Proposal: Development of a live-attenuated vaccine for the prevention of histomoniasis

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2 Proposal: Development of a live-attenuated vaccine for the prevention of histomoniasis Submitted to: U.S. Poultry & Egg Association Date Submitted: 28 January 2019 Submitted by: Christine N. Vuong, PhD Research Scientist (Principal Investigator) Billy M. Hargis, PhD & DVM Distinguished Professor (Co-Investigator) University of Arkansas: Division of Agriculture Department of Poultry Science J.K. Skeeles Poultry Health Laboratory 2652 N. McConnell Ave. Fayetteville, AR Total Funds Requested: $76,000 Duration of Project: June 2019 to May 2021 Make check payable to: University of Arkansas c/o Mike Sisco, Grants Officer 1120 West Maple Street Fayetteville, AR Keywords: histomoniasis, blackhead, vaccine, turkeys

3 Abstract Histomoniasis is a protozoal disease, primarily affecting turkeys and sometimes broiler breeders, characterized by the development of target-shaped lesions on the liver and development of cecal cores, eventually causing up to % mortality in some acutely-affected flocks. This disease has become a more prevalent issue in turkey production, and is reportedly more common in broiler breeders during the last decade, particularly after the voluntary removal of the last arsenical feed additive historically used to control histomoniasis infection. The need for an efficacious prophylaxis through vaccines or alternative treatments is urgent. Potential protective immunoprophylaxis against histomoniasis was demonstrated as early as 1963 by Joyner, wherein dimetridazole-rescued turkeys were found to be profoundly immune to subsequent wild-type challenge. Development of an attenuated vaccine strain would provide turkeys protection against histomoniasis, reducing losses caused by disease-induced performance suppression and mortality. Using a passaged Histomonas line developed in our lab which exhibited reduced virulence and partial protection in preliminary trials, the proposed project will undertake final selection of an attenuated passaged line for use as a vaccine candidate, cloning for single-cell stocks and genetic sequencing, and final protective efficacy testing in the field. Attenuation of virulence and protection trials will be completed by direct in vivo testing in turkeys. Critically limiting dilutions will be used to generate single-cell clones for use as a vaccine stock and will be in turn sequenced for vaccine genetic stock records. An approved attenuated histomoniasis vaccine for use in turkeys would both improve animal welfare and reduce economic losses caused by reduced performance or mortalities. 2

4 Development of a live-attenuated vaccine for the prevention of histomoniasis Christine N. Vuong and Billy M. Hargis Objectives 1) Attenuated Line Preliminary Testing: Confirm attenuation of cell culture passaged Histomonas meleagridis protozoa line via in vivo virulence testing in turkeys. Confirmation of protection against wild-type challenge will also be conducted. Preliminary age and route administration trials will be completed. 2) Line Isolation and Genetic Sequencing: The chosen attenuated Histomonas line will be cloned by critically limiting dilutions to create a singular cell origin line for exclusive use as a vaccine strain stock. Virulent wild-type and cloned attenuated strains will be sequenced for comparison of virulence domains and to serve as strain records. Selected Histomonas strains will be used for development of a diagnostic ELISA assay. 3) Vaccine Efficacy Testing: Final single-cloned strains will be tested to confirm attenuation and conferred protection are maintained in vivo against Histomonas wild-type challenge in turkeys. Optimization of route and dosage will be completed with the cloned strain to better suit industry practices. Justification Histomoniasis (also known as blackhead, infectious enterohepatitis, and histomonosis) is a protozoan disease affecting the gastrointestinal tract of turkeys, chickens, and other gallinaceous birds (van der Heijden et al., 2005; Hess et al., 2015). High mortality occurs in turkeys, while less severe damage occurs in chickens and other galliformes (Callait et al., 2002). Severe mortality results in turkeys with an estimated annual loss of over two million dollars and mortality approaching % of the flock (Callait et al., 2002; McDougald, 2005; Hess and McDougald, 2013). Although mortality is not as significant in chickens, economic losses are estimated to exceed those of turkeys due to greater frequency of disease and the larger number of flock infections within chickens (Callait et al., 2002). Chicken mortality can be 10-20% with a high morbidity, while an outbreak might often go unnoticed or result in increased condemnations at the broiler processing plant (McDougald, 2005). Beginning in the 1960s, research of this organism has wavered as the introduction of nitroimidazoles, nitrofurans, and arsenical compounds served as prophylaxis against outbreaks and solved the short term problem (van der Heijden et al., 2005; Hess et al., 2006). Regulatory action caused the removal of these compounds from the market, leaving no alternative treatments for poultry producers (Hu and McDougald, 2004). With this lack of prophylactic and treatment options, histomoniasis outbreaks continue to result in tremendous losses of broiler breeders, layer pullets, and young turkeys (Hu and McDougald, 2004). Finding methods to prevent and treat diseases such as histomoniasis are crucial for the overall well-being of animal husbandry and food production. 3

