Genes required for survival and proliferation of Mycoplasma bovis in association with host cells.

Mycoplasma bovis is an important emerging pathogen of cattle and bison, but our understanding of the genetic basis of its interactions with its host is limited. The aim of this study was to identify genes of M. bovis required for interaction and survival in association with host cells. One hundred transposon-induced mutants of the type strain PG45 were assessed for their capacity to survive and proliferate in Madin-Darby bovine kidney cell cultures. The growth of 19 mutants was completely abrogated, and 47 mutants had a prolonged doubling time compared to the parent strain. All these mutants had a similar growth pattern to the parent strain PG45 in the axenic media. Thirteen genes previously classified as dispensable for the axenic growth of M. bovis were found to be essential for the growth of M. bovis in association with host cells. In most of the mutants with a growth-deficient phenotype, the transposon was inserted into a gene involved in transportation or metabolism. This included genes coding for ABC transporters, proteins related to carbohydrate, nucleotide and protein metabolism, and membrane proteins essential for attachment. It is likely that these genes are essential not only in vitro but also for the survival of M. bovis in infected animals. IMPORTANCE: Mycoplasma bovis causes chronic bronchopneumonia, mastitis, arthritis, keratoconjunctivitis, and reproductive tract disease in cattle around the globe and is an emerging pathogen in bison. Control of mycoplasma infections is difficult in the absence of appropriate antimicrobial treatment or effective vaccines. A comprehensive understanding of host-pathogen interactions and virulence factors is important to implement more effective control methods against M. bovis. Recent studies of other mycoplasmas with in vitro cell culture models have identified essential virulence genes of mycoplasmas. Our study has identified genes of M. bovis required for survival in association with host cells, which will pave the way to a better understanding of host-pathogen interactions and the role of specific genes in the pathogenesis of disease caused by M. bovis.

A genome-wide transposon mutagenesis screening identifies LppB as a key factor associated with Mycoplasma bovis colonization and invasion into host cells.

Mycoplasma spp., the smallest self-replicating and genome-reduced organisms, have raised a great concern in both the medical and veterinary fields due to their pathogenicity. The molecular determinants of these wall-less bacterium efficiently use their limited genes to ensure successful infection of the host remain unclear. In the present study, we used the ruminant pathogen Mycoplasma bovis as a model to identify the key factors for colonization and invasion into host cells. We constructed a nonredundant fluorescent transposon mutant library of M. bovis using a modified transposon plasmid, and identified 34 novel adhesion-related genes based on a high-throughput screening approach. Among them, the ΔLppB mutant exhibited the most apparent decrease in adhesion to embryonic bovine lung (EBL) cells. The surface-localized lipoprotein LppB, which is highly conserved in Mycoplasma species, was then confirmed as a key factor for M. bovis adhesion with great immunogenicity. LppB interacted with various components (fibronectin, vitronectin, collagen IV, and laminin) of host extracellular matrix (ECM) and promoted plasminogen activation through tPA to degrade ECM. The 439-502 amino acid region of LppB is a critical domain, and F465 and Y493 are important residues for the plasminogen activation activity. We further revealed LppB as a key factor facilitating internalization through clathrin- and lipid raft-mediated endocytosis, which helps the Mycoplasma invade the host cells. Our study indicates that LppB plays a key role in Mycoplasma infection and is a potential new therapeutic and vaccine target for Mycoplasma species.

Diversity and antigenic potentials of Mycoplasmopsis bovis secreted and outer membrane proteins within a core genome of strains isolated from North American bison and cattle.

Mycoplasmopsis bovis is a worldwide economically important pathogen of cattle that can cause or indirectly contribute to bovine respiratory disease. M. bovis is also a primary etiological agent of respiratory disease in bison with high mortality rates. A major challenge in the development of an efficacious M. bovis vaccine is the design of antigens that contain both MHC-1 and MHC-2 T-cell epitopes, and that account for population level diversity within the species. Publicly available genomes and sequence read archive libraries of 381 M. bovis strains isolated from cattle (n = 202) and bison (n = 179) in North America were used to identify a core genome of 575 genes, including 38 that encode either known or predicted secreted or outer membrane proteins. The antigenic potentials of the proteins were characterized by the presence and strength of their T-cell epitopes, and their protein variant diversity at the population-level. The proteins had surprisingly low diversity and varying predictive levels of T-cell antigenicity. These results provide a reference for the selection or design of antigens for vaccine testing against strains infecting North American cattle and bison.

