key: cord-022383-pz0htccp authors: Kohn, Dennis F.; Barthold, Stephen W. title: Biology and Diseases of Rats date: 2013-11-17 journal: Laboratory Animal Medicine DOI: 10.1016/b978-0-12-263620-2.50010-0 sha: doc_id: 22383 cord_uid: pz0htccp nan The diversity of research for which the laboratory rat is used is probably greater than that associated with any other animal. The laboratory rat is a descendent of the wild rat, Rattus norvégiens, which originated in Asia and reached Europe in the early 1700s. Wild and albino mutants were first used for ex perimental purposes in Europe in the mid-1800s and in the United States shortly before 1900. The Wistar Institute in Phil adelphia was prominent in the development of the rat as a labo ratory animal, for here originated many of the rat strains now used worldwide. Henry Donaldson and his colleagues at the Wistar Institute used these early rat strains for a variety of stud ies dealing with neuroanatomy, nutrition, endocrinology, ge netics, and behavior. The history and evolution of the many rat strains used today have been recently summarized (Lindsey, 1979) . The most commonly used outbred rat stocks in North Amer ica are the Wistar, Sprague-Dawley, Long-Evans, and Holtzman. All are albino except the Long-Evans stock, which is usually marked with a black or gray hair coat over the shoul ders and is sometimes referred to as a "hooded rat." There are numerous inbred and mutant rat strains, although the number is less than that in the mouse. Table I lists the more commonly used strains. There are a rather large number of commercial vendors of laboratory rats in the United States. Most of the stocks and strains mentioned above can be obtained from more than one source. Although the origin of an outbred stock, such as the Sprague-Dawley, may have been the same for a number of vendors, in many cases it has been 20 to 30 years since such a stock has been removed from its original breeding colony. Ac cordingly, the genotype of outbred stocks and inbred strains may vary among sources and be reflected by differences in data when multiple sources of rats are used. A standardized scheme of identifying stocks and strains of rats has been devel oped and is now used by nearly all commercial vendors. More over, it is important that authors correctly identify stocks and strains that are used in their research since the success in re peating the work in another laboratory may be dependent upon the genotype (source of the rat). Table II summarizes the stan dardized nomenclature for outbred stocks as developed by the Table I Commonly Used Strains "National Institutes of Health (1981) . 1. Letters preceding the colon designate the supplier/breeder code consist ing of a capital and two or three lowercase letters 2. Capital letters following the colon are used by a breeder to identify his stock 3. Letters in parentheses denote origin of stock 4. Subscript symbols indicate rearing by means other than natural mother (f, fostered; fh, fostered by hand) International Committee on Laboratory Animals (ICLA). Table III contains the scheme for designating inbred strains of rats (National Institutes of Health, 1981) . "Animals for Re search" (National Academy of Sciences, 1979), a directory of sources for laboratory animals sold in the United States and Canada, lists all rodents according to standard nomenclature, and is a valuable aid in purchasing laboratory animals. Commercial production of rats has markedly changed since the 1960s due to the development of hysterectomy-derived and barrier-maintained breeding colonies. Prior to the application of this technology to production colonies, infectious diseases were ubiquitous in rats from most sources. Today, vendors can be selected who offer pathogen-defined animals for most stocks and strains. Concomitant with changes in commercial sources of rats are the major advances made in the design and construction of institutional animal resources and husbandry practices within them. Optimum housing of rats today includes provisions for quarantining and isolation of animals according to vendor subpopulations that have a similar microbial flora. There are various levels of sophistication to provide barriers to the spread of infections in rat colonies. Since many rat pathogens are spread by aerosol, ventilation control is very important. Nonrecirculating room air or high-efficiency panic ulate air (HEPA)-filtered air has become a design standard in modern animal facilities. As discussed in Chapter 17, clean/contaminated corridor-designed facilities aid in contain ment against the spread of pathogens by aerosol, personnel, Table III Nomenclature for Inbred Rats 1. The strain designation is given in capital letters followed by a slash 2. The substrain designation follows the slash and is given as numbers or as individual or company codes. Numbers are used to denote substrains that were derived from a common strain but separated before the completion of inbreeding 3. Subscript symbols indicate rearing by means other than natural mother and contaminated equipment. A more complete barrier system may include an entry area in which incoming supplies and equipment are sterilized and in which personnel shower and don sterile clothing and filter masks before entering animal rooms. More recently, laminar-flow (mass air displacement) rooms and mobile units have become popular because they can be incorporated in existing buildings that lack design charac teristics mentioned above. Environmental control in rat rooms is important to the com fort and health of the animals, as well as to the consistency of data derived from the rats. Room temperatures between 72 and 76°F are desirable, and the relative humidity should range be tween 40 and 60%. Daily fluctuations in temperature and hu midity act as significant Stressors. These fluctuations may be associated with the environmental control system of a building or may be induced by procedures such as cleaning floors with a water hose or high pressure sprayer. Twenty-four-hour tem perature/humidity recorders are useful in detecting changes in environmental conditions. Light intensity should be evenly distributed to all animals within a room. Seventy-five to 125 fc have often been suggested as an optimal range for light inten sity. However, recent evidence indicates that this intensity can induce retinal degeneration in albino rats (Anver and Cohen, 1979) . Light-timing devices are a convenient means to provide desired day/night cycles such as 12-12 or 14-10 hr. Caution should be exercised in the use of insecticides and air-deodorizing chemicals, since some have been shown to in duce hepatic microsomal enzymes in rats. Accordingly, their use in animal rooms is not usually recommended (Baker et al., 1979a) . Rats can be housed in either wire-or solid-bottom cages. Wire-bottom cages are more frequently used since they are less labor-intensive. Frequency of cage and litter pan changing is a function of animal density. Solid-bottom cages should be sani tized two to three times per week, while wire-bottom cages should be sanitized on a weekly or biweekly schedule with litter pans changed two or three times per week. Feed should be contained in hoppers. Either automatic systems or bottles are satisfactory for providing water to rats. Some caution is necessary when using automatic systems, since weanling and newly arrived rats may not drink initially from such devices. To avoid undesirable microbial contamination, water bottles should be sanitized before they are refilled and automatic sys tems should be drained and flushed when racks are sanitized. Acidification of water to a pH of 2.5 to 2.8 or chlorination at 8 to 12 ppm will control Pseudomonas aeruginosa contamina tion of water (Weisbroth, 1979) . However, this treatment is not necessary for immunocompetent animals. Wood shavings or chips are the most commonly used contact bedding mate rials. Hardwoods are preferred to softwoods, since the latter are capable of inducing hepatic microsomal enzymes (Baker et al, 1979a) . This section summarizes some of the anatomical characteris tics of the rat with emphasis on characteristics that are unique. The reader is advised to refer elsewhere in the literature for comprehensive descriptions (Bivin et al., 1979; Caster et al., 1956; Hebel and Stromberg, 1972; Smith and Calhoun, 1972; Zeman and Innés, 1963) . The rat dental formula is 2(1 1/1, C 0/0, PM 0/0, M 3/3) = 16. The incisors are well developed and grow continuously. The rat lacks tonsils and water taste receptors. The major pairs of salivary glands are the parotid, submandibular (submaxillary), and sublingual. The parotid gland is a serous gland consisting of three to four lobes and is located ventrolaterally from the caudal border of the mandible to the clavicle. The submandibular glands are mixed glands located ventrally between the caudal border of the mandibles and the thoracic inlet. The sublingual glands are mucous glands and are much smaller than the parotid and submandibular glands. They are located at the rostral pole of the submandibular glands to which they are closely associated. Brown fat deposits are present in the ventral cervical region. These multilocular deposits are well demarcated and can be confused with salivary glands or lymph nodes. The stomach of the rat is divided into two parts; the forestomach (nonglandular) and the corpus (glandular). The two portions are separated by a limiting ridge. The esophagus en ters at the lesser curvature of the stomach through a fold of the limiting ridge. This fold is responsible for the inability of the rat to