Location: Animal Parasitic Diseases Laboratory
2019 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 protozoan species in the genus Eimeria, can be partially controlled using anticoccidial drugs in poultry feed, however drug resistance limits the usefulness of these drugs; thus, rapid methods to assess sensitivity of Eimeria parasites in poultry houses is sorely needed. An in vitro method to test monensin 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 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. A 24-hour timepoint was found to be optimal for testing monensin sensitivity. Moreover, optimized methods for stabilizing E. maxima sporozoites during and after excystation were developed that allowed for time between sporozoite isolation and inoculation into cell culture. The impact is that a rapid method for determining the sensitivity of E. maxima to monensi is available for use by poultry companies and diagnostic laboratories.
As interest in growing chickens without 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 such chicks do not ingest the vaccine and thus remain susceptible to avian coccidiosis once placed in the poultry house. A method was developed that involves administering Eimeria oocysts to chicks through their drinking water. Vaccine uptake using water delivery was excellent, and chicks showed improved performance compared to vaccine administration at the hatchery. Attempts are now being made to identify commercial broiler farms that have a high incidence of coccidiosis and necrotic enteritis to compare water- and hatchery-delivery of protective vaccines.
While vaccination with live Eimeria oocyst vaccines represent a good alternative to anticoccidial drugs, this approach necessarily seeds poultry houses with virulent Eimeria. An alternative approach would entail immunizing chicks with recombinant Eimeria proteins 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 excellent protection in chickens raised in battery cages and floor pens. A similar approach was applied to IMP1 protein from E. acervulina and E. tenella. Methods for injecting embryonated chicken eggs were optimized that gave nearly 100% hatch. In ovo administration of NP-IMP1 protein is now being tested as a practical alternative to oral inoculation.
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. Moreover, C. perfringens DNA was analyzed for the presence of genes coding for netB- and alpha-toxins. 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. Also, a correlation was found between the levels of E. maxima oocysts or netB/alpha toxins and early chick mortality, pointing to the importance of coccidiosis and C. perfringens toxins in necrotic enteritis. This information suggests that 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.
Each avian Eimeria species strictly invades and develops in a particular portion of the gut. For instance, E. acervulina develops in the duodenum, E. maxima in the jejunum, and E. tenella in the cecum. We studied excystation (the release of sporozoites from oocysts) and found that the initial invasive stage sporozoites were viable for different lengths of time and that this could be temporarily overcome by treating them with reducing agents. This information may help devise ways to decrease the stability of E. tenella sporozoites, thereby preventing the parasite from reaching its natural site of infection.
Accomplishments
1. Vaccines against poultry disease improved using an antigen 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 current vaccines provide some protection, better vaccines would preclude virulent parasites from broiler houses. ARS scientists in Beltsville, Maryland, discovered that attaching a protective vaccine antigen to nanoparticles significantly improved efficacy when administering the vaccine orally to newly-hatched chicks, resulting in improved weight gain and feed conversion efficiency. They further established that in-ovo vaccination provided a flexible and effective means of vaccine delivery. 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, and reduce the occurrence of concomitant bacterial infections.
2. Improving the efficiency and uniformity of vaccination against avian coccidiosis. Currently, vaccinating broiler chickens against coccidiosis is accomplished by spraying newly-hatched chicks in their shipping container, with an aqueous solution containing a mixture of 3 different Eimeria species. While this method is easy to perform, USDA scientists in Beltsville, Maryland, determined that 50-70% of chicks do not ingest sufficient vaccine to become immune, rendering them fully susceptible to disease once they are placed in a chicken house. The USDA team therefore has explored other vaccination approaches, including a system feeding one-day-old chicks with gelatin beads containing vaccine. In small scale battery cage and floor pen studies, all chicks became vaccinated; subsequent, large-scale field trials (involving nearly 10 million commercial broiler chicks fed such gelatin beads, either at the hatchery or at the farm) resulted in improved feed conversion efficiencies. An even more promising method delivered vaccine through drinking water. These data suggest that drinking water administration may be a viable alternative way of vaccinating broiler chicks against coccidiosis.
Review Publications
Jenkins, M.C., Parker, C.C., Obrien, C.N., Ritter, D. 2019. Viable Eimeria oocysts in poultry house litter at time of chick placement. Poultry Science. https://doi.org/10.3382/ps/pez147.
Su, S., Miska, K.B., Fetterer, R., Jenkins, M.C., Lamont, S., Wong, E. 2018. Differential expression of intestinal nutrient transporters and host defense peptides in Eimeria maxima-infected Fayoumi and Ross chickens. Poultry Science. 97:4392-4400.
