The Use of Egg-Yolk Derived Antibodies (IGY) Against Campylobacter Jejuni Colonization Factors to Clear C. Jejuni from the Intestinal Tract of Broiler Chickens

NON-TECHNICAL SUMMARY: Campylobacter jejuni is one of the leading cause of food-borne gastroenteritis in humans. Poultry is the major reservoir host where C. jejuni can be found at concentrations as high as 108 colony forming unts (cfu) per gram of feces. Epidemiological studies have shown that 50 to 70% cases of human campylobacteriosis are caused due to consumption of raw and undercooked poultry meat. On-farm intervention measures such as brief period of feed withdrawal immediately prior to slaughter, competitive exclusion, vaccination and rearing of C. jejuni free chickens have shown limited success in the control of C. jejuni in broiler chickens. Interventions at the poultry meat processing plants such as rapid chilling of carcass can effectively reduce C. jejuni counts by 3 to 4 logs. However, due to the high initial contamination rates of carcass, a substantial portion (as much as 60% to 80%) of frozen and fresh retail chicken in the U.S.A. remain contaminated with C. jejuni with counts approaching 104 and 106 cfu, respectively. Given the very low infectious dose (100 to 500 cfu) for humans, further reduction or complete elimination of poultry contamination by C. jejuni will greatly decrease the risk of campylobacteriosis for public health. Passive immunization by oral administration of pathogen-specific hen egg yolk derived antibodies (IgY) should provide a useful and attractive alternative for on-farm (pre-harvest) control C. jejuni infection in broiler chickens. In the last decade, several colonization associated proteins (CAPs) of C. jejuni have been identified. We hypothesize that the chicken egg yolk derived antibodies (IgY) against Campylobacter jejuni colonization associated protein(s) will eliminate or reduce the intestinal colonization of C. jejuni in broiler chickens. I propose to test this hypothesis with the following specific aims: 1. Produce recombinant colonization associated proteins (CAPs) of C. jejuni. 2. Produce chicken egg yolk derived anti-C. jejuni CAP specific antibodies (IgY). 3. Evaluate the in vivo efficacy of egg yolk derived anti-C. jejuni CAP specific antibodies (IgY). All the C. jejuni CAPs are exposed on the cell surface of the bacterium. Our expectation is that IgY will bind to these C. jejuni CAPs and will either eliminate or significantly reduce C. jejuni loads in the intestine by inhibiting motility, attachment, and/or efficient uptake of nutrients and decrease the ability of C. jejuni to effectively compete with native microbial flora. The production and cost of egg yolk derived IgY is much less expensive and labor intensive than traditional antibody production practices. To our knowledge this is the first time anyone has proposed to test the efficacy of chicken egg yolk derived antibodies (IgY) against C. jejuni CAPs to reduce or eliminate the intestinal colonization of C. jejuni in broiler chickens immediately prior to slaughter. If successful, this study will identify a novel, economic and useful intervention tool to reduce the burden of C. jejuni in commercial broiler production settings.

<P>APPROACH: 1. Nine recombinant CAPs (His-tagged), most of which are known to play a role in the intestinal colonization of C. jejuni in chickens will be expressed in E. coli expression host and purified using affinity column chromatography. <P>
2. Approximately 100 ug of each purified protein will be mixed with equal volume of either Freund's complete (for 1st immunization) or incomplete adjuvant (subsequent booster immunizations). For each recombinant protein, two C. jejuni free (SPF) hens will be immunized four times (1 day, 2wk, 4 wk and 6 wk) intramuscularly at four sites in the breast muscle. Eggs will be collected from each hen at day 0 (before immunization) and every-day from 1 wk after 1st immunization up to 3 wk after final injection.<P>
3. Egg-yolks from each egg will be aseptically removed from albumen and freeze-dried to generate IgY (egg powder) as previously described and stored at 4-8C until further use. Eggs will be collected concurrently from non-immunized hens and processed identically for use as a negative control. The antibody response in hyperimmunized eggs will be determined by ELISA. We expect that the egg-yolks obtained from hens receiving at least three injections will have highest titer of specific antibodies against CAPs. We expect to collect a total of 50 to 60 large size eggs (60 g/egg) from each group of immunized hens. This will be sufficient to produce egg-yolk powder for a week long feeding of a group of seven 6 week-old chickens at the rate of 2.5% (wt/wt) of feed. <P>
4. 84 broiler chickens will be obtained from commercial source and randomly assigned to 12 groups (n=7 per group) in environmentally controlled isolator cages. Birds will be fed standard laboratory diet. Cloacal swabs and samples will be tested for C. jejuni at the day of arrival and before the day of infection as per the previously described procedures. At 2 wk, chickens in 11 groups will be orally inoculated with 10E8 cfu of wild-type C. jejuni strain whereas one group will serve as uninfected control. Three days post inoculation; infection will be confirmed by testing cloacal swab specimens and fecal samples to monitor bacterial level. Once the level in feces reach to 10E7 to 10E8 cfu/g, the birds will be monitored on weekly basis for the bacterial levels. <P>
At the beginning of week 6, two groups (uninfected and infected controls) will be fed standard laboratory diet, one group will be fed standard laboratory diet supplemented with 2.5% (wt/wt) egg-yolk powder derived from non-immunized hens and 9 groups will be fed with laboratory diet supplemented with 2.5% (wt/wt) egg-yolk powder derived from hens immunized with specific CAPs. This treatment will continue for 1wk during which the bacterial levels in feces will be monitored on a daily basis. At the end of 6 weeks (42 days of age), all chickens will be euthanized and the intestinal (caeca and crop) material will be processed for isolation and enumeration of C. jejuni. <P>
We expect that the treatment with egg-yolk powder containing anti-C. jejuni CAP specific antibodies will lead to either elimination or reduction of C. jejuni counts in infected chickens by at 4 to 5 logs immediately prior to slaughter.