Validation of an influenza virus A 5'Taq nuclease assay for the detection of equine influenza virus A RNA in nasal swab samples.

OBJECTIVE: Describe the in-house validation of a previously reported influenza virus type A 5'Taq nuclease assay for detecting equine influenza virus A RNA in nasal swab material. METHODS: The validation compares the 5'Taq nuclease assay with a gel-based reverse transcription nested polymerase chain reaction (PCR) previously reported by the Irish Equine Centre for detection of H3N8 and H7N7 equine influenza viruses. This test was chosen because it targets a different region of the viral genome to the real-time test, so it is not merely a repeat of the same test in a different format. Moreover, nested PCRs are commonly considered to have similar sensitivity to real-time PCRs and are therefore ideal for evaluation comparisons. RESULTS: The sensitivity of the nested PCR was comparable to the 5'Taq nuclease test. Known positive samples and known negative samples reacted with both tests with 100% correlation. Parallel testing of 276 nasal swab samples showed 98% agreement. CONCLUSION: The specificity of the nested amplicons was confirmed by nucleotide sequencing and showed >99.5% identity with the same region of previously published equine influenza virus A sequences. The results of this work are appropriate validation for the acceptance of the real-time PCR for equine influenza A virus in equine nasal swabs.

Isolation of Mucor circinelloides from a case of ulcerative mycosis of platypus (Ornithorhynchus anatinus), and a comparison of the response of Mucor circinelloides and Mucor amphibiorum to different culture temperatures.

The fungus Mucor circinelloides was isolated from a platypus (Ornithorhynchus anatinus) suffering from ulcerative mycosis. On horse blood agar at 20, 25 and 30 degrees C, the fungus formed sphaerule-like bodies, a morphology previously associated with Mucor amphibiorum, the species thought to be responsible for the disease in platypus. A biopsy taken from the ulcer was fixed, cut and stained. The sections were compared with sections taken from other platypuses suffering from ulcerative mycosis, and from which M. amphibiorum had been isolated. There were no discernible differences between the sphaerule-like bodies found in any of the sections. The presence of sphaerule-like bodies in tissues of ulcerated animals can, therefore, probably no longer be relied upon as a definitive method for the diagnosis of M. amphibiorum infection. It is possible that M. circinelloides is either a primary or a secondary pathogen of platypuses, and further work is required to resolve this point. The isolate of M. circinelloides grew at temperatures up to 38 degrees C, with an optimum temperature for growth of 30 degrees C. Of six isolates of M. amphibiorum derived from both platypus and amphibians, two grew well at 38 degrees C. The growth of one of these isolates at elevated temperatures may be explained by the hot climate of the area in Queensland in which it was found. All of the isolates tested had maximum temperatures for growth in excess of the body temperature of platypuses (32 degrees C).