Research Accomplishment Reports 2007

Investigation of the SnSAG gene family of surface antigens in the coccidian parasite Sarcocystis neurona

D. Howe
Department of Veterinary Sciences

 

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 by western blot using antibodies against the four major SnSAG surface antigens. This study demonstrated that these 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.

Recombinant protein and monospecific polyclonal antisera were produced to examine the S. neurona SnSAG5 gene, which was predicted to encode a merozoite surface antigen. Studies with these reagents have shown that the SnSAG5 protein is indeed a major merozoite surface antigen that is also expressed throughout intracellular development of the parasite, consistent with what has been observed for the other members of the SnSAG gene family. Interestingly, 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. An extensive collection of recombinant expression plasmids and DNA vaccine plasmids have been made for SnSAG2, SnSAG3, and SnSAG4. These plasmid constructs are 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).

A preliminary vaccination study utilizing the SnSAG3 as the immunogen was conducted in horses. Examination of the immune responses in the vaccinated animals revealed that very weak to non-existent responses were elicited, thus implying that either SnSAG3 is a poor candidate for vaccination or that our DNA vaccination protocol needs to be modified. Finally, a novel surface protein, SnSPR1, was identified and characterized in S. neurona merozoites. The SnSPR1 surface protein does not appear to be highly immunogenic, in contrast to the SnSAG surface proteins, but in vitro experiments suggested that SnSPR1 might be involved in the parasite's attachment or invasion of host cells.

Impact

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 will provide insight into the importance of these molecules during S. neurona infection and intracellular propagation. In turn, this information should prove useful for our efforts to develop methods for improved diagnostics and protective vaccination against the debilitating neurologic disease EPM.

Publications

Zhang, D., and Howe, D.K. (2007). Investigation of SnSPR1, a Novel and Abundant Surface Protein of Sarcocystis neurona Merozoites, Veterinary Parasitology (in press, doi: 10.1016/j.vetpar.2007.12.036).

Howe, D.K., Gaji, R.Y., Marsh, A.E., Patil, B.A., Saville, W.J., Lindsay, D.S., Dubey, J.P., and Granstrom, D.E. (2007) Strains of Sarcocystis neurona Exhibit Differences in Their Surface Antigens, Including the Absence of the Major Surface Antigen SnSAG1, International Journal for Parasitology (in press, doi: 10.1016/j.ijpara.2007.09.007).