Masters Theses

Date of Award

12-2013

Degree Type

Thesis

Degree Name

Master of Science

Major

Animal Science

Major Professor

Jun Lin

Committee Members

Irene B. Hanning, Raul A. Almeida

Abstract

Campylobacter is the leading bacterial cause of human enteritis in developed countries. Human campylobacteriosis is commonly associated with consumption of undercooked, contaminated chicken, a natural host of Campylobacter. Thus, control of Campylobacter colonization in poultry at the farm level would reduce the risk of human exposure to this pathogen. Vaccination is an attractive intervention measure to mitigate Campylobacter in poultry. Our recent studies have demonstrated that the outer membrane proteins CmeC (an essential component of CmeABC multidrug efflux pump) and CfrA (ferric enterobactin receptor) are feasible candidates for immune intervention against Campylobacter. By targeting these two promising vaccine candidates, three novel vaccines were developed for different vaccination strategies in this study. To construct DNA vaccines for in ovo and intranasal immunization, cfrA and cmeC genes were cloned into the eukaryotic expression vector pCAGGS; sequencing of the recombinant vectors confirmed the success of cloning. Transfection was also performed to determine the production of CfrA or CmeC in NIH 3T3-L1 and HEK-293 cell lines. To develop effective subunit vaccines for intranasal or oral vaccination, purification of recombinant CfrA (rCfrA) and CmeC (rCmeC) was optimized. Substantial quantities of highly purified rCfrA and rCmeC were produced through nickel-nitrilotriacetic acid (Ni- NTA) affinity chromatography. The purified rCfrA and rCmeC were further encapsulated into chitosan microsphere. Various encapsulation conditions were explored. To construct the attenuated Salmonella-vectored vaccine, cfrA and cmeC genes were cloned into vector pYA3493 and transferred into S. enterica serovar Typhimurium χ8914 [strain 8914], the USDA licensed live attenuated vaccine strain. The oral live Salmonella vaccines producing CfrA or CmeC (full length or truncated) were successfully constructed; expression of the target protein was confirmed by immunoblotting using specific antiserum. The efficacies of two live vaccines that produce CfrA or CmeC were evaluated using broiler chickens. Specific systemic and intestinal mucosal response was not significantly stimulated upon oral vaccination of chickens with the attenuated Salmonella derivatives. Together, three novel Campylobacter vaccines were developed in this study, which provides us a solid foundation to further develop and evaluate different vaccination regimens for effective mitigation of Campylobacter in poultry in the future.

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