Field evaluation of a quantitative polymerase chain reaction assay forMycoplasma hyorhinis

Abstract

Abstract. Mycoplasma hyorhinis has emerged as an important cause of systemic disease in nursery pigs. However, this bacterium can also be found in the upper respiratory tract of healthy swine. The current study describes the development of a quantitative polymerase chain reaction assay for the detection of M. hyorhinis and the evaluation of the assay in both disease diagnosis and disease surveillance using a large number of field samples. The analytical sensitivity was estimated to be 12 genome equivalents/μl. The assay was highly specific, detecting all 25 M. hyorhinis isolates tested and none of the 19 nontarget species tested. Assay repeatability was evaluated by testing different matrices spiked with known amounts of M. hyorhinis. Overall, assessment of the repeatability of the assay showed suitable precision within and between runs for all matrices. The coefficient of variation ranged from 10% to 24%. Mycoplasma hyorhinis DNA was detected in 48% of samples (pericardium, pleura, joints, nasal cavity, and lungs) from pigs with systemic disease. Mycoplasma hyorhinis was detected in nasal (92%) and oropharyngeal swabs (66%), as well as in oral fluids (100%). Potential uses of this tool involve the characterization of the prevalence of this pathogen in swine herds as well as bacterial quantification to evaluate intervention efficacy.

A new multiplex real‐time TaqMan®PCRfor quantification ofMycoplasma hyopneumoniae,M. hyorhinisandM. flocculare: exploratory epidemiological investigations to research mycoplasmal association in enzootic pneumonia‐like lesions in slaughtered pigs

“…() and was about one log 10 higher than the result of the 16S rRNA‐based qPCR developed by Clavijo et al . (). However, this estimate is arguable because the specificity was assessed by electrophoresis on 1% agarose gels (end‐point PCR) and not in real‐time conditions.…”

Genotyping of Mycoplasma hyorhinis using multiple-locus variable number tandem repeat analysis

“…pleura, pericardium, joint, peritoneum, nasal cavity, and lung. Isolates were propagated in modified Hayflick's medium (Kobisch and Friis, 1996) for 7-14 days at 37°C and confirmed by species specific real time PCR (Clavijo et al, 2014).…”

Quantitative real-time polymerase chain reaction for detectingMycoplasma hyosynoviaeandMycoplasma hyorhinisin pen-based oral, tonsillar, and nasal fluids

“…While previous research has demonstrated that a variety of other swine pathogens may be found in porcine oral fluids [ 1 3 5 6 28 ], the current study is the first to show that M. hyosynoviae can also be detected by qPCR in pen-based oral fluid samples. In our longitudinal study, delayed detection of either mycoplasma may have been caused by multiple factors such as protective circulating maternal antibodies, low rate of transmission, few infected animals at the time of weaning, or unknown host factors that confer temporary immunity.…”

“…In this study, the manual procedure using a spin column was shown to increase test sensitivity for both M. hyosynoviae and M. hyorhinis . As recently suggested [ 3 ], this step can affect detection of these mycoplasmas in pen-based oral fluids. As demonstrated here, this may be true for other specimens as well.…”

“…Since M. hyosynoviae and M. hyorhinis are presumably long-term colonizers of the upper respiratory tract of pigs, it was hypothesized that pen-based oral, tonsillar, and nasal fluids would yield positive test results provided that these organisms were known to be circulating in the population [ 3 13 17 32 ]. This criterion was established while taking into consideration that a herd with a clinical history of Mycoplasma -associated arthritis was selected for sampling.…”