candida associated diarrhea: a syndrome in search of credibility fungal diarrhoea: association of different fungi and seasonal variation in their incidence chromagar candida, a new differential isolation medium for presumptive identification of clinically important candida species feed contamination with candida krusei as a probable source of mycotic mastitis in dairy cows production of k , k , and p antigens by escherichia coli cultured on synthetic and complex media the prevalence of cryptosporidium in dairy calves in israel the influence of climate on the mortality of young calves in two different farming systems in israel the calf th ed enteric candidiasis. diagnosis and clinical considerations diarrhoea caused by candida besiedlung des verdaungstraktes vom kalb mit hefen philadelphia: lea & febiger zum vorkommen von hefepilzen im magen des menschen resistenz von hefen und hefeartige pilzen gegen schwefelsaure incidence of yeast-like fungi in gastric juice under normal and pathologic conditions systemic candidiasis in calves on prolonged antibiotic therapy incidence of mycotic gastritis in calves up to days of age candidiasis caused by candida glabrata in the forestomachs of a calf candida albicans infection of the forestomachs of a calf kimron veterinary institute key: cord- - hlbf authors: zwierzchowski, grzegorz; miciński, jan; wójcik, roman; nowakowski, jacek title: colostrum-supplemented transition milk positively affects serum biochemical parameters, humoral immunity indicators and the growth performance of calves date: - - journal: livest sci doi: . /j.livsci. . sha: doc_id: cord_uid: hlbf the aim of this study was to determine the effect of colostrum-supplemented transition milk on the growth rate of newborn calves. thirty-six day-old female holstein calves with a birth weight of . ± . kg were randomly assigned to a treatment group and a control group. on day , all calves received one portion ( kg) of colostrum within h after birth. between the second feeding and the age of days, control calves received only transition milk that had been collected from dams shortly before feeding, and the treatment group received . kg of colostrum collected from dams on day and . kg of transition milk from subsequent milkings. monthly body weight and average daily gain were used to measure growth performance. blood samples were collected from the jugular vein at multiple time points to evaluate the biochemical and immune status of calves. colostrum-supplemented transition milk increased the concentrations of serum immunoglobulins, triacylglycerols, cholesterol and total protein. the activity of lactate-dehydrogenase and ceruloplasmin decreased, whereas lysozyme activity increased with time in the control group. it can be concluded that additional five days of colostrum feeding positively influences non-specific humoral immunity indicators and serum biochemical parameters in dairy calves. high morbidity and mortality rates of calves can cause significant economic loss and reduce the profitability of milk production (raboisson et al., ) . thus, proper calf management and rearing are important considerations in livestock production. newborn calves are agammaglobulinemic and susceptible to infection. to prevent health problems, calves acquire immunity from colostrum which is a natural source of immune factors and nutrients (godden, ; raboisson et al., ; torsein et al., ) . in this paper, colostrum will be defined as a secretion collected from the first milking, while milk from subsequent milkings is referred to as "transition milk". it has been suggested that calves should be fed only high-quality colostrum containing at least g/l of immunoglobulins (igg) (mcguirk and collins, ) . however, igg concentrations in bovine colostrum can vary. several factors have been shown to affect igg concentrations in colostrum, including parity, breed, age, the metabolic status of dam, and the length of the dry period (gomez and chamorro, ; morin et al., ; quigley et al., ; weaver et al., ) . inadequate colostrum intake and its poor quality can result in the failure of passive transfer (fpt) which is a commonly encountered problem whose causes and consequences have been well documented (beam et al., ; chigerwe et al., ; furman-frątczak et al., ) . fpt is diagnosed when igg serum concentrations drop below g/l during the first h after birth. serum igg levels higher than g/l point to successful passive transfer of immunity. in calves, morbidity and mortality are minimized when igg serum levels exceed g/l (godden, ; güngör et al., ) . immunoglobulins are absorbed most effectively during the first h postpartum; therefore, calves should receive to l of colostrum during the first feeding within the first h postpartum. importantly, the volume