Proteomic and Lipidomic Profiling of Calves Experimentally Co-Infected with Influenza D Virus and Mycoplasma bovis: Insights into the Host-Pathogen Interactions.

The role of Influenza D virus (IDV) in bovine respiratory disease remains unclear. An in vivo experiment resulted in increased clinical signs, lesions, and pathogen replication in calves co-infected with IDV and Mycoplasma bovis (M. bovis), compared to single-infected calves. The present study aimed to elucidate the host-pathogen interactions and profile the kinetics of lipid mediators in the airways of these calves. Bronchoalveolar lavage (BAL) samples collected at 2 days post-infection (dpi) were used for proteomic analyses by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Additionally, lipidomic analyses were performed by LC-MS/MS on BAL samples collected at 2, 7 and 14 dpi. Whereas M. bovis induced the expression of proteins involved in fibrin formation, IDV co-infection counteracted this coagulation mechanism and downregulated other acute-phase response proteins, such as complement component 4 (C4) and plasminogen (PLG). The reduced inflammatory response against M. bovis likely resulted in increased M. bovis replication and delayed M. bovis clearance, which led to a significantly increased abundance of oxylipids in co-infected calves. The identified induced oxylipids mainly derived from arachidonic acid; were likely oxidized by COX-1, COX-2, and LOX-5; and peaked at 7 dpi. This paper presents the first characterization of BAL proteome and lipid mediator kinetics in response to IDV and M. bovis infection in cattle and raises hypotheses regarding how IDV acts as a co-pathogen in bovine respiratory disease.

MbovP0725, a secreted serine/threonine phosphatase, inhibits the host inflammatory response and affects metabolism in Mycoplasma bovis.

Mycoplasma species are able to produce and release secreted proteins, such as toxins, adhesins, and virulence-related enzymes, involved in bacteria adhesion, invasion, and immune evasion between the pathogen and host. Here, we investigated a novel secreted protein, MbovP0725, from Mycoplasma bovis encoding a putative haloacid dehalogenase (HAD) hydrolase function of a key serine/threonine phosphatase depending on Mg2+ for the dephosphorylation of its substrate pNPP, and it was most active at pH 8 to 9 and temperatures around 40°C. A transposon insertion mutant strain of M. bovis HB0801 that lacked the protein MbovP0725 induced a stronger inflammatory response but with a partial reduction of adhesion ability. Using transcriptome sequencing and quantitative reverse transcription polymerase chain reaction analysis, we found that the mutant was upregulated by the mRNA expression of genes from the glycolysis pathway, while downregulated by the genes enriched in ABC transporters and acetate kinase-phosphate acetyltransferase pathway. Untargeted metabolomics showed that the disruption of the Mbov_0725 gene caused the accumulation of 9-hydroxyoctadecadienoic acids and the consumption of cytidine 5'-monophosphate, uridine monophosphate, and adenosine monophosphate. Both the exogenous and endogenous MbvoP0725 protein created by purification and transfection inhibited lipopolysaccharide (LPS)-induced IL-1ß, IL-6, and TNF-α mRNA production and could also attenuate the activation of MAPK-associated pathways after LPS treatment. A pull-down assay identified MAPK p38 and ERK as potential substrates for MbovP0725. These findings define metabolism- and virulence-related roles for a HAD family phosphatase and reveal its ability to inhibit the host pro-inflammatory response. IMPORTANCE: Mycoplasma bovis (M. bovis) infection is characterized by chronic pneumonia, otitis, arthritis, and mastitis, among others, and tends to involve the suppression of the immune response via multiple strategies to avoid host cell immune clearance. This study found that MbovP0725, a haloacid dehalogenase (HAD) family phosphatase secreted by M. bovis, had the ability to inhibit the host pro-inflammatory response induced by lipopolysaccharide. Transcriptomic and metabolomic analyses were used to identify MbovP0725 as an important phosphatase involved in glycolysis and nucleotide metabolism. The M. bovis transposon mutant strain T8.66 lacking MbovP0725 induced a higher inflammatory response and exhibited weaker adhesion to host cells. Additionally, T8.66 attenuated the phosphorylation of MAPK P38 and ERK and interacted with the two targets. These results suggested that MbovP0725 had the virulence- and metabolism-related role of a HAD family phosphatase, performing an anti-inflammatory response during M. bovis infection.

