DEVELOPMENT AND TESTING OF SIMPLE (CRUDE) VACCINES AGAINST NECROTIC ENTERITIS OF CHICKENS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0223388
• Project Start Date: Oct 1, 2010
• Project End Date: Sep 30, 2012
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIV OF CONNECTICUT
438 WHITNEY RD EXTENSION UNIT 1133
STORRS,CT 06269

Performing Department
Pathobiology & Veterinary Science

Non Technical Summary
Necrotizing (necrotic) enteritis (NE) of chickens and turkeys which is caused by the bacterium C. perfringens, is a serious and often fatal disease. For many years, the disease has been well controlled, by the use of antibiotic "growth promoters" (AGPs) incorporated into feedstuffs. In spite of this, NE is currently estimated to cost the world poultry industry $2 billion/year, and in 2000 it was estimated to cost $0.05 for every broiler chicken raised in the US. Given a production rate of 8.9 billion broilers in 2007 for example (USDA/NASS), this represents losses of the order of $445 million annually to the US broiler industry. Furthermore, due to concerns about possible development of antibiotic resistance among bacteria that are pathogenic to humans, the use of AGPs to control NE (and other diseases) has been heavily criticized and is discouraged by the WHO. The use of AGPs has been banned by many countries, including all countries in the European Union. Their use is under threat in other countries including the US, not least because of consumer driven market pressures. The incidence of NE has risen in countries (and in US farms) which do not use AGPs, leading to animal suffering and serious economic losses. There is therefore a clear need to find alternative strategies to prevent the disease. Vaccination is one such strategy. Simple toxoid vaccines containing multiple toxins have been used very successfully in control of clostridial enteric disease in cattle, sheep and pigs but surprisingly such simple approaches have not yet been evaluated for control of NE in chickens. The current project investigates the ability of a toxoid containing all secreted proteins, either alone or in combination with a bacterin, to protect against NE, using a disease model for clinical necrotic enteritis in chickens. All secreted proteins will be used because studies to date have shown that at least 5 secreted proteins produce partial protection, and there are many more proteins which have not yet been evaluated for protective effect. The inclusion of all proteins will give an important indication as to whether the proteins needed for complete protection all lie within the secretome, and will provide important preliminary information about the potential to protect chickens against necrotic enteritis by use of non-purified toxoid vaccines of the type used successfully in other species. Inclusion of a bacterin will provide valuable first information about the role of bacterial cell surface protein in causation of disease. Since strain diversity often results in failure of vaccines prepared from one strain of a microorganism to protect against infection by another strain, heterologous challenge studies will also be performed. This will provide a preliminary indication as to whether strain diversity is likely to pose problems when attempting to protect against NE by vaccination.

Animal Health Component

10%

Research Effort Categories

Basic

30%

Applied

10%

Developmental

60%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3220	1100	10%
315	3220	1100	40%
315	3220	1160	10%
315	4010	1100	30%
315	4010	1160	10%

Knowledge Area
311 - Animal Diseases; 315 - Animal Welfare/Well-Being and Protection;

Subject Of Investigation
3220 - Meat-type chicken, live animal; 4010 - Bacteria;

Field Of Science
1160 - Pathology; 1100 - Bacteriology;

Keywords

clostridium perfringens

necrotic enteritis

chickens

poultry

vaccination

Goals / Objectives
Necrotic enteritis (NE) is a serious disease affecting the small intestine of chickens and turkeys. In very severe flock outbreaks, mortality rate has been as high as 50%. The liver may also be affected (cholangiohepatitis), usually subclinically, which often results in carcass condemnation in the processing plant. NE is caused by the bacterium Clostridium perfringens. The disease has been reasonably well controlled for many years by the use of the so-called antibiotic growth promoters. However, use of these products is now banned in many countries, and their use is under threat in other countries, including in the USA. Alternative strategies are needed to prevent the disease. Vaccination is one such strategy. Currently there is a commercially available vaccine, which is directed against the alpha toxin of C. perfringens. However, the literature indicates that vaccines against alpha toxin are only partially protective, and indeed alpha toxin negative mutants are capable of producing disease. In 2008, a new toxin, NetB, was identified in C. perfringens. The discoverers of NetB suggested that this new toxin is the factor responsible for disease development. However, work by the PI indicates that while NetB is very important, other factors are also involved and other workers have shown that some secreted proteins other than alpha toxin or NetB can induce partial protection. The current project will investigate the ability of a toxoid vaccine that contains all secreted toxins, to protect against NE, using a disease model for clinical necrotic enteritis in chickens. It will also examine whether or not the addition of a bacterin will enhance the protection provided. To examine the effect of diversity among disease producing strains of C. perfringens on protective capability of a vaccine, both homologous and heterologous challenge studies are planned. Thus, the specific objectives are 1. To test the protective effect of a multivalent toxoid vaccine, with or without bacterin, against necrotic enteritis of chickens 2. To test the best performing vaccination regime determined by Objective 1, against necrotic enteritis caused by heterologous strains of C. perfringens. Objective one will be performed in year 1, and objective in year 2 of the project. Activities and outputs will include preparing the simple vaccines and testing these for efficacy in animal disease challenge experiments, analyzing data and publishing the work in a peer reviewed journal. It will also provide training and research experience leading to completion of a Masters project for a graduate student, and research work experience opportunities for at least 2 undergraduate students.

