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Background: Mycoplasma bovis is a global pathogen of cattle but was detected for the first time in New Zealand in 2017, triggering a response under their Biosecurity Act as an "unwanted organism". Following a lengthy eradication campaign, the Ministry of Primary Industries (MPI) now requires all bovine semen destined for export to New Zealand to be screened with an M. bovis-specific real-time PCR (rtPCR) compliant with amended import health standard (IHS) test requirements aimed at preventing the accidental importation of M. bovis. The standard stipulates that semen samples cannot be centrifuged prior to DNA extraction. To comply with these strict requirements, one of the listed tests was validated together with different DNA preparation steps and compared with existing in-house procedures. DNA was extracted from semen straws using the current in-house semi-automated platform procedures for processing culture, tissue and body fluid sample submissions and was compared with the stipulated test requirements. DNA from centrifuged and unspun semen samples spiked with M. bovis was also compared., Results: The rtPCR had a sensitivity and specificity of 100% (95% confidence interval = 79-100% and 74-100%, respectively) when testing DNA from other Mycoplasma species or bovine semen spiked with the latter, with a high level of repeatability for within- and between- run replicates. The consistent limit of detection was 0.001 pg/µl M. bovis DNA and between 5.3 × 10 2 and 7.5 × 10 2 CFU/ml M. bovis when artificially spiked in semen. DNA extracted using the KingFisher Flex was detected with lower Cq values than the Maxwell 16, but the comparable improvements in sensitivity were mainly associated with non-centrifuged samples (p < 0.001). None of the procedures tested impeded the detection sensitivity of M. bovis in the presence of competitor organisms Acholeplasma laidlawii, Mycoplasma bovigenitalium and Ureaplasma diversum, confirming M. bovis specificity of the polC target., Conclusions: Under the experimental conditions applied, this rtPCR test efficiently detected M. bovis in extended bovine semen straw samples from DNA extracted using both semi-automated extraction platforms, irrespective of prior centrifugation of extended semen., (© 2024. Crown.)

Mycoplasma bovis (M. bovis) is capable of causing a range of diseases in cattle, encompassing calf pneumonia, arthritis, conjunctivitis, meningitis, and mastitis. It is widely recognized as one of the predominant pathogens posing a significant threat to the global cattle industry. Therefore, accurate and sensitive methods are urgently needed to detect M. bovis. This study aims to detect M. bovis by combining colloidal gold with biotin-labeled oligonucleotides to improve detection sensitivity and form a chromogenic detection probe based on signal amplification technology. Here, we developed a sensitive and specific polymerase chain reaction-lateral flow dipstick assay (PCR-LFD) strip for efficient nucleic acid detection of M. bovis. A pair of specific primers with 5' ends labeled with biotin and digoxigenin probes was designed for PCR experiments. Colloidal gold particles-labeled anti-digoxigenin IgG coated gold-labeled test strip was prepared, streptavidin was used as the detection probe, and nitrocellulose membrane coated goat anti-mouse IgG was used as the control line. Our results showed that the detection limit of the PCR-LFD was 89 fg/µL for the M. bovis DNA. The results from the test strip were highly consistent with those from real-time qPCR. This assay were highly specific for M. bovis, as there were no cross-reactions with other microorganisms tested and the detection sensitivity of the test was also relatively high (97.67%). The novel strips present a promising tool for the cost-effective and sensitive diagnosis of M. bovis., (© 2024. The Author(s).)

