key: cord-0040509-3qdaqjus
authors: Korhonen, H.J.; Marnila, P.
title: Bovine milk immunoglobulins against microbial human diseases
date: 2014-03-27
journal: Dairy-Derived Ingredients
DOI: 10.1533/9781845697198.2.269
sha: 6ba765c59c84e2482d86be23a73ce6900308ce33
doc_id: 40509
cord_uid: 3qdaqjus

Immunoglobulins (Igs), also called antibodies, are present in milk and colostrum of all lactating species. Igs are divided into classes having different physico-chemical structures and biological activities. The major Ig classes in bovine and human milk are IgA, IgG and IgM. Bovine colostral Igs provide the newborn calf with passive immune protection against microbial infections until the calf’s own immune system matures. Colostral Ig preparations designed for farm animals have been commercially available for many years. Potential health benefits attributed to bovine colostrum have increased manufacture and marketing of colostral Ig-based dietary supplements also for human use. Furthermore, specific anti-microbial antibodies can be produced into colostrum by immunizing cows with vaccines made of pathogenic microorganisms. These antibodies can be concentrated and used to formulate so-called immune milk preparations. Such preparations have proven effective in prevention of animal and human infections caused, e.g. by rotavirus, Shigella flexneri, Escherichia coli, Clostridium difficile, Streptococcus mutans, Cryptosporidium parvum and Helicobacter pylori. Their therapeutic efficacy, however, seems limited. A few immune milk products have been commercialized and more can be expected in the future for use, e.g. as a supportive means in antibiotic treatments and for prevention of hospital infections.

It has been recognized for more than 100 years that maternal colostrum and milk offer passive protection to a newborn infant against enteric pathogens, primarily via the transfer of immunoglobulins (Igs) and associated bioactive factors (Ehrlich, 1892) . Ruminant neonates are born virtually without Igs and therefore the colostral Igs are essential for survival. Probably due to this unique function, Igs represent the major protein fraction of colostrum, accounting for 70-80% of its total protein content (Butler, 1998; Marnila and Korhonen, 2002) .

The Ig fraction can be concentrated from colostrum or cheese whey and is marketed commercially as a feed supplement and replacer of colostrum, mainly for neonate calves and pigs, to prevent gastrointestinal infections (Scammell, 2001; Mehra et al., 2006) .

The advent of functional foods has increased interest in the bioactive components of bovine colostrum and milk as potential ingredients for health-promoting foodstuffs and even biopharmaceuticals. To this end, scientifi c and commercial attention has been focused on the development of bovine-derived Ig products that contain specifi c antibodies targeted for prevention or treatment of microbial infections in humans (Korhonen et al., 1998; . Such preparations have been described as 'immune milk'. This concept dates back to the 1950s when Petersen and Campbell fi rst suggested that orally administered bovine colostrum from hyperimmunized cows could provide passive immune protection for humans (Campbell and Petersen, 1963) . Since then, a large number of animal and human studies have been carried out to demonstrate that these preparations can be effective in the prevention or treatment of human and animal diseases caused by various pathogenic microbes (for reviews see Weiner et al., 1999; Korhonen et al., 2000a; Lilius and Marnila, 2001; Hoerr and Bostwick, 2002; Korhonen and Marnila, 2006; Mehra et al., 2006) . This article reviews the current state of knowledge about the properties of bovine Igs, their utilization as ingredients for immune milk preparations, and the application of these preparations for the prevention and treatment of various microbial infections in humans.

Igs, which carry the biological function of antibodies, are present in colostrum and milk of all lactating species. In mammals, all fi ve known classes of Igs have been characterized: IgG, IgM, IgA, IgD and IgE. The major Ig classes in both bovine and human milk are IgA, IgG and IgM. The basic chemical structure of all Igs is similar but their biological functions differ, although in principle they all contribute to the major defence mechanism against foreign materials recognized by the body's immune system. Igs account for up to 70-80% of the total protein content in colostrum, whereas in milk they account for only 1-2% of total protein. IgG1 is the predominant Ig class in bovine lacteal secretions as compared to IgA in human milk (Elfstrand et al., 2002; Marnila and Korhonen, 2002) . Table 10 .1 provides the concentrations of different Ig classes in bovine colostrum and milk.