offered to the calf should depend on the quality of colostrum. ideally, the portion should provide at least g of igg, which is regarded as sufficient for reaching igg serum concentration higher than g/l (raboisson et al., ; vasseur et al., ) . additionally, colostrum contains more vital ingredients such as maternal leukocytes, growth factors, hormones, cytokines and nonspecific antimicrobial factors (ontsouka et al., ) . these immunological and biochemical components also protect agammaglobulinemic calves and support the establishment and development of the gut microbiome (barrington and parrish, ; godden, ; gomez and chamorro, ) . despite the benefits of colostrum, artificial feeding (for example, with the use of an oroesophageal tube) is the method of choice in calves that experience problems with colostrum drinking to prevent fpt (godden, ) . therefore, in this study, we hypothesized that the supplementation of transition milk with colostrum could improve the immune status and blood biochemical parameters of calves and, consequently, their growth performance. the study was conducted on a commercial free-stall dairy farm located in the north-eastern region of poland (warmian-masurian voivodeship), where approximately polish holstein-fresian lactating cows were housed and fed according to the recommendations of the german agricultural society (deutsche landwirtschafts-gesellschaft, dlg). during the dry period, all cows were vaccinated against colibacillosis and rota-and coronaviruses. cows in maternity pens were monitored until calving. after birth, female calves born within two months (mean birth weight . ± . kg) to primiparous and multiparous dams [(the average parity of the dams was . ± . , mean ± sem for control group (cr) and . ± . , mean ± sem for treatment group, (trt)] were assigned to a cr (n = ) or a trt (n = ). after parturition calves were separated from dams and kept in individual pens until transfer (at the age of months) to another building within the farm where they were placed in group pens. additionally, after parturition, colostrum was harvested from each dam within h post partum. in the trt group, all colostrum collected on the first day (two milkings) was pooled in kg bottles separately for each calf, and kept in the freezer (− °c). calves were fed only from colostrum and/or transition milk milked from their dams. before feeding to calves, colostrum was thawed in a warm water bath (not exceeding °c). colostrum temperature was checked before feeding. the target temperature of colostrum was approximately °c. until d of age, all calves were fed twice a day ( kg/feeding; h and h) according to the described regimen. on d , all calves received one portion ( kg) of colostrum within h after birth. between the second feeding and the age of days, cr calves received only transition milk that had been collected from dams shortly before feeding, whereas trt calves received . kg of colostrum collected from dams on day and . kg of transition milk from subsequent milkings ( kg/d in total). beginning on d , all calves were placed on an identical feeding regimen. all calves received milk replacer (mr; all milk protein; % of cp and % of fat on a dm basis; polmass sa, bydgoszcz, poland) between d and d , that was formulated to obtain g dm/l. mr volume was decreased on subsequent days from . l/calf/day (between d to d ) to . l/calf/day (between d and ) and to . l/calf/day (between d and d ). calves had unlimited access to fresh water from d . between d and d , calves were fed solid feed (textured starter (cs); ground triticale and straw) ad libitum. from d , corn silage was introduced to the diet at . kg/d, whereas cs and triticale meal were limited to . kg/d. between d and d , calves received . kg of cs, . kg of ground triticale and . kg of corn silage. at d of age, all calves were fed a diet that met their nutrient requirements according to dlg recommendations and contained soybean meal ( . kg/d), rapeseed meal ( . kg/d), triticale ( . kg/d), vitamin premix ( . kg/d) and molasses pulp ( . kg/d). the mr and cs used in this study contained no antibiotics, and the quality of feedstuffs was controlled by a licensed nutritionist. the trial lasted until d of age, and it was conducted from september to april (table ) . blood was collected from the jugular vein of each calf on d , , , , and of age, into ml vacuette® serum separation probes. sampling was done h after morning feeding. blood was left to clot at °c and subsequently centrifuged ( min × rpm; mpw e centrifuge, mpw med. instruments, warsaw, poland). the serum was collected and frozen at − °c until analysis. immunological parameters (total igg (igg) content, activity of lysozyme (lys) and ceruloplasmin (cp)) and biochemical parameters (liver enzymes: aspartate