Diagnostic performance of Mycoplasmopsis bovis antibody ELISA tests on bulk tank milk from dairy herds.

BACKGROUND: Testing of bulk tank milk (BTM) for Mycoplasmopsis bovis (previously Mycoplasma bovis) antibodies is increasingly popular. However the performance of some commercially available tests is unknown, and cutoff values possibly need to be adjusted in light of the purpose. Therefore, the aim of this study was to compare the diagnostic performance of three commercially available M. bovis antibody ELISAs on BTM, and to explore optimal cutoff values for screening purposes. A prospective diagnostic test accuracy study was performed on 156 BTM samples from Belgian and Swiss dairy farms using Bayesian Latent Class Analysis. Samples were initially classified using manufacturer cutoff values, followed by generated values. RESULTS: Following the manufacturer's guidelines, sensitivity of 91.4%, 25.6%, 69.2%, and specificity of 67.2%, 96.8%, 85.8% were observed for ID-screen, Bio K432, and Bio K302, respectively. Optimization of cutoffs resulted in a sensitivity of 89.0%, 82.0%, and 85.5%, and a specificity of 83.4%, 75.1%, 77.2%, respectively. CONCLUSIONS: The ID-screen showed the highest diagnostic performance after optimization of cutoff values, and could be useful for screening. Both Bio-X tests may be of value for diagnostic or confirmation purposes due to their high specificity.

Within-herd transmission of Mycoplasma bovis infections after initial detection in dairy cows.

Mycoplasma bovis outbreaks in cattle, including pathogen spread between age groups, are not well understood. Our objective was to estimate within-herd transmission across adult dairy cows, youngstock, and calves. Results from 3 tests (PCR, ELISA, and culture) per cow and 2 tests (PCR and ELISA) per youngstock and calf were used in an age-stratified susceptible-infected-removed/recovered (SIR) model to estimate within-herd transmission parameters, pathways, and potential effects of farm management practices. A cohort of adult cows, youngstock, and calves on 20 Dutch dairy farms with a clinical outbreak of M. bovis in adult cows were sampled, with collection of blood, conjunctival fluid, and milk from cows, and blood and conjunctival fluid from calves and youngstock, 5 times over a time span of 12 wk. Any individual with at least one positive laboratory test was considered M. bovis-positive. Transmission dynamics were modeled using an age-stratified SIR model featuring 3 age strata. Associations with farm management practices were explored using Fisher's exact tests and Poisson regression. Estimated transmission parameters were highly variable among herds and cattle age groups. Notably, transmission from cows to cows, youngstock, or to calves was associated with R-values ranging from 1.0 to 80 secondarily infected cows per herd, 1.2 to 38 secondarily infected youngstock per herd, and 0.1 to 91 secondarily infected calves per herd, respectively. In case of transmission from youngstock to youngstock, calves or to cows, R-values were 0.7 to 96 secondarily infected youngstock per herd, 1.1 to 76 secondarily infected calves per herd, and 0.1 to 107 secondarily infected cows per herd. For transmission from calves to calves, youngstock or to cows, R-values were 0.5 to 60 secondarily infected calves per herd, 1.1 to 41 secondarily infected youngstock per herd, and 0.1 to 47 secondarily infected cows per herd. Among on-farm transmission pathways, cow-to-youngstock, cow-to-calf, and cow-to-cow were identified as most significant contributors, with calf-to-calf and calf-to-youngstock also having noteworthy roles. Youngstock-to-youngstock was also implicated, albeit to a lesser extent. Whereas the primary focus was a clinical outbreak of M. bovis among adult dairy cows, it was evident that transmission extended to calves and youngstock, contributing to overall spread. Factors influencing transmission and specific transmission pathways were associated with internal biosecurity (separate caretakers for various age groups, number of people involved), external biosecurity (contractors, external employees), as well as indirect transmission routes (number of feed and water stations).

LppA is a novel plasminogen receptor of Mycoplasma bovis that contributes to adhesion by binding the host extracellular matrix and Annexin A2.

