SEQUENCE-ANALYSIS AND POLYMERASE CHAIN-REACTION AMPLIFICATION OF SMALL-SUBUNIT RIBOSOMAL DNA FROM SARCOCYSTIS-NEURONA

Objectives-To identify Sarcocystis neurona-specific DNA sequences in t he nuclear small subunit ribosomal RNA (nss-rRNA) gene that could be u sed to distinguish S neurona from other closely related protozoal para sites, and to evaluate a polymerase chain reaction (PCR) test, using b road based primers and a unique species-specific probe on CSF for dete ction of S neurona in equids. Procedures-Sequencing of the nuclear sma ll subunit ribosomal RNA gene from a new S neurona isolate (UCD 1) was performed. The sequence was compared with that of other closely relat ed Sarcocystidae parasites. From this sequence, conserved DNA sequence primers were selected and an oligonucleotide probe was designed to hy bridize with a unique region of the S neurona gene. For clinical evalu ation, horses were considered test positive for S neurona infection on the basis of immunohistochemical detection of the parasites in the CN S. Results-Sensitivity of this PCR and probe-based detection system wa s approximately 1 to 5 merozoites. Cerebrospinal fluid from 2 of 5 hor ses with histologic lesions consistent with equine protozoal myeloence phalitis were PCR- and probe-positive in a blind test of this procedur e, and all uninfected horses were test negative. Conclusion and Clinic al Relevance-This PCR-based system is a useful method of confirming S neurona in CSF and has the advantage of facilitating detection of othe r apicomplexan protozoans that may be infective for horses. The useful ness of this test is limited by the presence of parasites free in the CSF of clinically affected horses.