2007 Annual Report 1a.Objectives (from AD-416) Discover vaccine candidates that are effective in preventing and controlling neosporosis. Determine the role of management practices and on-farm exposure to dogs, rodents, and wildlife in the risk of contracting neosporosis. Determine life cycle, epidemiology, and control of Sarcocystis neurona. 1b.Approach (from AD-416) Vaccine candidates will be discovered using genomics and proteomics tools and whole-cell and subunit vaccines will be tested in efficacy studies using the congenital and adult mouse model and the ovine model. Feed contamination with infective oocysts from the definitive canine host and rodent intermediate hosts will be determined as risk factors for horizontal transmission of neosporosis in cattle. Tissue parasitism in horses at different time periods after feeding various doses of S. neurona sporocysts will be evaluated. A search for additional intermediate and definitive hosts of S. neurona will also be conducted. The equine model will be characterized for testing vaccine efficacies and therapeutic interventions. Testing of ponazuril will continue in experimentally-infected horses. The gamma-interferon knock-out mouse model will continue to be used in the search for efficacious drugs against S. neurona. The study of immune basis of clinical EPM, S. neurona infections in various mouse models will continue. The genetic diversity and geographic distribution of S. neurona, microsatellite of new isolates from wildlife will continue. 4.Accomplishments Design and production of a high density oligo DNA microarray for N. caninum: Currently, there is no microarray to compare gene expression profiles among different strains of N. caninum. A high density oligo DNA microarray with about 6000 unique genes was designed and produced. This microarray enables us to compare pathogenic and non- or low-pathogenic strains of N. caninum and identify proteins responsible for Neospora virulence and pathogenesis. Some of these proteins will be qualified as vaccine candidates and be tested for efficacy in protection against neosporosis in animal models. The mouse model has been successfully used to test vaccine candidates. Impacts NP 103 Action Plan Component 5: Countermeasures to Prevent and Control Reproductive and Neonatal Diseases, Problem Statement 5B: Neosporosis Ponazuril reduces clinical signs and delays seroconversion in horses challenged with Sarcocystis neurona: The ability of ponazuril to prevent or limit clinical signs of equine protozoal myeloencephalitis (EPM) after infection with Sarcocystis neurona was evaluated. All horses in the control group developed neurologic signs. Only 71% or 40% of the horses treated with 2.5mg/kg or 5.0 mg/kg ponazuril developed neurologic abnormalities, respectively. Seroconversion was decreased in the 5.0 mg/kg group compared with the control horses (100% vs. 40%). Post-mortem examination showed that mild-to-moderate, multifocal signs of neuron-inflammation were observed. These results confirm that treatment with ponazuril at 5.0 mg/kg minimizes, but does not eliminate, infection and clinical signs of EPM in horses. Impacts NP 103 Action Plan Component 5: Countermeasures to Prevent and Control Reproductive and Neonatal Diseases, Problem Statement 5B: Neosporosis Demonstration of feral rodents as intermediate hosts of N. caninum: Using molecular techniques in conjunction with microscopic examination or histologic staining we showed that red foxes and coyotes are possible definitive hosts of Neospora caninum and that feral rodents, such as mice and rats, can serve as intermediate hosts of this parasite. These findings suggest that a sylvatic cycle of transmission between wild animals and domesticated animals on dairy farms may play a role in the epidemiology of neosporosis. Impacts NP 103 Action Plan Component 5: Countermeasures to Prevent and Control Reproductive and Neonatal Diseases, Problem Statement 5B: Neosporosis Analysis of