2008 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. 3.Progress Report Neospora caninum causes bovine neosporosis which results in high levels of abortion. Our recent study determined the protective efficacy of two Neospora antigens, Neospora cyclophilin (NcCyP) and NcSRS2. Mice vaccinated with rNcCyP, rNcSRS2, or both rNcCyP and rNcSRS2 produced high levels of NcCyP or NcSRS2 antibodies. Vaccines formulated with NcCyP alone, NcSRS2 alone, or NcCyP plus NcSRS2 had a protection rate (% animals free from infection) of 86%, 60%, or 79%, respectively, in the mouse model; this was much higher than the protection rate (less than 14%) in the control groups. These results indicate that NcCyP is an efficacious vaccine candidate that may be used to protect against Neospora infection in cattle. Clinical neosporosis was diagnosed in a litter of five pups born to a Beagle bitch; medication with Clindamycin was found to improve clinical conditions but not eliminate N. caninum infection. This is the first report of finding N. caninum DNA in feces of naturally infected dogs in Costa Rican dairy farms. By collaborating with scientists from India, antibodies to N. caninum were determined in 184 dogs (126 rural, 58 urban) and also found in 16.8% of the animals using a commercial ELISA. This is the first report of N. caninum infection in canids from India. Sarcocystis neurona is an important cause of neurological disease in horses (equine protozoal myeloencephalitis, EPM) and sea otters in the United States. By analyzing multiple isolates, we determined that geographic barriers may limit S. neurona dispersal and that only a limited subset of possible parasite genotypes may have been introduced to recently established opossum populations. Results confirm that diverse intermediate hosts share the opossum as a common infection source. A gene family of surface antigens is expressed by merozoites of S. neurona. These surface proteins, designated SnSAGs, are immunodominant and therefore excellent candidates for development of EPM diagnostics or vaccines. Our data indicated that there is heterogeneity in the surface antigen composition of different S. neurona isolates, which is an important consideration for the development of serologic tests and prospective vaccines for EPM. Furthermore, the diversity reported herein likely extends to other phenotypes reflecting strain virulence, and may have implications for the phylogeny of the Sarcocystis species that undergo sexual stages of their life cycle in opossums. Additionally, a new species of Sarcocystis was recently recognized. Histological sections of 374 black bears from Pennsylvania were examined for sarcocysts and we demonstrated 3 sarcocysts in 3 bears. Sarcocysts from 2 bears were considered a new species, Sarcocystis ursusi. Sarcocysts of S. ursusi n.sp. were microscopic and contained only bradyzoites. The sarcocyst from the third bear was structurally different from S. ursusi. This research relates to epidemiology and control of neospora caninum and related protozoa that aligns with NP103 objectives under Component 5, Countermeasures to Prevent and Control Reproductive and Neonatal Disease, and Problem Statement 5B: Neosporosis. 4.Accomplishments 1. Development of a vaccine against Neospora caninum infection in the mouse model: Neospora caninum is the causal agent of bovine neosporosis which results in high levels of abortion. The present study determined the protective efficacy of two Neospora antigens, Neospora cyclophilin (NcCyP) and NcSRS2. Recombinant NcCyP or NcSRS2 were tested either alone or in combination and formulated with adjuvant ImmuMax-SR and CpG. Mice vaccinated with rNcCyP, rNcSRS2, or both rNcCyP and rNcSRS2 responded with high levels of NcCyP or NcSRS2 specific antibodies. Vaccines formulated with NcCyP alone, NcSRS2 alone, or NcCyP plus NcSRS2 had a protection rate (% animals free from infection) of 86%, 60%, or 79%, respectively in the mouse model, which was much higher than the protection rate (less than 14%) in the control groups. The results of the present study clearly indicate that NcCyP is a highly efficacious vaccine candidate which may be useful in protection against Neospora infection in cattle. This work aligns with NP 103 Action Plan Component V: Countermeasures to Prevent and Control Reproductive and Neonatal Diseases, Problem Statement 5B: Neosporosis. 