transaminase and alanine transaminase (ast, alt), cholesterol (chol), glucose (gluc), urea (urea), alkaline phosphatase (ap), lactate dehydrogenase (ldh), triglycerides (tag), creatinine (crea) and total protein (tp) content) were analyzed. the concentrations of igg and tp were measured according to the colorimetric micromethod (sigma, diagnostic kits) proposed by lowry et al. ( ) and modified by siwicki and anderson ( ) . lysozyme activity was determined by the turbidimetric method developed by parry et al. ( ) and modified by siwicki and anderson ( ) , and cp activity was evaluated according to the method of siwicki and studnicka ( ) . the activity (u/l) of alt, ast, ap and ldh was determined by the kinetic method proposed by the international federation of clinical chemistry (ifcc). the concentrations (mg/dl) of crea, urea, tag, gluc, and chol were measured by the modified method of jaffe (crea), the urease-glutamate dehydrogenase uv method (urea), the glucose oxidase-peroxidase method (gluc), the glycerokinase peroxidase-peroxidase method (tag), and the cholesterol oxidase-peroxidase method (chol). all biochemical parameters were determined using the mindray bs- automatic biochemistry analyzer (mindray medical international ltd., shenzhen, china) and diagnostic kits (alpha diagnostics, warsaw, poland). additionally, igg concentration was estimated with commercial milk from dam, liter bovine igg elisa test (cat. no. e - ; bethyl laboratories inc., montgomery, tx, usa) according to the manufacturer protocol. briefly, µl of standards and samples were loaded onto a -well plate and left for h. the plate was then washed four times, followed by addition of detection antibodies into each well and incubated for h. after the incubation plate was washed, streptavidin-conjugated horseradish peroxidase (sa-hrp) was loaded to each well. after min of incubation substrate was loaded into the wells and left for min. next, the reaction was stopped with addition of sulfuric acid, and the absorbance was read at nm with tecan sunrise microplate reader (tecan trading ag, switzerland). samples were analyzed in duplicate and all steps were performed at room temperature. each calf was examined daily by one of the team members who was at the farm during the experimental period. all records were collected after morning feeding. diarrhea was diagnosed according to the protocol given by larson et al. ( ) . scoring included fecal fluidity (from to ; = normal, = watery) and consistency (from to ; = normal, = constipated). diarrhea was diagnosed if calf scored at least in fluidity but no more than in consistency for two days in a row. respiratory disease was diagnosed if at least two of given symptoms occurred simultaneously (coughing, serious nasal and lachrymal discharge, increased respiratory rate, increased breath sounds, and pyrexia), as described by pedersen et al. ( ) . additionally, the effect of colostrum-supplemented transition milk on the growth performance of calves was determined based on changes in body weight (bw) and average daily gain (adg). starting on d , all animals used in the experiment were weighed on a monthly basis until months of age. the data were analyzed using the mixed procedure developed by the sas institute ( ), using repeated measures with a first-order autoregressive covariance structure in time. the model included the fixed effects of treatment, time (d or week), and the interaction between treatment and time. the bonferroni correction method was used to adjust for multiple comparisons. the results are presented as least squares means and sem. the threshold of significance was set at p ≤ . . the mean concentration of colostral igg in dams was . g/l in the cr group and . g/l in the trt group (p < . ). twenty-four h after calving, only one out of calves enrolled to the study failed to reach the g/l serum igg threshold. in contrast, % of cr calves ( / ) and % of trt calves ( / ) had igg below g/l. in trt calves, average serum igg levels were higher than in the cr group (fig. a ) throughout the experiment. despite lower average igg concentrations in colostrum, one day-old trt calves had % higher serum igg concentrations relative to cr calves. it should be noted that serum igg concentrations on d exceeded g/l in both groups. at the end of the transition milk feeding period (d ), serum igg levels were % higher in trt calves than in cr animals (p < . ). however, on d the difference decreased to % in favor of trt calves (p < . ). of note, in trt group compared with cr, we recorded lower incidence of diarrhea ( vs cases, respectively) and respiratory disorders ( vs cases, respectively). colostrum-supplemented transition milk decreased lys activity in trt calves (p < . ) compared with cr calves (fig. b) . although