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Antibacterial factors in cows’ milk and colostrum : immunoglobulins and lactoferrin Facon, Michel Jean 1995

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ANTIBACTERIAL FACTORS IN COWS’ MILKAND COLOSTRUM:IMMUNOGLOBULINS AND LACTOFERRINbyMICHEL J. FACONM.Sc. (Genetics), Stanford University, 1971A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIES(Department of Food Science)We accept this thesis as conformingto the required standard.THE UMVERSITY OF BRITISH COLUMBIAApril 1995© Michel Jean Facon, 1995In presenting this thesis in partial fulfilmentof the requirements for an advanceddegree at the University of British Columbia,I agree that the Library shall make itfreely available for reference and study. I further agreethat permission for extensivecopying of this thesis for scholarly purposesmay be granted by the head of mydepartment or by his or her representatives.It is understood that copying orpublication of this thesis for financial gain shallnot be allowed without my writtenpermission.Department ofThe University of British ColumbiaVancouver, CanadaDateV(Signature)DE-6 (2/88)ABSTRACTThe objectives of this study were: 1) to test the antibacterial activity of a pepsindigest of bovine lactoferrin, containing the peptide lactoferricin, in complex media; 2) todetect specific antibodies to human enterotoxigenic Escherichia colt in bovine milk orcolostrum; and 3) to investigate the potential of a cell culture system to study and estimatethe biological activity of milk immunoglobulins.The pepsin digest of lactoferrin was bactericidal against Salmonella enteritidis in 1%peptone, but no substantial antibacterial activity could be demonstrated in trypticase soybroth or in some selected foods. Calcium at a concentration of 5 m?s’I was sufficient to inhibitthe antibacterial activity of the digest. Addition of lysozyme or EDTA enhanced theantibacterial activity of the digest, but not sufficiently to overcome the effect of inhibitors inthe foods of interest. The activity of the digest was also inhibited by bile salts. Thesefindings raise doubts about the potential for addition of lactoferricin to foods.Antibodies to the colonization factor antigen CFA 1 of enterotoxigenic E. colt weredetected in bovine colostrum by hemagglutination inhibition. Concentrations of antibodies toCFA 1, estimated by ELISA, ranged from 0.55 to 5.2 .tg/ml in colostrum samples of non-vaccinated cows. Samples of milk immune concentrates from vaccinated and non-vaccinatedcows were also tested. Vaccination increased the concentration of specific antibodies relativeto the total IgG content of the samples tested.Invasion of HeLa cells by Salmonella enteritidis, S. typhimurium andenteropathogenic E. coli was inhibited by addition of bovine colostrum to cell culturemedium. Inhibition levels ranged from 73% to over 99%. The immunoglobulin-containingfraction, isolated from colostrum by affinity chromatography on a protein G-agarose column,inhibited invasion by S. typhimurium. An unidentified high molecular weight factor in thenon-immunoglobulin fraction also inhibited invasion of HeLa cells. No inhibitory activity11was found in low molecular weight fractions. The results suggest that bovine colostrumcontains both immunoglobulin and non-immunoglobulin inbibitors of invasion of HeLa cellsby the bacteria tested.HeLa cell cultures have the potential to be a convenient method for the study andevaluation of antibacterial properties of bovine milk.111TABLE OF CONTENTSPageABSTRACT iiTABLE OF CONTENTS ivLIST OF TABLES viLIST OF FIGURES viiiACKNOWLEDGMENTS xI. INTRODUCTION 1II. LITERATURE REVIEW 3A. The problem of whey utilization 3B. The impact of gastrointestinal infections 5C. The enteropathogens 6D. Host defenses 91. Non-specific host defenses 92. Specific host defenses 103. Specific humoral immunity 104. Active vs passive immunity 125. Systemic vs mucosal immunity 16E. Milk and resistance to enteropathogens 171. Passive transfer in animals: experimental evidence 202. Passive transfer in humans: experimental evidence 22F. Assessment of the biological activity of immunoglobulin preparations 241. Animal models 26iv2. The study of bacterial adherence or invasion in cell cultures 263. Methods to estimate bacterial adherence or invasion 284. Use of cell culture systems to study inhibition of adherence 29G. Lactoferrin: antibacterial properties 30H. Lactoferricin 36I. Conclusion 39Ill. ANTIBACThRIAL ACTIVITY OF A PEPSIN DIGEST OFBOVINE LACTOFERRIN IN COMPLEX MEDIA 41A. Materials and methods 41B. Results 43C. Discussion 59IV. ANTIBODIES TO THE COLONIZATION FACTOR CFA 1 OFENTEROTOXIGENIC ESCHERJCHIA COLI IN BOVINE MILK AN])COLOSTRUM 67A. Materials and methods 68B. Results 71C. Discussion 78V. EFFECT OF BOVINE COLOSTRUM ON THE INVASIVENESSOF SALMONELLA ENTERITIDIS, S. TYPHIMURIUM AN]) E.COLI IN HeLa CELLS 82A. Materials and methods 83B. Results 88C. Discussion 118VI. CONCLUSION 125REFERENCES 127VLIST OF TABLESTable I. Effect of various concentrations of LFD on the growth or survival of S. enteritidis in1%peptone 48Table II. Effect of LFD or LFD plus lysozyme on the growth of S. enteritidis in infantfomula and chicken skin extract 49Table Ill. Concentrations of calcium and magnesium in some test media 51Table IV. Growth or survival of S. enteritidis in various concentrations of TSB with orwithout lactoferrin digest (LFD) and lysozyme (Lys) 57Table V. Effect of LFD and bile salts on the growth or survival of S. enteritidis in 1%peptone 60Table VI. Agglutination inhibition titers of various whey preparations against bovine (24 12-91) and human (H10407) E. coli strains 74Table VII. Estimates of total IgG concentration and of specific anti-CFA 1 IgG concentrationin various samples of cows colostrum and milk wheys 76Table VIII. Estimates of total IgG concentration and of specific anti-CFA 1 IgGconcentration in samples of bovine milk immune concentrates 77Table IX. Effect of bovine colostrum on the invasion of HeLa cell monolayers bySalmonella 90Table X. Effect of bovine colostrum on the invasion of HeLa cell monolayers by E. coliE2348/69 91Table XI. Invasion of HeLa cell monolayers by S. enteritidis ATCC13O76 or E. coliE2348/69 following preincubation of the cells with bovine colostrum 92Table XII. Effect of colostrum on the viability and invasiveness of S. typhimurium SL1344...94Table XIII. Inhibition of invasion of HeLa cell monolayers by bovine colostrum or dialysedcolostrum 96Table XIV. Inhibition of invasion of HeLa cell monolayers by S. typhimurium SL1344 or E.coli E2348/69 by bovine colostrum or colostrum fractions separated on the basis ofsize 97Table XV. Invasion of HeLa cell monolayers by SL1344 in the presence of fractions from apepsin digest of bovine colostrum 99Table XVI. Invasion of HeLa cell monolayers by SL1344 in the presence of heat treatedcolostrum 100viTable XVII. Invasiveness of SL1344 in the presence of concentrated pooled fractionsfollowing separation of colostrum on a protein G-agarose column 104Table XVIII. Invasiveness of SL 1344 in the presence of concentrated pooled fractionsfollowing separation of cottage cheese whey on a protein G-agarose column 105Table XIX. Invasiveness of SL1344 in the presence of bovine lactoferrin 107Table XX. Invasiveness of SL1344 in the presence of colostrum fractions obtained bychromatography on a protein G-agarose column, followed by separation on the basisof size 108Table XXI. Invasiveness of SL1344 in the presence of whey protein concentrate fractionsobtained by chromatography on a protein G agarose column, followed by separationon the basis of size 109Table XXII. Invasion of HeLa cell monolayers by SL1344 in the presence or absence ofbovine colostrum, peak I, peak I recycled or diluted peak II 112Table XXIII. Invasion of HeLa cell monolayers by SL1344 in the presence or absence ofbovine colostrum, peak I or peak I digested with pepsin 114viiLIST OF FIGURESFigure 1. The structure of immunoglobulins .13Figure 2. Model of the N-terminal lobe or domain of transferrins 32Figure 3. SDS-PAGE profiles of bovine lactoferrin and of pepsin digests of bovinelactoferrin 45Figure 4. Effect of lactoferrin (LF) and lactoferrin digest (LFD) on growth or survival ofSalmonella enteritidis in trypticase soy broth (A and B), and in 1% peptone (C andD) 46Figure 5. Effect of calcium on the antibacterial activity of LFD against S. enteritidis ATCC13076 52Figure 6. A: Effect of increasing concentration of lysozyme at a constant concentration oflactoferrin digest (LFD) (30 .tg/ml in terms of lactoferricin B) on the growth ofSalmonella enteritidis in TSB. B: Effect of increasing concentration of LFD at aconstant concentration of lysozyme (80 .ig/m1) 53Figure 7. Effect of increasing concentration of EDTA, with or without lactoferrin digest(LFD) and lysozyme (LYS) on growth or survival of Salmonella enteritidis intrypticase soy broth after 4 hours of exposure at 37°C 55Figure 8. Effect of lactoferrin digest (LFD, 30 .Lg/ml in terms of lactoferricin B), lysozyme(LYS, 80 jig/mI) and EDTA (0.25 mM), separately or in combination, on growth orsurvival of Salmonella enteritidis in three quarter strength trypticase soy broth (A andC), and in half strength trypticase soy broth (B and D), after 4 hours of exposure ....58Figure 9. A. Immunoglobulin G concentration of fractions of bovine colostrum wheyobtained by chromatography on a G100 Sephadex column. B. Agglutinationinhibition titers against E. coli H10407 of fractions of bovine colostrum whey andhuman millc whey obtained by chromatography on the same G100 Sephadex column.73Figure 10. Absorbance and relative IgG concentration of colostrum fractions obtained bychromatography on a protein G-agarose column 101Figure 11. SDS PAGE profiles of colostrum (UBC Col), and of Peak I and Peak II protein Gagarose fractions 103Figure 12. Immunoblots of bovine colostrum (UBC Col), Peak I, Peak II obtained bychromatography on a protein G agarose column, and a papain digest of Peak II 113Figure 13. A: Detection by TRITC immunofluorescence of S. typhimurium SL1344 on coverslips seeded with HeLa cells. B: Absence of non-specific binding of bovineviiiimmunoglobulins to cover slips seeded with HeLa cells 116Figure 14. A: FITC immunofluorescence with affmity purified antibody to bovine IgG of S.typhymurium SL1344 exposed to bovine colostrum on cover slips seeded with HeLacells. B: FITC immunofluorescence with affinity purified antibody to bovine IgG ofE. coil E2348/69 exposed to bovine colostrum on cover slips seeded with HeLa cells.117ixACKNOWLEDGMENTSI would like to thank Dr. B. Skura for his advice and support during this study.Thanks also to the members of my Research Committee: Dr. B. Finlay, Dr. D. Kitts and Dr.E. Li-Chan, as well as Dr. S. Nakai, who have contributed time, advice, supplies orequipment to this project.Many people have contributed to the progress of this work. In particular I would like to thankSherman Yee, Val Skura, Angela Kummer, Donna Smith and Sharon Ruchskowski for theirassistance.I would also like to acknowledge the participation and interest of Dairyworid Foods and theDairy Bureau of Canada. Financial support by the Science Council of British Columbia inthe form of a Graduate Research in Engineering and Technology Award is alsoacknowledged.x1I. INTRODUCTIONThe dairy industry is faced with the problem of cheese whey disposal or utilization.The recovery and commercialization of the whey solids has been seen as a possible solutionto this problem. Immunoglobulins and lactoferrin, which have antibacterial activity, arepresent in cheese whey at low levels. It has been suggested that they could be extracted,concentrated, and used for supplementation of infant formula, or foods for the elderly andimmunocompromised persons, in order to prevent diarrheal illnesses caused byenteropathogens.Supplementation would imitate a phenomenon commonly observed in nature: thepassive transfer of immunity from mothers to infants, which insures the survival of newbornsuntil their own immune system is sufficiently developed.In the literature review, the concept of passive transfer, which is the foundation of theproposal for supplementation, will be examined in the context of the main mechanisms ofhost defense against enteropathogens (humoral and mucosal immunity). The evidencesupporting a role for milk in the protection of infants against infectious gastroenteritis will bepresented. Experiments designed to demonstrate that colostrum or milk immune concentratesobtained from one species are effective in another species will then be reviewed. Similarstudies have been done with human infants whose diets have been supplemented withimmunoglobulins obtained from cows’ milk or eggs, and with adults, where protectionagainst challenge with enteropathogens was demonstrated. Finally, the reported in vitroantibacterial effects of lactoferrin and of lactoferricin, a recently discovered peptide obtainedby pepsin digestion of lactoferrin, will be reviewed. This peptide has been shown to have amuch greater antibacterial activity than lactoferrin in simple media.There is a long history of experimentation aimed at the use of immunoglobulins orlactoferrin in foods, and many problems remain to be addressed. In this thesis two general2questions were asked:- 1) can the bactericidal activity of a pepsin digest of bovine lactoferrin, containingthe peptide lactoferricin B, be demonstrated or enhanced in complex media?The results of experiments to compare the bactericidal activity of bovine lactoferricinin simple and complex media and in foods, and to examine the effect of added lysozyme orEDTA, will be presented.- 2) can the antibacterial activity of milk immunoglobulins be detected and estimatedin a relatively simple manner, using a method that would be more informativethan immunoassays but less cumbersome than in vivo experiments?The results of experiments to detect in bovine milk or colostrum, byhemagglutination and immunoassays, antibodies to the colonization factor antigen of a strainof human enterotoxigenic Escherichia coil will be presented. This is of interest because suchantibodies have been found to be very effective in animal studies. Adherence to or invasionof the intestinal epithelium are virulence characteristics of most enteropathogens. Butananti-adherent or anti-invasive effect of milk immunoglobulins cannot be testedbyimmunoassays.Experiments in vivo with human adult volunteers, with children and with a variety ofanimals are not routine. On the other hand, cell culture methods are in widespreaduse instudies of microbial pathogenesis, and adherence to or invasion of mammalian cellsinculture by pathogens has been correlated with virulence in vivo. The results of in Vitroexperiments, using HeLa cells, to test the anti-invasive properties of bovine colostrum andcolostrum fractions against Salmonella enteritidis, S. ryphimurium and Escherichiacoli willbe presented.3II. LITERATURE REVIEWA. The problem of whey utilizationWhey is the liquid fraction that remains following manufacture of cheese. It isproduced in very large amounts and its utilization has been a continuing challenge for theindustry.The problem can be appreciated by the following figures: More than half of the solidsin milk remain in the whey; the quantity of liquid whey produced is roughly ten times that ofcheese. In 1991, the amount of whey produced in North America was about 885 thousandmetric tons of solids, in Western Europe 1380 thousand metric tons, and in the Pacific Rimcountries 155 thousand metric tons (Horton, 1993). The utilization was 75% in Europe andprobably less than 50% in the rest of the world, and as a result a very large amount ofmaterial with potential value as food or feed is wasted. The corresponding volume of liquidcan be calculated on the basis that solids are about 6-7% of the whey. It can be seen that thedisposal of whey is a considerable problem. It has been calculated that simple discharge of1,000 gallons of whey into a river would require the dissolved oxygen contained in4,500,000 gallons of water for its oxidation (Gillies, 1974). Whey is comparativelyconcentrated compared to normal municipal waste and therefore puts great demand onmunicipal sewage systems, so that dairies have or will have to pay a surcharge tomunicipalities for disposal of their excess whey. The traditional methods of handling wheyhave been to feed it as is to livestock (pigs and cattle); to dry it for use in food or feeds; or touse it as fertilizer. The use of liquid whey as livestock feed is limited largely because ofhandling cost and labor, and excessive feeding may lead to digestive disturbances in someanimals. Whey is beneficial as a fertilizer as long as the risk of pollution is limited, and themain problem again is that of handling; in addition the demand is seasonal and limited bygeographical location (Gillies, 1974).4Since simple discharge of whey into waste disposal systems is expected to becomeincreasingly regulated, and since cheese production is increasing, research efforts havefocused on the recovery and use of whey solids. The dairy industry is also becoming moreconcentrated, and this creates another incentive to switch from simple disposal to large scaleprocessing. In practical terms this means ultrafiltration and separation of the liquid whey intoa permeate, mainly lactose, and a whey protein concentrate that may be spray-dried. In NorthAmerica about 500 thousand metric tons of permeate solids were produced in 1991 (Horton,1993). Some of the permeate can be spread on fields in very limited amounts because it isnot a balanced fertilizer (insufficient nitrogen). There is interest in industrial use of thelactose-permeate in such products as glues in plywood manufacture, as a fermentationmedium or in production of ethanol or lactic acid. Concentrated whey or whey proteinconcentrate are used in dairy and bakery products, in confectionery products, soups, softdrinks, and in meat products (Clark, 1987).In the whey protein concentrate, the main proteins are bovine serum albumin, xlactalbumin, 8-lactoglobulin, immunoglobulins, lactoferrin and lactoperoxidase, and thisfraction of the whey is attracting more interest. It shouldbe noted that a significant amountof research into utilization of whey appearsto be proprietary and therefore information islimited. Specific information on the economics of whey utilization is difficult to find but itappears that most uses of whey are often in competition against other established products,and therefore whether whey or whey fractions are profitable or havea market depends on themarket price of the competing products. As a result there is an incentiveto look for new enduses of whey with high value added, with products marketed to industry and productdevelopment supported by a high level of research (van Hoogstraten, 1987).Antimicrobial agents in milk whey have attracted attention as potential products forvalue-added processing of whey (Goldman, 1989), and of these, immunoglobulins andlactoferrin are the topic of this presentation.5B. The impact of gastrointestinal infectionsIntestinal infections are common in infants where conditions of hygiene are poor. Theresult is often diarrhea. In the case of infants in underdeveloped countries, as many as tenepisodes of diarrhea in the first year are not unusual (Black et al., 1989). Diarrhea is the maincause of infant deaths in some countries (Wadstrom, 1975). For example, it has beenreported that in Nigeria, about 300 children die every day from the consequences of infectionwith enteropathogens (Ogunsanya et al., 1994). Worldwide mortality has been estimated at 4to 6 million/year (Guerrant et a!., 1990).Travelers to underdeveloped countries are also at risk (Sack, 1990), as well asmilitary personnel during large scale operations (Oldfleld et al., 1991). An epidemiologicsurvey reported the incidence of diarrhea in travelers to tropical or subtropical areas favoredby tourists to be over 30% on average, up to 50% in selected locations, while the incidencewas about 5% for travelers to the United States and Canada (Steffen, 1986). Outbreaks inday care centers and nurseries in developed countries can result in very high rates ofincidence (Bower et al., 1989). In the United States, it has also become apparent that thegreatest annual number and rate of diarrheal deaths occur among the elderly (Lew et a!.,1991). Diarrheal diseases are a significant concern with hospital patientsandirnmunocompromised persons in developed countries (Guerrant et a!., 1990). In the UnitedStates, an incidence of between 6.5 million and 275 million cases of infectiousgastroenteritis per year have been estimated (Archer and Kvenberg, 1985; Hedberg et a!.,1994). This last number is rather astonishing and illustrates the uncertainties in estimatingthe incidence of illnesses that mostly go unreported.Treatment or prophylaxis with antibiotics is a possibility in the case of bacterialinfections, but there are problems: given the course of most episodes of gastroenteritis, it isoften not practical or possible to identify the pathogen or totest for sensitivity to antibiotics,so that often empirical treatment is practiced; grossabuse of antibiotics is common; in some6countries they can even be bought over the counter, and microbial resistance is widespread(Levy, 1982). Development of vaccines has been slow (Levine, 1991) and to date the mostsignificant success has been in the protection of calves or piglets against enterotoxigenic E.coil by passive transfer of immunoglobulins through the colostrum or milk of vaccinateddams (Tacket, 1991).The principles of prevention of diarrheal diseases in underdeveloped countries aresimple and well-known: improved hygiene, improved sanitation, safe water. Theirapplication has been hampered by a general lack of will to bring progress to these areas. Indeveloped countries where a high level of hygiene already exists with a resulting lowerincidence, improvements may be difficult to achieve because of the intractable nature ofsome of the problems: relative crowding in day care centres; increasing proportion of elderlypeople; a large number of immunocompromised persons and a high level of antibioticresistant microorganisms in hospitals.The understanding of the role of milk in the prevention of diarrheal diseases, whichwill be reviewed in a following section, has led to studies into the possibility ofsupplementing foods, targeted at some sections of the population, with antibacterial andantiviral factors isolated from the large supplies of whey produced by the dairy industry.C. The enteropathogensThe pathogens most frequently implicated are enterotoxigenic Escherichia coil(ETEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coil (EIEC), enterohemorragicE. coii (EHEC), Shigeiia, Vibrio choierce, Campylobacter, Saimoneiia and rotavirus (Blancoet al., 1991; Black et ai., 1989; Cravioto et al., 1990). Some of them are also found indomestic animals. Infection occurs through the food, water, feces, by contact with animals orperson-to-person. To give a few examples, infections with E. coil have been linked to groundbeef, V. choierce to unsanitary water supplies and Saimoneiia to eggs and poultry (IFT,71988). Prevalence of these pathogens varies depending on geographical location orenvironmental circumstances. For instance, the agent of travelers’ diarrhea isoverwhelmingly enterotoxigenic E. coil (Black, 1986), while Salmonella are a major causeof infections in hospitals, and rotavirus and Shigella are most frequent in day care centers(Guerrant et al., 1990). More detailed reviews of the relationship between these pathogensand foods can be found in the literature (IFT, 1988).Bacteria have been grouped in a variety of ways. Gram-negative or Gram-positivebacteria are defined by their ability to take up particular stains. All bacteria mentioned in theprevious paragraph are Gram-negative. The distinction between Gram-negative and Gram-positive bacteria is a reflection of their outer membrane structure, which affects theeffectiveness of many antibacterial agents. E. coil and Salmonella are also organized innumerous serogroups on the basis of the antigenicity of structures on their outer membrane.The groups of E. coil mentioned above (ETEC, EPEC, EHEC, EJEC) contain a variety ofserogroups and are defined by the mechanisms by which they cause illness.Enteropathogens cause disease by producing toxins, by damaging the microvilli or byinvading the cells of the intestinal epithelium. Adherence to the intestinal epithelium is arequirement for most if not all enteropathogens, and for some of them the mechanisms ofadherence have been studied in great detail.Adherence of enterotoxigenic E. coii (ETEC) is mediated by colonization factors oradhesins. Numerous fimbrial adhesins have been identified. These adhesins are proteins thatattach to glycoconjugate receptors, and exhibit organ and species specificity (Krogfelt,1991). Antibodies to ETEC fimbrial adhesins are highly protective and this has been thebasis of a successful vaccine against enteropathogens in cattle. Adherence of ETECto theintestinal epithelium is accompanied by production of toxins, proteins which are responsiblefor increased secretion of fluid and electrolytes and resulting diarrhea. Heat labile and heatstable toxins are produced. Only the heat labile toxin is ofsufficient size to be immunogenic.8Enteropathogenic E. coli exhibit several different patterns of adherence. Theadherence of some strains of enteropathogenic E. coli is correlated with the presence of anEPEC adherence