Mycoplasma bovis infections are responsible for substantial economic losses in the cattle industry, have significant welfare effects and increase antibiotic use. The pathogen is often introduced into naive herds through healthy carrier animals. In countries with a low prevalence of M. bovis, transmission from less common sources can be better explored as the pathogen has limited circulation compared to high prevalence populations. In this study, we describe how M. bovis was introduced into two closed and adequately biosecure dairy herds through the use of contaminated semen during artificial insemination (AI), leading to mastitis outbreak in both herds. Epidemiological analysis did not reveal an infection source other than semen. In both farms the primary clinical cases were M. bovis mastitis in cows inseminated with the semen of the same bull four weeks before the onset of the disease. One semen straw derived from the semen tank on the farm and other semen lots of this bull were positive for M. bovis. In contrast, semen samples were negative from other bulls that had been used for insemination in previous or later oestrus to those cows with M. bovis mastitis. Furthermore, cgMLST of M. bovis isolates supported the epidemiological results. To our knowledge this is the first study describing the introduction of M. bovis infection into a naive dairy herd via processed semen. The antibiotics used in semen extenders should be re-evaluated in order to provide farms with M. bovis-free semen or tested M. bovis-free semen should be available.

Mycoplasma bovis causes bovine respiratory disease, mastitis, arthritis and otitis. The importance of M. bovis has escalated because of recent outbreaks and introductions into countries previously free of M. bovis. We characterized the course of M. bovis infection on 19 recently infected dairy farms over 24 months. Our objective was to identify diagnostic tools to assess the efficacy of control measures to assess low risk infection status on M. bovis infected farms. PCR assays and culture were used to detect M. bovis, and in-house and BioX ELISAs were used to follow antibody responses. Cows and young stock were sampled on four separate occasions, and clinical cases were sampled when they arose. On 17 farms, a few cases of clinical mastitis were detected, mostly within the first eight weeks after the index case. Antibodies detected by in-house ELISA persisted in the serum of cows at least for 1.5 years on all farms, regardless of the M. bovis infection status or signs of clinical disease or subclinical mastitis on the farm. Six out of 19 farms became low risk as the infection was resolved. Our results suggest that, for biosecurity purposes, regular monitoring should be conducted on herds by screening for M. bovis in samples from cows with clinical mastitis and calves with pneumonia, in conjunction with testing young stock by screening longitudinally collected nasal swabs for M. bovis and sequential serum samples for antibody against recombinant antigen.3

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.

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Mycoplasma bovis is an important bovine pathogen. Artificial insemination (AI) using contaminated semen can introduce the agent into a naïve herd. Antibiotics, most often gentamycin, tylosin, lincomycin, spectinomycin (GTLS) combination are added to semen extender to prevent transmission of pathogenic bacteria and mycoplasmas. In a commercial AI straw production system with industrial scale procedures, we analyzed the mycoplasmacidal efficacy of GTLS and ofloxacin on M. bovis ATCC and wild type strain isolated from commercial AI straws. The strains were spiked at two concentrations (106 and 103 CFU/mL) into semen. Viable M. bovis in frozen semen straws was detected by enrichment culture and real-time PCR. We also compared different protocols to extract M. bovis DNA from spiked semen. None of the antibiotic protocols had any effect on the viability of either of the M. bovis strains at high spiking concentration. At low concentration, the wild type was inhibited by all other protocols, except low GTLS, whereas the ATCC strain was inhibited only by high GTLS. The InstaGene™ matrix was the most effective method to extract M. bovis DNA from semen. When there is a low M. bovis contamination level in semen, GTLS used at high concentrations, in accordance with Certified Semen Services requirements, is more efficient than GTLS used at concentrations stated in the OIE Terrestrial Code.