Location: Animal Parasitic Diseases Laboratory
2021 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, necessitating rapid means to assess sensitivity of Eimeria parasites in poultry houses. We developed in vitro culture methods that succeed in assessing drug sensitivity in Eimeria tenella, but the methods require too many sporozoites of E. acervulina and E. maxima to be of practical use because they do not replicate in culture. We therefore took another approach, continuously passaging E. maxima in chickens with or without anticoccidial drugs. We obtained, after 3 such passages, drug-resistant E. maxima clones that cause patent infection in drug-treated chickens. Drug-sensitive parasite fail to sporulate (form sporozoites). We are now determining the molecular basis for drug resistance in hopes of designing a rapid PCR-sequencing assay to identify resistant E. maxima on poultry farms. This work will benefit poultry producers and growers by pointing to which drugs to discontinue using to control coccidiosis.
As interest in growing chickens without antibiotics increases, producers are turning to alternative methods to prevent avian coccidiosis. Most deliver live Eimeria vaccines by spraying oocysts onto chicks in their shipping box, but we have demonstrated the failure of this method to vaccinate many such chicks, which remain susceptible to avian coccidiosis once placed in the poultry house. We devised a means to administer oocysts through drinking water, yielding excellent vaccine uptake and improved performance at the hatchery. We replicated this success several times on commercial broiler farms, hoping to encourage industry to adopt this improved method of vaccine delivery so as to stem coccidiosis outbreaks and curb the occurrence of necrotic enteritis.
Live Eimeria oocyst vaccines represent a good alternative to anticoccidial drugs; but their use necessarily seeds poultry houses with virulent Eimeria. Recombinant Eimeria proteins would not have this drawback. We therefore incorporated a protective Eimeria maxima antigen (EmaxIMP1) into nanoparticles (NP) and achieved excellent protection in chickens raised in battery cages and floor pens. Our innovation was awarded a patent. In order to make this approach practical, we injected these in ovo in the amnion. Injection at 18-19 days incubation conferred excellent protection against challenge. We also found evidence that air cell injection may not be as reproducible as amnion injection. We are testing if booster immunizations are necessary to establish complete protection. We are also determining whether immunity has early enough (2 weeks of age) to prevent outbreaks of necrotic enteritis. This work will benefit poultry producers and growers by providing a way to vaccinate chickens against coccidiosis without causing the release of virulent Eimeria oocysts into the poultry house.
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 using generic oligonucleotide primers designed to amplify single-copy genes (orthologues, SCO) from every Eimeria species. Using DNA from pure strains, a subset of these primers showed efficacy in distinguishing between E. acervulina, E. maxima, and E. tenella. PCR amplification of total Eimeria DNA from a commercial broiler farm was able to identify all Eimeria species present and identify strains of coccidia. This work will benefit poultry producers and growers by providing a way to rapidly identify Eimeria strains that may be associated with poor growth and performance, and thus suggest undertaking a complete clean-out of the house to remove highly virulent strains.
Understanding which genes are turned on and off during parasite development may help in the design of treatments (e.g. anticoccidial drugs) that can be used to affect gene expression in developing Eimeria. We therefore isolated RNA from Eimeria acervulina oocysts at various times during sporulation (formation of sporozoites) and then assayed for up- or down-regulation of specific genes. A number of genes, some known to mediate metabolism, vary in their expression as parasites mature. We hope to use this information to design drugs that prevent invasive stage sporozoites from forming. Genes we have identified that had no prior known function may play important roles in parasite development.
In a commercial hatcher, chicks typically hatch over a 30 hr “hatch window.” We were interested to learn whether different chicks hatching early grow at a different rate than chicks hatching late; differences might suggest benefits to removing chicks soon after they hatch. We therefore conducted several studies to measure body weights at hatch and after 7 days, observing no appreciable difference in broiler performance when comparing early and late hatchers. This information is valuable to poultry producers who may have otherwise elected to remove chicks from the hatcher at different times.