vomit. The forestomach, which is thinner than the cor pus, is linked with an epithelium similar to that of the esopha gus and extends from the cardia to a narrow band of cardiac glands at the junction of the glandular portion. The small intestine is composed of the duodenum (10 cm), jejunum (100 cm), and ileum (3 cm). The cecum is a thinwalled, comma-shaped pouch that has a prominent lymphoid mass in its apical portion. The colon is composed of the as cending colon, with prominent oblique mucosal ridges, trans verse and descending colons, with longitudinal mucosal folds; followed by a short rectum that is confined to the pelvic canal. The liver has four major lobes (median, right lateral, left, and caudate) and is capable of regeneration subsequent to par tial hepatectomy. The rat has no gallbladder. The bile ducts from each lobe form the common bile duct that enters the duo denum 25 mm from the pyloric sphincter. The pancreas is a lobulated, diffuse organ that extends from the duodenal loop to the gastrosplenic omentum. It can be dif ferentiated from adjacent adipose tissue by its darker color and firmer consistency. Up to 40 excretory ducts fuse into 2-8 large ducts, which empty into the common bile duct. The nasal cavity is not markedly different from that of other mammals. The rat has a maxillary recess (sinus) located be tween the maxillary bone and the lateral lamina of the ethmoid bone. The recess contains the lateral iiasal gland (Steno's gland) that secretes a watery product that is discharged at the rostral end of the nasal turbinate. It has been postulated that the nonviscous secretion contributes to the humidification of in spired air and acts to regulate the viscosity of the mucous layer overlying the nasal epithelium. The left lung has one large lobe, and the right lung is divided into four lobes (cranial, middle, accessory, and caudal). The pulmonary vein in the rat has cardiac striated muscle fibers within its wall that are contiguous with those in the heart. The rat does not have an adrenergic nerve supply to the bronchial musculature, and bronchoconstriction is controlled by vagai tone. Unlike the guinea pig, the rat lung has a low concentra tion of histamine (Bivin et al., 1979) . The heart and peripheral circulation in the rat differ little from that of other mammals. The blood supply to the heart is derived from both coronary and extracoronary arteries. The latter arise from the internal and subclavian arteries. The right kidney, which is more craniad than the left, has its cranial pole at the L, vertebra and its caudal pole at the level of L 3 . The rat kidney is unipapillate as are kidneys of other ro dents, lagomorphs, and insectivores. Having only one papillus and calyx makes the rat useful for studies in which cannulization of the kidney is done. The presence of superficial nephrons in the renal cortex has made the rat widely used as a model for studying nephron transport in an in vivo micropuncture system. The male reproductive system has a number of highly devel oped accessory sex glands. These include large seminal vesi cles, a bulbourethral gland, and a prostate gland composed of the coagulation gland (dorsocranial lobe) and ventral and dorsolateral lobes. The inguinal canal remains open throughout the life of a rat and testes descend initially by 40 days of age. The female rat has a bicornate uterus that is classified as the duplex-type because the lumina of the uterine horns are com pletely separate with paired ossa uteri and cervices. The female urethra does not communicate with the vagina or vulva, but rather exits at the base of the clitoris. The brain of the rat has very large olfactory bulbs and a nonconvoluted cerebrum. The hypophysis is behind the optic chiasma and is attached to the base of the brain by a thin hol low stalk, the infundibulum. The blood supply to the brain is from the internal carotid and vertebral arteries. Blood leaves the brain via a system of sinuses that are enclosed in the dura mater. The ventricular system is similar to that of other ani mals, but the rat lacks a foramen of Magendie. It must be recognized that many of the normal values deter mined for a specific group of rats may be accurate for only that rat stock/strain, source, and conditions under which they are held. Selected physiological, hematological, and clinical bio chemical parameters are listed in Tables IV-VII. More com plete information on biological values is available (Mitruka and Rawnsley, 1977; Ringler and Dabich, 1979) . Nutritionally adequate diets are readily available from com mercial sources. These standard rations are quite satisfactory for most applications. However, for some types of experimen tation there are factors, other than nutritional adequacy, which must be considered. The nutrient composition of diets and the contamination of feed components by mycotoxins, antibiotics, synthetic estrogens, heavy metals, and insecticides may have a profound impact on many studies. For instance, caloric intake and the percent of fat and protein in the diet of rats influence the incidence of neoplasia (Altman and Goodman, 1979) . Sim ilarly, various contaminants have an adverse effect on data from toxicologie, gerontological, and reproductive studies. Standard commercial diets are formulated from natural ingre dients and will vary in nutrient composition on a batch-tobatch basis due to differences in type and quality of ingredients used. Commercial makers of rodent feeds take precautions to preclude the presence of contaminants in feeds, but only a few products have a defined profile of maximal levels of heavy metals, aflatoxins, chlorinated hydrocarbons, and organophosphates. For some investigative purposes, feeds formulated with re fined ingredients (purified diets) or with chemically defined compounds are useful when control of nutrient concentrations is essential (National Research Council, 1978) . These diets are, however, too expensive for general use. Baker et al. (1979b) and Bivin et al. (1979). Rats are commonly fed ad libitum, and food intake will vary according to requirements for growth, gestation, and lactation. The nutritive requirements for the rat are listed in Table VIII. The duration of storage and the temperature at which feeds are stored prior to use effect the nutritive quality of diets. Com mercial diets are formulated to have a shelf life of up to 6 months. However, storage in a hot or damp environment will reduce this shelf-life. To help assure that only fresh diets are used, products should be used which have milling dates identi fied on their containers (see Chapter 17). Sexual maturity occurs between 6 and 8 weeks for both sexes, although the onset of first estrus in females occurs at about 5 weeks. The vagina opens between 34 and 109 days, and the testes descend between 15 and 51 days, although they remain fully retractable in adults. Rats ovulate spontaneously, but ovulation can also be induced by forced coitus during nonestrous intervals. Vaginal stimulation during mating is impor tant in rat reproductive physiology. The more often a male inserts his penis into the vagina prior to ejaculation, the greater the probability of a resulting pregnancy. However, natural or artificial stimulation of the vagina within 15 min of a first mat ing will abrogate pregnancy from the first mating by inhibition of sperm transport. A 12-hr estrous period recurs every 4 to 5 days and after parturition, without seasonal variation. Estrus can be suppressed when females are housed in groups and syn chronized in the presence of a male or its excreta (Whitten effect), but this effect is not as pronounced as in the mouse. Female fertility wanes at 600 to 650 days, but estrous cycles may continue through 32 months. Male fertility is lost between 16 and 20 months. Fertility of both sexes is generally regarded as maximal between 100 and 300 days of age (Adler and Zoluth, 1970; Baker, 1979; Farris, 1963; Lane-Petter, 1972; Leathern, 1979) . Males will mount estrous females numerous times with one or two rapid ejaculations in the course of 15 to 20 minutes. Ejaculated semen coagulates, forming a copulatory plug that remains in the distal vagina for a few hours, after which time it dissolves or is extruded. Copulation is usually nocturnal. Du ration of gestation varies with strain, age, litter size, and other variables, and ranges from 19 to 23 days, with an average of 21 or 22 days. Primiparous females tend to have a slightly longer gestation than multiparous females (Farris, 1963) . Estrus can be detected in a number of ways. Females in es trus are hyperactive and brace themselves when touched. Their ears quiver when they are stroked on the head or back, and stimulation of the pelvic region induces lordosis (Farris, 1963) . The vulva becomes swollen, and the vagina becomes dry in contrast to the moist pink wall during metestrus or diestrus. As proestrus occurs (approximately 12 hr), smears of va ginal cells contain nucleated epithelium, leukocytes, and occa sional cornified cells. Estrus (approximately 12 hr) begins with about 75% nucleated and 25% cornified cells, with cornified cells predominating as estrus continues. Metestrus follows (ap proximately 21 hr) with large numbers of leukocytes and corn ified cells, which form abundant caseous vaginal detritus. Metestrus is characterized by the presence of large flat nucle ated (pavement) cells. Diestrus persists for approximately 57 hr (Baker, 1979; Farris, 1963) . Breeding dates can be established by examination of vaginal swabs for spermatozoa or examining the distal vagina or cage pan for copulatory plugs. Timed