Development of polymerase chain reaction-lateral flow dipstick assay for detection of Mycoplasma bovis in cattle.

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.

De novo genome assembly resolving repetitive structures enables genomic analysis of 35 European Mycoplasmopsis bovis strains.

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_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.

Genomic features of Mycoplasma bovis subtypes currently circulating in France.

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.

An emerging role for cyclic dinucleotide phosphodiesterase and nanoRNase activities in Mycoplasma bovis: Securing survival in cell culture.

Mycoplasmas are host-restricted prokaryotes with a nearly minimal genome. To overcome their metabolic limitations, these wall-less bacteria establish intimate interactions with epithelial cells at mucosal surfaces. The alarming rate of antimicrobial resistance among pathogenic species is of particular concern in the medical and veterinary fields. Taking advantage of the reduced mycoplasma genome, random transposon mutagenesis was combined with high-throughput screening in order to identify key determinants of mycoplasma survival in the host-cell environment and potential targets for drug development. With the use of the ruminant pathogen Mycoplasma bovis as a model, three phosphodiesterases of the DHH superfamily were identified as essential for the proliferation of this species under cell culture conditions, while dispensable for axenic growth. Despite a similar domain architecture, recombinant Mbov_0327 and Mbov_0328 products displayed different substrate specificities. While rMbovP328 protein exhibited activity towards cyclic dinucleotides and nanoRNAs, rMbovP327 protein was only able to degrade nanoRNAs. The Mbov_0276 product was identified as a member of the membrane-associated GdpP family of phosphodiesterases that was found to participate in cyclic dinucleotide and nanoRNA degradation, an activity which might therefore be redundant in the genome-reduced M. bovis. Remarkably, all these enzymes were able to convert their substrates into mononucleotides, and medium supplementation with nucleoside monophosphates or nucleosides fully restored the capacity of a Mbov_0328/0327 knock-out mutant to grow under cell culture conditions. Since mycoplasmas are unable to synthesize DNA/RNA precursors de novo, cyclic dinucleotide and nanoRNA degradation are likely contributing to the survival of M. bovis by securing the recycling of purines and pyrimidines. These results point toward proteins of the DHH superfamily as promising targets for the development of new antimicrobials against multidrug-resistant pathogenic mycoplasma species.

Genome Mosaicism in Field Strains of Mycoplasma bovis as Footprints of In-Host Horizontal Chromosomal Transfer.

Horizontal gene transfer was long thought to be marginal in Mollicutes, but the capacity of some of these wall-less bacteria to exchange large chromosomal regions has been recently documented. Mycoplasma chromosomal transfer (MCT) is an unconventional mechanism that relies on the presence of a functional integrative conjugative element (ICE) in at least one partner and involves the horizontal acquisition of small and large chromosomal fragments from any part of the donor genome, which results in progenies composed of an infinite variety of mosaic genomes. The present study focuses on Mycoplasma bovis, an important pathogen of cattle responsible for major economic losses worldwide. By combining phylogenetic tree reconstructions and detailed comparative genome analyses of 36 isolates collected in Spain (2016 to 2018), we confirmed the mosaic nature of 16 field isolates and mapped chromosomal transfers exchanged between their hypothetical ancestors. This study provides evidence that MCT can take place in the field, most likely during coinfections by multiple strains. Because mobile genetic elements (MGEs) are classical contributors of genome plasticity, the presence of phages, insertion sequences (ISs), and ICEs was also investigated. Data revealed that these elements are widespread within the M. bovis species and evidenced classical horizontal transfer of phages and ICEs in addition to MCT. These events contribute to wide-genome diversity and reorganization within this species and may have a tremendous impact on diagnostic and disease control. IMPORTANCE Mycoplasma bovis is a major pathogen of cattle that has significant detrimental effects on economics and animal welfare in cattle rearing worldwide. Understanding the evolution and the adaptative potential of pathogenic mycoplasma species in the natural host is essential to combating them. In this study, we documented the occurrence of mycoplasma chromosomal transfer, an atypical mechanism of horizontal gene transfer, in field isolates of M. bovis that provide new insights into the evolution of this pathogenic species in their natural host. Although these events are expected to occur at low frequency, their impact is accountable for genome-wide variety and reorganization within M. bovis species, which may compromise both diagnostic and disease control.

Prevalence of three Mycoplasma sp. by multiplex PCR in cattle with and without respiratory disease in central Mexico.

