Transmission of Mycoplasma bovis infection in bovine in vitro embryo production.

Mycoplasma bovis (M. bovis) causes several costly diseases in cattle and has a negative effect on cattle welfare. There is no effective commercial vaccine, and antimicrobial resistance is common. Maintaining a closed herd is the best method to minimize the risk of introduction of M. bovis. Assisted reproduction is crucial in a closed herd to make genetic improvements. M. bovis has been found in commercial semen, and contaminated semen has been the source of disease in naïve dairy herds. The objective of this study was to evaluate M. bovis transmission in bovine in vitro embryo production (IVP) using several possible exposure routes. We used a wild-type M. bovis strain isolated from semen at a final concentration of 106 CFU/mL to infect cumulus-oocyte complexes, spermatozoa, and 5-day-old embryos. We also used naturally contaminated semen in fertilization. Blastocysts were collected on day 7-8 and zona pellucida (ZP)-intact embryos were either washed 12 times, including trypsin washes as recommended by the International Embryo Technology Society (IETS), or left unwashed. Washed and unwashed embryos, follicular fluids, maturation medium, cumulus cells, fertilization medium, and G1 and G2 culture media, as well as all wash media were analyzed using enrichment culture followed by real-time PCR detection of M. bovis. Altogether, 76 pools containing 363 unwashed embryos and 52 pools containing 261 IETS washed embryos were analyzed after oocytes, spermatozoa, or 5-day-old embryos were infected with M. bovis or naturally contaminated semen was used in fertilization. We could not detect M. bovis in any of the embryo pools. M. bovis was not found in any of 12 wash media from different exposure experiments. M. bovis did not affect the blastocyst rate, except when using experimentally infected semen. Contrary to an earlier study, which used a cell co-culture system, we could not demonstrate M. bovis in embryo wash media or tight adherence of M. bovis to ZP-intact embryos. Naturally infected semen did not transmit M. bovis to embryos. We conclude that by using our IVP system, the risk of M. bovis transmission via IVP embryos to recipient cows is very low.

Suitability of Nasal and Deep Nasopharyngeal Swab Sampling of Calves in the Mycoplasma bovis Control Program.

Mycoplasma bovis is an important cattle pathogen affecting animal health, welfare, and productivity. The main disease syndromes are mastitis, pneumonia, and otitis media in young stock, as well as arthritis. Response to antibiotic treatment is poor and no effective vaccine is available. Asymptomatic carriers are common and usually harbor the organism in the airways or mammary glands. Purchase of carrier animals is a major risk for the introduction of infection into naive herds. Following the detection of M. bovis in Finland in 2012, a voluntary control program was established. It aims to prevent the spread of the infection and to help farms attain certification of a low M. bovis risk. Among the diagnostic tools in the program, nasal swabs (NS) from young calves have been tested for M. bovis to indicate the infection status of the herd. In this study, we assessed the suitability of this test method. We analyzed the effectiveness of NS and deep nasopharyngeal swabs (NP) to detect M. bovis in pneumonic and healthy calves in dairy herds recently infected with M. bovis. In pneumonic calves, NP sampling followed by culture and real-time PCR demonstrated a proportion of positive agreement (PPA) of 0.91 compared with bronchoalveolar lavage (BAL), whereas NS showed only 0.5 PPA compared with BAL. Among healthy dairy calves, overall M. bovis prevalence in NS was 29.6%. The highest rate of shedding (43%) occurred in calves 31-60 days old. At the calf level, M. bovis prevalence in NP samples was 47% compared with 33% in NS samples among the 284 studied calves. However, at the herd level, NS sampling classified 51 out of 54 herds with a positive infection status as infected, whereas in NP sampling, the respective figure was 43 out of 54 herds (p = 0.061). In conclusion, NS sampling from calves under 6 months of age and analyzed by real-time PCR is a cost-efficient method for a control program to detect M. bovis in dairy herds, even if no M. bovis mastitis has been detected in the herd. For pneumonic calves, we recommend only NP or BAL sampling.

Mycoplasma bovis infection in dairy herds-Risk factors and effect of control measures.

As Mycoplasma bovis spreads to new countries and becomes increasingly recognized as a disease with major welfare and economic effects, control measures on dairy farms are needed. To minimize the risk of infection spread to naive herds, all possible risk factors for M. bovis infection should be identified and controlled. Mycoplasma bovis was first diagnosed in dairy cattle in Finland in 2012, and by January 2020, 86 Finnish dairy farms (<1.5%) supporting M. bovis infections were identified. We evaluated risk factors for M. bovis infection using a questionnaire provided to 40 infected and 30 control dairy farms. Control measures were advised for 19 of the infected dairy farms during visits by a veterinarian. The course of the infection on those farms was followed by analyzing calf nasal swabs with PCR for presence of M. bovis 4 times at 6-mo intervals. Control measures included culling of M. bovis mastitic cows, isolation of new calves from older animals after initial M. bovis mastitic cows had been culled, prevention of nose-to-nose contact with infected animals, early detection of mastitis cases using M. bovis PCR, and hygiene measures mainly related to milking, calf pens, feeding buckets, and teats. Farms implemented the control measures related to the isolation of calves or avoidance of nose-to-nose contact in various ways, according to farm structures and financial circumstances.In our study, the control measures recommended to the dairy farms appeared effective, such that 13 of 19 farms reached a low risk level during at least 3 consecutive negative samplings from calves, with no M. bovis mastitis detected subsequently. Among risk factors, insemination with an M. bovis-positive bull indicated a trend of increasing the odds of M. bovis infection on the farm in a multivariable logistic model. In contrast, higher herd average milk yield had an association with lower odds for M. bovis infection. Occurrence of other infectious diseases affecting several animals on the dairy farm in the previous 6 mo before M. bovis infection were more frequent on M. bovis-infected farms.

Efficacy of Two Antibiotic-Extender Combinations on Mycoplasma bovis in Bovine Semen Production.

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.

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

BACKGROUND: Several 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. RESULTS: The 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. CONCLUSION: With 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.

Semen as a source of Mycoplasma bovis mastitis in dairy herds.

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.