5 % Mortality % Mortality The research proposed directly addresses USPOULTRY s disease research priority #8 for the development of improved methods and pharmaceuticals for prevention and control of helminths and Histomonas. An efficacious Histomonas vaccine candidate, capable of controlling this disease would have immediate benefits in both improving animal welfare as well as decreasing animal losses during production. These benefits would be cumulative over time, yielding more turkeys reaching market age for processing and consumption. Reducing mortality losses and reducing disease-induced depressed performance would result in increased production efficacy. This translates to less feed/grain wasted, a reduced overall carbon footprint, and better sustainability while generating more animal protein. Procedures Our laboratory has successfully passaged Histomonas meleagridis protozoa collected from cecal contents of an outbreak mortality. This virulent, wild-type Histomonas has been passaged in vitro over 200 times, potentially creating an attenuated strain, which could be used for live vaccination. Initial trials challenged turkeys intracloacally with the virulent wild-type versus an 80-passaged Histomonas line have demonstrated successful attenuation of the line (reporting very low to no liver/cecal lesions in the passaged line compared to the extensive lesions and mortalities induced by the wild-type). When turkeys were vaccinated with the 80-passaged line at d14-of-age, the vaccinated turkeys exhibited a statistically significant reduction in mortalities (no mortalities) compared to the wild-type positive controls as recorded for two weeks postinoculation (see Figure 1a). This demonstrates successful attenuation and reduced virulence of the passaged line for up to two weeks after inoculation. When vaccinated turkeys are subsequently challenged with wild-type Histomonas, 97% of vaccinated turkeys survived to 11 days post-challenge (see Figure 1b). a 35 Post-Vaccination b 25 Challenge Phase * * * Negative Control Vaccine Wild-Type Positive Control Figure 1. Histomoniasis-induced mortalities a) caused by 80-passaged line versus wild-type positive control and b) after challenge with virulent wild-type. * indicates statistical differences based on chi-square test compared to the wild-type positive control group. 5 0 * Negative Control Vaccine Wild-Type Positive Control 4

6 % Liver Lesions % Cecal Lesions Vaccinated turkeys also possessed significantly milder lesions on the liver and ceca at necropsy compared to the wild-type positive challenge control at d11 post-challenge. (Data shown in Figure 2a-b; lesion scores ranged from 0 for normal/healthy to 3 for critically moribund lesions.) A minor amount of lesions were observed in the vaccinated birds d11 post-challenge, suggesting incomplete protection, but more passages of the attenuated line have been produced and have yet to be tested for candidacy. Optimization of dose and age have yet to be established. a 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 1 26 Liver Lesions P< P=< Negative Control Vaccine Wild-Type Positive Control Figure 2. Lesions score distribution d11 post-challenge with virulent wild-type for a) liver and b) ceca. (Score of 0 meaning no lesions/healthy and 3 meaning critically moribund classic lesions.) Both vaccinated and negative control groups exhibited statistically lower liver and cecal lesions compared to the wild-type positive control group. P-values indicated above respectively compared groups. Based on this preliminary data, we hypothesize that the passaged line would be efficacious as a potential live, attenuated vaccine. In Objective 1, trials will be completed using the passaged Histomonas line (currently having reached 200 passages) to screen for further attenuation via in vivo trials. Various candidate lines intended for testing include passage: 100, 150, and 200. Each passage line will be tested by intracloacal inoculation at 14 days-of-age, with necropsies conducted at 2, 3, and 4 weeks post-inoculation to confirm attenuation of disease virulence/safety of passaged Histomonas line. Using the selected passage line as a live vaccine at 14 days-of age, protective efficacy against wild-type challenge will also be completed 2 weeks post-vaccination to confirm maintained protective efficacy. As we have already observed and recorded an adequate level of attenuation and protection from the previously tested 80-passaged line, a high success rate is expected in these further passaged lines. The chosen passage line will immediately proceed to Objective 2, in which critically limiting dilutions will occur to create singular clone vaccine stocks. While the line is being cloned, in vivo trials will be conducted to screen for the effect of various immunization routes (cloacal versus oral) and ages (day-of-hatch versus 14 days-of-age) to optimize both ease of administration and inducible protection at an earlier age. b 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 2 25 Cecal Lesions P< P< Negative Control Vaccine Wild-Type Positive Control

