Location: Animal Parasitic Diseases Laboratory
2017 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
An in vitro method to test ionophore resistance in Eimeria maxima was developed using a ionophore-sensitive strain of E. maxima. This method relies on counting intracellular E. maxima sporozoites and quantitative PCR amplification of an E. maxima DNA sequence. An excellent dose response was observed such that increasing concentrations of salinomycin and monensin produced fewer intracellular sporozoites or lower PCR product. Studies are underway to expand ionophore-resistant E. maxima strains isolated from commercial broiler houses and test these in the in vitro assay.
Gel beads applied to commercial shipping boxes prior to deposit of 100 broiler chicks revealed excellent uptake of an Eimeria oocyst vaccine. While aqueous spray at the hatchery lead to less than 50% vaccine uptake, 100% chicks originating from shipping boxes containing Eimeria-impregnated gelatin beads had ingested the vaccine. Protective immunity in chicks receiving bead vaccination as measured by weight gain after an experimental challenge was much higher than in chicks given the same vaccine by aqueous spray.
A protective Eimeria maxima antigen, namely EmaxIMP1, was incorporated into nanoparticles (NP) using standard methodology. Chickens given NP-EmaxIMP1 showed excellent protection in 3 battery cage and 2 floor pen studies against E. maxima challenge infection compared to chickens given control NP as measured by weight gain and feed conversion efficiency. Histological studies showed that NP-EmaxIMP1 localized to various immune tissues of chicks given oral NP-EmaxIMP1.
Litter samples were collected from 7 poultry farms at 0, 2, 4, and 8 weeks of growout during anticoccidial drug and vaccine programs. Eimeria oocysts were enumerated by microscopy, and individual species ascertained by ITS1 PCR. In addition, Clostridium perfringens spores were recovered using standard techniques, extracted for DNA, and amplified in a C. perfringens-specific PCR. The data was related to mortality data from each house on every farm, and showed an excellent correlation between E. maxima levels and chick mortality at 2 weeks of age for vaccine control programs, but not for anticoccidial drug programs. These data suggest that high levels of E. maxima possibly arising from incomplete, non-uniform vaccination lead to reduced livability in the early phases of broiler growout. These data provide further support for the need to achieve 100% vaccine coverage in newly-hatched chicks.
Gene sequences coding for protective Eimeria antigens were inserted into Salmonella expression plasmids, used to transform harmless AroA Salmonella mutants, that were then tested for secretion of recombinant Eimeria protein and ability to protect chickens against coccidiosis challenge infection. Strong cellular immune responses and significant protection against Eimeria infection as measured weight gain was achieved with the recombinant Salmonella.
Litter samples from broiler houses using a proprietary coccidiosis vaccine were evaluated for the level and species composition of Eimeria therein. The data showed that species composition was similar to that found in the commercial vaccine, yet non-vaccine strains were also present suggesting that incorporating additional Eimeria is warranted.
Studies were conducted comparing commercial aqueous spray of coccidiosis vaccine to gelatin beads as a means of achieving efficient and uniform vaccination of newly-hatched chicks against Eimeria infection. The data showed that gel bead vaccination gave 100% uptake compared to 30-60% uptake with aqueous spray vaccination. Field studies conducted using nearly 10 million broiler chicks during 2 weeks of vaccination with gelatin beads provided good feed conversion efficiency.
Different preparations of the ionophore drug monensin were tested for efficacy against mixed Eimeria acervulina, E. maxima, and E. tenella infection in broiler chickens. All drug preparations prevented clinical signs of coccidiosis, such as weight loss and intestinal lesions, compared to non-medicated controls.
Accomplishments
1. Vaccines against poultry disease improved by administering an antigen 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 and although vaccines exist to prevent avian coccidiosis, these vaccines are comprised of low doses of virulent organisms. Better vaccines are needed to avoid introducing virulent parasites into broiler houses. ARS scientists in Beltsville, Maryland, discovered that attaching a protective vaccine antigen to nanoparticles significantly improved efficacy. Chickens administered the vaccine by oral inoculation at hatch showed improved weight gain and feed conversion efficiency, as compared to chickens vaccinated with the same antigen but without nanoparticles. A patent disclosure describing the use of nanoparticle-conjugated EmaxIMP1 was approved by the ARS Patent Committee. If licensed and adopted, the resulting technology may markedly improve the health of poultry flocks, reduce the occurrence of concomitant bacterial infections which compromise food safety, and reduce costs of poultry production.