This study aimed to identify Mycoplasma bovis, Myc. dispar, and Myc. bovirhinis, which are involved in bovine respiratory disease through a multiplex PCR as an alternative to culture's features that hamper Mycoplasma isolation. Nasal swabs were taken from 335 cattle with and without respiratory disease background (RDB) from dairy herds in the central region of Mexico. Each sample was divided in two; the first part was processed for the direct DNA extraction of the nasal swab and the second for Mycoplasma isolation, culture, and then the multiplex PCR was performed. In the nasal swabs, Myc. bovis was identified in 21.1%; Myc. dispar, in 11.8%; and Myc. bovirhinis, in 10.8% in cattle with RDB. Isolates were identified as Myc. bovis, 20.1%; Myc. dispar, 11.8%; and Myc. bovirhinis, 6.1%. There is a strong correlation between the presence of Mycoplasma identified by PCR and the clinical history of the disease (ρ < 0.0000). In animals without RDB, Myc. bovirhinis was the only species detected in 6.1% of the samples processed directly for multiplex PCR, and in 2% of the isolates. There is an excellent correlation (kappa 0.803) between the isolation and the 16S PCR and a high correlation (kappa 0.75) between the isolation and the multiplex PCR. Therefore, we conclude that the PCR multiplex test is highly sensitive and may be used for the diagnosis and surveillance of the three species in biological samples and mycoplasma isolates.

Application of Four Genotyping Methods to Mycoplasma bovis Isolates Derived from Western Canadian Feedlot Cattle.

Mycoplasma bovis is a significant pathogen of feedlot cattle, responsible for chronic pneumonia and polyarthritis syndrome (CPPS). M. bovis isolates (n = 129) were used to compare four methods of phylogenetic analysis and to determine if the isolates' genotypes were associated with phenotypes. Metadata included the health status of the animal from which an isolate was derived (healthy, diseased, or dead), anatomical location (nasopharynx, lung, or joint), feedlot, and production year (2006 to 2018). Four in silico phylogenetic typing methods were used: multilocus sequence typing (MLST), core genome MLST (cgMLST), core genome single nucleotide variant (cgSNV) analysis, and whole-genome SNV (wgSNV) analysis. Using Simpson's diversity index (D) as a proxy for resolution, MLST had the lowest resolution (D = 0.932); cgSNV (D = 0.984) and cgMLST (D = 0.987) generated comparable results; and wgSNV (D = 1.000) provided the highest resolution. Visual inspection of the minimum spanning trees found that the memberships of the clonal complexes and clades had similar structural appearances. Although MLST had the lowest resolution, this methodology was intuitive and easy to apply, and the PubMLST database facilitates the comparison of sequence types across studies. The cg methods had higher resolution than MLST, and the graphical interface software was user-friendly for nonbioinformaticians, but the proprietary software is relatively expensive. The wgSNV approach was the most robust for processing poor-quality sequence data while offering the highest resolution; however, application of its software requires specialized training. None of the four methods could associate genotypes with phenotypes.

Evaluation of Nanopore Sequencing as a Diagnostic Tool for the Rapid Identification of Mycoplasma bovis from Individual and Pooled Respiratory Tract Samples.

Rapid identification of Mycoplasma bovis infections in cattle is a key factor to guide antimicrobial therapy and biosecurity measures. Recently, Nanopore sequencing became an affordable diagnostic tool for both clinically relevant viruses and bacteria, but the diagnostic accuracy for M. bovis identification is undocumented. Therefore, in this study Nanopore sequencing was compared to rapid identification of M. bovis with matrix-assisted laser desorption ionization-time of flight mass spectrometry (RIMM) and a triplex real-time PCR assay in a Bayesian latent class model (BLCM) for M. bovis in bronchoalveolar lavage fluid (BALf) samples obtained from calves. In practice, pooling of samples is often used to save money, but the influence on diagnostic accuracy has not been described for M. bovis. Therefore, a convenience sample of 17 pooled samples containing 5 individual BALf samples per farm was analyzed as well. The results for the pooled samples were compared with those for the individual samples to determine sensitivity and specificity. The BLCM showed good sensitivity (77.3% [95% credible interval, 57.8 to 92.8%]) and high specificity (97.4% [91.5 to 99.7%]) for Nanopore sequencing, compared to RIMM (sensitivity, 93.0% [76.8 to 99.5%]; specificity, 91.3% [82.5 to 97.0%]) and real-time PCR (sensitivity, 94.6% [89.7 to 97.7%]; specificity, 86.0% [76.1 to 93.6%]). Sensitivity and specificity of pooled analysis for M. bovis were 85.7% (95% confidence interval, 59.8 to 111.6%) and 90.0% (71.4 to 108.6%%), respectively, for Nanopore sequencing and 100% (100% to 100%) and 88.9% (68.4 to 109.4%) for RIMM. In conclusion, Nanopore sequencing is a rapid, reliable tool for the identification of M. bovis. To reduce costs and increase the chance of M. bovis identification, pooling of 5 samples for Nanopore sequencing and RIMM is possible.