7 In Objective 2, the chosen passaged Histomonas line, along with the virulent wild-type for comparison purposes, will be cloned by critically limiting dilutions to generate single-cell origin stocks. These clones will be used for in vivo confirmation testing in Objective 3, which will occur concurrently with the remaining Objective 2 goals. Clones which maintain reduced virulence and high protective efficacy in Objective 3 testing will have their genomes sequenced in order to investigate mutant regions which could have attributed to the attenuated virulence. This information will further scientific knowledge in virulence domains of this pathogen, as well as serve as a genome record of the vaccine candidate for quality assurance and control. Should effective gene manipulation and transformation techniques be developed for Histomonas/protozoa species in the future, these virulence regions can be targeted to force attenuation without the need for prolonged cell culture passages. The genome information will also pave way for genetic analysis of potential protective epitopes for further development of subunit or peptide vaccines which do not rely on administration of live pathogens for vaccination. These chosen lines of Histomonas will also serve as targets for diagnostic immunoassay development to detect the pathogen itself or seroconversion against the Histomonas. Specifically, an enzyme-linked immunosorbent assay (ELISA) will be designed, which would detect Histomonas protozoa in tissues samples as well as measure circulating antibodies developed against the protozoa (to both measure vaccine efficacy as well as serve as a surveillance method for prior exposure in field flocks). Data and assays generated from these studies would drive progression of this vaccine candidate through commercialization as well as contribute to the knowledge available on the pathogen itself. From the single-origin stocks generated, 5-8 selected clones will be tested in Objective 3 to confirm retained vaccine efficacy via in vivo immunogenicity and challenge trials. Vaccination with the single-origin stock will occur at 14 days-of-age, and morbidity observed for two weeks to confirm reduced virulence. Turkeys will then be challenged with 10 5 wild-type Histomonas to confirm retention of conferred protective immunity, as measured by reduced mortality and reduced liver/cecal lesions as compared to wild-type challenge without previous vaccination. Two single-origin clones which perform well in protective efficacy trials will be chosen for advancement and designated as final vaccine candidates. These two final candidates will be sent to Dr. John Barta s laboratory (University of Guelph) for next generation sequencing of their entire genomes, as mentioned in Objective 2. While sequencing is performed, the vaccine candidates will be optimized for dosage (10 3, 10 4, and 10 5 ) to reduce per dose costs and depending on company request, potential for drinking water or spray administration will also be tested. One of the confirmed clone candidates will proceed to field trials with two regional turkey producers (per company: two farms of different integrator area blocks with history of histomoniasis breaks, with minimum of two barns/farm). An additional non-vaccinated control barn will be included from each farm as a reference. Serum samples will be collected monthly post-vaccination for 6 months minimum, at which time grower/integrator willingness to continue sampling will be re-evaluated for further longevity testing. 6

