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Evaluation of immunofluorescent antibody and immunoblot tests for the detection of antibodies against Besnoitia bennetti tachyzoites and bradyzoites in donkeys
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9th ICEID Abstracts / Journal of Equine Veterinary Science 32 (2012) S3-S95
takes 72h to complete and requires certain laboratory facilities, equipment, and technical expertise to perform. Furthermore, non-specific cellular cytotoxicity of particular samples and inter-laboratory variation in SN results have been reported. For these reasons an alternative serologic test is desirable. None of the previously reported tests have shown equivalent sensitivity and specificity compared to the SN. In attempting to produce a better alternative assay, a cELISA was developed using EAV gp5-specific neutralizing monoclonal antibody (MAb) 4B2 which performed moderately well. This cELISA has been significantly improved by substituting 4B2 antibody with the non-neutralizing monoclonal antibody 17B7. In the present study, the improved cELISA was validated according to the OIE-recommended validation protocol. As part of an in-house validation procedure of the EAV antibody cELISA, the following five analyses were performed: 1. the primary assay was calibrated with the OIE approved reference serum panel for EVA, 2. repeatability of the assay was evaluated within and between runs, 3. analytical specificity was evaluated using sera specific to related viruses, 4. analytical sensitivity was evaluated with sera collected from horses vaccinated with the modified live virus vaccine against EVA (ArvacÒ, Pfizer Animal Health), and 5. the duration of the positive cELISA antibody detection was evaluated following EVA vaccination. The outside validation of the cELISA utilized three laboratories including one OIE reference laboratory and two AAVLD-accredited state laboratories. Each laboratory assayed their panel of field sera (150-200 sera) to evaluate diagnostic specificity and sensitivity of the cELISA, and VMRD prepared an inter-dependency panel (25 sera in duplicate) to evaluate the robustness of the cELISA in all three laboratories. The cut-off of the cELISA was evaluated by ROC plot analysis. As a result, the original cELISA using MAb 4B2 was significantly improved by switching to 17B7 and incorporating other test procedures. The analytical sensitivity of the new cELISA was comparable to the SN assay in that it detected EAV-specific antibody as early as 6 days postvaccination. The duration of EAV-specific antibody detected by cELISA was over six years post-vaccination. In the field trial, the relative specificity of the new cELISA was 99.5% and the relative sensitivity was 98.2%. This field trial data also showed a significant correlation between SN and cELISA results (r2¼0.79, P < 0.0001). These results indicate that new EAV antibody cELISA is a reliable, simple alternative to the SN assay for detecting EAV-specific antibodies in horses.
Evaluation of immunofluorescent antibody and immunoblot tests for the detection of antibodies against Besnoitia bennetti tachyzoites and bradyzoites in donkeys S.L. Ness 1, G. Schares 5, J. Peters 2, J.P. Dubey 6, L.D. Mittel 3, H.O. Mohammed 3, D.D. Bowman 4, and T.J. Divers 1 1 Departments of Clinical Sciences, 2 Biomedical Sciences, 3 Population Medicine and Diagnostic Sciences, 4 Microbiology, Cornell University College of Veterinary Medicine, Ithaca, NY 14853, 5 Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Wusterhausen, Germany, 6 Animal Parasitic Diseases
Laboratory, United States Department of Agriculture, Beltsville, MD 20705 Besnoitiosis is caused by infection with protozoan Besnoitia spp, which are cyst-forming coccidian parasites that affect multiple host species worldwide. Besnoitia bennetti is the species known to infect equids and has been reported in both horses and donkeys in Africa and Asia, and more recently, donkeys in the United States. Clinical disease is characterized by a miliary dermatitis with parasitic cysts in the skin, mucous membranes, and sclera (Figure 1). Several serologic assays have been validated for the diagnosis of bovine besnoitiosis, caused by infection with Besnoitia besnoiti. We hypothesize that assays validated for the diagnosis of bovine besnoitiosis will also be effective for identifying equine infection. The goal of the present study was to evaluate the utility of three serologic assays for the detection of B. bennetti antibodies in donkeys. Sera from donkeys confirmed to be infected with B. bennetti via histopathology (cases; n ¼ 13) and donkeys with no clinical signs of besnoitiosis (controls; n ¼ 126) were tested for antibodies against B. bennetti using an immunofluorescent antibody test and 2 immunoblot assays specific for bradyzoite and tachyzoite antigens, respectively. For all tested hypotheses, values of P < 0.05 were considered significant. Donkeys with besnoitiosis had significantly higher geometric mean immunofluorescent antibody titers than control donkeys (case mean geometric titer 2.87 [reciprocal titer 741] vs control mean geometric titer 2.0 [reciprocal titer 100]). There was a significant difference in the median number of bradyzoite immunoblot bands between cases and controls (7 vs. 1), as well as in the median number of tachyzoite immunoblot bands between cases and controls (6 vs. 0). There were positive correlations among geometric titer and number of bradyzoite and tachyzoite immunoblots bands. Immunofluorescent antibody and immunoblot tests are effective at identifying donkeys with besnoitiosis and provide a more efficient and less invasive diagnostic alternative to histopathology.
Figure 1. Besnoitia bennetti lesions on the sclera (A) and muzzle (B) of an infected donkey.