Ig molecules of all classes are symmetrical, multi-chain glycoproteins composed of two identical glycosylated heavy chains and two identical non-glycosylated light chains. The basic structure of all monomeric Igs is similar (see Fig. 10 .1). The IgG class can be considered as a general model of a monomeric Ig. The molecular weight of each light chain is around 23 kDa and of the heavy chains 53 kDa. The molecular weight of the complete Ig molecule varies around 160 kDa. Both the light and heavy chains contain domains referred as constant regions (C L , C H ) and variable regions (V L , V H ). The light chains are attached to the heavy chains by a disulphide bond, and also the two heavy chains are held together by two disulphide bonds near a hinge region which gives the molecule structural fl exibility needed in antibody-antigen interactions (Nezlin, 1998a) . The two identical antigen-binding sites needed in these interactions are formed by the N-terminal region of one heavy chain and the variable region of one light chain. The V L -region determines the immunological specifi city. Antigen binding occurs by the interactions between the antigen and these regions. The Ig classes and subclasses are determined by the genes encoding the constant regions of heavy chains (Butler, 1998) . The bovine IgG molecule occurs dominantly in two sub-classes: IgG 1 and IgG 2 .

Monomeric IgM and IgA have a similar basic structure to IgG except for the differences in heavy chain structures and the addition of a C-terminal octapeptide to the heavy chain of IgA (Butler, 1998; Nezlin, 1998b) . Monomeric IgA occurs in serum, but in milk it is present as a dimer comprising two IgA molecules joined together by a polypeptide J-chain and an additional 75 kD secretory component. This secretory IgA (SIgA) has a molecular weight of about 380 kDa, and is more resistant to proteolysis and therefore more stable in the gastrointestinal tract than antibodies without the secretory component. IgM is a circular pentamer consisting of fi ve subunits similar to those of monomeric IgA or IgG, which are linked together in a circular mode by disulphide bonds and a J chain: the molecular weight of IgM is approximately 900 kDa (see Fig. 10 .2). The physicochemical properties of Igs are presented in Table 10 .2. Proteolysis of an Ig with papain cleaves it near the hinge region into two antigen binding-sites (Fab-fragments), which are formed by the N-terminal part of one heavy and one light chain, and to the third C-terminal fragment, termed the Fc-part. The differences between Ig classes impact upon the cleavage patterns in enzymatic proteolysis. For instance, cleavage of bovine IgG2a(A2) by pepsin results to two Fab and one Fc parts, whereas in IgG2a(A1) the Fab parts remain together by one disulphur bridge resulting in one F(ab′) 2 and one Fc fragment which is cleaved further by pepsin (Heyermann and Butler, 1987) .

Igs are produced by B lymphocytes and plasma cells diversifi ed from B cells. The soluble Igs function as fl exible adaptors between cellular and humoral immunity by binding to antigens and exhibiting one or more effector functions. The Fab parts attach the antigens while other parts (mostly the Fc region) interact with other elements of the immune system. For many pathogenic organisms, the attachment to the epithelial lining is an obligatory fi rst step in the establishment of colonization and infection. Many pathogens have receptors, e.g. fi mbriae for epithelial surfaces. Thus, the prevention of the microbial adhesion to epithelial linings is an important mechanism of SIgA antibodies in protecting the host (Woof and Kerr, 2006) . Colostrum and human milk protect the neonate intestinal mucosa against EPEC infections by inhibiting bacterial adhesion to epithelial cells (Carbonare et al., 1997) . This effect has been shown to be mediated by colostrum and milk SIgA (Cámara et al., 1994; Carbonare et al., 1997 Carbonare et al., , 2005 Fernandes et al., 2001; Corrêa et al., 2006) , and by oligosaccharides present in human milk (Cravioto et al., 1991) . M-cells in intestinal epithelia have specifi c receptors for SIgA, and antigen-SIgA-receptor complex can be transcytosed by M-cells into lymphoid tissue for antigen presenting and induction of mucosal immune responses (Woof and Kerr, 2006) .

