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ARS Home Northeast Area Beltsville, Maryland (BARC) Beltsville Agricultural Research Center Animal Parasitic Diseases Laboratory Research





- Jenkins, Mark
- 2023
- 2022
- Publications
- Animal Health (103)

Research Project: Developing Improved Control Strategies for Avian Coccidosis **Location:
                            Animal Parasitic Diseases Laboratory** 2023 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**
                                

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), Eimeria maxima sensitive or resistant to zoalene were produced and extracted for high quality deoxyribonucleic acid (DNA). Sequencing of zoalene-resistant and zoalene-sensitive E. maxima genomes was carried out using Minion Nanopore and Illumina sequencing technologies. HiC analysis of the parental E. maxima genome is being carried out to assign these sequences to individual chromosomes, which will allow scaffolding of sequences from zoalene-sensitive and zoalene-resistant lines and the eventual identification of genetic markers of zoalene resistance. 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, 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 using the water system in poultry houses was optimized to deliver Eimeria oocysts to young broiler chicks. Vaccine uptake by young chicks via water delivery was excellent which should lead to lower incidence of necrotic enteritis. Parameters such as volume of stock solution and number of Eimeria doses were refined during several field trials with a commercial broiler company. Patent protection for this technology is being sought. 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.

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. One technology that has been found useful for delivering Eimeria vaccines is to incorporate recombinant Eimeria proteins into harmless mutant Salmonella. A National Institute of Food and Agriculture (NIFA) grant was awarded to University of Florida in conjunction with a member of this project to further develop Salmonella delivery vectors. This technology has been found effective in immunizing chicks against Clostridium perfringens for prevention 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 species on poultry farms may help explain why some farms experience more frequent outbreaks of coccidiosis and necrotic enteritis (NE). Litter samples were collected from 8 poultry farms at 0, 2, and 4 weeks of growout during a vaccine program. Eimeria oocysts were enumerated by microscopy while DNA was extracted for PCR directed to ITS1 (7 different PCRs) or SCO (metagenomics using single-copy orthologues (SCO). The latter was found useful for determining the Eimeria species diversity in a litter sample. This work will benefit poultry producers and growers by providing a technique to characterize the Eimeria population in litter and inform poultry producers whether complete clean-out of the house to remove highly virulent or drug-resistant 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) revealed several genes that were either up- or down-regulated during parasite development. The gene (EAH33530) coding for a protein associated with the parasite outer membrane was cloned and expressed in Escherichia coli using recombinant DNA technology. Staining of 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.

Producing recombinant Eimeria proteins in Escherichia coli is used as a means of avoiding purifying a native antigen away from the thousands of proteins in Eimeria. The gene coding for a particular protein is inserted in E. coli, but only after the sequence has been adjusted to use codons preferred by E. coli in a method called codon optimization. In theory, codon optimization should lead to high expression levels in the bacterial host. However, our research has shown that often the level of recombinant protein remains low regardless of whether the sequence was codon-optimized or not. These data suggest that codon usage in Eimeria, as has been observed for other microorganisms, is more complicated than originally thought. These findings should be helpful to researchers attempting similar cloning studies. **Accomplishments**
                                

1. Improving the efficiency and uniformity of vaccination against avian coccidiosis. Currently, vaccinating broiler chickens against coccidiosis is accomplished by spraying newly-hatched chicks, while in a shipping container, with an aqueous solution containing a mixture of 3 different Eimeria species. While this method is easy to perform, 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 delivering this vaccine through drinking water. Using this 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. At least 6 months of the year, typically Fall and Winter, broiler chickens are grown on feed containing anticoccidial drugs to prevent outbreaks of coccidiosis and associated necrotic enteritis (NE). The problem for poultry growers is that there is no prior knowledge of the drug sensitivity of Eimeria in the poultry house prior to chicks being placed therein. If Eimeria are resistant to an ionophore, such as monensin, then adding monensin to feed will be ineffective. Current methods of assessing Eimeria drug-sensitivity is time-consuming and labor intensive, involving the use of susceptible chickens grown in the presence or absence of a given drug. These assays take at least 2 months to obtain a drug-sensitivity profile; this is too long to be useful for the poultry grower who has a window of opportunity of no more than 3 weeks before another set of chicks in placed in the broiler house. 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. Replication of drug-resistant strains of E. maxima in the presence of the drug was not affected by drug treatment. The genomic DNA sequence from drug-resistant or drug-sensitive E. maxima strains was obtained and DNA sequencing identified a number of genetic changes that may be responsible for drug resistance. This information will provide a rapid way to determine whether drug-resistant Eimeria are present in litter will provide ample time for making decisions on whether to switch to another drug prior to chicks arriving at a particular broiler farm. **Review Publications**
                            *Jenkins, M.C., Tucker, M., Parker, C.C., Obrien, C.N., Miska, K.B. 2022. Cloning and expression of a cDNA coding for Eimeria acervulina 25 kDa protein associated with oocyst and sporocyst walls. Veterinary Parasitology. 309.Article 109762. https://doi.org/10.1016/j.vetpar.2022.109762.*
                                *Jenkins, M.C., Cline, J., Parker, C.C., Obrien, C.N., Burleson, M., Schaefer, J. 2022. Administering Eimeria maxima oocysts through drinking water improves coccidiosis vaccine uptake in broiler chickens. Journal of Applied Poultry Research. 32(1).Article e100312. https://doi.org/10.1016/j.japr.2022.100312.*
                                *Jenkins, M.C., Parker, C.C., Obrien, C.N., Campos, P., Tucker, M.S., Miska, K.B. 2023. Effects of codon optimization on expression in Escherichia coli of protein-coding DNA sequences from the protozoan Eimeria. Journal of Microbiological Methods. 211. Article 106750. https://doi.org/10.1016/j.mimet.2023.106750.*


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