Upper respiratory tract detection of Mycoplasma ovipneumoniae employing nasopharyngeal swabs

Abstract

Abstract Background Flock-level prevalence and characterization of Mycoplasma ovipneumoniae is determined almost exclusively using nasal swabbing followed by molecular detection with either quantitative PCR or multi-locus sequence typing. However, the diagnostic performance and efficiency of swabbing the nasal passage compared to other anatomical locations has not been determined within sheep populations. The goal of this research was to assess the diagnostic capability of nasopharyngeal swabs in comparison to nasal swabs for the detection of Mycoplasma ovipneumoniae. Results Nasal and nasopharyngeal swabs were collected during a controlled exposure study of domestic sheep with Mycoplasma ovipneumoniae. Both swab types were then analyzed via conventional and quantitative PCR. This dataset showed that the use of nasopharyngeal swabs in lieu of nasal swabs resulted in higher sensitivity, reduced inhibition during quantitative PCR, and higher bacterial copy numbers per swab. Moreover, it was demonstrated that diagnostic sensitivity could be further increased during quantitative PCR via ten-fold dilution of the extracted DNA. To confirm these observations in naturally infected animals, we conducted a field study employing a production flock of domestic sheep using both nasal and nasopharyngeal swabbing techniques. Extracted DNA was assessed using the same molecular techniques, where detection of Mycoplasma ovipneumoniae was confirmed by sequencing of either the rpoB or 16S rRNA gene. Similar improvements were observed for nasopharyngeal swabs and template treatment methods within the naturally infected flock. Conclusions Results demonstrate increased diagnostic sensitivity and specificity when sampling with nasopharyngeal swabs as compared to nasal swabs. Therefore, alternate field-testing strategies employing nasopharyngeal swabs should be considered for diagnosis of the presence of M. ovipneumoniae. Importantly, sample treatment following acquisition was found to affect the sensitivity of quantitative PCR, where dilution of eluted DNA template doubled the calculated sensitivity. This demonstrates that, in addition to anatomical location, the presence of inhibitory components in swab extracts also strongly influences diagnostic performance.

Keywords

• Mycoplasma ovipneumoniae
• Nasopharyngeal
• Nasal swab
• Inhibition
• Quantitative PCR
• Diagnostic test