WO2004093561A1 - Prevention and treatment of pathogenic infection in poultry and eggs - Google Patents

Abstract

The invention relates to the use of yolk, yolk protein or egg white fractions for prevention or treatment of pathogenic infection, such as Salmonella enteritidis, Salmonella typhimurium, Camplyobacter jejuni or Escherichia coli 0157:H7 infection in poultry or eggs. The yolk, yolk protein or egg white fraction may be supplemented in the diet of poultry at a level of up to 15 %. Further, a method for preventing or treating Salmonella enteritidis, Salmonella typhimurium, Camplyobacter jejuni or Escherichia coli 0157:H7 infection in poultry or eggs is disclosed, comprising the step of feeding a diet supplemented with yolk, yolk protein, or egg white fractions to poultry in need thereof.

Description

Prevention and Treatment of Pathogenic Infection in Poultry and Eggs

FIELD OF THE INVENTION

Th^e present invention relates generally to a method for prevention and treatment of pathogens in Poultry and Eggs.

BACKGROUND OF THE INVENTION

Pathogen infection of poultry and eggs is an ongoing problem. Salmonella spp. infection is a problem for poultry and eggs. Many methods have attempted to alleviate this problem, some of which involve feeding animals fractions derived from eggs, as well as feeding animals other dietary components in order to prevent or eliminate S. enteritidis infection in poultry.

Perhaps the most important problem affecting the poultry industry today is the increasing incidence of food borne human salmonellosis caused by S. enteritidis associated with table eggs (Angulo et al., 1998). The serotype of Salmonella spp. that is responsible for the most cases of reported Salmonellosis does change over the years. Until mid 1980s, Salmonella typhimurium (S. typhimurium) was commonly the reported serotype in North America, but S. enteritidis has overtaken S. typhimurium in more recent years due to a large rise, particularly in egg-related outbreaks and incidents of

Salmonellosis involving in S. enteritidis PT4 (Bell et al, 2002). Since the 1970s, the incidence of Salmonella spp. serotype enteritidis infections has increased in European and North American countries (Rodrigue et al, 1990). In the USA from 1972 to 1996, the proportion of Salmonella spp. isolates that were identified S. enteritidis increased from 6% to 25%ι making S enteritidis the predominant Salmonella spp. serotype (Angulo et al.,

1998). Investigations of outbreaks (Morse et al, 1994; St. Louis et al, 1988) and sporadic infections (Shmid et al, 1996) have most frequently identified eating raw or undercooked eggs as the principle risk factor for human illness. During 1985-1998, state and territorial health departments in North America reported 796 S. enteritidis outbreaks that associated for 28,689 illnesses, 2839 hospitalization and 79 deaths (MMWR, 2000); 82% of the outbreaks were associated with shell eggs. The integrity of eggshell formation is vital for the propagation of all avian species, since the shell and associated membrane are a physical and microbial barrier against the external environment. Flaws in eggshell fabrication can permit microbial contamination of the egg contents, and are believed to be responsible for about 10,000 cases of Salmonella spp. poisoning due to cracked eggshell annually in Canada. In number of recent outbreaks, the infection dose was found to be low <10- 100

CFU/shell (Bell et ah, 2002). As few as'lO CFU/shell of appropriate strains of Salmonella spp. are known to initiate infection in susceptible individuals. Broilers are widely acknowledged to be a considerable reservoir for Salmonella spp. infections in man, due to the ability oϊ Salmonella spp. to proliferate in the gastrointestinal tract of chickens and subsequently survive on commercially processed broiler carcasses and edible giblets (Todd, 1980).

Salmonella spp. are gram negative flagellated, facultatively anaerobic bacilli possessing three major antigens: H or flagellar antigen, O or somatic antigen and Ni antigens (possessed by only a few serovars mainly S. typhimurium). H antigen may occur in either or both of two forms called phase 1 and phase 2. O antigens occur on the surface of the outer membrane and were determined by specific sugar sequences as the cell surface. Antigenic analysis of Salmonella spp. by using specific antisera offers clinical and epidemiological advantages (Board et al, 1994).

S. enteritidis was vertically transmitted to eggs through the ovary of infected hens (Benson et al, 1991 ; St Louis et al, 1988) and can be isolated from either the shells or contents of the egg. Eggshells could become contaminated with Salmonella spp. as a result of infection of the oviduct. Colonization of the intestinal tract commonly occurs after the consumption of contaminated feed and chickens can acquire a wide range of Salmonella spp. serotypes by this route. In a recent survey in Canada, 4, 850 Salmonella spp. serovars were identified; 58

S. enteritidis strains were typed, among the 34 S. enteritidis isolates from chickens, 2 were from dust from the environment of chickens, 1 from egg contents, 15 were from feces, 5 from fluff samples, 1 from organ, 1 from semi-prepared meat, 32 from shell eggs, and 6 from swabs taken from chickens or from chicken environmental sources (Poppe, 2001). Faecal carriage can also lead to eggshells becoming contaminated with S. enteritidis and Gast et al. (1997) reported that there was an apparent relationship between faeces positivity and eggshell contamination in hens artificially infected with Salmoenlla enteritidis. Infection of the reproductive tissue also appears to be important. Egg contents can become contaminated with Salmonella spp. as a result of either infection in reproductive tissue or the passage, through the shell, of organisms derived from either intestinal tract or the environment. Hence, the news media have focused on eggs and other poultry products to the extent that Salmonella spp. enteritidis related disease raises issues of urgent political and economic concern to the poultry farmer. S. enteritidis is recognized as a frequent and important pathogen for poultry and has been isolated from broilers, breeders and commercial egg laying flocks (Board, 1994). Both poultry meat and eggs are often mentioned to cause Salmonellosis in man.

In summary, laying hens can be infected easily by the following common routes; contaminated feed; crop inoculation; contaminated aerosols via the conjunction; spread to contact birds; cloacal inoculation; and vertical transmission. S. enteritidis must be regarded as being highly invasive in chickens, can spread easily from bird to bird (Board, 1994) and can be isolated from tissues.

Realizing that a low number of cells oϊ Salmonella spp. can be capable of causing illness serves to underline the importance of all the measures required to be taken to minimize the incidence. Therefore, to control S. enteritidis contamination in field poultry flocks, accurate knowledge of the state of infection has been thought to be important. However, to do accurate monitoring in the field is very difficult because the prevalence of S. enteritidis contamination in shell eggs is very low (Board, 1994).

It was suggested that S. enteritidis is vertically transmitted to eggs through the ovary of infected hens (Benson et al, 1991; St Louis et al, 1988) and can be isolated from either the shells or contents of the egg. Eggshells can become contaminated with Salmonella spp. as a result of infection of the oviduct or faecal carriage. Colonization of the intestinal tract commonly occurs after the consumption of contaminated feed and chickens can acquire a wide range oϊ Salmonella spp. serotypes by this route.