Mycoplasma bovis is responsible for various inflammatory diseases in cattle. The prevention and control of M. bovis are complicated by the absence of effective vaccines and the emergence of multidrug-resistant strains, resulting in substantial economic losses worldwide in the cattle industry. Lipoproteins, vital components of the Mycoplasmas cell membrane, are deemed potent antigens for eliciting immune responses in the host upon infection. However, the functions of lipoproteins in M. bovis remain underexplored due to their low sequence similarity with those of other bacteria and the scarcity of genetic manipulation tools for M. bovis. In this study, the lipoprotein LppA was identified in all examined M. bovis strains. Utilizing immunoelectron microscopy and Western blotting, it was observed that LppA localizes to the surface membrane. Recombinant LppA demonstrated dose-dependent adherence to the membrane of embryonic bovine lung (EBL) cells, and this adhesion was inhibited by anti-LppA serum. In vitro binding assays confirmed LppA's ability to associate with fibronectin, collagen IV, laminin, vitronectin, plasminogen, and tPA, thereby facilitating the conversion of plasminogen to plasmin. Moreover, LppA was found to bind and enhance the accumulation of Annexin A2 (ANXA2) on the cell membrane. Disrupting LppA in M. bovis significantly diminished the bacterium's capacity to adhere to EBL cells, underscoring LppA's function as a bacterial adhesin. In conclusion, LppA emerges as a novel adhesion protein that interacts with multiple host extracellular matrix proteins and ANXA2, playing a crucial role in M. bovis's adherence to host cells and dissemination. These insights substantially deepen our comprehension of the molecular pathogenesis of M. bovis.

Prevalence and antimicrobial susceptibility of Mycoplasma bovis from the upper and lower respiratory tracts of healthy feedlot cattle and those diagnosed with bovine respiratory disease.

Mycoplasma bovis is an important respiratory pathogen of cattle. In this study, the prevalence and antimicrobial susceptibility of M. bovis were evaluated from two Cohorts of feedlot cattle spanning an 8-year period. In the first study conducted in 2008-2009, nasopharyngeal swabs from cattle sampled at feedlot entry and after 60 days on feed were collected (Cohort 1). In a second study conducted in 2015-2016, nasopharyngeal and trans-tracheal samples were collected from cattle diagnosed with bovine respiratory disease (BRD) and matching healthy controls (Cohort 2). For Cohort 1, the prevalence of M. bovis was lower in cattle at entry compared to when the same individuals were sampled ≥60 days later (P < 0.05). For Cohort 2, the prevalence of M. bovis was greater in both nasopharyngeal and tracheal samples from cattle diagnosed with BRD, compared to controls (P < 0.05). In both Cohorts, almost all isolates were resistant to tilmicosin. Compared to M. bovis from Cohort 1, isolates of Cohort 2 exhibited increased resistance to clindamycin, enrofloxacin, florfenicol, tylosin, and tulathromycin, with the latter showing resistance levels >90 %. These data suggest that antimicrobials used to prevent and treat BRD selected for resistance in M. bovis over the 8-year period. For macrolides, cross-resistance occurred and M. bovis can retain resistance even when antimicrobial selection pressure is removed. Within 9 years of commercial availability of tulathromycin, the majority of M. bovis displayed resistance. Therefore, longitudinal evaluation of resistance in respiratory pathogens is important to ensure efficacious treatment of BRD.

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.

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.

Prevalence, molecular characterization and antibiogram of Mycoplasma bovis isolated from milk in Pakistan.