factor (EAF) which results in a typical pattern of microcolonies adhering tocultured epithelial cells. In addition, such EPEC produce a characteristic lesion calledadhering-effacing, which is believed to be associated with diarrhea as a result of theeffacement of the intestinal brush border (Levine et al., 1985; Knutton et al., 1987). Thesestrains of enteropathogenic E. coli have also been shown to be invasive (Donnenberg et al.,1989), and the invasiveness is correlated with the presence of a 94 kDa outer membraneprotein (Francis et al., 1991).Adherence of invasive enteropathogens such as Yersinia, Salmonella or Shigella isalso mediated by proteins secreted or present on the outer membrane. The interaction ofthese proteins with host cell membrane receptors results in invasion by the bacteria. The bestcharacterized of these proteins is the Yersinia pseudotuberculosis invasin (Isberg et al.,1987). In Shigella, a number of outer membrane proteins associated with a virulence plasmidhave been identified (Oaks et al., 1986), while in Salmonella gene products associated withinvasiveness have not yet been characterized (Finlay, 1994).While this thesis is concerned with protection against bacterial pathogens, it shouldbe noted that rotaviruses are responsible for a large proportion of intestinal illnesses inchildren (Hilpert et al., 1987). At least as many studies have been carried out on theprotective effects of milk against rotavirus infections as on its antibacterial effects. Theresults of some of these studies will be presented in later sections to illustrate some aspectsof this topic, since the same principles are involved.9D. Host DefensesPathogens in the digestive tract need to survive, to colonize by adherence toreceptors, and for some of them to enter the intestinal epithelium (invasion) or to generatetoxins. These steps are potential targets for the antibacterial factors that may be present inmilk.To fight pathogens, the hosts have evolved many different mechanisms that have abactericidal or bacteriostatic effect: they may deprive the bacteria of required nutrients, orgenerate toxic products, or prevent adhesion or invasion. Resistance to infections can bedefined in different ways:- specific vs non-specific host defense or immunity,- active vs passive immunity,- cellular vs humoral immunity,- mucosal vs systemic immunity.It is possible to combine these groups to further define various aspects of immunityor host defense; they will be discussed in greater or lesser detail to the extent that they relateto the topic of this presentation.1. Non-specific host defensesThe organism is protected against infectious agents by a variety of “non-immune”mechanisms. Such mechanisms are of a general nature and do not depend on previousexposure to a pathogen or toxin, and are sometimes called “native immunity”. The skin andmucous membranes provide a physical barrier to the penetration of microorganisms, andsecretions associated with the mucous membranes contain chemicals such as lysozymewhich are harmful to the bacteria, or create an unfavorable environment such as low pHwhich inhibits colonization. Lactoferrin and lactoperoxidase in milk or saliva are otherexamples of chemicals that may havea protective effect. Lactoferrin has attracted a great10deal of attention as a possible candidate for supplementation of foods and will be discussedin greater detail in another section. A protective effect has also been attributed tooligosaccharides in milk and this will be discussed briefly later. At the cellular level,phagocytosis of bacteria by neutrophils and macrophages is another important defensemechanism.2. Specific host defensesInfection leads to the development of an immune response that is specific to theinfectious agent. The immune response involves both cellular and non-cellular (humoral)components (Tizard, 1992). A main feature of the immune response is the production ofantibodies against antigenic determinants present on the pathogen. This aspect of theimmune response is most relevant to the topic of this presentation3. Specific humoral immunity: the immunoglobulinsThe humoral components of specific immunity are the immunoglobulins (Igs), whichare found in the circulatory system and in secretions such as milk. Antibodies areimmunoglobulins with specificity for an antigen. Immunoglobulins are glycoproteinsoriginally defined by their electrophoretic mobility at pH 8.6 (y globulins). They all have abasic structure of four polypeptide chains: two heavy chains (approximately 450 aminoacids, 50 kDa) and two light chains (approximately 220 amino acids, 23 kDa). The fourchains are held together by disulfide bonds. Multiples of this basic structure exist and in partdefine classes of Igs. The general structure of Igs was elucidated by clever experimentsinvolving separation of the chains by reduction and ailcylation, as well as by papain digestionof the immunoglobulins and characterization of the corresponding fragments (Porter, 1959;Fleischman et al., 1962). It was also possible to group immunoglobulins into several classes.Classes were defined by serological methods, by their biological activity and by some11structural differences; for example in humans the following classes have been identified:IgG, IgA, 1gM, IgD and IgE. Subclasses of IgGs are present. Similar classes are found inother mammals. An immunoglobulin class is defined by a particular class of heavy chain.Two classes of light chains also exist but they associate with heavy chains of any class. Theimmunoglobulins classes in different species are sufficiently different antigenically that theycan be distinguished by serological methods.IgGs are the most abundant class of immunoglobulins in human serum and areinvolved in neutralization of toxins and viruses and binding and opsonization of bacteria.IgMs are made up of five basic units, and are the first class of antibody to appear followingoriginal stimulation by an antigen (primary response) while IgGs appear later (secondaryresponse). IgMs are very efficient at fixing complement, a group of proteins involved in lysisof bacteria. IgAs are present at low concentration in serum but are the predominantimmunoglobulins in milk and other secretions where they exist as dimers to which isattached a secretory component (slgAs). The secretory component is attached to theIgAdimer during transport through the epithelium; it also appears to protect IgAs fromdegradation by proteolytic enzymes, which is beneficial to its protective role in the digestivetract. The main role of IgAs is to prevent adherence of bacteria to the intestinal epithelium.IgDs are present in very small amounts in the serum and are associated with cells of thelymphoid system. IgEs are also present in extremely low amounts in the serum but are ofgreat importance because of their property of binding to basophils and mast cells andsubsequently to participate in allergic reactions. Similar classes of immunoglobulins arefound in other mammals. In cattle, a subclass of IgG called IgGi is the predominantimmunoglobulin in milk, while IgG2 is predominant in serum (Tizard, 1992; Kimball, 1986).The enzymatic digestion of immunoglobulins by papain permitted the identificationof three fragments: two identical fragments called Fab or antigen binding fragments, whichwere later determined to be made up of the light chains and the N-terminal half of theheavy12chains, and the Fc fragment, which is made up of the C-terminal half of the heavy chains(Figure 1). An intact antibody molecule is bivalent, which means it can bind to two antigensor two antigenic sites, whereas the Fab fragments are monovalent. Agglutination orprecipitation reactions which are the most visible sign of an antigen-antibody reaction in thelaboratory, require an intact antibody molecule. The monovalent Fab fragment, however,retains the ability to bind to the antigen.The specificity of immunoglobulins is determined by the primary structure of someportions of their light chains and heavy chains. Light chains are madeup of two domains ofequal lengths, and heavy chains are made up of four or five domains. Homology between thesequences of the domains reflect the evolutionary origins of immunoglobulins. The N-terminal domains of both heavy chains and light chains are called the variable regionsbecause of their great variability in amino acid sequence. This diversity is generated bymultiple germ-line genes for the variable regions, by gene rearrangement and by somaticmutations (Tizard, 1992). The variable regions define the specificity of a given antibodymolecule for an antigen. The other domains define the constant region of the light and heavychains. They also define the classes to which they belong. The heterogeneity ofimmunoglobulins was quite an obstacle to their analysis: for instance it would have beenimpossible to obtain a complete amino acid sequence ofa given immunoglobulin because ofthe great heterogeneity of the variable regions. The study of myeloma proteins, whicharehomogeneous immunoglobulins produced in large amounts by clones of malignant plasmacells, allowed for the sequencing of many different immunoglobulins and the understandingof the basis of the specificity of antibodies.4. Active vs passive immunityThe distinction between active and passive immunity is important and is thefoundation from which the idea of supplementation of diets with antibacterial factorswas13Fab fragmentsLight chain_________Site of papaindigestionFe fragmentHeavy chain\1ariable regionsConstant regionsInterchain disulfide bridgesFigure 1. The basic structure of immunoglobulins.14developed. Active immunity refers to the immune response that develops following exposureto an antigen, and which results in expansion of cell lines with a specific interest in theparticular antigen, or in increased production of immunoglobulins. Both infection (natural)and vaccination (artificial) may produce an active immune response, the result of which is toprovide the individual with protection against the particular pathogen for a period of time, ofvarying duration depending on the antigen. Active immunity, however, requires time todevelop, and time may be a critical factor in the defense of the body against an infection, inthe case of a first exposure to an antigen. Passive immunity refers to the transfer ofimmunoglobulins from a donor, who has been immunized against an antigen, to a recipientwho may be at risk of exposure, or who has been exposed to the antigen but for some reasonis not immune to it. The classic example of passive immunity is that of the use of antiserumagainst the tetanus toxin. A person infected with the tetanus bacillus, and who is not immune,may succumb to the toxin before being able to develop active immunity. The passive transferof immune globulins provides the recipient with temporary protection against the toxin. Thetetanus antitoxin has been produced by immunization of horses, but injection of horseimmunoglobulins, which are antigenic in humans, may have negative consequences for therecipient in the long term. A better approach has been to use human immunoglobulins isolatedfrom pooled plasma, which have the advantage of being less immunogenic to humans thanforeign proteins (Kimball, 1986).This type of passive transfer is of course an artificial situation but nature has provideda striking and widespread example of successful passive transfer. Ironically, the therapeuticuse of antiserum was developed long before there was any understanding of the passivetransfer of immunity in mammals. A newborn has had no exposure to pathogens and thereforewhile he may be able to mount an active immune response, this response may be too slow. Aprimary immune response following first exposure to an antigen requires several days beforeproduction of an effective amount of specific antibodies, by which time an infection may have15taken a fatal course. While newborns are rapidly colonized by bacteria after birth, and areexposed to pathogens in the environment, most survive as a result of the passive transfer ofimmunity from the mother to the infant. Several variations on this mechanism exist inmammals. Humoral immunity can be transferred from the mother in three different waysduring late pregnancy and after birth: 1) transfer of immunoglobulins in utero to thecirculation of the fetus; 2) immediately following birth with the colostrum, by gut absorptioninto the circulation of the newborn; and 3) later with the milk, where there may or may notbe selective gut absorption of the immunoglobulins. Combinations of these mechanisms arefound in nature, so that it has been possible to classify mammals into three different groups(Butler et al., 1985): Group I mammals are those where the transfer of Igs is strictly in utero(humans and rabbits); group Ill mammals where the transfer is strictly through the colostrumand gut absorption for a very limited period following birth (pigs, cows, sheep, goats); andgroup II mammals where both mechanisms are found (dogs, rodents) and where selectiveabsorption through the gut may continue for a significant length of time (18 days in mice andrats). Newborns of group Ill rarely survive in a natural environment without colostrum. In allgroups, passive transfer of immunoglobulins continues during lactation, the main componentof milk Igs being secretory IgAs, except in bovines where IgGi is the main component.Group Ill mammals, being born without any detectable amount of immunoglobulins, haveprovided an excellent system for the study of the development of humoral immunity. Inpiglets, following normal colostrum intake, immunoglobulin concentrationin serum, whichis highest 12 hours after birth, steadily declines for several weeks before de novo synthesisresults in increasing concentrations of Igs. For instance, 1gM concentration is at the lowestafter 2 weeks, IgG after 4 to 5 weeks and IgA after 2to 3 weeks (Klobasa et al., 1981). Theconcentration of IgAs in sows milk at that time is higher than that of the piglets serum. It hasalso been reported that the daily oral intake of immunoglobulinsby a seven day old piglet isgreater than the immunoglobulin content of its entire circulatory system (Wilson, 1974),16which illustrates the importance of passive transfer.While this appears to be a rather gratifying picture of nature at work in a beneficialway, it is complicated by the fact that newborns are unresponsive for a period of time tosome antigens even though they are generally immunocompetent, and it is thought that thepresence of Igs transferred from the mother may contributeto the slow development ofactive immunity (Kiobasa et a!., 1981).5. Systemic vs mucosal immunityThe distinction between systemic and mucosal immunity is of interest in this contextbecause while much of the knowledge of the immune system was obtained by the study ofsystemic immunity, the exposure of the body to the environment and to microorganisms isgreatest at the mucosal level, for instance the lungs and the digestive tract. Obviously, it iseasier to obtain serum from the circulatory system in a reproducible manner than togetsamples from the intestinal epithelium or from the lungs. Also, it has been found easiertoimmunize an animal or a person in a way that would result ina significant systemic immuneresponse, whereas it is still rather difficult to immunize an individual so as to achieve agoodlevel of mucosal immunity (de Aizpurua and Russell-Jones, 1988), and this has been one ofthe reasons why vaccines to enteropathogens have been slow to appear.The concept of compartmentalization of the immune system derives in part fromtheobservation that in humans and other mammals the classes of immunoglobulins that arepredominant in the circulation (IgGs) are different from the classes predominant on mucosalsurfaces (slgAs). The passive transfer of immunity in the newborn is a good example of thedistinction between mucosal and systemic immunity. In the human,IgGs are transferredthrough the placenta from the circulation of the motherto the circulation of the fetus, andafter birth slgAs are transferred with the milkto the digestive tract of the infant. In domesticanimals, the situation is not so obvious because thereis no placental transfer of immunity,17but studies of colostrum and milk composition in sows have shown that IgGs arepredominant in colostrum, while slgAs become predominant in the milk after a few days.The IgGs from the colostrum can be absorbed from the intestine into the circulation, therebyfulfilling the same function as placental IgGs in humans, while after a short time the pigletintestine becomes impermeable to immunoglobulins and therefore the slgAs then present inthe milk remain in the intestine. An interesting point is that the IgGs in the colostrum havebeen found to originate predominantly in the serum of the sow, while the IgAs in the milkare synthesized in the mammary gland (Salmon, 1989). Antibody-producing cells mature inthe lymphoid tissues of the intestine, from where they migrate to other tissues on bodysurfaces, for instance the mammary gland, thereby establishing a connection betweenantigen stimulation in the intestine and antibody secretion in the milk (Tizard, 1992).Even though this presentation is concerned with the use of immunoglobulins andother factors isolated from milk, it should be noted that hens eggs are another potentialsupply of immunoglobulins that could be used in passive transfer of immunity. Research tostudy the protective effect of immunoglobulins obtained from eggs of vaccinated chickenshas demonstrated their efficacy, and methods have been developed to purifyimmunoglobulins from eggs (Fichtali et al., 1992). While this will not be reviewed in detail,evidence from a number of experiments will later be presented briefly in support of theconcept of passive transfer of immunity across species.E. Milk and resistance to enteropathogens in humansIn humans, placental transfer of immunoglobulins reduces the dependency of thenewborn on the intake of milk or colostrum, and therefore the use of infant formula has beena practical or even popular alternative to breast feeding. There has been a long-standingcontroversy over the advantages of one or the other,and the evidence supporting a protective18effect of milk is mostly circumstantial.Colonization by pathogens does not necessarily result in diarrhea. In a study of 315children in Nigeria, it was found that 75% of children with diarrhea were infected withenteropathogens, while of the control group, with no symptoms of diarrhea, 28% wereinfected with enteropathogens (Ogunsanya et al., 1994). Cravioto et at. (1990) studied apopulation of 75 infants in a Mexican village for one year, and found an average of 4episodes of diarrhea per child in the first year, during which the children were colonized withthe following pathogens: enteropathogenic, enterotoxigenic, enteroinvasive andenterohemorrhagic E. coli; Salmonella, Shigella, Campylobacter jejuni and rotavirus.Incidence of colonization did not necessarily correlate with incidence of diarrhea. In otherwords, it is possible for a child to be colonized and not show symptoms. It was suggestedthat consumption of milk by breast-fed infants conferred some protection against appearanceof symptoms of disease, while colonization did occur and led to development of immunity.Studies have shown that there is an inverse correlation between levels of specificantibodies in mothers milk and the likelihood of the child showing symptoms of infection.For example, Cruz et al.(1988) surveyed a number of infants in a poor urban area ofGuatemala and found that among the infants infected with enterotoxigenic E. coli, those whobecame sick ingested milk with significantly lower titers of antibody to the heat labile toxinof ETEC than the children who remained asymptomatic. Glass et al. (1983), following up onan observation that children past the breast feeding age are hospitalized more frequently forcholera than children who are breast fed, found a lower incidence of symptoms in childrenreceiving milk with a high level of specific antibodies, while there was no relationshipbetween antibody levels and colonization. Production of specific antibodies in the milk hasbeen observed following natural infections. Stoll et at., (1986) found that milk antibody titersto cholera toxin increased in 80 to 90% of Bangladeshi patients following natural infection;anti-LPS titers also increased. High titers of anti-Ca,npylobacter-flagellin IgAantibodies19were found in the milk of all women tested in an area of Central Africa (Renom et al., 1992).Similarly, an association between Campylobacter antibodies in human milk and protectionfrom diarrhea caused by Campylobacter was found in a prospective study of Mexicanchildren (Ruiz-Palacios et al., 1990).Antibodies to viruses can also be found in human milk: For instance, in a survey of49 mothers, McLean and Holmes (1980) found evidence of secretory IgA specific forrotavirus in every sample of milk.Antibodies are thought to inhibit adherence of bacteria to the intestinal epithelium, orto have some antitoxin action. However, levels of immunoglobulins in milk are variable(Cruz et al., 1982), levels of specific antibodies in any individual mother’s milk are alsohighly variable over time (Cruz & Arevalo, 1985) and some children are raised on infantformuke, which are devoid of biologically active immunoglobulins.Other agents in human milk that may have a protective effect were reviewed byGoldman (1989). Of these, lactoferrin will be discussed later. A variety of oligosaccharides,which are believed to act as receptor analogs for various adherence factors ofenteropathogens and enterotoxins, have been found in human milk (Cravioto et a!., 1991;Ashkenazi and Mirelman,1987; Newburg et a!., 1992). Legrid et al. (1986) showed thatgangliosides from human milk completely inhibited fluid accumulation caused by choleratoxin in rabbit ileal loops. As far as bovine milk oligosaccharides are concerned, few in vivoexperiments have been done. In one study, glycoconjugates obtained from adult bovineplasma were shown to protect colostrum deprived calves against lethal doses ofenterotoxigenic E. coli (Mouricout et al., 1990). While milk oligosaccharides are not part ofthe research presented here, they may be potential factors in the interpretation of results andtherefore deserve this brief mention.201. Passive transfer in animals: exyerimental evidenceMany experiments have shown that immunoglobulins obtained from domesticanimals can protect against enteric pathogens in the same or another species. Uncontrolledexperiments axe practiced by some farmers who freeze excess colostrum for later use in anemergency, and at times colostrum from one species is fed to a newborn of another specieson the same farm. Failureto receive colostrum results in a well-defined end point fordomestic animals and thereforesuccess is easily measured.Besides such circumstantial evidence, experiments have demonstrated the role ofmilk immunoglobulins in passive transfer of immunity. For instance, newbornseronegativecalves fed milk replacer supplemented with colostrum from vaccinatedcows were fullyprotected against challenge with rotavirus, whereas control calves were not (Saifet aL,1983). Similar results were obtainedby Castrucci et al. (1984).Gnotobiotic piglets infected orally with E. coli and treated with colostrum or milkfrom vaccinated sows survivedsignificantly longer than control piglets. Survival dependedon continuous intake of milk or colostrum (Wilson, 1974)Experimental evidence is also available to support the concept of passivetransferacross species. Boesman-Finlcelstein et a!. (1989) immunized pregnantcows with choleratoxin and Vibrio cholerce outer membranes and collected the colostrum forup to four dayspost partum. The recovery of purified colostrum immunoglobulins ranged from 143to 794 gper cow. Titers were determinedagainst the respective antigens. The immunoglobulinpreparations were fed to 6-day-old rabbits priorto and following challenge with virulent Vcholerce. The rabbits who received the immunoglobulinsshowed significantly delayed onsetof diarrhea compared to the control. Rivier and Sobotka (1978) showedthat a serumpreparation from rabbits vaccinated with enterotoxigenic E. coli gave 100%protection torats challenged orally with thebacteria. Interestingly, the serum had only weak bactericidaleffect in vitro and none couldbe detected in vivo. Yoshiyama and Brown (1987) immunized21pregnant rabbits with live virulent V cholerce. Milk collected from the rabbits inhibited Vcholerce induced water secretion in rat ileal loops (the cholera toxin induces fluid secretion inligated loops). Immunoabsorption studies showed that the immunoglobulins were theprotective agent, and that they were specific for the enterotoxin. Lecce et a!. (1991) foundthat antibody from cows immunized with a simian strain of rotavirus protected pigletschallenged with a porcine strain of rotavirus. Gnotobiotic rats were protected against dentalcaries by ingestion of whey obtained from the milk of cows hyperimmunized with severalstrains of Streptococcus mutans (Michalek et al., 1987).Calves and piglets fed cow colostrum were protected against rotavirus (Bridger andBrown, 1981). Dose-dependent protection of piglets by bovine antibodies against humanrotavirus was demonstrated in one study (Schaller et al., 1992).Colostrum-deprived piglets have been raised fairly successfully by supplementingtheir milk replacer with immunoglobulins isolated from bovine serum: the 21-day survivalrate of piglets receiving milk replacer supplemented with bovine serum Igs was 75%,compared to 92% for the group receiving porcine Igs and 22% for a control group receivingonly milk replacer (Drew and Owen, 1988).Similar results have been obtained with egg yolk immunoglobulins (IgY). Mice wereprotected against challenge with human rotavirus by IgY immunoglobulins isolated fromtheyolk of eggs from immunized hens (Ebina et al., 1990). Colostrum deprived piglets wereprotected against enterotoxigenic E. coli infection by an antibody powder obtained fromeggs of chicken immunized with K88, K99 and 987P fimbrial adhesins. Piglets receiving thehighest titer preparations had a 100% survival rate following challenge, while controlpigletshad mortality from 80 to 100% (Yokoyama et al., 1992). In a similar study, Peralta et al.