pregnancies are best achieved by placing the female in the male's cage in the afternoon and examining her for a plug or spermatozoa the following morning. Abdominal enlargement becomes evident at about 2 weeks. Pseudopregnancy is rare (Lane-Petter, 1972) . Rats reproduce successfully under a variety of conditions, but husbandry practices can significantly influence fecundity. Rats can be bred as monogamous pairs, taking advantage of postpartem estrus for maximal breeding efficiency. Polyg amous breeding is more economical, since only one male can be kept with 6 to 9 females. Females are often removed to a separate cage prior to whelping, since they may not tolerate other females in the cage while nursing. They will tolerate their mates, however. Females with litters do best on clean dust-free wood shavings in solid-bottom cages. Due to heat regulation, pups neither thrive in overly spacious cages with wide flutuations in ambient temperature, nor in overly crowded cages where they cannot dissipate heat. The recom mended cage floor area for a female and her litter is 150 in. 2 . Ambient room temperature and humidity should be within the acceptable range with minimal fluctuation. High ambient tem perature can cause male infertility (Baker, 1979; Baker et al., 1979a; Lane-Petter, 1972) . The rat estrous cycle is particularly sensitive to variations in light. Daily lighting at an average of 100 fc with a spectrum approximating natural light for 12 to 16 hr is best for breeding. Constant light for as few as 3 days may induce persistent es trus, hyperestrogenism, polycystic ovaries and endometrial hy pertrophy or metaplasia (Baker et ai, 1979a; Gralla, 1981) . Nutrition may also affect reproductive performance. Re quirements for certain components are increased during preg nancy and growth, but overfeeding is deleterious. Caloric re striction may actually improve fertility and possibly reproduc tive life of the female (Leathern, 1979) . Excess dietary protein can adversely affect female sexual development. Vitamin defi ciencies can cause infertility, particularly those vitamins (A, E, riboflavin, and thiamin) that are most labile to autoclaving or deterioration (Baker, 1979) . It is not necessary to add nesting material to bedding for successful breeding, but rats will utilize it if offered. Shredded paper or cotton nesting material will be readily accepted and used by prepartem and nursing dams. Parturition is heralded by pronounced postural stretching and rear leg extension. A vagi nal discharge may be noted li-4 hr prepartum. Parturition is usually complete in 1 or 2 hr, but can range from a few minutes to several hours depending on litter size. Dystocia is exceed ingly rare. Litters average between 6 and 12 pups, with highest fecundity through the sixth litter. Inbred rats tend to produce smaller litters. Although infrequent, cannibalism is most apt to occur with nervous or primiparous females subjected to stress (Farris, 1963; Lane-Petter, 1972; Leathern, 1979) . The neonate weighs about 5i gm, depending on litter size, sex, strain, and physical condition of the dam. Pups are born hairless, blind, with closed ears, undeveloped limbs, and short They are fully haired between 7 and 10 days (Baker, 1979; Farris, 1963; Lane-Petter, 1972) . Maternal antibody is trans ferred in utero, via the yolk sac and by intestinal absorption of colostrum by the neonate for up to 18 days after birth (Chev ille, 1976) . Optimal weaning age is 20-21 days, although pups can be weaned as early as 17 days. Differentiation of sex in adult rats is relatively easy after the testes descend. The adult testes can be readily retracted through large inguinal canals. Male neonates have a larger genital papillus and the anogenital space is greater in males than females. From National Research Council (1978) . h Adequate to support growth, gestation, and lactation; based on 90% dry matter. ( Linoleic acid, 0.6%, is required. ^One-third to one-half can be supplied by L-cystine. ^One-third to one-half can be supplied by L-tyrosine. ^Mixture of glycine, L-alanine, and L-serine. ^Vitamin A, 1 IU = 0.300 ìg retinol, 0.344 ìg retinyl acetate, 0.550 ìg retinyl palmitate. Vitamin D, 1 IU = 0.025 ì£ ergocalciferol. Vitamin E, 1 IU = 1 mg DL-a-tocopheryl acetate. Artificial insemination can be achieved in rats, but the major obstacle is the coagulative properties of their semen. Sperm can be obtained by maceration of the epididymis and vasa or by electroejaculation, although the latter method is unreliable and the semen often rapidly coagulates. Coagulation can be eliminated by prior surgical extirpation of the seminal vesicles and coagulating glands without significant effect on fertility. Semen can be diluted with a number of media but frozen stor age of rodent semen has met with little success. Insemination can be achieved surgically by direct injection of seminal fluid into the uterus and by nonsurgical means. Successful concep tion seems to require not only insemination during estrus but also induction of pseudopregnancy by mating with a vasectomized male or mechanical stimulation within a few hours (before or after) insemination. Egg harvest for transfer can be accomplished by excision of the preovulatory ovaries and teas ing from gravid follicles or recovery from the oviduct or uterus by flushing with transfer medium. Superovulation by injection of gonadotropisms may enhance yield, but is usually not nec essary. Eggs are generally injected directly into the uterus but the recipient uterus must be at the same stage of the uterine cycle (Bennet and Vickery, 1970) . Synchronization of estrus can be achieved by vaginal inser tion of polyurethane sponges containing 0.75 mg medroxyprogesterone for 7 days. Females are then put in a cage previously occupied by male rats, sponges are removed, and the rats are injected with 3 IU of pregnant mare's serum. Within 34 hr, 93% will be in estrus. This can also be attained by administer ing 40 mg medroxyprogesterone in 200 ml ethanol/liter drink ing water, prepared fresh daily for 6 days, then intramuscular injection of 1 IU of pregnant mare's serum (Bennet and Vick ery, 1970 ). The rat has been utilized extensively in a variety of research fields, including behavioral science. Rats are docile, adapt to new surroundings, tend to explore, and are easily trained to a variety of sensory cues by positive or negative reinforcement. Rats sleep during daylight hours and activity, including feed ing, is greater during the night and early morning. Laboratory rats are easily handled, but strain differences exist. Sprague-Dawley and LEW rats tend to be less fractious than Long Evans or F344 rats. Docility is improved with routine and proper handling. Rats become nervous and refractory to han dling when they hear others squeal. Nutritional deficiency, particularly hypovitaminosis A, and mishandling can make rats vicious. Rats seek entry into small openings, a trait that is utilized for coaxing them into restrait apparatus. Like other rodents, rats are coprophagic, which must be taken into con sideration when administering drugs, measuring fecal output, or performing nutritional studies. Unlike mice, rats are less apt to fight, and males can be housed together. In addition, rats are not gregarious like mice, and seem to tolerate single caging well. Experimental studies indicate significant changes in plasma corticosteroid levels, depending on cage cohort size. Levels tend to be least in rats housed singly, to increase in groups up to 5, to decrease in larger groups up to 10-12, then rise again in groups up to 30 (Lane-Petter, 1972). Infectious agents constitute a significant environmental vari able that impacts on research data derived from laboratory rats. As is the case with other species, infectious agents induce a wide range of diseases in the rat that vary from inapparent to overt clinical disease. Most investigations use large numbers of rats in which a specific group or colony consists of several to hundreds of rats. Accordingly, emphasis on disease is one of prevention and placed at the colony level rather than on a sin gle or a few animals. Curative use of antibiotics, which is important in the treatment of bacterial diseases of nonrodent species, is rarely useful in the laboratory rat. Administration of drugs to obtain therapeutic blood levels is difficult to achieve in a colony; also some animals may improve clinically but re main colonized by the pathogen and serve as carriers, reinfecting other animals. Rats seldom show clinical signs of disease upon arrival to the laboratory from commercial sources. However, these rats may harbor pathogens that are of low to moderate virulence and that are capable of severely compromising the health of animals when the rats are exposed to various types of experimental stress. Moreover, some of these pathogens may never cause clinical disease, yet induce microscopic lesions or biochemical aberrations that can have profound effects on research data. For these reasons, investigators and clinicians should be aware of the pathogen status of the animals used in studies, both ini tially and throughout the course of the studies. This section on infectious diseases contains those agents that are of principal importance to the investigative use of the rat. a. Streptococcosis. The causative organism, Streptococcus pneumoniae, is a gram-positive coccus that is rather ubiq uitous among humans and animals. Streptococcus pneumoniae is frequently recovered from respiratory tract lesions in guinea pigs, nonhuman primates, and some domestic animals. In hu mans, it is often