The ability of Igs to form cross-links between surface antigens results in a network of cells that can be removed mechanically from the body. This agglutination of microbes reduces their capability to adhere to surfaces. Agglutinated microbes are usually not able to release toxins or to colonize the host. Normal colostrum and milk are known to contain natural antibodies which can agglutinate a large number of pathogenic and non-pathogenic micro-organisms (Bostwick et al., 2000; Korhonen et al., 2000b) .

Igs can bind bacterial toxins which, thereafter, can be recognized more effectively by phagocytic leukocytes. Many bacterial toxins must fi rst be actively transported via receptors inside the host cells to cause cell death. Blocking the toxin may prevent its internalization in host cells. Also, Igs can inhibit or reduce the production of toxins and other harmful components by inhibiting bacterial metabolism and by blocking enzymes and receptors. This blocking may also reduce the ability of pathogens to produce structures needed in adherence to epithelia. Specifi c Igs can protect against viral infections by binding viruses and preventing the virus replication by blocking the receptor mediated internalisation of viruses in the host cells. Specifi c Igs augment the recognition and phagocytosis of antigens by leukocytes (opsonisation). Divalent or polyvalent binding of an Ig to antigenic structure results in conformational change of the Ig molecule, which process again enables the Fc portion to bind to the corresponding Fc receptors on the leukocyte surface. This receptor binding leads to various immune cell effector functions depending on the cell Ig and leukocyte type. Leukocytes are an integral part of normal milk and colostrum and are of vital importance in defending the mammary gland against pathogens (Korhonen et al., 2000b) .

Specifi c Igs are rarely cidal to micro-organisms but may disturb cellular metabolism by blocking receptors and enzymes, and cause structural alterations leading to immobilization, increased membrane permeability and impaired cell growth. Igs may contribute to the killing of microbes by activating the classical pathway of complement. In blood and tissues, the activation of complement mediated bacteriolytic reactions may be the most important function of Igs, but the signifi cance of this mechanism in milk remains obscure. Bovine and human colostrum contain an active complement system participating in the immune defense of the udder (Butler, 1998) .

IgM antibodies, although produced in smaller amounts than IgG, are considerably more effi cient than IgG with regard to most of the above activities, including complement fi xation, opsonization and agglutination of bacteria. Bovine IgG 2 B is more effective than IgG 2 A in activating the classical pathway of complement (Bastida-Corquera, 1999) . Specifi c IgM can prevent effectively the migration of bacteria by binding the fl agellas used in the movement. Bovine IgA does not fi x complement or opsonize bacteria, but it agglutinates antigens, neutralizes viruses and bacterial toxins. The SIgA dimer is found in mucosal secretions and its main function is to protect the mucosal barriers by binding microbes and preventing their attachment to the epithelium, and to contribute to the antigen presenting. The milk Igs have also been found to exert a synergistic effect on the activity of non-specifi c antimicrobial factors in milk, such as lactoferrin and lysozyme, as well as the lactoperoxidase-thiocyanate-hydrogen peroxide system (Loimaranta et al., 1998a; Bostwick et al., 2000) .