2. Neosporosis in Beagle dogs demonstrated and characterized: Clinical neosporosis was diagnosed in a litter of five pups born to a Beagle bitch from Virginia, USA. The dogs were suspected to have neosporosis based on clinical signs and empirically treated with Clindamycin (75 mg, oral, twice daily, total 150 mg) starting at 9 weeks of age and the dosage was doubled at 13 weeks of age. Viable N. caninum was isolated in gamma interferon gene knock out (KO) mice and in cell culture from the pup killed at 137 days of age. Tissue cysts, but no tachyzoites, were found in histological sections of brain and muscles. The isolate was also identified as N. caninum by PCR and sequence analysis and designated NC-9. N. caninum was neither isolated by bioassay in KO mice nor found in histological sections of tissues of the bitch. Clinical signs in the remaining three pups improved considerably after a 6-month treatment with Clindamycin; N. caninum antibody titers were still persistent in these pups at 23 months of age. Results indicate that medication with Clindamycin can improve clinical condition but not eliminate N. caninum infection. Impacts NP 103 Action Plan Component V: Countermeasures to Prevent and Control Reproductive and Neonatal Diseases, Problem Statement 5B: Neosporosis. 3. Neospora caninum in farm dogs in Costa Rica determined: Neospora caninum oocysts in dog feces of dairy farms in Costa Rica was determined. N. caninum DNA was detected by PCR in four fecal samples, twice from one dog, but oocysts were not detected microscopically in these dogs. Fifteen (48.4%) of 31 dogs had antibodies to N. caninum by ELISA. Seroconversion was not found in 28 dogs. Only one dog tested positive to N. caninum by both ELISA and PCR. This is the first report of finding N. caninum DNA in feces of naturally infected dogs in Costa Rican dairy farms. Impacts NP 103 Action Plan Component V: Countermeasures to Prevent and Control Reproductive and Neonatal Diseases, Problem Statement 5B: Neosporosis. 4. Seroprevalence of Neospora caninum antibodies in dogs in India: Neospora caninum is one of most important causes of abortion in cattle worldwide, and dogs are an important risk factor for N. caninum infection in cattle. Antibodies to N. caninum were determined in 184 (126 rural, 58 urban) dogs from the Punjab State, India, and found in 16.8% of the animals using a commercial ELISA. The prevalence of N. caninum antibodies was significantly higher in rural dogs (21.4%, 27 of 126) than city dogs (6.9%, 4 of 58). This is the first report of N. caninum infection in canines from India. Impacts NP 103 Action Plan Component V: Countermeasures to Prevent and Control Reproductive and Neonatal Diseases, Problem Statement 5B: Neosporosis. 5. Genetic diversity among S. neurona isolates recognized: Sarcocystis neurona is an important cause of neurological disease in horses (equine protozoal myeloencephalitis, EPM) and sea otters in the United States. In addition, EPM-like disease has been diagnosed in several other land and marine mammals. Opossums are its only definitive host. Little genetic diversity among isolates of S. neurona from different hosts has been reported. We used 11 microsatellites to characterize S. neurona DNA isolated from natural infections in 22 sea otters (Enhydra lutris) from California and Washington and in 11 raccoons (Procyon lotor) and one striped skunk (Mephitis mephitis) from Wisconsin. By jointly analyzing these 34 isolates with 26 isolates previously reported, we determined that geographic barriers may limit S. neurona dispersal and that only a limited subset of possible parasite genotypes may have been introduced to recently established opossum populations. Results confirm that diverse intermediate hosts share a common infection source, the opossum. Impacts NP 103 Action Plan Component V: Countermeasures to Prevent and Control Reproductive and Neonatal Diseases, Problem Statement 5B: Neosporosis. 6. Antigenic variations among S. neurona isolates recognized: A gene family of surface antigens is expressed by merozoites of Sarcocystis neurona, the primary cause of equine protozoal myeloencephalitis (EPM). These surface proteins, designated SnSAGs, are immunodominant and therefore excellent candidates for development of EPM diagnostics or vaccines. However, prior work had identified an EPM isolate that lacked the major surface antigen SnSAG1, thus suggesting there may be some variation in the SnSAGs expressed by different S. neurona isolates. Examination of an expressed sequence tag (EST) database revealed the presence of several notable SnSAG polymorphisms. In particular, the EST information implied that the EPM strain SN4 lacked the major surface antigen SnSAG1, as originally described in the