A European interlaboratory trial to evaluate the performance of different PCR methods for Mycoplasma bovis diagnosis

Abstract

BackgroundSeveral species-specific PCR assays, based on a variety of target genes are currently used in the diagnosis of Mycoplasma bovis infections in cattle herds with respiratory diseases and/or mastitis. With this diversity of methods, and the development of new methods and formats, regular performance comparisons are required to ascertain diagnostic quality. The present study compares PCR methods that are currently used in six national veterinary institutes across Europe. Three different sample panels were compiled and analysed to assess the analytical specificity, analytical sensitivity and comparability of the different PCR methods. The results were also compared, when appropriate, to those obtained through isolation by culture. The sensitivity and comparability panels were composed of samples from bronchoalveolar fluids of veal calves, artificially contaminated or naturally infected, and hence the comparison of the different methods included the whole workflow from DNA extraction to PCR analysis.ResultsThe participating laboratories used i) five different DNA extraction methods, ii) seven different real-time and/or end-point PCRs targeting four different genes and iii) six different real-time PCR platforms. Only one commercial kit was assessed; all other PCR assays were in-house tests adapted from published methods. The analytical specificity of the different PCR methods was comparable except for one laboratory where Mycoplasma agalactiae was tested positive. Frequently, weak-positive results with Ct values between 37 and 40 were obtained for non-target Mycoplasma strains. The limit of detection (LOD) varied from 10 to 103 CFU/ml to 103 and 106 CFU/ml for the real-time and end-point assays, respectively. Cultures were also shown to detect concentrations down to 102 CFU/ml. Although Ct values showed considerable variation with naturally infected samples, both between laboratories and tests, the final result interpretation of the samples (positive versus negative) was essentially the same between the different laboratories.ConclusionWith a few exceptions, all methods used routinely in the participating laboratories showed comparable performance, which assures the quality of diagnosis, despite the multiplicity of the methods.Electronic supplementary materialThe online version of this article (10.1186/s12917-019-1819-7) contains supplementary material, which is available to authorized users.

Mycoplasma bovis Infections—Occurrence, Diagnosis and Control

“…M. bovis causes serious health problems in cattle herds almost all over the world, but its detection is not harmonised as yet and relies on different diagnostic methods, often in-house molecular techniques based on a variety of target genes and various different DNA extraction methods. There was conducted a European interlaboratory comparison of the diagnostic utility of the molecular tests for M. bovis detection [ 71 ]. Six laboratories from different countries were included in the study.…”

Monitoring Mycoplasma bovis Diversity and Antimicrobial Susceptibility in Calf Feedlots Undergoing a Respiratory Disease Outbreak

“…Real-time PCR (rtPCR) was performed on these bulk extracts using LSI VetMAX Screening pack Ruminant Respiratory Pathogens (ThermoFisher) to detect the various pathogens responsible for BRD ( M. bovis , Histophilus somni , Pasteurella multocida , Mannheimia haemolytica , Coronavirus, Respiratory Syncytial Virus and Parainfluenza 3) (at T1), or with VetMAX™ M. bovis Kit (ThermoFisher) to only assess M. bovis presence (at T0 and T2), according to the manufacturer’s recommendations. The Ct cut-off for M. bovis -positiveness was set at ≤ 37 according to the recommendation of Wisselink et al [ 45 ].…”

The presence of Mycoplasma bovis in colostrum

“…(Table 2). High Ct values could indicate the presence of other Mycoplasma species [14], and very high Ct values may also indicate carryover of DNA between samples [15]. Even though all farmers were instructed to take milk samples as aseptically as possible through an on-site demonstration, it is possible the actual sampling was not done lege artis in every case.…”

“…This might point to the fact that some individual herds are more affected, depending on the timing of infection of periparturient cows. Alternatively, it might be the consequence of false positive PCR results due to sample contamination or the presence of other Mycoplasma species [14,15].…”