Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695
Performing Department
Population Health and Pathobiology
Non Technical Summary
In an effort to stem the growing antibiotics resistance in bacteria, the U.S. Food and Drug Administration instituted new regulations banning, as of January 1, 2017, the use of medically important antibiotics as feed supplements. This has led to an increased incidence of necrotic enteritis (NE) in poultry, particularly in broiler flocks. This necessitates urgent efforts by the research community to develop effective NE control measures and thus, significantly steering towards judicious use of antibiotics and antimicrobial stewardship in poultry production.Necrotic enteritis is a multifactorial disease caused by toxin-producing, anaerobic virulent strains of Clostridium perfringens bacteria. The disease is of utmost importance to the poultry industry as the economic annual losses to the global poultry industry due to NE are estimated to be $6 billion [1]. For many decades, use of sub-therapeutic doses of antimicrobials in feed, as growth promoters in food animal production, had kept NE under control. However, an increased demand for raising poultry without antibiotics has intensified research to find antimicrobial alternatives. Many means of NE control such as the use of acidifiers, plant-based products, prebiotics, probiotics and vaccines have been tested in research settings with varying degree of success. Amongst these tested strategies, vaccination seems to offer an effective antibiotic alternative means of NE control. Significant work carried out previously by the PI and others has shown that the protection against NE in broilers is associated with the virulence capacity of C. perfringens [2]. This work showed that it is possible to protect against NE by immunizing broilers orally using C. perfringens secreted proteins [3, 4]. Therefore, there is a consensus amongst the NE researchers that the secretory protein component of these bacteria has a critical role in immunity to NE in chickens [5]. To this end, one of our recent pilot studies here at CVM investigated the cellular immune responses to C. perfringens secreted proteins in chicken spleen and cecal tonsils. Mononuclear splenocytes and tonsillocytes ex-vivo stimulated with the secreted proteins from C. perfringens isolates that varied in their virulence capacity showed that chickens can mount robust immune responses to C. perfringens antigens, including CD4+, CD8+ T cell and macrophage responses (Kulkarni et al., unpublished). Interestingly, the secreted proteins from the virulent isolates showed better immunostimulatory properties compared to non-virulent controls. This finding suggests that chickens can be immunized using C. perfringens secretory antigens. In commercial poultry setting, the route of delivery is an important aspect of vaccine development. In-ovo vaccination offers many advantages over post-hatch vaccination that include an early induction of immunity, reduction in bird stress, precise and uniform injection and reduced labor costs. The proposed work would undertake an in-ovo vaccination strategy to develop an adjuvanted C. perfringens secreted toxoid protein-based vaccine to induce immunity to NE in chickens.
Animal Health Component
37%
Research Effort Categories
Basic
25%
Applied
37%
Developmental
38%
Goals / Objectives
Hypothesis: C. perfringens secreted proteins can induce protective immune response in chickens.Aims: The overall aim is to develop an in-ovo delivered secretory component-derived toxoid vaccine that generates robust protection against NE in broiler chickens.Specific aims:1.To develop a C. perfringens supernatant toxoid vaccine and assess the safety of in-ovoadministered CpG-ODN adjuvanted vaccine by measuring hatchability.2. To assess the cellular immune responses in newly hatched chicks vaccinated in-ovo withthe adjuvanted toxoid vaccine preparations.3. To test the protective efficacy of in-ovo vaccination against an experimental NE challengein broiler chickens post-hatch and characterize the determinants of protective immunity.
Project Methods
Experiment-1 will involve preparation of toxoid vaccine for in-ovo delivery to optimize a safe andsuitable dose-range. A virulent C. perfringens isolate (PI Lab collection) will be chosen for thisstudy. C. perfringens will be grown and the preparation of supernatant toxoid will be carried outas described by us previously [2, 6]. A range of toxoid doses (5, 10, 25, 50, 75 and 100ug/egg)will be used to inject eggs. CpG-ODN, an adjuvant molecule, shown to possess potentimmunostimulatory activity in chickens [7-9], will be used at a dose of 50ug/egg. Followingadministration at ED18, the safety of the toxoid/ adjuvant will be determined based on the embryoexamination, mortality and hatchability.Based on the experiment-1 findings, Experiment-2 will select 2-3 doses of toxoids for in-ovoimmunization with and without CpG-ODN adjuvant. Spleen, cecal tonsils and bursa of Fabriciuswill be harvested from day-old chicks and we will evaluate cellular responses using immune geneexpression (real-time PCR) [10] and immunophenotyping (flow cytometry) techniques [11]. Thetarget genes chosen for these studies will be cytokines (IL-1b, IL-6, IFNg, TGFb, IL-4 and IL-13)and toll-like receptor (TLR-21). Cells of interest in immunophenotyping will be T cells (CD4+,CD8+ and gamma delta T), B cells and macrophages.Experiment-3 will assess the protective efficacy of vaccines against NE in an in-house establishedexperimental challenge model. Based on the findings of experiment-2, an optimal adjuvantedvaccine dose will be chosen for in-ovo vaccination. The in-ovo vaccinated chicks will be grownto 17 days with weekly body weight gain measurement followed by a challenge for 5 days (d18-22) and protection assessment, as described by us previously [12]. Serum will be collected atweekly intervals for anti-C. perfringens antibody evaluation using ELISA technique. Immune response evaluation to determine determinants of protective immunity will also be performedusing gene expression and immunophenotyping techniques, as stated above.