Mycoplasma bovis causes a range of diseases in cattle, including mastitis, arthritis, and pneumonia. However, accurate serological diagnosis of infection remains problematic. The studies described here aimed to identify an antigen that might be used to develop a more specific and sensitive diagnostic assay. A 226-kDa immunogenic protein was consistently detected in Western blots by antibodies in sera from calves experimentally infected with M. bovis. This protein was shown to be a membrane protein with lipase activity and was named mycoplasma immunogenic lipase A (MilA). Different regions of MilA were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins and recombinant products from the amino-terminal end shown to have strong immunoreactivity with M. bovis-specific bovine sera. The most immunoreactive fusion protein, GST-MilA-ab, was used to develop indirect IgM and IgG enzyme-linked immunosorbent assays (ELISAs). The IgM ELISA detected M. bovisspecific IgM antibody 2 weeks after infection with 97.1% sensitivity and had a specificity of 63.3%, while the IgG ELISA detected M. bovis-specific IgG 3 weeks after infection with 92.86% sensitivity and had a specificity of 98.7%, demonstrating that the IgG ELISA has potential for use as a sensitive and specific assay for detecting infection in cattle.

dMycoplasma bovis is a pathogen of emerging significance in cattle throughout the world that is causing a range of diseases, including mastitis, arthritis, and pneumonia. The limited availability and efficacy of current diagnostic and prophylactic tools for its control and its increasing antimicrobial resistance are contributing to its increasing importance in beef and dairy cattle. We have developed an indirect IgG enzyme-linked immunosorbent assay (ELISA) based on a recombinant fragment of the MilA protein and have shown its potential as an effective diagnostic tool. To more comprehensively estimate the diagnostic sensitivity and specificity of this IgG ELISA for detection of infection with M. bovis in cattle and to define a suitable cutoff for use in the field, we further assessed its performance in experimentally infected calves in a closed beef herd and by applying Bayesian latent class modeling to laboratory testing results from 7,448 cattle entering Australian feedlots. The most effective cutoff points were estimated to be 68.6 antibody units (AU) for experimentally infected calves and to be 58.7 AU for a closed adult herd. Under field conditions, in feedlot cattle the globally optimal cutoff was estimated to be 105 AU. At this cutoff, the diagnostic sensitivity was 94.3% (95% probability interval [PI], 89.9% to 99.6%) with a diagnostic specificity of 94.4% (95% PI, 90.3% to 99.6%). Applying this 105 AU cutoff, 13.1% of cattle were seropositive for infection with M. bovis on entry into feedlots, and 73.5% were seropositive when followed up approximately 6 weeks later suggesting a high risk of infection shortly after entry into feedlots. Mycoplasma bovis is regarded to be an emerging cause of bovine respiratory disease (BRD) in veal calves, and infection can also cause polyarthritis and/or otitis media. In adult cattle, M. bovis is mainly associated with mastitis, keratoconjunctivitis, and reproductive tract disorders. Outbreaks of pneumonia induced by M. bovis have been reported throughout the world (1-3) and cause economic loss due to reduced weight gain and increased medication costs (4). The importance of M. bovis has been increasing because of increasing resistance to antimicrobial agents (5) and the lack of an effective vaccine (6). Transmission can occur through fomites, contact, or aerosol, and subclinically infected calves are believed to be carriers for life, so comingling of animals from different farms and age groups, combined with transport stress, results in outbreaks shortly after arrival in feedlots (7). Therefore, identification of infected source herds before introduction of animals to a naive group may help to prevent outbreaks (6).The gold standard for diagnosis of M. bovis infection is culture of the organism, but this is time-consuming and laborious. A M. bovis-specific PCR assay has been developed (8), but such assays are unlikely to be useful for herd screening, as excretion is only detectable for a limited time after infection. Serological diagnosis can be sensitive and inexpensive...