(1994) immunized chickens with a preparation of purified fimbrlie from S. enteritidis. Testmice fed the egg-yolk antibody preparations had a survival rate of 78% compared to 32% forcontrol mice, when challenged with the homologous strain of S. enteritidis.222. Passive transfer in humans: exnerimental evidenceBesides the correlation between presence of specific Igs in human milk andresistance to enteric infections found by surveys, a few experiments have been carried outwith human subjects, primarily studying protection against E. coli and rotavirus by passivetransfer of immunoglobulins obtained from milk or eggs. In an early experiment, cows werevaccinated with a mixture of pathogenic E. coli (Mietens and Keinhorst, 1979). A milkimmune concentrate was obtained, containing about 45% immunoglobulins, mostly IgGl.The concentrate was fed to infants suffering from diarrhea caused by pathogenic E. coli.Stool cultures in 84% of the patients in the treatment group became negative, whereas8 outof 9 patients in a control group remained positive.Milk immunoglobulin concentrates obtained from vaccinated cows were used to treat75 infants, up to two years of age, against rotavirus infection (Hilpert et al., 1987); theinfants had been admitted to hospital with acute gastroenteritis. Following identification ofrotavirus as the causative agent, the infants received 2g of concentrate per kg of bodyweight per day for five days. Concentrates with three different levels of neutralizing activitywere used. A therapeutic effect was defined as the reduction in excretion time of rotavirus. Ahigh neutralizing activity titer was required for the concentrateto achieve this therapeuticeffect. Ebina et al. (1985) found that infants fed 20 ml daily ofcow colostrum obtained fromvaccinated cows were protected against infection during an outbreak of rotavirus in anorphanage, but that the colostrum had no therapeutic effect on infants suffering fromdiarrhea prior to receiving the colostrum. Feeding of cow colostrum containingantibodies torotavirus also protected infants during a prospective study (Davidson et aL, 1989). Itappears, from the various studies, that the effect of ingesting colostrum was protective, ratherthan therapeutic.Tacket et a!. (1988) immunized pregnant cows during the last eight weeks ofgestation with enterotoxigenic E. coli belongingto 14 different serogroups, with cholera23toxin and with heat labile toxin. After removal of casein, fat, lactose and salt, the milk wasconcentrated and lyophilized. The resulting milk immune concentrate, containing 45%immunoglobulins, was fed three times a day, with antacid, to 10 adult volunteers in anamount equivalent to a total daily dose of 4.8g of immunoglobulinsfor 7 days. A controlgroup received a control milk concentrate. On the third day, the volunteers were challengedwith an enterotoxigenic strain of E. coli (1110407). None of the volunteers in the test grouphad diarrhea, while 9 out of 10 controls did. All volunteers excreted the pathogen for thesame number of days, even though at a lower level for the group receiving the immuneconcentrate, indicating that the protective activity was not entirely bactericidal. While thestudy by Tacket et al. (1988) was at that time the most detailed study of supplementation inadults, there was no determination of minimum effective dose, or of the antibody specificitythat was most protective (anti-toxin, anti-LPS, anti-fimbri, etc).It is interesting to speculate on the number of doses of immune concentrate that couldbe obtained from one immunized cow on the basis of the above results combined with datafrom another paper. According to the data of Boesman-Finkelstein et al. (1989) it is possibleto project an average recovery of about 400 g of IgGs during the first four days of lactation.If a cow produces about 8,000 liters of milk a year with an IgG concentration of 0.5 mg/mi,one is looking at a total production of over 4,400g of IgG, approximately 10% of which isproduced during the first few days of lactation. This amount would translate into about 1,000doses from each cow per year.In a similar experiment, Tacket et a!. (1992) immunized pregnant cows with apreparation of formalin treated Shigella flexneri. The resulting milk immune concentrateswere used to protect adult volunteers against challenge with S. flexneri. A preparation withahigh titer against S. flexneri LPS provided full protection, whereas a preparation with a lowertiter only provided limited protection. Again the preparations were not tested for thepresence of antibodies with different specificities.24Supplementation with bovine colostrum or immunoglobulins of immunodeficientpatients suffering from gastroenteritis has been reported. Malnourished children sufferingfrom prolonged infantile diarrhea associated with low level of intestinal IgA were reported toimprove following feeding of bovine colostrum (Bustos Fernandez et al., 1978). Twochildren with immunodeficiency and chronic diarrhea were reported to improve followingextended supplementation with immune globulins (presumably of human origin) (Melamedet a!., 1991). Three immunodeficient children suffering from chronic diarrhea associatedwith the presence of rotavirus were treated with human serum globulin possessingantirotaviral activity. It was found that the immunoglobulins were capable of binding torotavirus in the digestive tract so that excreted rotavirus was in the form of immunecomplexes (Losonsky et al., 1985). Finally, AIDS patients suffering from Cryptosporidium -associated diarrhea have been treated with hyperimmune bovine colostrum (Nord et a!.,1990; Ungar et al., 1990).These experiments are evidence that passive transfer of immunoglobulins obtainedfrom vaccinated cows or eggs provides protection against enteric pathogens. One recurringshortcoming has been a lack of information on the effective amounts and specificity ofantibodies in the preparations.F. Assessment of biological activity of immunoglobulin preparationsShould a preparation of antibacterial agents be obtained from milk or whey, it isnecessary to estimate its biological activity. For example, it would be necessary todetermineor define a dose of antibody that achieveda desired therapeutic or preventive effect. Giventhe heterogeneity of immunoglobulins,a dose may have to be defined by a specific effect ina controlled laboratory situation and related to the desired result in vivo.Measurement of total immunoglobulin content is not sufficient because it does notprovide information on the specificity of any antibodypresent. Measurements of antibodies25against a specific antigen (colonization factor antigen, lipopolysaccharide, enterotoxin) ismore informative but may not reflect activity given the possible effects of processing onactivity, or differing affinities of the antibodies for the antigen. Few of the experimentswhere humans or animals have been protected against an infection by colostrum or a milkimmune concentrate have attempted to relate dose to effect, or to identify the antigensagainst which the immunoglobulins were effective. Ebina et a!. (1985) protected infantsagainst infection with rotavirus by feeding colostrum from vaccinated cows. Antibodyneutralizing titers were determined but all infants were fed the same amount of colostrum.Total immunoglobulins levels were also measuredbut no relationship was apparent with theantibody neutralizing titer. In another experimentto protect calves against rotavirus usingcolostrum from immunized cows, the antibody titer only was determined and all calvesreceived the same amount of colostrum (Castrucci et a!., 1984). In experiments with ETECE. coli, Tacket et al. (1988) measured antibody titers against LPS, enterotoxin andcolonization factors, by ELISA or hemagglutination, in an immune concentrate obtainedfrom the milk of vaccinated cows. No dose response relationship was presented, and therespective effectiveness of the various antibodies could not be detennined. Schaller et a!.(1992), in a study on the prevention of rotavirus induced diarrhea in gnotobiotic piglets usingbovine antibody, defined an in vitro virus neutralizing titer from which in turn a functionalantibody dose was determined. They established a dose dependent relationship between viralshedding, diarrhea and antibody. The functional antibody dose was defined as that requiredto reduce the incidence of diarrhea or viral shedding by 50%.The problems associated with the use of human volunteers to test antibacterials areobvious, and a need for practical substitutes has led to the use of animal models or organsobtained from animals, or cell culture models.261. Animal models.The virulence characteristics of enteropathogens (invasion, adherence or enterotoxinproduction) can be studied in laboratory animals. Mice are good models to study infectionand invasion by Salmonella (Takeuchi, 1967; Hohmann era!., 1978). An infant mouse modelhas been used to study ETEC virulence, but mouse strains showed great differences insusceptibility to ETEC of various origins (bovine, porcine or humans) and generally showedonly weak susceptibility to human ETEC (Duchet-Suchauxet al., 1990). Guinea pigs havebeen used to study adherence of ETEC (Ashkenazi and Mirelman, 1987), and rabbits havebeen used to study adherence of rabbit and human EPEC strains (Demierre etal., 1975;Moon et al., 1983). Ligated loops models can be used to study the effect of enterotoxins(Svennerholm, 1975).Problems associated with the use of laboratory animals are numerous: cost,inconvenience, low productivity, variability and ethical considerations. Alternativemodelsthat are less controversial have been developed.2. The study of bacterial adherence or invasion in cell culturesCell culture systems have been used to study bacterial adherence, bacterial invasionand the effect of enterotoxins. The systems differ in the origin of the cells used: either cellsuspensions obtained directly from the organ of interest, or permanent cell lines. Each haveadvantages and disadvantages related to ease of use and similarity to actual in vivosituations.Enterocytes have been obtained from human biopsies to study adhesion ofenterotoxigenic E. coli (Knutton et al., 1984) or of enteropathogenic E. coli (Knutton et al.,1987). The advantage of cells suspensions is that they are obtained from the organ ofinterest. The problem with the use of cell suspensions is the need and inconvenience togoback to the original source, animal or human, when required.27Permanent cell lines have the advantage of relative ease of use compared to human oranimal subjects, uniformity, control of experimental conditions and availability. A variety ofpermanent cell lines such as chinese hamster ovary cells (CHO), Madin-Darby canine kidneycells (MDCK), human cervical epithelium cells (HeLa) or human larynx epithelium cells(HEp-2) have been used to study invasion or adherence by enteropathogens. Some cell linessuch as Caco-2 cells (human colon) and HT-29 cells (human colon) have the abilitytodifferentiate in culture and display characteristics of intestinal cells, which is convenient forthe study of the pathogens that require an enterocyte brush border for adherence.Campylobacter jejuni was shown to adhere to and invade HEp-2 cells (Konkel andJoens, 1989); the invasiveness of S. cholera-suis, S. flexneri and Yersinia enterocolitica werestudied in CHO, HEp-2 and MDCK cells (Finlay and Fallcow, 1988); the invasiveness of S.typhimurium has been studied in Caco-2 cells (Gahring et al., 1990); the invasiveness ofListeria monocytogenes was studied in Caco-2 cells (Gaillard et al., 1987) and RPMI-4788and HT-29 cells (Meyer et al., 1992) as well as the invasiveness of Shigeila fiexneri(Mounier et al., 1992) and the adherence of enterotoxigenic E. coil (Darfeuille-Michaud e tal., 1990). Heat labile enterotoxins from E. coii can be detected with the use of Yl mouseadrenal cortex tumor cells ((Donta et al., 1974, Sack and Sack, 1975).The validity of the cell culture models relies on the ability to demonstrate that i nvitro invasion of, or adherence to permanent cell lines is correlated with the samephenomenon either in primary cell suspensions of the corresponding in vivo target or withvirulence in vivo. This has been accomplished in many cases. For example, Giannella et a!.(1973) correlated invasion of HeLa cells by S. lyphimurium with invasion of rabbit ilealmucosa. Strains that were able to invade HeLa cells were also able to invade rabbit ilealmucosa, while strains that did not invade HeLa cells could not invade rabbit ileal mucosa.Day et ai. (1981) compared the invasiveness of strains of Shigelia and E. coil in 1{Ep-2 cellswith the results of the Sereny test. Of 63 strains tested, 37 were positive in both tests, 2528were negative in both and only one strain was positive in one test and negative in the other.Similarly, the ability to adhere to cultured cell lines correlated with virulence for EPEC andETEC. Levine et a!. (1985) demonstrated that EPEC strainE2348/69, isolated during anoutbreak of infant diarrhea, adhered to HEp-2 cells and to the intestinal mucosa of colostrumdeprived piglets, whereas strain MAR 20, derived fromE2348/69 and cured of a 60-MDaplasmid, could not adhere to HEp-2 cells or to piglets’ intestinal mucosa. Ingestion of theparent strain by volunteers resulted in diarrhea and production of antibodies against aplasmid associated 94-KDa outer membrane protein, whereas ingestion of the derived strainresulted in a lower proportion of cases of diarrhea and no detectable production of antibodiesagainst the outer membrane protein. The outer membrane protein was called EPECadherence factor (EAF) and was found in other EPEC strains but not in ETEC. Similarresults were obtained by Knutton et al. (1987). Darfeuille-Michaud et a!. (1990)demonstrated that ETEC H10407 (CFA 1), Pb 176 (CFA II), 1373 (CFA II), and 2230adhered to differentiated Caco-2 cells with a brush border, but not to other cell lines.Adherence to the Caco-2 cells was correlated to the presence of the respective plasmidsinvolved in the production of the adhesion factors, and to adherence to the brush border ofhuman enterocytes. Inhibition studies with purified antigens and with antisera to the purifiedantigens also demonstrated the specificity of adherence to Caco-2 cells and it was concludedthat Caco-2 cells behaved in the same wayas human enterocytes.3. Methods to estimate adherence or invasiveness of bacteria in cell culturesWhile adherence of bacteria to cultured cells has most often been estimated visuallyby microscopy following staining of the monolayers, invasion of cell cultures by bacteria hasbeen evaluated by plating techniques and by microscopy. Two general schemes to studyinvasion, one relying on bacterial enumeration by plating techniques, the other onobservation by microscopy, will be described.29Bacteria within mammalian cells are protected from the effect of some antibiotics. Ifmonolayers of mammalian cells are exposed to the bacteria of interest for a sufficient lengthof time to allow invasion of the cells, it is possible to eliminate the extracellular bacteria byincubation with the antibiotic gentamicin; intracellular bacteria are then released bytreatment with a detergent and counted by dilution and plating on bacteriological medium(Failcow et al., 1992). This method is relatively easy and efficient.Bacterial adherence can be determined by running a parallel experiment in somewells in which the monolayers are washed, prior to lysis of the cells, instead of being treatedwith gentamicin. This treatment will give an estimate of the total number of adhering andinvading bacteria, from which the number of adherent bacteria can be obtained bysubtracting the number of invasive bacteria obtained with the gentamicin treatment.Alternatively, it is possible to block invasion by treatment of the mammalian cells withcytochalasins, which prevent uptake of the bacteria (Finlay and Failcow, 1988), and adherentbacteria can be estimated by enumeration or microscopy. From a methodological point ofview, it is apparent that invasion can be evaluated with more confidence than adherence.The second method, microscopy, relies on the fact that antibodies cannot normallyenter mammalian cells (Heeseman and Laufs, 1985), so that external (adherent) bacteria canbe stained with a fluorescein labeled antibody, for instance, and then, followingpermeabilization of the mammalian cells with a mild detergent, the internalized bacteria canbe stained with a rhodamine antibody. The monolayer of mammalian cells can then beexamined with a fluorescence microscope. Internal and external bacteria can also bequantitated with this method, even though it is not as efficient as the gentamicin assay.4. Use of cell culture systems to study inhibition of adherence or invasionCell culture systems are also suitable to study or test factors that may inhibit30adherence or invasion. In particular, cell culture systems have been used to study the anti-adherence properties of human milk or colostrum againsta variety of pathogens, andmammalian cell suspensions and permanent cell lines have been used. Attachment ofStreptococcus pneumonke and Hcemophilus influenza to human buccal epithelial cells wasinhibited by human milk and receptor oligosaccharides (Andersson et al., 1986). Adhesionof EPEC E. coli to HEp-2 cells was inhibited by IgAs or oligosaccharides purified fromhuman colostrum (Cravioto et al., 1991). In other studies, adhesion of human EPECto HeLacells was inhibited by human colostrum or high molecular weight fraction of colostrum,butnot by any low molecular weight fraction (Camara et al., 1994; Silva and Giampaglia, 1992).Adherence of enterotoxigenic E. coli to porcine duodenal and ileal cells in vitro wasinhibited by anti-fimbri2e antibodies isolated from egg yolk (Yokoyama eta!., 1992).Adherence of Salmonella enteritidis to mouse intestinal epithelial cells in vitrowas alsoreduced by anti-fimbrial antibody (Peralta et al., 1994).No examples of the use of cell culture systems to test for anti-invasive effect ofhuman milk, or for anti-adherent or anti-invasive properties of bovine milk havebeen found.Therefore one of the goals of the research reported here was to investigate the suitabilityof acell culture method to determine whether bovine milk, or colostrum, or immunoglobulinshad any anti-invasive effect against some enteropathogens,so that at some point in thefuture, the in vitro and in vivo antibacterial activity of bovinemilk immunoglobulins couldbe correlated.G. Lactoferrin: antibacterial propertiesLactoferrin is a glycoprotein related to the iron binding transferrins. It isfound inmost external secretions as well as in the granules of neutrophils, where it is presentat veryhigh concentrations (Lebrer and Ganz,1990). Lactoferrin is present in the milk of some, butnot all, mammals. It was reportedthat a one month old breast-fed infant receives around311200 mg of lactoferrin a day (Butte et al, 1984). The concentration of lactoferrin in humancolostrum is about 7 mg/nil and in milk approximately 1 to 2 mg/mi. (Hennart et aL, 1991).The concentration in cows milk is much lower: approximately 0.1 to 0.3 mg/mi (Nonneckeand Smith, 1984). Even though lactoferrin has been studied extensively, its biological roleremains undefined. The various functions that have been suggested for lactoferrin werereviewed by Sanchez et al. (1992).The structure of lactoferrin is now well understood. Similar to all transferrins, thelactoferrin molecule is made up of two lobes, each witha binding site for iron (Figure 2).Binding of iron requires bicarbonate ions and is inhibitedby citrate ions (Brock, 1985). Theoutstanding feature of transferrins and lactoferrin is their high binding affinity for iron. Theexistence of transferrin receptors on mammalian cell surfaces, and of lactoferrin receptors onintestinal microvillous membranes in some species, point to a role of transferrins andlactoferrins in the transport and absorption of iron. However, there does not seemto be anyin vivo experimental evidence at this point that supports a role for lactoferrin in ironabsorption, or that demonstrates improved iron absorption by supplementation of diets withlactoferrin (Sanchez et al., 1992).Transferrins maintain the extracellular concentration of unbound iron at a level whichis too low to sustain the growth of bacteria, and injection of iron compounds reducesthelethal dose of infectious pathogens (Williams and Griffiths, 1992). Such finding wouldsuggest an antibacterial role for lactoferrin, but for the fact that pathogens grow well in bodyfluids. Bacteria have developed mechanismsto compensate for the unavailability of freeiron: the iron scavenging siderophores, whichremove iron from the transferrins, and thebacterial transferrin or lactoferrin receptors similarto the mammalian receptors (Griffiths,1993; Williams and Griffiths, 1992; Modun et a!., 1994). These bacterial receptors oftenshow species specificity for the host transferrin or lactoferrin.In spite of this paradoxical situation, lactoferrin has repeatedly been shown to have32Lactoferricin B157Figure 2: Model of the N-terminal lobe or domain of transferrins(adapted from Brock,1985).The most conserved disulfide bridges are shown,with numbers indicating their position inbovine lactoferrin. The iron binding aminoacids are Asp6O, Tyr92, Tyr192, His253 (Pierceetal., 1991)The location of the lactoferricin B peptide is shown.The sequence of the peptide is: Phel7-Lys-Cys-Arg-Arg-Trp-Gln-Trp-Arg-Met-Lys-Lys-Leu-Gly-Ala-Pro-Ser-Ile-Thr-Cys-Val-Arg-Arg-Ala-Phe4l (Yamauchiet al., 1993).231 2453619833some bacteriostatic activity in vitro. Reiter et at. (1975) found that dialysed colostrum had abacteriostatic effect on two strains of E. coli. A bacteriostatic effect of human miik or bovinecolostrum was attributed to lactoferrin by Griffiths and Humphreys (1977). In both casescitrate and iron were reported to be inhibitors of the lactoferrin activity, and addition ofbicarbonate was beneficial. Law and Reiter (1977) demonstrated bacteriostatic activity ofbovine lactoferrin isolated from cheese whey. Saturation with iron inhibited the effect oflactoferrin. Spik et at. (1978) found inconsistent activity of iron binding proteins in 1%peptone, except for human lactoferrin, which was strongly bacteriostatic. There was goodactivity in heat treated human milk, in conjunction with IgA. The addition of bicarbonatewas without effect. Stephens et at. (1980) found that human lactofenin and sIgA individuallyhad no effect on the growth of E. coti. In combination,they had significant bacteriostaticactivity on enteropathogenic serotypes, but not on commensal serotypes. On the other hand,Arnold et at. (1980) showed that a virulent culture of Streptococcus pneumonite was lesssensitive to lactoferrin that the avirulent strain. From other reports it appears that the effectof lactoferrin is highly dependent on the medium composition and on the condition of thebacteria. For instance, Rainard (1986a) demonstrated a strong reduction of growth of E. coliB 117 and 11 other E. coti isolates from mastitic cows using only 0.1 mg/mi of bovineapolactoferrin. The bacteria had been grown in an iron-poor, semi-defined medium, anda longincubation time in the presence of lactoferrin was required, presumablyto deplete the storediron in the bacteria. Addition of IgGi resulted in little improvement to the inhibiting effect oflactoferrin. In another experiment, Rainard (1986b) found that addition of 10% brain-heartinfusion broth to the medium abolished the bacteriostatic effect of lactoferrin. Giventhatiron-saturated lactoferrin has no bacteriostatic effect, it was proposed that it is effectivebymaking iron unavailable to microorganisms (Finkelsteinet al., 1983). However,experimental evidence that lactoferrin is bactericidal independently of its ability to removeiron from the growth medium was presented (Arnoldet at., 1982).34More recent work has suggested that lactoferrin may act by causing damage to theouter membrane of Gram-negative bacteria. The outer membrane of Gram-negative bacteriais made up of a bilayer with lipopolysaccharide (LPS) molecules occupying most of theouter layer (Nikaido and Vaara, 1987). Ellison et al. (1990) found that lactoferrin at aconcentration of 2 mg/mi caused the release of LPS from S. typhimurium SL696. Ethylenediamine tetraacetic acid (EDTA) (2 x 10-5 M) had the same effect. The LPS release could beblocked by the addition of calcium or magnesium. They also observed that, similar to EDTA,lactoferrin increased the susceptibility of a wild type E. coli strain to rifampicin and that thiseffect was reversed by the addition of calcium. It was suggested that the action of lactoferrinon the LPS was similar to that of EDTA or to that of