2. Better quantification and characterization of poultry parasites lead to improved monitoring and control. By knowing what strains of parasites are in a poultry house, poultry companies can tailor vaccination strategies to prevent disease outbreaks; further, new methods were developed and tested that better specified the composition and abundance of parasites on broiler farms. Improved accuracy enabled prediction of incomplete vaccination that led to poor growth and increased mortality because the parasite allows infection of the gut by harmful bacteria, such as Clostridium perfringens. The methods are now being used to ascertain uniformity and efficiency of coccidiosis vaccine administration. These methods also allow “problem” houses on a farm to be identified, characterized by especially abundant numbers of parasites. These advances will assist all entities involved in poultry production from individual growers to poultry companies to vaccine and drug manufacturers in developing effective strategies against avian coccidiosis.
3. A problem in developing practical vaccines against a poultry disease such as avian coccidiosis is to figure out how to deliver the parasite protein to the chicken immune system. Although one might identify a vaccine protein, it has to elicit an appropriate immune response in the host. A practical way of protecting chickens against coccidiosis was developed using harmless Salmonella delivery vectors to express an immunoprotective Eimeria antigen. The Salmonella are harmless double-mutants that cannot replicate outside of the laboratory, but can be used to stimulate an immune response in chickens by virtue of releasing recombinant protein either before or after being taken up by immune cells. This approach may be feasible because similar to avian parasites such as Eimeria, the Salmonella bacteria migrate to the gut and stimulate an immune response that is effective against not only Eimeria, but possibly against pathogenic Salmonella, which have been implicated in human food poisoning.
4. Commercial coccidiosis vaccines consist of a mixture of low numbers of Eimeria parasites. These parasites are similar to those in litter that can cause outbreaks of coccidiosis in young chickens. However, many of the parasites in litter have developed resistance to drugs, and vaccines may be a way of replacing drug-resistant parasites with drug-sensitive ones. Baseline data was gathered on the species composition of Eimeria present in poultry houses containing broilers that had been vaccinated with a commercial coccidiosis vaccine. These data confirmed anecdotal evidence that vaccine oocysts can supplant field strains of the same Eimeria species. However, the data showed that Eimeria species were present in litter that were not in the vaccine possibly reflecting a lack of immunity against nonvaccine strains. This data provides evidence for a need to know the Eimeria species composition in poultry litter to prevent outbreaks of coccidiosis during growout.
5. Current methods of vaccinating broiler chickens against coccidiosis involving spraying an aqueous solution containing a mixture of 3 different Eimeria species onto newly-hatched chicks in a shipping container. While this method is easy to perform, often 50-70% of chicks do not ingest sufficient vaccine to become immune to coccidiosis and this is a problem because non-immune chicks are fully susceptible to disease once placed in a chicken house. Our research over the last few years has taken another approach that involves incorporating Eimeria oocysts into gelatin beads, and applying these to the chick box or directly to feed so that chicks ingest the vaccine during the first day of life. In small scale battery cage and floor pen studies, uptake of gelatin beads containing Eimeria oocyst was 100% compared to 50-70% from aqueous spray; further, large scale field trials involving nearly 10 million commercial broiler chicks given Eimeria-gelatin beads at either the hatchery or at the farm revealed improved feed conversion efficiencies. These data suggest that gel bead technology may be a viable alternative way of vaccinating broiler chicks against coccidiosis. Efforts are now being directed to make this technology practical and adaptable to the poultry industry.
Review Publications
Su, S., Miska, K.B., Fetterer, R.H., Jenkins, M.C., Wong, E. 2015. Expression of digestive enzymes and nutrient transporters in Eimeria-challenged broilers. Experimental Parasitology. 150:13-21.
Su, S., Dwyer, D., Miska, K.B., Fetterer, R.H., Jenkins, M.C., Wong, E.A. 2017. Expression of avian beta-defensins in the intestine of Eimeria-challenged chickens. Poultry Science. doi: 10.3382/ps/pew468.
Lin, H., Summers, L., Dalloul, R., Miska, K., Fetterer, R.H., Jenkins, M.C., Zhu, Q., Wong, E. 2015. Expression of an antimicrobial peptide, digestive enzymes and nutrient transporters in the intestine of E. praecox-infected chickens. Poultry Science. 94:1521-1526.
Jenkins, M.C., Parker, C.C., Ritter, D. 2017. Eimeria oocyst concentrations and species composition in litter from commercial broiler farms during anticoccidial drug or live Eimeria oocyst vaccine control programs. Avian Diseases. 61:214-220.
