2006 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Foodborne parasites cause significant disease in humans, domestic stock and companion animals resulting in great economic loss. Rapid, accurate, and sensitive methods are needed to better identify and quantify coccidian parasites of zoonotic potential, such as Toxoplasma gondii, and to discriminate these from related species in the genera Sarcocystis, Neospora, Hammondia, and Goussia that may pose little or no zoonotic risk. More completely understanding parasite diversity, phylogeny, and population genetics will foster more effective interventions. Given the diminishing acceptability of antibiotics to promote food animal growth and the growing consumer interest in organic food production, such information is urgently needed. The genome of T. spiralis, soon to be sequenced in its entirety, needs to be exploited as a source of genetic markers useful in tracking outbreaks and improving interventions. And future efforts to enumerate and diagnose other parasitic helminthes need to be "barcoded" using genetic signatures that provide immediate comparability to similar efforts with other organisms. To define food safety and security threats posed by certain parasites, and to improve their detection and identification, progress in the molecular systematics and comparative population genetics will be accomplished through four integrated research objectives:. 1)molecular means will be developed to diagnose an increasing diversity of coccidian parasites in major food animals, employing single-gene and genomic approaches;. 2) the genetic diversity and evolutionary relationships among coccidia of fish will be established in order to better define their potential risk to food safety and security, and in order to better understand their relationship to the Eimeriidae (including the agents of avian coccidiosis) and the Sarcocystidae (including the agent of human toxoplasmosis);. 3) the historical and ongoing interactions among wildlife and livestock reservoirs of Toxoplasma gondii will be clarified through comparative population genetic analysis and. 4)population genetic tools for key parasitic helminthes will be developed. The Project Plan develops diagnostic tools for detecting pathogenic microorganisms under Sections 1.2.1 Detection and 1.2.9 Food Security. Additionally, it exploits sequence and annotation information, genomics and proteomics to elucidate the phylogenetic relationships and relative risk among parasites of critical food safety concern under Sections 1.2.5 (Omics); 1.2.7 Risk Assessment as described in the 2006-2010 NP 108 Action Plan. The research also addresses Agency Performance Measure 3.1.2: Develop and transfer to Federal agencies and the private sector systems that rapidly and accurately detect, identify, and differentiate the most critical and economically important food-borne microbial pathogens. 2.List by year the currently approved milestones (indicators of research progress) Year 1 (2006) Provide molecular phylogenetic context for parasite reports and redescriptions (continues in years 2-5). Determine sensitivity of PCR detection of S. cruzi DNA in local retail meats. Document variation in the small subunit rDNA from morphologically defined Goussia from cyprinid and percid freshwater hosts. Complete analysis of Sarcocystis neurona microsatellite variation and initially characterize markers for Besnoitia tarandi. Refine methods for amplifying COXI from selected nematodes; identify T. spiralis repeats as genome matures. Year 2 (2007) Explore generalized quantitative PCR assays for coccidian food borne parasites. Determine whether phylogenetic patterns indicated by SSU rDNA in preliminary sample are corroborated by other genetic loci. Assess the diversity and geographic structure of B. tarandi; ascertain whether these markers may also be used to study cattle and horse parasites. If T. spiralis assembly permits, verify that candidate loci are distributed across chromosomes; optimize amplification and labeling of msat loci. Year 3 (2008) Refine microdissection methods for isolating encysted coccidian parasites; explore the application of whole genome amplification methods to such material. Conduct sensitivity analysis of parasite DNA in various fish tissues with samples "spiked" with known templates. Optimize assays as needed. Evaluate high throughput, parallel T. gondii genotyping systems. Analyze diverse geographic collection of T. spiralis isolates using markers developed in years 1 and 2. Year 4 (FY 2009) Pilot methods to clone and sequence libraries generated by whole genome amplification from microdissected tissue cysts. Commence survey of major food fish for "molecular evidence" of coccidian parasitism. Evaluate whether available T. gondii classification systems identify strains also identifiable using statistical methods free of a priori assumptions. Identify whether other genotypes of Trichinella may be characterized by subset of loci cloned in silico from T. spiralis. Year 5 (FY 2010) Commence development of a reference collection of genomic libraries from tissue cyst forming coccidia for immediate or eventual sequencing (depending on current and projected costs). Complete survey of major food fish for "molecular evidence" of coccidian parasitism. Assess and compare the stability and epidemiological prominence of T. gondii, S. neurona, and B. tarandi strains. Describe markers useful in tracking T. spiralis molecular epidemiology; complete studies of T. spiralis biogeography. 4a.List the single most significant research accomplishment during FY 2006. The single most important accomplishment during FY 2006 was completing an analysis of population genetic variation in Sarcocystis neurona and related coccidia. This brought to fruition the efforts of the former postdoctoral fellow in the laboratory, and represented a "proof of principal" that informative markers could be derived, de-novo, from parasites about which we lacked a priori genomic information. This study provided the first evidence that Sarcocystis neurona represents a cohesive, relatively homogeneous population, that parasites causing illness in certain marine mammals are derived from the same population causing illness in horses, that gene flow among sexually reproducing parasites occurs at appreciable rates in North America, and that certain strains appear capable of perpetuating as asexual clones. 