Development of a recombinant protein for the identification of Sarcocystis neurona infections in horses

Equine protozoal myeloencephalitis is an infectious parasitic infection of horses caused by Sarcocystis neurona that results in devastating neurologic disease. Factors surrounding the pathogenesis of infection in the horse are unclear, but it is currently believed the horse is an aberrant host. The diagnosis of equine protozoal myeloencephalitis has been based on serum and cerebral spinal fluid antibodies to Sarcocystis neurona derived antigens demonstrated by immunoblot. The antigens used in the diagnosis of equine protozoal myeloencephalitis have not been demonstrated to be species specific or diagnostic.; Sarcocystis neurona merozoites were grown in cultured cells and a novel method for parasite release was developed. Parasite derived antigens were separated by electrophoresis and used in immunoassays to identify antigens recognized by antibodies from infected horses, but not recognized by those from uninfected horses. A major surface antigen was characterized. The nucleotide sequence of a gene encoding a major surface antigen of S. neurona was cloned and sequenced. Oligonucleotides were developed to amplify an RT-PCR product from Sarcocystis neurona RNA.; A cDNA library was prepared from the mRNA of S. neurona and cloned into an expression vector. A cDNA clone encoding a putative surface antigen was identified among 96 clones characterized by preparing sequence tags. The sequence tag encoded a peptide with significant sequence similarity to a segment of the major surface antigen of S. muris. The cDNA library was screened with the partial nucleotide sequence as a probe, and a clone containing the sequence of the full coding region of SnMSA-1 was obtained. Southern and Northern analysis of the SnMSA-1 gene revealed a single copy in the genomic DNA of S. neurona cultured merozoites and an abundance of mRNA in the merozoite. A recombinant form was produced, rSnMSA-1, and purified using an affinity column. Monoclonal antibodies raised against merozoites recognized the recombinant protein overexpressed in pET 14b. Studies of the native and recombinant protein confirmed that the antigen was a 29 kDa protein with a pI of 7.3 located on the surface of the parasite. Immunoblotted rSnMSA-1 confirmed the presence of antibodies to this antigen in serum and cerebrospinal fluids from horses with clinical EPM. Antibodies raised against rSNMSA-1 were useful for the quantitation of antigen in preparative samples and in the identification of parasites in clinical tissues.