Project Methods
For objective 1, a NetB positive strain of C. perfringens type A that is known to consistently produce clinical necrotic enteritis will be used to prepare both a toxoid vaccine and vaccine containing both toxoid and bacterin. Bacteria will be grown overnight in protein free media and bacteria will be pelleted by centrifugation and supernatants will be tested to confirm the production of exotoxins. The supernatant will be concentrated by centrifugation through 3KDa filters, and protein concentration determined spectrophotometrically. The concentrated toxins will be inactivated using formaldehyde using previously published methods, to form toxoid. The preparation will be tested for inactivation of toxins as previously described. The toxoid will be combined with Quil A as an adjuvant. A combined bacterin-toxoid vaccine will be made by re-suspending the bacterial pellet obtained by the earlier described centrifugation step, in the concentrated supernatant prepared as described above. The bacteria-toxin mixture will be inactivated by formaldehyde treatment as described above and formulated into a vaccine by addition of Quil A as before. The vaccine will be tested for the absence of viable bacteria by plating on blood agar. Absence of toxic activity will be assayed as described above for the toxoid vaccine. The vaccines will be tested for efficacy in a NE disease challenge model in chickens. An experiment will be conducted with 7 groups of birds. Chickens in Groups 2- 6 will be vaccinated twice (14 days apart). Group 1 will be given adjuvant only. Group 2 will receive low dose toxoid vaccine. Group 3 will receive low dose toxoid - bacterin vaccine. Group 4 will receive low dose toxoid vaccine followed by low dose of non-inactivated supernatant. Group 5 will receive high dose toxoid vaccine. Group 6 will receive high dose toxoid - bacterin vaccine. Group 1 will receive adjuvant only. One week after the booster vaccination, Groups 1-6 will be exposed to an NE disease challenge system. The NetB positive C. perfringens type A isolate that was used to make the vaccine (Strain 1) will be used to challenge the chickens. Group 7 will serve as negative controls (non vaccinated, non challenged). Chickens will be monitored before and after challenge for any signs of illness. When Group 1 chickens (adjuvant only) begin to show signs of illness, all chickens will be euthanized, and necropsied to look for necrotic lesions in the small intestine. A lesion score will be assigned. Semiquantive measurement of C. perfringens numbers in the intestine will also be made. Lesion scores will be compared between groups as indicators of vaccine efficacy. Numbers of C. perfringens recovered from the small intestine of chickens in each group will also be compared. For the second objective, the most successful vaccine formulation and regime identified in Experiment 1 will be adopted and tested against homologous and 3 heterologous challenge strains during 2 experiments. A Masters student will conduct much of the work assisted and supervised by the PI. Undergraduate students will assist with experiments and at necropsies, providing practical research experience.

Progress 10/01/10 to 09/30/12

Outputs
OUTPUTS: This 2 year project had 2 objectives. The Year 1 objective was to test the protective effect of a multivalent toxoid vaccine, with or without bacterin, against necrotic enteritis of chickens. The year 2 objective was to test the best performing vaccination regime as determine in objective 1 against necrotic enteritis caused by heterologous strains of C. perfringens. However, since the first vaccine constructs did not provide an encouraging level of protection, the contingency plan Objective 2 outlined in the original project proposal (to examine different adjuvants and doses) was carried out. Multiple vaccine formulations containing different amounts of antigen and 3 different adjuvants were prepared. One vaccine was given by the oral route and the rest were given parenterally (subcutaneous or intramuscular). These were tested in 2 animal experiments involving 244 chickens. Each experiment required the obtaining of day-old birds which were brooded and reared for an average of 5 weeks they were vaccinated twice 14 days apart and then challenged in a disease model by experimental infections. Outcomes were determined by performing clinical, gross pathological, and bacteriological examinations on all chickens. Blood samples were collected from all chickens at 3 time points. Selected samples were also collected and processed for histological examination. Two Masters students worked on this project and one has graduated. Some of the work was presented at Graduate Seminar series. Nine undergraduate students have been given extensive experiential learning opportunities working with the PI on this project PARTICIPANTS: Joan A Smyth (PI) designed vaccines, prepared the second batch of vaccines, designed experiments, performed clinical and pathological examinations on all experimental birds, collected some blood samples, interpreted and analyzed data and supervised the work of all students. Alicia Sobczynski (Graduate Assistant), prepared the vaccines and infectious inocula for experimental birds of Objective 1, assisted in the set-up and execution the experiment, did all bacterial cultures on the experimental birds, looked after birds once infections commenced, oversaw the experimental data logs. Masters student 2 (Abigail Brodsky) was trained in processes but left the Masters program and the project on grounds of ill health. She is now a lab technician. Nine undergraduate students were involved in the project over the course of the 2 years. They gained experience in multiple or all of the following: rearing, management and handing of chickens, injecting vaccines, collecting blood samples, serum separation, bacterial growth and recovery, preparing samples for histological examination, determining protein concentrations, assisting at necropsy examinations performed by a certified pathologist, ELISA development and testing, data management and record keeping, biological and chemical health and safety TARGET AUDIENCES: Scientific researchers and wider community will be targeted through eventual publication of the findings. Students have been educated through participation in the work program. Laboratory and practicum experiences were provided to 2 graduate students and 9 undergraduate students during the course of this project. PROJECT MODIFICATIONS: Since the first vaccine constructs did not provide an encouraging level of protection, the original objective 2 which was to test the best performing vaccination regime determined by Objective 1, against necrotic enteritis caused by heterologous strains of C. perfringens, the contingency plan Objective 2 outlined in the original project proposal (to examine different adjuvants and doses) was executed in year 2.

