Improved detection of equine antibodies against Sarcocystis neurona using polyvalent ELISAs based on the parasite SnSAG surface antigens.
Abstract: Equine protozoal myeloencephalitis (EPM) is a common neurologic disease of horses that is caused by the apicomplexan pathogen Sarcocystis neurona. To help improve serologic diagnosis of S. neurona infection, we have modified existing enzyme-linked immunosorbent assays (ELISAs) based on the immunogenic parasite surface antigens SnSAG2, SnSAG3, and SnSAG4 to make the assays polyvalent, thereby circumventing difficulties associated with parasite antigenic variants and diversity in equine immune responses. Two approaches were utilized to achieve polyvalence: (1) mixtures of the individual recombinant SnSAGs (rSnSAGs) were included in single ELISAs; (2) a collection of unique SnSAG chimeras that fused protein domains from different SnSAG surface antigens into a single recombinant protein were generated for use in the ELISAs. These new assays were assessed using a defined sample set of equine sera and cerebrospinal fluids (CSFs) that had been characterized by Western blot and/or were from confirmed EPM horses. While all of the polyvalent ELISAs performed relatively well, the highest sensitivity and specificity (100%/100%) were achieved with assays containing the rSnSAG4/2 chimera (Domain 1 of SnSAG4 fused to SnSAG2) or using a mixture of rSnSAG3 and rSnSAG4. The rSnSAG4 antigen alone and the rSnSAG4/3 chimera (Domain 1 of SnSAG4 fused to Domain 2 of SnSAG3) exhibited the next best accuracy at 95.2% sensitivity and 100% specificity. Binding ratios and percent positivity (PP) ratios, determined by comparing the mean values for positive versus negative samples, showed that the most advantageous signal to noise ratios were provided by rSnSAG4 and the rSnSAG4/3 chimera. Collectively, our results imply that a polyvalent ELISA based on SnSAG4 and SnSAG3, whether as a cocktail of two proteins or as a single chimeric protein, can give optimal results in serologic testing of serum or CSF for the presence of antibodies against S. neurona. The use of polyvalent SnSAG ELISAs will enhance the reliability of serologic testing for S. neurona infection, which should lead to improved diagnosis of EPM.
Copyright © 2010 Elsevier B.V. All rights reserved.
Publication Date: 2010-11-13 PubMed ID: 21075532DOI: 10.1016/j.vetpar.2010.10.034Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
- Antibodies
- Diagnosis
- Diagnostic Technique
- Disease
- Disease Diagnosis
- Enzyme-Linked Immunosorbent Assay (ELISA)
- Equine Diseases
- Equine Health
- Equine Protozoal Myeloencephalitis
- Horses
- Immunology
- Infection
- Laboratory Methods
- Neurological Diseases
- Parasites
- Pathogens
- Protein
- Sarcocystis
- Serodiagnosis
- Veterinary Medicine
- Veterinary Research
Summary
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The study presents improved methods for detecting antibodies against Sarcocystis neurona, a pathogen causing a common neurological disease in horses, equine protozoal myeloencephalitis (EPM). The researchers developed polyvalent enzyme-linked immunosorbent assays (ELISAs) based on the parasite’s surface antigens which enhanced the reliability of serologic testing and improved the diagnosis of EPM.
Background and Objectives
- The research was conducted to overcome the issues related to antigenic variants of the Sarcocystis neurona parasite and diversity in equine immune responses impacting the diagnosis of equine protozoal myeloencephalitis (EPM), a common neurological disorder in horses.
- The objective was to modify the existing enzyme-linked immunosorbent assays (ELISAs) that are based on the parasite’s immunogenic surface antigens (SnSAG2, SnSAG3, and SnSAG4) and make these assays polyvalent, multiplying their sensitivity to different antigens.
Methods and Approaches
- The researchers used two approaches to achieve polyvalence: (1) including mixtures of the individual recombinant SnSAGs (rSnSAGs) in single ELISAs, and (2) creating a collection of unique SnSAG chimeras that fused protein domains from different SnSAG surface antigens into a single recombinant protein.
- The newly developed ELISAs were then assessed using a defined sample set of equine sera and cerebrospinal fluids (CSFs) which had been previously characterized by Western blot and/or were from confirmed EPM horses.
Results and Findings
- All of the newly developed polyvalent ELISAs performed relatively well. However, the highest sensitivity and specificity (100% each) were achieved with assays containing the rSnSAG4/2 chimera (Domain 1 of SnSAG4 fused to SnSAG2) or using a mixture of rSnSAG3 and rSnSAG4.
- The rSnSAG4 antigen alone and the rSnSAG4/3 chimera (Domain 1 of SnSAG4 fused to Domain 2 of SnSAG3) exhibited the next best accuracy with 95.2% sensitivity and 100% specificity.
- Furthermore, when comparing the positive and negative sample values, best signal to noise ratios were provided by rSnSAG4 and the rSnSAG4/3 chimera.
Implications
- This study strongly implies that a polyvalent ELISA based on SnSAG4 and SnSAG3, whether mixed as two proteins or as a single chimeric protein, can offer optimal results in serologic testing for the presence of antibodies against S. neurona in horse serum or CSF.
- These polyvalent SnSAG ELISAs can enhance the reliability of serologic testing and result in improved diagnosis of EPM in horses.
Cite This Article
APA
Yeargan MR, Howe DK.
(2010).
Improved detection of equine antibodies against Sarcocystis neurona using polyvalent ELISAs based on the parasite SnSAG surface antigens.
Vet Parasitol, 176(1), 16-22.
https://doi.org/10.1016/j.vetpar.2010.10.034 Publication
Researcher Affiliations
- Department of Veterinary Science, M.H. Gluck Equine Research Center, University of Kentucky, Lexington, KY 40546-0099, USA.
MeSH Terms
- Animals
- Antibodies, Protozoan / blood
- Antibodies, Protozoan / immunology
- Antigens, Protozoan / immunology
- Enzyme-Linked Immunosorbent Assay / veterinary
- Horse Diseases / blood
- Horse Diseases / diagnosis
- Horse Diseases / immunology
- Horses
- Protozoan Proteins / immunology
- Recombinant Proteins / immunology
- Sarcocystis / immunology
- Sarcocystosis / diagnosis
- Sarcocystosis / immunology
- Sarcocystosis / veterinary
- Sensitivity and Specificity
Citations
This article has been cited 4 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.
- 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.
- Yeargan MR, Alvarado-Esquivel C, Dubey JP, Howe DK. Prevalence of antibodies to Sarcocystis neurona and Neospora hughesi in horses from Mexico. Parasite 2013;20:29.
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