8 Literature Review Histomonas meleagridis, the causative agent of histomoniasis, is a protozoa which penetrates the cecal epithelial lining, replicates, enters the bloodstream, and parasitizes the liver (McDougald, 2005; Hess and McDougald, 2013). Although commonly referred to as blackhead within industry and laymen s terms due to the indicative of cyanosis of the head, initial symptoms include declining feed consumption, drooping wings, unkempt feathers, and inactivity (Duffy et al., 2005). Necrotic thyphlitis (necrosis of the cecum), hepatitis, sulfuric fecal droppings, and high mortality are also characteristic pathological lesions (Callait et al., 2002; Hess et al., 2015). The life cycle and disease spread by H. meleagridis is not fully understood, although the cecal worm Heterakis gallinarum has been identified as an important vector and reservoir for disease transmission (Hess et al., 2015). Protozoan cells have been observed within the intestinal wall, larvae, and eggs of this cecal nematode (Hess et al., 2015). Histomoniasis infection can result from the ingestion of embryonated Heterakid eggs or the adult cecal worms (Hu et al., 2004). Additionally, regular earthworms can ingest infected cecal worm eggs, harboring the histomonads, and thereby acting as a transport host for the infected Heterakid eggs (Hu et al., 2004). Histomonads are delicate in nature and cannot withstand long periods of time outside of a host unless protected by a vector such as the Heterakid or earthworm (McDougald and Fuller, 2005). Direct ingestion of histomonads has not reliably initiated the disease in previous research, presumably due to the acidity and mechanical action within the crop and ventriculus (gizzard) of the bird (Hu et al., 2004). Heterakis gallinarum would appear to create a protective mechanism to transfer the infective material to the gastrointestinal tract of poultry, leading to establishment of disease through vectors (Hu et al., 2004). Rapid transmission by direct contact between infected birds or fecal droppings can occur without an intermediate host or vector (Sentíes-Cué et al., 2009). Once a turkey is infected, it can transmit disease to others in the flock within two or three days, causing an outbreak (Hess and McDougald, 2013). Interestingly, this method of transmission occurs via the cloacal drinking phenomenon rather than a fecal-oral transmission or respiratory-respiratory passage. This cloacal drop or cloacal drinking phenomenon transfers disease from the cloaca to the ceca via rhythmic contractions that draw the infectious material inside the vent (Hu et al., 2004; McDougald and Fuller, 2005). To further complicate the understanding of this disease, bacterial flora are found to be important in disease development (McDougald, 2005). The cecal environment wherein H. meleagridis resides is constantly in a state of flux, further indicating the difficult nature in understanding and controlling this disease (Callait et al., 2002). The ban of effective prophylactic and therapeutic compounds resulted in control measures focused upon prevention and control rather than treatment of the disease (Hess and McDougald, 2013). Overall effectiveness is limited, but outbreak prevention can be moderated with careful animal management practices. The disease is generally more prevalent when birds are housed in environments favoring the coexistence with the cecal nematode Heterakis gallinarum (Hess and McDougald, 2013). Although the disease tends to be sporadic within turkey flocks, chronic exposure may occur within chicken flocks (Hess and McDougald, 2013). As chickens often serve as reservoirs for the cecal worm and potential infection, separate rearing should occur between poultry species to prevent resulting cross-transmission outbreaks (McDougald, 2005; Hess and McDougald, 2013). 7

9 Early studies by Tyzzer (1934) expressed the reduction of virulence within long-term in vitro cultivated H. meleagridis, although immunization results yielded conflicting success. Previous studies within chickens and turkeys have shown reduction of liver and cecal lesions following administration of in vitro attenuated Histomonas (Mitra et al., 2017). Recently, vaccination with attenuated histomonads resulted in lowered variation of T and B cell subsets, while histomoniasis mortality in turkeys was demonstrated to be coherently associated with increased cellular immune response in comparison to chickens (Mitra et al., 2017). Taken together with Joyner s (1963) observation that dimetridazole-rescued acutely-afflicted histomoniasis turkeys were found to be protected against subsequent wild-type challenge, the previous research hints at possible adaptive immune protection against disease with the proper vaccine formulation. The lack of interest and research in parasitology within the past 30 years has left numerous unanswered questions about H. meleagridis as well as the resulting histomoniasis disease. Recent research has gained attention in the areas of antiprotozoal compounds, antibiotics, vaccines, and plant products with chemical activity (Hess et al., 2015). The research proposed in this grant differs from previous and current research focused primarily in finding feed supplements or probiotic direct fed microbials to prevent histomoniasis. Of the feed supplements and probiotics tested, an effective and practical option for industry use has yet to be found. The development of a viable vaccine candidate does not depend on long-term supplementation of the diet, but instead focuses on inducing the turkey s own immune memory to protect against disease. As feed supplementation trials rarely show full prevention against disease and typically only reduce disease severity, even a partially protective vaccine candidate would be comparable to a moderately effective feed supplement, should a worthwhile supplement even be found. Preliminary trials with the passaged Histomonas have indicated reduced disease severity compared to wild-type, suggesting this passaged line of Histomonas will perform well as a vaccine. 8