Protection against Mycoplasma bovis infection in calves following intranasal vaccination with modified-live Mannheimia haemolytica expressing Mycoplasma antigens.

Novel live vaccine strains of Mannheimia haemolytica serotypes (St)1 and St6, expressing and secreting inactive yet immunogenic leukotoxin (leukotoxoid) fused to antigenic domains of Mycoplasma bovis Elongation Factor Tu (EFTu) and Heat shock protein (Hsp) 70 were constructed and tested for efficacy in cattle. Control calves were administered an intranasal mixture of M. haemolytica St1 and St6 mutants (ΔlktCAV4) expressing and secreting leukotoxoid while vaccinated calves were administered an intranasal mixture of like M. haemolytica St1 and St6 leukotoxoid mutants coupled to M. bovis antigens (EFTu-Hsp70-ΔlktCAV4). Both M. haemolytica strains were recovered from palatine tonsils up to 34 days post intranasal exposure. On day 35 all calves were exposed to bovine herpes virus-1, four days later lung challenged with virulent M. bovis, then euthanized up to 20 days post-challenge. Results showed all cattle produced systemic antibody responses against M. haemolytica. The vaccinates also produced systemic antibody responses to M. bovis antigen, and concurrent reductions in temperatures, middle ear infections, joint infection and lung lesions versus the control group. Notably, dramatically decreased lung loads of M. bovis were detected in the vaccinated cattle. These observations indicate that the attenuated M. haemolytica vaccine strains expressing Mycoplasma antigens can control M. bovis infection and disease symptoms in a controlled setting.

Isolation, identification and biological characteristics of Mycoplasma bovis in yaks.

Mycoplasma bovis (M. bovis) is one of the important pathogens which may cause bovine respiratory disease syndrome (BRDS), and results in huge economic losses for yaks (Bos gaurus) breeding industry. However, there is limited information about M. bovis in yaks. In our study, 145 nasal mucus samples from yaks with pneumonia were collected to clarify. Bacteriological determination was carried out through biochemical identification and Polymerase Chain Reaction (PCR) detection. And ten strains of Mycoplasma bovis (M. bovis) were found from collected samples. Then, the growth curve of isolated strains was determined by the change of optical density (OD630), pH value and Color Change Cnit (CCU). K-B disk method was also used for antimicrobial susceptibility testing. Results of colony morphology and biochemical testing were consistent with the biological characters of M. bovis. The nucleotide sequences of uvrC specific gene and 16S rRNA gene among the 10 strains were highly homologous. The growth curve assay showed that the isolates cultured in PPLO medium were in lag phase for 24 h, entered stable period in 42 h, and entered decline phase after 78 h. The isolates were found resistant to macrolides, aminoglycosides and lincomycin at various degrees, but they were sensitive or moderately sensitive to doxycycline and kanamycin under antimicrobial susceptibility analysis. In conclusion, the results provided certain reference for the follow-up research and guiding for the treatment of M. bovis in yaks.

MALDI-TOF mass spectrometry and high-resolution melting PCR for the identification of Mycoplasma bovis isolates.

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.

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.

Mycoplasma bovis mbfN Encodes a Novel LRR Lipoprotein That Undergoes Proteolytic Processing and Binds Host Extracellular Matrix Components.