Traditionally, the globulin fraction was isolated from colostral whey by precipitation with either ammonium sulphate or ethanol. These chemical methods are, however, economically not suitable for large scale-production of Igs although they yield rather pure Ig fractions. Other challenges in isolation of active Igs from colostrum has been its complex composition and the sensitivity of Igs to heat treatments. With the rapid development of new chromatographic and membrane-separation technologies in the last thirty years, it is now possible to isolate individual milk proteins on a large-scale. Based on these techniques, a great number of pilot-or industrial-scale methods have been developed and patented for fractionation and isolation of Igs from colostral or cheese whey (for reviews see Korhonen, 2004; Korhonen and Pihlanto, 2007) . With these methods, the recovery rate of Igs has varied from 40% to 70% of the level present in the starting material (Elfstrand et al., 2002) . Specifi c chromatographic techniques, such as immobilized metal chelate chromatography, immunoaffi nity chromatography and cation-exchange chromatography have been applied to improve the yield and purity of immunoglobulin preparations further (Fukumoto et al., 1994; Akita and Li-Chan, 1998) . Microfi ltration (MF) combined with ultrafi ltration (UF) of bovine, equine and caprine colostrum has led to IgG/total solids purity of more than 90% (Piot et al., 2004) . Using cheese whey, a 0.1 µm membrane and the concept of selective membrane separation through pH manipulation, Mehra and Kelly (2004) produced an Ig-rich preparation with a protein composition similar to that produced from colostral whey. Korhonen et al. (1998) used various MF methods, such as UF, MF and reverse osmosis, and a cation-exchange resin as a molecular sieve, to concentrate Igs from colostral whey. The Ig level of the fi nal freeze-dried concentrates varied from 45% to 75%. Expanded Bed Adsorption Chromatography (EBAC) is a new technology which has been applied to isolate Igs from cheese whey using an adsorbent with tailored ligand chemistry (Nielsen et al., 2002) . EBAC provides signifi cant advantages over conventional packed bed column chromatography, and an Ig-purity from 50% to 70% can be achieved with this up-scalable method.

The technological properties of Igs have been studied extensively, as these proteins are known to affect many dairy processes (see review by Mehra et al., 2006) . Igs may inhibit or retard the activity of renneting enzymes of bacterial origin. In normal milk this is not noticed due to the relatively low Ig concentration but in case of mastitic milk, or if the Ig concentration of milk is increased, e.g. by adding colostrum, renneting may be retarded. The antimicrobial properties of Igs may adversely impact upon fermentation processes. A retarded fermentation by dairy starters is noted in colostrum and mastitic milk, which contain increased amounts of Igs. Also, high Ig concentrations may adversely affect the antibiotic residual tests based on microbial growth, causing false positive results (Korhonen, 2004) . Igs contribute to cream formation by agglutinating fat globules, which process accelerates the ascent of cream to the surface. This phenomenon is attributed primarily to IgM, which have been termed cryoglobulins or 'cold agglutinins'. The agglutination property of Igs can, however, be inactivated by heat treatment (pasteurization) and mechanical agitation.

Among dairy processes, the properties of Igs are most affected by the thermal treatments. In high temperature/short time (HTST) pasteurization (72°C/15 sec) about 25-40% of the Ig activity is lost, whereas ultra high temperature (UHT) treatment (138°C/4 sec) and evaporation processing destroy most of the specifi c immune activity of milk due to Ig denaturation (Li-Chan et al., 1995) . In contrast, other studies (Mainer et al., 1997 (Mainer et al., , 1999 have reported that bovine milk Igs could resist the HTST pasteurization treatment without affecting their structure. Only 1% of IgG, 2% of IgA, and 14% of IgM concentrations were denatured in laboratory experiments. Also, the HTST pasteurization had little effect on the activity of bovine colostral IgG as the original rotavirus neutralizing activity was reduced by only 0.5%. In a recent study by McMartin et al. (2006) , it was demonstrated that a rapid heat inactivation of IgG started at temperatures higher than 65°C, and at 81°C, as much as 90% of the virus neutralization activity of Igs was lost in less than two minutes. On the other hand, heating moderate or high quality colostrum at 60°C for at least 120 min had no effect on mean IgG concentration or titer of neutralizing antibodies against bovine viral diarrhea virus Type 1. In storage stability studies, it has been observed that bovine IgG added into UHT-treated milk retained its specifi c immune activity for over several months (Fukumoto et al., 1994) . Also, Ig molecules seem to retain their specifi c activity well in milk powder, irrespective of the storage temperature (Mehra et al., 2006) . Further, bacterial fermentation of milk by yoghurt or probiotic bacteria have not been found to reduce signifi cantly the activity of colostral antibodies added in milk during a storage period of 50 days of at 4°C. (Wei et al., 2002) .