Mycoplasmopsis (M.) bovis, the agent of mastitis, pneumonia, and arthritis in cattle, harbors a small genome of approximately 1 Mbp. Combining data from Illumina and Nanopore technologies, we sequenced and assembled the genomes of 35 European strains and isolate DL422&#95;88 from Cuba. While the high proportion of repetitive structures in M. bovis genomes represent a particular challenge, implementation of our own pipeline Mycovista (available on GitHub www.github.com/sandraTriebel/mycovista ) in a hybrid approach enabled contiguous assembly of the genomes and, consequently, improved annotation rates considerably. To put our European strain panel in a global context, we analyzed the new genome sequences together with 175 genome assemblies from public databases. Construction of a phylogenetic tree based on core genes of these 219 strains revealed a clustering pattern according to geographical origin, with European isolates positioned on clades 4 and 5. Genomic data allowing assignment of strains to tissue specificity or certain disease manifestations could not be identified. Seven strains isolated from cattle with systemic circular condition (SCC), still a largely unknown manifestation of M. bovis disease, were located on both clades 4 and 5. Pairwise association analysis revealed 108 genomic elements associated with a particular clade of the phylogenetic tree. Further analyzing these hits, 25 genes are functionally annotated and could be linked to a M. bovis protein, e.g. various proteases and nucleases, as well as ten variable surface lipoproteins (Vsps) and other surface proteins. These clade-specific genes could serve as useful markers in epidemiological and clinical surveys., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: Mycoplasma (M.) bovis is a major etiological agent of bovine respiratory disease, which is the most economically costly disease of cattle worldwide. Cattle disease surveillance on M. bovis is increasingly using gene-based techniques, such as multilocus sequence typing (MLST), or genome-based techniques such as core genome MLST that both require only partial genomic data. However, accurate up-to-date surveillance also demands complete, circular genomes that can be used as reference to track the evolution of the different lineages. Yet, in France, two of the main subtypes currently circulating still have no representing genome in public databases. Here, to address this gap, we provide and compare three new complete M. bovis genomes obtained from recent clinical isolates that represent major subtypes circulating in France and Europe., Results: Genomes were obtained using a hybrid assembly strategy (Illumina and Nanopore) with fine-tuning of settings and inputs used in the Unicycler assembly pipeline, such as size selection of reads and quality trimming of the FASTQ files. The main characteristics and synteny of the genomes were compared. The three genomes mainly differed by their content in terms of mobile genetic elements, i.e. integrative conjugative elements (ICE) and insertion sequences (IS), a feature that impacts their structure. For instance, strain L15527, representing subtype3 (st3), harbours an exceptionally high number of ICEs, which results in a bigger-sized genome than all those previously described and could be associated with the propensity of st3 to gain and fix mutations through chromosomal transfer mechanisms. In contrast, strain F9160, of st1, is very close to the PG45 type strain isolated in 1961 in the USA, and harbours a huge number of IS. These features may be associated with an evolution towards a host-restricted state or in a "closed" host or environment reservoir until a recent re-emergence., Conclusions: Whole-genome comparison of the three French M. bovis subtypes provides valuable resources for future studies combining epidemiology, phylogenetic data, and phylodynamic methods., (© 2022. The Author(s).)

Background: Mycoplasma bovis is an important pathogen of cattle worldwide. Many different clinical manifestations of infection can occur, including respiratory disease, arthritis, and mastitis, causing heavy losses to beef and dairy industries. Because Mycoplasma species are slow-growing and fastidious, traditional identification methods are not cost- or time-effective, and improved methods are sought to streamline laboratory processes. High-resolution melting PCR (HRM-PCR) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) are 2 relatively recent tools that are rapid and inexpensive to use; we tested 9 isolates of M. bovis using both assays. The HRM-PCR assay used universal mycoplasma primers for the 16S-23S intergenic spacer region (IGSR)., Results: The resulting melting profiles of the field isolates were indistinguishable from the reference strain, indicating accurate identification. For the MALDI-TOF MS, each M. bovis isolate was accurately identified. Mycoplasma arginini and Mycoplasma alkalescens isolates did not identify as M. bovis when tested by either assay., Conclusions: Our work shows that either assay could be used to identify unknown M. bovis isolates. For future work, the MALDI-TOF MS library should be expanded to include more mycoplasmas, and the HRM-PCR assay should be tested on additional mycoplasmas to ensure that the melting profiles are sufficiently distinctive.

