Isolation of Mycoplasma bovis from bovine clinical samples in the Republic of Ireland

Mycoplasma bovis was detected in 134 (18 per cent) of 736 samples of bovine lung tissue collected from fatal pneumonia cases in the Republic of Ireland between April 1995 and December 1998. The cases occurred in 95 herds and recurred in four of them. Other respiratory pathogens were identified in 66 per cent of the M bovis-positive cases, with Pasteurella species, infectious bovine rhinotracheitis and parainfluenza 3 virus being most frequently detected. Mastitis and arthritis were less common clinical signs associated with M bovis infection; 22 cases of M bovis mastitis and five cases of M bovis arthritis were diagnosed in five herds.     

Adherence of Moraxella bovis to cell cultures of bovine origin

The adherence of five strains of Moraxella bovis to cell cultures was investigated. M bovis adhered to cultures of bovine corneal epithelial and Madin-Darby bovine kidney cells but not to cell types of non-bovine origin. Both piliated and unpiliated strains adhered but piliated strains adhered to a greater extent than unpiliated strains. Antiserum against pili of one strain inhibited adherence of piliated strains but caused only slight inhibition of adherence to the unpiliated strains. Treatment of bacteria with magnesium chloride caused detachment of pili from the bacterial cell and markedly inhibited adherence of piliated strains but caused only slight inhibition of adherence by the unpiliated strains. The results suggested that adhesion of piliated strains to cell cultures was mediated via pili but that adhesins other than pili may be involved in the attachment of unpiliated strains of M bovis to cells.     

Immunofluorescent Studies of Bovine Para-Influenza 3 Virus I. Cell Cultures and Experimentally Infected Hamsters

Immunofluorescent techniques were used to follow the replication of the SF-4 strain of bovine para-influenza 3 (PI-3) virus in Madin-Darby Bovine Kidney (MDBK), secondary bovine kidney (BK), Embryonic Bovine Trachea (EBTr) cell cultures and experimentally infected hamsters. The virus replicated equally well in MDBK and secondary BK cell cultures but less successfully in EBTr cultures. Nuclear fluorescence was not observed in the cell systems studied.

Infection was limited to the epithelial lining of the upper respiratory tract, trachea and bronchi of experimentally infected hamsters. Virus was most easily identified in smears prepared from turbinate mucosa suggesting a potential diagnostic technique for use in identifying PI-3 infected animals.

     

Mycoplasma bovis: disease,diagnosis, and control

Mycoplasma bovis is a major, but often overlooked, pathogen causing respiratory disease, mastitis, and arthritis in cattle. It is found worldwide and has spread into new areas, including Ireland and parts of South America, in the last decade. In Europe, it is responsible for at least a quarter to a third of all calf pneumonia although this may be an underestimate as few laboratories regularly monitor for mycoplasmas. Like all mollicutes, M. bovis is inherently refractory to certain groups of antibiotics because it does not possess a cell wall; furthermore evidence is accumulating that strains of M. bovis are becoming resistant to antibiotics, including tetracycline, tilmicosin and spectinomycin, traditionally used for their control. No vaccines are presently available for the control of M. bovis infections.     

牛支原体引起奶牛乳房炎的诊断

牛支原体（Mycoplasma bovis）是引起成年牛乳房炎和犊牛肺炎、关节炎的主要病原之一。本文从有乳房炎临床症状的奶牛中采集牛奶样本,经细菌培养和支原体培养、特异性PCR与环介导等温扩增（LAMP扩增）和16S rRNA测序等病原学检测证实为牛支原体感染,同时还有其他细菌的混合感染。     

Assessment of Bdellovibrio bacteriovorus 109J killing of Moraxella bovis in an in vitro model of infectious bovine keratoconjunctivitis

