Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) To refine the Sarcocystis neurona model for equine protozoal myeloencephalitis. Approach (from AD-416) Interferon gamma gene knockout mice will be used for bioassay to detect Sarcocystis neurona in feces of opossums and tissues of horses that will be fed graded doses of S. neurona sporocysts. Significant Activities that Support Special Target Populations This report documents research conducted under a trust agreement between ARS and the Ohio State University. Additional details of the research can be found in the report for the Parent Project 1265-32000-082-00D, �Epidemiology and Control of Neospora Caninum and Related Protozoa�. Sarcocystis neurona is the primary causative agent of equine protozoal myeloencephalitis (EPM), a common neurologic disease of horses in the Americas. We have developed a set of enzyme-linked immunosorbent assays (ELISAs) based on the four major surface antigens of S. neurona (SnSAGs) to analyze the equine antibody response to S. neurona. The SnSAG ELISAs were optimized and standardized with a sample set of 50 equine sera that had been characterized by Western blotting against total S. neurona parasite antigen, the current gold standard for S. neurona serology. The recombinant SnSAG2 (rSnSAG2) ELISA showed the highest sensitivity and specificity at 95.5% and 92.9%, respectively. In contrast, only 68.2% sensitivity and 71.4% specificity were achieved with the rSnSAG1 ELISA, indicating that this antigen may not be a reliable serological marker for analyzing antibodies against S. neurona in horses. Importantly, the ELISA antigens did not show cross-reactivity with antisera to Sarcocystis fayeri or Neospora hughesi, two other equine parasites. The accuracy and reliability is being investigated using a larger sample size. The plans and progress for this grant were discussed in frequent phone conversations, emails and periodic encounters at scientific meetings.
Impacts
(N/A)
Publications
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Progress 10/01/02 to 09/01/07
Outputs
Progress Report Objectives (from AD-416) To refine the Sarcocystis neurona model for equine protozoal myeloencephalitis. Approach (from AD-416) Interferon gamma gene knockout mice will be used for bioassay to detect Sarcocystis neurona in feces of opossums and tissues of horses that will be fed graded doses of S. neurona sporocysts. Significant Activities that Support Special Target Populations Sarcocystis neurona is the primary causative agent of equine protozoal myeloencephalitis (EPM), a common neurologic disease of horses in the Americas.The EPM equine model was improved for testing of pathogenesis and therapy. Ponazuril was found to be effective as a prophylactic for EPM in horses. An ELISA test was modified for diagnosis of EPM. We developed a set of enzyme-linked immunosorbent assays (ELISAs) based on the four major surface antigens of S. neurona (SnSAGs) to analyze the equine antibody response to S. neurona. The SnSAG ELISAs were optimized and standardized with a sample set of 50 equine sera that had been characterized by Western blotting against total S. neurona parasite antigen, the current gold standard for S. neurona serology. The recombinant SnSAG2 (rSnSAG2) ELISA showed the highest sensitivity and specificity at 95.5% and 92.9%, respectively. In contrast, only 68.2% sensitivity and 71.4% specificity were achieved with the rSnSAG1 ELISA, indicating that this antigen may not be a reliable serological marker for analyzing antibodies against S. neurona in horses. Importantly, the ELISA antigens did not show cross-reactivity with antisera to Sarcocystis fayeri or Neospora hughesi, two other equine parasites. The plans and progress for this grant were discussed in frequent phone conversations, emails and periodic encounters at scientific meetings.