Mycoplasma bovis causes serious infections in ruminants, leading to huge economic losses. Lipoproteins are key components of the mycoplasma membrane and are believed to function in nutrient acquisition, adherence, enzymatic interactions with the host, and induction of the host's immune response to infection. Many genes of M. bovis have not been assigned functions, in part because of their low sequence similarity with other bacteria, making it difficult to extrapolate gene functions. This study examined functions of a surface-localized leucine-rich repeat (LRR) lipoprotein encoded by mbfN of M. bovis PG45. Homologs of MbfN were detected as 48-kDa peptides by Western blotting in all the strains of M. bovis included in this study, with the predicted 70-kDa full-length polypeptide detected in some strains. Sequence analysis of the gene revealed the absence in some strains of a region encoding the carboxyl-terminal 147 amino acids found in strain PG45, which could account for the variation detected by immunoblotting. In silico analysis of MbfN suggested that it may have an adhesion-related function. In vitro binding assays confirmed MbfN to be a fibronectin and heparin-binding protein. Disruption of mbfN in M. bovis PG45 significantly reduced (P = 0.033) the adherence of M. bovis PG45 to MDBK cells in vitro, demonstrating the role of MbfN as an adhesin.IMPORTANCE Experimental validation of the putative functions of genes in M. bovis will advance our understanding of the basic biology of this economically important pathogen and is crucial in developing prevention strategies. This study demonstrated the extracellular matrix binding ability of a novel immunogenic lipoprotein of M. bovis, and the role of this protein in adhesion by M. bovis suggests that it could play a role in virulence.

High quality genome assemblies of Mycoplasma bovis using a taxon-specific Bonito basecaller for MinION and Flongle long-read nanopore sequencing.

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.

Genomics-based epidemiology of bovine Mycoplasma bovis strains in Israel.

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.

Mycoplasma bovis Infections in Free-Ranging Pronghorn, Wyoming, USA.

Mycoplasma bovis is 1 of several bacterial pathogens associated with pneumonia in cattle. Its role in pneumonia of free-ranging ungulates has not been established. Over a 3-month period in early 2019, ¼60 free-ranging pronghorn with signs of respiratory disease died in northeast Wyoming, USA. A consistent finding in submitted carcasses was severe fibrinosuppurative pleuropneumonia and detection of M. bovis by PCR and immunohistochemical analysis. Multilocus sequence typing of isolates from 4 animals revealed that all have a deletion in 1 of the target genes, adh-1. A retrospective survey by PCR and immunohistochemical analysis of paraffin-embedded lung from 20 pronghorn that died with and without pneumonia during 2007-2018 yielded negative results. These findings indicate that a distinct strain of M. bovis was associated with fatal pneumonia in this group of pronghorn.

Rapid Identification of Mycoplasma bovis Strains from Bovine Bronchoalveolar Lavage Fluid with Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry after Enrichment Procedure.

Mycoplasma bovis is a leading cause of pneumonia in modern calf rearing. Fast identification is essential to ensure appropriate antimicrobial therapy. Therefore, the objective of this study was to develop a protocol to identify M. bovis from bronchoalveolar lavage fluid (BALf) with matrix-assisted laser desorption ionization-time of flight mass spectrometry MALDI-TOF MS and to determine the diagnostic accuracy in comparison with other techniques. BALf was obtained from 104 cattle, and the presence of M. bovis was determined in the following three ways: (i) rapid identification of M. bovis with MALDI-TOF MS (RIMM) (BALf was enriched and after 24, 48, and 72 h of incubation and was analyzed using MALDI-TOF MS), (ii) triplex real-time PCR for M. bovis, Mycoplasma bovirhinis, and Mycoplasma dispar, and (iii) 10-day incubation on selective-indicative agar. The diagnostic accuracy of the three tests was determined with Bayesian latent class modeling (BLCM). After 24 h of enrichment, M. bovis was identified with MALDI-TOF MS in 3 out of 104 BALf samples. After 48 and 72 h of enrichment, 32/104 and 38/100 samples, respectively, were M. bovis positive. Lipase-positive Mycoplasma-like colonies were seen in 28 of 104 samples. Real-time PCR resulted in 28/104 positive and 12/104 doubtful results for M. bovis The BLCM showed a sensitivity (Se) and specificity (Sp) of 86.6% (95% credible interval [CI], 69.4% to 97.6%) and 86.4% (CI, 76.1 to 93.8) for RIMM. For real-time PCR, Se was 94.8% (CI, 89.9 to 97.9) and Sp was 88.9% (CI, 78.0 to 97.4). For selective-indicative agar, Se and Sp were 70.5% (CI, 52.1 to 87.1) and 93.9% (CI, 85.9 to 98.4), respectively. These results suggest that rapid identification of M. bovis with MALDI-TOF MS after an enrichment procedure is a promising test for routine diagnostics in veterinary laboratories.