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Investigation of the SnSAG gene family of surface antigens in the coccidian parasite Sarcocystis neurona
Department of Veterinary Sciences
Equine protozoal myeloencephalitis (EPM) is a common and debilitating neurolgic disease of horses that is caused by the protozoan pathogen Sarcocystis neurona. Efforts to correctly diagnose, treat, and prevent EPM have been partly hampered by a lack of information about the pathogen. This project will investigate a group of parasite surface proteins that are very immunogenic and likely important for the pathogen's survival. The information gained from these studies could lead to better EPM diagnostics and a protective vaccine.
2011 Project Description
A collection of 14 Sarcocystis neurona isolates that included nine strains isolated from EPM horses and one from a diseased sea otter were examined for the presence of the four major SnSAG surface antigens. Reagents were produced to allow investigation of the SnSAG5 gene, which was predicted to encode a novel S. neurona merozoite surface antigen. An additional novel surface protein, SnSPR1, was identified and characterized in S. neurona merozoites.
An extensive collection of recombinant expression plasmids were designed to express the SnSAGs as full-length proteins, as partial proteins (single domain of the prototypic SnSAG two-domain structure), and as chimeras (e.g., SnSAG2-SnSAG3 fusions). Various combinations of these recombinant proteins have been tested in enzyme-linked immunosorbent assays (ELISAs) to assess whether improved diagnostic accuracy can be achieved over our previous ELISAs that incorporated single SnSAG proteins.
Extensive sample sets of equine serum and cerebrospinal fluid were tested to assess the utility of the modified SnSAG ELISAs as ancillary tests for diagnosis of equine protozoal myeloencephalitis (EPM). Equine serum samples from Spain, a region devoid of S. neurona, were examined to address the specificity of the SnSAG ELISAs. Information about the ELISAs and their use for EPM diagnosis was disseminated via multiple abstracts and presentations at the 2010 American College of Veterinary Internal Medicine meeting. The assays have been licensed by EDS and are now offered for commercial testing of serum/CSF samples submitted by veterinary practitioners.
A preliminary vaccination study utilizing the SnSAG3 as the immunogen was conducted in horses. In collaboration with Dr. Udeni Balasuriya at the M.H. Gluck Equine Research Center, recombinant equine arteritis virus cDNAs were designed and constructed to express the SnSAGs. Preliminary analyses of the recombinant viruses suggested that infectious viral particles were produced from the cDNAs and the incorporated SnSAG was faithfully expressed in infected cells. These EAV:SnSAG recombinant viruses will be tested for their ability to elicit immune responses against the parasite proteins.
Expression of the Sarcocystis neurona SnSAGs was examined during the three major life cycle stages of the parasite (merozoites, bradyzoites, sporozoites). Finally, the S. neurona genome sequence was searched to identify potential new SnSAG genes.
The SnSAG surface antigens are abundant and highly immunogenic parasite proteins expressed by the equine pathogen Sarcocystis neurona. In-depth characterization of the SnSAGs and the identification of new parasite surface proteins is providing insight into the importance of these molecules during S. neurona infection and intracellular propagation.
This project demonstrated that the SnSAG surface antigens exhibit polymorphism in the population of strains tested, including the absence of the major surface antigen SnSAG1 in half of the strains. Genetic analyses of the strains confirmed that the SnSAG1 gene locus was absent from the strains that did not express the protein. Examination of the collection of S. neurona strains demonstrated that SnSAG5 is expressed by only half of the strains and its presence is mutually exclusive to the previously described major surface antigen SnSAG1. One parasite strain from a diseased sea otter was found to lack both SnSAG1 and SnSAG5.
Collaboration with investigators at Virginia Tech, Rood and Riddle Equine Hospital and the equine testing company Equine Diagnostic Solutions (EDS), LLC has demonstrated the utility of the modified SnSAG ELISAs as ancillary tests for diagnosis of equine protozoal myeloencephalitis (EPM). Examination of equine serum samples from Europe confirmed that the SnSAG ELISAs were highly specific for detection of antibodies against S. neurona.
Analyses of the three major life cycle stages demonstrated that the SnSAG surface proteins are differentially regulated in each of the stages. Western blot analyses of S. neurona strain SN138 sporozoites indicated that SnSAG2, SnSAG3, and SnSAG4 are present during this life cycle stage but the SnSAG5 major surface antigen of this strain is not expressed or is significantly down-regulated relative to the other SnSAGs. Immunohistochemical staining of SN138 strain S. neurona bradyzoites implied that all four major SnSAGs expressed by this strain (SnSAG2-SnSAG5) are down-regulated during this latent parasite stage, which is consistent with what has been found for the TgSAGs of the related parasite Toxoplasma gondii.
Interrogation of the newly-generated S. neurona genome sequence revealed less than 10 gene sequences for novel SnSAGs/SAG-related sequences (SRSs), which is far less than the 161 SAG/SRS gene family members that have been described in T. gondii. Collectively, the results obtained from this research project have provided insight into the importance of the SnSAG surface molecules during S. neurona infection. As well, this information has been used to improve diagnosis of the debilitating neurologic disease EPM, and it will further enhance efforts to develop protective vaccination strategies.
Specifically, the findings of this project have shown that there is variation in the profile of SnSAGs expressed in different strains of S. neurona and during different life cycle stages. These parasite antigenic variations will impact the immune response in infected horses and must be considered when developing and using new diagnostic tests or vaccines.
Arias, M., M. Yeargan, I. Francisco, S. Dangoudoubiyam, P. Becerra, R. Sanchez-Andrade, A. Paz-Silva and D.K. Howe. 2011. Exposure to Sarcocystis spp. in horses from Spain determined by Western blot analysis using Sarcocystis neurona merozoites as heterologous antigen. Veterinary Parasitology, doi:10.1016/j.vetpar.2011.09.042.
Gautam, A., J.P. Dubey, W.J. Saville, and D.K. Howe. 2011. The SnSAG merozoite surface antigens of Sarcocystis neurona are expressed differentially during the bradyzoite and sporozoite life cycle stages. Veterinary Parasitology 183:37-42.
Dangoudoubiyam, S., J. B. Oliveira, C. Viquez, A. Gomez-Garcia, O. Gonzalez, J. J. Romero, O. C. H. Kwok, J. P. Dubey, and D. K. Howe. 2011. Detection of antibodies against Sarcocystis neurona, Neospora spp., and Toxoplasma gondii in horses from Costa Rica. Journal of Parasitology 97(3):522-524.