The objective of this study was to determine the potential of Bdellovibrio bacteriovorus 109J as an alternative non-chemotherapeutic treatment of infectious bovine keratoconjunctivitis (IBK). To accomplish this, various parameters of B. bacteriovorus predation of Moraxella bovis were determined in vitro. Initial passage of B. bacteriovorus using M. bovis as prey required 10 d for active cultures to develop compared with 2 d for culture on normal Escherichia coli prey; however by the 5th passage, time to active predatory morphology was reduced to 2 d. This high passage B. bacteriovorus culture [1 × 10(10) plaque forming units (PFU)/mL] killed 76% of M. bovis [1 × 10(7) colony forming units (CFU)/mL] present in suspension broth in a 4 h assay. The minimal level of M. bovis supporting B. bacteriovorus predation was 1 × 10(4) CFU/mL. To assess the ability of B. bacteriovorus to kill M. bovis on an epithelial surface mimicking IBK, an in vitro assay with Madin-Darby bovine kidney (MDBK) cells inoculated with 4 × 10(7) CFU/mL M. bovis was used. Treatment with a B. bacteriovorus suspension (1.6 × 10(11) PFU/mL) decreased adherence of M. bovis to MDBK cells by 6-fold at 12 h of treatment, as well as decreased the number of unattached M. bovis cells by 1.4-fold. This study demonstrates that B. bacteriovorus has potential as an effective biological control of M. bovis at levels likely present in IBK-infected corneal epithelia and ocular secretions.     

Incidence and transmission of Mycoplasma bovis mastitis in Holstein dairy cows in a hospital pen: A case study

The objective was to determine the incidence and transmission of mycoplasma mastitis in the hospital pen in a dairy herd of 650 lactating cows after a hospital pen was established following an outbreak of this disease. Mycoplasma mastitis status was monitored for 3 months through repeated collection of milk samples from cows with clinical mastitis (CM) and from bulk tank milk. During the outbreak 13 cows were diagnosed with Mycoplasma bovis CM, 1 cow with Mycoplasma sp. mastitis and 8 cows showed signs of arthritis, 3 of which were confirmed as having M. bovis arthritis. M. bovis isolates from cows with CM, arthritis and bulk tank milk had indistinguishable chromosomal digest pattern fingerprints. Incidence rates of M. bovis CM cases in the milking and hospital pens were 0.01 and 1.7 cases per 100 cow-days at risk. Approximately 70% of cows with M. bovis CM became infected within 12 days of entering the hospital pen. Transmission of M. bovis in the hospital pen occurred as 3 episodes. Each episode corresponded to the introduction of a cow with M. bovis CM from a milking pen. Evidence indicates that cows with M. bovis CM from milking pens were the source of transmission of the disease in the hospital pen and thus their presence in the hospital pen appeared to be a risk factor for transmission of M. bovis mastitis in this single case study herd.     

牛支原体病研究进展

牛支原体是一种非常重要但容易被忽视的病原,能导致牛肺炎、乳腺炎、关节炎等多种疾病。国外已证实,该病给养牛业造成巨大的经济损失。目前,对牛支原体的毒力因子及免疫机理均不十分清楚,缺少其相应的有效疫苗和检测方法,而且,牛支原体对许多常用抗生素不敏感,从而,给临床防治带来很大困难。我国自2008年首次报道了牛支原体肺炎,但对牛支原体病危害的认识还很不全面,妨碍了对该病的有效防治。论文分析归纳了牛支原体病的国内外研究概况,从病原学、流行病学、临床症状与病理变化、诊断、毒力因子、免疫与疫苗、防治措施等方面进行了系统综述,以期为我国牛支原体病的防治提供参考。     

Herd-specific strains of Mycoplasma bovis in outbreaks of mycoplasmal mastitis and pneumonia

Mycoplasma bovis causes severe economic losses in livestock production, particularly on the Northern American continent and more recently also in continental Europe. The aim of the current study was to evaluate whether the recently emerging outbreaks were due to a particular clone or strain of M. bovis or whether these outbreaks are due to multiple infectious strains of M. bovis. The study is based on the analysis M. bovis isolated from cattle of herds with outbreaks of mycoplasmal mastitis or pneumonia from geographically non related parts of Switzerland. M. bovis isolates were typed by insertion sequence (IS) element analysis based upon ISMbov1 and ISMbov2 southern-blot hybridization. We observed a strong divergence of M. bovis strains among affected herds which mostly were herd specific. This argues against the assumption that a recent infiltration of a particular clone of M. bovis is the cause of the perilous emerging outbreaks. The study suggests that transmission occurs from animal to animal most probably via milk.     

