Location: Animal Parasitic Diseases Laboratory
2018 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
Avian coccidiosis caused by protozoa Eimeria is controlled in part by application of anticoccidial drugs to poultry feed. However, drug resistance limits the usefulness of these drugs, and thus rapid methods to assess sensitivity of Eimeria parasites in poultry houses is sorely needed. An in vitro method to test ionophore resistance in Eimeria maxima was applied to ionophore-resistant E. maxima isolated from commercial broiler houses. Earlier studies in chickens grown on feed containing different ionophores showed that these E. maxima were resistant to salinomycin and monensin. The in vitro studies corroborated these findings in that ionophore-resistant E. maxima field strains, unlike ionophore-sensitive E. maxima laboratory strains, displayed lower levels of invasion and development in cell culture as measured by parasite counts and molecular assays. Moreover, different timepoints of culture harvest were found necessary for monensin compared to salinomycin with the former being optimal at 24 hr and the latter optimal at 48 hr. The impact is that a rapid method for determining the sensitivity of E. maxima to ionophore drugs is available for use by poultry companies and diagnostic laboratories.
As interest in growing chickens in the absence of antibiotics increases, alternative methods of preventing avian coccidiosis are being applied. At present, live Eimeria oocyst vaccines are delivered by spraying the vaccine onto 100 chicks in a shipping box, but our research has shown that a high percentage of chicks do not ingest the vaccine and thus remain susceptible to avian coccidiosis once placed in the poultry house. Edible gel beads were developed to contain the Eimeria vaccine and showed excellent uptake and conferred good protection in broiler chickens. This approach was tested in over 10 million broiler chicks in collaboration with a commercial poultry company on the Maryland Eastern Shore. Generally improved performance was observed in chicks given gel bead vaccine compared to spray vaccination. Efforts are now being applied to make this or similar approaches more practical for use in the poultry industry.
While vaccination with live Eimeria oocyst vaccines represent a good alternative to anticoccidial drugs, this approach necessarily leads to seeding poultry houses with virulent Eimeria. An alternative approach is to immunize chicks with a recombinant Eimeria protein to induce protective immunity against Eimeria challenge infection. A protective Eimeria maxima antigen, namely EmaxIMP1, was incorporated into nanoparticles (NP) using standard methodology and showed excellen protection in chickens raised in battery cages and floor pens. A similar approach was applied to IMP1 protein from another Eimeria, namely E. acervulina. Preliminary studies in battery cages showed promising results in protecting chickens against E. acervulina infection.
Understanding the population dynamics of Eimeria and Clostridium on poultry farms may help explain why some farms experience more frequent outbreaks of coccidiosis and necrotic enteritis (NE) caused by the respective pathogens. Litter samples were collected from 9 poultry farms at 0, 2, and 4 weeks of growout during anticoccidial drug and vaccine programs. Eimeria oocysts were enumerated by microscopy, and individual species ascertained by PCR. In addition, Clostridium perfringens spores were recovered using standard techniques and were grown on selective media and extracted for DNA, and amplified in a C. perfringens-specific PCR. It was found that high numbers of Eimeria oocysts and Clostridium spores are present in poultry litter, and are viable even at day of placement suggesting that techniques to treat litter is not effective at destroying these organisms. Thus, the poultry industry must either ensure that newly-placed chicks are protected against Eimeria and Clostridium infection or that new methods of treat litter are developed to eliminate these pathogens.
Controlling avian coccidiosis depends in part on understanding the details of how Eimeria develop inside the chicken host, and thus if a control strategy can target certain phases of the parasite life cycle. Studies were conducted to determine where in the gut and what type of cells E. maxima life stages invade and develop during the course of infection. Tissue samples from E. maxima-infected broiler chicks were harvested every 12 hours and examined for developmental stages. The study revealed new findings in that the initial invasive stage sporozoites are present up to 48 hr, and that there are 6 additional stages including sexual stages that fuse to form oocysts. All of these stages are found in the middle intestine which explains the effect of E. maxima on nutrient absorption in the gut.
Accomplishments
1. Vaccines against poultry disease improved by administering an antigen using small nanometer-size particles (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. Although vaccines exist to prevent avian coccidiosis, these vaccines are comprised of low doses of virulent (highly infectious) 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 given the vaccine by oral administration at hatch showed improved weight gain and feed conversion efficiency, as compared to chickens vaccinated with the same antigen but without nanoparticles. This 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. Improved monitoring and control of poultry parasites and bacteria. By knowing what strains of parasites (Eimeria) and bacteria (Clostridium) are in a poultry house, poultry companies can tailor treatment strategies to prevent disease outbreaks. New methods were developed and tested by ARS scientists at Beltsville, Maryland, that better specified the composition and abundance of Eimeria and Clostridium on broiler farms. Incomplete vaccination lead to poor growth and increased mortality because the parasite allows infection of the gut by harmful bacteria, such as the strain Clostridium perfringens. The new methods are now being used to ascertain uniformity and efficiency of coccidiosis vaccine administration.
5. New method of vaccination against avian coccidiosis. 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 has taken another approach that involves administering the Eimeria parasites directly to chicks in the poultry house a few days after placement. This new method lead to 100% vaccine uptake by chicks.
Review Publications
Jenkins, M.C., Stevens, L., Obrien, C.N., Parker, C.C., Miska, K.B., Konjufca, V. 2018. Incorporation of a recombinant Eimeria maxima IMP1 antigen into nanoparticles confers protective immunity against E. maxima challenge infection. Vaccine. 36(8):1126-1131.
Dubey, J.P., Jenkins, M.C. 2017. Re-evaluation of the life cycle of Eimeria maxima Tyzzer, 1929 in chickens (Gallus domesticus). Parasitology. https://doi.org/10.1017/S0031182017002153