Much effort is also directed towards the inhibition and the elimination of Salmonella spp. infection in poultry. Preventing S. enteritidis infection and colonization in poultry is a major concern for both the poultry and food processing industries as well as for the consumer (Roberts, et al, 1990). The intestinal colonization of S. enteritidis plays a significant role in carcass contamination during processing. Therefore, reducing intestinal colonization of S. enteritidis during the grow-out period is crucial in improving carcass microbiological quality during processing and reducing the potential carcass contamination during slaughter to provide a safer meat product. Furthermore, it can reduce the contamination of eggs via the trans-shell route. In trans-shell contamination, the organism gains access to the egg after oviposition, as it passes through the intestinal tract and by penetration to the shell. The contamination of eggs has important economic implications to the poultry industry. In the case of table eggs, spoilage may occur and if the organism is of public health significance, like S. enteritidis, the affected eggs may cause the spread of disease. Consequently, the contamination of hatching eggs may reduce hatchability, be responsible for transmission of poultry pathogens and impair the quality of chicks produced.

The crop and cecum are the major sites oϊ Salmonella spp. colonization in the chicken (Brownell at al, 1969, 1970). These sites are responsible for the movement of Salmonella spp. into intestinal tissues (Nagaraja et al, 1991). However, the preferred site oϊ Salmonella spp. colonization is in the caudal intestinal region (Turnbull et al, 91 A) where S. enteritidis have a predilection to establish chronic infection in the ceca.

Feed supplemented with various components to eliminate S. enteritidis from the intestine has been investigated. D-mannose has been reported to remarkably suppress the colonization of S. typhimurium in vitro (McHan et al, 1989) and in vivo (Deloach, 1989); however, it is too expensive to be used in feed applications. Allen at al, (1997) has reported that palm kernel meal, which contains considerable amounts of structural carbohydrates such as arabinans, galactans, glucans, mannans, and pectins is effective in preventing Salmonella spp. infection; however, its preparation on an industrial scale has not been established, it may be difficult to put this into practical use. Furthermore, lactose has also been reported to be effective in the prevention of S. typhimurium infection (Corner et al, 1990), but it has also been suggested that the dosage of lactose used causes diarrhea and decreases the daily gain (Kogut, 1994). For these reasons the search and development of a new, practical, low cost method for the elimination and prevention of S. enteritidis infection is important for maintaining public health.

Sugita-Konishi et al. (2000) describes the use of egg-derived antibodies (anti-S. enteritidis antibodies) from hens immunized with formalized Salmonelle enteritidis. After hens were immunized, egg yolk from the hens was obtained, and a fraction containing the IgG antibody was derived. This fraction was used in vivo to prevent adherence of S. enteritidis to Caco-2 cell monolayers. This study showed that the anti-Salmonella spp. antibodies blocked penetration oϊ Salmonella spp. into the epithelial cells. Fulton et al (2002) studied the effect of egg-derived antibodies against S. enteritidis infection in ducklings. The egg-derived antibody was obtained from immunizing chickens with S. enteritidis and isolating a fraction from the eggs laid by those chickens. Although the yolk was purified to some degree, it was merely processed in a food blender, with the superficial lipid layer being removed, and the supernatant was considered as the antibody source. The supernatant was not further purified, but the presence of the antibody was confirmed by ELISA. Although the researchers concluded that anti-Salmonella enteritidis antibody derived from chicken eggs prevented Salmonella spp. infection in ducklings when provided in advance, these findings may be attributable to any of the other components that were present in the non-purified yolk supernatant. Sugita-Konishi et al. (Journal of Agricultural and Food Chemistry, 2002) examined the effect of a sialyoligosaccharide and its derivatives on the binding of Salmonella spp. to epithelial intestinal cells. The oligosaccharide tested was derived from chicken egg yolk, but the purification method is not described in detail. The results showed that the oligosaccharide had inhibitory effects on the binding of S. enteritidis on these cells. Although the authors do not imply any direct link to whether a non- fractionated egg yolk would have a similar effect, or whether an effect would be seen in poultry, this document does serve to indicate that there is an effective component within the egg yolk that can be used to counter Salmonella spp. infection.

Other documents describe a nutritional component that may be used to prevent or counter Salmonella spp. infection in chickens. In particular, Oyofo et al. (1989) discloses the use of mannose in chickens, Line et α .(1997) discloses the use of yeast treatment in broiler chickens to prevent Salmonella spp. infection, Ishihara et al. (2000) investigated the use of guar gum in preventing S. enteritidis infection, Tellez et al. (1993) investigated the use of lactose in inhibiting S. enteritidis invasion in chicks, and Massa et al. (1998) discussed a probiotic approach to protection of chicks against Salmonella spp. infection. It is clear that a number of approaches involving either nutritional supplementation or probiotic use have been researched in an effort to overcome the problem oϊ Salmonella spp. infection in poultry.

United States Patent No. 5,985,336 (Ivey et al.) issued November 16, 1999, discloses a formulation of feed for poultry comprising an egg-derived high protein mixture aimed at improving growth in young animals. This document does not teach that Salmonella spp. infection can be prevented or treated with the formulation.

Other pathogens, such as Salmonella typhimurium, Campylobacter jejuni and Escherichia coli 0157:H7 are problematic in poultry infection, or infection of laying hens, which ultimately may result in egg infection. There remains a need for an inexpensive nutritional supplement capable of treating or preventing Salmonella spp. and other pathogenic infections in poultry and eggs.

Background references include:

Allen, V.M. et al. (1997). Evaluation of the influence of supplementing the diet with mannose or palm kernel meal on Salmonella spp. colonisation in poultry. Poult. Sci. 38; 485-488

Angulo, F J., Swerdlow, D.L.(1998). Salmonella enteritidis infections in the United States. J.Am. Vet. Med. Assoc. 12; 1729-31

Bell, C. and Kyriakides, A. (2002). Salmonella: A practical approach to the organism and its control in foods. Blackwell Science Ltd. Oxford, UK Benson, C.E. and Eckroade, J.R. (1991). Virulence properties oϊ Salmonella enteritidis isolates In: Colonization control of human bacterial enteropathogens in poultry. L.C. Blankenship, ed. Academic Press, San Diego, Calif. P. 149-160

Beard.and Gast (1990). In Microbiology of Avian Egg. Chapman and Hall. London, UK Board, G.R. and Fuller, R. (1994). Microbiology of Avian Egg. Chapman and Hall.