The progress in understanding the mechanisms of Ig mediated immune functions and the rapid development of industrial fractionation technologies have raised interest in developing formulations supplemented with bovine colostral or cheese whey derived Igs. Most of the current commercial Ig products are prepared from colostrum of non-immunized cows by removing the fat, followed by microfi ltration or pasteurization under conditions that retain the biological activity of Igs. These products are usually in the form of spray-dried and freeze-dried powders, but some are in the form of fi ltered colostral whey liquids or concentrates. Dried products include whole colostrum powder, skim powders, skim colostrum protein concentrate and colostrum whey concentrates. Some of above preparations have been tested clinically for certain physiological functions or prevention/ treatment of microbial infections. Accordingly, a few products boast specifi c health or nutrition function claims, such as boosting immunity against microbial infections or speeding recovery from physical endurance exercises. (Scammel, 2001; Kelly, 2003; Tripathi and Vashishtha, 2006) . However, in most cases, the clinical evidence related to these products is very limited or not available.

The specifi city of natural antibodies found in milk and colostrum of different cows refl ects the wide spectrum of antigens the animals have encountered in the past in their environment and in ingested feedstuffs (Korhonen et al., 2000a; Kelly, 2003) . The antibody titre against certain antigenic pathogens or structure, e.g. virulence factor, can be raised up to several hundred times by immunizing the cow before parturition with vaccines containing the antigens (Korhonen et al., 1995) . The resulting immune colostra and products made thereof have fundamentally different antimicrobial properties and effi cacies against pathogens than normal colostrum and these two concepts have, therefore, to be differentiated. This is emphasized by the fact that the normal colostrum and preparations made from it are, in most countries, regarded as food or dietary supplement, whereas the Ig containing preparations from immunized cows are often regarded as pharmaceuticals, e.g. in the EU and USA, or their regulatory status is not defi ned (Scammel, 2001; Hoerr and Bostwick, 2002; Mehra et al., 2006) . Table 10 .3 lists commercial Ig and immune milk preparations developed over the last decade.

It is well known that the low pH of gastric acid reduces signifi cantly the activities of ingested Igs. Furthermore, the ingested Igs are subjected to degradation by intestinal proteases. In the stomach, the enzymatic hydrolysis by pepsin, fragments the IgG molecule to F(ab′) 2 , Fab/c, pFc and Fv fragments (Nezlin, 1998a) . In the small intestine, trypsin, chymotrypsin, carboxypeptidase and elastase initially degrade the antibodies to F(ab′) 2 , Fab and Fc fragments. The secretory piece of milk SIgA protects this Ig form against digestion by proteolytic enzymes. Of the SIgA present in human colostrum, 20-80% passes undegraded through the gut of the human infant. Also, bovine IgG 1 makes an exception since it is rather resistant to trypsin. Bovine colostrum and mastitic milk contain a compound which inhibits trypsin activity. The resulting F(ab′) 2 and Fab fragments retain at least part of the neutralizing and adhesion inhibiting activities in the intestine. Pacyna et al. (2001) supplemented whole milk with immune colostrum containing specifi c Igs against rotavirus. When 100 ml of this supplemented whole milk was administered to 105 children three times a day for six days, the anti-rotavirus Ig activity was detected by the ELISA method in 521 of 602 fecal specimens obtained during the study. Kelly et al. (1997) demonstrated that 10%-30% of orally administered bovine anti-Clostridium diffi cile Ig concentrate could be recovered from the stools of human infants and adults in the form of F(ab′) 2 and Fab fragments. The survival of IgG increased remarkably by encapsulation with gelatin.

