Location: Animal Parasitic Diseases Laboratory
2022 Annual Report
Objectives
Objective 1: Develop improved vaccines and immunization strategies for chickens that are consistent with industry practices to provide lasting protection against coccidiosis.
Subobjective 1a: Develop in-house vaccination of newly-placed broiler chicks using gelatin beads containing a mixture of Eimeria spp. oocysts.
Subobjective 1b: Develop in ovo immunization of chickens against avian coccidiosis using recombinant Eimeria proteins linked to nanoparticles (NP).
Objective 2: Characterize the molecular epidemiology of Eimeria on poultry farms to determine antigenic variation in and between farms and determine differences in pathogenicity of Eimeria strains for the targeted development of more efficacious therapeutic and preventive strategies.
Objective 3: Sequence and annotate the genomes and transcriptomes of major Eimeria species, including characterizing genetic markers that allow for the development of drug resistance in Eimeria species.
Approach
Incorporate Eimeria oocysts into gelatin beads as a way to vaccinate day-old chickens against coccidiosis. Imunize chickens in ovo with recombinant Eimeria proteins that have been linked to nanoparticiles and then test for efficacy of vaccination by challenging chickens with Eimeria parasites. Evaluate the population structure of Eimeria on commercial poultry farms by collecting litter and processing for Eimeria oocysts followed by oocyst enumeration and molecular analyses to possibly identify virulent strains of the parasite that are responsible for necrotic enteritis and decreased performance. Obtain the complete genome sequence of the major Eimeria, specifically E. acervulina, E. maxima, and E. tenella. Attempt to isolate drug-resistant strains of Eimeria and compare the genome sequences of these to drug-sensitive strains to identify markers of drug resistance toward the goal of producing a rapid molecular test for anticoccidial drug resistance.
Progress Report
The project hired a permanent full-time biological technician to support work on Objectives #2 and #3. Additionally, a new lab space was prepared with the assistance of the new technician to further genomic research activities related to generation of Eimeria metagenomic and genomic research objectives.
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, a rapid method to assess sensitivity of Eimeria parasites in poultry houses is sorely needed. Based on the success in drug-sensitivity testing of malaria parasites (Plasmodium resistance to chloroquine), we passaged Eimeria maxima 9 times in the presence or absence of zoalene. We thereby produced 3 lines of stable zoalene-resistant E. maxima, producing similar numbers of oocysts production in the presence or absence of zoalene. We are now seeking to identify genetic markers of zoalene resistance by comparing genomes of these and wild type strains. This work will benefit poultry producers and growers by pointing to which drugs to discontinue using to control coccidiosis.
Consumers and industry increasingly seek chickens produced without antibiotics. These require alternative methods to prevent avian coccidiosis, such as vaccination. At present, hatcheries spray live Eimeria oocyst vaccines onto 100 chicks in a shipping box; but we have 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. Our scientific team, mentoring undergraduate engineering students supported by an ARS Innovation Fund grant, designed automated gel bead-making and delivery devices. We submitted a patent disclosure to the ARS Mechanical Patent Committee, but the committee advised that we instead transfer the technology to producers through a Cooperative Research and Development Agreement (CRADA) with an equipment manufacturer.
We optimized a method to deliver Eimeria vaccine oocysts to young broiler chicks via watering systems. We demonstrated excellent vaccine uptake by young chicks, which should lead to lower incidence of necrotic enteritis. We refined parameters such as volume of stock solution and number of Eimeria doses during several field trials with a commercial broiler company. We are seeking patent protection for this technology. This work will benefit poultry producers and growers by providing a way to efficiently vaccinate chickens against coccidiosis and thereby prevent outbreaks of coccidiosis and associated diseases like necrotic enteritis.
Although 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 challenge infection. We therefore incorporated a protective Eimeria maxima antigen, namely EmaxIMP1, into nanoparticles (NP) using standard methodology and showed excellent protection in chickens raised in battery cages and floor pens. To make this approach practical, we undertook studies to inject NP-EmaxIMP1 in ovo in the amnion or air-cell. Numerous vaccination studies conferred partial protection against challenge but suggest that booster immunizations may be required. ARS is negotiating with a private animal health company to develop alternative ways of delivering EmaxIMP1. 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 species and Clostridium perfringens on poultry farms may help explain why some farms experience more frequent outbreaks of coccidiosis and necrotic enteritis (NE) caused by the respective pathogens. We collected litter samples from 8 poultry farms at 0, 2, and 4 weeks of growout during a vaccine program. We enumerated Eimeria oocysts by microscopy and evaluated the presence of C. perfringens toxin by polymerase chain reaction (PCR). Statistical analyses revealed a direct correlation between the presence and relative abundance of netB or Tpel toxin genes in the C. perfringens population and NE-associated mortality in young chicks. This work will benefit poultry producers and growers by providing a technique to characterize the C. perfringens population in litter and inform poultry producers whether complete clean-out of the house to remove highly virulent strains is warranted.