London, UK

Brownell, J.R., Sadier, W.W., Fanelli, MJ. (1969). Factors influencing the intestinal infection of chickens with Salmonella typhimurium. Avian Dis. 13; 804-816

Brownell, J.R., Sadier, W.W., Fanew, MJ. (1970). Role of the ceca in intestinal infection of chickens with Salmonella typhimurium. Avian Dis. 14; 106-116

Corner, D.E., Hinton, R.L. et al. (1990). Effect of dietary lactose on Salmonella colonization of market-age broiler chickens. Avian Dis. 34; 668-676 Deloach J.R.(1989). Salmonella prevention with carbohydrates. Broiler Ind. 52; 8- 10

Fey, H. (1979). A novel method for the production of Salmonella flagellar antigen. II. Further purification for the preparation of H antisera. J. abr. Orig. A. 245; 55-56 Fulton et al. (2002) Poultry Science 81 :34-40

Gast, R.K., R. E. Porter Jr. and P.S. Holt (1997). Applying tests for specific yolk antibodies to predict contamination by Salmonella enteritidis in eggs from experimentally infected laying hens. Avian Dis. 41; 195-202

Ishihara et al. (2000) Poultry Science, 79:689-697 Kogut, M.H., et al. (1994). Effect of Eimeria tenella infection on resistance to

Salmonella typhimurium colonization in broiler chicks inoculated wit anaerobic cecal flora and fed dietary lactose. Avian Dis. 38; 59-64

Line et α/.(1997) Poultry Science, 76:1227-1231

Massa et al. (1998) International Journal of Food Microbiology 40:123-126 McHan, F.N., Blankenship, C.L., Bailey, J.S. (1989). In vitro attachment of

Salmonella typhimurium in chick ceca exposed to selected carbohydrates. Avian Dis. 33; 340-344

Morse, D.L., Birkhead, G.S., et /.(1994). Outbreak and sporadic egg-associated cases oϊ Salmonella enteritidis: New York's experience. Am. J. Public Health. 84; 859-60 Nagaraja, K.Y., Pomeroy, B.S., Williams, J.E.(1991). Paratyphoid infections. P.99-

130. In Calnek, J.H. et al. Disease of Poultry. 9th ed. Iowa State University Press, Ames, Iowa

Oyofo et al. (1989) Avian Diseases, 33:531-534, 1989

Poppe, C. (2001). Epidemiology of Salmonella enterica serovar enteritidis. In: Salmonella enterica serovar enteritidis in Humans and Animals; Saeed M. A. Chapter 1 ; p. 3-16. Iowa State University Press

Roberts, D. (1990). Sources of infection: food. Lancet 336; 859-861

Rodrigue, D.C., Tauxe, R.N., Rowe, B. (1990). International increase in Salmonella enteritidis: a new pandemic? Epidemiol. Infect. 105; 21-7 Shmid H., Burnens, A.P., et al. (1996). Risk factors for sporadic salmonellosis in

Switzerland. Eur. J. Clin. Microbiol. Infect. Dis. 15; 725- 32 St. Louis, M.E., Morse, L.D., Potter, E. M., Demali, T.M., Guzewich, J.J., Tauxe, R.N., Blake, P.A. (1988). The emergence of Grade A eggs as a major source of Salmonella enteritidis infections: new implications for the control of salmonellosis. J. Am. Med. Assoc. 259; 2103-2107 Sugita-Konishi et al. (2000) Microbiol. Immunol. 44 (6), 473-479

Sugita-Konishi et al. (2002) Journal of Agricultural and Food Chemistry Sugita-Konishi, Y., M. Ogawa, S. Arai, S. Kumagai, S. Igimi, and M. Shimizu. (2000). Blockade of Salmonella enteritidis passage across the basolateral barriers of human intestinal epithelial cells by specific antibody. Microbiol Immunol 44:473-9. Sugita-Konishi, Y., S. Sakanaka, K. Sasaki, L. R. Juneja, T. Νoda, and F. Amano (2002). Inhibition of bacterial adhesion and salmonella infection in BALB/c mice by sialyloligosaccharides and their derivatives from chicken egg yolk. J Agric Food Chem 50:3607-13.

Tellez, G., N. M. Petrone, M. Escorcia, T. Y. Morishita, C. W. Cobb, L. Nillasenor, and B. Promsopone (2001). Evaluation of avian-specific probiotic and Salmonella enteritidis-, Salmonella typhimurium-, and Salmonella heidelberg-specific antibodies on cecal colonization and organ invasion of Salmonella enteritidis in broilers. J Food Prot 64:287-91

Tellez et al. (1993) Poultry Science, 72:636-642 Todd, E. C.(1980), Poultry-associated foodborne disease-its occurrence, cost, sources, and prevention. J. Food Prot. 43; 129-139

Turnbull, P.C., Snoeyenbos, G.H. (1974). Experimental salmonellosis in the chickens. 1. Fate and host response in alimentary canal, liver and spleen. Avian Dis. 18;

153-177

SUMMARY OF THE INVENTION

It is an object of the present invention to overcome or mitigate at least one disadvantage of previous methods of treating bacterial infection in poultry and eggs.

In a first aspect, the present invention provides the use of yolk, yolk protein or egg white fractions as a poultry feed supplement for prevention of pathogenic bacterial infection by organisms such as Salmonella enteritidis, Salmonella typhimurium, Campylobacter jejuni or Escherichia coli 0157:H7. A method of preventing or treating pathogenic bacterial infection by such organisms as Salmonella enteritidis, Salmonella typhimurium, Campylobacter jejuni ox Escherichia coli 0157:H7 is provided comprising feeding a diet supplemented with yolk, yolk protein, or egg white fractions to an avian animal in need thereof. The diet may be supplemented to a level of up to 15%. Other aspects and features of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS Embodiments of the present invention will now be described, by way of example only, with reference to the attached Figures, wherein:

Figure 1 illustrates S. enteritidis counts in fecal samples when chickens were infected with S. enteritidis then fed egg yolk powder, egg yolk proteins, egg white, and the control (regular feed). Figure 2 shows S. enteritidis counts in fecal samples after chickens were fed feed supplemented with egg yolk powder or egg yolk protein for 4 weeks then orally infected with S. enteritidis.

Figure 3 shows S. enteritidis counts in fecal samples after chickens were fed control (regular feed) or feed supplemented with egg white for 4 weeks then orally infected with S. enteritidis.

Figure 4 shows total counts of microflora in fecal samples throughout the feed supplementation period.

Figure 5 illustrates bacterial counts in fecal samples from animals after consuming egg yolk supplemented feed with and without specific antibody (IgY) to S. enteritidis. Figure 6 shows the effect of feeding non-immunized egg yolk powder on the elimination oϊ Salmonella typhimurium from the intestine of laying hens. ^± Standard error of the mean. ** P < 0.05 compared with the control.

Figure 7 illustrates the effect of feeding non-immunized egg yolk powder on the prevention oϊ Salmonella typhimurium from the intestine of laying hens. ^x Standard error of the mean. Figure 8 shows the effect of feeding non-immunized egg yolk powder on the elimination of Campylobacter jejuni from the intestine of laying hens. Standard error of the mean. ** P < 0.05 compared with the control.

Figure 9 illustrates the effect of feeding non-immunized egg yolk powder on the prevention of Campylobacter jejuni from the intestine of laying hens. Standard error of the mean. ** P < 0.05 compared with the control.

Figure 10 shows the effect of feeding non-immunized egg yolk powder on the elimination oϊ Escherichia coli 0157:H7 from the intestine of laying hens. ¹ Standard error of the mean. ** P < 0.05 compared with the control. Figure 11 demonstrates the effect of feeding non-immunized egg yolk powder on the prevention oϊ Escherichia coli 0157:H7 from the intestine of laying hens. ^x Standard error of the mean. ** P < 0.05 compared with the control.

Figure 12 shows the effect of feeding non-immunized egg yolk powder on the weight of laying hens. ^x Standard error of the mean. ** p < 0.05 compared with the control.