Development of a rapid isothermal assay to detect the causative agent of strangles S.E. North, P. Das, P.R. Wakeley, and J. Sawyer Technology Transfer Unit, Specialist Scientific Support Department, Animal Health Veterinary Laboratories Agency – Weybridge, United Kingdom We have developed a Loop mediated AMPlification (LAMP) assay to detect Streptococcus equi subspecies equi (S. equi), the causative agent of equine strangles. The
9th ICEID Abstracts / Journal of Equine Veterinary Science 32 (2012) S3-S95
takes 72h to complete and requires certain laboratory facilities, equipment, and technical expertise to perform. Furthermore, non-specific cellular cytotoxicity of particular samples and inter-laboratory variation in SN results have been reported. For these reasons an alternative serologic test is desirable. None of the previously reported tests have shown equivalent sensitivity and specificity compared to the SN. In attempting to produce a better alternative assay, a cELISA was developed using EAV gp5-specific neutralizing monoclonal antibody (MAb) 4B2 which performed moderately well. This cELISA has been significantly improved by substituting 4B2 antibody with the non-neutralizing monoclonal antibody 17B7. In the present study, the improved cELISA was validated according to the OIE-recommended validation protocol. As part of an in-house validation procedure of the EAV antibody cELISA, the following five analyses were performed: 1. the primary assay was calibrated with the OIE approved reference serum panel for EVA, 2. repeatability of the assay was evaluated within and between runs, 3. analytical specificity was evaluated using sera specific to related viruses, 4. analytical sensitivity was evaluated with sera collected from horses vaccinated with the modified live virus vaccine against EVA (ArvacÒ, Pfizer Animal Health), and 5. the duration of the positive cELISA antibody detection was evaluated following EVA vaccination. The outside validation of the cELISA utilized three laboratories including one OIE reference laboratory and two AAVLD-accredited state laboratories. Each laboratory assayed their panel of field sera (150-200 sera) to evaluate diagnostic specificity and sensitivity of the cELISA, and VMRD prepared an inter-dependency panel (25 sera in duplicate) to evaluate the robustness of the cELISA in all three laboratories. The cut-off of the cELISA was evaluated by ROC plot analysis. As a result, the original cELISA using MAb 4B2 was significantly improved by switching to 17B7 and incorporating other test procedures. The analytical sensitivity of the new cELISA was comparable to the SN assay in that it detected EAV-specific antibody as early as 6 days postvaccination. The duration of EAV-specific antibody detected by cELISA was over six years post-vaccination. In the field trial, the relative specificity of the new cELISA was 99.5% and the relative sensitivity was 98.2%. This field trial data also showed a significant correlation between SN and cELISA results (r2¼0.79, P < 0.0001). These results indicate that new EAV antibody cELISA is a reliable, simple alternative to the SN assay for detecting EAV-specific antibodies in horses.
Evaluation of immunofluorescent antibody and immunoblot tests for the detection of antibodies against Besnoitia bennetti tachyzoites and bradyzoites in donkeys S.L. Ness 1, G. Schares 5, J. Peters 2, J.P. Dubey 6, L.D. Mittel 3, H.O. Mohammed 3, D.D. Bowman 4, and T.J. Divers 1 1 Departments of Clinical Sciences, 2 Biomedical Sciences, 3 Population Medicine and Diagnostic Sciences, 4 Microbiology, Cornell University College of Veterinary Medicine, Ithaca, NY 14853, 5 Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Wusterhausen, Germany, 6 Animal Parasitic Diseases
Laboratory, United States Department of Agriculture, Beltsville, MD 20705 Besnoitiosis is caused by infection with protozoan Besnoitia spp, which are cyst-forming coccidian parasites that affect multiple host species worldwide. Besnoitia bennetti is the species known to infect equids and has been reported in both horses and donkeys in Africa and Asia, and more recently, donkeys in the United States. Clinical disease is characterized by a miliary dermatitis with parasitic cysts in the skin, mucous membranes, and sclera (Figure 1). Several serologic assays have been validated for the diagnosis of bovine besnoitiosis, caused by infection with Besnoitia besnoiti. We hypothesize that assays validated for the diagnosis of bovine besnoitiosis will also be effective for identifying equine infection. The goal of the present study was to evaluate the utility of three serologic assays for the detection of B. bennetti antibodies in donkeys. Sera from donkeys confirmed to be infected with B. bennetti via histopathology (cases; n ¼ 13) and donkeys with no clinical signs of besnoitiosis (controls; n ¼ 126) were tested for antibodies against B. bennetti using an immunofluorescent antibody test and 2 immunoblot assays specific for bradyzoite and tachyzoite antigens, respectively. For all tested hypotheses, values of P < 0.05 were considered significant. Donkeys with besnoitiosis had significantly higher geometric mean immunofluorescent antibody titers than control donkeys (case mean geometric titer 2.87 [reciprocal titer 741] vs control mean geometric titer 2.0 [reciprocal titer 100]). There was a significant difference in the median number of bradyzoite immunoblot bands between cases and controls (7 vs. 1), as well as in the median number of tachyzoite immunoblot bands between cases and controls (6 vs. 0). There were positive correlations among geometric titer and number of bradyzoite and tachyzoite immunoblots bands. Immunofluorescent antibody and immunoblot tests are effective at identifying donkeys with besnoitiosis and provide a more efficient and less invasive diagnostic alternative to histopathology.
Figure 1. Besnoitia bennetti lesions on the sclera (A) and muzzle (B) of an infected donkey.
Development of a rapid isothermal assay to detect the causative agent of strangles S.E. North, P. Das, P.R. Wakeley, and J. Sawyer Technology Transfer Unit, Specialist Scientific Support Department, Animal Health Veterinary Laboratories Agency – Weybridge, United Kingdom We have developed a Loop mediated AMPlification (LAMP) assay to detect Streptococcus equi subspecies equi (S. equi), the causative agent of equine strangles. The