Accomplishments
1. Vaccine against Eimeria using recombinant protein linked to nanoparticles. Chicks immunized at hatch or in ovo at 18d incubation were protected against coccidiosis challenge infection. 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 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 describing the use of nanoparticle-conjugated EmaxIMP1 was granted by the US Patent Office. A private company is seeking license of the nanoparticle recombinant vaccine which, if adopted industry-wide, 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. Improving delivery of Eimeria live vaccines. Poultry producers vaccinate broiler chickens against coccidiosis by spraying newly-hatched chicks, while in a shipping container, with an aqueous solution containing a mixture of 3 different Eimeria species. ARS scientists in Beltsville, Maryland, determined that 50-70% of chicks do not ingest sufficient vaccine to become immune to coccidiosis using this method, rendering them fully susceptible to disease once they are placed in a chicken house. The team therefore explored other vaccination approaches, including a system of feeding one-day-old chicks with gelatin beads containing vaccine. In small scale battery cage and floor pen studies, all chicks became vaccinated; subsequently, large scale field trials (involving nearly 10 million commercial broiler chicks fed such gelatin beads, either at the hatchery or at the farm) were conducted and resulted in improved feed conversion efficiencies. An even more promising method, delivering this vaccine through drinking water administration is being pursued as a viable alternative. These efforts should provide the poultry industry with an effective coccidiosis vaccine that is practical and adaptable to current industry practices.
3. Improved method for assessing drug sensitivity of Eimeria tenella. Many poultry producers augment the feed of broiler chickens with anticoccidial drugs to prevent outbreaks of coccidiosis and necrotic enteritis (NE) in Fall and Winter. Unfortunately, they do so without any means to know, ahead of time, whether the parasites in the house remain sensitive to these drugs. Resistance to ionophores, such as monensin, renders such feed addition ineffective. Current methods of assessing drug-sensitivity are costly, labor-intensive, and require at least 2 months to complete. Thus, available methods provide not help to growers who typically have less than three weeks to make a management decision. Therefore, ARS scientists at Beltsville, Maryland, developed a highly-reproducible molecular assay that distinguish between drug-sensitive and drug-resistant isolates, providing a rapid result when applied to Eimeria tenella. The team then overcame a technical challenge that prevented applying this approach to other important parasites (E. maxima and E. acervulina), adding a reducing agent to the culture medium and thereby extending the viability of sporozoites. This accomplishment enables the drug-sensitivity profile of parasites to be defined within 2 weeks, providing ample time for making decisions on the most efficacious drug for growing chickens on any particular broiler farm.
Review Publications
Jenkins, M.C., Parker, C.C., Obrien, C.N., Ritter, D. 2020. Correlation between Clostridium perfringens alpha- and netB-toxin and chick mortality in commercial broiler farms during different anticoccidial control programs. Avian Diseases. 64(3):401-406. https://doi.org/10.1637/aviandiseases-D-19-00118.
Cervantes, H.M., McDougald, L.R., Jenkins, M.C. 2020. Coccidiosis. In Diseases of Poultry. (D.E. Swayne, ed.) Wiley Blackwell Press, Hoboken, NJ. P. 1193-1216.
Jenkins, M.C. 2019. Chapter 4: Vaccination. In Dubey, J.P., editor. Coccidiosis in Livestock, Poultry, Companion Animals, and Humans. Boca Raton, LA: CRC Press. P. 51-58.
Proszkowiec-Wegla, M.K., Miska, K.B., Schreier, L.L., Grim, C.J., Jarvis, K.G., Shao, J.Y., Vaessen, S., Sygall, R., Jenkins, M.C., Kahl, S., Russell, B.A. 2020. Effect of butyric acid glycerol esters on ileal and cecal mucosal and luminal microbiota in chickens challenged with Eimeria maxima. Poultry Science. https://doi.org/10.1016/j.psj.2020.06.022.
Hansen, V.L., Kahl, S., Proszkowiec-Wegla, M.K., Jiménez, S.C., Vaessen, S., Schreier, L.L., Jenkins, M.C., Russell, B.A., Miska, K.B. 2021. The effects of tributyrin supplementation on weight gain and intestinal gene expression in broiler chickens during Eimeria maxima-induced coccidiosis. Poultry Science. https://doi.org/10.1016/j.psj.2021.01.007.
