2008 Annual Report 1a.Objectives (from AD-416) 1. Conduct comparative genomics studies of Eimeria species of economic importance and identify genetic determinants of infection and virulence. 1-1. Discover biological determinants that drive drug resistance and immunovariability. Hypothesis: Biological determinants exist in strains and species of Eimeria that can be used as a tool for precision application of controls. 2. Identify genetic sequences and gene products of developmental stages of Eimeria that are associated with host responses during infection. 2-1. Genomics driven methods of Eimeria protein discovery: Identification of genes that are expressed in distinct developmental stages of Eimeria using high throughput EST analysis. Hypothesis: Genes that are expressed by the intracellular and/or invasive stages of the Eimeria life-cycle encode proteins that will serve as good vaccine target candidates, immunomodulators, or targets for novel control strategies. 2-2. Clone, express, and characterize proteins that by their structure or function or biological properties suggest they may be important in regulation of parasite or host processes. Hypothesis: Parasite proteins exist that interact with the host to modify the host environment or to adapt the parasite to the host so as to aid development of the parasite within host cells. These proteins can be used to design novel anti-coccidia control measures. 1b.Approach (from AD-416) Obtain nucleic acid sequences from Eimeria species that have different phenotypic characteristics, such as infection and virulence, and identify genes or functional mutations associated with these variances. Using molecular methods that can identify gene sequences that are expressed during the various stages of Eimeria development, upregulated during intracellular development, or are associated with parasite-host interactions. Study the functions of these expressed gene products in parasite development and determine parasite factors that are associated with immune evasion or induction of protective immunity. Conduct studies on antigenic variation and drug resistance in Eimeria with the goal of developing genetic markers for these traits. 3.Progress Report Using rapid isolation and detection methods, a species of Eimeria, namely E. praecox that was thought to represent a minor component of the coccidia population was found to be present in a large number of commercial broiler farms. Studies of pathology associated with infection were carried out, and showed substantial weight gain depression in infected chickens. Immune studies revealed that E. praecox elicits a non-specific response in the region of the gut invaded by E. maxima, and thus prevents intestinal lesions, but does not inhibit E. maxima immunity. Inoculation of day-old chickens with harmless Salmonella bacteria that produce an Eimeria protein elicited immunity against challenge infection with two Eimeria species- E. acervulina and E. tenella. Booster immunization with a second strain of Salmonella expressing a different Eimeria protein enhanced the immunity that developed, such that chickens receiving the primary and booster immunization showed no weight gain depression associated with E. acervulina challenge infection. Composition of Eimeria present in high as well as low performing poultry farms was surveyed using three genetic markers. Based on our preliminary data it appears that low performing facilities appear to contain more Eimeria species compared to the high producing farms. Additionally a highly pathogenic strain of E. maxima was isolated from the low production facilities. In an attempt to identify genetic markers of drug-resistance in Eimeria, chickens were infected with E. acervulina oocysts in the presence or absence of an ionophore drug that is regularly used by the poultry industry to control avian coccidiosis. At present, 4 passages of E. acervulina in the presence or absence of the drug has led to a strain that produces nearly the same number of oocysts that are excreted by chickens that have been challenged with E. acervulina in the absence of the ionophore. The macrophage inhibitory factor (MIF) from E. tenella and E. acervulina has been fully characterized at the gene level. Because this represents the first description of homologues of vertebrate cytokines in a parasitic single celled organism, MIFs from both Eimeria species were expressed as recombinant proteins. These proteins will now be tested on their effect in stimulating chicken macrophage cell lines. Eimeria from two species of gamebirds, chukars and pheasants, were characterized using molecular means. The sequences from these species have helped resolve the evolutionary history of Eimeria in galliforme birds (which include chickens). Our findings indicate that Eimeria in chickens is paraphyletic and may be more closely related to Eimeria in turkeys or gamebirds, rather than to other chicken Eimeria. This finding will have serious implications on devising control strategies in treating coccidiosis. The research relates to delivering improved control methods and interventions against mucosal diseases that are the most critical under NP 103, Component 2, Genetic and Biological Determinants of Disease susceptibility, Problem Satement 2C: Mucosal Diseases of Livestock and Poultry. 4.Accomplishments 1. Discovery of a species of Eimeria prevalent on commercial broiler farms. Live oocysts vaccines are being used with increasing frequency by the poultry industry to combat drug-resistance in Eimeria and to reduce the use of ionophores in poultry feed. A rapid recovery and detection method was used to assay litter samples from numerous commercial U.S. broiler farms, and revealed that Eimeria praecox was a major species present. Eimeria praecox was isolated and propagated, and used to study its pathogenicity. This species counteract the pathogenic effects of another Eimeria species, namely E. maxima, without affecting immunity. These findings indicate that E. praecox should be incorporated in live oocyst vaccines, and may prevent “vaccine effects” associated with vaccine delivery. The research relates to delivering improved control methods and interventions against mucosal diseases that are the most critical under Component 2, Genetic and Biological Determinants of Disease Susceptibility, Problem Statement 2C: Mucosal Diseases of Livestock and Poultry. 