The concept of 'immune milk', i.e. the transfer of passive immunity by milk Igs, dates back to the 1950s. Since then, a great number of clinical studies have been published on the effi cacy of various immune and non-immune milk Ig preparations in the prevention and treatment of various gastrointestinal microbial diseases. Recently, these studies have been reviewed by Mehra et al. (2006) , Korhonen and Marnila (2006) and Hammarström and Weiner (2008) . Thus, the protocols and results of these numerous studies are not discussed in detail in this article. Briefl y, the general tendency of these studies has been that orally administered bovine milk or colostral Igs have, in most cases, proved to be effective in the prevention of orally mediated infections. Colostral or milk Igs can effectively agglutinate bacteria (Xu et al., 2006) , and bovine colostrum and colostral preparations prevent effectively the attachment of a pathogen to the cells in epithelial lining. However, since bovine colostrum contain more than one adhesion inhibiting factor, the importance of specifi c Igs is often not known. Palmeira et al. (2001) observed that inhibition of enteropathogenic E. coli adherence to HEp-2 cultured cells by bovine colostrum was not caused by IgG fraction and was mediated by a high molecular weight fraction. However, in some studies the inhibition of pathogenic adhesion to epithelial cells has been attributed to presence of specifi c Igs. Casswall et al. (2002) showed that a specifi c bovine immune colostral preparation blocked almost 90% of Helicobacter pylori attachment to human gastric mucosal tissue in vitro and 95% of binding to Lewis b glycoconjugate, while a control colostral preparation from nonimmunized cows was ineffi cient. Doyle et al. (1993) reported that hyperimmune bovine colostral Ig inhibited Cryptosporidium parvum adherence to epithelial MDCK cells and infectivity in an in vitro assay, and this inhibition was correlated with the protective capacity of the bovine colostrum in vivo. No signifi cant adhesion inhibition was observed for control preparation from colostra of sham immunized cows. Bojsen et al. (2007) studied the effi cacy of bovine macromolecular whey protein fractions in prevention of infection of two human intestinal cell lines (Caco-2 and FHs 74 Int) by four different rotavirus strains (Wa, RRV, YM, RF). The major component of the protein fraction that inhibited effectively the rotavirus infectivity in vitro was shown to be bovine IgG. The same fraction was the only one affecting rotavirus shedding in an in vivo mouse model.

In the treatment of already established infections, promising therapeutic effects have been reported, mainly in such diseases where the infection is maintained through a reattachment and reinfection, e.g. inside the oral cavity or the gastrointestinal lumen, and where the secretion of toxins or other infl ammatory compounds is involved, which can be neutralized by the specifi c colostral Igs. Diseases caused by enterotoxigenic Escherichia coli strains, Cryptosporidium parvum, Candida spp., Shigella fl exneri and rotavirus are examples. As instances of clinical studies, a closer look is taken hereunder at recent progress in developing bovine immune milks targeted against Streptoccus mutans and Clostridium diffi cile. Table 10 .4 provides data on recent clinical studies carried out with colostral Ig and immune milk preparations.

The pathogenesis of dental caries caused by Streptococcus mutans involves a series of attachment and binding events that lead to accumulation of cariogenic bacteria suffi cient to cause the caries. Immunization strategies against streptococcal adhesins or glucosyltransferase enzymes (GTFs) have been shown to effectively interfere with the pathogenesis of mutans streptococci (Smith et al., 2001) . Blocking of the activity of S. mutans glucan binding proteins serves another effective strategy. Since humans cannot be immunized actively against caries bacteria due to the risk of side effects, bovine colostral antibodies have been studied in local passive immunization to prevent dental caries (for a review see Koga et al., 2002) .

An immune milk preparation from cows immunized with a S. mutans and Streptococcus sobrinus whole cell vaccine inhibited in vitro glucosyltransferase and fructosyltransferase enzyme activities of S. mutans (Loimaranta et al., 1997) , promoted aggregation of S. mutans, and inhibited adherence of the bacteria to saliva-coated hydroxyapatite particles (Loimaranta et al., 1998b) . The same immune preparation resulted in a higher resting pH in dental plaque of adult volunteers as compared to control groups, when used as a mouth rinse for three days (Loimaranta et al., 1999) . After the rinsing period with the immune product, the relative number of mutans streptococci had decreased signifi cantly as compared to the controls.