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

BackgroundInexpensive and convenient diagnostic tests for use in clinical work and for the surveillance of infection with Mycoplasma bovis are in demand. The objective of this longitudinal field study was to gain knowledge about the dynamics of antibodies against M. bovis in sera from naturally exposed calves with and without different clinical signs, measured by two different ELISA tests.ResultsA total of 83 calves were subject to between one and five blood samples and clinical examinations using a standard protocol during five herd visits to each of four outbreak dairy herds. The blood samples were analysed for the presence of antibodies against M. bovis using the commercial IgG ELISA test BioX K302 (BioX) and an in-house indirect IgG ELISA test (MilA ELISA).Linear mixed models were used to describe and compare the antibody dynamics as measured by the two tests in relation to the disease status and age of the animals.The BioX ELISA response was below the recommended cut-off (37 ODC%) for the entire study period in many of the calves. The estimated mean ODC% increased slowly but did not reach the recommended individual animal cut-off in three of the four herds. The highest estimated ODC% was not reached until the calf was 110–130 days old. The MilA ELISA response rose above the recommended cut-off (135 antibody units (AU)) in almost all calves, and in two herds, the estimated mean was above the individual animal cut-off shortly after the birth of the calf. The highest estimated antibody concentration was reached when the calf was approximately 60 days old. Disease status of the calf was not significantly associated with the results of either test.ConclusionsWe conclude that the BioX ELISA cannot be recommended for use in calves below 3 months of age. The MilA ELISA was able to detect antibodies shortly after birth (i.e. from approximately 3 weeks of age and onwards) and is therefore a more sensitive test for M. bovis exposure in young calves. Neither ELISA seemed able to differentiate between calves with arthritis and/or otitis media, and respiratory disease.

Mycoplasma gallisepticum is the primary etiological agent of chronic respiratory disease in chickens. Live attenuated vaccines are most commonly used in the field to control the disease, but current vaccines have some limitations. Vaxsafe MG (strain ts-304) is a new vaccine candidate that is efficacious at a lower dose than the current commercial vaccine strain ts-11, from which it is derived. In this study, the transcriptional profiles of the trachea of unvaccinated chickens and chickens vaccinated with strain ts-304 were compared 2 weeks after challenge with M. gallisepticum strain Ap3AS during the chronic stage of infection. After challenge, genes, gene ontologies, pathways, and protein classes involved in inflammation, cytokine production and signaling, and cell proliferation were upregulated, while those involved in formation and motor movement of cilia, formation of intercellular junctional complexes, and formation of the cytoskeleton were downregulated in the unvaccinated birds compared to the vaccinated birds, reflecting immune dysregulation and the pathological changes induced in the trachea by infection with M. gallisepticum. Vaccination appears to protect the structural and functional integrity of the tracheal mucosa 2 weeks after infection with M. gallisepticum.

Mycoplasma bovis is an important pathogen of cattle, causing pneumonia, arthritis and otitis media in young calves, and mastitis in lactating cows, resulting in increased morbidity and, in some instances, mortality. The objective of this study was to evaluate the survival of a M. bovis isolate following nebulisation and to establish whether respiratory disease similar to that seen in the field could be induced in calves by exposing them to an aerosolised culture of M. bovis. A group of eight M. bovis-free calves 14-28days old were exposed to an aerosolised culture of a field isolate of M. bovis that had originally been recovered from a joint lesion in a calf. Three weeks after aerosol exposure necropsies were conducted on all calves. Lung lesions were seen in 7 of 8 calves exposed to the aerosol of M. bovis, whilst calves exposed to the culture medium alone did not develop lesions. Two calves in the infected group had detectable concentrations of serum antibody against M. bovis on day 7 post infection and 4 calves had detectable concentrations of serum antibody against M. bovis on day 21 post infection when tested by MilA IgG ELISA. M. bovis was reisolated from the upper trachea of 6 of the 8 infected calves. The infection method described here appeared to induce lung lesions typical of naturally occurring disease associated with infection with M. bovis and should be applicable to testing the safety and efficacy of attenuated vaccine candidates to control disease caused by this pathogen.

Mycoplasma bovis is associated with several clinical syndromes of cattle. Currently, limited information is available on the sensitivity (Se) and specificity (Sp) of serological assays used for the detection of M. bovis-specific antibodies. Consequently, it is difficult to critically evaluate the outcomes of studies that use these assays. Therefore, the current study used bovine sera sourced from M. bovis exposure studies from three countries to estimate the Se and Sp of two commercial M. bovis enzyme-linked immunosorbent assays (ELISA), BIO K302 and BIO K260, and Western blotting. Western blotting had the highest Se estimate of 74% (95% confidence interval (CI): 16–98%), compared to the BIO K302: 47% (95% CI: 10–87%) and BIO K260: 28% (95% CI: 1–92%). However, for Sp, the BIO K302: 96% (95% CI: 87–99%) and the BIO K260: 100% (95% CI: 93–100%) out-performed Western blotting: 88% (95% CI: 56–98%). Western blotting was the best assay for detecting seroconversion, correctly identifying 61% (95% CI: 29–86%) of exposed animals compared to 35% for BIO K302 (95% CI: 21–54%) and 8% for BIO K260 (95% CI: 0–87%). While none of the methods assessed had high Se and Sp, the availability of these estimates will aid in the interpretation of studies that use these assays. The results of this study highlight the difficulties encountered when using serology to detect exposure to M. bovis in cattle.

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