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. Analysis of RNA was isolated from Eimeria acervulina oocysts at various times during sporulation (formation of sporozoites), revealing several genes that were either up- or down-regulated during parasite development. The gene coding for a protein associated with the parasite outer membrane was cloned and expressed in Escherichia coli using recombinant DNA technology. Staining E. acervulina confirmed the presence of this protein on the parasite’s surface. Moreover, this protein appears to elicit antibodies in chickens infected with E. acervulina. This information will be useful in designing drugs that can dampen gene expression and thereby prevent invasive stage sporozoites from forming. This work will benefit poultry producers by providing a target for drugs or as a candidate vaccine against coccidiosis infection.
Poultry diagnostic laboratories identify coccidiosis problems by performing necropsies on dead chickens submitted to them by poultry companies. Diagnosticians smear intestinal samples onto glass slides for examination via microscopy using a technique called micro-oocyst counting. This technique is time-consuming and requires highly experienced personnel to enumerate Eimeria oocysts that are extremely small (10-30 um in diameter). Moreover, this method fails to differentiate among species of Eimeria. We therefore developed a procedure to extract DNA from Eimeria-infected intestinal tissue and use PCR to identify those Eimeria present. This technique will provide an objective measure of Eimeria infection intensity in chickens possibly afflicted with coccidiosis. This technique will help poultry producers by identifying Eimeria that may have developed drug-resistance or have evaded vaccination, and thus indicate a change in coccidiosis control.
Accomplishments
1. Improving the efficiency and uniformity of vaccination against avian coccidiosis. Currently, poultry producers vaccinate broiler chickens against coccidiosis by spraying newly hatched chicks, in a shipping container, with an aqueous solution containing a mixture of 3 different Eimeria species. ARS scientists in Beltsville, Maryland, determined, however, that 50-70% of these 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 delivering this vaccine through drinking water. This alternative delivery method led to greater than 90% of chicks ingesting the Eimeria vaccine. These efforts should provide the poultry industry with an effective coccidiosis vaccine that is practical and adaptable to current industry practices.
2. Improving sensitivity testing of Eimeria parasites to anticoccidial drugs. To prevent outbreaks of coccidiosis, most producers grow broiler chickens on feed containing anticoccidial drugs to prevent in the fall and winter months. Where parasites resist ionophores such as monensin, this approach fails. Unfortunately, growers do not know, ahead of time, whether these drugs will control growth of the parasites in their facility because current methods to assess drug-sensitivity are labor intensive and requires at least 2 months to complete. A poultry grower has no more than 3 weeks to make a management decision for the next broiler flock. Therefore, ARS scientists at Beltsville, Maryland, developed lines of Eimeria maxima that are resistant or sensitive to a commonly used anti-coccidial drug by repeatedly passaging the parasite in the presence or absence of the drug. These strains provide the means to seek adaptations that explain drug resistance. A rapid means to identify drug resistance would provide timely means to make management decisions and reduce overuse of anticoccidial drugs.
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.
Campos, P., Miska, K.B., Kahl, S., Jenkins, M.C., Shao, J.Y., Proszkowiec-Wegla, M.K. 2022. Effects of Eimeria tenella on cecal luminal and mucosal microbiota in broiler chickens. Avian Diseases. 66(1):1-14. https://doi.org/10.1637/21-00068.
Jenkins, M.C., Obrien, C.N., Parker, C.C., Thompson, P.C., Fitzcoy, S., Bautista, D. 2022. Polymerase chain reaction directed to Eimeria ITS1 rDNA or single copy orthologues corroborates standard micro-oocyst analysis of intestinal tissue from E. acervulina, E. maxima, or E. tenella-infected chickens. Avian Diseases. 66(2). https://doi.org/10.1637/aviandiseases-D-22-00001.