other permeabilizing agents of theGram-negative outer membrane. Further studies by Ellison and Giehi (1991) showed thatlactoferrin did not have any ability to chelate calcium, and the antibacterial effect requireddirect contact between lactoferrin and the bacteria.Whether lactoferrin is bacteriostatic in two different ways (binding of iron or releaseof LPS) appears to still be an open question. It has been suggested that the binding of iron,which results in a conformational change of the protein (Anderson et al., 1990; Grossman e tal., 1992) prevents binding of lactoferrin to the LPS and thereby inhibits its bacteriostaticactivity.The effect of lactoferrin on the bacterial membrane led Ellison and Giehi (1991) toinvestigate a potential synergistic effect of lactoferrin and lysozyme on Gram negativebacteria. Lysozyme, which is used as a food preservative in some countries (Proctor andCunningham, 1988) is another antibacterial agent that causes lysis of some bacteria byenzymatic hydrolysis of the peptidoglycans found in the cell wall of both Gram-positive andGram-negative bacteria. The peptidoglycans of Gram-negative bacteria, however, areprotected by a layer of lipopolysaccharide (LPS), and therefore are not accessible tolysozyme under normal conditions. The LPS are strongly anionic and are stabilized by35calcium and magnesium. They bind to lysozyme and inactivate its enzymatic activity (Ohnoand Morrison, 1989). The activity of lysozyme against Gram-negative bacteria can bepromoted by EDTA in tris buffer at pH 8.0 (Wooley and Blue, 1975). Disruption of thebacterial outer membrane by polycations or chelating agents such as EDTA can bedemonstrated by the increased sensitivity of the bacteria to antibiotics, lysozyme or bile salts(Nikaido and Vaara, 1987).Ellison and Giehl (1991) found that lactoferrin and lysozyme in combination had aslight bactericidal effect in 1% peptone or similar simple medium, while individuallylactoferrin was only bacteriostatic and lysozyme had little or no effect against strains ofV.cholerce, E. coli and S. typhimurium. Binding studies showed that lactoferrin and poly-llysine had similar abilityto bind LPS and in general the properties of lactoferrin in thoseexperiments appeared to be similar to those of polycationic agents. Synergy betweenapolactoferrin and lysozyme was also demonstratedby Suzuki et a!., (1989) in 1% peptone.As a result of these laboratory studies and of its presence in various secretions andwithin the neutrophils, lactoferrin has been widely presented in the literatureas offeringprotection against infectious microorganisms (Hennart et al., 1991). Goldman (1989)suggested that lactoferrin could be addedto foods as a preservative agent or to protectindividuals against gastrointestinal infections. Ambitious projects have been undertakentoproduce lactoferrin on a large scale. A biotechnology company is planningto build a herd ofseveral hundred transgenic cows and toextract from their milk several tons of humanlactoferrin every year, which will be used for oral supplementation of immunocompromisedpatients (Seltzer, 1994; Hodgson, 1992).It is interesting that, to this writer’s knowledge, little if any direct experimentalevidence for an in vivo antibacterial effectof lactoferrin has been presented. In anexperiment designed to study the in vivo effects of lactoferrin, Moreauet a!. (1983),attempted to set up conditions that wouldbe most favorable to the effect of lactoferrin, such36as addition of sodium bicarbonate and trypsin inhibitors to a mouse diet deficient in iron.The gnotobiotic mice were inoculated with a strain of E. coli that had been found to besensitive to lactoferrin in vitro. No differences were found in the extent of colonization of thetest mice compared to control mice, as determined by ftecal bacteria counts. Similarly nodifferences were found in fecal counts of infants receiving either breast milk, infant formulaor formula supplemented with lactoferrin. Balmer and Wharton (1989) found that breast fedand formula fed infants had significantly different fiecal flora. To test the hypothesis that thedifference was caused by a lack of lactoferrin in the bovine milk derived formula, infantswere fed a basic formula or a formula supplemented with bovine lactoferrin. The infantswere observed for 14 days. It was found that addition of lactoferrin to the formula had noeffect on the infants’ fiecal flora (Balmer et al., 1989). On the other hand, Teraguchi et al.(1993) showed that supplementing the diet of specific pathogen free mice with bovinelactoferrin prevented the large increase in the number of fiecal Enterobacteriacea thatotherwise followed a switch from a solid to a milk based diet. This effect was not dependenton the iron content of lactofeffin, and was attributed to the bactericidal domain of lactoferrinnamed lactoferricin, which will be described in the following section. This hypothesis is notlikely to be correct, and data addressing this question will be presented in a later chapter.Finally, with regard to the supplementation of infant formula with lactoferrin, Drewet al. (1990) calculated that 833g of lactoferrin are required to bind 1 g of iron. On thisbasis, approximately 10g of lactoferrin would be required to bind the 12 mg of iron in 1 literof a soy based brand of infant formula. The total protein content of the infant formula is 18gil, and as a result lactoferrin would have to make up the majority of the protein fraction ofthe formula.H. LactoferricinRecently, it has been reported that peptides obtained by pepsin digestion of bovine37lactoferrinhave a strongbactericidalactivity againsta varietyof Grampositive andGramnegative bacteriain 1% peptone.Tomita etal. (1991) foundthat a pepsindigest of bovinelactoferrininhibited the growthof E. coil 0111at concentrationsof 0.25 mg/mior more (interms of lactoferrin),whereas aminimumconcentrationof 2.0 mg/miwas requiredforundigestedlactoferrin toachieve thesame inhibitoryeffect. Inaddition, it wasdemonstratedthat the lactoferrinhydrolysateretained itsactivity underconcentrationsof iron whichabolished theactivity of undigestedlactoferrin.The antibacterialactivity ofthe digest wasretained evenfollowing heatingat 121°Cfor 15 mm atpH 3 to 7.Hydrolysatesof both humanand bovinelactoferrinwere fractionatedby reverse-phase HPLC andthe active peptideswere sequenced(Bellamy etai., 1992b).The peptideobtained fromhuman lactoferrinhad a calculatedmolecular weightof 5,558 Da,whereas thepeptide obtainedfrom bovine lactoferrinhad a molecularweight of 3,126Da. The peptideswere called lactoferricinH and lactoferricinB respectively.LactoferricinB was found tobe25 amino-acidslong, whilelactoferricinH was 47amino-acidslong. Theprimary structureof each peptidewas foundto correspondexactly to asequence ofresiduesnear the N-terminal endof the correspondinglactoferrin,and it wasdeterminedthat disulfidebondsbetween cysteineresidues formeda ioop of18 amino-acidresidues(Figure 2).A highproportion ofthe residuesin each peptidewere lysinesor arginines.The peptidesare alsolocated awayfrom the iron-bindingresiduesof undigestedlactoferrin,leading totheconclusionthat the antibacterialactivity ofthe peptidesis not relatedto the iron-bindingability of lactoferrin.Iron seemed tohave little effecton the activityof lactoferricinagainstE. coli 0111(Bellamy et al.,1992b). Yamauchiet al. (1993)demonstratedthat 80 tMferricchloride inhibitedthe antibacterialeffect of 2 iMbovine lactoferricintoward E. coilCL991-2, but not of18 .tM bovinelactoferricin,while 20 .tMlactoferrinwas completelyinhibitedby the same concentrationof ferric chloride.Pepsin digestionof relatedproteins such ashuman transferrin,murine lactoferrinor ovotransferrinfailed toproduce peptideswith38antibacterial activity (Bellamy et al., 1992b). The corresponding sequences of these proteinshave a lower content of lysines and arginines than lactoferricin H or lactoferricin B.The antibacterial spectrum of lactoferricin B was studied in some detail (Bellamy e tal., 1992a). Most gram-positive or gram-negative bacteria tested, including many foodbornepathogens, were susceptible to inhibition by lactoferricin B, with only Pseudomonasfluorescens IFO-14 160 and Bfidobacterium bfidum ATCC 15696 being completely resistantto the action of lactoferricin B. The activity of lactoferricin B was not affected by addition ofvarious carbohydrates and protein, but was sensitive to the addition of calcium ormagnesium, as well as to the presence of citrate, succinate, lactate or acetate. The inhibitionby calcium or magnesium appeared to vary depending on the target strain (two E. coli strainswere tested).Wakabayashi et al. (1992) demonstrated that 4 strains of Listeria monocytogeneswere highly susceptible to inhibition by lactoferricin B in 1% peptone (minimum inhibitoryconcentration, 0.6 .tg/ml) and that at a concentration of 31 pg/ml, bacteria numbers werereduced from over106CPU/mi to less than 100 CFU/ml in 30 mm at 37°C.Finally, Yamauchi et al. (1993) showed that lactoferricin bound to and releasedlabelled LPS from strains of E. coli and Salmonella and compared this effect to that of othercationic peptides which also produce alterations in the outer membrane of Gram-negativebacteria.Bellamy et a!. (1992a) and Tomita et a! (1991) suggested the use of lactoferricin B asa “natural preservative agent in foods and cosmetics” or in some “clinical foods and productsfor prevention or treatment of infectious diseases”. The results published so far show thateven though lactoferricin is a potent antibacterial agent under some well-defined laboratoryconditions it may be susceptible to environmental effects that could make its applicationproblematic. Should this be the case, the possibility of enhancing the antibacterial effect oflactoferricin with lysozyme and EDTA is worth investigating.39ConclusionTo conclude this brief survey of some of the mechanisms of host defense againstenteropathogens, the supplementation of diet with antibacterial factors obtained from cowsmilk can be defined as:- protection at the mucosal level;- passive transfer of immunity;- humoral rather than cellular;- specific (immunoglobulins) or- non-specific (lactoferrin or lactoferricin).For supplementation by immunoglobulins to be implemented, a number of questionsremain to be answered. For instance:- how to extract immunoglobulins economically on large scale from milk or eggs;- to what extent is biological activity affected after processing;- how to measure the amount and effectiveness of any specific antibody in a sampleof purified Igs;- what amount of specific antibody is necessary to provide protection against a givenpathogen in a given population;- is it necessary or economical to immunize the donor animal;- of the variety of antibodies produced against a given pathogen, which is the mosteffective (i.e. anti-LPS, anti-adhesins, anti-toxins, others?);- is there a problem with allergenicity of milk Igs;- is there a synergistic effect between antibodies and other constituents of the milk;- how can sterile, safe and stable preparations of Igs be obtained without losing theiractivity;40- which is the target population that would benefit most from the supplementation,with the minimum of risks?With regard to a food industry application of lactoferrin or lactoferricin, the mainquestion remains: do they have any antibacterial effect in vivo or in more complexenvironments than those tested to date?The experiments reported on the following pages addressed a few of these questions.41III. ANTIBACTERIAL ACTIVITY OF A PEPSIN DIGEST OF BOVINELACTOFERRIN IN COMPLEX MEDIA.Lactoferricin H and lactoferricin B, cationic peptides derived by enzymatic digestionof human and bovine lactoferrin respectively, have been shown to have strong bactericidalactivity against a variety of pathogens in simple media, and it has been proposed that theycould be used as food additives, either as preservatives or for the prevention ofgastroenteritis (Tomita et al., 1991). At the time the experiments reported below wereundertaken the antibacterial effect of lactoferricin had not been tested in more complexmedia. The purpose of the present study was: to determine whether bovine lactoferricin, inthe form of a pepsin digest of bovine lactoferrin, had a bactericidal or bacteriostatic effect infoods or similar complex media; to determine the limits on this effect; and whether the use oflysozyme and EDTA in conjunction with lactoferricin could improve its antibacterialactivity.A. Materials and methodsBacteria.S. enteritidis ATCC 13076 were obtained from the American Type CultureCollection, Rockville, MD, and were maintained on TSA slants at 4 °C.LysozymeChicken egg white lysozyme (Sigma L-6876) (E.C. 3.2.1.17) was used at a finalconcentration of 0.08 mg/mi, unless otherwise indicated. The lytic activity of lysozyme wasconfirmed by spectrophotometric assay of lysis of Micrococcus lysodeikticus according tothe supplier’s directions.42EDTAEDTA was used at a final concentration of 0.25 mM, unless otherwise stated. This isin the range of concentration of EDTA allowed for use in some foods in Canada (Anon.,1988).Pepsin digestion of lactoferrinBovine lactoferrin (LF) (Sigma L4765) was dissolved in 0.05 M KCI/HC1 pH 2.0buffer and porcine pepsin (E.C. 3.4.23.1) (Sigma P-7012)added at a concentration of 3%(w/w of substrate). The original concentration of lactoferrin was determined byspectrophotometry at 280 nm using an extinction coefficient at 1% and 1 cm of 15.1(Fasman, 1976). The mixture was incubated at 37°C for 45 miii, followedby heating at 80°Cfor 15 mm. NaOH (1 M) was added to bring the pHto 7.0 and the digest was filtered througha 0.45 p. cellulose acetate filter (Corning 21053-25) and stored frozen at -20 °C. Digestionwas confirmed by SDS-PAGE electrophoresis using a PhastSystem electrophoresis unit(Pharmacia LKB Biotechnology, Uppsala, Sweden); to avoid repetition, the procedure isdescribed in detail in Chapter V. The digest (LFD) was used without further purification. Theconcentration of lactoferricin was calculated from the original lactoferrin concentrationassuming a molecular weight for bovine lactoferricin of 3,126 daltons (Bellamyet al.,1992b) and 83,000 for bovine lactoferrin.Culture conditionS. enteritidis were grown overnight in 50 ml of trypticase soy broth (TSB), in a 125ml flask, in a water bath at 37°C, at80 oscillations per minute. Bacteria were diluted 1:1000in 1% peptone and further diluted1:10 in the test medium. To the test medium were alsoadded the lactoferrin digest at a fmal concentration of 0.8 mg/nil interms of lactoferrin,unless otherwise stated (30 jig/mI in terms oflactoferricin B). Lysozyme and EDTA were43also added separately or together, at the required concentrations. The final volume was 200i.il.Samples were incubated for 4 hours at 37°C and aliquots removed at the beginning andthe end of the incubation period. Serial 1:10 dilutions prepared in 0.1% peptone were platedin duplicate on Difco plate count agar (PCA), or plate count agarto which had been addedDifco bile salts #3 at a final concentration of 1.5 g/l (PCA-BS). The drop plate method(ICMSF, 1978) was employed with 10 jil inocula applied to the media. Results of typicalexperiments are presented.Calcium and magnesium concentrations were determined by the complexometricmethod of Ntaiianas and Whitney (1964). This is a back titration method where calcium andmagnesium in the test sample complex with an excess of EDTA. The excess of EDTA isdetermined by titration with a solution of calcium of known concentration in the presence ofcalcein, an indicator that gives a green fluorescent color inthe presence of free calcium.Titrations at pH 12 and 13 respectively make it possibleto determine amounts of calciumand magnesium in the same sample. An aliquot of the sampleto be tested was added to 50ml of distilled water, to which were added 5 ml ofa 0.025 M solution of EDTA (in distilledwater with 2 g/l of NaOH. Sufficient potassium hydroxide(8 N) was added to bring the pHto 12, followed by three drops of calcein (0.2g in 100 ml of 0.025 N NaOH). The solutionwas back titrated with the standard calcium chloride solution (0.025 M) to obtain the firstend point. The combined amount of calcium and magnesium is calculated from this value.The pH is then raised to 13, and the next end point isused to calculate the amount ofcalcium. Two determinations were performed for each sample, the firstto approximate theend point and develop the color; this is used asa reference for the second determination, theresults of which are used in the calculations.B. ResultsDigestion of bovine lactoferrin by pepsin was confirmedby SDS-PAGE44electrophoresis. Two preparations were tested. The single band of lactoferrin was no longerpresent after peptic digestion of lactoferrin (Figure 3) and fast moving bands of smallpeptides appeared.The comparative activities against S. enteritidis of lactoferrin (LF) and the lactoferrindigest (LFD), added at equivalent molar concentrations, in 1% peptone and trypticase soybroth (TSB) were determined by incubating the bacteria in the respective media for 4 hoursat 37 °C followed by plating on plate count agar (PCA) and on plate count agarsupplemented with bile salts (PCA-BS). Bile salts have been used as probes to detectincreases in permeability of the Gram-negative outer membrane (Vaara, 1992) and fordifferentiation of injured and uninjured bacteria. Unlike Gram-positive bacteria, Gram-negative bacteria are protected against the effects of bile salts by the LPS of the outermembrane.Lactoferrin at a concentration of 0.8 mg/mi only slowed the growth of the bacteria in1% peptone, but caused some injury which was demonstrated when S. enteritidis were platedon PCA-bile salts agar, whereas LFD at a concentration of 30 tg/m1 in terms of lactoferricinB showed bactericidal activity in 1% peptone (Figure 4). Bactericidal activity is defined as areduction in the original number of bacteria determined by plating on PCA, and injuryas areduction in the number of bacteria growing on PCA-BS compared to that growing on PCA.Both compounds had little effect on S. enteritidis in TSB. The results suggest apermeabilizing effect of both lactoferrin and the lactoferrin hydrolysate in 1% peptone.Ellison et al. (1990) could not demonstrate a sensitizing effect of human lactoferrin todesoxycholate against a serotype 026 E. co/i while they could demonstrate sensitization of awild type E. coli to the antibiotic rifampicin, a rather contradictory finding. In retrospectthestudy of the effects of human lactoferrin on sensitization to desoxycholate may have beenconducted under conditions (high NaC1 concentration) where lactoferrin may not have hadany effect.Figure 3. SDS-PAGE profiles of bovine lactoferrin (LF) and of two pepsin digests ofbovine lactoferrin (LFD).MW= Molecular weight markers (Sigma M4038)45-y84,00014,200 —.--6,500 £LIiIMW LFDLFD LF4684-.0 6a)4-.0C.)200Figure 4. Effect of lactoferrin (LF) and lactoferrin digest (LFD) on growth orsurvival of Salmonella enteritidis in trypticase soy broth (A and B), and in 1%peptone (C and D).Bacteria were exposed to the various conditions for 4 hr at 37°C.Counts, expressed as log CFUs in 0.2 ml, obtained by plating on plate count agar(A and C), or on plate count agar + bile salts (B and D). Concentration oflactoferrin: 0.8 mg/mi. Concentration of lactoferrin digest: 30 j.tg/ml in terms oflactoferricin B.Log CFUs at start of incubation: 4.7 (dotted line).Detection limit: 2 log CFUs.*:<2 log CFUs.IControlLFLEDA B C D47The minimum inhibitory concentration was determined by incubating S. enteritidisfor 4 hours with different levels of LFD. Two preparations were tested. The lactoferrin digestat a concentration as low as 3 .tg/m1 in terms of lactoferricin B inhibited the growth of S.enteritidis ATCC 13076 in 1% peptone (Table I). This compares with concentrations of 7.8j.ig/ml (Jones et al., 1994b) and 12 J.tg/ml (Bellamy et al., 1992a) determined for other strainsof S. enteritidis using higher bacterial inocula. By comparing the results in Figure 4 andTable I, it can be seen that in 1% peptone, the minimum inhibitory concentration oflactoferrin was higher than 0.8mg/mi, whereas the minimum inhibitory concentration of thedigest was less than 0.08 mg/nil in terms of lactoferrin (3 .tg/ml in terms of lactoferricin B),a tenfold improvement or more in potency. This compares to increases in potency of fromeight to twentyfold reported by Tomita et al. (1991) for various Gram-negative and Gram-positive bacteria.Given that LFD showed a diminished activity in TSB compared to 1% peptone, andthat a synergistic effect of lactoferrin and lysozyme had previously been demonstrated inbacto-peptone or proteose peptone (Ellison and Giehl, 1991; Suzuki et al., 1989), and thatactivity only in 1% peptone would be of little use in terms of practical application,experiments were carried out to determine whether LFD alone or in combination withlysozyme would have an effect on bacterial growth or survival in some complex foods ormedia, some of which could be potential candidates for addition of LFD or lysozymeasantibacterial agents.Table II shows that LFD alone or in combination with lysozyme (80 .tg/ml) had noantibacterial action against S. enteritidis ATCC 13076 in a milk based and a soy basedinfant formula and in a chicken skin extract. The concentration of lysozyme in human milkhas been reported to be about 65 p.g/ ml and in gastric juice about 75 ig/ml (Hankiewicz andSwierczek, 1974).Bellamy et al. (1992a) previously showed that concentrations of calcium and48TABLE I. Effect of various concentrations of LFD on the growth orsurvival of S. enteritidis in 1%peptonea.LFD concentration(pg/ml)bLog CFtJs60-c2.830 2.9 3.415 3.5 3.66 4.0 4.23 45-c0 5.9 6.4a:Bacteria were exposed to the various concentrations of LFD for 4 hr at 37 °C.Two different pepsin digests were tested.Counts, expressed as log CFUs in 0.2 ml, were obtained by plating serialdilutions on plate count agar. Log CFUs at the start of incubation: 4.8 and 4.5respectively.b:Calculated in terms of the lactoferricin peptide.C:Data not available49TABLE II. Effect of LFD or LFD plus lysozyme on the growth of S.enteritidis in infant formula and chicken skin extract.log CFUs/0.2 mlatt=Oaatplated on plated onGrowth medium PCA PCA-BSSIMILACC49 7.0 6.9SIMILAC + LFD 4.9 7.0 6.5SIMILAC + LFD + Lys 4.9 6.7 7.0ISOMUf1 5.1 7.4 7.2ISOMIL÷LFD 5.1 7.3 7.1ISOMIL + LFD + Lys 5.1 7.6 7.4Chicken skinextracte4.6 7.6 7.6tt“ “+LFD 46 76 77+ LFD + Lys 4.6 7.6 7.9a: log CFUs/0.2 nil at the beginning of the experiment.b:log CFUs/0.2 ml after 4 hours of exposure in the respective growth mediaat 37 °C, followed by enumeration on PCA or PCA-BS.cSIMILAC®:a milk based liquid infant formula.d: ISOMIL®:a soy based liquid infant formula.e :prepared by homogenizing 1g of chicken skin in 1 ml of distilledH20.50magnesium in a range from 2 to 5 mM greatly inhibited the antibacterial activity oflactoferricin B against Escherichia coil 110-861, but had only a minimal effect on theactivity against E. coil 0111. Inhibition of the antibacterial activity of lactoferricin B againstE. coil 10418 by similar concentrations of calcium was also recently reported by Jones et al.(1994b).Concentrations of calcium and magnesium were determined in 1% peptone, TSB,chicken skin and infant formula (Table III). The infant formulas had concentrations ofcalcium and magnesium substantially higher than those reported to inhibit the action oflactoferricin B against the E. coil strains. The foods tested were chosen because they aresusceptible to microbial contamination either at the consumer level (infant formula) or at theprocessing level (chicken).Addition of calcium (as calcium chloride) to 1% peptone, to a concentrationequivalent to that found in milk (25mM), completely inhibited the activity of LFD againstS. enteritidis. Addition of calcium following 2 hours of incubation with LFD in 1 % peptonealso reversed the antibacterial effect of LFD (Figure 5). It was also found that LFD had nobactericidal or bacteriostatic activity against S. enteritidis when calcium was added to 1%peptone at a final concentration of 5 mM. The chicken skin extract had concentrations ofcalcium and magnesium of the same orderas those found in TSB. These levels of calciumand magnesium, presumably in conjunction with other factors, therefore appear to besufficient to inhibit the effect of lactoferricin B against S. enteritidis ATCC 13076.It was of interest to know whether it is possible to extend the range of effectivenessof a mixture of LFD and lysozyme in TSBby increasing the concentration of eitherindependently. TSB was used for convenience and because some preliminary observationsindicated that the concentration of inhibitors of lactoferricin in TSB might bejust lowenough that an improvement in antibacterial activity couldbe detected by the combinedeffect of LFD and lysozyme. It can be seen from Figure6A that increasing the concentration51Table Ill. Concentrations of calcium and magnesium in some test media.Calcium (mM) Magnesium (mM)1%Peptone 0.6 0.1TSB 3.1 0.4Chicken skinextracta33 0.3SIMILAC 12.3 1.7 (from label)ISOMIL 17.5 2.0 (from label)a:prepared by homogenizing 1g of chicken skin in 1 ml of distilledH20 at room temperature with a tissue homogenizer (Pyrex 7715).52760DUC)0-J32TimeFigure 5: Effect of calcium on the antibacterial activity of LFD against S. enteritidis ATCC13076.—°-—— A: 1% peptone.B: 1% peptone + calcium (25 mM).C: 1% peptone + LFD (30 .ig/m1).—0--—D: 1% peptone + calcium + LFD.