Performing Department
Infectious Diseases and Vaccin
Non Technical Summary
One element of traditional commercial poultry rearing is the addition of sub-therapeutic levels of antibiotics in the feed to promote growth. Due to concerns that this practice contributes to an increase in antibiotic resistant human bacterial pathogens, there is a movement in the US to reduce or eliminate all use of antibiotics in the food chain. Based on experiences in Europe and Scandinavia, one negative outcome of eliminating antibiotic use in poultry is an increase in the incidence of necrotic enteritis caused by the bacterium Clostridium perfringens. Birds with necrotic enteritis display a range of symptoms. Subclinical infections result in decreased digestion and adsorption of food, while more severe, clinical infections lead to a variety of symptoms including severe depression, decreased appetite, reluctance to move, diarrhea and ruffled feathers, often leading to death.Fortunately, as an alternative to antibiotics, necrotic enteritis can be prevented by vaccination. The goal of this project is to develop a low cost, highly efficacious necrotic enteritis vaccine. We will use novel attenuated (i.e. genetically modified to be incapable of causing disease) Salmonella strains to deliver multiple protective C. perfringens antigens. Use of Salmonella for antigen delivery will allow for cost-effective, mass vaccination by coarse spray or in water. As an added safety feature, the Salmonella strain used is designed to "self-destruct" once it has accomplished its objective of inducing a protective immunity against C. perfringens. In addition, we anticipate that birds vaccinated with this necrotic enteritis vaccine will also be immunized against the human pathogen Salmonella, adding an increased level of safety for the consumer.
Animal Health Component
100%
Research Effort Categories
Basic
20%
Applied
60%
Developmental
20%
Goals / Objectives
Our major goal is to develop a vaccine that can be applied in water or by coarse spray, to protect broiler birds against necrotic enteritis. The approach involves the use of novel, attenuated Salmonella Typhimurium strains for antigen delivery, thereby providing immunity against Salmonella as well.WWe will be using a novel S. Typhimurium antigen delivery strain, c11802 ((del)PmurA25::TT araC PBAD murA (del)asdA27::TT araC PBAD c2 (del)(wza-wcaM)-8 (del)pmi-2426 (del)relA198::araC PBAD lacI TT (del)recF126). Relevant phenotypes include lysis in the absence of arabinose and diaminopimelic acid (DAP) and mannose-dependent O-antigen synthesis. In the vaccine (c11802 + C. perfringens antigens), the DAP requirement is complemented by addition of an AsdA+ antigen-encoding plasmid, but the arabinose requirement is not complemented, leading to lysis of the vaccine in vivo.ObjectivesIdentify and clone new C. perfringens antigens, in addition to the ones we have already cloned. Leading candidates include fructose 1,6-biphosphate aldolase (FBA), a hypothetical protein, HP, with proven efficacy, fimbriae and/or adhesins, and peptides derived from the large clostridial toxin, TpeL.Peptide mapping of TpeL to identify immunogenic regions suitable for cloning and expression in the vaccine strain.Construct live attenuated S. Typhimurium strains that produce at least three protective proteins from Clostridium perfringens. If this is not possible, we will construct multiple vaccine strains producing one or two antigens that can be co-administered.Characterize vaccine strains in vitro. This will include demonstrating arabinose-regulated antigen production, mannose-regulated O-antigen synthesis and plasmid stability over 50 generations of bacterial growth. Vaccine lysis in the absence of arabinose will also be tested.Evaluate the immunogenicity of vaccine constructs in broiler birds. This will include evaluations of serum, mucosal and cellular immunity elicited by the vaccines against relevant Clostridial and Salmonella antigens.Evaluation of protection against virulent C. perfringens challenge after vaccination. Primary outputs measured will be survival (though most birds survive challenge) and intestinal lesions.Evaluation of protection against colonization by wild-type S. Typhimurium. This assay will be done with the vaccine strains providing the best protection against C. perfringens challenge.
Project Methods
Candidate Clostridium genes will be synthesized and the genetic code optimized for expression in Salmonella. Gene cloning will be done using standard methods.Arabinose-regulated antigen synthesis will be evaluated by western blot. Plasmid stability is assessed by passaging vaccine strains for 50 generations in LB broth containing diaminopimelic acid (DAP). After the final passage, the strain is plated on medium containing DAP. 50 - 100 colonies are then patched on medium without DAP to determine the percentage of cells that have lost the plasmid (plasmid loss results in DAP-dependency for growth). At least ten to twenty colonies will be evaluated by western blot to confirm antigen expression.For vaccine efficacy trials, two-day old broiler chicks will be randomized and placed in cages with litter. Three randomly selected birds will be euthanized and necropsied to establish Salmonella-free status of the group. Birds will be orally inoculated with candidate vaccine strains, or mock inoculated with phosphate buffered saline. Fourteen days later, birds are orally boosted. During the course of each study, we will evaluate colonization by the Salmonella vaccine, typically ten days after the primary immunization.We will take intestinal scrapings and perform blood draws at various times after the boost to evaluated antibody responses. Antigen-specific IgY (serum and intestinal), IgA and IgM (both intestinal) titers will be determined by enzyme linked immunosorbent assay (ELISA). Spleens will be collected after the boost to determine cellular responses via a splenocyte proliferation assay.Birds will be challenged with virulent C. perfringens. Intestinal lesions will be examined after challenge. Blinded scoring of lesions will be performed using published criteria.The significance of differences in lesion score sums (i.e. rank sums) between treatment groups will be tested using a nonparametric Kruskal-Wallis test. If the overall test is significant, then each treatment group will be compared to the control group using Dunn's tests. Statistical analyses of immune parameters will also be performed using 1 way ANOVA.We plan to test three new antigens, in addition to the two antigens that we already have. One of the antigens, TpeL, is a very large protein not suitable for production in a live Salmonella vaccine. We will first establish that TpeL is a protective antigen by injecting chickens with purified protein. If the birds are protected from challenge with a C. perfringens strain expressing tpeL, we will use sera from protected birds for peptide mapping. We will perform peptide mapping to determine which regions of the protein are most immunogenic. The DNA sequence encoding immunogenic regions of TpeL will be synthesized, cloned, expressed in Salmonella and evaluated for efficacy in chickens.Experiments to determine protective efficacy against S. Typhimurium will be performed with leading candidates. In this case, protection is measured as a statistical decrease in organ colonization by a wild type challenge strain given 14 days after the boost.Our primary goal is to develop a practical oral vaccine against necrotic enteritis for use in the poultry industry. We hope to achieve reproducible, statistically significant protection against both C. perfringens and S. Typhimurium challenge. In the case that the vaccine is not protective against S. Typhimurium challenge, the vaccine will still be useful for protecting against C. perfringens.