present in the nasopharynx in the absence of clinical symptoms of infection. Upper respiratory tract infec tion of conventionally raised rats has been reported to be com mon. However, it is seldom present in barrier-maintained, commercial rat sources. As in pneumococcal disease in hu mans, a number of serological types have been associated with respiratory disease in rats. Streptococcus pneumoniae infection in rats often remains lo calized in the nasopharynx without the development of overt disease. A shift in the host-parasite balance due to stress or concurrent infection with another pathogen may result in bronchopneumonia and bacteremia. The most common signs of respiratory disease are serous to mucopurulent nasal discharge and "red tears" due to porphyrin pigments secreted from the Harderian glands, dyspnea, rales, and depressed activity. Ani mals will often die within a few days after the onset of pneu monic signs. The severity and prevalence of clinical disease within an infected colony are associated with environmental conditions that induce stress (e.g., experimental manipulation, overcrowding, fluctuations in ambient temperature and humid ity, and copathogens). Although all age groups are susceptible to infection and clinical disease, young animals are more apt to be clinically affected. Transmission between rats is by aerosol droplet. Although both humans and rats can carry the same serotypes of S. pneumoniae, the authors are unaware of evi dence indicating zoonotic or human-to-animal transmission. The most characteristic gross lesions are pulmonary consol idation and fibrinopurulent pleuritis and pericarditis ( Fig. 1 ). An extensive fibrinopurulent peritonitis, orchitis, or meningitis may occur as well. If a bacteremia occurs early, the disease may be acute with few gross lesions. Streptococcus pneu moniae induces an outpouring of exudate rich in fibrin, neutrophilic leukocytes, and erythrocytes into the alveoli. Bron chioles are filled with neutrophilic leukocytes. Embolie lesions may occur in multiple tissues which include the spleen, liver, kidneys, joints, and brain. Streptococcosis is diagnosed by clinical signs, characteristic lesions, and isolation of S. pneumoniae from lesions. The per icarditis, pleuritis, and pleural effusion noted above differenti ate pneumococcal disease from pneumonia due to Mycoplasma, although the two pathogens often are superimposed. This organism produces an á-hemolysis on blood agar plates similar to that of the Streptococcus viridans group. Streptococ cus pneumoniae isolates are most commonly differentiated from nonpathogenic S. viridans by the sensitivity of the former organism to Optochin (hydrocuprein hydrochloride). Optochin-impregnated discs are placed on a blood agar plate which has been inoculated with a pure culture of the clinical isolate. If the isolate is S. pneumoniae, a distinct zone of growth inhibition will be present around the disc. Although typing of S. pneumoniae isolates is seldom done today, one can type an isolate by reacting known specific S. pneumoniae antisera with S. pneumoniae isolates. This serological test is the Neufeld-Quellung reaction and is based on the capsular swelling that is induced by specific antiserum. There is no effective means to control S. pneumoniae infec tion once it is enzootic in a colony. Benzathine penicillin (30,000 units/200 gm body weight) may be helpful in reducing the severity of the disease and as an aid in limiting infections to a subclinical mode in some animals. However, antibiotics will not eliminate the organism from rat colonies. Hysterectomy rederivation of breeding stock from infected colonies is an ef fective method of initiating new stock free from pneumococcal infection (Weisbroth, 1979) . b. Pseudotuberculosis (Corynebacteriosis). The causative agent of pseudotuberculosis is the gram-positive bacillus, Corynebacterium kutscheri. On occasion, other Corynebacterium species can cause similar syndromes in rats. Typically, the or ganism causes inapparent infections in rats, with exacerbation of respiratory disease under conditions of stress. When clinically ill, the most commonly seen signs include serous oculonasal discharge, dyspnea, anorexia, and loss of weight or retarded growth. Animals with severe pulmonary signs usually succumb within several weeks, while rats with less severe signs often survive much longer. Most rats will have inapparent infections in which C. kutscheri cannot be isolated from internal organs. Little is known concerning how C. kutscheri is carried or transmitted within a colony. It has been suggested that the organism is transmitted via aerosol droplet or direct contact. Once rats are infected, a hematogenous spread may be involved, since lung lesions are initially interstitial and not bronchial. Gross lesions are characterized by a variable number of grayish-yellow foci surrounded by red zones, particularly in the lung (Fig. 2) . In longer-standing cases, individual foci co alesce into raised lesions 1 cm or larger in diameter. Occasion ally, fibrous adhesions occur between the lungs and thoracic walls. Similar lesions may be seen in other organs, including the liver, brain, and kidneys. The hepatic lesions resemble tu bercles and have caseous centers and fibrous capsules. Prepucial adenitis, arthritis, and otitis media may also be caused by C. kutscheri. The lesions in various target organs appear to be due to septic emboli. Pulmonary lesions initially consist of a polymorphonuclear cell and macrophage infiltrate of the bronchioles and interstitial tissue with a round cell infiltrate occurring later. Bronchi become impacted with polymorphonuclear cells and necrotic leukocytes. Giemsa or Gram staining of infected tissues will reveal the rod-shaped C. kutscheri organisms. Diagnosis of C. kutscheri infection is made on clinical signs, gross and microscopic lesions, and isolation of the bacterium from infected tissues. Although the respiratory signs are simi lar to those present with mycoplasmosis, the rapidity with which C. kutscheri clinically affected rats succumb helps dif ferentiate it from Mycoplasma pulmonis-'mduced disease. Un like streptococcosis, fibrinopurulent pericarditis, peritonitis, and pleural effusion are not seen. Whereas peribronchial lymphoid hyperplasia is a dominant lesion in mycoplasmosis, it is unremarkable in C. kutscheri infections. Corynebacterium kutscheri is easily recovered from lesions and upper respiratory tract exudates by culturing on blood agar plates incubated aerobically at 37°C. Epizootics of pseudotuberculosis may occur in conven tionally raised breeding colonies, but rarely occur in barrierraised colonies. Epizootics often can be retrospectively associ ated with an environmental stress (e.g., fluctuation in ambient temperature or ventilation). Culling of ill animals will not eliminate C. kutscheri from animals remaining in a colony. Isolation of the organism from animals with subclinical infec tions is not usually successful. For this reason, cortisone ad ministration has been advocated as a means for surveillance of infection in colonies prior to necropsy and culturing for C. kutscheri. In the past, most serological methods have been un satisfactory in detecting antibody in animals with inapparent infections (Weisbroth, 1979) . Recently, however, enzymelinked immunoabsorbant assay (ELISA) has been shown to be capable of detecting antibody in animals without clinical signs of infection (Ackerman et al., 1984) . Hysterectomy derivation is an effective means to establish a C. kutscheri-free colony. Antibiotic therapy will not eliminate C. kutscheri from a colo ny, but a 7-day regimen of penicillin has been reported to be effective in curtailing an epidemic of C. kutscheri-'mductd pneumonia (Fox et al., 1979) . Since C. kutscheri infection is, in most cases, inapparent and manifests itself whenever the host is sufficiently stressed, it can be a significant problem in experimentally stressed rats. c. Tyzzer's Disease. Tyzzer's disease is caused by the gram-negative, spore-forming rod, Bacillus piliformis. This organism, which is not a true Bacillus, is an intracellular pathogen that has not been cultivated on artificial media, and is, as yet, taxonomically undefined. In the laboratory, B. piliformis is propagated in the yolk sac of embryonated chick eggs. This disease occurs in other rodent species and appears to be widely distributed in many nonrodent species, but there ap pears to be a degree of species specificity among B. piliformis strains. It occurs occasionally in conventionally raised rat colo nies. The vegetative form of B. piliformis is unstable in the environment. However, spores of the organism are relatively stable and are believed to be the source of transmission among animals. Clinical signs associated with Tyzzer's disease are not partic ularly distinctive and, accordingly, only suggestive in making a diagnosis. Typically, affected rats are apt to be adolescents with signs such as lethargy, weight loss, and distended abdo mens. Diarrhea is not a common sign in rats with B. piliformis infection. Animals displaying clinical signs generally die with in several weeks. Clinically inapparent infections occur and are most probably responsible for transmission of the organism within a colony. Clinically evident Tyzzer's disease is usually associated with experimentation that compromises the immunocompetence of rats. The most remarkable gross lesions involve the liver, ileum, and myocardium. Hepatic lesions consist of numerous small, pale foci on the surface and within the parenchyma. The intes tinal lesion has