Identification of opossums (Didelphis virginiana) as the putative definitive host of Sarcocystis neurona

Sarcocystis neurona is an apicomplexan that causes equine protozoal myeloencephalitis (EPM) in North and South America. Horses appear to be an aberrant host, because the merozoites continually divide in the central nervous system, without encysting. The natural host species has not previously been identified. The small subunit ribosomal RNA (SSURNA) gene of S. neurona was compared to those of Sarcocystis muris, Sarcocystis cruzi, Toxoplasma gondii, and Cryptosporidium parvum to identify a unique region suitable for a species-specific amplification primer. The S. neurona SSURNA primer was used in a polymerase chain reaction (PCR) assay for the purpose of identifying this organism in feces and intestinal digest of wildlife specimens. Sporocysts were isolated from 4 raccoons (Procyon lotor), 2 opossums (Didelphis virginiana), 7 skunks (Mephitis mephitis), 6 cats (Felis catus), 1 hawk (Accipiter sp.), and 1 coyote (Canis latrans). The S, neurona SSURNA PCR assay and a control PCR assay using protist-specific primers were applied to all sporocyst DNA samples. All sporocyst DNA samples tested positive on the control assay. The SSURNA PCR assay yielded a 484-bp product only when applied to opossum samples. The SSURNA gene of both opossum sporocyst samples was sequenced to determine its relationship to the S. neurona SSURNA gene. The sequence had 99.89% similarity with S. neurona. This suggests that opossums are the definitive host of S, neurona.