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

Antoinette E. Marsh From the Departments of Pathology, Microbiology, and Immunology (Marsh, Barr, Conrad), Medicine and Epidemiology (Madigan), and Anatomy, Physiology, and Cell Biology (Lakritz), School of Veterinary Medicine, University of California, Davis, CA 95616.

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Bradd C. Barr From the Departments of Pathology, Microbiology, and Immunology (Marsh, Barr, Conrad), Medicine and Epidemiology (Madigan), and Anatomy, Physiology, and Cell Biology (Lakritz), School of Veterinary Medicine, University of California, Davis, CA 95616.

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John Madigan From the Departments of Pathology, Microbiology, and Immunology (Marsh, Barr, Conrad), Medicine and Epidemiology (Madigan), and Anatomy, Physiology, and Cell Biology (Lakritz), School of Veterinary Medicine, University of California, Davis, CA 95616.

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Jeffrey Lakritz From the Departments of Pathology, Microbiology, and Immunology (Marsh, Barr, Conrad), Medicine and Epidemiology (Madigan), and Anatomy, Physiology, and Cell Biology (Lakritz), School of Veterinary Medicine, University of California, Davis, CA 95616.

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Patricia A. Conrad From the Departments of Pathology, Microbiology, and Immunology (Marsh, Barr, Conrad), Medicine and Epidemiology (Madigan), and Anatomy, Physiology, and Cell Biology (Lakritz), School of Veterinary Medicine, University of California, Davis, CA 95616.

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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. (Am J Vet Res 1996;57:975–981)

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. (Am J Vet Res 1996;57:975–981)

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