Figure 13 shows the effect of feeding non-immunized egg yolk powder on the mean number of eggs laid. ^x Standard error of the mean. ** p < 0.05 compared with the control.

Figure 14 shows the effect of feeding non-immunized egg yolk powder on the mean weight of eggs laid. ^x Standard error of the mean. ** P < 0.05 compared with the control.

Figure 15 demonstrates the effect of feeding non-immunized egg yolk powder on Salmonella typhimurium in the ovary, oviduct, intestine and spleen of laying hens. ** P < 0.05. Figure 16 shows the effect of feeding non-immunized egg yolk powder on

Campylobacter jejuni in the ovary, oviduct, intestine and spleen of laying hens. ** P < 0.05.

Figure 17 shows the effect of feeding non-immunized egg yolk powder on Escherichia coli 0157:H7 in the ovary, oviduct, intestine and spleen of laying hens. ** P < 0.05. DETAILED DESCRIPTION

The invention relates to a method for prevention or treatment of pathogenic bacterial infection by microorganisms such as Salmonella enteritidis, Salmonella typhimurium, Camplyobacter jejuni and Escherichia coli 0157:H7 in poultry and eggs. The method involves feeding egg yolks, egg yolk proteins, or egg whites to chickens as a feed supplement at levels up to 15%. According to the invention, it was found that four weeks of egg yolk powder feeding was adequate to prevent Salmonella enteritidis from colonizing the intestinal tract of chickens at concentrations as low as 5%. Further, the invention allows exclusion or elimination oϊ Salmonella enteritidis from animals in which the intestinal tract has already been colonized, using egg yolk powder supplementation in the diet at concentrations as low as 5%. For the prevention or treatment of Salmonella typhimurium, Camplyobacter jejuni and Escherichia coli 0157:H7, prevention and elimination were significant with concentrations as low as 1.5%.

The invention finds some basis on the observation that the preventative effect feeding egg yolk was not significantly altered whether or not IgY to S. enteritidis was supplemented with the yolk. This illustrates that it is not this particular antibody which is responsible for the effect of egg yolk feeding on Salmonella enteritidis elimination.

Based on these data, the invention provides a method of preventing colonization or eliminating Salmonella enteritidis infection in poultry by supplementing the animals feed with egg yolk, egg yolk protein, or egg whites. The method can also be used to prevent or eliminate other pathogenic bacterial infections, such as Salmonella typhimurium, Camplyobacter jejuni and Escherichia coli 0157:H7 infection. _t

The significant epidemiological association between human illness caused by S. enteritidis and the consumption of contaminated eggs and poultry meat has made the urgent issue to eliminate and prevent the colonization in the intestinal tract of the birds. According to this invention, egg yolk powder and its protein components in addition to egg white were tested as a potential solution. In the pre-feeding infection study to determine the possibility of elimination of the organism, the chickens were orally infected with S. enteritidis then given the supplemented feed with 5, 10 or 15% of each of the test samples. Fecal samples tested weekly showed a substantial decrease in the bacterial counts when feeding 5, 10 or 15% egg yolk powder only 4 days after supplementation. A dramatic change was detected with 10 and 15 % of egg yolk proteins and egg white; however chickens still retained bacteria after supplementation of feed with 5 % yolk powder. In the post feeding-study aimed to determine whether any of the feed supplements can prevent S. enteritidis from colonizing in the intestinal tract, Salmonella spp. free chickens were fed the supplemented feed for 4 weeks then infected orally and given the regular feed. Fecal samples tested for 4 weeks showed that egg yolk powder was able to prevent S. enteritidis from colonizing in the intestinal tract even at a concentration as low as 5% throughout the experimental period. With the egg yolk proteins (10 and 15%) a delay in the colonization occurred, but only for up to 2 weeks.

Example 1

Reduction or Elimination of Infection: Pre-Feeding Infection Followed by Egg Yolk

Supplementation

22-24 weeks old SPF white leghorn hens, SPF were housed at the Isolation Unit,

Pathobiology Dept, University of Guelph. Each chicken was housed in a separate cage. Chickens were originally fed by the regular feed (Soy-based) without antibiotics.

The bacterial strain used for infection was, the S. enteritidis PT4 SA992212. One ml of 10⁹ colony forming units per ml (CFU/ml) overnight culture and 1 ml of sterile trypticase soy broth (TSB), Difco, as the control were used for infection.

Prior to infection fecal samples were collected and tested for S. enteritidis by the pre-enrichment/enrichment selective plating conventional method. Brilliant Green Agar

(BGA) plates and XLD culture plates were used and suspected Salmonella spp. colonies were identified biochemically by the LSI/TSI biotyping technique and serologically by the somatic O- group agglutination for serogrouping and finally serotyping using flagellar antigens. 24 hours prior to infection the feed and water were withheld. On the infection day chickens were orally given 1 ml (10⁹ CFU) inoculum. A booster infection of another 1ml was given 1 weeks after the first infection.

3 days post-inoculation, the infection was confirmed by taking cloacal swabs.

Fecal samples were also tested prior feeding to monitor S. enteritidis level. Once the level of S. enteritidis in feces reached a constant level of 10⁴ CFU/ml, feeding study commenced. Of the U S. enteritidis infected leghorn laying hens at Isolation Unit. Nine were fed for 4 weeks, each 3 a different supplemented feed with either egg yolk proteins, egg yolk powder, and egg white at concentrations of 5%, 10%, or 15%. The remaining 2 control leghorn hens infected with S. enteritidis and used as control were fed the regular feed All egg supplements were prepared in the lab. The feeding procedure was as follows: chickens were fed the supplemented feed over a period of 4 weeks.

S. enteritidis Detection after Feeding. Fecal samples were collected once a week starting 1 week after feeding the supplemented feed throughout the experimental period. S. enteritidis tested, detected and enumerated as before according to previous prepared protocols. Data were reported and analysed to determine the effect of each supplement on eliminating S. enteritidis that was colonized in the intestinal tract of each hen. Results. Figure 1 : S. enteritidis counts in fecal samples when chickens were infected with S. enteritidis then fed egg yolk powder, egg yolk proteins, egg white, and the control (regular feed).

Discussion. S. enteritidis colonizes in the intestinal tract of poultry and causes food borne illness in humans either through the consumption of contaminated meat or eggs. Egg yolk powder and its protein components in addition to egg white have been tested as a potential solution in overcoming the drawbacks in the other suggested supplemental methods. In the pre-feeding infection study, the chickens were orally infected with 1 ml of 10⁹ CFU/ml of S. enteritidis and a booster was given 2 weeks later. Infection was confirmed by fecal and cecal bacteriological testing then were given the supplemented feed with the various concentrations (5, 10, 15%) of the test samples.

Fecal samples collected weekly and tested microbiologically for S. enteritidis, showed a substantial decrease in the bacterial counts when feeding 5, 10 or 15% egg yolk powder only 4 days after commencing the supplemented feed. A gradual decrease was detected when feeding 10 and 15 % of egg yolk proteins and egg white; however no effect with 5 % supplementation. Therefore, although egg yolk proteins do have an elimination effect, egg yolk powder overweighs this effect probably by providing a synergistic effect with the other components.