been termed "megaloileitis" due to a segmen tai dilatation and inflammation of the ileum (Fig. 3) (Jonas et ai, 1970) . Heal distension is not always present. In some rats, circumscribed gray foci also occur in the myocardium. The pathogenesis of the disease is believed to involve a pri mary intestinal infection with spread to the liver via the portal circulation. Bacillus piliformis invades enterocytes, resulting in villus shortening, inflammation, necrosis, and hemorrhage. Intracellular organisms are demonstratable in epithelium of crypts and villi. The necrotic foci in the liver are most often present near vessels. Surrounding these foci are varying num bers of leukocytes, macrophages, and fibroblasts. Intracytoplasmic bacteria may be seen in hepatocytes at the periph ery of the lesions, but may be present in very small numbers and thus be hard to find. Organisms are also found in myocar dium around foci of necrosis (Weisbroth, 1979) . A presumptive diagnosis can be made by the gross lesions, but a definitive diagnosis is dependent upon observation of the organism within hepatocytes, intestinal epithelium, or myocar dium. Impression smears of liver taken at necropsy and stained with Gram, Giemsa, or méthylène blue stains may be useful for a rapid diagnosis. However, formalin-fixed specimens stained by Giemsa or Warthin-Starry methods are usually per formed to confirm a diagnosis. The ileal distension seen in rat Tyzzer's disease must be differentrated from other causes of adynamic ileus, particularly chloral hydrate-induced lesions. Prevention of Tyzzer's disease in a colony is dependent upon a barrier that excludes entry of the agent by contaminated cages, equipment, and infected animals. Routine cage sanita tion probably is ineffective in killing the spores of B. piliformis, but exposure of spores to 80°C for 30 min has been shown to inactivate them. Sodium hypochlorite (0.3%) is an effective disinfectant (Ganaway, 1980) . Although antibiotics have been shown to be effective under experimental conditions in mice, there is no evidence to indicate that antibiotic therapy can be of value under natural conditions within a colony of rats (Weisbroth, 1979) . d. Pasteur elio sis. Pasteur ella pneumotropica frequently infects conventionally raised rats and has been recovered occa sionally in rats from barrier-and axenic-maintained colonies. It is a pathogen of very low virulence, and most infections remain clinically inapparent. Only a relatively few reports doc ument P. pneumotropica as a primary pathogen in cases of penumonia, otitis media, and conjunctivitis. As a copathogen with either M'ycoplasma pulmonis or Sendai virus, it has a con tributory role in the resultant respiratory lesions and otitis. Its localization is not limited to the respiratory tract, since it is frequently isolated from the oral cavity, intestinal tract, and uterus. It also has been associated with mastitis and furunculosis in rats. It has been suggested that P. pneumotropica is essentially an enterotropic rather than a pneumotropic orga nism. The intestinal tract is probably the primary site for local ization of the organism in subclinical infections. Horizontal transmission is by the oral-fecal route and direct contact. Since P. pneumotropica is frequently carried in the uterus, vertical transmission can occur, and, accordingly, this can compromise the microbial status of axenic and gnotobiotic colonies. Distinctive clinical signs and lesions do not occur with P. pneumotropica-induoed disease. Accordingly, a diagnosis must be based upon its isolation as the sole pathogen or, as in many cases, as a copathogen within lesions. Blood agar medi um is satisfactory for primary isolation from nonenteric sites. However, for recovery from the intestinal tract, enrichment in a medium such as GN broth is recommended before isolation is attempted on blood agar plates (Weisbroth, 1979) . Hysterectomy derivation and barrier maintenance are the only means to control infection. However, particular attention must be made to ensure that hysterectomy-derived young came from dams that had culturally negative uteri. Antibiotic thera py is not effective in eliminating the organism from a colony. e. Salmonellosis. Salmonella species that infect rats in clude Salmonella enteritidis, S. typhimurium, S. dublin, and S. meleagridis. Salmonellosis, which was once a major cause of disease in laboratory rat and mouse colonies, is rarely reported in either species today. However, it still exists in wild popula tions of rodents and, therefore, remains a potential threat to laboratory rodents. Infection in an immunologically naive colony typically re sults in an epizootic of clinically affected rats and a varying proportion of animals with inapparent infection. These latter animals act as subclinical carriers to render the infection as enzootic in a colony. Acute outbreaks will occur intermittently whenever immunological and other host defense mechanisms are altered. Signs associated with salmonellosis in the rat are anorexia, depressed activity, starry hair coats, and soft to formless feces. Affected animals die in 1 to 2 weeks. Lesions that occur in salmonellosis differ depending on the stage of the disease. Salmonellae penetrate the intestinal mucosa at the level of the ileum and cecum. The earliest le sions occur in this locale and consist of a mild dilatation, thick ened intestinal walls, and a granular mucosal surface. Involve ment of the reticuloendothelial system is reflected by enlarged Peyer's patches, mesenteric lymph nodes, and spleen. In some infected animals, a bacteremic state occurs that results in the demise of the host before the development of further lesions. However, in animals not succumbing to septicemia, ulcération of the ileal, colonie, and cecal mucosa occurs. Histologically, the villus epithelium of the ileum is markedly degenerated, and the lamina propria is infiltrated with neutrophils and mac rophages. Concomitant with intestinal lesions is the develop ment of focal necrosis and granulomas in the spleen and liver due to hematogenous spread of the organism (Buchbinder et al, 1935; Maenzae/fl/., 1970) . In rats who are intermittent or chronic shedders of salmonel la, the most remarkable lesions are lymphadenitis of the mes enteric lymph nodes and ulcération of the cecal mucosa. Rats from which salmonella is chronically shed have more ad vanced lesions than do intermittent shedders of the organism. A diagnosis of salmonellosis relies upon identification of an isolate as a Salmonella sp. Recovery of salmonella from the intestines, spleen, and liver is readily accomplished in rats clinically affected during an epizootic. However, this is not true for asymptomatic carriers, since some will shed the orga nism intermittently in the feces, and recovery from tissues is difficult. Recovery in carrier animals is best accomplished by initial incubation of fecal pellets in an enrichment broth, such as selenite F plus cystine broth, followed by streaking onto brilliant green agar (Weisbroth, 1979) . From this medium, possible salmonella colonies are inoculated into triple-sugariron slants. Final identification is then made by biochemical tests and serotyping. Prevention of this disease is based upon the exclusion of wild rodents from laboratory animal facilities and the use of only feed and bedding that has been properly processed and pack aged to ensure against salmonella contamination. /. Pseudomoniasis. Pseudomonas aeruginosa, a ubiq uitous gram-negative bacterium found in soil and water, colo nizes plants, insects, animals, and humans. It often colonizes the oropharynx and can be isolated from the intestinal tract of rodents. Infection with this organism in immunocompetent rats is nearly always inapparent. However, when rats are immunosuppressed, P. aeruginosa invades the upper respiratory mucosa and cervical lymph nodes, becomes bacteremic and induces an acute, lethal disease. In some cases, rats develop facial edema, conjunctivitis, and nasal discharge. In genet ically thymic-deficient rats (nude), retro-orbital abscesses may occur prior to bacteremia. Transmission in laboratory rodents occurs primarily by direct contact and contaminated water bottles and automatic watering systems. Phenolics are usually effective disinfectants, but quaternary ammonium compounds may actually support its growth. Diagnosis of pseudomoniasis is based upon a history of immunosuppression associated with an epizootic of acute disease and isolation of P. aeruginosa from the blood and organs of affected rats. Facial edema in affected rats must be differenti ated from viral sialodacryoadenitis. Pseudomonas aeruginosa grows well on blood agar and most other standard laboratory media. Most strains are ß-hemolytic and produce a bluish-green pigment, pyocyanin, as well as fluorescein. The use of specialized media (Pseudomonas P agar) enhances pigment production. The organism derives energy from carbohydrates via oxidation rather than fermentative me tabolism. Identification of isolates as P. aeruginosa is easily made by the above characteristics and appropriate biochemical reactions (Weisbroth, 1979) . In most research applications, P. aeruginosa-free rats are not necessary for the conduct of the work. It is a major problem, however, in rats used for burn research and in studies in which drugs or radiation induce immunosuppression. Infection can be relatively well controlled in a colony by hyperchlorinating drinking water at 12 ppm or by acidification of water to a pH of 2.5-2.8. In a closed colony, it is also advisable to remove rats that remain culturally