archetypal UCD1 and SN3 strains. The absence of this surface antigen from the SN4 strain was confirmed by both western blot and Southern blot. SnSAG2, SnSAG3, and SnSAG4 are present in all 14 S. neurona strains tested. Importantly, SnSAG1 was not detected in seven of the strains, which included isolates from four cases of EPM and a case of fatal meningoencephalitis in a sea otter. Collectively, the data indicated that there is heterogeneity in the surface antigen composition of different S. neurona isolates, which is an important consideration for the development of serologic tests and prospective vaccines for EPM. Furthermore, the diversity reported herein likely extends to other phenotypes, such as strain virulence, and may have implications for the phylogeny of the various Sarcocystis that undergo sexual stages of their life cycle in opossums. Impacts NP 103 Action Plan Component V: Countermeasures to Prevent and Control Reproductive and Neonatal Diseases, Problem Statement 5B: Neosporosis. 7. A new species of Sarcocystis recognized: Infection with Sarcocystis species is common in herbivores, but is rare in bears. Histological sections of 374 black bears (Ursus americanus) from Pennsylvania were examined for sarcocysts. In total, 3 sarcocysts were found in 3 bears, with 1 sarcocyst per section. Sarcocysts from 2 bears were considered a new species, Sarcocystis ursusi. Sarcocysts of S. ursusi n.sp. were microscopic and contained only bradyzoites. By light microscopy, the sarcocyst wall was thin (<0.5 µm thick) and had minute serrations. Ultrastructurally, the serrations on the sarcocyst wall consisted of villar protrusions (Vp) that were mostly 0.5 µm long. The Vp had bundles of electron-dense microtubules that were as wide as long; these microtubules extended deep in to the ground substance layer, a feature that distinguished this species from unnamed sarcocysts from black bear. Bradyzoites were 4.8-6.0 µm long. The sarcocyst from the third bear was structurally different from S. ursusi; its sarcocyst wall was approximately 2 µm thick and had finger-like villi on the cyst wall giving the sarcocyst wall a striated appearance. Impacts NP 103 Action Plan Component V: 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 Commercial Licenses Executed 1 Review Publications Dubey, J.P., Humphreys, G.T., Fritz, D. 2008. Sarcocystis ursusi, n. sp (apicomplexa; sarcocystidae) from the black bear (ursus americanus). Journal of Parasitology. 94:496-499. Dubey, J.P., Morales, J.A., Rosenthal, B.M. 2008. Molecular phylogeny implicates new world opossums (Didelphidae) as the definitive hosts of Sarcocystis ramphastosi, a parasite of the keel-billed toucan (Ramphasotos sulfuratus). Acta Parasitologica. 47:55-61. Dubey, J.P., Rosenthal, B.M., Sundar, N., Velmurugan, G.V., Beckman, K.B. 2007. Sarcocystis arctosi, n. sp. (Apicomplexans: Sarcocystidae) from the brown bear (Ursus arctos), and its genetic similarity to schizonts of Sarcocystis canis-like parasite associated with fatal hepatitis in polar bears. Acta Parasitologica. 52:299-304. Dubey, J.P., Vianna M, C.R., Kwok, O.C., Hill, D.E., Miska, K.B., Tuo, W., Velmurugan, G.V., Conors, M., Jenkins, M.C. 2007. Neosporosis in Beagle dogs clinical signs, diagnosis, treatment, isolation and genetic characterization of Neospora caninum. Veterinary Parasitology. 149:158-166. Howe, D.K., Gaji, R.Y., Marsh, A.E., Patil, B.A., Saville, W.J., Lindsay, D.S., Dubey, J.P., Granstrom, D.E. 2008. Strains of Sarcocystis neurona exhibit variation in their surface antigens, including the absence of the major surface antigen SnSAG1. International Journal for Parasitology. 38:623-631. Meenakshi, R.H., Sandhu, K.S., Singh, J., Sharma, S., Sidhu, P.K., Sreekumar, C., Dubey, J.P. 2007. Neospora caninum infections in cattle in india seroprevalence of neospora caninum antibodies in cattle and water buffaoes in india Journal of Parasitology. 93:1374-1377. Olson, E.J., Wunschnann, A., Dubey, J.P. 2007. Sarcocystis-associated meningoencephalitis in a bald eagle (Haliaeetus leucocephalus). Journal of Veterinary Diagnostic Investigation. 19:564-568. Palavicini, P., Romero, J.J., Dolz, G., Jiminez, A.E., Hill, D.E., Dubey, J.P. 2007. Fecal and serological survey of Neospora caninum in farm dogs in Costa Rica. Veterinary Parasitology. 149:265-270. Sundar, N., Asmundsson, I.M., Thomas, N.J., Samuel, M.D., Dubey, J.P., Rosenthal, B.M. 2007. Modest genetic differentiation among North American populations of Sarcocystis neurona may reflect expansion in its geographic range. Veterinary Parasitology. 152:8-15.