—0-—E: 1% peptone + LFD; calcium added after two hours of incubation.Bacteria were incubated at 37 °C in the media indicated. Bacteria countswere determined after 2 hr and 4 hr of incubation, by dilution and plating on plate countagar.Numbers of S. enteritidis expressed as log CFUs/0.2 ml (average of two experiments)*:<2.3logCFusDBAE0 1 2 3 4 5538•10Di.i_4C.)0,0200 80 160320800Lysozyme ig/m1Figure 6. A: Effect of increasing concentration of lysozyme at a constant concentration oflactoferrin digest (LFD) (30 .tg/ml in terms of lactoferricin B) on the growth ofSalmonella enteritidis in TSB. B: Effect of increasing concentration of LFD at a constantconcentration of lysozyme (80 tg/ml). Counts of Salmonella enteritidis, after 4 hours ofexposure in trypticase soy broth at 37°C, were obtained by plating on plate count agar(PCA), or on plate count agar + bile salts (PCA + BS).Log CFUs at start of incubation: 4.8 (dotted line)PCAPCA+BS0 30 60 150LED gfm154of lysozyme up to 800 .tg/m1 while maintaining the concentration of LED (equivalent to 30ig/ml of lactoferricin) had minimal effect on the potency of the mixture, whereas increasingthe LFD concentration above 60 tg/ml while maintaining a constant concentration oflysozyme (80 j.ig/ml) increased the effectiveness of the mixture, as shown by a smallerincrease in numbers and by injury to S. enteritidis (as evidenced by the decreased number ofCFUs on PCA-BS) (Figure 6B). However, the improvement was not great and even at 150pg/mi of lactoferricin B, only a bacteriostatic effect could be demonstrated. Moreover thedata did not allow for the contribution of lysozyme, if any, to be estimated. The resultspresented in Figure 6 show that first, lysozyme had little effect on S. enteritidis under theconditions of the experiments, and second that any additional effect due to lysozyme willonly be evident under conditions where LFD itself has some degree of effectiveness.To further attempt to counteract the effect of TSB on the activity of LFD, EDTA wasadded to TSB in increasing amounts, while maintaining the concentrations of LFD andlysozyme. EDTA is an outer membrane permeabilizer that requires the presence of Tris atpH 8.0 to be most effective. It is not as effective in nutrient broth but may be able to chelatethe calcium and magnesium in the medium and thereby reduce their effect on lactoferricin.Increasing the concentration of EDTA alone in TSB progressively resulted in a diminishedincrease in bacteria count, even though the effect was small, while addition of LFD (30jig/mi) and lysozyme (80 jig/mI) together with EDTA had greater effect. However, EDTA ata concentration of 1.25 mM in combination with LFD and lysozyme was required to preventan increase in the number of bacteria from the initial inoculum level (Figure 7A). Someinjury to S. enteritidis could be demonstrated by plating on PCA-BS (Figure 7B).Considering that TSB at normal concentration appears to inhibit the action of LED ata concentration equivalent to 30 jig/mI of lactoferricin, the possible synergy of LED andlysozyme could then be studied either by increasing the concentration of LED, or by dilutingthe growth medium to reduce the concentration of inhibitors.550C)4-.0DC.)00EDTA (mM) EDTA (mM)Figure 7. Effect of increasing concentration of EDTA, with or without lactoferrin digest(LFD) and lysozyme (LYS) on growth or survival of Salmonella enteritidis in trypticasesoy broth after 4 hours of exposure at 37°C.Counts obtained by plating on plate count agar (A), or on plate count agar + bile salts(B). Lactoferrin digest concentration: 30 ig/ml in terms of lactoferricin B. Lysozymeconcentration: 80 .tg/m1. Log CFUs at start of incubation: 4.6 (dotted line).864ControlLFD + LYS864200 0.25 0.5 1.25 1.88 0 0.25 0.51.25 1.8856In view of the results in Figure 6, it was felt that the latter would be more productive,and the antibacterial activity of LFD with and without lysozyme was tested in variousstrengths of TSB.Table TV shows that S. enteritidis grew equally well in TSB or TSB diluted to 1/4normal concentration, that the effect of LFD increased as the TSB concentration decreased,and that as well an additional effect with lysozymewas apparent as the concentration of TSBdecreased. The effect of LFD, lysozyme and EDTA was further studied by reducing theconcentration of the growth medium, so that EDTA could be tested at a concentration of 25mM, which is approved in some foods in Canada (Anon, 1988). Figure 8 shows thatlysozyme at 80 .tg/ml or EDTA individually had little if any effect on the growth of S.enteritidis. On the other hand, addition of lysozyme, or E1)TA plus lysozyme, to the mediumtogether with LFI) resulted in increased antibacterial action, which again was dependent onthe concentration of the growth medium. The results in Figure 8 compare well with theresults presented in Table IV and in Figure7. For instance, the data in the columns LFD orLFD + Lys in Figure 8 are a replicate of a similar test in Table IV, while the data with EDTAare an extension of some of the results presented in Figure 7. Certainly in the combinationswhere lysozyme or lysozyme and EDTA were added to LFD an increased effect was foundcompared to the activity of LFD alone. It can also be seen that while in TSB a concentrationof EDTA of 1.25 mM was required to achieve a bacteriostatic effect in conjunction withLFD and lysozyme, in 3/4 strength TSB, a bactericidal effect was achieved when EDTAwas used in this combination at 0.25 mlvi.Considering that exposure to lactoferricin appears to result in some injury to thebacteria, so that they become sensitive to bile salts, the effect of lactoferricin in combinationwith bile salts was tested, in contrast to the previous experiments where the bacteria whereexposed first to lactoferricin, then plated on bilesalts agar. If lactoferricin were to be usedin vivo as suggested to prevent gastrointestinal infection, it would be present in an57TABLE IV. Growth or survival of S. enteritidis in various concentrations ofTSB with or without lactoferrin digest (LFD) and lysozyme(Lys)a.log CFUsGrowth medium on PCA on PCA-BS1xTSB 6.8 7.20.25xTSB 6.7 6.70.75xTSB + LFD 5.7 3.00.75xTSB + LFD + Lys 5.0 2.80.5OxTSB + LFD 5.0 2.30.5OxTSB + LFD + Lys 4.0 <2.00.25xTSB + LFD 3.6 <2.00.25xTSB + LFD + Lys <2.0 <2.0a:bacteria were incubated for 4 hr at 37 °C in the various test media.Bacteria counts, expressed as log CFLJs in 0.2 ml, were determined byplating serial dilutions on plate count agar (PCA) and plate count agar plusbile salts (PCA-BS).Log CFUs at start of incubation: 4.558ControlLYSEDTALEDLED + LYSLED + LYS + EDTAFigure 8. Effect of lactoferrin digest (LFD, 30 g/m1 in terms of lactoferricin B), lysozyme(LYS, 80 .ig/m1) and EDTA (0.25 mM), separately or in combination, on growth or survivalof Salmonella enteritidis ATCC 13076 in three quarter strength trypticase soy broth (A andC), and in half strength trypticase soy broth (B and D), after 4 hours of exposure. Counts,expressed as log CFLTs/O.2 ml, obtained by plating on plate count agar (A and B), or on platecount agar + bile salts (C and D). Log CFUs at start of incubation: 4.7 (dotted line).Detection limit: 1 log CFUs.•10C’,DLIC-)a,0DQA B C D59environment where bile salts are abundant. Some incidental observations had raised doubtsabout a beneficial effect of bile salts on the antibacterial activity of lactoferricin. The bacteriawere incubated for 4 hours at 37 °C in 1% peptone with LFD (30 .tg/m1) and bile salts (1.5mg/mi). While LF]) showed the expected bactericidal effect at that concentration, addition ofbile salts inhibited the effect of LFD. Bile salts alone only slowed the growth of Senteritidis (Table V).C. DiscussionThe purpose of this work was to determine whether LFD had any antibacterial effectagainst S. enteritidis in some selected foods, and whether a synergistic effect between LFD,lysozyme and EDTA could be demonstrated under conditions approximating those that maybe found in foods.The antibacterial effects can be defined in different ways:- a bactericidal effect, where a drop in the number of bacteria is observed at sometime following the addition of the agent,- a bacteriostatic effect, where the bacteria are prevented from increasing in number,- a sensitizing effect, which in the case of Gram-negative bacteria refers to a loss ofresistance to agents such as lysozyme, bile salts, or some antibiotics which are effectiveagainst Gram-positive bacteria, but from which Gram-negative bacteria are protected by thenature of their outer membrane. The sensitizingagents have been called permeabilizers(Vaara, 1992; Hancock and Wong, 1984), and some of the agents whose entry is facilitatedhave been used as probes to demonstrate the effects of the permeabilizers. Evidently thedifferent antibacterial effects are not necessarily independent from each other.A number of experiments were carried out and the results are presented in severaltables and figures which when related to each other give a good indication of the potential oflactoferricin as a food additive. For example, data on the effect of lactoferricin in TSB or60TABLE V. Effect of LFD and bile salts on the growth or survival of S.enteritidis in 1% peptone.Medium LogCFUsa1% peptone 6.41% peptone+ LFDb2.51% peptone + LFD+ BSC451%peptone+BS 5.8a:Log CFUs in 0.2 ml after 4 hours of exposure (average of 2 experiments).Log CFUs at the start of incubation: 4.8Counts determined by plating on PCA and overnight incubation at 37 °C.b:30 .tg/ml in terms of lactoferricin B.C:Bile salts at 1.5 mg/mi.61complex media were presented in six different instances; the data on the effect in 1%peptone were presented in five different instances; two different experiments were reportedon the effect of lactoferricin in reduced strength TSB; the interaction of LFD and lysozymewas studied in four different experiments; and the interaction with EDTA in two differentexperiments; finally the effect of LFD on sensitization of S. enteritidis to bile salts wasexamined in six different instances.The results showed that the pepsin hydrolysate of bovine lactoferrin at aconcentration of 30 jig/mi in terms of lactoferricin B had a bactericidal effect against S.enteritidis ATCC 13076 in 1% peptone, and that a definite bacteriostatic effect could bedemonstrated at a concentration as low as 3 jig/mi. In contrast, bovine lactoferrin at aconcentration of 0.8 mg/mi barely showed a bacteriostatic effect in 1% peptone. Therefore itis apparent that at equivalent molar concentration, LFD was approximately 10 times aspotent as lactoferrin. Both lactoferrin and LFD sensitized S. enteritidis to the bile salts,which suggested that the use of lysozyme in conjunction with LFD might be beneficial.However, neither lactoferrin nor lactoferricin had any detectable effect in TSB, a morecomplex medium. Testing of LFD alone or with lysozyme in infant formulas or a chickenskin extract showed a complete lack of antibacterial activity at the concentrations tested. Itwas found that addition of calcium at a concentration of 25mM was sufficient to inhibit theactivity of LFD and that the concentrations of calcium in the foods and media tested, withthe exception of 1% peptone were at least equal to the level that had been found to inhibit theantibacterial activity of lactoferricin against one strain of E. coli (Bellamy et al., 1992a).Addition of calcium to 1% peptone after two hours of exposure to LFD was sufficient toreverse the effect of LFD, so that the surviving bacteria were then able to increase in number.Attempts to overcome the inhibition of LFD in TSB by combinations of LFD,lysozyme and EDTA, and by raising their respective concentrations, were generally notsuccessful. Increasing the concentration of lysozyme to 800 jig/mi had no effect even in the62presence of LFD; it was necessary to increase the concentration of LFD to 60 jig/mI in thepresence of lysozyme to detect a reduction in the rate of increase of S. enteritidis and somesensitization of the bacteria to bile salts; increasing the concentration of EDTA alone hadminimal effect, while in combination with LFD and lysozyme a bacteriostatic effect wasachieved at a concentration of 1.25 mM EDTA.To demonstrate a bactericidal effect of LFD with lysozyme, or of LFD, lysozyme andEDTA at a concentration of EDTA that is allowed in foods in Canada, it was necesary toreduce the strength of the medium.These results raise doubts about the potential for addition of lactoferricin to foods,where calcium is often present at high levels and is an important nutrient. Other inhibitorssuch as magnesium or citrate are also likely to be present. Suggestions for an in vivo use oflactoferricin suffer from the same problem. Moreover, while the sensitizing effect oflactoferricin to bile salts may be thought of as beneficial, it is evidently useful only whenbacteria are exposed to lactoferricin prior to being exposed to bile salts, since it was shownthat bile salts inhibit lactoferricin when both are present at the same time in the test medium.Bile salts are secreted in large amounts in the intestine, and the conditions in the digestivetract appear to be most favorable to the inhibitors of lactoferrin and lactoferricin. Any effectof lactoferricin in vivo remains to be demonstrated, and the fact that lactoferrin or fragmentsof lactoferrin have been found in stools is no proof of efficacy. Careful experimentsbyMoreau et al. (1983) failed to show any effect of lactoferrin on the intestinal bacterial floracomposition in gnotobiotic mice and in infants. It is just as doubtful that increasing theconcentration of lactoferricin will help to overcome the inhibition by food components.While a concentration of lactoferricin of about 3 jig/mi is sufficient to showa bacteriostaticeffect in 1% peptone against S. enteritidis, the concentration of lactoferricin has to goup toat least 150 jig/mi to achieve the same result in TSB,a factor of 50, which does not seem63commensurate with the increase in concentration of the medium. Jones et al. (1994b) alsoobserved that addition of milk or infant formula to 1% peptone to a level of only 5% (vlv)was sufficient to increase the minimum inhibitory concentration of lactoferricin against Ecoli 10418 to over 500 .tg/m1. In the case of the two strains of E. coli where curves ofminimum inhibitory concentrations of lactoferricin versus calcium levels have beenproduced (Jones et al., 1994b; Bellamy et al., 1992a), the curves rise very steeply atconcentrations of calcium above 5 mM. This would suggest that increasing the amounts oflactoferricin in attempts to overcome the inhibitors in the foods is not likely to be productiveor economical. A prime requirement of any agent that may be added to foods as apreservative would be the ability to be effective under a wide range of conditions. This doesnot appear to be the case with lactoferricin.The results presented here do not provide an answer to the question of whetherlactoferricin has potential for an external application, for example in sanitation or, as hasbeen suggested, in cosmetics (Tomita et al., 1991). The use of lactoferrin has been proposedin toothpaste, and in the treatment of pinkeye in cattle (Borgstrom, 1990). The experimentspresented here were not designed to address this question directly, and it remains apossibility that the conditions may be more favorable to the use of lactoferricin, possibly inconjunction with lysozyme and EDTA. Experiments to control Salmonella on poultry meat(Samuelson et al., 1985), or coliforms in bladder infections (Goldschmidt et al., 1975), withlysozyme in combination with EDTA used much higher concentrations of either compoundsthan presented here. The addition of lactoferricin in such experiments could make itpossibleto reduce the concentrations of the other components. It would be worthwhile to investigatewhether lactoferricin has any antibacterial activity at low temperature or under conditionswhere bacteria may not be actively metabolizing.A number of experiments reported here showedthat under conditions where LFD64was active, it not only had a bactericidal effect, but it also had a permeabilizing effect so thatlysozyme, which otherwise had no effect against Gram-negative bacteria, becamebactericidal. Ellison and Giehl (1991) using human lactoferrin and human lysozyme, andYamauchi et al. (1993) with lactoferricin B and human lysozyme, reported the samesituation, with the difference that lactofenin was not bactericidal. A permeabilizing effect ofa large bacteriostatic molecule like lactoferrin that favors the effect of lysozyme might beviewed as beneficial. On the other hand, the use of lysozyme in conjunction with a smallbactericidal molecule like lactoferricin might be of little practical interest, as the same resultmight be obtained simply by increasing the concentration of lactoferricin itself. It could beargued that a mixture of these two compounds might have a broader effect than individually.Ellison et al., (1990) reported that bacteria grown in calcium rich medium were moresusceptible to the LPS releasing effect of human lactoferrin than bacteria grown in a lowcalcium medium. Yamauchi et al., (1993) confirmed this finding with bovine lactoferrin andadvanced the hypothesis that increasing numbers of cations are incorporated into the outermembrane when bacteria are grown in medium with high concentration of cations, and thatthis high numbers of cations in the outer membrane would make it more susceptible todamage by agents such as lactoferrin. No mechanism for this effect was offered. However,they also found that lactoferricin releases the same amount of LPS independently of theconcentration of cations in the growth medium, and moreover the amount of LPS released bylactoferricin is similar to the maximum amount released by lactoferrin from bacteria grownin a medium with high concentration of cations. This would tend to imply that, at the samemolar concentration, lactoferrin and lactoferricin have the same maximum capacity torelease LPS, but that when bacteria are grown in medium that is low in cations, themembrane is assembled in such a way that the LPS are not accessible to lactoferrin, whereasthey are accessible to lactoferricin. At the same time, Yamauchi et al. (1993) found that65under the conditions where both lactoferrin and lactoferricin released the same highproportion of LPS, lactoferrin had no effect on the viability of the bacteria, whereaslactoferricin caused a 99% decrease in CFUs. This may be a reflection of the ability of thesmaller lactoferricin molecule to penetrate the bacterial membrane. It would have beeninteresting to study the effect of probes like bile salts, lysozyme or actinomycin D in such anexperiment. This may be an indication that the antibacterial effect is a two steps process(permeabilization with the release of LPS, followed by penetration), and that lactoferricin isable to perform the two steps, but that lactoferrin, presumably restricted by its size, can onlyeffect perineabilization.Weiss et al. (1986) showed that changes in the sensitivity of E. coli to the neutrophilbactericidal/permeability increasing factor (BPI) could be significantly influenced by thecomposition of the growth medium. The BPI is a cationic protein with bactericidal activityagainst Gram-negative bacteria. Weiss et al., (1986), suggested that the effect of BPI isdirectly related to its binding to the bacteria, and that the binding is dependent on the chainlength of the 0-antigenic polysaccharides of the LPS. Longer chain polysaccharidesresulting from growth in a richer medium inhibited binding of BPI to the bacteria. It isdifficult to reconcile these findings with those of Yamauchi et al.(1993). Rana et a!., (1991),in a study of the effect of the cationic peptides magainins on Salmonella typhimuriwn,concluded that in addition to the length of the polysaccharides side chains, the charge of theLPS is a factor in the interaction between magainins and LPS.Whether this situation applies to lactoferricin is not known, but theaboveobservations suggest that the effect of a richer medium may be more complex than simplyproviding inhibitors of the binding of lactoferricinto the LPS. It may be that the synthesis bythe bacteria of longer chain polysaccharidesin rich media provides another obstacle tolactoferricin binding. Experiments with isogenic rough mutantsthat have various lengths ofO-polysaccharides side chains may be informative in this respect.66Regarding other future studies that may be undertaken with lactoferricin, it isdoubtful that further research with the intention of using it as a general food additive will beproductive until the properties of this peptide have been better defined, with respect to thenature of its binding to the bacteria and the nature of its bactericidal effect. It may beworthwhile to investigate a possible application in the areas of sanitation or treatment ofsurfaces of some food commodities provided that the limitations defined in the aboveexperiments are kept in mind. It may be interesting to investigate applications wherepermissible levels of EDTA or other chelators would be higher than in foods. Properties ofthe peptide that may confer an advantage in the area of sanitation are its stability (resistanceto heat or to further enzymatic degradation) and its potential for a residual effect. Forexample, recent experiments in this laboratory point to a bactericidal effect of the lactoferrindigest in water at room temperature at a concentration of about 3 j.tg/ml in terms oflactoferricin for at least 3 days (C. Chong, unpublished data).The above discussion applies only to the effect of lactoferricin on S. enteritidis andGram-negative bacteria. A bactericidal effect has also been demonstrated with Gram-positivebacteria (Bellamy et at., 1992a) and particularly on L. monocytogenes (Wakabayashi et al.,1992). No information has been published to-date on the effect, if any, of media componentson the bactericidal activity of lactoferricin on Gram-positive bacteria.67IV. ANTIBODIES TO THE COLONIZATION FACTOR CFA 1 OFENTEROTOXIGENIC ESCHERICHIA COLI IN BOVINE MILK ANDCOLOSTRUMWhile antibodies to bacterial LPS have been found in the milk of non-vaccinatedcows (Li-Chan et a!., 1994; Losso et a!., 1993; Al-Mashiki et a!., 1988), and whileimmunoglobulin preparations containing antibodies against bacterial LPS have been shownto protect human volunteers against challenge with enteropathogens (Tacket et al., 1988;Tacket et al., 1992), this does not demonstrate that antibodies to LPS are the best or onlyprotection. In the experiment by Tacket et at. (1992), the immune concentrates were obtainedby vaccinating cows with formalin killed bacteria. Antibodies to structures on the bacteriaother than LPS could have been produced, but it is not easy to estimate them. There isevidence that with enterotoxigenic E. coli, antibodies against the colonization factors are themost effective. In the case of passive transfer of immunity against EThC in cattle, it wasfound that protection was correlated to titers against the colonization factor K99, but not totiters against the 0 antigen (LPS) (Acres et al., 1979). Similarly, in trials with human ETEC,it has been reported that vaccines against the CFA 1 colonization factor appeared to be morepromising than vaccines against other bacterial products, such as LPS or heat labile toxin(Tacket, 1991).With this in mind, it was thought useful to look for the presence of antibodies to theCFA 1 antigen of human ETEC in bovine colostrum. Some enteric pathogens are able toagglutinate various types of red blood cells (Evans et al., 1979a). This ability correlates withthe presence of structures (colonization factors) on the bacteria that are involved inadherence (Evans et al. 1979b). It is known that components of human milk (antibodies andoligosaccharides) inhibit agglutination of erythrocytes by the bacteria and also inhibitadherence of the bacteria (Holmgren et al., 1981). Therefore, hemagglutination inhibition68was used to detect anti-CFA 1 antibodies in cows colostrum. Subsequently, a purifiedpreparation of CFA 1 antigen became available and it was then possible to test for anti-CFA1 antibodies by immunoassays. The results are presented below.A. Materials and methodsColostrum samples.Colostrum samples were obtained from the University of British Columbia DairyFarm or from Dr L.A. Babiuk, Veterinary Infectious Diseases Organization, Saskatoon, SK.Samples of bovine milk were obtained from Dr. L. Fisher, Agriculture and Agri-FoodCanada Research Station, Agazziz, B.C. Human milk samples were obtained from a localhospital.Wheys were prepared by acidification of the milk to pH 4.6 with iN HC1 in an icebath. The preparations were then brought to 30°C, centrifuged for 20 mm at 10,000 x g andthe supernatant adjusted to pH 7 prior to use.Samples of lyophilized milk immunoglobulin concentrate from cows immunizedwith a purified CFA 1 preparation, or with a heat killed whole cell preparation of CFAi E.coli H 10407, and from non-immunized cows were obtained from Dr. D. Maneval, Center forVaccine Development, Baltimore, MD.Bacteria.Enterotoxigenic E. coli H10407 (a CFA 1 positive strain), was obtained from Dr. M.Levine, Center for Vaccine Development, Baltimore, MD. Enterotoxigenic E. coli 2412-91(a K99 positive strain) was obtained from Dr. M. Schoonderwoerd, Alberta Agriculture,Edmonton, AB. ETEC 2412-9 1 were maintained at 4 °C on E medium Difco agar slants(Vogel and Bonner, 1956), while ETEC H10407 were maintained at 4 °C on CFA Difco agarslants (Evans et al., 1979b). The bacteria were subcultured monthly. Growth on these media69promotes production of fimbrie.Hemagglutination inhibition.Hemagglutination experiments were done in round bottom 96-well microtiter plates(Corning # 430245) according to the method of Korhonen et al. (1985), in the presence of0.5% mannose. Briefly, the test proceeded in two steps: titration of the bacterial suspension,followed by the test of inhibition. For titration, serial doubling dilutions of the bacterialsuspension in PBS-mannose (0.05 M phosphate buffered saline, pH 7.2, with 0.5% dmannose) were made in duplicate in wells 1-11 of the microtiter plate. The volume in eachwell was 25i.