Mycoplasma bovis is a highly contagious pathogen that causes clinical or subclinical mastitis. The present study was aimed for the isolation, molecular characterization and antibiogram determination of M. bovis from raw milk samples. Milk samples were collected randomly from lactating cows and buffaloes from different tehsils of district Faisalabad, Pakistan. Samples were inoculated on modified Hayflick medium and biochemical tests were performed for further confirmation of isolated M. bovis. Out of total 400 milk samples, 184 (46%) samples were found positive for culture method. The 16S-rRNA gene polymerase chain reaction was performed for molecular characterization of isolated M. bovis strains. Out of total 400 milk samples, 240 (60%) positive for M. bovis through PCR method were examined. The 16S-rRNA gene PCR positive isolated M. bovis strains were sequenced and results were compared using Maximum-likelihood method and sequenced strains of M. bovis were aligned and analyzed by Clustal W software. Antibiogram of isolated M. bovis strains was analyzed by disc diffusion assay against eight commonly used antibiotics. Tylosin (30µg) and Tilmicosin (15ug) showed inhibition zones of 32.34 ± 1.10 mm and 17.12 ± 0.93 mm respectively against isolated M. bovis which were found sensitive. Isolated M. bovis was found resistant to other commonly used antibiotics. Statistical analysis revealed that p-value was < 0.05 and the odds ratio was >1.0 at 95% CI. This study complemented the lack of epidemiological knowledge of molecular characterization, comparative effectiveness and resistance trends of isolated M. bovis strains against commonly used antibiotics.

Toward a Method for Harmonized Susceptibility Testing of Mycoplasma bovis by Broth Microdilution.

Mycoplasma bovis is a fastidious pathogen of cattle causing massive economic losses in the calf and dairy industries worldwide. Since there is no approved standard method for antimicrobial susceptibility testing (AST) of M. bovis, the Clinical and Laboratory Standards Institute has requested the development of a suitable method. Therefore, this study aimed at developing a method for harmonized broth microdilution AST of M. bovis. For this, 131 M. bovis field isolates and M. bovis strain DSM 22781T were collected and macrorestriction analysis was performed to select 15 epidemiologically unrelated M. bovis strains for method validation steps. To select a suitable broth for AST of M. bovis, growth determinations were performed using five media and growth curves were compiled. Then, susceptibility testing was performed considering the exact (precondition of five identical MICs) and essential (MIC mode, accepting a deviation of ±1 dilution step) MIC agreements to evaluate the reproducibility of MIC values using a panel of 16 antimicrobial agents. Subsequently, the remaining field isolates were tested and the suitability of quality control (QC) strains was assessed. Growth experiments showed that SP4 broth was the only one of the five media that yielded sufficient growth of M. bovis. Therefore, it was selected as the test medium for AST and homogeneous MIC values were obtained (exact and essential agreements of 36 to 100% and 92 to 100%, respectively). For all other isolates tested, easy-to-read MIC endpoints were determined with this medium. High overall MIC50 and/or MIC90 values were observed for aminoglycosides and macrolides, and some isolates showed elevated MICs of fluoroquinolones, gentamicin, and/or tiamulin. Since the MICs of four commonly used QC strains were partially not within their ranges, a 20-fold MIC testing of M. bovis DSM 22781T was performed and met the criteria for a new QC strain. For harmonized AST of M. bovis, SP4 broth seems to be suitable with an incubation time of 72 ± 2 h and further validation of M. bovis DSM 22781T as a future QC strain is recommended.

Genome-wide phylodynamic approach reveals the epidemic dynamics of the main Mycoplasma bovis subtype circulating in France.

Mycoplasma bovis is a major aetiological agent of bovine respiratory disease worldwide. Genome-based analyses are increasingly being used to monitor the genetic diversity and global distribution of M. bovis, complementing existing subtyping schemes based on locus sequencing. However, these analyses have so far provided limited information on the spatiotemporal and population dynamics of circulating subtypes. Here we applied a genome-wide phylodynamic approach to explore the epidemic dynamics of 88 French M. bovis strains collected between 2000 and 2019 in France and belonging to the currently dominant polC subtype 2 (st2). A strong molecular clock signal detected in the genomic data enabled robust phylodynamic inferences, which estimated that the M. bovis st2 population in France is composed of two lineages that successively emerged from independent introductions of international strains. The first lineage appeared around 2000 and supplanted the previously established antimicrobial-susceptible polC subtype 1. The second lineage, which is likely more transmissible, progressively replaced the first M. bovis st2 lineage population from 2005 onward and became predominant after 2010. Analyses also showed a brief decline in this second M. bovis st2 lineage population in around 2011, possibly due to the challenge from the concurrent emergence of M. bovis polC subtype 3 in France. Finally, we identified non-synonymous mutations in genes associated with lineages, which raises prospects for identifying new surveillance molecular markers. A genome-wide phylodynamic approach provides valuable resources for monitoring the evolution and epidemic dynamics of circulating M. bovis subtypes, and may prove critical for developing more effective surveillance systems and disease control strategies.