Development of a real-time PCR for detection of Mycoplasma bovis in bovine milk and lung samples.

A real-time polymerase chain reaction (PCR) assay using hybridization probes on a LightCycler platform was developed for detection of Mycoplasma bovis from individual bovine mastitis milk and pneumonic lung tissues. The detection limit was 550 colony forming units (cfu)/ml of milk and 650 cfu/25 mg of lung tissue. A panel of bovine Mycoplasma and of other bovine-origin bacteria were tested; only M. bovis strains were positive, with a melting peak of 66.6 degrees C. Mycoplasma agalactiae PG2 was also positive and could be distinguished because it had a melting peak of 63.1 degrees C. In validation testing of clinical samples, the relative sensitivity and specificity were 100% and 99.3% for individual milks and 96.6% and 100% for the lung tissue. Using M. bovis real-time PCR, the M. bovis culture-positive milk samples were estimated to contain between 5 x 10(4) and 7.7 x 10(8) cfu/ml and the M. bovis culture-positive lungs between 1 x 10(3) and 1 x 10(9) cfu/25 mg. Isolation, confirmed with the real-time PCR and colony fluorescent antibody test, showed that at the herd level, the proportion of samples positive for M. bovis isolation in mastitis milk samples submitted to the Mastitis Laboratory, Animal Health Laboratory, University of Guelph, Ontario, Canada, was 2.4% (5/201). We conclude that this probe-based real-time PCR assay is a sensitive, specific, and rapid method to identify M. bovis infection in bovine milk and pneumonic lungs.     

Preparation and characterization of monoclonal antibodies against Mycoplasma bovis.

Monoclonal antibodies (mabs) against Mycoplasma (M.) bovis were prepared for use in diagnosis of bovine mastitis. From the original 32 hybridomas actively secreting mabs against M. bovis, 6 stable lines were cloned. Two of them, Mb 5D8 and Mb 4F6, recognized M. bovis antigens of estimated molecular weights of 33 and 26 kDa, respectively. They showed no cross-reaction to other bovine mycoplasmas, thus rendering them useful for specific detection of this pathogen. All mabs investigated cross-reacted with M. agalactiae which is known to be closely related to M. bovis, but does not occur in cattle. Two other mabs, Mb 5D4 and Mb 1F6, exhibited further cross-reactions to a number of bovine mycoplasma species. Finally, mabs Mb 5D5 and Mb 2G5 reacted with all mycoplasmas tested. The possibility that they recognized constituents of the broth culture medium is discussed.     

牛支原体疫苗的研究进展

牛支原体（Mycoplasma bovis,M.bovis）是一种造成全世界范围内育肥牛和奶牛多种疾病综合征的重要病原体。临床症状主要包括长期性肺炎及多发性关节炎（CPPS）、呼吸道疾病综合征（BRD）、乳腺炎及生殖器官疾病。牛支原体能感染多种组织和器官,也能从健康的牛体内分离,是威胁畜牧业生产的主要病原体。由于临床上抗生素治疗效果不佳,预防或控制牛支原体感染最好的选择是研发有效的商业可用的疫苗。牛支原体疫苗的研究已历经多年,虽然存在很多问题,但也取得了一定进展。文章对牛支原体弱毒疫苗、灭活疫苗及亚单位疫苗的研究进展进行了总结,并讨论了疫苗设计的优化方案,为合理设计和研发有效的牛支原体防控技术提供参考。     

Naturally occurring Mycoplasma bovis-associated pneumonia and polyarthritis in feedlot beef calves.

