Sequence analysis and polymerase chain reaction amplification of small subunit ribosomal DNA from Sarcocystis neurona

Abstract

Objectives: To identify Sarcocystis neurona-specific DNA sequences in the nuclear small subunit ribosomal RNA (nss-rRNA) gene that could be used to distinguish S neurona from other closely related protozoal parasites, and to evaluate a polymerase chain reaction (PCR) test, using broad based primers and a unique species-specific probe on CSF for detection of S neurona in equids.

Procedures: Sequencing of the nuclear small subunit ribosomal RNA gene from a new S neurona isolate (UCD 1) was performed. The sequence was compared with that of other closely related Sarcocystidae parasites. From this sequence, conserved DNA sequence primers were selected and an oligonucleotide probe was designed to hybridize with a unique region of the S neurona gene. For clinical evaluation, horses were considered test positive for S neurona infection on the basis of immunohistochemical detection of the parasites in the CNS.

Results: Sensitivity of this PCR and probe-based detection system was approximately 1 to 5 merozoites. Cerebrospinal fluid from 2 of 5 horses with histologic lesions consistent with equine protozoal myeloencephalitis were PCR- and probe-positive in a blind test of this procedure, and all uninfected horses were test negative.

Conclusion and clinical relevance: This PCR-based system is a useful method of confirming S neurona in CSF and has the advantage of facilitating detection of other apicomplexan protozoans that may be infective for horses. The usefulness of this test is limited by the presence of parasites free in the CSF of clinically affected horses.