positive after water treatment has been instituted. In studies requiring pseudomonas-free rats, isolators are useful in which a gnotobiotic environment can be achieved. Alternatively, laminar flow units may suffice if supplies and equipment are sterilized and personnel wear sterile garments. g. Streptobacillosis. Streptobacillus moniliformis is a commensal bacterium often present in the nasopharynx of con ventionally raised rats. Although it may be involved occasion ally as a secondary invader within inflammatory lesions of the rat, the chief importance of S. moniliformis is that it is the principal agent causing rat-bite fever in humans (Anderson et ai, 1983) . The other bacterium associated with this clinical syndrome is Spirillum minus. Clinical signs in humans usually occur within 10 days of a rat bite and consist of headache, weakness, fever, a generalized rash, and arthritis. Often clini cal signs subside in several days but then recur at irregular intervals for weeks or months (see Chapter 22). a. Murine Respiratory Mycoplasmosis. Murine respirato ry mycoplasmosis (MRM) is the term now accepted for a dis ease which, for many years, had an undefined etiology and a number of synonyms [i.e., infectious catarrh, enzootic bronchiectasis, chronic respiratory disease (CRD), and chronic murine pneumonia]. Since 1969, the causal relationship of Mycoplasma pulmonis with this disease has become well estab lished (Kohn and Kirk, 1969; Lindsey et al., 1971; Whittlestone et al., 1972) . Of all the pathogens occurring in laboratory rats, M. pulmonis has had the greatest negative im pact on studies. This has been primarily due to the chronicity of the disease, which often manifests itself only after months of infection. Long-term studies in areas of toxicology, carcinogenesis, nutrition, and gerontology, in particular, have been affected. Prior to the use of gnotobiotic techniques and barrier maintenance in rat production colonies, M. pulmonis was enzootic in nearly all commercial and institutional colo nies. Today, vendors can be selected who offer mycoplasmafree rats. My coplasma pulmonis is highly contagious and in duces a disease that frequently results in debilitation or demise of the host after a long period of time. The clinical signs associated with MRM range from negligi ble upper respiratory tract signs to systemic signs associated with pneumonia. The earliest and most common signs include snuffling and serous or mucopurulent oculonasal discharge. Extension of M. pulmonis infection from the nasopharynx via the eustachian tubes to the middle ears is common. However, torticollis and circling due to involvement of the inner ear are infrequently observed, even though one or both middle ear bullae may be impacted with exudate. The onset of upper res piratory signs is variable, but often occurs within several weeks postinfection. Signs of penumonia include dyspnea, rales, and systemic effects such as weight loss, starry hair coat, and hunched posture. Characteristically, signs of pneumonia occur 3-6 months postinfection, but this is quite variable and is a function of environmental influences, such as intracage ammonia levels and the immune competence of the host. In a small percentage of cases, the disease will be nearly subclinical even in the presence of extensive pulmonary lesions. Mycoplasma pulmonis is transmitted both horizontally and vertically from dams to their litters. In most instances, trans mission from the female occurs postpartum by direct contact, but if the genital tract of the dam is infected, antenatal infec tion can occur. Horizontal transmission between postweanling rats of any age readily occurs, and there appears to be no sig nificant age-related resistance to either infection or disease. Although little is known about differences in resistance among rat stocks and strains, the LEW rat has been shown to be more susceptible to MRM than the F344 rat. There is little evidence available to indicate that transmission occurs through fomites such as caging equipment and garments worn by personnel. Since aerosol droplet and direct contact appear to be the prima ry modes by which M. pulmonis infections are spread, the rapidity with which the organism is transmitted is dependent upon environmental factors, such as ventilation rates, degree of recirculation of air, and animal density within rooms. The basis for the pathogenicity of M. pulmonis is not well understood. Mycoplasma pulmonis adsorbs to the cell mem brane of the ciliated, columnar or cuboidal epithelia in the res piratory tract (Fig. 4) . It has been suggested that adsorption is a means by which mycoplasmas damage host cells by uptake of essential cellular metabolites; release of cytotoxic products, such as H 2 0 2 ; or cross reaction of antibody with cell mem brane components that are antigenically similar to or altered by mycoplasmas. Infection severely distorts or ablates ciliary structures (Fig. 4) , interfering with mucociliary clearance mechanisms. The gross lesions in the upper respiratory tract include mucopurulent exudate in the nasal cavity, sinuses, and middle ear bullae. Later, the exudate becomes caseous within the bul lae. Lesions in the lower respiratory tract reflect those of a bronchopneumonia. The earliest lesion is a mucopurulent exu date within the trachea, bronchi, and bronchioles. This pre cedes grossly evident lesions of the lung parenchyma that ini tially consist of atelectasis due to bronchial occlusion. Later, bronchiectatic lesions appear as numerous cream-colored nod ular abscesses on the surface of the lung. These lesions may be restricted to only a portion of a lobe or may involve nearly all of the parenchyma (Fig. 5) . Microscopically, the inflammatory response is characterized by a lymphocyte and plasma cell infiltrate in the submucosa and neutrophilic leukocyte response within the lumina of the epithelium. nasal cavity, eustachian tubes, middle ears, and tracheobronchial tree. A consistent and prominent lesion in the lung is the peribronchial lymphoid hyperplasia that often be comes quite massive. Within the lumina of the bronchi and bronchioles, mucin and neutrophil exudation increases during the course of the disease to the point of bronchiectasis. Con comitant with the impaction of bronchi is a change in the epithelia from a ciliated, columnar type to a squamoid type. This change in epithelial architecture is likely associated with cytotoxic enzymes from autolyzed neutrophils, although a di rect cytotoxic effect from mycoplasmas could be involved. A tentative diagnosis of MRM can usually be made by obser vance of the clinical signs and gross lesions described above. Clinical signs alone are not particularly helpful, since nasal exudates are present in bacterial infections such as S. pneumoniae. In addition, the reddish porphyrin deposition seen in the nares and periorbitally in sialodacryoadenitis virus infec tion and water deprivation may be confused with exudation. The gross lesions of otitis media and bronchiectasis are rather distinct. However, C. kutschen lung lesions may grossly mim ic those of MRM. Histopathology and serological evidence will differentiate MRM from Sendai virus infection, although the two infections are often superimposed. Recently a filamen tous bacterium has been associated with bronchiectasis in wild and laboratory rats (MacKenzie et al., 1981) . However, the causal relationship of this organism with lesions is undefined since the rats were also infected with M. pulmonis. This fila mentous bacterium has not been successfully grown on artifi cial media, and its presence is best verified by either histology, using the Warthin-Starry Stain, or electron microscopy (Fig. 6) . Although a definitive diagnosis of MRM is made by isola tion of M. pulmonis from involved tissues, it is evident that the existence of other agents must be evaluated to determine if copathogens are contributory to lesions. Prevention of MRM in either breeding or experimental colo-nies is dependent upon barrier systems that preclude the entry of M. pulmonis into the facility. Hysterectomy derivation is the only means of establishing an M. pulmonis-frtt breeding colo ny from a previously infected stock. Due to the frequent local ization of this microorganism in the uterus, it is necessary to ensure that neonates taken by hysterectomy have not been in fected in utero. Rats used in research animal facilities are ob tained from various commercial and institutional sources. Ac cordingly, it is essential that the mycoplasma status of these sources is known and that the rats are housed by vendor or in groups with a similar microbial status. For assessment of whether a group of rats is M. pulmonisfree, the best sites for isolation in animals without gross lesions are the nasal cavity, middle ear, trachea, and uterus-oviduct. Mycoplasma pulmonis is not particularly fastidious and grows well in several types of mycoplasma media (Cassell et al., 1979; Lentsch et al., 1979) . Most formulations have a pH indi cator that is useful since M. pulmonis ferments glucose. In broth media, moderate to heavy growth is reflected by pH and color of the broth. In broth cultures in which the titer is low, a perceptable pH change may not occur. Tissue and washing samples should be placed in broth rather than agar media, since recovery of the organism is more likely in those samples con taining few mycoplasmas. Samples from broth cultures are transferred to agar media when a pH change is readily evident or at 7-10 days if no pH change occurs. Mycoplasma colonies are evident in 3-4 days by observation with 40 x stereoscopic