10 References Callait, M., C. Granier, C. Chauve, and L. Zenner In vitro activity of therapeutic drugs against Histomonas meleagridis. Poultry Science 81: Van der Heijden, H., L. R. McDougald, and W. J. M. Landman High yield of parasites and prolonged in vitro culture of Histomonas meleagridis. Avian Pathol. 34: Hess, M., T. Kolbe, E. Grabensteiner, and H. Prosl Clonal cultures of Histomonas meleagridis, Tetratrichomonas gallinarum and a Blastocystis sp. established through micromanipulation. Parasitology 133: Hess, M., D. Liebhart, I. Bilic, and P. Ganas Histomonas meleagridis - new insights into an old pathogen. Veterinary Parasitology 208: Hess, M., and L. McDougald Histomoniasis (blackhead) and other protozoan diseases of the intestinal tract. Diseases of Poultry, 13th edn. Wiley, Ames: Hu, J., L. Fuller, and L. R. McDougald Infection of turkeys with Histomonas meleagridis by the cloacal drop method. Avian Diseases 48: Hu, J., and L. McDougald The efficacy of some drugs with known antiprotozoal activity against Histomonas meleagridis in chickens. Veterinary Parasitology 121: Joyner, L Immunity to Histomoniasis in turkeys following treatment with dimetridazole. Journal of Comparative Pathology and Therapeutics 73: McDougald, L. R Blackhead disease (histomoniasis) in poultry: a critical review. Avian Diseases 49: McDougald, L., and L. Fuller Blackhead disease in turkeys: direct transmission of Histomonas meleagridis from bird to bird in a laboratory model. Avian Diseases 49: Mitra, T., W. Gerner, F. A. Kidane, P. Wernsdorf, M. Hess, A. Saalmüller, and D. Liebhart Vaccination against histomonosis limits pronounced changes of B cells and T-cell subsets in turkeys and chickens. Vaccine 35: Sentíes-Cué, G., R. Chin, and H. Shivaprasad Systemic histomoniasis associated with high mortality and unusual lesions in the bursa of Fabricius, kidneys, and lungs in commercial turkeys. Avian Diseases 53: Tyzzer, E. E Studies on histomoniasis, or blackhead infection, in the chicken and the turkey. Pages in Proceedings of the American Academy of Arts and Sciences. 9

11 Resume of Investigator: CHRISTINE N. VUONG EDUCATION PhD in Veterinary Pathobiology (Poultry Immunology) Texas A&M University 2017 MS in Biomedical Sciences (Poultry Virology) Texas A&M University 2012 POSITIONS/RESEARCH Research Scientist University of Arkansas Present Post-Doctoral Associate 2018 Manage research on development of vaccines and immunoassays to improve poultry welfare and gut health. Histomoniasis attenuated vaccine development and prophylaxis testing. Established challenge model for inclusion body hepatitis (IBH) and developed inactivated autogenous vaccine. Led immunoassay development for vaccine quality control or detection of pathogens (e.g. IBH, cellulitis). Recombinant vaccine vector development against: necrotic enteritis and coccidiosis. Research Assistant (MS & PhD) Texas A&M University Directed research laboratory and projects focused on developing poultry vaccines, immunoassays, and characterization of avian immune cells. Led research and development of patented antibody-guided smart vaccine delivery systems against various pathogens e.g. avian influenza, Clostridium perfringens, and Eimeria spp. Designed immunoassays for vaccine quality control and assurance. Led development and live trials of recombinant Sindbis-virus vector as a universal avian influenza vaccine. Classified Newcastle Disease Virus isolates collected from wild waterfowl via multiplex TaqMan qpcr. SELECTED PUBLICATIONS/PATENTS Delivering inactivated avian influenza to antigen presenting cells by targeting CD40 for enhanced protection against lethal challenge. Vuong et al. Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 37: doi: /mab , A fast and inexpensive protocol for empirical verification of neutralizing epitopes in microbial toxins and enzymes. Vuong et al. Frontiers in Veterinary Science: Veterinary Infectious Diseases, 4: 19. doi: /fvests , Significant mucosal siga production after a single oral or parenteral immunogen administration using in vivo CD40 targeting in the chicken. Chou et al. Research in Veterinary Science, 108: , Anti-Clostridium vaccine for control of necrotic enteritis in poultry. United States Patent No. 62/663,798. Antibody-guided, smart vaccines for generation of rapid immune responses. PCT International Patent Publication No. WO2105/ SELECTED AWARDED/COMPLETED GRANTS Development and evaluation of experimental autogenous viral and bacterial vaccines to control emergent diseases for Arkansas poultry industry. Arkansas Biosciences Institute Grant Program (awarded $48K) 2018 A platform technology for the isolation of anti-cancer monoclonal antibodies from chickens. Cancer Prevention & Research Institute of Texas: High Risk/High Impact (awarded $100K) 2016 Antibody-guided vaccination for the sustainable prevention of chicken coccidiosis correlation with xanthophyll. Texas A&M CONACYT Research Grant (primary investigator, awarded $24K) 2015 SELECTED HONORS, AWARDS, AND PROFESSIONAL AFFILIATIONS Merck/MSD Animal Health High Quality Poultry Science Awards (Americas regions) 2018 Poultry Science Association Certificate of Excellence (Immunology, Health, and Disease) 2017 Poultry Science Association Certificate of Excellence (Immunology, Health, and Disease) 2016 Poultry Science Association Certificate of Excellence (Immunology, Health, and Disease) 2015 Poultry Science Association, member 2014-present Manuscript reviewer for Poultry Science Journal (Immunology, Health, and Disease section) 2018-present Cold Spring Harbor Laboratories Scholarship (Antibody Engineering) 2013 Texas A&M Graduate Student Diversity Fellowship