NAHMS Dairy 1996, 2002, and 2007 for N. caninum: NAHMS Dairy 1996, 2002, and 2007 surveyed ~20 participating states representing 83% of the U.S. milk cows and 80% of U.S. dairy operations. Prevalence of N. caninum was not evaluated during these studies. Sera and collected farm management surveys are being evaluated for N. caninum prevalence and associated on-farm risk factors. Farm management and biosecurity measures which can be implemented by producers to maintain a barrier between N. caninum infective stages and dairy cattle in the U.S. will be identified. A risk management protocol based on identification of critical control points for on-farm exposure to N. caninum will be developed and provided to producers. Impacts NP 103 Action Plan Component 5: Countermeasures to Prevent and Control Reproductive and Neonatal Diseases, Problem Statement 5B: Neosporosis The kinetics of S. neurona invasion: The kinetics of S. neurona invasion is determined in the equine model. Six ponies were orally inoculated with 250 x 106 S. neurona sporocysts via nasogastric intubation and killed on days 1, 2, 3, 5, 7, and 9 post-inoculation (PI). At necropsy, tissue samples were examined for S. neurona infection. The parasite was isolated from the mesenteric lymph nodes at 1, 2, and 7 days PI, the liver at 2, 5, and 7 days PI, and the lungs at 5, 7, and 9 days PI by bioassays in interferon gamma gene knock out mice (KO) and from cell culture. Microscopic lesions consistent with an EPM infection were observed in brain and spinal cord of ponies killed 7 and 9 days PI. Results suggest that S. neurona disseminates quickly in tissue of ponies. Impacts NP 103 Action Plan Component 5: Countermeasures to Prevent and Control Reproductive and Neonatal Diseases, Problem Statement 5B: Neosporosis N. caninum in wildlife species: N. caninum infects many mammalian species. To thoroughly understand the reservoir of N. caninum in wildlife species nationally and internationally, a number of wild animals were studied collaboratively. Results indicate that in certain areas of Spain, N. caninum is present in wildlife, especially in red deer. These results suggest that sylvatic cycles may influence the prevalence of infection in cattle farms in those areas. To our knowledge, this is the first report of antibodies to N. caninum in wildlife from Spain and the first report of N. caninum antibodies in barbary sheep and wild boar. Impacts NP 103 Action Plan Component 5: Countermeasures to Prevent and Control Reproductive and Neonatal Diseases, Problem Statement 5B: Neosporosis N. caninum in white-tailed deer, foxes and coyotes: White-tailed deer serve to maintain the N. caninum life cycle in the wild. Sera from white-tailed deer from south central Wisconsin and southeastern Missouri, USA were tested for antibodies to N. caninum by Western blot analyses and two indirect ELISAs. The results indicate the existence of N. caninum antibodies in MO and WI deer, and that Western blot is superior to ELISA for serologic testing when using degraded blood samples collected from deer carcasses. In another study, the performance and agreement of serological assays (ELISA, IFAT, Neospora caninum agglutination test and immunoblot) using reference sera and field sera from foxes and coyotes were evaluated and the N. caninum seroprevalence in foxes and coyotes on Prince Edward Island, Canada was estimated. Although the seroprevalence observed was low, N. caninum antibodies were present in foxes and coyotes on Prince Edward Island (PEI) and their role in the N. caninum epidemiology needs further study. Impacts NP 103 Action Plan Component 5: Countermeasures to Prevent and Control Reproductive and Neonatal Diseases, Problem Statement 5B: Neosporosis Comparison of commercial and in-house N. caninum antibody assays: The aims of this study were to evaluate the performance and agreement of various commercial and in-house Neospora caninum antibody assays used in dairy cattle in North