Background: Mycoplasma bovis (M. bovis) is a highly contagious cattle pathogen spreading worldwide and especially in Ningxia Hui Autonomous Region in China., Results: Two types of ST, ST10and ST134, were identified in Ningxia Hui Autonomous Region. Thirty-seven strains belonged to ST10 and 28 strains belonged to ST134. ST134 was a new ST and first found in 2009 and was only widely distributed in Ningxia Hui Autonomous Region at present. The M. bovis ST10 was widely spread in many provinces in China and was widespread in Ningxia Hui Autonomous Region since 2010. It is speculated that the prevalence of M. bovis ST10 in Ningxia Hui Autonomous Region began in 2010., Conclusions: This study is the first report on the genetic diversity of M. bovis from 2009 to 2018 in Ningxia Hui Autonomous Region and provides the epidemiological information. These results may help further our understanding of the evolution of M. bovis and provide information that may be useful for the development of novel vaccines.

Background: Implementation of Third-Generation Sequencing approaches for Whole Genome Sequencing (WGS) all-in-one diagnostics in human and veterinary medicine, requires the rapid and accurate generation of consensus genomes. Over the last years, Oxford Nanopore Technologies (ONT) released various new devices (e.g. the Flongle R9.4.1 flow cell) and bioinformatics tools (e.g. the in 2019-released Bonito basecaller), allowing cheap and user-friendly cost-efficient introduction in various NGS workflows. While single read, overall consensus accuracies, and completeness of genome sequences has been improved dramatically, further improvements are required when working with non-frequently sequenced organisms like Mycoplasma bovis. As an important primary respiratory pathogen in cattle, rapid M. bovis diagnostics is crucial to allow timely and targeted disease control and prevention. Current complete diagnostics (including identification, strain typing, and antimicrobial resistance (AMR) detection) require combined culture-based and molecular approaches, of which the first can take 1-2 weeks. At present, cheap and quick long read all-in-one WGS approaches can only be implemented if increased accuracies and genome completeness can be obtained., Results: Here, a taxon-specific custom-trained Bonito v.0.1.3 basecalling model (custom-pg45) was implemented in various WGS assembly bioinformatics pipelines. Using MinION sequencing data, we showed improved consensus accuracies up to Q45.2 and Q46.7 for reference-based and Canu de novo assembled M. bovis genomes, respectively. Furthermore, the custom-pg45 model resulted in mean consensus accuracies of Q45.0 and genome completeness of 94.6% for nine M. bovis field strains. Improvements were also observed for the single-use Flongle sequencer (mean Q36.0 accuracies and 80.3% genome completeness)., Conclusions: These results implicate that taxon-specific basecalling of MinION and single-use Flongle Nanopore long reads are of great value to be implemented in rapid all-in-one WGS tools as evidenced for Mycoplasma bovis as an example.

M. bovis is one of the leading causes of respiratory disease and antimicrobial use in cattle. The pathogen is widespread in different cattle industries worldwide, but highest prevalence is found in the veal industry. Knowledge on M. bovis strain distribution over the dairy, beef and veal industries is crucial for the design of effective control and prevention programs, but currently undocumented. Therefore, the present study evaluated the molecular epidemiology and genetic relatedness of M. bovis isolates obtained from Belgian beef, dairy and veal farms, and how these relate to M. bovis strains obtained worldwide. Full genomes of one hundred Belgian M. bovis isolates collected over a 5-year period (2014-2019), obtained from 27 dairy, 38 beef and 29 veal farms, were sequenced by long-read nanopore sequencing. Consensus sequences were used to generate a phylogenetic tree in order to associate genetic clusters with cattle sector, geographical area and year of isolation. The phylogenetic analysis of the Belgian M. bovis isolates resulted in 5 major clusters and 1 outlier. No sector-specific M. bovis clustering was identified. On a world scale, Belgian isolates clustered with Israeli, European and American strains. Different M. bovis clusters circulated for at least 1.5 consecutive years throughout the country, affecting all observed industries. Therefore, the high prevalence in the veal industry is more likely the consequence of frequent purchase from the dairy and beef industry, than that a reservoir of veal specific strains on farm would exist. These results emphasize the importance of biosecurity in M. bovis control and prevention.