Impacts
For Objective 1, preparation of toxoid and bacterin was completed, birds vaccinated and challenged. However, none of the vaccines produced satisfactory level of protection as determined by lesion scoring at necropsy. This led to a change from the original Objective 2 experiment to the contingency Objective 2. For the new Objective 2 experiment, different adjuvants were used, and one group was also inoculated by the oral route with an oral adjuvant. In some test groups of this second experiment, some but not all chickens were completely protected based on gross and histological examination. Two graduate students and 9 undergraduate students had extensive experiential learning by working closely with the PI on the project for between 1 and 4 semesters. Both graduate students are now working as lab technicians, one of these in a veterinary diagnostic laboratory. The undergraduate students gained experience in rearing, management and handing of chickens, injecting vaccines, collecting blood samples, serum separation, bacterial growth and recovery, preparing samples for histological examination, determining protein concentrations, assisting at necropsy examinations performed by a certified pathologist, data management and record keeping, biological and chemical health and safety. Two of these students will enter vet school in Fall, one is farming part time, the others continue their undergraduate studies.

Publications

• No publications reported this period

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: This 2 year project has 2 objectives. The Year 1 objective was to test the protective effect of a multivalent toxoid vaccine, with or without bacterin, against necrotic enteritis of chickens. The Year 2 objective was to test the best performing vaccination regime as determine in objective 1 against necrotic enteritis caused by heterologous strains of C. perfringens. The year 1 objective has been accomplished by the following activities. A C. perfringens type A strain known to consistently produce clinical necrotic enteritis was grown in a protein free medium. Bacteria were pelleted by centrifugation and inactivated by treatment with formaldehyde to create bacterin. Inactivation was confirmed by culture of the treated cells. Supernatants were filtered, concentrated , and inactivated using formaldehyde to create toxoid. Inactivation was confirmed by examining for loss of hemolytic activity. 125 day-old chickens were obtained and divided into 7 groups. Two groups of birds were vaccinated twice each with toxoid (50 or 200ug of protein per dose respectively) combined with Quil A as an adjuvant. Two groups were vaccinated with the same doses of toxoid but combined with bacterin. One group received a priming does of toxoid but a booster of filtered non-formaldehyde treated supernatant. A further group served as a positive control for the disease challenge system and the remaining group as a negative control. Birds were blood sampled at 14 days post first vaccine dose, and 7 days post the second vaccination, which was just prior to disease challenge. Disease challenge took place over the next 5 days. Birds were then observed for onset of disease, then subjected to necropsy and lesion scoring. Results were analyzed. An ELISA test was developed for detection of antibodies to alphatoxin, and blood samples were tested for antibodies to alphatoxin. A graduate student and 5 undergraduate students were trained in multiple laboratory techniques and experimental animal manipulations. PARTICIPANTS: The PI closely supervised a graduate student in the preparation of the vaccines, and actively carried out (and trained others in) vaccinations, bleedings and experimental vaccinations of birds. The PI, a pathologist, performed all necropsy examinations. A Graduate student (Masters) prepared the vaccines under supervision of the PI, prepared inocula for the disease challenge, worked with undergraduate students to set up experimental accommodation and care for experimental animals, and assisted the PI at necropsies. Undergraduate students gained experience by working closely with the PI, in experimental design, preparation of inocula, handling of chickens for tagging, weighing, intramuscular injections and bleeding, as well as assisting the PI at necropsies, data recording and data management. TARGET AUDIENCES: The work in this project will be of benefit to the poultry industry, and is of interest to scientists studying the disease necrotic enteritis. The work has provided substantial learning opportunities for a graduate student and 5 undergraduate students to date through laboratory instruction and practicum experience. PROJECT MODIFICATIONS: Since the first vaccine constructs did not provide an encouraging level of protection, the original objective 2 which was to test the best performing vaccination regime determined by Objective 1, against necrotic enteritis caused by heterologous strains of C. perfringens, the contingency plan Objective 2 outlined in the original project proposal (to examine different adjuvants and doses) will be executed.

Impacts
Preparation of toxoid and bacterin was completed, birds vaccinated and challenged. However, none of the vaccines produced satisfactory level of protection as determined by lesion scoring at necropsy. ELISA testing revealed an antibody response in response to vaccination, but in lower dose groups, response was low. A graduate student and 5 undergraduate students had extensive experiential learning by working closely with the PI on the project for 1 to 2 semesters.

Publications

• No publications reported this period