on d , lys activity was % lower in trt calves than in cr calves, the difference between groups was minimized to % on d (p < . ). at the end of the experiment, lys activity was % higher in cr calves than in trt calves. most importantly, trt calves had lower overall lys activity throughout the experiment (p < . ). although cp concentration was higher in trt calves than in cr calves, significant differences were observed only at and d of age ( % and %, respectively). the effect of colostrum-supplemented transition milk on selected serum biochemical variables (alt, ast, ap, ldh, gluc, chol, tag, urea, crea and tp) is presented in table . the treatment influenced ldh activity and the blood concentrations of chol, tag, tp and urea (p < . ), but not the activity of alt, ast and ap, or gluc and crea levels. all analyzed biochemical parameters were affected by sampling time (p < . ). the time × treatment effect was observed for ast, ap, chol, tag, urea and crea (p < . ). in comparison with calves from the cr group, calves from the trt group had lower alt activity between d and d , but the noted difference was not significant (fig. a) . ast activity was % higher in trt calves on d , but it decreased on d and d relative to cr calves (p < . ) (by % and %, respectively) (fig. b) . on d , average ast activity was similar in both groups. ap activity varied throughout the experiment. on d , ap activity was % higher in trt calves, whereas on d and d it decreased by % and %, respectively (p < . ). on subsequent days, ap activity increased in trt calves to reach % of the average value determined in the cr group, but on d , the difference fell to % in favor of trt calves (p < . ). although ldh activity was lower in the cr group throughout the experiment, differences between groups were observed only on d and d (fig. d ) (p < . ). the effect of colostrum-supplemented transition milk on the serum concentrations of gluc, chol, tag and tp at different time points is presented in fig. a -d. trt calves had higher average concentrations of chol and tp compared with cr calves (by % and %, respectively; p < . ) at each time point, but colostrum-supplemented transition milk had a significant effect only on chol levels. in trt calves, average tag concentrations were % higher between d and d , and % lower on d and d relative to cr calves (p < . ). colostrumsupplemented transition milk had no significant effect on serum gluc concentrations in calves. in contrast, urea and crea levels were affected by colostrum-supplemented transition milk (fig. ) . on average, the treatment led to a % decrease in serum urea concentrations in the trt group (p < . ). the treatment also lowered crea concentrations (fig. b) in trt calves between d and d , but a significant treatment effect was reported only on d , and . colostrum-supplemented transition milk influenced bw and adg (p < . ) (fig. a-b) . the average birth weight was kg ( kg in the cr group and kg in the trt group), without significant differences between groups. between weaning and months of age, cr calves were characterized by higher adg than trt calves. however, after weaning, adg was % higher in the trt group between and months of age (p < . ). differences in bw were not observed between groups until months of age. however, at months of age, trt calves were % heavier than cr calves ( kg vs. kg; p < . ). blood igg concentrations in newborn calves are closely related to the quality of colostrum. according to the current industrial recommendations, colostrum should contain at least g/l of immunoglobulins to prevent fpt (morrill et al., ; vasseur et al., ). in the present study, average igg concentration in colostrum determined for the entire herd was . g/l, which points to its high quality. the calculated average concentration of igg was higher than that noted by furman-frątczak et al. ( ) ( . g/l) but lower than that reported by kinal et al. ( ) ( . g/l). according to several authors (faber et al., ; swan et al., ; weaver et al., ) , fpt is diagnosed if serum igg falls below g/l within - h after birth. in our experiment, only one cr calf failed to reach the recommended threshold within h after birth. moreover, despite lower colostrum quality, colostrum-supplemented transition milk increased serum igg concentrations in calves receiving the treatment. in this study, colostrum-supplemented transition milk seem to offer more effective immunological protection due to increased serum igg concentrations on d . in the cr group, % of calves were treated for diarrhea. additionally, the incidence of respiratory disorders in this group reached %. our results correspond with those reported by Żychlińska-buczek et al. ( ) , but are higher than those noted by svensson et al. ( ) and gulliksen et al. ( ) . in