•The last wells in each row were used as negative control and received onlyPBS-mannose. Then 25 .tl of PBS-mannose were added to each well and mixed. This step isrequired to account for the volume of inhibitor that was later used in the test of inhibition.Then 25 p1 of a 2% suspension of erythrocytes in PBS-mannose were added and mixed. Thesealed plate was then incubated at 4 °C for 2 hours. Agglutination gives an even covering ofred cells, whereas lack of agglutination is shown by a small tight button of red cells at thebottom of the wells. The highest dilution showing agglutination is the titer of the bacterialsuspension. This titer is required to get the right proportion of bacteria, red cells andinhibitor in the second step. To test for inhibition, 25 p1 of serial doubling dilutions, in PBSmannose of the colostrum or milk samples were made in wells 1 to 11 of the microtiter plate.The last well was used as positive control. Bacteria (25 p.1) were then added at aconcentration four times that determined in the first step. Erythrocytes were added and theassay carried out as in the first step.Samples of human or cow milk whey were used to inhibit the agglutination ofindicator red blood cells by human or bovine enterotoxigenic E coli. The highest dilutionthat inhibited agglutination provided a titer and allowed for comparison of inhibitory powerof samples of different origins. Sheep red blood cells (obtained from the Animal Care70Centre, UBC) were used to determine the hemagglutination inhibition titer of wheys againstthe bovine pathogen E. coli 2412-9 1. Human type A blood cells (obtained from a localhospital) were used with the human pathogen E. coli H10407.Gel filtrationA sample of human milk whey and a sample of cow colostrum whey were applied toa 10 cm x 1.5 cm Sephadex G100 column (Pharmacia Biotech, Uppsala, Sweden), elutedwith 0.05 M phosphate buffer pH 7.4, to determine whether the inhibitory activity wasassociated with a large molecular weight compound such as Igs, or a low molecular weightcompound, such as oligosaccharides. Exclusion volume and retained volume weredetermined with Dextran Blue 2000 and potassium dichromate, respectively, and 1 mlfractions were collected.Determination of total and specific immunoglobulins levelsThe following buffers were used in immunoassays: Carbonate coating buffer, pH 9.6(1.59 g Na2CO3;2.93g NaHCO3;2 g NaN3;11 H20); PBS, pH 7.4 (8.0 g NaC1; 0.2 gKH2PO4;1.15 g Na2HPO4;0.2 g KC1; 0.2 g NaN3;11 H20) with 0.5 ml of Tween 20added for PBS-Tween; blocking buffer (0.25% ovalbumin in PBS); 10% diethanolaminebuffer pH 9.8 (97.0 ml diethanolamine; 0.1gMgC12.6H0); 0.2 g NaN3;H20 to 11)(Kummer et al., 1992). The ovalbumin was a gift from Canadian Lysozyme, Abbotsford,B.C.Amounts of total antibodies in milk and colostrum samples, or in immuneconcentrate samples were estimated by enzyme linked immunosorbent assay (ELISA).Ninety-six well microtiter plates (Immulon 2, Dynatech Labs, Chantilly, Va) were firstcoated for one hour at 37 °C with 100 .tl of rabbit anti-bovine Ig (Sigma B7265), at aconcentration of 1 .tg/m1, in carbonate buffer. The plates were then washed 3 times with71PBS, and 250 j.il blocking buffer were added to each well. After 30 mm of incubation at 37°C, the plates were washed with PBS, and serial dilutions of the samples in PBS + 0.05%Tween-20 were added in duplicate and incubated for one hr at 37 °C. The plates were thenwashed with PBS-Tween, and 100i.tlof anti-bovine Ig alkaline phosphatase conjugate(Sigma A7914, diluted 1:1000 in PBS-Tween) were added. After one hour of incubation at37 °C, followed by washing with PBS-Tween and distilled water, 100 j.tl of substrate (pnitrophenyl phosphate, Sigma 104) in diethanolamine buffer were added. Following colordevelopment, absorbances were read on a Bio Rad 450 microplate reader (BioRad,Richmond, CA) at 405 nm. A titration curve for each test sample was compared to a standardcurve obtained from a reference milk sample, the concentration of which had beenpreviously determined by radial immunodiffusion. The values from at least two dilutions induplicate were used in the calculation.Amounts of anti-CFA 1 antibodies in cows milk wheys and milk immuneconcentrates were estimated by enzyme linked immunosorbent assay (ELISA), using apurified CFA 1 preparation (obtained from Dr D. Maneval, Center for Vaccine Development,Baltimore, MD) as coating antigen at a concentration of 2.5 jig/mi. Otherwise, the assay wasidentical to the above. Since no standard for specific anti-CFA 1 Igs was available, theabsorbance values were compared to a standard curve for total Igs, obtained as above, in thesame microtiter plate.B. ResultsExperiments were conducted to determine the extent to which cows milk orcolostrum whey and human milk whey inhibit agglutination of sheep red blood cells byenterotoxigenic E. coli 2412-9 1, and of human type A red blood cells by enterotoxigenic E.coil H10407.Results of an initial experiment indicated that both a human milk whey sample and a72cow colostrum whey sample inhibited agglutination of red cells by human (H10407 CFA1)and bovine (2412-9 1 K99j E. coli strains. The inhibition titers against H10407 were 128forthe human milk whey and 64 for the bovine colostrum whey, while the titers against24 12-91 were 64 for the human milk whey and 32 for the bovine colostrum whey. Dialysis of thewheys, using a membrane with a molecular weight cutoff of 6,000 to 8,000, did not result ina decrease in agglutination inhibition titers.Fractionation of the wheys on a Sephadex G100 column showed that all thedetectable agglutination inhibition activity was associated with the high molecularweightfractions. The immunoglobulins, as determined by ELISA, eluted in the exclusion volumeofthe column. No activity was detected in the late fractions (Figure 9).Further experiments were conducted to determine whether this activity could befound in samples of different origins. Serial dilutions of several samples of human or cowsmilk whey were tested for inhibition of agglutination of red blood cells by human or bovinepathogenic strains of E. coli.Nine samples of cows milk or colostrum whey, as well as four samples of humanwhey, were tested. The titers of the preparations are shown in Table VI.The human whey samples as well as the bovine whey samples inhibited red bloodcell agglutination by both the human and bovine strains of E. coli; the titers of the bovinecolostrum samples were comparable to the titers of the human whey samples, while the titersof the bovine milk whey samples were lower.At that time, a sample of purified CFA 1 antigen was donated by Dt D. Maneval, aswell as samples of milk immune concentrates from cows immunized with CFA 1 or wholecell heat killed E. coli H10407. It then became possible to estimate the specific antibodyactivities of the bovine whey samples against the human CFA 1 antigen by usinganimmunoassay technique (ELISA). The specific activities of lyophiized samples of milkimmune concentrates from cows immunized with this antigen or with a whole cell E. coli73A20000):iCl)0)0--- •I. I0 1020Fractions.4 . 4I IIB80-Human milk whey60--Cows colostrum whey40200-—— H •I•• I0 10 20FractionsFigure 9. A. Immunoglobulin G concentration of fractions of bovinecolostrum whey obtained by chromatography on a GlOO Sephadex column.B. Agglutination inhibition titers against E. coli H10407 of fractions ofbovine colostrum whey and human milk whey obtained bychromatography on the same GlO0 Sephadex column.Exclusion volume (I) and retained volume (II) are shown.Table VI. Agglutination inhibition titers of various wheypreparations against bovine (2412-91) and human (H10407) E. colistrains.Samples 2412-91 titers H10407 titersja256 1281024 512w...a1024 51264 1285795b256 2567317b128 12864 3264 12864 64128 1288 6464 128UBCCold128 128a: Human milk samplesb: Colostrums obtained from Dr. L. Babiuk, Veterinary InfectiousDiseases Organization, Saskatoon, SK.C: Cows milk samples obtained from Dr. L. Fisher, Agazziz ResearchStation, Agriculture and Agri-Food Canada, Agazziz, B.C.d: Colostrum from the UBC dairy herd.7475preparation were also estimated. Since no preparation of purified, specific and standardizedbovine antibodies to the CFA 1 antigen was available to us, it was necessary to compareabsorbances obtained from the titration of anti-CFA 1 antibodies to a standard curve obtainedfrom the titration of total IgGs of a milk sample whose concentration had been determinedby immunodiffusion.The amounts of specific antibody to the colonization factor antigen 1 (CFA 1), insamples of colostrum from cows that had not been vaccinated, ranged from 0.55 to 5.2 j.tg/ml(Table VII). The amount in milk was lower and barely detectable. Total immunoglobulinslevels were also estimated and a ratio of specific anti-CFA 1 antibodies to totalimmunoglobulins was calculated. Similar immunoassays of the reconstituted milk immuneconcentrates showed that vaccination increased the ratio of specific antibodies to total Igs inmilk by factors of about 10 to 25 (TableVIII). For comparison, the amount of anti-CFA 1antibodies in human serum (from asymptomatic individuals), was reported to average about6 jig/mi (Clegg et a!., 1980). This amount increased on average by a factor of 40 in theserum of patients infected with a CFA 1 positive enterotoxigenic E. coli, but very largedifferences in responses were found between patients, with some individuals having nodetectable specific anti-CFA 1 antibodies, even after infection (Clegg et a!., 1980).76Table VII. Estimates of total IgG concentration and of specific antiCFA 1 IgG concentration in various samples of cows colostrum andmilk wheys.Samples Total IgGs Anti-CFA IgGs % anti-CFA 1 IgGsmg/mi jig/mi1127a>80 1.0 >0.001579..5a79 5.2 0.007958a60 1.4 0.00281a43 0.8 0.0027317a40 1.0 0.003838a33 1.1 0.0043458a14 0.6 0.004UBCCoib18 1.3 0.0078738c0.86 0.1 0.012a:Colostrums obtained from Dr L. Babiuk, Veterinary InfectiousDiseases Organization, Saskatoon, SK.b:Colostrum from the UBC dairy herd.C: Milk obtained from Dr L. Fisher, Agazziz Research Station,Agriculture and Agri-Food Canada, Agazziz, B.C.77Table VIII. Estimates of total IgG concentration and of specific antiCFA 1 IgG concentration in samples of bovine milk immuneconcentrates.Samples Total Igs Anti-CFA 1 Igs % anti-CFA 1 Igsmg/mi ig/mlwc MIC 30 25.0 0.083CFAMICb31 12.0 0.039ControlMICc31 0.93 0.003aA sample of milk immune concentrate, lyophilized andreconstituted to 1 ml in PBS, from cows vaccinated with a whole cellpreparation of E. coil H 10407.b: A sample of milk immune concentrate, lyophilized andreconstituted to 1 ml, from cows vaccinated with the CFA-1 antigen.c: A sample of milk immune concentrate, lyophilized andreconstituted to 1 ml, from non vaccinated cows.The above samples were a generous gift from Dr. D. Maneval, Centerfor Vaccine Development, Baltimore, MD.78C. DiscussionThe purpose of the experiments reported above was to detect and estimate specificantibodies to the colonization factor CFA 1 of human ETEC in bovine milk or colostrum.Anti-CFA 1 antibodies were detected in bovine milk and colosirum wheys, and inhuman milk wheys, by inhibition of hemagglutination. Anti-K99 antibodies were alsodetected. Testing for anti-K99 antibodies provided a check of the method and of thehypothesis: if antibodies to CFA 1 were to be found in cows milk or colostrum, conversely itwould be reasonable to look for antibodies to K99 in human milk. No low molecular weightfactors were found that inhibited hemagglutination.Determination of amounts of total IgGs and particularly of specific Igs in milk byimmunoassays can be a rather difficult task. For determination of total IgGs, the dilutionsrequired are sufficiently high that background levels due to non-specific binding are notmuch of a problem. This is not the case for the determination of specific antibodies and attimes high background may make this work impossible. Other difficulties arise from theheterogeneous nature of the immunoglobulins to be estimated. This heterogeneity may causeproblems with the use of the standard curve method of data analysis, where the titrationcurve of the test sample is compared with that of the reference sample (Peterman and Butler,1989). There may be some difficulty in finding a suitable standard, as there is some evidencethat purified IgGs do not necessarily behave in the ELISA in the same way as IgGs in milk;one solution to this problem has been to use a sample of milk whose IgG concentration hasbeen determined by another method (Kummer et at., 1992). This was the method adoptedhere. Obviously this relies on the accuracy of the original measurement.The problem is more difficult in the case of determination of specific antibodyconcentration. Generally, a sample of specific antibody of known concentration, from whichto construct a standard curve, is not available. Various methods of expressing the results havebeen used. For instance, the optical density at a given dilution can be stated (Li-Chan et at.,791995). The results can be expressed as titers, the inverse of the dilution giving a minimumarbitrary optical density. Results have been expressed as relative fluorescence at a givendilution in Particle Concentration Fluorescence Immunoassays (PCFIA) assays (Losso et al.,1993). Results have been expressed as relative values of a reference sample; in one examplea range of concentration of specific Igs for the reference sample was estimated to be 7.5 to60 ng/ml by determining dilutions that would give comparable absorbance values in thespecific ELISA and in the total ELISA (Li-Chan et al., 1994). A similar method has beenused in this presentation except that rather than expressing the results as a percentage of thereference sample that would give the same aborbance as the test sample, the calculation ofconcentration has been done for every sample tested rather than just for the reference sampleand the results expressed in Lg/ml rather than as a percentage. This is not ideal but doesprovide an easier way to visualize a range of values for comparison purposes. Expressed inthis manner, it was found that the ratio of specific anti-CFA 1 antibodies to total IgGs incolostrum of non-vaccinated cows was at least 1:15000. Li-Chan et al. (1994) found ratiosaround 1:5000 for specific anti-LPS antibodies in cows milk.It may be surprising to find antibodies to a human pathogen in cows milk, orantibodies to a bovine pathogen in human milk, but recently published work suggests apossible explanation: it has been found that colonization factor antigens of human ETEC canprime and boost immune response against heterologous, serologically distinct (byimmunoassay) colonization factor antigens (Rudin and Svennerholm, 1994). Even thoughthe CFAs are distinct antigenically, some homology has been demonstrated at the level ofamino acid sequence. A similar situation has been found with rotavirus (Taniguchi et al,1991). Yolken et al. (1985) also found antibodies to human rotavirus in cows’ milk.It appears, from this and from other work cited above, that antibodies to humanpathogens are commonly found at low levels in cows milk. A survey of milk samples fromvarious areas of the Province of British Columbia was conducted to determine the total80immunoglobulin G content and the levels of specific antibodies to the LPS of E. coli011 1:B4, 0128:B 12, Shigella flexnerii 1A, Salmonella enteritidis and S. typhimurium ELiChan et a!., 1994). Specific antibodies against the same bacterial LPS were also estimated insamples of human milk (Losso et aL, 1993). It could be argued from these results thatantibodies will be found in milk or colostrum against any bacterial antigen that one tests for.Fukumoto (1992) discussed in detail the proposition that immunoglobulins could beseparated from whey obtained from the milk of non-immunized cows: even though the IgGconcentration is very low, the volumes are so high that considerable amounts could beobtained. Of course specific antibodies need to be present, and the studies cited in theprevious paragraph, as well as the results presented in this chapter, show this to be the case.This proposal has had some experimental support. Stott and Lucas (1989) described aprocess to concentrate whey obtained from the milk of non-immunized cows, and presenteddata showing that feeding approximately 1 g/kg bodyweight to colostrum deprived calvesimmediately after birth resulted in performance equal to or better than that of calvesreceiving colostrum.While the findings of specific antibodies to human pathogens in bovine milk areuseful and interesting, the limitations of this approach are also becoming evident. Certainly,antibodies to the above bacterial LPS and CFA 1 were found first because the correspondingantigens were available for immunoassays, not necessarily because these antigens were themost significant; specific antibodies cannot be found by this method unless thecorresponding antigen is known and available, or a workable immunoassay exists. Forexample, attempts at detecting antibodies to the enterotoxin (LT) of E. coli in bovinecolostrum were unsuccessful because while a system is available to capture the antibodies,using the GM1 ganglioside and cholera toxin (CT), problems of high background, and cross-reactions with the commercially available antisera made it impossible to develop a workingimmunoassay.81Immunoassays are useful for the detection and estimation of levels of antibodies, butthey provide no information on their biological activity or on the relative efficacies ofspecific antibodies. For example, Apter et al. (1993) showed by challenge of neonatal micethat anti-LPS antibodies are much more effective at preventing V cholercz induced diarrheathan anti-cholera toxin antibodies. On the other hand, a study of breast-fed Mexican infantsfound that the milk concentration of sIgA against Shigella virulence plasmid-associatedantigen was a better predictor of symptom status than the milk concentration of antiShigella antibodies (Hayani et al., 1992). Other factors with antibacterial activity that may bepresent in milk or colostrum may not lend themselves to detection by this method.The next chapter will report on the use of a cell culture method to estimate theantibacterial activity of bovine colostrum.82V. EFFECT OF BOVINE COLOSTRUMON THE INVASIVENESS OFSALMONELLA ENTERITIDIS, S. TYPHIMURIUM AND ESCHERICHIA COLI INHeLa CELLS.Model systems are required to study the biological activity of antibacterial factorsthat may be isolated from milk or colostrum. In thecase of antibodies to enteropathogens,studies of bactericidal activity in vitro are not likelyto be informative given indications thatprotection by milk antibodies in the intestinemay not be the result of agglutination orreduced viability of the bacteria, but is ratherthe result of inhibition of adherence of thebacteria to the intestinal epithelium. Some of the problems associated with immunoassayshave been presented in the previous chapter. Animal modelsmay be available, but sufferfrom many drawbacks, such as cost, reproducibilityor low productivity. The alternative wastherefore to use cell cultures.The purpose of the experiments presented belowwas to determine whether bovinecolostrum could inhibit invasion of HeLa cells by invasiveenteropathogens. Results ofexperiments with Salmonella enteritidis, S. typhimurium and enteropathogenicEscherichiacoli will be presented.While cell culture systems have been usedto test for anti-adherent activity of humanmilk or human milk components against E. coli strains,to theauthor?sknowledge no suchstudies have been reported with bovine milk or colostrum. In addition no reports ofanti-invasive activity of humanor bovine milk have been found in the literature.Antibodies to the colonization factor antigen 1 of ETEC have been found incowscolostrum (previous chapter), and it would have been logicalto test these samples ofcolostrum for anti-adherent activity ina cell culture system. However, it was deemedpreferable to postpone this type of experimentin favor of anti-invasion assays for thefollowing reasons: 1) ETEC adherencehas been demonstrated only in Caco-2 cells that are83fully differentiated, requiring lengthy cultures and slower turnover of experiments. 2) From amethodological point of view, the assessment of adherence or lack of it appeared to besubject to uncertainties that are not present in assessment of invasion. 3) A wider range ofcell lines are available to test for invasion with a greater range of bacterial strains. While wehave no interest in the fate of the bacteria following invasion, the method simply appearedbetter suited to initial experiments of this kind with bovine colostrum.HeLa cells were therefore chosen to test the anti-invasive ability of bovine colostrum;colostrum rather than milk was chosen to make the study more efficient: any antibacterialfactor in milk would be expected to be present in colosirum at a much higher concentration.A. Materials and methodsColostrum samples.Colostrum samples from individual cows were obtained from the University ofBritish Columbia Dairy Farm (UBC Col) or from Dr L.A. Babiuk, Veterinary InfectiousDiseases Organization, Saskatoon, SK (Samples # 112-7, 83-8, 8-1, 95-8, 579-5, 73 1-7).Cottage cheeese whey was obtained from Dairyworld Foods, Burnaby, B.C.A whey protein concentrate (WPC) in powder form was obtained from StolleBiologicals, Cincinnati, OH. The milk from which this concentrate was obtained had beenproduced by cows vaccinated against a variety of pathogens (Stolle and Beck, 1987).Bacteria.Salmonella enteritidis ATCC 13076 was obtained from the American Type CultureCollection, Rockvile, MD. S. enteritidis CD5, S. typhimurium SL1344 and enteropathogenicEscherichia coli E2348/69 were obtained from Dr. B. Finlay, Biotechnology Laboratory,UBC. The bacteria were maintained at 4 °C on irypticsoy agar and subeultured monthly.84Cell culturesHeLa cells were obtainedfrom Dr. B. Finlay,Biotechnology Laboratory,UBC andused between passages12 and 65. The cells weremaintained inMinimum EssentialMediumwith Earle’s salts, L-glutamineand non-essentialamino-acids (Gibco410-I500EB),supplemented withsodium bicarbonate (2.2g/l), penicillin G (100U/mI), streptomycin(100pg/ml) (Gibco 600-5140AG)and 10% fetal calfserum (Gibco 200-6140AJ).The cells werepassaged as needed bytrypsinization with trypsin-EDTA(Gibco 610-5300AJ),seeded at a 1to 5 dilution in 75 cm2flasks (Falcon 3023) andgrown in a 5% CO2environment at 37 C°.In all experiments, except immunoassays,Dulbecco’s PBS(pH 7.4) was used(0.1 gCaC12;0.2 g KC1; 0.2 gKH2PO4;0.1 g MgCl2.6H0;2.16 gNa2HPO4.7H0;8.0 g NaC1;to 1.01H20).Invasion assays.Invasion assays were doneaccording to Bettsand Finlay (1992).HeLa cells wereharvested by trypsinizationand the cell density adjustedto 1x105/ml. Oneml of cellsuspension was addedto each well of a 24well microplate andincubated overnight asabove.The monolayers werethen washed twice withpenicillin and streptomycinfree medium, and300 .tl of this mediumwere added to each well,followed by 30 ilof colostrum or other testsample, and finally 10 i1of an overnight standingculture of bacteriain trypticase soy broth(TSB) (Salmonella)or LB broth (E. coli). Afterone hour ofincubation as above, thecellsexposed to Salmonellawere washed twicewith PBS, and 500p.1 of culture mediumcontaining 100 p.g/ml ofgentamicin (Sigma G1272)were added. Thecondition of the cellmonolayer was checked bylight microscopy at everystep of the experiment.After one hourof incubation at 37 °Cin a 5% CO2 incubator thecells were washedtwice with PBS, and100 p.1 of 1% Triton X100in PBS were added.After 5 mm of incubation,400 p.1 of TSBwere added; followingmixing, 10 p.1 of serial dilutionswere drop platedin duplicate on plate85count agar which was then incubated overnight at 37 °C.In the case of the cells exposed to E. coli, the wells were washed twice with PBS onehour after addition of colostrum and bacteria, and medium and test colostrum replenished fora further period of incubation. After two hours, the cells were treated with gentamicin asabove. In any given experiment, each control or colostrum sample was tested inquadruplicate, unless otherwise noted.Immunofluorescence.Hela cells were seeded in a 24-well microplate as described above, on microscopecover slips. After overnight incubation, the cells were washed as above and medium withoutantibiotics was added. Following addition of 10i.tlof an overnight culture of bacteria(SL1344 or E2348169) the plates were incubated for one hour at 37°C in 5% C02. The cellswere then washed twice with PBS and exposed to 2% paraformaldehyde in PBS for 30 mm.Following washing, the cells were permeabilized with 0.1% (vlv) Triton Xl00 for 5 mm.During subsequent steps, the cells were exposed for 60 mm at room temperature first to thetest colostrum samples, then to a fluorescein isothiocyanate (F1TC) anti-bovine IgG affinitypurified antibody (Jackson Immunoresearch 301-095-003). The cover slips were thenmounted on slides on a drop of mounting fluid, sealed with nail polish and examined with anepifluorescent Zeiss Axioskop microscope. Photographs were taken with Kodak T-Max 400film.Immunoassays.The following buffers were used in immunoassays: Carbonate coating buffer, pH 9.6(1.59 g Na2CO3;2.93g Na}1C03;2 g NaN3;11 H20); PBS, pH 7.4 (8.0 g NaCl; 0.2 gKH2PO4;1.15 g Na2HPO4;0.2g KC1; 0.2 g NaN3;11 H20) with 0.5 ml of Tween 20added for PBS-Tween; blocking buffer (0.25% ovalbumin in PBS); 10% diethanolamine86 buffer pH 9.8 (97.0 ml diethanolamine; 0.1 g MgCl2-6H20); 0.2 g NaN3; H20 to 1 1) (Kummer et al, 1992). Ovalbumin was a gift from Canadian Lysozyme, Abbotsford, B.C. a) Determination of bovine IgGs: An enzyme linked immunoassay (ELISA) to estimate the relative concentration of bovine Igs in milk or colostrum or colostrum fractions was conducted in 96 well microtiter plates (Immulon U, Dynatech Labs, Chantilly, Va.). The plates were first coated with an affinity purified rabbit antibody to bovine IgGs (Sigma B7265) in 100 jxl of carbonate buffer. Following one hr incubation at 37 °C, the plates were washed with PBS and 250 \i\ of blocking solution were added. After 30 min of incubation the plates were washed with PBS-Tween, and 100 fil of serial dilutions of colostrum or other test samples in PBS-Tween were added. Following one hr incubation, the plates were washed and 100 (il of a rabbit anti-bovine IgG alkaline phosphatase conjugate (Sigma A7914, 1:1000 dilution) was added to each well, and the plates incubated for another hour. The plates were then washed with PBS and distilled water, and 100 (0.1 of substrate (p-nitrophenyl phosphate, Sigma 104) in diethanolamine buffer were added. Following color development, the plates were read in a BioRad 450 microplate reader at 405 nm. b) Determination of F(ab')2 fragments: An ELISA to estimate bovine F(ab')2 was carried out as above using a rabbit antibody to F(ab')2 as coating (Jackson Immunoresearch 301-005-006), and the corresponding conjugate (Jackson Immunoresearch 301-055-006). The assays were done in duplicate. A reference sample in any one assay was used to establish a standard curve. The absorbance values of at least two dilutions in duplicate for each sample were used to determine the IgG concentration relative to the reference sample. 