2. Preventing avian coccidiosis with harmless Salmonella. Alternatives to drug-treatment or live oocyst vaccines are desired to control coccidiosis on broiler farms. DNA coding for two different Eimeria proteins were inserted into harmless Salmonella, which were then used to immunize day-old chicks against coccidiosis infection. Chickens inoculated with these recombinant Salmonella bacteria were protected against weight loss associated with Eimeria acervulina challenge infection. This discovery may provide a vaccine that is effective against Eimeria and possibly against some strains of Salmonella. The research relates to delivering improved control methods and interventions against mucosal diseases that are the most critical under Component 2, Genetic and Biological Determinants of Disease Susceptibility, Problem Statement 2C: Mucosal Diseases of Livestock and Poultry. 3. Successful immunization of chickens against avian coccidiosis with a recombinant Eimeria protein. Although a number of recombinant proteins have been tested for protective effects against coccidiosis, none have shown complete efficacy in terms of clinical parameters, such as weight loss and intestinal lesions. The effectiveness of immunostimulatory complexes (ISCOMs) for immunization of chickens against avian coccidiosis was tested by incorporating a recombinant antigen (SO7) and novel compounds from plants native to Kazakhstan to immunize chicks by various routes of administration. It was found that ISCOMs were very effective in reducing pathology and weight gain reduction caused by E. tenella infection. In ovo immunization was most effective route of administration suggesting that ISCOMs could be developed into a very cost effective and efficient vaccination system for protecting of chickens against avian coccidiosis. The research relates to delivering improved control methods and interventions against mucosal diseases that are the most critical under Component 2, Genetic and Biological Determinants of Disease Susceptibility, Problem Statement 2C: Mucosal Diseases of Livestock and Poultry. 4. Identified a protein of Eimeria parasites that may be involved in the invasion of host cells. Inhibiting invasion of intestinal cells by Eimeria parasites would stop infection at an early stage and thereby prevent clinical features associated with this protozoal disease. Seminal studies were carried out on Serpin, a protein serine proteases inhibitor, in developmental stages of the coccidian parasite Eimera acervulina. Gene cloning and expression of the recombinant Serpin protein in Escherichia coli allowed for localization of the protein in unique vesicles inside the parasite. This is the first report of a Serpin in E. acervulina, and suggests that serine proteases and serine protease inhibitors are important in the biology of Eimeria and may serve as potential targets for controlling the parasite. The research relates to delivering improved control methods and interventions against mucosal diseases that are the most critical under Component 2, Genetic and Biological Determinants of Disease Susceptibility, Problem Statement 2C: Mucosal Diseases of Livestock and Poultry. 5. Genomic analysis of Eimeria parasites. Determining which gene sequences are turned on in different developmental stages of the major species of Eimeria would greatly assist in figuring out common and unique intracellular processes. The DNA sequence of approximately 3,000 expressed sequence tags (ESTs) from second generation E. acervulina merozoites was determined and analyzed. A database and a pipeline for sequence deposition and annotation was developed to assist ARS and other researchers in discovering new genes. This is the first large scale effort in identification of genes expressed in E. acervulina. The preliminary analysis suggests that there is a large amount of genetic divergence between E. acervulina and E. tenella suggesting that control measures may need to be tailored to each species. The research relates to delivering improved control methods and interventions against mucosal diseases that are the most critical under Component 2, Genetic and Biological Determinants of Disease Susceptibility, Problem Statement 2C: Mucosal Diseases of Livestock and Poultry. 6. Molecular analysis of an Eimeria parasite in wild game birds. Avian coccidiosis is not restricted to broiler and layer chickens, but is prevalent in a wide variety of avian species, particularly those raised in close proximity to one another. In recent years, a number of wild game bird producers throughout the US have been plagued with high bird mortalities which they ascribed to coccidiosis. Eimeria from chukar partridges housed in a commercial wild-game bird operation were isolated and examined by both morphometric and molecular analysis. The results indicate high numbers of a single Eimeria species in these operations, and indicate that preventive measures to control coccidiosis (feed medication, biosecurity) should help to prevent future outbreaks. The research relates to delivering improved control methods and interventions against mucosal diseases that are the most critical under Component 2, Genetic and Biological Determinants of Disease Susceptibility, Problem Statement 2C: Mucosal Diseases of Livestock and Poultry. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of Active CRADAs 1 Number of New Commercial Licenses Executed 5 Number of Non-Peer Reviewed Presentations and Proceedings 5 Review Publications Jenkins, M.C., Allen, P., Wilkins, G., Klopp, S., Miska, K.B. 2008. Eimeria praecox infection ameliorates effects of e. maxima infection in chickens. 155:10-14. Kniel, K., Shearer, A., Cascarino, J., Wilkins, G., Jenkins, M.C. 2007. High hydrostatic pressure and uv light treatment of produce contaminated with eimeria acervulina as a cyclospora cayetanensis surrogate. Journal of Food Protection. 70:2837-2842.