Background: Mycoplasma bovis is an important etiologic agent of bovine mycoplasmosis affecting cattle production and animal welfare. In the past in Israel, M. bovis has been most frequently associated with bovine respiratory disease (BRD) and was rarely isolated from mastitis. This situation changed in 2008 when M. bovis-associated mastitis emerged in Israel. The aim of this study was to utilize whole genome sequencing to evaluate the molecular epidemiology and genomic diversity of M. bovis mastitis-associated strains and their genetic relatedness to M. bovis strains isolated from BRD in local feedlot calves and those imported to Israel from different European countries and Australia., Results: Phylogeny based on total single nucleotide polymorphism (SNP) analysis of 225 M. bovis genomes clearly showed clustering of isolates on the basis of geographical origin: strains isolated from European countries clustered together and separately from Australian and Chinese isolates, while Israeli isolates were found in the both groups. The dominant genotype was identified among local mastitis-associated M. bovis isolates. This genotype showed a close genomic relatedness to M. bovis strains isolated from calves imported to Israel from Australia, to original Australian M. bovis strains, as well as to strains isolated in China., Conclusions: This study represents the first comprehensive high-resolution genome-based epidemiological analysis of M. bovis in Israel and illustrates the possible dissemination of the pathogen across the globe by cattle trade.

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 10 3  CFU/ml to 10 3 and 10 6  CFU/ml for the real-time and end-point assays, respectively. Cultures were also shown to detect concentrations down to 10 2  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.

Background: Mycoplasma bovis (M. bovis) is a major etiological agent of bovine mycoplasmosis around the world. Point-of-care testing in the field is lacking owing to the requirement for a simple, robust field applicable test that does not require professional laboratory equipment. The recombinase polymerase amplification (RPA) technique has become a promising isothermal DNA amplify assay for use in rapid and low-resource diagnostics., Results: Here, a method for specific detection of M. bovis DNA was established, which was RPA combined with lateral flow dipstick (LFD). First, the analytical specificity and sensitivity of the RPA primer and LF-probe sets were evaluated. The assay successfully detected M. bovis DNA in 30 min at 39 °C, with detection limit of 20 copies per reaction, which it was compared the real-time quantitative PCR (qPCR) assay. This method was specific because it did not detect a selection of other bacterial pathogens in cattle. Both qPCR and RPA-LFD assays were used to detect M. bovis 442 field samples from 42 different dairy farms in Shandong Province of China, also the established RPA-LFD assay obtained 99.00% sensitivity, 95.61% specificity, and 0.902 kappa coefficient compared with the qPCR., Conclusions: To the author's knowledge, this is the first report using an RPA-FLD assay to visualise and detect M. bovis. Comparative analysis with qPCR indicates the potential of this assay for rapid diagnosis of bovine mycoplasmosis in resource limited settings.

Background: Mycoplasma bovis is an important pathogen causing pneumonia, mastitis and arthritis in cattle worldwide. As this agent is primarily transmitted by direct contact and spread through animal movements, efficient genotyping systems are essential for the monitoring of the disease and for epidemiological investigations. The aim of this study was to compare and evaluate the multi locus sequence typing (MLST) and the multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) through the genetic characterization of M. bovis isolates from Hungary., Results: Thirty one Hungarian M. bovis isolates grouped into two clades by MLST. Two strains had the same sequence type (ST) as reference strain PG45, while the other twenty nine Hungarian isolates formed a novel clade comprising five subclades. Isolates originating from the same herds had the same STs except for one case. The same isolates formed two main clades and several subclades and branches by MLVA. One clade contained the reference strain PG45 and three isolates, while the other main clade comprised the rest of the strains. Within-herd strain divergence was also detected by MLVA. Little congruence was found between the results of the two typing systems., Conclusions: MLST is generally considered an intermediate scale typing method and it was found to be discriminatory among the Hungarian M. bovis isolates. MLVA proved to be an appropriate fine scale typing tool for M. bovis as this method was able to distinguish closely related strains isolated from the same farm. We recommend the combined use of the two methods for the genotyping of M. bovis isolates. Strains have to be characterized first by MLST followed by the fine scale typing of identical STs with MLVA.