Improvement of western blot test specificity for detecting equine serum antibodies to Sarcocystis neurona.
Abstract: Equine protozoal myeloencephalitis (EPM) is a neurological disease of horses and ponies caused by the apicomplexan protozoan parasite Sarcocystis neurona. The purposes of this study were to develop the most stringent criteria possible for a positive test result, to estimate the sensitivity and specificity of the EPM Western blot antibody test, and to assess the ability of bovine antibodies to Sarcocystis cruzi to act as a blocking agent to minimize false-positive results in the western blot test for S. neurona. Sarcocystis neurona merozoites harvested from equine dermal cell culture were heat denatured, and the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a 12-20% linear gradient gel. Separated proteins were electrophoretically transferred to polyvinylidene fluoride membranes and blocked in 1% bovine serum albumin and 0.5% Tween-Tris-buffered saline. Serum samples from 6 horses with S. neurona infections (confirmed by culture from neural tissue) and 57 horses without infections (horses from the Eastern Hemisphere, where S. neurona does not exist) were tested by Western blot. Horses from both groups had reactivity to the 62-, 30-, 16-, 13-, 11-, 10.5-, and 10-kD bands. Testing was repeated with another step. Blots were treated with bovine S. cruzi antibodies prior to loading the equine samples. After this modification of the Western blot test, positive infection status was significantly associated with reactivity to the 30- and 16-kD bands (P<0.001, Fisher's exact test). The S. cruzi antibody-blocked Western blot had a sample sensitivity of 100% and sample specificity of 98%. It is concluded that the specificity of the Western blot test is improved by blocking proteins not specific to S. neurona and using reactivity to the 30- and 16-kD bands as the criterion for a positive test.
Publication Date: 2000-02-26 PubMed ID: 10690772DOI: 10.1177/104063870001200105Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
- Antibodies
- Clinical Pathology
- Clinical Study
- Diagnosis
- Diagnostic Technique
- Disease Diagnosis
- Disease Treatment
- Epidemiology
- Equine Diseases
- Equine Health
- Equine Protozoal Myeloencephalitis
- Horses
- Immunology
- Infection
- Infectious Disease
- Laboratory Methods
- Protozoa
- Sarcocystis
- Veterinary Medicine
- Veterinary Research
- Western Blot
Summary
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This research endeavoured to improve the specificity of the Western blot test, which is used to detect antibodies of Sarcocystis neurona, a parasite causing equine neurological disease. The researchers used bovine antibodies to block proteins unlinked to S.neurona and deemed the 30- and 16-kD bands as positive test markers, which led to a high specificity rate in their tests.
Research Objectives and Methodology
- The main objective was to develop stringent criteria for a positive Western blot test result to detect antibodies to Sarcocystis neurona in equine serum.
- The study assessed the ability of bovine antibodies to Sarcocystis cruzi to act as a blocking agent to minimize false-positive results.
- The methodology involved harvesting Sarcocystis neurona merozoites from equine dermal cell culture, heat denaturing, and separating the proteins via sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
- These separated proteins were transferred to polyvinylidene fluoride membranes and blocked in a mixture of 1% bovine serum albumin and 0.5% Tween-Tris-buffered saline.
- Serum samples from horses with confirmed S. neurona infections and horses without infections were tested using Western blot.
Findings and Conclusion
- Reactivity to the 62-, 30-, 16-, 13-, 11-, 10.5-, and 10-kD bands was observed in horses from both infection and non-infections groups.
- The tests were repeated by treating blots with bovine S. cruzi antibodies before loading the equine samples. After this modification, positive infection status was significantly associated with reactivity to the 30- and 16-kD bands (P<0.001, Fisher's exact test).
- When these new criteria were applied, the Western blot test had a sample sensitivity of 100% and sample specificity of 98%.
- Thus, it was concluded that the specificity of the Western blot test for detecting S. neurona in equine serum is greatly improved by blocking proteins irrelevant to the parasite and using reactivity to the 30- and 16-kD bands as indicators of a positive test.
Cite This Article
APA
Rossano MG, Mansfield LS, Kaneene JB, Murphy AJ, Brown CM, Schott HC, Fox JC.
(2000).
Improvement of western blot test specificity for detecting equine serum antibodies to Sarcocystis neurona.
J Vet Diagn Invest, 12(1), 28-32.
https://doi.org/10.1177/104063870001200105 Publication
Researcher Affiliations
- Animal Health Diagnostic Laboratory, The Population Medicine Center, Michigan State University, East Lansing 48824, USA.
MeSH Terms
- Animals
- Antibodies, Protozoan / analysis
- Blotting, Western / standards
- Cattle
- Cattle Diseases / genetics
- Cattle Diseases / immunology
- Cattle Diseases / parasitology
- Encephalomyelitis, Equine / genetics
- Encephalomyelitis, Equine / immunology
- Encephalomyelitis, Equine / virology
- Sarcocystis / genetics
- Sarcocystis / immunology
- Sarcocystosis / genetics
- Sarcocystosis / immunology
- Sarcocystosis / veterinary
- Sensitivity and Specificity
Citations
This article has been cited 6 times.- Borges-Silva W, de Jesus RF, Ferreira R, Gondim LFP. Reactivity of Horse Sera to Antigens Derived From Sarcocystis falcatula-Like and Sarcocystis neurona.. Front Vet Sci 2020;7:573016.
- McElroy A, Rashmir A, Manfredi J, Sledge D, Carr E, Stopa E, Klinge P. Evaluation of the Structure of Myodural Bridges in an Equine Model of Ehlers-Danlos Syndromes.. Sci Rep 2019 Jul 10;9(1):9978.
- Zoll WM, Prakoso D, Dark M, Liu J, Stockdale-Walden H, Long MT. Histologic characterization of eosinophilic encephalitis in horses in Florida.. J Vet Diagn Invest 2018 May;30(3):442-446.
- Reed SM, Furr M, Howe DK, Johnson AL, MacKay RJ, Morrow JK, Pusterla N, Witonsky S. Equine Protozoal Myeloencephalitis: An Updated Consensus Statement with a Focus on Parasite Biology, Diagnosis, Treatment, and Prevention.. J Vet Intern Med 2016 Mar-Apr;30(2):491-502.
- Dubey JP, Howe DK, Furr M, Saville WJ, Marsh AE, Reed SM, Grigg ME. An update on Sarcocystis neurona infections in animals and equine protozoal myeloencephalitis (EPM).. Vet Parasitol 2015 Apr 15;209(1-2):1-42.
- Hoane JS, Morrow JK, Saville WJ, Dubey JP, Granstrom DE, Howe DK. Enzyme-linked immunosorbent assays for detection of equine antibodies specific to Sarcocystis neurona surface antigens.. Clin Diagn Lab Immunol 2005 Sep;12(9):1050-6.
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