Impacts
(N/A)
Publications
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Progress 10/01/05 to 09/30/06
Outputs
Progress Report 4d Progress report. This report serves to document research conducted under a trust agreement between ARS and the Ohio State University. Additional details of research can be found in the report for the parent project 1265-32000-071- 00D. Sarcocystis neurona is the primary causative agent of equine protozoal myeloencephalitis (EPM), a common neurologic disease of horses in the Americas. We have developed a set of enzyme-linked immunosorbent assays (ELISAs) based on the four major surface antigens of S. neurona (SnSAGs) to analyze the equine antibody response to S. neurona. The SnSAG ELISAs were optimized and standardized with a sample set of 36 equine sera that had been characterized by Western blotting against total S. neurona parasite antigen, the current gold standard for S. neurona serology. The recombinant SnSAG2 (rSnSAG2) ELISA showed the highest sensitivity and specificity at 95.5% and 92.9%, respectively. In contrast, only 68.2% sensitivity and
71.4% specificity were achieved with the rSnSAG1 ELISA, indicating that this antigen may not be a reliable serological marker for analyzing antibodies against S. neurona in horses. Importantly, the ELISA antigens did not show cross-reactivity with antisera to Sarcocystis fayeri or Neospora hughesi, two other equine parasites. The accuracy and reliability exhibited by the SnSAG ELISAs suggest that these assays will be valuable tools for examining the equine immune response against S. neurona infection, which may help in understanding the pathobiology of this accidental parasite-host interaction. Moreover, with modification and further investigation, the SnSAG ELISAs have potential for use as immunodiagnostic tests to aid in the identification of horses affected by EPM.
Impacts
(N/A)
Publications
- Sofaly, C.D., Reed, S.M., Gordon, J.C., Dubey, J.P., Oglesbee, M.J., Njoku, C.J., Grover, D.L., Saville, W.J.A. Experimental induction of equine protozoal myeloencephalitis (EPM) in the horse: effect of Sarcocystis neurona sporocyst inoculation dose on the development of clinical neurologic disease. Journal of Parasitology. 2002. v. 88. p. 1164-1170.
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Progress 10/01/04 to 09/30/05
Outputs
4d Progress report. This report serves to document research conducted under a Trust Agreement between ARS and Ohio State University. Additional detailes of research can be found in the report for the parent project 1265-32000-071-00D Biology and Epidemiology of Neospora caninum and Related Protozoa. Equine protozoan myeloencephalitis (EPM) is a serious neurologic disease of horses in the Americas, and Sarcocystis neurona is the protozoan parasite most frequently associated with EPM. There is no efficient horse model to study EPM. Previous challenge studies performed at Ohio State University involved a transport-stress model where the study animals were dosed with Sarcocystis neurona sporocysts on the day of arrival. This study was to test a second transportation of horses following oral inoculation with S. neurona sporocysts. Horses were randomly assigned to groups: Group 1 were transported 4 days after inoculation (DAI); Group 2 at 11 DAI; Group 3 at 18 DAI; and
Group 4 horses were not transported a second time (controls). An overall neurologic score was determined based on a standard numbering system used by veterinarians. All scores are out of 5 which is the most severely affected animal. The mean score for the Group 1 horses was 2.42; Group 2 horses was 2.5; Group 3 horses was 2.75; and Group 4 horses was 3.25. Since the Group 4 horses did not have a second transport, they were compared to all other groups. Statistically different scores were present between Group 4 and Groups 1 and 2. There were no differences in the time of seroconversion between groups. There was a difference between the time of onset of first clinical signs between Groups 1 and 4. This difference was likely due to the different examination days. Differences in housing and handling were likely the reason for the differences in severity of clinical signs. This model results in consistent, significant clinical signs in all horses at approximately the same time period
post inoculation but was most severe in horses that did not experience a second transport. Sarcocystis neurona and Sarcocystis fayeri infections are common in horses in Americas. Their antemortem diagnosis is important because the former causes a neurological disorder in horses whereas the latter is considered nonpathogenic. There is a concern that equine antibodies to S. fayeri might react with S. neurona antigens in diagnostic tests. In the present study 4 ponies without demonstrable serum antibodies to S. neurona by Western immunoblot were used. Three ponies were fed 105 to 107 sporocysts of S. fayeri obtained from dogs that were fed naturally- infected horse muscles. All ponies remained asymptomatic until the termination of the experiment day 79 post inoculation (PI). All serum samples collected were negative for antibodies to S. neurona using the Western blot at the initial screening, just prior to inoculation with S. fayeri (day 2) and weekly until day 79 PI. Cerebrospinal
fluid (CSF) samples from each pony were negative for S. neurona antibodies. Using the S. neurona agglutination test, antibodies to S. neurona were not detected in 1:25 dilution of sera from any samples except pony no. 4 on day 28; this pony had received 107 sporocysts. Seven serum samples using indirect fluorescent antibody tests (IFAT) were positive for S. neurona antibodies from 1:25 to 1:400 dilutions. Sarcocystis fayeri sarcocysts were found in striated muscles of all inoculated ponies with heaviest infections in tongue. All sarcocysts examined histologically appeared to contain only metrocytes. Ultrastructurally, S. fayeri sarcocysts could be differentiated from S. neurona sarcocysts by the microtubules (mt) in villar protrusions on sarcocyst walls; in S. fayeri the mt extended from the villar tips to the pellicle of zoites whereas in S. neurona the mt were restricted to the middle of the cyst wall. Results indicate that horses with S. fayeri infections may be misdiagnosed as