Chicken egg yolk Igs obtained from immunized hens with S. mutans glucan binding protein had a clear protective effect against S. mutans infection and caries development in a rat model (Smith et al., 2001) . Mitoma et al. (2002) immunized cows with a fusion protein prepared by a fusion of a saliva-binding alanine-rich region of a cell surface protein antigen (Pac) and a glucan binding domain of the glucosyltransferase-I cell surface protein from S. mutans. The immune colostrum preparation effectively prevented dental caries development in a rat model when given as concentrate once a day for 55 days, together with cariogenic diet. Shimazaki et al. (2001) examined the effect of immune colostrum containing Igs against the same fusion protein on adult subjects after an antibiotic (cetylpyridinium chloride) treatment. The immune preparation inhibited signifi cantly recolonization of S. mutans in the saliva and plaque as compared to the control group, and the ratios of S. mutans to total streptococci in saliva and plaque were lower than in the control group. Wei et al. (2002) studied the combined effect of specifi c colostral Ig preparation against caries streptococci with the probiotic bacterium Lactobacillus rhamnosus GG, ATCC 53103 (LGG).

LGG added in milk was earlier shown to reduce the risk of caries in day-care children (Näse et al., 2001) . The LGG-bacteria and specifi c Igs in LGG-fermented milk synergistically inhibited adhesion of S. mutans to saliva-coated hydroxyapatite particles. The Igs remained active in UHT milk and in LGG fermented milk over the whole period of expected shelf-life of these products. Thus, the rinsing with bovine immune whey indicates favourable effects on human dental plaque by controlling S. mutans in the human oral cavity and it may be benefi cial to combine specifi c bovine milk antibodies against mutans streptococci to probiotic LGGcontaining milk products.

Clostridium diffi cile immune milk Colonization of Clostridium diffi cile in the intestine may cause severe infectious diarrhea and colitis. The most important virulence factors are the toxins A and B, which are associated with the development of the disease. Outbreak of C. diffi cile infection often results from antibiotic treatments. In animal models, bovine immune colostrum preparations have been effective in the treatment of experimental C. diffi cile diarrhea. Van Dissel et al. (2005) used a preparation made of milk from cows immunized against C. diffi cile toxins and whole cell C. diffi cile as a supportive treatment for two weeks after a standard antibiotic treatment in an uncontrolled cohort study. Nine of 16 patients had a history of relapsing C. diffi cile diarrhea. After the regimen in 15 patients, the C. diffi cile toxins had disappeared from feces, and during the follow-up period of 11 months, none of the patients had another episode of C. diffi cile diarrhea. Numan et al. (2007) assessed the effi cacy of C. diffi cile immune milk preparation in aiding the prevention of relapses in C. diffi cile patients. The immune milk was administered orally 5 g/day, divided into three equal doses for 14 days starting two weeks after standard treatment with oral metronidazole or vancomycin. In 109 disease episodes, 11 (about 10%) were followed by a relapse, whereas in contemporary controls the relapse rate was 20-25%. Young et al. (2007) evaluated the safety of the same immune milk administered to 77 C. diffi cile patients, similarly as in the previous study. Adverse effect monitoring, physical examinations and haematological and biochemical assessments showed no adverse effects in this group. Mattila et al. (2008) immunized cows with a whole-cell vaccine made of two toxigenic C. diffi cile strains and the immune whey preparation was made from the colostra of these cows. In a controlled double-blind randomized study, 18 patients with C. diffi cile associated diarrhea received orally 1.6 g of Ig in 200 ml volume twice per day for 2 weeks, and 20 patients received 400 mg of metronidazole twice per day. A C. diffi cile culture and toxin test was made on days 0, 14 and 28, respectively. At day 14, 100% of the metronidazole group and 83% of the immune colostrum group, respectively, responded positively to the treatment. The authors concluded that the immune colostrum treatment is somewhat less effective than the standard treatment with antibiotics but, on the other hand, it does not cause antibiotic resistance problems and does not alter the normal colonic bacterial fl ora as antibiotics do. In future, by optimizing the dosage, the treatment time, and the antigens used in immunizing the cows, the effi cacy of bovine milk Ig preparations could be improved and this treatment strategy could also be used as a supportive treatment together with conventional antibiotics.