Example 2

Prevention of Infection: Egg Yolk Supplementation and Post-Feeding Infection

While the pre-feeding infection aimed to determine whether the supplemented feed can eliminate S. enteritidis from the intestinal tract, the post-feeding is aimed to determine whether such supplements can prevent S. enteritidis from reinfecting or recolonizing the intestinal tract if the hens were reexposed to the organism.

Based on results from the preinfection study, the concentrations of each supplement that showed some degree of elimination were picked for the post-infection study. These were the 5, 10% egg yolk powder, 10, 15% egg yolk proteins, and 10, 15% egg white powder. S. enteritidis free chickens were fed their designated feed supplement daily for 4 weeks and the 2 control chickens their regular feed. Fecal samples were collected once a week and tested bacteriologically for S. enteritidis. After the 4 weeks of feeding, feed and water were withheld for 24 hours and all the chickens were orally infected with 1 ml of 10⁹ CFU/ml of S. enteritidis using a sterile 1ml syringe. After the infection, chickens were fed the regular feed.

S. enteritidis Detection after Infection. Fecal samples were collected once a week starting 2 days after infecting and throughout the experimental period. Samples were tested for S. enteritidis bacteriologically as before. At the end of the experimental period, chickens were euthenized.

Results. Figures 2 and 3 show S. enteritidis counts in fecal samples after chickens were fed egg yolk powder and egg yolk protein (Figure 2), and after chickens were fed control (regular feed) and egg white (Figure 3) for 4 weeks, and then orally infected with S. enteritidis. The arrow indicates commencement of the feeding period.

Figure 4 shows total counts of microflora in fecal samples throughout the supplemented feed period.

Discussion. In the post feeding- study Salmonella spp. free chickens were fed the supplemented feed for 4 weeks then infected orally with 1 ml of the 10⁹ CFU/ml S. enteritidis and given the regular feed afterwards. This experiment was aimed to determine whether any of the feed supplements can prevent S. enteritidis from colonizing in the intestinal tract if the birds were exposed to the organism. Fecal samples were collected weekly and tested for S. enteritidis as before. Interesting results showed that egg yolk powder was capable of preventing Salmoenlla enteritidis from colonizing in the intestinal tract even at a concentration as low as 5%. No evidence of the organism even 4 weeks from terminating the supplemented feed and orally infecting the chickens. With the egg yolk proteins (10 and 15%), a prevention effect or delay in S. enteritidis colonization occurred, but only for 2 weeks from the supplement termination day and infection, then S. enteritidis was able to gradually colonize and grow in the intestine. This same effect was also observed with the 10 % egg white. However at a higher concentration of 15% egg white was capable of preventing S. enteritidis from colonizing even after the 4th week. In conclusion, egg yolk powder was the substance that could both eliminate and prevent S. enteritidis intestinal colonization at a concentration as low as 5 %. Since egg yolk protein did show an elimination effect, this suggests that the proteins in the egg yolk play a role in the elimination and prevention but there are other components present in the egg yolk that also might have an activity and most probably a synergistic one. To determine the active component and its function as well as the possibility of any synergistic effect, further experiments will be conducted using a cell culture model system.

The feed supplements need to be not only readily available, practical and economic; but also must not disrupt the natural microflora that is naturally present in the birds' intestine. Therefore, monitoring the level during the feeding study was essential. When fecal samples were collected and bacteriologically tested throughout the study, none of the fed supplements at all the concentrations had a negative effect on the natural intestinal microflora of the chickens, the counts remained constant. This suggests that the use of the egg yolk or its components as supplements is safe for the birds' general wellbeing.

Example 3

Effect of Supplementing Egg Yolk Powder with IgY To illustrate that the effects of egg yolk powder supplementation (such as those effects illustrated in Examples 1 and 2) are not attributable solely to the presence of IgY, an experiment was designed in which animals were fed egg yolk with or without IgY supplementation.

The experimental conditions were as follows: Chickens: 4 S. enteritidis infected leghorn laying hens (40 wks of age , numbered as 1 to 4) at Isolation Unit, University of Guelph. These were infected orally with 10⁹ CFU/ml ). 1 kg of feed was required per chicken per week. For Chickens 1 and 2, feed was formulated to contain 150 g yolk powder (containing 1.5 g of specific IgY to S. enteritidis). Chickens 3 and 4, as control, were fed a supplemented feed of 15% egg yolk powder (150 g of yolk powder per kg) without specific antibody.

Housing was in the Isolation Unit of the Pathobiology Department, University of Guelph, Canada. Each chicken in a separate cage in room. Plastic sheets used under the wire floor of each cage to catch droppings. Feed Preparation: Feed used was pelleted and contained no antibiotics. Figure 5 illustrates bacterial counts in fecal samples from animals after consuming egg yolk supplemented feed. Clearly, the effect of IgY supplementation did not significantly enhance the beneficial effect of egg yolk supplementation. Example 4

Non-Immunized Egg Yolk Powder as a Feed Supplement Alters Colonization of Salmonella typhimurium, E coli 0157:H7 and Campylobacter jejuni in laying hens The purpose of the present example was to determine the extent to which non- immunized egg yolk powder according to the invention could be equally effective in reducing or inhibiting intestinal colonization by populations of other pathogens, such as Salmonella typhimurium, Camplyobacter jejuni and Escherichia coli 0157:H7 in laying hens. Materials and Methods. All animal experiments were performed with the knowledge and approval of the University of Guelph Animal Care and Ethics Committee and complied with the Canadian Code of Practice for the care and use of animals for scientific purposes.

Bacterial Strains. The isolates used for infection were Salmonella typhimurium var Copenhagen PT 10 SA992416 from turkey (provided by Dr. Cornelius Poppe, Health Canada, Guelph, ON, Canada), Campylobacter jejuni ATCC 29428 (provided by Dr. Joseph Odumeru, Laboratory Services, University of Guelph, Guelph, ON, Canada) from chickens, and Escherichia coli 0157:H7 920005 from chickens (CRIFS, Guelph, ON, Canada). The inocula for Salmonella typhimurium infectious challenge was prepared from an overnight culture in tryptic soy (TS) broth (D4552) (BD diagnostic system, Oakville ON., Canada) incubated at 37°C for 24 hr, then serially diluted to the specified viable cell concentration of 10⁹ CFU/ml. Inoculum was estimated by spectrophotometry, optical density at 660nm, and confirmed by colony counts on Xylose Lysine Deoxycholate (XLD) agar (B211838) (BD diagnostic system) plates. 1.0 ml of the challenge inoculum and 1.0 ml of sterile TS broth as control, were used for the oral infection. The inocula for