87 SDS-PAGE electrophoresis. To each sample were added 20 JULI of 10% SDS, 2 (J.1 of mercaptoethanol and 5 |xl of 0.05% bromophenol blue, to a total volume of 100 (J,l in 10 mM Tris/HCl, 1 mM EDTA. The samples were then heated for 5 min in a boiling water bath. Electrophoresis of fully reduced samples was performed on a PhastSystem (Pharmacia LKB Biotechnology AB, Uppsala, Sweden), with PhastGel gradient 10-15 acrylamide gels. The gels were run at 250 V, 10 mA, 15 °C for 63 volt hours. The gels were stained with Coomassie blue (0.1% PhastGel Blue R solution in 30% methanol and 10% acetic acid). The gels were destained with 30% methanol and 10% acetic acid, and preserved in 10% acetic acid and 5% glycerol. Immunoblotting. Colostrum fractions were analysed by SDS-PAGE in 12% gels on a Mini PROTEAN II Electrophoresis System (BioRad 165-2940) at 100 volts according to the method of Laemmli (1970) and the bands transferred to nitrocellulose for 1 hr at 100 volts with a Mini Trans-Blot Electophoretic Transfer Cell (BioRad 170-3930) according to the method of Towbin et al. (1979). Blots were blocked for 1 hr with 0.25% ovalbumin in PBS and exposed using rabbit alkaline phosphatase antibody conjugates to bovine IgG and F(ab')2 fragments (1:1000 in PBS-ovalbumin). The blots were developed with a 5-bromo-4-chloro-indolyl phosphate (0.2 mg/ml) and nitroblue tetrazolium (0.4 mg/ml) (BCIP/NBT) substrate (McGadey, 1970) in diethanolamine buffer.. Affinity chromatography. Colostrum (250 ^1) was applied to a 1.5 ml column of immobilized protein G (Pierce 20398) in 0.2 M acetate buffer pH 5.0. Following washing of the unbound fractions, the bound material was eluted with 0.05 M glycine buffer, pH 2.8. Fractions were monitored by UV spectrophotometry at 280 nm and by ELISA. 88Following chromatography, Centricon concentrators (Amicon Inc. Beverly, MA,USA) were used, according to manufacturer’s directions, in a refrigerated centrifuge, toconcentrate the fractions and to exchange buffers prior to the invasion assay. In someexperiments, fractions were separated sequentially according to size on Centricon 100, 30and 10, with respective cut-off sizes of 100, 30, and 10 kDa. The respective retentates orfiltrates wereused in the invasion assay.Enzyme digestion.Colostrum or colostrum fractions were digested with pepsin (Sigma P70 12) (1:25w:w) at pH 2.8. Following digestion for 4 hrs at 37°C, the pH was raised to neutrality with 1N NaOH and the samples dialysed overnight in PBS at 4 °C with a Spectrapor membrane(Spectrum Medical Industries, Los Angeles, CA) with a cut-off of 6,000-8,000, orfractionated on Centricon C30. Colostrum or colostrum fractions dialysed in PBS weredigested with papain (Sigma P4762) (1:50 w:w) in the presence of mercaptoethanol(0.015M) and EDTA (0.O1M). Following digestion for 4 hours at 37°C, iodoacetamidewasadded to a final concentration of 0.03M. The samples were dlialysed in PBS as above prior touse.B. ResultsInhibitory effect of colostrumThe inhibitory capacity of various bovine colostrum samples was determinedbychallenging monolayers of HeLa cells with invasive bacteria inthe presence of colostrum.Approximately 1 x to 2 x CFUs were added to each well. A normal level of invasionwas determined by control wells to which no colostrum was added. The extracellularbacteria were eliminated by treatment with the antibiotic gentamicin. Followinglysis of themonolayer, the intracellular bacteriawere diluted as needed and plated for determination ofcolony forming units. A number ofinitial experiments were done with S. enteritidis ATCC8913076. Even though colostrum was found to effectively reduce the levels of invasioncompared to control, it was observed that the invasiveness of ATCC 13076 varied greatlyfrom day to day. One experiment with S. enteritidis CD5 gave a very low level of invasion.A number of experiments with S. lyphimurium SL1344 gave consistently acceptable levelsof invasion, in the order of 5 x i0 to 2 x 10 colony forming units per well, and thereforemost subsequent experiments with Salmonella were done with SL1344. Results will bepresented as percent invasion in test wells compared to invasion in control wells.Five experiments to test different colostrum samples were done with S. enteritidisATCC 13076, one with S. enteritidis CD5, and four with S. ryphimurium SL1344. Inhibitionranged from 97.7% to 87.3%. The results of typical challenges with the three strains ofSalmonella are presented in Table IX.The results of two challenge experiments to determine the inhibitory ability ofcolostrum samples against E. coli E2348/69, performed on two different days, are presentedin Table X. Inhibition ranged from 99.6% to 73%. All samples of colostrum tested inhibitedinvasion of the HeLa cells by the bacteria. One sample (UBC col) which was a goodinhibitor of invasion by Salmonella, was a weaker inhibitor of E. coli E2348/69 than theother samples. This may be a reflection of the origin of this particular sample, or of the timepost partum at which the various samples of colostrum were obtained. Visual examination ofthe samples would lead to the conclusion that the samples originating from Saskatoon wereobtained from the first milking after calving, while the sample originating from UBCappeared to have been obtained from a later milking. The concentration of colostrum rapidlydeclines over the first 24 hours of lactation.To determine whether the presence of colostrum had an effect on HeLa cells thatcould make them less susceptible to invasion, or whether the inhibitory factors fromcolostrum could bind to the mammalian cells, HeLa monolayers were exposed to colostrumfor 30 mm, then washed with medium three times prior to challenge with the bacteria. TheTable IX. Effect of bovine colostrum on the invasion of HeLa cell monolayers by Salmonella. Samples Control UBC Col. 112-7 83-8 8-1 95-8 S. enteritidis ATCC 13076 100.0a 6.9 4.2 9.5 6.1 (48.9) (2.3) (2.2) (1.6) (3.3) S. enteritidis CD 5 100.0 (35.9) 2.3 (1.6) 3.5 (1.9) S. typhimurium SL1344 100.0 (24.4) 12.7 (11.2) 5.0 (2.0) a: Percent of intemalyzed bacteria relative to control, averages of 4 wells, standard deviations in parentheses. 91Table X. Effect of bovine colostrum on the invasion of HeLa cells monolayers by E.coli E2348/69.Samples Exptl Expt2Control100•0a(12.2) 100.0 (38.3)UBC Col. 26.8 (9.5)345-8 1.2 (0.8)8-1 1.5 (0.5)112-7 0.8 (0.4)579-5 0.4 (0.3)731-7 2.1 (0.8)a:Percent of intemalyzed bacteria relative to control, averages of 4 wells, standarddeviations in parentheses.Experiments 1 and 2 were conducted on different days.92results of such a test are presented in Table XI. An effect of bovine colostrum on the HeLacells could not be detected under such conditions, therefore the inhibiting agents ofcolostrum do not appear to bind strongly to the mammalian cells or to have an effect on theirability to be invaded by the bacteria.To establish whether the reduced invasiveness was a reflection of reduced viabilityor agglutination of the bacteria, aliquots of medium were taken from wells with and withoutcolostrum after one hour of incubation, and bacterial counts determined by serial dilutionand drop plating. Counts of internalized bacteria were also obtained. As a further control,some wells were treated with a rabbit antiserum specific for the LPS of SL1344 (Difco2948-47-6). The results (Table XII) show that addition of colostrum did not result in adecrease in the number of colony forming units recovered from the medium even though itcaused a significant reduction in invasiveness, whereas the rabbit antiserum, which was veryeffective at preventing invasion, caused a reduction in the number of colony forming units inthe medium. All samples tested were subsequently checked for their effect on the viability ofSL 1344, and no instances of reduced viability were detected. However, this does notpreclude the involvement of immunoglobulins in this phenomenon. Immunoglobulins incolostrum could be inhibiting adherence to the mammalian cells without having abactericidal effect or agglutinating the bacteria.Are low molecular weight fractions responsible for the inhibition of invasion?Both immunoglobulins (slgAs) and oligosaccharides in human colostrum and breastmilk have been reported to inhibit adhesion of some EPEC strains to HEp-2 cells (Craviotoet a!., 1991), while in a similar system using HeLa cells, only an effect of slgAs couldbedemonstrated (Camara et al., 1994; Silva and Giampaglia, 1992). In a study of inhibition ofattachment by human milk of Streptococcus pneumonia and Hcemophilus influenzce, theinhibitory activity was attributed to a non-immunoglobulinhigh molecular weight fraction,93Table XL Invasion of HeLa cells monolayers by S. enteritidis ATCC 13076 or E. coliE2348/69 following preincubation of the cells with bovinecolostruma.S. enteritidis E. coliSamples ATCC 13076 E2348/69Control100•0b(48.9) 100.0 (28.1)UBC Col. 141.2 (51.9)112-7 86.1 (11.7)a:HeLa cell monolayers were exposed to colostrum for 30 mm, then washed 3x withmedium prior to challenge with the bacteria.b:Percent of intemalyzed bacteria relative to control, averages of 4 wells, standarddeviations in parentheses.94Table XII. Effect of colostrum on the viability of S. zyphimurium SL1344.Sample InternalCFUsaS External CFUs SControl 6.8x 10 2.3x 10 1.32x 10 0.32x l0Difcobl/10<25 0.27x 10 0.14x 10UBC Colostrum 1.2 x103 2.9 x1021.16 x i0 0.28 x i07a:colony forming units per well. Averages and standard deviations (S) of quadruplicatedeterminations.b:a specific rabbit antiserum.95and the hypothesis was put forward that oligosaccharides on glycoproteins could be thefactors involved in the inhibition (Andersson et al., 1986). In the same study, inhibition ofattachment of S. pneumonke was also attributed to a low molecular weight fraction,tentatively identified as a glycolipid. Holmgren et al. (1981) and Ashkenazi and Mirelman(1987) similarly observed that a non-immunoglobulin fraction of human milk inhibitedadherence of enterotoxigenic E. coli.To test whether a low molecular weight fraction of bovine colostrum wasresponsible for inhibition of invasion, samples of colostrum were separated into fractions ofvarious molecular weight either by dialysis at 4°C in PBS with a Spectrapor membrane witha cut-off of 6000-8000, or by sequential filtration through Centricon filters. The respectivefractions were then tested by the gentamicin resistance assay.Table XIII shows that dialysis did not reduce the inhibitory activity of the samplestested. To confirm this finding, fractions of high or low molecular weight were obtained byfiltration with Centricon separators. Centricon filters separate fractions on the basis of sizeby centrifugation so that the filtrate is collected and changes in volume are minimized. Thisprocess yielded a retentate (C3OR) of fractions above30 kDa and a filtrate after filtrationwith Centricon C30. This filtrate was again run through Centricon ClO yielding a retentate(C1OR) of fractions between 10 kDa and 30 kDa, and a filtrate (C1OF) of fractions below 10kDa. Volumes of retentate were adjusted to account for the increase in concentration of theretained colostrum components. The respective fractions weretested for inhibition ofinvasion by SL1344 and E2348/69. All the inhibitory activitywas found in the highmolecular weight fractions (Table XIV).To determine whether a low molecular weight fraction with inhibitory activity couldbe generated from colostrum by enzymatic digestion, colostrum was digested with pepsin. Aparallel control was run with no pepsinadded. Both samples were fractionated withCentricon C30. The retentates and filtrates weretested for inhibitory activity against S.96Table XIII. Inhibition of invasion of HeLa cells monolayers by bovine colostrum ordialysed colostrum.S. typhimurium E. coliSamples SL1344 E2348/69Control100•0a(24.4) 100 (12.2)UBCCo1. 8.8 (2.0)UBC Col dialysed 3.2 (2.4)112-70.8 (0.4)112-7 dialysed 1.6 (0.7)a:percent of internalyzed bacteria relative to control, averages of 4 wells, standarddeviations in parentheses.97Table XIV. Inhibition of invasion of HeLa cells monolayers by S. typhimurium SL1344or E. coli E2348/69 by bovine colostrum or colostrum fractions separated on the basis ofsize.S. typhimurium E. coliSamples SL1344E2348/69Control1000a(214)1000a(310)UBCCo1 2.4 (1.1)UBC Col C30R’ 1.4(0.4)UBC ColC1ORC142.4 (18.7)UBC Col C1OF’ 150.6(34.1)8-19.0 (3.4)8-1 C30R’ 12.6(3.5)8-1C1ORC162.7 (82.7)8-1 C10 125.4 (47.5)a:percent of internalyzed bacteria relative to control, averages of 4 wells, standarddeviations in parentheses.b:retentate after filtration with Centricon 30.C:retentate after filtration of the Centricon30 filtrate with Centricon 10.d:filtrate after filtration with Centricon 10.98typhimurium SL1344. Following pepsin digestion, the inhibitory activity of the highmolecular weight fraction was reduced compared to the original sample and to a control thatwas treated in the same manner but without pepsin. No anti-invasion activity appeared in theC30 filtrate (Table XV).To determine whether the inhibitory activity was sensitive to heat, colostrum washeated at 80°C for 30 mm. Heating resulted in extensive coagulation of the sample, butthesoluble fraction following heat treatment was extracted and tested for inhibitory activity(Table XVI). Heat treatment reduced the activity of colostrum.Inhibitory effect of immunoglobulin and non-immunoglobulin fractionsChromatography on Protein A or protein G agarose is a convenient method to purifyimmunoglobulins. Protein A and protein G are bacterial proteins, obtained from some strainsof Staphylococcus and Streptococcus, which have the ability to bind specificallyto IgGs ofvarious animal species. Protein G binds more strongly to bovine IgGs than protein A andtherefore was used to obtain purified bovine colostrumIgGs (Björck and Kronvall, 1984).Samples of colostrum (UBC Col) were applied to a protein G agarose column.Unbound fractions of colostrum were eluted at pH 5.0 while IgGs were elutedby loweringthe pH to 2.8. Fractions were monitored by spectrophotometry at 280 nm.Levels ofimmunoglobulin G in the respective peaks were detennined by ELISA in order to establishthe volume of sample that could be applied without exceedingthe column capacity forbinding immunoglobulins, in orderto achieve a good separation of the IgGs. A typicalelution pattern is presented.Immunoglobulins were retained on the column and appeared inthe second peak (Figure 10). A relatively low level of immunoglobulinswas detected in thefirst peak. Recycling, on protein G agarose, ofthe pooled first peaks from several columnsshowed that only about 4% ofthe protein was then recovered in the second peak. Pooledfractions of each peak were concentrated with Centricon30 concentrators, washed with PBS,99Table XV. Invasion of HeLa cells monolayers by SL1344 in the presence of fractionsfrom a pepsin digest of bovine colostrum.Samples % InvasionControl1yJ0a(26.8)UBCCo1 2.8 (1.4)Pepsin digestC3ORb33.8 (5.8)Pepsin digestC3OFC85.6 (17.7)Control no pepsinC3ORb3.1 (2.3)Control no pepsinC3OFc84.5 (34.7)a:Percent of internalyzed bacteria relative to control, averages of 4 wells,standarddeviations in parentheses.b:Retentate after filtration with Centricon 30.c:Filtrate after filtration with Centricon 30.100Table XVI. Invasion of HeLa cells monolayers by SL 1344 in the presence of heat treatedcolostrum.Samples % InvasionControl1000a(28.1)UBCCo1 10.1 (5.8)Heat treatedcolostrumb79.8 (15.0)a:Percent of intemalyzed bacteria relative to control, averages of 4 wells, standarddeviations in parentheses.b:Colostrum heated at 80°C for 30 mm.101C04-I________4-C0UC0U0>4-0Figure 10. Absorbance and relativeIgG concentration of colostrum fractionsobtained by chromatography ona protein G-agarose column.Column volume: 1.5 ml; samplevolume: 0.25 ml; fraction size: 0.25 ml.120010008006004002000—a-—— Relative Igs concentrationA28004010 20FractionsPeak I- .Peak II102and their IgG concentration relative to the original colostrum sample estimated by ELISA.SDS PAGE electrophoresis showed minimal contamination of the respective peaks by theother (Figure 11). However, some of the strongest bands in Peak I had a mobility similar towhat would be expected of Fab fragments of immunoglobulins in their reduced form. Peak Iand Peak II were then tested for their ability to inhibit invasion of HeLa cells by SL1344(Table XVII).Table XVII shows that while inhibition of invasion (about 13% of control) wasassociated with the IgG containing peak, a high level of inhibitory activity (about 9% ofcontrol) was also found in the first peak, where the IgG concentration was approximately 1%of that of the second peak. These results of this experiment were confirmed by three otherindependent fractionations of this sample of colostrum.This test was repeated with a sample of cottage cheese whey, obtained from thepooled milk of a large number of cows. Since protein concentration in whey is quite low, atotal volume of 90 ml of whey was used, and the fractions obtained followingchromatography on protein G-agarose required extensive concentration with Centricon 30separators. Approximately 20 mg of IgG were recovered. Table XVIII shows the results of achallenge of HeLa cells with S. typhimurium SL1344 in the presence of the Peak I and PeakII obtained from this cottage cheese whey. Both Peak I and Peak II inhibited invasion. Peak Ihad not been concentrated to the same extent as Peak II and therefore the effect was smaller,but nevertheless substantial. Therefore an inhibiting effect of the immunoglobulin fractionand the non-immunoglobulin fraction was found in samples of very different origins.Longhi et al. (1993) reported an anti-invasive effect of human lactoferrin when HeLacells were challenged with E. coli HB 101 (pRi 203), which is capable of invading culturedepithelial cells. Lactoferrin would be expected to elute in Peak I on the protein G-agarosecolumn. To check on the activity of bovine lactoferrin in this system, HeLa cells werechallenged with S. typhimurium SL1344 in the presence of bovine lactoferrinat a final103.IIColostrum Peak IPeak IIFigure 11. SDS PAGE profiles ofcolostrum (UBCCol),and of Peak I and Peak IIprotein G agarosefractions.104Table XVII. Invasiveness of SL1344 in the presence of concentrated pooled fractionsfollowing separation of colostrum on a protein G-agarose column.SampleIgGsa%invasionbControl 100.0 (44.2)UBC Colostrum 100.0 8.3 (3.2)Protein G Peak 1 0.44 9.6 (2.8)Protein G Peak II 53.6 13.3 (2.4)a:relative concentration of IgGs in Peaks I and II as % of the original colostrum sample(UBC Col). Peak I and Peak II reconstituted in PBS to approximately the originalvolume.b:percent of intemalyzed bacteria relative to control, averages of 4 wells, standarddeviations in parentheses.105Table XVIII. Invasiveness of SL1344 in the presence of concentrated pooled fractionsfollowing separation of cottage cheese whey on a protein G-agarose column.SampleIgGsajjçjjjbConcentration factor’Control 100 (18.0)UBC Colostrum 100 4.1 (1.6)Whey Peak I 0.2 16.5 (2.9)30Whey Peak II 57.9 6.9 (1.7)300a:relative concentration of IgGs as % of the UBC colosirum sample. Approximately 20mg of IgG were recovered in Peak II, as determinedby spectrophotometry, from 90 mlof whey.b:percent of intemalyzed bacteria relative to control, averages of 4 wells, standarddeviations in parentheses.C:factor by which the volume of the whey sample was reduced followingconcentration.106concentration of 2.5 mg/mL The results presented in Table XIX do not support an anti-invasive effect for bovine lactoferrin at that concentration. A similar finding with E. coli hasalso been reported by Longhi et al. (1994), who also found that lactoferrin had nobactericidal or cytotoxic effect at that concentration.A number of tests were then done to characterize the inhibiting factors in each peak,and particularly to determine whether the activity in Peak I was associated withcontaminating immunoglobulins or with immunoglobulin fragments that could have beengenerated by proteolytic activity in the colostrum. Following fractionation on a proteinGagarose column, the pooled fractions from each of Peak 1 and Peak 2 were sequentiallyseparated with Centricon concentrators C100, C30 and ClO. The respective fractions wereassayed for IgG and (Fab’)2 contentby ELISA. It was speculated that if immunoglobulinfragments such as Fab were somehow present in colostrum, it might be possible todetectthem in Peak I with an anti-F(ab’)2antibody. HeLa cells werechallenged with SL1344 aspreviously described. The results (Table XX) complement thedata from Tables XVII andXVIII and in addition show that mostif not all the activity in both Peak 1 and Peak 2 wascontained in the fraction above 100 kDa. No great differences were found intheimmunoassays for IgGs and for F(ab’)2fragments.A sample of whey protein concentrate (WPC) (200 mg) was reconstituted andtreatedas above (fractionation on protein G-agarose, separation on the basis of size withCentriconconcentrators). HeLa cells were challenged with SL1344 in the presence or absence of thefractions which were also assayed for their IgG and F(ab’)2 relative concentrationsbyELISA (Table XXI). Results similarto those presented in Table XX were obtained. Theresults in Tables XX and XXI do not totally preclude activity in the lower MWfractions;however most of the activity was recovered in the high MW fraction, in agreementwith theresults presented in Table XIVFrom the results presented in Tables XVII, XVIII, XX and XXI, it can beconcluded107Table XIX. Invasiveness of SL1344 in the presence of bovine lactofeffin.Samples %invasionaControl 100.0 (31.4)UECColostrum 3.4 (2.1)Lactoferrin (2.5 mg/mi) 76.0 (15.2)a:percent of internalyzed bacteria relative to control, averages of 4 wells, standarddeviations in parentheses.108Table XX. Invasiveness of SL1344 in the presence of colostrum fractions obtained bychromatography on a protein G-agarose column, followed by separation on the basis ofsize.ConcentrationaSamples IgGs F(ab’)2Control 100.0 (41.6)UBC Col 100.0 100.0 2.5(0.8)PeakIC100R’ 6.6 5.6 4.7 (3.2)Peak I C3OR 0.0650.073 48.6 (15.4)PeakiClOR <0.018 <0.019 27.6 (6.6)Peak II C100R 133.0 97.0 4.4 (2.3)Peak II C3OR 0.157 0.112 46.5 (26.6)Peak II C1OR <0.018 0.023 109.6 (53.0)a:relative concentration of IgGs or F(ab’)2 fragments in the various fractions as % of theoriginal colostrum sample (UBC Col). Approximately 20 mg of IgG were recovered inPeak II, as determined by spectrophotometry.b:percent of internalyzed bacteria relative to control, averages of 3 wells, standarddeviations in parentheses.C:R: retentates following concentration with the respective Centricons, toapproximately 50% of the original volume.109Table XXI. Invasiveness of SL1344 in the presence of whey protein concentratefractions obtained by chromatography on a protein G agarose column, followed byseparation on the basis of size.Concentrationa%Samples IgGs F(ab’)2Control 100.0 (57.0)UBC Col 100.0 100.0 4.3 (2.6)Peak IClOoRc1.0 2.9 6.3 (2.6)Peak I C3OR 0.06 0.05 40.2 (10.5)Peak I C1OR <0.014 <0.028 36.6 (4.4)Peak II C100R 22.7 20.7 3.4 (1.0)Peak II C3OR 0.09 0.075 39.7 (15.2)Peak II C1OR <0.016 0.029 69.1 (19.2)a:relative concentration of IgGs or F(ab’)2 fragments in the various fractions as % of thereference colostrum sample (UBC Col). Approximately 