Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001
Performing Department
Poultry Science
Non Technical Summary
Necrotic enteritis in broilers due to Clostridium perfringens results in almost 30% mortality, costing the poultry industry upwards of US $2 billion annually. There is an urgent need for strategies other than antimicrobial growth promoters that is currently used to control necrotic enteritis. The goal is to develop efficacious immuno-modulators using E-Beam to protect against Cl. perfringens infections. Vaccination of broiler chickens with a cocktail of Electron Beam (E-Beam) inactivated Cl. perfringens strains is hypothesized to protect broiler chickens from necrotic enteritis. The specific objectives are (1) Use E-beam technology to develop a cocktail of killed Cl. perfringens strains that have effective immuno-modulation potential. (2) Conduct bird challenge studies to determine whether vaccination with the E-Beam inactivated strains would protect birds from necrotic enteritis. (3) Conduct bird challenge studies to determine whether vaccination with a cocktail of E-Beam inactivated strain would cross-protect against necrotic enteritis. The outcome of this project will be a novel vaccine to control necrotic enteritis in broiler chickens.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The long-term goal of this project is to control/prevent necrotic enteritis in poultry and it is within the scope of the Formula Animal Health Funds-section 1433, which includes improving poultry health and preventing poultry diseases. The objective of the proposed research is to use electron beam (E-beam) technology to develop a suite of killed Clostridium perfringens strains that could be used as immuno-modulators and to test the efficacy of these E-Beam inactivated strains both individually and as multiple strain cocktails in bird challenge studies. The central hypothesis of the proposed research is that vaccination of broiler chickens with a cocktail of E-beam-inactivated Cl. perfringens strains will protect the chickens from necrotic enteritis. The following specific objectives will be pursued to test our hypothesis: (1) Develop a cocktail of E-Beam inactivated Cl. perfringens strains that have effective immuno-modulation potential. (2) Conduct bird challenge studies to determine whether vaccination with the E-Beam inactivated strains would protect birds from necrotic enteritis. (3) Conduct bird challenge studies to determine whether vaccination with a cocktail of E-Beam inactivated strain would cross-protect against necrotic enteritis
Project Methods
Vaccine preparation: Cl. perfringens cultures will be pooled in approximately equal proportion (~10^8) and exposed to defined E-Beam doses. To find out the efficacy of individual strains verses multiple strains, individual strains at a concentration of ~10^8 will also be irradiated at defined dose. E-Beam irradiation will be performed at the E-Beam Center on campus. The cells will be diluted to ~ 1x10^4 CFU/embryo, and stored at 4 degree C until administered. Embryo Vaccination: The E-beam-inactivated Cl. perfringens vaccine will be delivered by in ovo injection on day 18 embryos to simulate commercial hatchery injection practices. Either E-beam inactivated Cl. perfringens alone, or CpG ODN alone, or E-beam inactivated Cl. perfringens combined with and CpG ODN will be administered in physiologic saline, into the amnion using a 1 cc tuberculin syringe and a 25-gauge needle equipped with a modified needle guard to limit all injections to a depth of 3 cm. Following treatment injection to individual embryos, injection sites on all eggs will be covered with melted paraffin using a cotton swab. Upon hatch, experimental and control chickens will be placed in individual pens in separate rooms. Bird Challenge Studies: Chickens used as negative control will be given fluid thioglycollate (FTG) medium via oral gavage from day 17, twice daily for three consecutive days. Vaccinated chickens will be challenged with a virulent strain A8854-11-1 by oral gavage with 10^7 CFU twice daily from day 17 for three consecutive days. Serology and Colonization Assays Serum samples will be collected from both experimental and control groups before challenge (on day 15). On day 25 (post-challenge), serum will be collected from both experimental and control groups. The serum will be used for ELISA assays for antibody titers. We will also verify the extent of Cl. perfringens colonization in the vaccinated and non-vaccinated birds. Briefly, a 6 inch section of the small intestine cranial to Meckel's diverticulum will be removed. The sample will be placed in 10 mL of anaerobic FTG, stomached, and 0.5 mL of gut contents will be removed and placed into 4.5 mL of thioglycollate medium. Serial dilutions will be made and dilutions plated on thioglycollate agar and incubated anaerobically at 37 degree C for 24 h. Colonies exhibiting typical colony morphology will be counted and used in data analysis. Examination of NE lesions: Birds will be examined for gross lesions associated with NE and lesion scores will be recorded. The feed conversion rate, mortality rate will be assessed and then all chickens will be euthanized. Data Analysis: Mortality and log10 values of vaccinated and the non-vaccinated will be subjected to ANOVA. Nonparametric analysis will be performed by ranking the scores, applying the mean to ties, and running an ANOVA on the ranks, allowing the treatment groups to be compared by the mean ranks. Expected Results: E-Beam inactivated strains will stimulate the immune system and protect broilers against NE. In ovo vaccination will provide protection against NE and therefore would be an efficacious method of vaccination since birds are protected from day1.