Campylobacter jejuni was prepared by inoculating one hundred microliter from the - 70°C glycerol broth stock into 10 ml of Brucella Broth (11088) (BD diagnostic system) containing 0.02% cysteine HC1 (BD diagnostic system), at 37°C for 24 hr under microaerophilic conditions (Gaspak, Becton Dickenson Co., MD 21030). It was then subcultured onto Campylobacter agar base (1820-07) (BD diagnostic system) containing 10% sterile defibrinated sheep blood and supplemented with 1% campylobacter Antimicrobic supplement B (3279-32) (BD diagnostic system) and placed at 42°C for 24 hr under microaerophilic conditions. One colony was transferred into Brucella broth and was incubated at 37°C under microaerophilic conditions overnight, then serially diluted to the specified viable cell concentration of 10⁹ CFU/ml. Inoculum was estimated by spectrophotometry, optical density at 660nm. 1.0 ml of the challenge inoculum and 1.0 ml of sterile Brucella broth as control, were used for the oral infection. Finally, the challenge inocula oϊ Escherichia coli 0157:H7 was prepared from an overnight culture in LB Lennox broth (B240-230) (BD diagnostic system) incubated at 37°C for 24 hr, then serially diluted to the specified viable cell concentration of 10⁹ CFU/ml. Inoculum was estimated by spectrophotometry, optical density at 660nm, and confirmed by colony counts on Tellurite-Cefixine-Sorbitol MacConkey (TC-SMAC) agar (DF0079-17-7) (BD diagnostic system) plates 1.0 ml of the challenge inoculum and 1.0 ml of sterile LB broth as control, were used for the oral infection.

Methods of the FDA Bacteriological Analytical Manual (Wallace et al, 1995) were used for the detection and identification oϊ Salmonella typhimurium, Campylobacter jejuni and Escherichia coli 0157:H7 in the feces of test samples.

Birds. In the elimination and prevention studies, respectively, fifty four and twenty four 22-24 weeks old white leghorn hens; specific pathogen free (SPF), were obtained from University Research Station (Arkell Poultry Research Unit, Guelph, Canada). Cloacal swabs and fecal samples were collected and tested for Campylobacter jejuni, Salmonella typhimurium and Escherishia coli 0157:H7 by the selective plating and identification methods (Wallace et al, 1995). In testing for Campylobacter jejuni, serial dilutions of the fecal suspensions were prepared and plated onto Campylobacter agar base containing 10% sterile defibrinated sheep blood and supplemented with 1% campylobacter Antimicrobic supplement B. Plates were placed at 42°C for 24 hr under microaerophilic conditions. After incubation, plates were observed for presumptive Campylobacter spp colonies, which appear as non-hemolytic, mucoid, and gray on the above media. Representative colonies were subjected to a latex agglutination assay (Dr. Cornelius Poppe, Health Canada, Guelph, ON, Canada) specific for Campylobacter jejuni to confirm the identity. Salmonella typhimurium infection was tested by the selective enrichment of individual cloacal swabs and fecal suspensions in tefrathionate broth (TB) (Dr. Joseph Odumeru, Laboratory Services, University of Guelph, Guelph, ON, Canada) for 24 h at 37°C. This was followed by subculturing onto XLD agar at 37°C for 24 h. The identity of Salmonella typhimurium was confirmed biochemically by the lysine iron (LI) agar (B211363)/tryptic sugar iron (TSI) agar (D4402)² slants biotyping technique. Then Salmonella typhimurium was positively identified serologically by slide agglutination using both polyvalent and specific O and H antigen antisera (BD diagnostic system) (Wallace et α/.,1995). Fecal samples were also collected for the testing of E. coli 0157:H7 ^' by the enrichment/isolation procedure using the TC-SMAC medium (Wallace et α/.,1995). Inoculated plates were incubated at 37°C overnight and presumptive E. coli 0157:H7 colonies were confirmed by latex agglutination (E. coli 0157 test kit) (Dr. Joseph Odumeru). Prior to infection, eggs from each bird were cracked and egg yolks were aseptically separated from the albumen. Egg yolk samples were tested for antibodies to Campylobacter jejuni, Salmonella typhimurium and Escherishia coli 0157:H7 by the enzyme-linked immunosorbent assay (ELISA) using formalin treated whole cells as coating antigens and primary and secondary antibodies for the colorimetric identification as described previously (Mine, 1997). All birds were negative for the aforementioned organism.

Housing. Birds were housed in separate wire-bottom, covered metal cage systems in the isolation unit (Isolation Unit, Pathobiology Dept. University of Guelph, Canada)- One bird per cage and cages were raised in single layers on metal tables with each 3 (i.e 3 cages of 3 birds) that represented 3 replicates of one treatment were placed on one table keeping a distance of approximately 60 cm between one cage and another and a distance of 100 cm between the tables carrying the 3 cages. Cages of each challenge organism were placed in a separate room to avoid any cross-contamination with the other organisms. Plastic sheets were used under the wire floors of each cage to catch droppings. They were cleaned and replaced on daily bases. Food and water were provided ad libitum.

Feed Preparation. The regular untreated feed (Arkell Poultry Research Unit, Guelph, Canada) was soy-based, pelleted and contained no antibiotics. Prior to each feeding study, the feed of each chicken was prepared and stored separately by mixing the regular untreated feed thoroughly with the egg yolk powder supplement at the required concentration. This non-immunized egg yolk powder was prepared from eggs obtained from the University research station (Arkell Poultry Research Unit) that were cracked after the disinfection of the exterior shell surface and the egg yolks aseptically separated from albumen. Pooled egg yolks were freeze-dried and crushed into a fine powder. All egg yolk samples were tested for antibodies to Salmonella typhimurium, Campylobacter jejuni and Escherichia coli 0157:H7 by the enzyme-linked immunosorbent assay (ELISA) using formalin-killed whole cells as coating antigens. Samples were negative for the aforementioned antibodies.

Elimination Study. Feed and water were withheld for 24 hours prior to infection. Of the 54 birds, 18 were inoculated orally with 1.0 ml of 10⁹ CFU/ml of Salmonella typhimurium and 2, negative control, with 1.0 ml of sterile TS broth using a syringe and a blunt-end catheter. Eighteen chickens were also infected with 1.0 ml of 10⁹ CFU/ml of Campylobacter jejuni and 2, negative control, with 1.0 ml of sterile Brucella Broth; and 12 with 1.0 ml of 10⁹ CFU/ml oϊ Escherishia coli 0157:H7 and 2, negative control, with 1.0 ml of sterile LB Lennox broth. A booster infection of another 1ml was given 1 week after the first infection. Three days post-inoculation, the infection was confirmed by testing cloacal swab specimens and fecal samples to monitor bacterial level. Once the level in the feces reached 10⁴-10⁵ CFU/g, supplemented feeding commenced. For 4 weeks, each three birds in 3 separate cages representing 3 replicates of one treatment were fed a supplemented feed of non-immunized egg yolk powder at concentrations of 1%, 2.5%, 5%, 7.5%) or 10% (w/w) for Salmonella typhimurium and Campylobacter jejuni challenge tests; and 5%, 7.5% or 10% (w/w) for Escherishia coli 0157:H7 challenge test. The 3 positive controls of each challenge test (challenged with either Salmonella typhimurium, Campylobacter jejuni, or Escherishia coli 0157:H7) and the 2 negative control (unchallenged) were fed the untreated regular feed. [3 birds per concentration per strain x 5 concentrations (Salmonella typhimurium, Campylobacter jejuni) OR x 3 concentrations (Escherishia coli 0157:H7) + 3 positive control + 2 negative control = 20 birds/ Salmonella typhimurium + 20 birds/ 'Campylobacter jejuni and 14 birds/ Escherishia coli 0157:H7 = 54 total birds].