contrast, the incidence of diarrhea and upper respiratory tract infections in trt calves was % and %, respectively, supporting a positive effect of colostrum-supplemented transition milk on the health status of calves. although igg are absorbed most effectively within the first h postpartum, the efficiency of this process drops rapidly h after delivery (ontsouka et al., ; suh et al., ) , it seems that the applied treatment enhanced acquiring immunity in supplemented calves. in our study, calves from the trt group had lower lys activity than calves from the cr group. lys is an antimicrobial enzyme that is found in cells and tissues and is involved in non-specific innate immunity; therefore, increased lys activity in early stages of life might be considered beneficial (firth et al., ; gueorguiev et al., ) . calves from the cr group were characterized by lower values of growth parameters than calves from the trt group. the absence of clinical disease symptoms and improved growth in the trt group point to reduced absorption of lys from colostrum, but are not indicative of subclinical infections (piantedosi et al., ) . previous research (li et al., ) indicates that glycation, a posttranslational protein modification which requires the presence of proteins and sugars in the same matrix, significantly decreases lys activity. however, the above conclusions should be formulated with caution due to limited data on changes in lys activity in newborn calves. the activity of cp, a non-specific humoral immunity indicator, is yet another health status parameter that was analyzed in this study. according to calamari et al. ( ) , an increase in cp activity during the first days of life is linked with liver function after birth. thus, our observations of changes in cp activity, accompanied by higher igg concentrations and lower lys activity, indicate that alterations in cp levels are associated with cu release from the liver (abeni et al., ) . the above phenomenon as well as the lower incidence of clinical symptoms of infection and a fast growth rate of calves can be attributed to the healthy development of the gastrointestinal tract (zwierzchowski et al., ) . multiple blood variables were measured during the experiment. the effect of aging on changes in biochemical parameters has been widely discussed (knowles et al., ; mohri et al., ; pavlik et al., ; zwierzchowski et al., ) . however, the aim of this study was to determine alterations in selected parameters associated with gastrointestinal function as a natural consequence of experimental diet manipulation. alt, ast, and ap are non-specific enzymatic disease markers associated with liver function, growth and development of skeletal muscles and bone mineralization (mohri et al., ) . in our study, table the effect of the applied treatment on the serum concentrations of selected blood variables in holstein calves. colostrum-supplemented transition milk had no effect on alt and ast activity (table ) , which can be considered desirable. any changes in the activity of those enzymes should be treated with caution and approached as a potential marker of liver dysfunction. it should be noted that increased ast activity in ruminants was associated with liver damage caused by high-grain diets (castillo et al., ) . according to egli and blum ( ) , increased ap and ldh activity in the first stage of life is related to colostrum ingestion. our findings confirm this hypothesis. the average activity of ap and ldh was higher in trt calves that gained weight at a faster rate. rapid weight gain requires better bone mineralization which stimulates ap release. ldh is also considered as a non-specific marker of disease since there are many factors contributing to its activity variations. additionally, ldh activity differ between tissues, cells, fluids and those differences can be enhanced depending on the disease the animal is suffering from (mohri et al., ; nagy et al., ) . for instance, some authors (Żarczyńska et al., ) link increased ldh activity with muscular dystrophy, but it was not the case in our study since we did not observe any symptoms of this disease. on the contrary, there are reports suggesting ldh fluctuations during respiratory diseases (nagy et al., ) . however, we did not analyze each isoform separately, therefore any conclusion coming from our study should be drawn with caution. calves from the trt group were characterized by higher values of glu, chol, tag, and tp. according to reynolds et al. ( ) , in the first stage of life, glu levels are associated with colostrum intake, whereas in later stages, an increase in glu concentrations is linked with up-regulated corticosteroid secretion due to intensified growth. the latter increases chol and tag levels (cavestany et al., ) . the