Pathogenicity and molecular characterization of Mycoplasma bovis isolate from calves in Morocco.

Bovine respiratory disease caused by Mycoplasma bovis (M. bovis) represents a major health problem for cattle worldwide that causes considerable financial losses. This study reports for the first time the molecular and pathogenic characterization of a strain of M. bovis isolated from a dead local calf with respiratory symptoms in Morocco. M. bovis was isolated from lung tissue, purified by cloning, and subtyped using MLST analysis. Experimental infection was conducted in naïve calves to evaluate pathogenicity. The isolate was identified as a new subtype ST-204 that shares similarities with the 2019-2021 Spanish strains (ST-169, ST-170, ST-171) and the 2018 Algeria isolate (ST-4). Experimental infection resulted in fever and respiratory symptoms with serous nasal discharge. At postmortem, lung lesions of congestion and hepatization were observed with lymph node enlargement and foci of necrosis. The study confirms the high pathogenicity of the isolate and the important role of M. bovis in bovine respiratory disease.

Mannheimia haemolytica increases Mycoplasma bovis disease in a bovine experimental model of BRD.

Amongst the bacterial pathogens associated with the bovine respiratory disease syndrome (BRD) in cattle are Mannheimia haemolytica and Mycoplasma bovis. The interaction between these two pathogens has not been investigated before; thus, there are gaps in the knowledge of why and how a previous infection with M. haemolytica allows the development of M. bovis-related lesions. We hypothesized that upon M. haemolytica infection, inflammatory products are produced in the lung and that these inflammatory products stimulate M. bovis to produce proteases and lipases that degrade lipids and proteins important for lung function. In this work, we identified several M. bovis proteases and lipases whose expression was modulated by M. haemolytica products in vitro. We performed co-infection animal challenges to develop a model to test vaccine protection. A prior exposure to BHV-1 followed by infection with M. bovis and M. haemolytica resulted in severe pathology and the BHV-1 infection was abandoned. When M. bovis and M. haemolytica were introduced into the lungs by bronchoscopy, we found that M. haemolytica resulted in worsening of the respiratory disease caused by M. bovis. We performed a proof-of-concept trial where animals were immunized with the M. bovis proteins identified in this study and challenged with both pathogens. Despite detecting significant humoral immune responses to the antigens, the experimental vaccine failed to protect against M. bovis disease.

Evaluation of the accuracy of the IDvet serological test for Mycoplasma bovis infection in cattle using latent class analysis of paired serum ELISA and quantitative real-time PCR on tonsillar swabs sampled at slaughter.

Mycoplasma bovis (Mbovis) was first detected in cattle in New Zealand (NZ) in July 2017. To prevent further spread, NZ launched a world-first National Eradication Programme in May 2018. Existing diagnostic tests for Mbovis have been applied in countries where Mbovis is endemic, for detecting infection following outbreaks of clinical disease. Diagnostic test evaluation (DTE) under NZ conditions was thus required to inform the Programme. We used Bayesian Latent Class Analysis on paired serum ELISA (ID Screen Mycoplasma bovis Indirect from IDvet) and tonsillar swabs (qPCR) for DTE in the absence of a gold standard. Tested samples were collected at slaughter between June 2018 and November 2019, from infected herds depopulated by the Programme. A first set of models evaluated the detection of active infection, i.e. the presence of Mbovis in the host. At a modified serology positivity threshold of SP%> = 90, estimates of animal-level ELISA sensitivity was 72.8% (95% credible interval 68.5%-77.4%), respectively 97.7% (95% credible interval 97.3%-98.1%) for specificity, while the qPCR sensitivity was 45.2% (95% credible interval 41.0%-49.8%), respectively 99.6% (95% credible interval 99.4%-99.8%) for specificity. In a second set of models, prior information about ELISA specificity was obtained from the National Beef Cattle Surveillance Programme, a population theoretically free-or very low prevalence-of Mbovis. These analyses aimed to evaluate the accuracy of the ELISA test targeting prior exposure to Mbovis, rather than active infection. The specificity of the ELISA for detecting exposure to Mbovis was 99.9% (95% credible interval 99.7%-100.0%), hence near perfect at the threshold SP%=90. This specificity estimate, considerably higher than in the first set of models, was equivalent to the manufacturer's estimate. The corresponding ELISA sensitivity estimate was 66.0% (95% credible interval 62.7%-70.7%). These results confirm that the IDvet ELISA test is an appropriate tool for determining exposure and infection status of herds, both to delimit and confirm the absence of Mbovis.