8 mg of IgG were recovered inPeak II, as determined by spectrophotometry.b:percent of internalyzed bacteria relative to control, averages of 3 wells, standarddeviations in parentheses.c:R: retentates following concentration with the respective Centricons, toapproximately 50% of the original volume.110that inhibiting activity was associated with both the immunoglobulin rich fraction, and withthe non-immunoglobulin fraction obtained by chromatography on the protein G agarosecolumn.The inhibitory activity of the IgG fraction isolated from the whey proteinconcentrate appears to be relatively higher than that of the IgG fraction isolated from thecolostrum. The cows that produced the milk from which the whey concentrate was obtainedhad been vaccinated with a proprietary mixture of bacteria, among which were S. enteritidisATCC 13076 and S. typhimurium ATCC 13311 (Stolle and Beck, 1987).The results presented in Tables XX and XXI do not support the hypothesis that theactivity in Peak I is due to immunoglobulin fragments such as F(ab’)2 or similar types offragments. In a situation where both whole immunoglobulins and F(ab’)2fragments may bepresent, immunoassays using affinity purified antibodies to the whole immunoglobulinwould be expected to be more efficient at detecting whole immunoglobulins thanF(ab’)2 fragments, because of the high inimunogenicity of the Fe fragment. The oppositewould be true of antibodies to F(ab’)2,which would be more efficient at detectingimmunoglobulin fragments containing light chains, and quantitative ELISAs done side byside using such antibodies for capture and detection would be expected to give widelydiffering results if one sample (Peak I) happenedto contain an amount of fragmentssufficient to cause inhibition of invasion of the same magnitude as Peak II. This was notfound to be the case.To determine whether the inhibitory activity of Peak I could be due to contaminationby IgGs, in spite of their low level as determined by ELISA, a sample of pooled Peak I,concentrated with Centricon separators, was recycled on a proteinG agarose column. Therecycled Peak I fraction was concentrated again in the same manner, assayed for Igs leveland tested for inhibitory activity. Even though about 75% of the contaminating Igs wereremoved by the second passage on the column, the inhibitory activity of peak I was notreduced. Conversely, dilution of the Peak II fraction to bring the IgG concentrationto a level111similar to that of Peak I prior to recycling resulted in a decrease in inhibitory activity (TableXXII).The data presented in Tables XVII, XVIII, XX, XXI and XXII do not support thehypothesis that the activity in Peak I is related to the presence of contaminating wholeimmunoglobulins or even of immunoglobulin fragments. This last point was furtherexamined by immunoblotting of colostrum, Peak I, Peak II, and a papain digest of Peak II,using affinity purified antibodies to whole bovine IgG or to bovine F(ab’)2 fragments fordetection. In neither type of blot were bands in Peak I stained with the respective antibodiesbeyond barely visible indication of slight contamination with Peak II, while colostrum, PeakII and the papain digest of Peak II showed all the bands corresponding to reducedimmunoglobulins or their fragments (Figures 12A, 12B).Incidentally, the intensity of the bands in the blots respectively exposed to the anti-bovine IgG or the anti-F(ab’)2antibodies confirms the operating hypothesis on which theexperiments presented in Tables XX and XXI were based.Finally, to further characterize Peak I, pooled concentrated Peak I was digested withpepsin at pH 2.8 for 4 hours at 37°C, followed by dialysis in PBS. A control was done inparallel without pepsin. The results of an invasion assay show that pepsin digestion reducedthe activity of Peak I (Table XXIII).To confirm the presence in colostrum of antibody to the bacteria, HeLa cells seededon cover slips were infected with SL1344 orE2348/69. Following incubation in medium forone hour, the cells were washed, fixed and permeabilized as described in the method section.Colostrum was added to each cover slip, and following incubation for one hour and washingof the test samples, bovine antibodies boundto bacteria were detected with an affinitypurified anti-bovine IgG fluorescent antibody. A control where HeLa cells were not infectedwith bacteria was included todetect any non specific binding of bovine antibody to the cells,112Table XXII. Invasion of HeLa cells monolayers by SL1344 in the presence or absence ofbovine colostrum, Peak I, Peak I recycled or diluted Peak II.SamplesIgGsaControl 100.0 (26.2)UBCCoI 100.0 2.1 (1.8)Peak I 7.8 1.0 (0.7)Peak I recycled 1.9 0.5 (0.2)Peak II 136.9 1.8 (0.5)Peakllh/20 6.8 18.0 (2.8)a:relative concentration of IgGs in the various fractions comparedto the originalcolostrum sample (LTBC Col).percent of internalyzed bacteria relative to control, averages of 4 wells, standarddeviations in parentheses.113HPeak II Peak I Pap. Colostrum Peak II Peak I Pap. ColostrumDigest DigestA BFigure 12. Immunoblots of bovine colostrum (UBCCol), Peak I,Peak II obtained by chromatographyon a protein G agarosecolumn, and a papain digest of Peak II.A: blot detected with an affinity purified antibodyto bovine IgG.B: blot detected with an affinity purified antibody to bovine F(ab)2fragments.H= Heavy chains; L= Light chains.114Table XXIII. Invasion of HeLa cells monolayers by SL1344 in the presence orabsence of bovine colostrum, Peak I or Peak I digested with pepsin.Samples % InvasionControl1yj0a(19.1)UI3C Col 2.4(0.4)Peak I control” 0.7 (0.2)Peak I pepsin digest 54.8 (8.7)a:percent of intemalyzed bacteria relative to control, averages of 4 wells, standarddeviations in parentheses.b:IgG concentration in Peak I was determined by ELISA to be about 3.2% of that ofUBC Col prior to pepsin digestion. Peak I control was treated identically to Peak Ipepsin digest, in the absence of pepsin.115as well as a control (in the case of SL1344) where the bacteria were detected with a specificanti-LPS typing antiserum. Examination of the control cover slips showed that SL1344 wereassociated with the cells, and that no non-specific binding of bovine antibody could bedetected (Figures 13A, 13B). Fluorescence associated with the bacteria was evident on thecover slips exposed to colostrum. However, while fluorescence was found evenly distributedaround the bacteria exposed to the specific anti-LPS antiserum, fluorescence on the bacteriaexposed to colostrum or colostrum fractions was not so evenly distributed around thebacteria or appeared to be directed at fimbri. On the other hand, in the case of E2348/69,the fluorescence was associated strictly with the bacteria, which themselves appeared asclumps, or microcolonies (Figures 14A, 14B). No specific antiserum was available to us toindependently detect the bacteria, but the microcolonies were clearly visible by phasecontrast microscopy. Microcolonies are a typical adherence pattern to HeLa cells or to 1{Ep-2 cells for this type of EPEC (Levine eta!., 1985).116AFigure 13. A: Detection by TRITC immunofluorescence of S.typhimurium SL1344 on cover slips seeded with HeLa cells.B: Absence of non-specific binding of bovineimmunoglobulins to cover slips seeded with HeLa cells.FITC immunofluorescence with affinity purified antibody tobovine IgG. Scale: i i = 10 .Lm.B117AFigure 14. A: FITC immunofluorescence with affinity purifiedantibody to bovine IgG of S. typhimurium SL1344 exposed tobovine colostrum on cover slips seeded with HeLa cells.B: FITC immunofluorescence with affinity purified antibody tobovine IgG of E. coli E2348/69 exposed to bovine colostrum oncover slips seeded with HeLa cells. Scale: I I = 10 Jim.B118 C. Discussion The purposes of the experiments reported above were: 1) to evaluate the suitability of cell cultures methods for in vitro testing of the antibacterial activity of fractions that may in the future be purified from cows milk or whey, and: 2) to determine whether any antibacterial effect that may be found could be attributed to immunoglobulins. In these experiments, the anti-invasive properties of bovine colostrum or whey against S. enteritidis, S. typhimurium and E. coli were tested. Colostrum was tested first in this investigation because of its very high content of antibacterial factors, thereby alleviating the need for extensive purification and concentration. The results presented here showed that the fraction of bovine colostrum remaining after removal of fat or insoluble matter inhibited the invasion of HeLa cells by S. enteritidis ATCC13076 and CD5, S. typhimurium SL1344 and E. coli E2348/69. All samples of colostrum tested showed inhibitory activity. One sample was from B.C. and the others from Saskatchewan; all were from individual cows. To the best of this author's knowledge, it is the first time that an anti-invasive effect of bovine colostrum, or milk or whey has been reported. So far only reports of anti-adherent effects of human milk against some strains of E. coli have been found in the literature. The inhibitory activity was not reduced by dialysis and was not found in low molecular weight fractions. The inhibition did not seem to be related to reduced viability of the bacteria. A similar finding with human milk IgAs was reported by Cravioto et ah, 1991. The colostrum factors did not appear to become associated with the HeLa cells. The low molecular weight fractions of colostrum, able to pass through Centricon 30 and 10 membranes, had no activity. Similarly, no low molecular weight fractions with anti-invasive properties were generated following digestion of the colostrum with pepsin. Digestion with pepsin and heat treatment both reduced the anti-invasive ability of the colostrum. Fractionation of one colostrum sample, one cottage cheese whey sample and one 119 reconstituted whey protein concentrate sample on a protein G-agarose column showed that both the immunoglobulin fraction (Peak II) and the IgG depleted fraction (Peak I) were able to inhibit invasion of HeLa cells by S. typhimurium SL1344. Separation on the basis of size showed that the inhibitory activity of both fractions was retained on Centricon 100. The immunofluorescence studies showed that bovine IgGs bound to S. typhimurium SL1344 and E. coli E2348/69, but the pattern of binding was different. In the case of SL1344, bovine IgGs bound to the bacteria and to filamentous structures (fimbriae), while with E2348/69, bovine IgGs appeared to be associated strictly with the bacteria. Human subjects infected with this type of EPEC are known to produce antibodies specific for a 94 kD bacterial outer membrane protein associated with localized adherence to epithelial cells (Levine et al., 1985), and human milk antibodies have also been shown by immunoblotting to react with the 94 kD outer membrane protein, and to inhibit adherence to HEp-2 cells (Cravioto et al, 1991; Camara et al., 1994). Whether bovine immunoglobulins react with the same outer membrane protein has not been determined. With regard to the effect against Salmonella, the gene products responsible for adherence and invasion of 5. typhimurium are not known (Finlay, 1994). Peralta et al. (1994) showed that oral administration of egg antibodies specific for a 14-kDa fimbriae protected mice against experimental salmonellosis with S. enteritidis, and also that the same antibodies reduced adhesion of S. enteritidis to mouse intestinal epithelial cells in vitro. The factor(s) in Peak I responsible for its anti-invasive properties have not been identified. It is not known whether the inhibitory activity is caused by more than one compound. The findings presented here point to the factor(s) being predominantly protein(s) of a large size. The question was whether the factors were fragments of immunoglobulins resulting from enzymatic degradation by milk proteinases. The Fc fragments of bovine IgGs are known to be particularly susceptible to enzymatic action with resulting production of fragments made up of various portions of heavy and light chains (Wie et al., 1978; 120 Heyermann and Butler, 1987). SDS PAGE electrophoresis showed some major components of Peak I to be found approximately where one would expect to see bands for the reduced Fab fragment of IgGs, and association of four chains or parts of chains (similar to F(ab')2) would be large enough to be retained on Centricon 100. It is known that Fab fragments from specific antibodies are able to block adherence of enterotoxigenic E. coli to Caco-2 cells (Darfeuille-Michaud et al., 1990) or to block invasion of HEp-2 cells by invasin-producing bacteria (Leong et al, 1990). The evidence presented above, obtained by immunoassays and immunoblots, does not support the hypothesis that the activity of Peak I is due to the presence of immunoglobulin fragments. Other incidental evidence derives from attempts to separate Fab fragments, obtained by papain digestion of Peak II, by protein G-agarose chromatography: it was found that a considerable proportion of the fragments bind to the column and therefore are not likely to be efficiently recovered in the first peak. While it is difficult at this point to speculate on the nature of the factor(s) involved, studies by Longhi et al. (1994) as well as results presented above, showed that bovine lactoferrin at concentration of 2 mg/ml did not prevent invasion of HeLa cells by E. coli. There are several examples in the literature of non-immunoglobulin fractions of human milk having anti-adherent activity. The anti-adherent activities have been attributed to glycoproteins, glycolipids or oligosaccharides (Andersson et al., 1986; Ashkenazi and Mirelman, 1987; Holmgren et al., 1981). Cravioto et al. (1991) showed that an oligosaccharide fraction of human milk inhibited adherence of E. coli E2348/69 to HEP-2 cells. However Camara et al. (1994) were not able to confirm this finding. To this writer's knowledge, no example of a non-immunoglobulin fraction of bovine milk or colostrum that inhibits invasion of mammalian cells by Salmonella has been published. Adherence, the interaction between an adhesin and a receptor, could be inhibited in several ways: by blocking of the adhesin on the bacteria with antibodies or with receptor analogs; by flooding the receptors on the host cells with an adhesin analog; and last by 121 blocking the receptor with antibodies. Adhesins analogs have been shown to block invasion experimentally (Leong et al., 1990). But blocking of receptors does not appear to be a likely scenario in nature, considering that they probably have other more important functions than binding to bacteria. Blocking of adhesins is a more desirable strategy, and the results presented above suggest that the effect of bovine colostrum is probably on the bacteria, not on the host cells. Antibodies against adhesins of course have been found in milk or serum as a result of experimental or natural infections (Tacket et al., 1988; Tacket, 1991). The presence of specific oligosaccharide receptor analogs has been demonstrated in milk. For example the GM1 gangliosides, the cell membrane receptors for CT (cholera toxin) and LT (heat labile toxin), are present in human and bovine milk and have been shown to block enterotoxin binding. Milk gangliosides are derived from mammary gland cell membranes (Laegrid et al., 1986). Whether the non-immunoglobulin fraction with anti-invasive activity reported above, which is probably a protein, is also a receptor analog, remains to be demonstrated. The receptor for the invasin of Yersinia pseudotuberculosis is known to be part of the protein family of integrins (Leong et ah. 1990). Could protein cell receptors analogs, derived from mammary gland cell membranes, also be found in milk? Somatic cells are present in significant numbers in milk and colostrum (Tizard, 1992). The work reported in this chapter opens many avenues for further research on the antibacterial factors of cows milk. With regard to the unidentified factor in Peak I obtained by protein G-agarose chromatography, the work is still at the stage where confimation of its presence in a greater number of samples of milk, cheese whey or colostrum is required. The fact that it has been detected in three samples (one colostrum and two whey samples) of widely different origins is encouraging. Moreover, it is relatively easy to separate from the immunoglobulins, and the gentamicin assay provides a test system to track this factor through the further purification steps that will be required to identify it. However, it would be quite useful to have some other more sensitive means to track this factor, which would 122facilitate its recovery from the wheys whereit is present at low concentration,or to havesome purification protocol which would efficientlyseparate it from most of the otherwheyproteins. A confirmation of its size would be the firststep, so that preliminaryfractionationby membrane filtration would achieve that goal andincrease its concentration at thesametime. Scaling up the chromatography on protein G-agarosewould greatly increase theefficiency of the process, which, while satisfactory sofar, will require much largeramountsof separated Peak I and Peak II in any attempt to further characterizetheir activity. The effectof the Peak I factor(s) on bacteria other than S. typhymuriumSL1344, or in other cellsystems, also remains to be investigated. frrespectiveof the results of such experiments,questions will be raised about the specificity, the homogeneityand the mode of action of thefactor(s).The anti-invasive activity of bovine immunoglobulins alsoneeds to be investigatedfurther. Samples of different origins should be tested, againstother enteropathogens, andusing other cell lines, to demonstrate the generality of the phenomenon.Of particular interestwould be experiments to compare the anti-invasive ability ofbovine colostrum with that ofhuman milk. It is rather puzzling that only an anti-adherenceeffect against E. coli has beendemonstrated with human milk to-date, and that no resultsof experiments to investigate ananti-invasive effect of human milk, for instance against Salmonella,have been reported. Itwould also be useful to identify the targets of the colostrumantibodies, with particularattention to the bacterial components that are known to either induce antibodyproduction inthe course of natural infection, or that are associated with adherenceand invasion, forinstance: the 94 kDa outer membrane protein of EPEC; the S.enteritidis fimbrie identifiedby Peralta et al. (1994); or, in the case of bacteria for which theeffect of bovine colostrum incell cultures remains to be tested, the outer membrane proteins of Shigellaidentified by Oakset at. (1986); the Yersinia invasin, or the outer membrane proteinsof E. coli 0157:H7identified by Sherman et a!. (1991). The finding of antibodies to the CFA1 of ETEC has123been reported in this thesis, and these could also be tested for anti-adherence activity withthe cell culture system used by Darfeuille-Michaud et al. (1990), who demonstrated thatspecific rabbit antisera to the colonization factors antigens of human ETEC preventedadherence to Caco-2 cells by the homologous bacteria.In future work it will also be of interest to determine whether colostrum antibodies orother factors inhibit adherence, or do not affect adherence but inhibit invasion. This maydepend on whether invasion follows adherence automatically, or whether adherence andinvasion are separate steps in the process of entry into the mammalian cell. In E. coilE2348/69 and related strains, there is evidence that invasion is a two-step process (Franciset al., 1991) and it may be possible to test for both an anti-adherence effect and an anti-invasion effect of colostrum. Francis et al. (1991) found that the localized adherence patternof this type of EPEC was mediated by the EPEC adherence factor (EAF), while anotherfactor (eae) was required for effacement of microvilli and entry into mammalian cells. Thiseae factor has been correlated with the presence of the 94 kDa outer membrane proteinreferred to above (Jerse and Kaper, 1991). Efficacy in vivo could be tested with the ligatedloop model, which was used by Jones et al. (1994a) to demonstrate invasion of the intestinalPeyer’s patches by S. typhimurium.To end with a more general and speculative discussion, it may be that the question ofthe specificity of the milk inhibitors will be of great interest. Recent studies of bacterialpathogenesis have uncovered an intriguing conservation of structures and functions ofproteins involved in transport of virulence determinants in a variety of pathogens, amongwhich are Salmonella, Shigeila and Yersinia (Ginocchio and Galan, 1995; Collazo et al.,1995). However, the determinants exportedby these systems appear to be very specific tothe various pathogens. If the role of milk in the protection of infants against enteropathogensis accepted, then the question arises whether this protection is directed only at the vastnumber of antigenic determinants present on the various species of bacteria, or wheher there124 also exists some general mechanism that would interfere with the export system mentioned above. If this export system is activated or enhanced by contact of the bacteria with the host cell, as may be the case with Yersinia (Rosqvist et al., 1994), then a prime target of the milk inhibitors, besides preventing contact between the bacteria and host cells, could be whatever structure on the bacteria generates a signal to activate the secretion sytem. There is no evidence for such a mechanism at this point, but this should be kept in mind when investigating the identity of the immunoglobulin or non-immunoglobulin milk inhibitors or their targets on the bacteria. And finally, to return to the original question of whey utilization, more samples of a variety of cheese wheys need to be studied to determine their antibacterial properties by the above methods, and in particular to investigate the influence of various manufacturing processes on their antibacterial activity. The suitability of cell cultures to study the antibacterial effect of cows milk and whey was demonstrated by the experiments in the present study, particularly in this case by providing a test system for anti-invasive activity by a non-immunoglobulin fraction which could not have been detected by immunoassays. It may be that continuation of this work will provide results that would make further processing of cheese whey a more attractive proposition. In any case it should provide added understanding of the mechanisms by which milk contributes to protection against enteropathogens. VI. CONCLUSION. 125 The results of a study of the antibacterial activity of a pepsin digest of bovine lactoferrin (LFD) were reported. It was found that LFD is bactericidal against Salmonella enteritidis ATCC 13076 in 1% peptone at a concentration of 30 |ig/ml, and bacteriostatic at a concentration as low as 3 (ig/ml. It was also found that some bacteria were injured by the effect of LFD and became susceptible to killing by bile salts. LFD had no activity in trypticase soy broth (TSB) or in milk-based or soy-based infant formulae. Calcium at a concentration of approximately 5 mM was sufficient to inhibit the activity of LFD in 1% peptone. Addition of lysozyme or EDTA to LFD in TSB was not beneficial unless the concentration of EDTA was significandy higher than that permissible in foods. Even though lysozyme (80 fig/ml) or EDTA (0.25 mM) alone had no inhibitory effect on S. enteritidis, they showed a synergistic effect with LFD provided that the concentration of TSB was reduced to 75% of its recommended strength. It was also found that bile salts inhibited the activity of LFD in 1% peptone. It can be concluded that while the bovine lactoferrin digest showed some considerable antibacterial activity under some limited conditions, there is no evidence that it had any effect against S. enteritidis ATCC 13076 in the foods tested. These findings raise doubts about the application of lactoferricin in foods. Antibodies to the Colonization Factor Antigen 1 of enterotoxigenic Escherichia coli HI0407 were detected in cows milk and colostrum by hemagglutination inhibition and immunoassays. The concentration of antibodies was determined by ELISA and found to range approximately from 0.5 to 5 |ig/ml in colostrum samples of non-vaccinated cows. 126 Testing of milk immune concentrates showed that vaccination with a purified antigen or a whole cell preparation increased the concentration of specific antibodies relative to the total immunoglobulin G concentration of the samples. Finally, it was found that bovine colostrum inhibited invasion of HeLa cells by S . enteritidis ATCC 13076 and CD5, S. typhimurium SL1344, and E. coli E2348/69. Inhibition levels ranged from 73% to over 99% when colostrum was added to a final concentration of 10% of the culture medium. Seven samples of colostrum were tested. An effect of colostrum on the number of viable bacteria in the medium or on the mammalian cells could not be detected. The inhibitory activity of colostrum was diminished by heat treatment above 80 °C or by digestion with pepsin, and was not found in low molecular weight fractions. The immunoglobulin-containing fraction, isolated from colostrum by affinity chromatography on a protein G-agarose column, inhibited invasion by SL1344. An unidentified high molecular weight factor in the non-immunoglobulin fraction also inhibited invasion of HeLa cells by SL1344. 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