concentrations of glu, chol, and tag can be altered by infection or diet (bozukluhan et al., ) . this observation is consistent with our results because the incidence of diarrhea was higher in the cr group than in the trt group ( vs. diarrhea cases, respectively). calloway et al. ( ) suggested that tp is a potential fpt diagnostic tool. in the proposed approach, serum tp levels should not decrease below g/l h postpartum. in our study, the average tp concentration in cr calves at d of age was . g/l, which indicates that the suggested safety threshold was not reached. indeed tp was correlated with igg concentration (p < . ), however correlation coefficient indicated moderate link between both parameters (r = . ; figure s ). in out of calves in the cr group tp was below the recommended level of g/l. in contrast, only one trt calf failed to reach the tp concentration of g/l. these results corroborate previous research findings (cuttance et al., ; ježek et al., ; knowles et al., ; mohri et al., ; zwierzchowski et al., ) . urea and crea were monitored as the biochemical indicators of hydration (stockham and scott, ). an increase in urea levels that is not accompanied by changes in crea levels is associated with diarrhea incidence rates (pekcan et al., ) . this observation is consistent with fig. . activity of serum enzymes (alanine aminotransferase -alt (a), aspartate aminotransferase -ast (b), alkaline phosphatase -ap (c), lactate dehydrogenase -ldh (d)) in polish holstein-fresian calves at , , , , and days of age. cr (■; dotted line) and trt calves fed with a mixture of colostrum and transition milk ( : (v/v)) from d to d (◆; dashed line). asterisks indicate differences between groups at the respective time points (p < . ). our findings. all cases of diarrhea were recorded until d of age, thus reflecting changes in urea and crea. interestingly, calves from the trt group were diagnosed with diarrhea at an earlier stage of life, but the supplementation of transition milk with colostrum delivered health benefits at later stages. in previous studies (adams et al., ; thrall et al., ) , elevated crea levels were noted in calves when skeletal muscles were used as a source of energy. this is a highly undesirable situation which negatively affects the growth and development of calves. a decline in urea concentrations was also reported as a potential indicator of accelerated growth in calves (otto et al., ) . in conclusion, this study revealed the benefits of transition milk supplementation with colostrum. additional volumes of colostrum in the diet positively influenced non-specific humoral immunity indicators and serum biochemical parameters in holstein calves. colostrum-supplemented transition milk decreased the prevalence of upper respiratory tract infections and diarrhea incidence. an alternative strategy of calf feeding when high-quality colostrum is unavailable was also proposed in the study. this research received no external funding. i wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. i confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. i further confirm that the order of authors listed in the manuscript has been approved by all of us. i confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. in so doing i confirm that we have followed the regulations of our institutions concerning intellectual property. i further confirm that any aspect of the work covered in this manuscript that has involved experimental animals has been conducted with the ethical approval of all relevant bodies and that such approvals are acknowledged within the manuscript. i understand that as the corresponding author i am the sole contact for the editorial process (including editorial manager and direct communications with the office). i am responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. the authors declare no conflict of interest. fig. . average weight (a) and daily gain (b) of polish holstein-fresian calves at birth and at the age of , , , , and months. cr (■; dotted line) and trt calves fed with a mixture of colostrum and transition milk ( : (v/v)) from d to d (◆; dashed line). asterisks indicate differences between groups at the respective time points (p < . ). effect of average daily gain on body size, metabolism, and milk production of italian holstein heifers raised on two different planes of nutrition and calving at two different ages physiologic differences between twin and single born beef calves in the first two days of life bovine neonatal immunology prevalence of failure of passive transfer of immunity in newborn heifer calves and associated management practices on us dairy operations alterations in lipid profile in neonatal calves affected by diarrhea evoluzione di alcuni parametri ematochimici