Prevention Study. For 4 weeks, each three Salmonella typhimurium,

Campylobacter jejuni, or Escherishia coli 0157:H7 free birds in 3 separate cages representing 3 replicates of one treatment were fed a supplemented feed of 10% (w/w) non-immunized egg yolk powder,, while the 3 positive control (to be challenged with Salmonella typhimurium, Campylobacter jejuni, or Escherishia coli 0157:H7) and the 2 negative control (to remain unchallenged) were fed the untreated regular feed. [3 birds per concentration per strain + 3 positive control + 2 negative control = 8 birds/bacterial strain x 3 bacterial strains = 24 total birds]. The supplement concentration in this study was determined based on results obtained from the elimination study above. At the end of 4 weeks post- feeding, feed and water were withheld for 24 hours and all the chickens except for the 2 negative control were orally infected with 1.0 ml of 10⁹ CFU/ml of either Salmonella typhimurium, Campylobacter jejuni or Escherichia coli 0157:H7. Post-infection, the chickens were fed the untreated regular feed.

Bacteriologic Examination. Fecal samples were collected once a week starting 4 days post-feeding in the elimination study or 4 days post-infection in the prevention study for 4 weeks. Salmonella typhimurium, Campylobacter jejuni and Escherichia coli 0157:H7 were detected, identified and enumerated as outlined above.

Internal Organs Test. At the end of the prevention study period, the 3 positive control chickens and the 3 chickens that were fed 10 % (w/w) egg yolk powder of each challenged group were euthanized by cervical dislocation and samples of each chicken's intestine, spleen, oviduct, and ovary were collected aseptically. The samples were then minced with scissors and processed to determine the presence and numbers of the challenged organisms per gram of tissue. The method and material used for each organism was the same as outlined above.

Weight Determination. Before and throughout the feeding and infection periods, the body weight of chickens in all groups were measured on day 1 and after 3, 8 and 10 weeks. In addition the average weight and number of eggs laid per chicken were monitored weekly to ascertain if the addition of egg yolk powder to the diet had any effect on growth and egg laying patterns in comparison to that of the two negative control chickens in each challenge test. Statistical Analysis. Statistical analysis of differences between treatment means and that of the control was performed on all data following log₁₀ transformation, using a one-way analysis of variance, ANON A, (SPSS version 8.0 for Microsoft Windows; SPSS, Chicago, IL). Means were separated by Duncan's multiple range test. Statistical significance was set at P < 0.05. Results. As shown in Figures 6-11, non-immunized egg yolk powder contributes significantly the elimination (Figures 6, 8 and 10) and prevention (Figures 7, 9 and 11) of Salmonella typhimurium, Campylobacter jejuni and Escherichia coli 0157:H7 infection in the intestine of laying hens. Birds were infected (at point 'a') with 10⁹ CFU/ ml of Salmonella typhimurium (Figures 6 and 7), Campylobacter jejuni (Figures 8 and 9) or Escherichia coli 0157:H7 (Figures 10 and 11). Supplemented feeding with the various concentrations were commenced at point 'b\ Figures 12, 13 and 14 show the effect of feeding non-immunized egg yolk powder on the weight of laying hens, mean number of eggs laid, and the mean weight of eggs laid, respectively. Positive and negative control groups were fed untreated regular feed.

Figures 15, 16 and 17 demonstrate the effect of feeding non-immunized egg yolk powder on Salmonella typhimurium, Campylobacter jejuni and Escherichia coli 0157:H7, respectively, in the ovary, oviduct, intestine and spleen of laying hens. "After infection" ('a') refers to after birds were infected with 10 CFU/ml of Salmonella typhimurium, Campylobacter jejuni or Escherichia coli 0157:H7. "After feeding" ('b') refers to after the supplemented feeding of 10% egg yolk powder for 4 weeks.

In conclusion, this study demonstrated that the inclusion of non-immunized egg yolk powder in the poultry feed may become a useful tool in controlling Salmonella, Campylobacter jejuni and E. coli 0157:H7 colonization of chickens, and so the consumer's exposure to potential agents of food-borne disease via either the contaminated carcasses or eggs will be diminished. Egg yolk powder can also reduce the possibility of egg contamination by reducing the pathogen colonization in the internal organs; notably the ovary, oviduct and intestine. Furthermore, findings from this and our previous studies suggest that egg yolk possess novel anti-adhesive factors yet to be discovered. Additional studies are being conducted in our laboratory to further investigate the active components in the egg yolk powder and the possible mechanism responsible for this inhibitory effect. It may be either by binding to receptor sites on epithelial cells or by interacting with fimbriae on the bacteria or still another undetermined mechanism.

Example 5

In Vitro Identification of Novel Anti-adhesive Component/s in Non-immunized Egg Yolk Powder Based on the feeding study results of egg yolk powder and egg yolk proteins, a further investigation was needed in order to determine the functional anti-adhesive component/s that was/were capable of eliminating or preventing Salmonella spp. and the other pathogens from colonizing the intestinal tracts of poultry.

An in vitro Caco-2 epithelial cell line was used. Since the egg yolk powder showed the highest effect in the elimination/prevention study of Salmonella spp. and the other pathogens, its various components were separated and used in the model study.

Bacterial Strains The isolates used for this assay were the same as before except for Campylobacter jejuni that was eliminated from this study because its microaerophilic requirements made it difficult to control in such assays. All inocula were prepared from overnight cultures, then serially diluted to the specified viable cell concentration of 10⁵ CFU/ml. Inocula were estimated by spectrophotometry, optical density at 660nm, and confirmed by colony counts on selective agar plates. 100 μl of the challenge inocula and 100 μl of sterile broth as control, were used in the assays.

Test Samples The phosvitin and the HDL of the granules and the low density lipoprotein (LDL) fraction and livetins (IgY) of the plasma were separated and purified. All prepared fractions were analyzed for protein concentration by the Lowry assay method (Bio.Rad) and by SDS-PAGE with 12.5% polyacrylamide gel. The egg yolk components used in the assays, in addition to non-immunized egg yolk were, plasma, granule, IgY, HDL, phosvitin, LDL, and whey proteins (control) The amount used of each was adjusted based on the protein concentration in order to have a final concentration of 0.5% (w/w) in the assay. All samples were subjected to pepsin and trypsin digestion to mimic the gastrointestinal tract conditions; and so 0.15N HC1 + 0.03M NaCl buffer, which was used in the pepsin digestion, was also used as another control.

Experimental Design. Confluent monolayered intestinal cells grown on 24-well culture plates were used in the model assay. The growth media (DMEM + 20% FCS) was replaced with 1.0 ml of the same media but without antibiotics and then stabilized at 37° C in 5 % CO₂.

