Location: Animal Parasitic Diseases Laboratory
2020 Annual Report
Objectives
Objective 1: Develop improved in vitro assays to assess the level of resistance by the various species of Eimeria to coccidiostats.
Objective 2: Develop and evaluate the efficacy of new vaccine regimens and new vaccine candidates using novel vaccine vector systems and platforms
Subobjective 2.A. Improve gel-bead delivery of Eimeria oocysts to vaccinate chickens against aviancoccidiosis.
Subobjective 2.B. Evaluate recombinant Eimeria proteins expressed by recombinant attenuated Salmonella vaccine (RASV) strains or incorporated into nanoparticles (NP) for eliciting protective immunity against coccidiosis infection.
Objective 3: Assess the epidemiology of the population dynamics of Eimeria on poultry farms that will provide needed information on vaccine and treatment strategies as well as identification of the various strains on farms including potential new emerging species.
Approach
Live Eimeria oocysts vaccines will be improved by testing the efficacy of alternative delivery systems, including gel bead application to broiler and layer chicks at the hatchery or at the poultry farm. Successful vaccination will be determined by measuring Eimeria oocyst uptake and effects on chick performance after placement in poultry houses. The epidemiology of Eimeria will be studied during different coccidiosis control programs to provide insight on the efficacy of anticoccidial drug treatment or vaccination. Rapid methods for assessing anticoccidial drug sensitivity will be developed in order to obtain timely information on the drug resistance profiles of Eimeria in poultry houses. These methods include in vitro culture of Eimeria in the presence of various concentrations of ionophore drugs or synthetic chemicals used by the poultry industry to control avian coccidiosis. Alternative vaccination approaches including the development of recombinant Eimeria proteins for protecting chickens against coccidiosis will be explored as well as different methods for delivery these antigens to the chicken immune system.
Progress Report
Protozoan species in the genus Eimeria cause poultry coccidiosis. Adding anti-coccidial drugs to poultry feed can partially control the disease. However, growing drug resistance necessitates rapid methods to assess drug sensitivity of the parasites. Currently, it may take two months to determine if field-derived parasites are susceptible to these drugs, because the parasites need to be isolated and administered to chickens. In vitro assays could speed this process, but natural substances impede such assays by preventing parasite development in cell culture. We therefore improved methods to purify sporozoites by passing mixtures contaminated with oocysts through a nylon wool-glass bead column. The columns retain oocyst constituents. Pure sporozoites pass through. The short life-span of E. maxima and E. acervulina sporozoites also impedes cell culture assays. We discovered that adding a reducing agent extends the viability of such sporozoites. These two improvements enabled us to culture E. maxima sporozoites. We are testing the sensitivity of various strains to ionophore drugs. This progress speeds results from two months to two weeks.
Raising chickens without antibiotics requires new ways to prevent avian coccidiosis. Hatchery employees now spray vaccines (made of live Eimeria oocysts) onto chicks shipped to growers. We discovered that many such chicks do not ingest the vaccine. They thus remain susceptible to the disease. We therefore devised a way to administer vaccine to chicks through their drinking water. Vaccine uptake and chick performance improved. We therefore began testing this approach under commercial conditions by working with a commercial broiler company raising antibiotic-free chickens. Preliminary data indicates that vaccination through water is safe and efficacious under commercial conditions.
Vaccinating chickens with live Eimeria oocyst vaccines reduces reliance on anticoccidial drugs. Unfortunately, this approach seeds poultry houses with parasites. Immunizing chicks with recombinant Eimeria proteins might avoid this risk. We therefore incorporated a protective Eimeria maxima antigen, EmaxIMP1, into nanoparticles (NP). Excellent protection resulted in chickens raised in battery cages and floor pens. To make this approach practical, we evaluated whether NP-EmaxIMP1 could confer protection when administered in ovo. Injecting the amnion 18-19 days after incubation conferred excellent protection. We are now evaluating air cell injection to avoid the need to include antibiotics, such as gentamycin, when formulating the vaccine.
Why do some farms experience more frequent outbreaks of coccidiosis and necrotic enteritis? Industry needs better tools to diagnose which parasites are locally abundant. We evaluated new diagnostic techniques using PCR metagenomics. We evaluated samples collected from litter on 9 poultry farms at 0, 2, and 4 weeks of growout. These farms used either anticoccidial drugs or vaccines to control the disease. We counted Eimeria oocysts by microscopy and evaluated various PCR primers for their power to diagnosed parasite species. Certain primers distinguished infections with E. acervulina, E. maxima, or E. tenella. We also commenced sequencing the entire ~ 46 mB genome of E. acervulina using Minion and PacBio technology.
Accomplishments
1. Improved vaccines against poultry disease using nanoparticles. Coccidiosis, a gut disease of poultry, costs U.S. producers $350 million annually due to poor weight gain in affected animals and the costs of treatment. The vaccines that prevent avian coccidiosis deliver low doses of virulent organisms. Better vaccines would not introduce virulent parasites to broiler houses. ARS scientists in Beltsville, Maryland, improved the efficacy of a protective antigen (EmaxIMP1) by attaching it to nanoparticles. Nanoparticle delivery improved weight gain and feed conversion efficiency. This technology may markedly improve the health of poultry flocks and also reduce bacterial infections.
2. Improved vaccine delivery to combat avian coccidiosis. Hatcheries spray newborn chicks in their shipping containers with an aqueous solution containing three species of Eimeria to vaccinate broiler chickens against coccidiosis. ARS scientists in Beltsville, Maryland, determined that 50 to 70 percent of chicks do not ingest sufficient vaccine to become immune to coccidiosis. Our team explored other vaccination approaches, including a system of feeding one-day-old chicks with gelatin beads containing vaccine. A large-scale field trial involving nearly 10 million commercial broiler chicks fed gelatin beads with the vaccine, showed improved feed conversion efficiency. Delivering this vaccine through drinking water may further protect birds. These efforts provide the poultry industry with effective coccidiosis vaccines that are practical and adaptable to changing industry practices.
3. A faster test for anticoccidial drug sensitivity. In fall and winter, broiler producers typically administer feed containing anticoccidial drugs to prevent outbreaks of coccidiosis and necrotic enteritis (NE). They do so without knowing whether the parasites in their houses are sensitive to these drugs. It may take two full months to assess drug sensitivity using a current method. However, a grower typically places the next chicks in a house within 3 weeks. ARS scientists at Beltsville, Maryland, previously established a much faster method to assess drug sensitivity for E. tenella, in vitro. However, obstacles prevented assaying E. maxima or E. acervulina in this way. The team overcame these obstacles by improving parasite purification and by discovering that a reducing agent prolongs sporozoite viability in culture. Drug-sensitivity can now be established in less than two weeks, enabling timely decisions as to which drugs should work on any given farm.
Review Publications
Jenkins, M.C. 2019. Vaccination. In: Dubey, J.P., editor. Coccidiosis in Livestock, Poultry, Companion Animals and Humans. 1st Edition. Boca Raton, Florida: CRC Press. p. 51-58.
Avendano, C., Jenkins, M.C., Mendez-Callejas, G., Oviedo, J., Guzman, F., Patarroyo, M., Sanchez-Acedo, C., Quilez, J. 2018. Cryptosporidium spp. CP15 and CSL protein-derived synthetic peptides. Vaccine. 36(45):6703-6710. https://doi.org/10.1016/j.vaccine.2018.09.044.
Dubey, J.P. (Ed.) 2020. Coccidiosis in Livestock, Poultry, Companion Animals, and Humans. 381 pages.