As a result of progress made in membrane separation and chromatographic techniques it is now possible to isolate Igs from bovine colostrum and cheese whey on a large scale. This progress has facilitated and boosted manufacture of Ig concentrates as dietary supplements or ingredients. Some of these preparations are being marketed with health or nutrition function claims attached, even though the clinical evidence is limited or not reported at all. More scientifi c research is therefore needed in this fi eld to substantiate the claimed health benefi ts. As for immune milk preparations, there are already a few commercial products on the market with quite well established clinical evidence about the effi cacy. The main limitation of the clinical use of bovine milk antibodies for humans is that, as proteins of another species, the Igs can be used only orally against gastrointestinal pathogens. An interesting attempt to overcome this limitation is a transchromosomic calf that has been cloned for producing humanized polyclonal Igs. As a result of fi ve sequential genetic modifi cations and seven consecutive cloning events, this calf was reported to have full human IgG 10-20% of total serum IgG. After hyperimmunization of this calf with an anthrax protective antigen, both full human IgG and chimeric IgG were found to be effective in a toxin-neutralization assay (Kuroiwa et al., 2009) . The regulatory status regarding products containing specifi c Igs may be considered as the main reason why products (for instance those containing specifi c Igs against cariogenic bacteria) are not on the market in most countries. The regulatory status of immune colostral preparations was undetermined for a long time and for some years these products have been classifi ed as pharmaceuticals in the EU and USA. In the case of diseases that can be controlled also by conventional means, e.g. dental caries, this regulatory approach has slowed down the development of immune milk preparations in many countries. However, in the case of severe diseases caused by bacterial strains resistant to antibiotics, such as C. diffi cile, the regulatory status of a pharmaceutical has delayed but not hampered completely the progress of such product development (Hoerr and Bostwick, 2002; Mattila et al., 2008) . Currently, the US Food and Drug Administration (FDA) have accepted the safety of hyperimmune milks on the basis of clinical studies that show no adverse health effects from these products (Gingerich and McPhillips, 2005; Krissansen, 2007) .

Incidences if diarrhea and mortality rates among children in many areas of developing countries are high. The prevention of diarrhea would improve the nutritional status and health in general. The emergence of antibiotic resistant pathogen strains puts emphasis on the need to develop alternative ways to prevent and treat gastrointestinal infections. In developed countries, the control of gastrointestinal microbial fl ora has become an integral part of health promotion. It is concluded that synergistic effects of Igs with probiotics and other milk bioactives, such as lactoferrin, may open new prospects for developing novel means to prevent microbial diseases by nutritional intervention. 

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Milk immunoglobulins for health promotion: A review

Passive immunization with bovine milk containing antibodies to a cell surface protein antigen-glucosyltransferase fusion protein protects rats against dental caries

Effect of long-term consumption of a probiotic bacterium, Lactobacillus rhamnosus GG, in milk on dental caries and caries risk in children

Scandinavian Dairy Information, Issue 2. numan s c, veldkamp p, kuijper e j, van den berg r j, van dissel j t

Survival of rotavirus antibody activity derived from bovine colostrum after passage through the human gastrointestinal tract

Inhibition of enteropathogenic Escherichia coli (EPEC) adherence to HEp-2 cells by bovine colostrum and milk

Preparation of serocolostrum by membrane microfi ltration

Production and uses of colostrum

Passive immunization with milk produced from an immunized cow prevents oral recolonization by Streptococcus mutans

Passive transfer of immunoglobulin Y antibody to Streptococcus mutans glucan binding protein B can confer protection against experimental dental caries

Effi cacy of an infant formula containing anti-Escherichia coli colostral antibodies from hyperimmunized cows in preventing diarrhea in infants and children: A fi eld trial

Bioactive compounds of colostrum and its application

Bovine antibody-enriched whey to aid in the prevention of a relapse of Clostridium diffi cile-associated diarrhoea: Preclinical and preliminary clinical data

Stability and activity of specifi c antibodies against Streptococcus mutans and Streptococcus sobrinus in bovine milk fermented with Lactobacillus rhamnosus strain GG or treated at ultra-high temperature

Passive immunity against human pathogens using bovine antibodies

The function of immunoglobulin A in immunity

Bovine immune colostrum against 17 strains of diarrhea bacteria and in vitro and in vivo effects of its specifi c IgG

The safety of whey protein concentrate derived from the milk of cows immunized against Clostridium diffi cile