S. neurona infections using the IFAT and further research is needed on the serologic diagnosis of S. neurona infections.
Impacts
(N/A)
Publications
- Sofaly, C.D., Reed, S.M., Gordon, J.C., Dubey, J.P., Oglesbee, M.J., Njoku, C.J., Grover, D.L., Saville, W.J.A. Experimental induction of equine protozoal myeloencephalitis (EPM) in the horse: effect of Sarcocystis neurona sporocyst inoculation dose on the development of clinical neurologic disease. Journal of Parasitology. 2002. v. 88. p. 1164-1170.
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Progress 10/01/03 to 09/30/04
Outputs
4. What were the most significant accomplishments this past year? Question 4D: This report serves to document research conducted under a trust agreement between ARS and Ohio State University. Additional details of the research can be found in the report for the parent project 1265-332000- 071-00D, Biology and Epidemiology of Neospora caninum and Related Protozoa. As yet, there is no reliable method to induce EPM in horses. Previous challenge studies performed at Ohio State University involved a transport-stress model where the study animals were dosed with Sarcocystis neurona sporocysts on the day of arrival. This study was to test a second transportation of horses following oral inoculation with S. neurona sprocosysts. Horses were randomly assigned to groups: Group 1 were transported 4 days after inculation (DAI); Group 2 at 11 DAI; Group 3 at 18 DAI; and Group 4 horses were not transported a second time (controls). An overall neurologic score was determined
based on a standard numbering system used by veterinarians. All scores are out of 5 which is the most severely affected animal. The mean score for the Group 1 horses was 2.42; Group 2 horses was 2.5; Group 3 horses was 2.75; and Group 4 horses was 3.25. Since the Group 4 horses did not have a second transport, they were compared to all other groups. Statistically different scores were present between Group 4 and Groups 1 and 2. There were no differences in the time of seroconversion between groups. There was a difference between the time of onset of first clinical signs between Groups 1 and 4. The difference was likely due to the different examination days. Differences in housing and handling were likely the reason for the differences in severity of clinical signs. This model results in consistent, significant clinical signs in all horses at approximately the same time period post inoculation but was most severe in horses that did not experience a second transport.
Sarcocystis neurona and Sarcocystis fayeri infections are common in horses in Americas. Their antemortem diagnosis is important because the former causes a neurological disorder in horses whereas the latter is considered nonpathogenic. There is a concern that equine antibodies to S. fayeri might react with S. neurona antigens in diagnostic tests. In the present study 4 ponies without demonstrable serum antibodies to S. neurona by Western immunoblot were used. Three ponies were fed 105 to 107 sporocysts of S. fayeri obtained from dogs that were fed naturally- infected horse muscles. All ponies remained asymptomatic until the termination of the experiment day 79 post inoculation (PI). All serum samples collected were negative for antibodies to S. neurona using the Western blot at the initial screening, just prior to inoculation with S. fayeri (day 2) and weekly until day 79 PI. Cerebrospinal fluid (CSF) samples from each pony were negative for S. neurona antibodies. Using the S.