Bayesian diagnostic test evaluation and true prevalence estimation of mycoplasma bovis in dairy herds.

The true prevalence of dairy cattle herds with M. bovis infections in the Netherlands is unknown. Previous attempts to estimate prevalences were hampered by the absence of a diagnostic serological test that was validated under field conditions. This study estimated sensitivity and specificity of two commercial serum ELISAs and the true M. bovis herd prevalence using different Bayesian latent class models. A total of 7305 serum samples from 415 randomly chosen dairy herds were collected in fall/winter 2019 and investigated for presence of antibodies against M. bovis using the BIO-K-260 ELISA from Bio-X. Serum samples from 100 of these herds were also tested with a second ELISA, from IDvet. A Bayesian latent class model using the paired test results estimated a sensitivity of 14.1% (95% Bayesian probability interval (BPI): 11.6-16.7%) for the Bio-X ELISA and a specificity of 97.2% (95% BPI: 95.9-98.4%). Sensitivity and specificity for the IDvet ELISA were estimated at 92.5% (95% BPI: 88.3-96.5%) and 99.3% (95% BPI: 98.7-99.8%), respectively. Also, Bio-X ELISA sensitivity was considerably higher with data from calves only and with data from a selection of herds with a clinical outbreak, whereas the IDvet ELISA sensitivity was fairly constant under these conditions. These differences in test sensitivity is expected to be related to an effect of time since infection. A second Bayesian model, applied on test results of all 415 herds, estimated a true herd prevalence of 69.9% (95% BPI: 62.7-77.6%), suggesting M. bovis in endemic amongst dairy cattle herds in the Netherlands. To what extent seropositive herds have experienced a clinical outbreak needs further investigation.

Characteristics of Mycoplasma bovis, Mycoplasma arginini, and Mycoplasma californicum on immunological response of bovine synovial cells.

Mycoplasma arthritis in calves caused by M. bovis exhibits joint swelling, lameness, and immobility. In contrast to M. bovis, M. arginini, and M. californicum which were similarly isolated from the affected joints, only induced mild inflammation. The changes in pathogenesis that depended on species, however, remained unknown. This investigation aims to examine the characteristics of immune responses to M. bovis, M. arginini, and M. californicum in synovial cells. Intracellular M. bovis was detected by gentamicin assay, but M. arginini and M. californicum were not detected. M. bovis-infected synovial cells were encouraged to proliferate and had their apoptosis suppressed. We suggest that M. bovis invaded and inhibited apoptosis of synovial to evade host immunity, which led to long term survival in joints. M. bovis infection significantly increased IL-6 mRNA expression compared to control, although M. arginini and M. californicum infection were comparable to control. We suggest that M. arginini and M. californicum have low abilities to induce inflammation in joints and therefore do not cause severe pathology. Our findings are the first to show the variations in synovial cell immune responses to M. bovis, M. arginini, and M. californicum, which are thought to be related to the pathogenicity of arthritis.

Antimicrobial susceptibility and molecular characteristics of Mycoplasma bovis isolated from cases of bovine respiratory disease in Australian feedlot cattle.