nel primo mese di vita in vitelli frisoni e meticci comparison of refractometers and test endpoints in the measurement of serum protein concentration to assess passive transfer status in calves effect of moderate cu supplementation on serum metabolites, enzymes and redox state in feedlot calves studies of the transition cow under a pasture-based milk production system: metabolic profiles effect of colostrum administration by use of oroesophageal intubation on serum igg concentrations in holstein bull calves prevalence and calf-level risk factors for failure of passive transfer in dairy calves in new zealand clinical, hematological, metabolic and endocrine traits during the first three months of life of suckling simmentaler calves held in a cow-calf operation case study: effects of colostrum ingestion on lactational performance passive and active components of neonatal innate immune defenses the influence of colostral immunoglobulin concentration in heifer calves serum on their health and growth colostrum management for dairy calves the importance of colostrum for dairy calves. revista colombiana de ciencias pecuarias serum lysozyme activity in new-born calves and relationship to milk yield of their mothers enteropathogens and risk factors for diarrhea in norwegian dairy calves the usefulness of the γ-glutamyltransferase activity and total proteinemia in serum for detection of the failure of immune passive transfer in neonatal calves influence of age on biochemical parameters in calves mineral bioplex supplementation of diets for cows affects colostrum quality and immunoglobulins in calf blood serum changes in the blood biochemical and haematological profile of neonatal calves with age guidelines toward more uniformity in measuring and reporting calf experimental data antibacterial activity of lysozyme and lactoferrin is inhibited by binding of advanced glycation modified proteins to a conserved motif protein measurements with the folin phenol reagent managing the production, storage, and delivery of colostrum hematology and serum biochemistry of holstein dairy calves: age related changes and comparison with blood composition in adults effect of colostral volume, interval between calving and first milking, and photoperiod on colostral igg concentrations in dairy cows nationwide evaluation of quality and composition of colostrum on dairy farms in the united states the effect of respiratory diseases on serum lactate dehydrogenase and its isoenzyme patterns in calves invited review: growth-promoting effects of colostrum in calves based on interaction with intestinal cell surface receptors and receptor-like transporters biochemical blood profile of angoni cattle in mozambique a rapid and sensitive assay of muramidase blood plasma metabolic profile of aberdeen angus bulls during postnatal ontogenesis how milk-fed dairy calves perform in stable versus dynamic groups serum biochemistry and native protein electrophoresis in diarrheic calves with arthritis colostrum and serum lysozyme levels in mediterranean buffaloes (bubalus bubalis) and in their newborn calves evaluation of the brix refractometer to estimate immunoglobulin g concentration in bovine colostrum failure of passive immune transfer in calves: a meta-analysis on the consequences and assessment of the economic impact splanchnic metabolism of dairy cows during the transition from late gestation through early lactation immunostimulation in fish: measuring the effects of stimulants by serological and immunological methods ceruloplasmin activity in carp (cyprinus carpio) urinary system differences in the serum immunoglobulin concentrations between dairy and beef calves from birth to days of age mortality in swedish dairy calves and replacement heifers passive transfer of immunoglobulin g and preweaning health in holstein calves fed a commercial colostrum replacer veterinary haematology and clinical chemistry risk factors for calf mortality in large swedish dairy herds a survey of dairy calf management practices in canada that affect animal welfare does a calf's motivation to ingest colostrum depend on time since birth, calf vigor, or provision of heat passive transfer of colostral immunoglobulins in calves changes of selected biochemical indicators and electrocardiography indicators in nutritional muscular dystrophy in calves influence of a diet containing β-carotene and omega- fatty acids on the biochemical and nonspecific humoral immunity indicators and on the results of experimental calf rearing the main causes of calf mortality in dairy farms in poland the authors thank monika zwierzchowska for the preparation of graphs and the graphic abstract. supplementary material associated with this article can be found, in the online version, at doi: . /j.livsci. . .