4b.List other significant research accomplishment(s), if any. A groundbreaking study on the systematics of parasites in the genus Trichinella was completed and published in the Proceedings of the National Academy of Science, USA (192192). This paper reconstructed the dissemination of this important zoonotic pathogen through space and among animal hosts, inferred that human infections likely preceded the domestication of swine, and proposed that T. spiralis established its current global distribution as people transported livestock. Significant progress was made in establishing high-resolution genotyping methods for parasites in the genus Trichinella. Manuscripts defining genes of ungulate lungworms (181556), canine coccidia (187108), an agent of equine besnoitiosis (178837), and of avian sarcocystosis (194971) were accepted for publication. 4c.List significant activities that support special target populations. Better understanding the array of potentially zoonotic intracellular parasites is of particular importance to pregnant women and to individuals with suppressed T-cell immunity such as induced by AIDS. Improving the ability to understand and manage parasitic threats may be especially useful to producers employing organic production methods that preclude antibiotic use. 4d.Progress report. The Project Plan was developed, submitted, and accepted "without revision" by OSQR's review panel. A travel fellowship was funded by the Cooperative Research Directorate, Directorate for Food, Agriculture, and Fisheries, of the Organization for Economic Cooperation and Development and completed during the Spring of 2006 in Budapest and Rome. An NRI grant proposal was submitted to the NRI with the objective of augmenting project objectives. Not funded. 5.Describe the major accomplishments to date and their predicted or actual impact. Identified previously unknown diversity among parasites of the genus Sarcocystis that infect livestock and wildlife when they ingest oocysts excreted by opossums and established means by which to differentiate pathogenic from nonpathogenic forms. Action Plan components: 1.1.1.1 and 1.1.1.2. Provided the molecular criteria that enabled the first experimental completion of the life cycle of Sarocystis. neurona, the causative agent of Equine Protozoal Myeloencephalitis, paving the way for subsequent identification of several natural intermediate hosts. This helped explain the broadening geographic distribution of this emerging parasitic disease by identifying this infection as prevalent in raccoons. Action Plan component 1.1.1.2. Documented symptomatic Sarcocystis neurona infections in Southern Sea Otters, underscoring the risk that such parasites pose not only to domestic animals such as the horse, but also to wildlife (in this case an endangered species). Identified a second, genetically distinct species of Sarcocystis infecting such Sea Otters. Described a new parasite species, Sarcocystis lindsayi, in opossums. Action Plan components 1.1.1.2 and 1.1.1.4. Developed the first microsatellite markers for defining strain variation and population structure of Sarcocystis neurona, an important veterinary parasite and an important comparative model for Toxoplasma gondii. When completed, this study will help elucidate why Toxoplasma lacks the genetic diversity expected for such a cosmopolitan and highly prevalent species. Action Plan components 1.2.1.2 and 1.3.1.1. Developed the first robust phylogenetic hypothesis for the genus Trichinella based on variation in several genetic loci using a comprehensive sample of all ecologically and genetically recognized parasite forms. From its first description in 1835 through much of the following century, the Genus Trichinella was considered monospecific. However, our analysis of variation in nuclear and mitochondrial ribosomal DNA and mitochondrial COXI indicates strong support for at least six encapsulated forms, including T. spiralis, as well as an additional three unencapsulated forms which represent a related, but distinct, evolutionary lineage. Our hypothesis provides a unique historical perspective on the biogeography and epizootiology of the group, indicating:. 1)all members of the genus share a close evolutionary relationship to the exclusion of other known nematodes;. 2)capsules evolved once in the history of the genus, and;. 3)both the encapsulated and non-encapsulated clades contain species that are now cosmopolitan, as well as species inhabiting more geographically and ecologically restricted ranges. Action Plan component 1.1.1.2. Documented genetic variation among isolates presumed to represent Hammondia heydorni, elucidating the diversity of coccidian parasites excreted by dogs that are morphologically identical to Neospora caninum, a causative agent of epidemic bovine abortion. Action Plan components 1.1.1.1, 1.1.1.2, and 1.3.1.1. Described or redescribed several coccidian parasitic species important to veterinary health and of potential zoonotic importance. 1.1.1, 1.1.1.2, and 1.3.1.1. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Genetic sequences were deposited in GenBank, making them available to the global research community free of charge. An efficient publication pace has made significant scientific findings widely known to the research community. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). McBride, J. May, 2002. Defining Risk from Meat-borne Parasites. Agricultural Research Magazine. Kuehn, B. July 2002. American Journal of Veterinary Research. Review Publications Asmundsson, I.M., Dubey, J.P., Rosenthal, B.M. 2006. A genetically diverse but distinct north american population of sarcocystis neurona includes an overrepresented clone described by twelve microsatellite alleles; infection, genetics and evolution. Infection, Genetics and Evolution. 6(5):352-360.