J Vet Sci.  2015 Jun;16(2):195-201. 10.4142/jvs.2015.16.2.195.

Quantitative real-time polymerase chain reaction for detecting Mycoplasma hyosynoviae and Mycoplasma hyorhinis in pen-based oral, tonsillar, and nasal fluids

• Gomes Neto JC
• Bower L
• Erickson B
• Wang C
• Raymond M
• Strait EL

• KMID: 2164529
• DOI: http://doi.org/10.4142/jvs.2015.16.2.195

Abstract

Mycoplasma (M.) hyorhinis and M. hyosynoviae are pathogens known to cause disease in pigs post-weaning. Due to their fastidious nature, there is increased need for culture-independent diagnostic platforms to detect these microorganisms. Therefore, this study was performed to develop and optimize quantitative real-time PCR (qPCR) assays to rapidly detect M. hyorhinis and M. hyosynoviae in pen-based oral fluids as well as nasal and tonsillar fluids as proxies for samples used in swine herd surveillance. Two methods of genomic DNA extraction, automated versus manual, were used to compare diagnostic test performance. A wean-to-finish longitudinal study was also carried out to demonstrate the reproducibility of using pen-based oral fluids. Overall, pen-based oral and tonsillar fluids were more likely to be positive for both types of bacteria whereas only M. hyorhinis was detected in nasal fluids. DNA extraction protocols were shown to significantly influence test result. Although the initial detection time somewhat differed, both organisms were repeatedly detected in the longitudinal study. Overall, this study evaluated two qPCR methods for rapid and specific detection of either mycoplasma. Results from the present investigation can serve as a foundation for future studies to determine the prevalence of the two microorganisms, environmental load, and effectiveness of veterinary interventions for infection control.

Keyword

Mycoplasma hyorhinis; Mycoplasma hyosynoviae; real-time PCR; swine

MeSH Terms

Animals
Diagnostic Tests, Routine/methods/*veterinary
Female
Longitudinal Studies
Mouth/microbiology
Mycoplasma Infections/diagnosis/microbiology/*veterinary
Mycoplasma hyorhinis/*isolation & purification
Mycoplasma hyosynoviae/*isolation & purification
Nose/microbiology
Palatine Tonsil/microbiology
Real-Time Polymerase Chain Reaction/*veterinary
Reproducibility of Results
Swine
Swine Diseases/*diagnosis/microbiology

Figure

• 

  Fig. 1 M. hyosynoviae load in tonsillar, pen-based oral, and nasal fluids according to two distinct methods of genomic DNA extraction (magnetic beads versus spin column). The box-and-whisker plot includes only samples that were positive in order to demonstrate the range of bacterial load among diagnostic specimens and DNA extraction protocols. Asterisks indicate significant differences for the log10 number of bacteria when comparing the two methods of DNA extraction (p < 0.05). The limit of detection for M. hyosynoviae-specific qPCR is indicated in the figure. TSSC: tonsil scrapping and spin column, TSMB: tonsil scrapping and magnetic beads, OFSC: oral fluids and spin column, OFMB: oral fluids and magnetic beads, NSSC: nasal swab and spin column, NSMB: nasal swab and magnetic beads.

• 

  Fig. 2 M. hyorhinis load in tonsillar, pen-based oral, and nasal fluids according to two distinct methods of genomic DNA extraction (magnetic beads versus spin column). The box-and-whisker plot only includes samples that were positive in order to demonstrate the range of bacterial load among diagnostic specimens and DNA extraction protocols. Asterisks indicate significant differences for the log10 number of bacteria when comparing the two methods of DNA extraction (p < 0.05). The limit of detection for M. hyorhinis-specific qPCR is indicated in the figure.

• 

  Fig. 3 M. hyosynoviae and M. hyorhinis load in pen-based oral fluids serially sampled from wean-to-finish pigs in a longitudinal study. Sampling was performed for the same five pens initially selected. The box-and-whisker plot includes only samples that were positive in order to demonstrate the range of bacterial load in the pen-based oral fluids over time. The limit of qPCR detection for both types of mycoplasma is indicated in the figure.

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