microscopy. Although culturing and histopathology have been the usual means to survey rat colonies, ELISA testing has recently been shown to be a very sensitive serological assay and one that can be performed quickly in most clinical laboratories (Cassell et al., 1981a) . In vitro sensitivity tests show M. pulmonis to be susceptible to tetracycline and tylosin. Tetracyline, given at 5 mg/ml drinking water, may be useful in some situations (Lindsey et al., 1971) . However, treatment with antibiotics seldom influences the disease course of MRM in a colony situation. b. Murine Genital Mycoplasmosis. Mycoplasma pulmonis recently has become recognized as an important pathogen in the female genital tract of rats, and thus is being treated here as a distinct disease rather than as a sequella to MRM. Infection of the genital tract is usually inapparent. However, reduced fertility and fetal deaths can occur. Infection of the oviduct and uterus occurs frequently in rats who have respiratory my coplasmosis. It is unknown whether localization in the genital tract occurs due to a hematogenous spread or to an ascending infection of the genital tract. It has been shown that subsequent to intravenous inoculation, M. pulmonis almost invariably lo calizes in the female oviduct-uterus. Gross lesions, when present, consist of a purulent oophoritis, salpingitis (Fig. 7) , and pyometra. The LEW strain is particu-Ä^SfcVVV ( Ä ß F/g. 6. Electron micrograph of filamentous bacterium (large arrow) and M. pulmonis (small arrow) attached to epithelium of respiratory mucosa. The morphology of size of the filamentous bacterium are similar to that of the cilia. (Courtesy of Dr. W. F. MacKenzie.) larly prone to develop gross lesions. Mycoplasmapulmonis ad sorbs to the epithelial cells in the genital tract in a manner similar to that seen in the respiratory tract. Salpingitis occurs most frequently and is characterized by exudation of neutrophils into the lumen, hyperplasia of oviductal epithelium, and a lymphoid response in the submucosa. The lesions in the ovarian bursa include edema and inflammation. Uterine le sions can vary from a mild inflammatory change to pyometra (Casselle/fl/., 1981b). Genital mycoplasmosis in the male rat has not been well doc umented. However, it is known that experimental inoculation can include an inflammatory response in the ductus efferens and epididymis. Moreover, it is known that M. pulmonis is capable of adherence to spermatozoa in an in vitro system. Since Pasteur ella pneumotropica can also induce similar le sions in the female rat, a diagnosis of mycoplasmosis is depen dent upon isolation of M. pulmonis from the lesions. Methods for culturing and identification are similar to those used for respiratory mycoplasmosis. Because the rat is widely used in various types of reproduc tive biology research, M. pulmonis colonization, even without gross lesions, would probably impact on the validity of data. The grossly evident caseous lesions in the ovary and oviduct can be mistaken for neoplasia if microscopy is not done. c. Mycoplasmal Arthritis. The etiological agent of this disease is Mycoplasma arthritidis. This mycoplasma species colonizes the pharynx, middle ears, and lungs of rats, although few studies have been done to document the relative frequency of this mycoplasma in rat sources. Within the respiratory tract, M. arthritidis colonization is thought to induce negligible le sions, and it has been shown to coexist with M. pulmonis. Although it is often considered to be the principal agent in volved in arthritis in rats, the disease has been rarely reported. Nearly all reports of its involvement in clinically apparent ar thritis have been made prior to 1960. It has been suggested that poor cage sanitation and abrasions of the extremities are in volved in entry of the organism to the joints by hematogenous spread or extension from surrounding tissues (Ward and Cole, 1970) . Since the organism appears to be of low virulence, the immunocompetence of the host may be a major factor in the outcome of infection. Arthritic animals limp and move with difficulty due to pain associated with the polyarthritis. Any of the joints in the limbs and vertebrae can be affected, but the tibiotarsal and radiocarpal joints are most often involved. Affected joints are hyperemic and swollen. Incised joints reveal a purulent exudate in both articular and periarticular tissues. Microscopically, there is exudation of neutrophils into the synovial spaces, and a lym phocyte and plasma cell infiltration in the synovial mem branes. Destruction of the articular cartilage occurs subsequent to the inflammatory response. Since polyarthritis can occur subsequent to septicemias asso ciated with other bacteria, particularly C. kutschen, a diag nosis of M. arthritidis-'mduced arthritis is contingent upon the demonstration of M. arthritidis by isolation or immunofluorescence techniques. This mycoplasma species grows well in me dia used to isolate M. pulmonis if arginine is added to the for mulation (Cassell et al., 1979) . Tetracyclines have been used to prevent the onset of arthritis when the organism has been inoculated intravenously, but there are no reports of its efficacy in spontaneous cases. Mycoplasma arthritidis, like M. pulmonis, may contaminate transmissible tumors and caution should be exercised to ensure transplanted tissues are not contaminated. Hemobartonellosis. The causative agent of this rickettsial disease is Hemobartonella muris. This organism is an extra cellular parasite of erythrocytes and induces inapparent infec tions that may persist for long periods. The ability of the host to restrict the infection to a subclinical mode rests with the integrity of the reticuloendothelial system. Evidence of infec tion is usually limited to splenomegaly and laboratory findings of mild parasitemia and reticulocytosis. Transmission of H. muris involves the blood-sucking louse, Polyplax spinulosa. Transmission can occur during a blood meal or when rats crush infected lice and are inoculated via pruritis-induced abrasions. The organism can also be transmit ted inadvertantly with transplantable tumors and other biolog ical products. Diagnosis of hemobartonellosis is dependent upon identifica tion of the organism in the peripheral blood of infected ani mals. The usual method of detection is by splenectomizing rats suspected of harboring the organism. In these rats, severe para sitemia and hemolytic anemia occur within 2 weeks after sur gery. Hemobartonella muris can be visualized on the surface of erythrocytes in Romanowsky-stained blood smears as coc- coid bodies arranged singly, in clusters, or chains (Cassell et al., 1979) . The rarity of reported cases would indicate H. mûris is no longer a significant problem in barrier-maintained colonies. However, conventionally maintained colonies may be exposed to infected wild rats and P. spinulosa and, accordingly, the disease still is of importance in the laboratory rat. The disease has had a negative impact on investigations of various types, but principally with those in which the host's immune compe tence has been impaired. a. Parvoviral Syndromes. Parvoviruses that can infect rats include rat virus (RV), Toolan H-l (H-l) virus, and minute virus of mice (MVM). Parvoviruses are small nonenveloped viruses that resist extremes in temperature, pH, and drying. Rat virus, or Kilham rat virus (KRV), has several antigenically related strains (RV, H-3, X-14, L5, HB, SpRV, HER, HHP, Kirk), all of which have been isolated as inadvertant contami nants of rat tissue or rat-passaged biological material. Toolan H-l related serotypes (H-l and H-T) are antigenically distinct from RV serotypes. Both RV and H-l are experimentally pathogenic, producing similar lesions, but only RV has been associated with natural disease. Neonatal rats can be experi mentally infected with MVM, but the virus does not seem to cause natural infection. Minute virus of mice antibody reac tivity can be present in rat serum, but this is probably non specific, since it can be found in germfree rat serum and is reduced or eliminated by receptor destroying enzyme. Rat virus infection is usually subclinical or latent, but a num ber of clinical syndromes have been associated with it. Infec tion of pregnant females can cause fetal résorption and birth of small litters. Pups are runted, atactic, or jaundiced. Neonates develop similar signs following postpartem exposure. Rats in troduced to an infected colony can develop ruffled fur, de hydration, and sudden high mortality. A similar syndrome oc curs in latently infected adults subjected to immunosuppressive regimens. The rat is the only natural host for RV and H-1, although experimental infection can be established in a number of other species. Seroconversion to both RV and H-l virus is common, with a high prevalence of infection within an enzootically in fected colony. Horizontal transmission is achieved by the oral and probably respiratory routes, with virus excretion primarily in the feces. Some strains of RV can be excreted in the milk or in utero. Clinical signs are manifest transiently upon introduc tion of RV into a previously uninfected population, but, there after, the virus spreads rapidly to produce subclinical or inapparent enzootic infection. Rat virus can persist as a true latent infection in the presence of high circulating antibody, but dis ease can be activated by immunosuppression. It must, there- fore, be assumed that seropositive rats are persistently infected and can serve as a source of infection to other rats. Pups infected in utero or as neonates