In the elimination model assay, 100 μl of 10⁵ CFU/ml of either Salmonella enteritidis, Salmonella typhimurium, ox Escherishia coli 0157:H7 were added to the Caco- 2 monolayer cells. After incubation for lhr, 100 μl of the various samples were added. Two wells per sample were used.

In the prevention model assay, on the other hand, the epithelial cells were mixed with 100 μl of the various samples first then after incubation they were inoculated with 100 μl of 10⁵ CFU/ml of either one of the organisms used previously. The effect of each of the components on the colonization of the various pathogens was determined after the cell wells were washed with 1.0 ml PBS for 3-4 times to remove any unattached organisms, then harvested with 0.1% Triton X-100 in 1.0 ml PBS. Serial dilutions were made and 100 μl of each were selectively plated and representative colonies were identified and enumerated.

Anti-adhesive Versus Anti-microbial effect Assay. This was to confirm that the elimination and/or preventive effect of the egg yolk components against the various enteropathogens was an anti-adhesive rather than an anti-microbial one. Therefore, this was to determine that the functional activity of the hen-egg yolk components was actually by blocking or inhibiting bacterial adhesion to the epithelial cells, which is the prerequisite step for infection, rather than by exhibiting a bacteriostatic or a bactericidal effect.

In this assay, the same procedure and conditions as above were followed except that the test samples were mixed with the bacterial strains without using the epithelial cell line.

Statistical Analysis. Statistical analysis of differences between treatment means and that of the control was performed on all data following log₁₀ transformation, using a one-way analysis of variance, ANON A, (SPSS version 8.0 for Microsoft Windows; SPSS, Chicago, IL). Means were separated by Duncan's multiple range test. Statistical significance was set at P < 0.05.

Identification of Novel Anti-adhesive Component/s in Non-immunized Egg Yolk Powder. Results from this assay indicate that the novel anti-adhesive factor in the non- immunized egg yolk powder is the granule fraction and notably the HDL component of the granule. These two fractions exhibited the most reduction in the various enteropathgen assays, but the most effect was observed against Salmonella enteritidis (Tables 2 & 3). This organism's adhesion was reduced two log cycles when compared with the positive control by the granule and HDL, pepsin and trypsin digested. The IgY, which was pointed out by many other researchers as the active component, in our study it only showed a reduction effect when it was not subjected to pepsin or trypin digestion, after being digested no significant effect was noted. The whey protein and the Buffer A (low pH) that were used as controls did not exert any anti-adhesive effect, this supports our hypothesis that the granule fraction of the egg yolk does have a specific anti-adhesive mechanism against Salmonella spp. which is not achieved by any other protein nor effected by other chemicals used in the procedure.

* Values of significant reduction (P< 0.005) in Mean log₁₀ Salmonella^ compared with the positive control of each study as illustrated within columns

-¹- Mean + Standard Deviation of 12 trials ^a 0.15N HC1 + 0.03M NaCl buffer used in the pepsin digestion ^b Elimination Study ^: Inoculate with 10⁵CFU/ml of Salmonella ^SPP ^men treat with the samples ^c Prevention Study" Treat with the samples then inoculate with 10⁵CFU/ml of Salmonella ^SPP- ^d Anti-microbial study ^: Samples + 10⁵ CFU/ml of Salmonella ^SPP- without CacoS cells

0.5% HDL Trypsin 2.64 + 0.41 ^» 2.92 + 0.06* 4.07 + 0.18

* Values of significant reduction (P< 0.05) in Mean log₁₀ E_{, o}ulrcλ compared with the positive control of each study as illustrated within columns

¹ Mean ± Standard Deviation of 12 trials ^a 0.15N HC1 + 0.03M NaCl buffer used in the pepsin digestion ^b Elimination Study ^: Inoculate with 10⁵CFU/ml of E.coli ^men reat with the samples ^c Prevention Study ^{: :} Treat with the samples then inoculate with 10⁵CFU/ml of . _cou. ^d Anti-microbial study^: Samples + 10⁵ CFU/ml of E.coli- without Caco-2 cells

Furthermore, this anti-adhesive vs. anti-microbial assay illustrates that the observed effect may be attributable to anti-adhesive/anti-infectious causes, in which the organism's attachment to the epithelial cells is being prevented in the infection process. It was observed that none of the protein fractions had any reduction or inhibitory effect on the bacterial counts, when compared with the positive control, in the absence of the epithelial cells for attachment. Therefore, the egg yolk granule fraction does not appear to be antimicrobial in a generalized sense, but rather illustrates an anti-adhesive mechanism that may be responsible for the prevention or treatment of pathogen infection. In conclusion, these findings demonstrate that the inclusion of dried non- immunized egg yolk powder in the poultry feed at relatively low concentrations can help to control Salmonella spp. Campylobacter jejuni and E. coli 0157.H7 colonization in the intestinal tract of laying hens. Our results also indicate that the administration of egg yolk powder does not disrupt the intestinal microflora balance, but can reduce egg contamination by reducing pathogen colonization in the internal organs; notably the ovary, oviduct and intestine. Therefore, this method has utility as a practical intervention method for reducing the human exposure to agents of food-borne disease via either the contaminated carcasses or eggs. Furthermore, results from the in vitro assay illustrate that the egg yolk's granule fraction and its HDL component possess anti-infectious/anti- adhesive charactaristics. These non-immunolglobulin constituents of egg yolk provide one explanation of a mechanism behind the protective effects observed in the laying hens against food-borne pathogens. The above-described embodiments of the present invention are intended to be examples only. Alterations, modifications and variations may be effected to the particular embodiments by those of skill in the art without departing from the scope of the invention, which is defined solely by the claims appended hereto.

Claims

CLAIMS:

1. Use of yolk, yolk protein or egg white fractions for prevention or treatment of pathogenic bacterial infection in poultry or eggs.

2. The use of claim 1 wherein said pathogenic bacterial infection comprises an enteropathogenic bacterial infection.

3. The use of claim 1 wherein said pathogenic bacterial infection comprises infection by Salmonella spp.

4. The use according to claim 1, wherein said pathogenic bacterial infection comprises infection by a pathogen selected from the group consisting of: Salmonella enteritidis., Salmonella typhimurium, Camplyobacter jejuni and Escherichia coli 0157.H7.

5. The use according to claim 1 wherein the yolk, yolk protein or egg white fraction is supplemented in the diet of poultry at a level of up to 15%.

6. A method of preventing or treating pathogenic bacterial infection in poultry or eggs comprising the step of feeding a diet supplemented with yolk, yolk protein, or egg white fractions to poultry in need thereof.

7. The method of claim 6 wherein said pathogenic bacterial infection comprises an enteropathogenic bacterial infection.

8. The method of claim 6 wherein said pathogenic bacterial infection comprises infection by Salmonella spp.

9. The method according to claim 6, wherein said pathogenic bacterial infection comprises infection by a pathogen selected from the group consisting of: Salmonella enteritidis., Salmonella typhimurium, Camplyobacter jejuni and Escherichia coli 0157:H7.

10. The method according to claim 6 wherein the diet is supplemented with yolk, yolk protein, or egg white fractions at a level of up to 15%.