12 Current or Previous Research on Subject (if any) by Investigator: The project leader is experienced in vaccine development and has led and completed multiple poultry vaccine development projects in the past 7 years, of various designs and disease targets. Poultry vaccines, immunoassay development, and cloning techniques are the project leader s primary field of expertise and the proposed project fits within this purview. The co-investigator also has extensive experience in poultry health and disease, spanning over 30 years of research in poultry gut health. Histomoniasis research has been completed within this laboratory for several years, leading to the technical development of this in vitro passaged Histomonas line and has led to the proposed project outlined. Facilities and Equipment Required and Available for This Project: Biosafety Level 2 animal facilities are available within the Poultry Health Laboratory for live animal trials to test vaccine immunogenicity and protective efficacy. This facility is also furnished for the laboratory benchtop equipment needs for the passage and culture of Histomonas in vitro. Sequencing will be completed in collaboration with leading parasitologist Dr. John Barta (University of Guelph). Final candidate field testing will be completed in collaboration with local turkey producers whose attending veterinarians have already expressed interest in field testing the vaccine candidate. Research Timetable: (a) Date project is scheduled to begin: June 2019 (b) Date project is scheduled to end: May 2021 Months Year 1 (June 2019 to May 2020) Objective 1: Attenuated Line Preliminary Testing Animal Trial #1: Attenuated Line Selection Animal Trial #2: Protective Efficacy/Challenge Testing Animal Trial #3: Optimization of Administration Objective 2: Line Isolation/Genetic Sequencing Single-Cell Cloning/Propagation of Attenuated & Wild-type Lines Immunoassay Development for Diagnostics Objective 3: Vaccine Efficacy Testing Animal Trial #4: Chosen Isolate(s) Efficacy Re-confirmation #1 Year 2 (June 2020 to May 2021) Objective 2: Line Isolation/Genetic Sequencing Immunoassay Development for Seroconversion Diagnostics Sequencing of Chosen Isolates Objective 3: Vaccine Efficacy Testing Animal Trial #4: Chosen Isolate(s) Efficacy Re-confirmation #1 Animal Trial #5: Chosen Isolate(s) Efficacy Re-confirmation #2 Animal Trial #6: Field Trials/Long-term Vaccine Efficacy 11

13 Personnel Support Provided by the University: There will be an advising research scientist and professor (Drs. Christine N. Vuong and Billy M. Hargis) at hand to manage project progression as well as participation with collaborating professors from other universities. The university will provide full salary support for the project leader and the co-investigator. This project will be assigned to two graduate students. Aid will be provided on major trial dates for live animal procedures from over 5 other graduate students and 3 support staff. Financial Support: (a) From the university: Project reagent costs and animal housing costs will be covered by the supervising laboratory s internal funds. (b) From other sources: This research will be completed with support from local turkey producers/industries for aid in supplying poults for in vivo trials and facilitating vaccine efficacy field trials. Institutional Units Involved: Work proposed in this project will be conducted using facilities and personnel within the J.K. Skeeles Poultry Health Laboratory of the University of Arkansas: Division of Agriculture (Fayetteville, AR). Clone sequencing will be conducted in collaboration with Dr. John Barta from the University of Guelph. Budget: Funds will be used to cover two graduate students stipends (at $16,150/student per year) as well as the 15% indirect costs ($5,700/year). Project reagent expenses and associated animal housing/care expenses will be covered by the supervising laboratory. Total Funds Requested: $76,000 total ($64,600 direct + $11,400 indirect). Indirect Costs: $11,400 (15%) will be allocated for indirect costs to the university ($5,700/year). Receipt of Funds Needed: Quarterly (based on 2 year project timeline)/every 6 months. Make Check Payable To: University of Arkansas c/o Mike Sisco, Grants Officer 1120 West Maple Street Fayetteville, AR

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