To date, antimicrobial susceptibility has not been reported for Australian Mycoplasma bovis isolates. This study determined minimal inhibitory concentrations (MICs) for 12 different antimicrobials against Australian M. bovis isolates and used whole genome sequencing to screen those showing high macrolide MICs for point mutations in target genes. Most lung tissue/swab samples from bovine respiratory disease cases (61/76, 80.3%) tested positive for M. bovis. A set of 50 representative isolates (50/61, 82.0%) that showed adequate growth, was used for MIC testing. Uniformly, low MIC values were confirmed for enrofloxacin (≤ 4 µg/mL), florfenicol (≤ 8 µg/mL), gamithromycin (≤ 2 µg/mL), spectinomycin (≤ 4 µg/mL), tetracycline (≤ 8 µg/mL), tiamulin (≤ 4 µg/mL), and tulathromycin (≤ 0.5 µg/mL). A small proportion (10%) of isolates exhibited high MICs (≥ 32 µg/mL) for tildipirosin, tilmicosin, tylosin, and lincomycin, which were above the epidemiological cut-off values for each antimicrobial (≥ 4 µg/mL). These isolates, originating from three Australian states, underwent whole genome sequencing/multilocus sequencing typing and were compared with the reference strain PG45 to investigate mutations that might be linked with the high macrolide/lincosamide MICs. All five belonged to ST52 and two macrolide associated mutations were identified within the 23 S rRNA gene (A2058G in two sequenced isolates and G748A in all sequenced isolates). Four additional 23 S rRNA gene mutations did not appear to be linked to macrolide resistance. Whilst the majority of Australian M. bovis isolates appear susceptible to the tested antimicrobials, emerging macrolide resistance was detected in three Australian states and requires continued monitoring.

Mycoplasma bovis testing for the screening of semen imported into New Zealand.

AIMS: To evaluate the fitness of three PCR assays for the detection of Mycoplasma bovis in dilute (extended) bovine semen, and a reverse transcriptase-PCR (RT-PCR) adaptation as a proxy for viability. MATERIALS AND METHODS: Four commercial kit-based methods for nucleic acid extraction were compared to test for the presence of PCR inhibitors in nucleic acid extracted from undiluted and diluted semen. Then, analytical sensitivity, analytical specificity, and diagnostic specificity of two real-time PCR and one conventional PCR were evaluated for the detection of M. bovis DNA in semen and compared against microbial culture. Furthermore, an RT-PCR was adapted to detect RNA only and tested on viable and non-viable M. bovis to establish its ability to discriminate between the two. RESULTS: No significant PCR inhibition was detected from the dilute semen. All DNA extraction methods except one were equivalent, regardless of semen dilution. The analytical sensitivity of the real-time PCR assays was estimated as 45.6 cfu per 200 µL semen straw (2.2 × 102 cfu/mL). The conventional PCR was 10 times less sensitive. No cross-reactivity was observed for the real-time PCR for any of the bacteria tested and the diagnostic specificity was estimated as 100 (95% CI = 94.04-100) %. The RT-PCR was poor in distinguishing between viable and non-viable M. bovis. The mean quantification cycle (Cq) values for RNA extracted from different treatments to kill M. bovis remained unchanged 0-48 hours after inactivation. CONCLUSION AND CLINICAL RELEVANCE: The real-time PCR were fit for the purpose of screening dilute semen for the detection of M. bovis to prevent incursion via importation of infected semen. The real-time PCR assays can be used interchangeably. The RT-PCR could not reliably indicate the viability of M. bovis. Based on the results from this study, a protocol and guidelines have been produced for laboratories elsewhere that wish to test bovine semen for M. bovis.

Complement-mediated killing of Mycoplasma bovis does not play a role in the protection of animals against an experimental challenge.

Despite numerous efforts, developing recombinant vaccines for the control of M. bovis infections has not been successful. Many factors are contributing to the lack of success including the identification of protective antigens, use of effective adjuvants, and relatively limited information on the quality of immune responses needed for protection. Experimental trials using vaccination with many M. bovis proteins resulted in significant humoral immune responses before and after the challenges, however these responses were not enough to confer protection. We explored the role of complement-fixing antibodies in the killing of M. bovis in-vitro and whether animals vaccinated with proteins that elicit antibodies capable of complement-fixing would be protected against an experimental challenge. We found that antibodies against some of these proteins fixed complement and killed M. bovis in-vitro. Vaccination and challenge experiments with proteins whose cognate antibodies either fixed complement or not resulted in lack of protection against a M. bovis experimental challenge suggesting that complement fixation does not play a role in protection.