neurona agglutination test, antibodies to S. neurona were not detected in 1:25 dilution of sera from any samples except pony no. 4 on day 28; this pony had received 107 sporocysts. Seven serum samples using indirect fluorescent antibody tests (IFA) were positive for S. neurona antibodies from 1:25 to 1:400 dilutions. Sarcocystis fayeri sarcocysts were found in striated muscles of all inoculated ponies with heaviest infections in tongue. All sarcocysts examined histologically appeared to contain only metrocytes. Ultrastructurally, S. fayeri sarcocsyts could be differentiated from S. neurona sarcocysts by the microtubules (mt) in villar protrustions on sarcocyst walls; in S. fayeri the mt extended from the villar tips to the pellicle of zoites whereas in S. neurona the mt were restricted to the middle of the cyst wall. Results indicate that horses with S. fayeri infections may be misdiagnosed as S. neurona infections using the IFAT and further research is needed on the serologic
diagnosis of S. neurona infections.
Impacts
(N/A)
Publications
- Sofaly, C.D., Reed, S.M., Gordon, J.C., Dubey, J.P., Oglesbee, M.J., Njoku, C.J., Grover, D.L., Saville, W.J.A. Experimental induction of equine protozoal myeloencephalitis (EPM) in the horse: effect of Sarcocystis neurona sporocyst inoculation dose on the development of clinical neurologic disease. Journal of Parasitology. 2002. v. 88. p. 1164-1170.
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Progress 10/01/02 to 09/30/03
Outputs
4. What were the most significant accomplishments this past year? D. This report serves to document research conducted under a trust agreement between ARS and Ohio State University. Additional details of research can be found in the report for the parent CRIS 1265-32000-071- 00D. Equine protozoan myeloencephalitis (EPM) is a serious neurologic disease of horses in the Americas, and Sarcocystis neurona is the protozoan parasite most frequently associated with EPM. Evaluation of the efficacy of treatments and preventive medications has been hampered by the lack of a reliable equine model for EPM. The effect of inoculation dose of Sarcocystis neurona sporocysts on the development of clinical neurologic disease in horses was investigated. Twenty-four seronegative weanling horses were subjected to the natural stress of transport and then randomly assigned to 6 treatment groups of 4 horses each. Horses were then immediately inoculated with either 102, 103, 104, 105, or
106 S. neurona sporocysts or placebo using nasogastric tube and housed indoors. Weekly neurologic examinations were performed by a blinded observer. Blood was collected weekly for antibody determination by Western blot analysis. Cerebrospinal fluid was collected before inoculation and before euthanasia for S. neurona antibody determination. Horses were killed and necropsied between 4 and 5 wk after inoculation. Differences were detected among dose groups based on seroconversion times, severity of clinical neurologic signs, and presence of microscopic lesions. Seroconversion of challenged horses was observed as early as 14 days postinfection in the 106 sporocyst dose group. Mild to moderate clinical signs of neurologic disease were produced in challenged horses from all groups, with the most consistent signs seen in the 106 sporocyst dose group. Histologic lesions suggestive of S. neurona infection were detected in 4 of the 20 horses fed sporocysts. Parasites were not detected
in equine tissues by light microscopy, immunohistochemistry, or bioassay in gamma-interferon gene knockout mice. Control horses remained seronegative for the duration of the study and had no histologic evidence of protozoal infection.
Impacts
(N/A)
Publications
- Sofaly, C.D., Reed, S.M., Gordon, J.C., Dubey, J.P., Oglesbee, M.J., Njoku, C.J., Grover, D.L., Saville, W.J.A. Experimental induction of equine protozoal myeloencephalitis (EPM) in the horse: effect of Sarcocystis neurona sporocyst inoculation dose on the development of clinical neurologic disease. Journal of Parasitology. 2002. v. 88. p. 1164-1170.
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