develop intranuclear inclusions and necrosis in the outer germinal cell layer of the cerebellum. The recovered animal has severe depletion of the internal granular layer and disorganized Purkinje cells. Intra nuclear inclusions are also in hepatocytes, Kupffer cells, endothelial cells, and biliary epithelial cells, resulting in necrotizing hepatitis and the sequellae thereof (bile retention, jaundice, peleosis, bile ductal hyperplasia, parenchymal collapse, nodu lar hyperplasia). In adults, infection is usually inapparent, but when acute disease is precipitated, RV injures vascular walls and hematopoietic elements, causing coagulative disorders, thrombosis, hemorrhage, and infarction within the central ner vous system (hemorrhagic encephalomyelopathy). Hemorrhagic and necrotic lesions have also been noted in the per itoneum, testis, and epididymis. Rat virus has broad tissue tropism and lesions or clinical signs may potentially be varied, depending on virus and host factors (Coleman et al., 1982; Jacoby et al, 1979) . Infertility and unthrifty pups caused by RV must be differenti ated from environmental and husbandry factors or infectious agents such as Mycoplasma or Sendai virus. Adult disease must be differentiated from toxicity, nutritional deficiency, and trau ma. Diagnosis is made by the typical lesions, if present, virus isolation, and serology. Seroconversion to each virus (RV or H-l) can be detected by serum neutralization, hemagglutination inhibition, complement fixation, and immunofluorescence. Hemagglutination inhibition is currently the most commonly used means of antibody determination (Jacoby et ai, 1979) . Since RV infection is usually silent and persistent and can be transmitted either vertically or horizontally, effective control is best achieved by destroying the entire population, decon taminating, and repopulating with clean stock. Virus-free rats can be obtained from selected commercial vendors or by caesarean rederivation. Rederived progeny must be tested for vertically transmitted strains of virus. Colonies can be kept virus-free by limiting entry to seronegative, virus-free rats (as well as transplantable rat neoplasms or tissues), periodic serological testing, and adequate physical containment. Although parvovirus infection of rats is usually inapparent, there can be adverse effects on the research usefulness of in fected rats. Immunosuppression may exacerbate illness and mortality in latent carriers. The viruses often contaminate transplantable tumors and cell lines, can modify immune re sponsiveness or cause teratological effects. A decision to work with infected animals should be made carefully. b. Other DNA Virus Infections. Rats are susceptible to rat cytomegalovirus, which has a predilection for the salivary and lacrimai glands. Infection is widespread among wild, but not laboratory rats (Jacoby et al., 1979) . Rats also seroconvert to mouse adenovirus, but it is not known if infection is due to a mouse or rat strain of virus. Adenovirus-like inclusions have been reported in the intestine of rats treated with cancer chemotherapeutic agents (Ward and Young, 1976) . c. Siaiodacryoadenitis Virus and Related Coronaviral Infections. Two strains of coronavirus have been identified as pathogens of laboratory rats: siaiodacryoadenitis virus (SDAV) and rat coronavirus (RCV). Furthermore, rats are experimen tally susceptible to the coronavirus of mice, mouse hepatitis virus (MHV). Coronaviruses are large, pleomorphic enveloped RNA viruses with surface peplomers or spikes that confer a corona-like appearance to the virion. Viruses of this group have complex antigenic interrelationships and cross-react ex tensively. Common antigens are shared by SDAV, RCV, and MHV, particularly by complement fixation, but antibody reac tivity is highest with homologous virus. Siaiodacryoadenitis virus and RCV represent different strains of the same virus, but whether different strains of the same virus or separate viruses, they are both important natural pathogens in rats. The signifi cance of MHV for rats is not known, but the virus can replicate in the respiratory tract of intranasally inoculated rats (Taguchi et al., 1979) . Natural antibodies to MHV can occur in rats, but this is probably due to the closely related antigenicity of MHV to SDAV and RCV rather than natural MHV infection of rats (Barthold, 1984) . Clinical signs of SDAV infection vary widely in severity, but include blepharospasm, sneezing, porphyrin-pigmented nasal and ocular discharge, and cervical edema (Fig. 8) . Some rats develop keratoconjunctivitis and other ocular lesions. Signs persist approximately one week, but ocular sequellae can be permanent. Acutely infected rats become anorectic, and estrus can cease temporarily. Infection is subclinical in weanling or older rats, but intranasally inoculated neonates die and suck lings develop lower respiratory disease. Siaiodacryoadenitis virus is highly contagious and spreads rapidly among susceptible rats by contact, aerosol, or fomite. Susceptible rats of any age can be infected. When enzootic within a colony, clinical disease occurs only in sucklings, since adults are immune. Infection is acute, lasting only about 1 week, at which time rats seroconvert with no carrier state. Maintenance of SDAV in a colony requires continuous intro duction of susceptible stock as weanlings or newly introduced rats. The epizootiology of RCV is presumed to be similar to SDAV. Within 2 days of intranasal inoculation, SDAV causes rhi nitis followed by necrosis of the ductular and acinar epithelium of salivary and lacrimai glands, accompanied by intense in flammation and edema. Tracheitis and peribronchial lymphoid hyperplasia can also be found. Salivary glands appear swollen, pale, with interlobular and periglandular edema. Harderian glands are flecked with yellow-gray foci. One, some, or all of the salivary or lacrimai glands can be affected, with the excep tion of the sublingual glands, which are spared. Cervical lymph nodes become enlarged. Glandular repair ensues within 1 week, with squamous metaplasia of ductular epithelium and hyperplasia of acinar epithelium. The repair phase subsides within 30 days with minimal residual lesions. Interstitial pneu monia can occur in suckling, but not adult rats. Conjunctivitis, keratitis, corneal ulcers, synechia, hypopyon, and hyphema can arise due to lacrimai dysfunction. Eye lesions usually re solve, but can proceed to chronic keratitis, megaloglobus (Fig. 9) , and retinal degeneration. Rat coronavirus infection causes rhinotracheitis and focal interstitial pneumonia. Salivary but not lacrimai gland infection is rare, but when present, resem bles wild SDAV lesions. Infection with RCV also lasts approx imately 1 week (Barthold, 1984; Jacoby et al., 1979) . Nasal and ocular signs must be differentiated from those caused by mycoplasma, Sendai virus, pathogenic bacteria, ex cess ammonia, or hypovitaminosis A. Cervical swelling may Fig. 8 . Epiphora and swelling of the ventral neck in a rat naturally infected with SDAV. (From Barthold, 1984 ; courtesy of Hemisphere Publishing Corp.) Fig. 9 . Megaloglobus and hyphema in a young rat naturally infected with SDAV. (From Barthoid, 1984; courtesy of Hemisphere Publishing Corp.) also occur in immunosuppressed rats infected with P. aeruginosa. Microscopic SDAV lesions are characteristic. Mild lower respiratory tract lesions associated with RCV must be differentiated from those of Sendai virus or pneumonea virus of mice (PVM). Seroconversion or rising complement fixing antibody titers following acute disease is confirmatory. How ever, antibody may be low or undetectable with this method. Serum neutralization is another test that can be used, but the most sensitive antibody tests are immunofluorescence or ELISA. Either mouse or rat coronaviruses are used as antigen in these latter tests (Smith, 1983) . Rats can be kept free of SDAV and RCV if they are isolated and if newly introduced rats are immune or unexposed. Intro duction of a single subclinically infected rat can precipitate epizootic disease among naive rats. If an outbreak occurs, the infection will run its course and die out within 3-4 weeks if new rats are not introduced into the room and if breeding is temporarily ceased. Routine disinfection of rooms and equip ment is sufficient to destroy environmental sources of virus. Sialodacryoadenitis virus lesions can be confused with or contribute to changes induced by test compounds or nutritional deficiencies, particularly vitamin A. Sialodoacyoadenitis virus disease can predispose to anesthetic death due to airway hypersecretion. Eye lesions resulting from SDAV infection can in terfere with eye research. Both SDAV and RCV can potentiate other respiratory infections. d. Sendai Viral Infection. Sendai virus commonly infects laboratory rats, but its clinical significance is less than in mice. Sendai virus is a parainfluenza 1 virus of the paramyxovirus family. Paramyxoviruses are pleomorphic, enveloped, labile RNA viruses. Sendai virus infection in rats is usually subclinical, but can be manifested as ruffled fur, dyspnea, or anorexia. A decrease in average litter size and runted pups is common during outbreaks in breeding colonies. Sendai virus is highly contagious and disseminates rapidly. Outbreaks subside following development of an immune popu lation, with the potential of recurrence several months later as the susceptible population enlarges. Sendai virus induces an acute respiratory infection with no natural carrier state. Excre tion and transmission of virus occurs via the respiratory tract (Jacoby