A review of mycoplasma diagnostics in cattle.

Mycoplasma species have a global distribution causing serious diseases in cattle worldwide including mastitis, arthritis, pneumonia, otitis media and reproductive disorders. Mycoplasma species are typically highly contagious, are capable of causing severe disease, and are difficult infections to resolve requiring rapid and accurate diagnosis to prevent and control disease outbreaks. This review discusses the development and use of different diagnostic methods to identify Mycoplasma species relevant to cattle, with a particular focus on Mycoplasma bovis. Traditionally, the identification and diagnosis of mycoplasma has been performed via microbial culture. More recently, the use of polymerase chain reaction to detect Mycoplasma species from various bovine samples has increased. Polymerase chain reaction has a higher efficiency, specificity, and sensitivity for laboratory diagnosis when compared with conventional culture-based methods. Several tools are now available for typing Mycoplasma spp. isolates, allowing for genetic characterization in disease outbreak investigations. Serological diagnosis through the use of indirect ELISA allows the detection of antimycoplasma antibodies in sera and milk, with their use demonstrated on individual animal samples as well as BTM samples. While each testing method has strengths and limitations, their combined use provides complementary information, which when interpreted in conjunction with clinical signs and herd history, facilitates pathogen detection, and characterization of the disease status of cattle populations.

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

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.

Genetic characterization of Australian Mycoplasma bovis isolates through whole genome sequencing analysis.

Mycoplasma bovis is a major pathogen in cattle causing mastitis, arthritis and pneumonia. First isolated in Australian cattle in 1970, M. bovis has persisted causing serious disease in infected herds. To date, genetic analysis of Australian M. bovis isolates has not been performed. With whole genome sequencing (WGS) becoming a common tool for genetic characterization, this method was utilized to determine the degree of genetic diversity among Australian M. bovis isolates collected over a nine year period (2006-2015) from various geographical locations, anatomical sites, and from clinically affected and non-clinical carrier animals. Eighty-two M. bovis isolates underwent WGS from which single nucleotide polymorphism (SNP) analysis, comparative genomics and analysis of virulence genes was completed. SNP analysis identified a single M. bovis strain circulating throughout Australia with marked genomic similarity. Comparative genomics suggested minimal variation in gene content between isolates from clinical and carrier animals, and between isolates recovered from different anatomical sites. A total of 50 virulence genes from the virulence factors database (VFDB) were identified as highly similar in the Australian isolates, while the presence of variable surface lipoprotein (vsp) genes was greatly reduced compared to reference strain M. bovis PG45. These results highlight that, while the introduction of multiple M. bovis strains has been prevented, elimination of the current strain has not been successful. The persistence of this strain may be due to the significant role that carrier animals play in harboring the pathogen. The similarity of clinical and non-clinical isolates suggests host and environmental factors play a significant role in determining host pathogen outcomes.