America, and to investigate reproducibility of two assays performed in different laboratories. From 1998 to 2005, three enzyme linked immunosorbent assays (ELISAs, a competitive ELISA-VMRD Inc., an indirect ELISA-Biovet Inc., and another indirect ELISA-Herdchek IDEXX Corp.), two indirect fluorescent antibody tests (IFATs, VMRD Inc., and in-house USDA) and one N. caninum agglutination test (NAT, in-house USDA) were utilized to test 397 randomly selected dairy cattle serum samples from 34 herds in eastern Canada for antibodies to N. caninum. Sensitivity was high for all assays except the NAT. Specificity was high for all assays except for one indirect ELISA. The performance characteristics observed for most assays in this study make them useful for screening antibodies to N. caninum in cattle. Impacts NP 103 Action Plan Component 5: Countermeasures to Prevent and Control Reproductive and Neonatal Diseases, Problem Statement 5B: Neosporosis. Sarcocystis sp. from the African grey parrot: A Sarcocystis sp. is reported from a naturally infected African grey parrot from Costa Rica. Only mature sarcocysts were found measuring up to 2 mm in length and up to 750 um in width. Bradyzoites in sections were 5.4- 6.6 x 1.3-2.0 um in size. Sequencing of the small subunit and first internal transcribed spacer portions of ribosomal DNA related this parasite to, but distinguished it from, previously characterized species of Sarcocystis that encyst in the musculature of birds and complete their sexual development in New World opossums. Impacts NP 103 Action Plan Component 5: Countermeasures to Prevent and Control Reproductive and Neonatal Diseases, Problem Statement 5B: Neosporosis 5.Significant Activities that Support Special Target Populations None 6.Technology Transfer Number of new CRADAs and MTAs 1 Number of invention disclosures submitted 1 Number of non-peer reviewed presentations and proceedings 3 Review Publications Almeria, S., Vidal, D., Ferrer, D., Pabon, M., Fernandez, M., Ruiz-Fons, F., Alzaga, V., Marco, I., Calvete, C., Lavin, S., Gortazar, C., Lopez-Gatius, F., Dubey, J.P. 2006. Seroprevalence of Neospora caninum in non-carnivorous wildlife from Spain. Veterinary Parasitology. 143:21-28. Anderson, T., Dejardin, A., Howe, Dubey, J.P., Michalski, M.L. 2007. Neospora caninum antibodies detected in Midwestern white-tailed deer. Veterinary Parasitology. 145:152-155. Dubey, J.P., Lindsay, D.S. 2006. Neosporosis, Toxoplasmosis, Sarcocystosis. Veterinary Clinics of North America. 22:645-671. Dubey, J.P., Schares, G., Ortega-Mora, L.M. 2007. Epidemiology and control of neosporosis and neospora caninum. Clinical Microbiological Reviews. 20:323-367. Dubey, J.P., Rosenthal, B.M., Morales, J.A. 2006. Morphologic and genetic characterization of Sarcocystis sp sarcocysts from the African grey parrot, psittacus erithacus. Acta Parasitologica. 51(3):161-168. Furr, M., Mckenzie, H., Saville, W.J., Dubey, J.P., Reed, S.M., Davis, W. 2006. Prophylactic administration of ponazuril reduces clinical signs and delays seroconversion in horses challenged with sarcocystis neurona. Journal of Parasitology 92:637-643. Jenkins, M.C., Parker, C.C., Hill, D.E., Pinckney, R.D., Dubey, J.P. 2007. Neospora caninum detected in wild rodents. Veterinary Parasitology. 143:161-165. Sellon, D.C. and J.P. Dubey. 2007. Equine protozoal myeloencephalitis. In: Sellon, D.C. and Long, M.T., editors. Equine Infectious Diseases. St. Louis, MO:Elsevier Inc. p. 453-464. Wapenaar, W., Barkema, H.W., Schares, G., Rouvinen-Watt, K., Zeijlemaker, L., Poorter, B., O¿Handley, R.M., Kwok, O.C., Dubey, J.P. 2007. Evaluation of four serological techniques to determine the seroprevalence of Neospora caninum in foxes (Vulpes vulpes) and coyotes (Canis latrans) on Prince Edward Island, Canada. Veterinary Parasitology. 145:51-58. Wouda, W., Snoep, J.J., Dubey, J.P. 2006. Eosinophilic myositis due to Sarcocystis hominis in a beef cow. Journal of Comparative Pathology. 135:249-253.