In situ hybridisation for identification and differentiation of Mycoplasma hyopneumoniae, Mycoplasma hyosynoviae and Mycoplasma hyorhinis in formalin‐fixed porcine tissue sections^Note

Abstract

Oligonucleotide probes targeting 16S ribosomal RNA were designed for species-specific identification of the porcine mycoplasmas Mycoplasma hyopneumoniae, Mycoplasma hyorhinis and Mycoplasma hyosynoviae using a fluorescent in situ hybridisation assay. The specificity of the probes was evaluated using pure cultures as well as porcine tissue sections with artificial presence of mycoplasma, and the probes were found specific for the target organisms. The assay was applied on sections of 28 tissue samples from pigs infected with one or more of the three Mycoplasma species as determined by cultivation. M. hyopneumoniae and M. hyorhinis were identified in accordance with cultivation in lung sections from nine pigs affected by catarrhal to purulent bronchopneumonia. Likewise, in eight cases of fibrinous pericarditis, M. hyopneumoniae, M. hyorhinis and M. hyosynoviae were the infectious agents according to cultivation and were correctly identified by in situ hybridisation. Out of 11 joints cultivation positive for M. hyosynoviae, the probe was only able to identify M. hyosynoviae in eight cases probably due to a low number of microorganisms in the tissue sections. The in situ hybridisation assay is well suited for use in diagnostic and experimental work as well as a tool for pathogenesis studies.

Mycoplasma hyorhinis infection in early cases of mycoplasmal pneumonia in swine and evaluation of diagnostic assays

“…In our study, similar to observations by Boye et al (2001), FISH had no cross-reaction between the two Mycoplasma species studied, showing 100% specificity.…”

“…The FISH procedure was performed according to Boye et al (2001), using individual probes for the detection of the Mycoplasmas, with oligonucleotides sequences that target the 16s portion of the rRNA: 5'CCGTCAA-GACTAGAGCAT 3' for M. hyopneumoniae and 5'GCTGTGAAGC-TCCTTTCT 3' for M. hyorhinis. Tissue sections were deparaffinized by heating in an incubator at 65°C, immersed in xylene and hydrated in decreasing ethanol solutions and distilled water for 5 minutes.…”

A Multiplex PCR to Identify Porcine Mycoplasmas Present in Broth Cultures

“…In contrast to the other three Mycoplasma species, this bacterium is grown in media enriched with arginine (Friis, 1974). Di¡erent techniques, including PCR, have already been reported to di¡erentiate these porcine Mycoplasma species (Dussurget and Roulland-Dussoix, 1994;Stemke et al, 1994;Mattsson et al, 1995;Blanchard et al, 1996;Baumeister et al, 1998;Caron et al, 2000;Boye et al, 2001), but no single PCR test has been described that simultaneously distinguishes M. hyopneumoniae, M. hyorhinis and M. £occulare. The aim of this study was to develop such a multiplex PCR to identify these Mycoplasma species in broth culture.…”

Development of In Situ Hybridization for the Detection of Mycoplasma hyorhinis in Formalin-Fixed Paraffin-Embedded Tissues from Naturally Infected Pigs with Polyserositis

“…In situ hybridization using a fluorescein-labelled probe had been reported for the differentiation between M. hyopneumoniae and M. hyorhinis in formalin-fixed tissues [3]. Although M. hyorhinis DNA can be detected in cells by fluorescent in situ hybridization, definitive localization of the mycoplasma DNA within various cells has not been possible, especially when the amount of DNA is small.…”