Comparison of culture and a multiplex probe PCR for identifying Mycoplasma species in bovine milk, semen and swab samples

Abstract

Mycoplasma spp. are a major cause of mastitis, arthritis and pneumonia in cattle, and have been associated with reproductive disorders in cows. While culture is the traditional method of identification the use of PCR has become more common. Several investigators have developed PCR protocols to detect M. bovis in milk, yet few studies have evaluated other sample types or other important Mycoplasma species. Therefore the objective of this study was to develop a multiplex PCR assay to detect M. bovis, M. californicum and M. bovigenitalium, and evaluate its analytical performance against traditional culture of bovine milk, semen and swab samples. The PCR specificity was determined and the limit of detection evaluated in spiked milk, semen and swabs. The PCR was then compared to culture on 474 field samples from individual milk, bulk tank milk (BTM), semen and swab (vaginal, preputial, nose and eye) samples. Specificity analysis produced appropriate amplification for all M. bovis, M. californicum and M. bovigenitalium isolates. Amplification was not seen for any of the other Mollicutes or eubacterial isolates. The limit of detection of the PCR was best in milk, followed by semen and swabs. When all three Mycoplasma species were present in a sample, the limit of detection increased. When comparing culture and PCR, overall there was no significant difference in the proportion of culture and PCR positive samples. Culture could detect significantly more positive swab samples. No significant differences were identified for semen, individual milk or BTM samples. PCR identified five samples with two species present. Culture followed by 16S-23S rRNA sequencing did not enable identification of more than one species. Therefore, the superior method for identification of M. bovis, M. californicum and M. bovigenitalium may be dependent on the sample type being analysed, and whether the identification of multiple target species is required.

Detection of Mycoplasma bovis in Pneumonic Calves

“…Depending on the obtained results, the PCR technique was appeared as an excellent directly method in the diagnosis of Mycoplasma and principally M. bovis from pneumonic lungs, without the demands for culturing microorganism, which is accurate method and will reduce the consuming time and efforts. Many global studies supported the use of PCR instead of the culturing or together for diagnosis of Mycoplasma and particularly M. bovis [39][40][41][42][43][44][45] .…”

Gap analysis ofMycoplasma bovisdisease, diagnosis and control: An aid to identify future development requirements

“…In general, molecular methods can be divided into those allowing specific detection of M. bovis (using either conventional or real‐time PCRs; Cai, Bell‐Rogers, Parker, & Prescott, ; Ghadersohi, ; Hotzel, Sachse, & Pfützner, ; Rossetti, Frey, & Pilo, ; Sachse et al., ; Subramaniam et al., ) and those allowing simultaneous detection of M. bovis with other mycoplasma species or Mollicutes . The latter includes multiplex PCRs (Boonyayatra et al., ; Cornelissen et al., ; Gioia, Werner, Nydam, & Moroni, ; Justice‐Allen, Trujillo, Goodell, & Wilson, ; Parker, House, Hazelton, Bosward, & Sheehy, ; Righter, Rurangirwa, Call, & McElwain, ), PCR with denaturing gradient gel electrophoresis (DGGE); (McAuliffe, Ellis, Lawes, Ayling, & Nicholas, ) and a DNA microarray based on oligonucleotide probes derived from the 23S rRNA gene and species‐specific probes from the tuf gene target (Bottinelli et al., ; Schnee et al., ). It is important to note that although multiplex detection may help in the diagnosis of mixed mollicute infections, it may introduce additional complexity into the determination of disease aetiology; multiple mycoplasma species may be found, for example, in the upper respiratory or genital tracts that are apparent commensals with questionable significance in disease (Brown et al., ).…”

Short communication: Shedding of Mycoplasma bovis and antibody responses in cows recently diagnosed with clinical infection

“…First, isolates were processed using a Mycoplasma spp. conventional PCR assay as described by Parker et al (2017). This was followed by an Acholeplasma laidlawii conventional PCR assay designed using the Primer3 software program (Untergasser et al, 2012; Table 1).…”