Mycoplasma bovis is perceived as an emerging cause of mortality in feedlot beef cattle. This study examined the lesions and infectious agents in naturally occurring M. bovis-associated bronchopneumonia and arthritis and the relationship of this condition with bovine viral diarrhea virus (BVDV) infection. Standardized pathologic, immunohistochemical, and microbiologic investigations were conducted on 99 calves that died or were euthanized within 60 days after arrival in 72 feedlots. Cranioventral bronchopneumonia with multiple foci of caseous necrosis was identified in 54 of 99 calves, including 30 with concurrent fibrinosuppurative bronchopneumonia typical of pneumonic pasteurellosis. Mycoplasma bovis was consistently identified in these lesions by culture and immunohistochemistry, but also commonly in healthy lungs and those with pneumonia of other causes. Focal lesions of coagulation necrosis, typical of pneumonic pasteurellosis, were often infected with both Mannheimia haemolytica and M. bovis. Arthritis was present in 25 of 54 (46%) calves with M. bovis pneumonia, and all calves with arthritis had pneumonia. BVDV infection was more common in calves with lesions of bacterial pneumonia than in those dying of other causes, but BVDV infection was not more common in calves with caseonecrotic bronchopneumonia than those with fibrinosuppurative bronchopneumonia. Retrospective analysis identified cases of M. bovis pneumonia in the early 1980s that had milder lesions than the current cases. The findings suggest that, in at least some calves, M. bovis induces caseonecrotic bronchopneumonia within the lesions of pneumonic pasteurellosis.     

Mycoplasma agalactiae subsp. bovis in pneumonia and arthritis of the bovine.

The pneumonic lungs of 42 cattle from 26 feedlots were examined for the presence of mycoplasma, pathogenic bacteria and viruses. Four animals representative of two lots failed to yield mycoplasma. One of these yielded the virus of infectious bovine rhinotracheitis and Pasteurella hemolytica, the other yielded only P. P. multocida. Nine animals in eight lots yielded Mycoplasma sp.: five of these were M. bovirhinis, two were M. arginini and two were untypable. All of these animals yielded one or more of P. hemolytica, P. multiocida, infectious bovine rhinotracheitis virus or bovine virus diarrhea virus. Twenty-five of 29 animals in 16 lots yieled M. agalactiae subsp. bovis from lung tissues. The same organism was recovered from the arthritic joints of 12 of these animals. Eight of the 25 animals yielded no other pathogen and all of these had not received any treatment. Nine of the 25 M. agalactiae subsp. bovis positive animals also yielded one or more of P. hemolytica, P. multocida, Corynebacterium pyogenes or infectious bovine rhinotracheitis virus. Bacteriological and virological studies were not completed for the remaining eight of the 25 positive animals. In five lots of cattle which had not received medication for pneumonia and for arthritis only M. agalactiae subsp. bovis was recovered. Twenty-five grossly normal lungs obtained from normal cattle at the time of slaughter were cultured and all were negative. The possible role of M. agalactiae subsp. bovis in pneumonia and arthritis was discussed.     

牛支原体生物学特性及其膜蛋白的研究进展

牛支原体(Mycoplasma bovis,Mb)是引起牛肺炎、关节炎、乳房炎和中耳炎等众多疾病的主要病原之一,牛支原体引起的疾病在欧美地区广泛蔓延并造成巨大的经济损失。中国2008年首次报道在犊牛肺炎中分离到牛支原体,目前国内对牛支原体的认识还很欠缺,现对牛支原体生物学特性及其膜蛋白进行简要综述,以期为牛支原体病的防控与治疗提供一定的参考依据。     

Adherence to bovine neutrophils and suppression of neutrophil chemiluminescence by Mycoplasma bovis

The adherence of viable and heat-treated Mycoplasma bovis to bovine peripheral blood neutrophils was studied by specific immunofluorescence staining and flow cytometry. Viable and heat-treated M. bovis cells, adhered to bovine neutrophils in dose-dependent fashion within a 30 min incubation. Fluorescence quenching using crystal violet indicated that unopsonized M. bovis cells remained on the surface of bovine neutrophils without experiencing significant ingestion. The effect of M. bovis adherence on neutrophil microbicidal function was examined by measuring luminol enhanced chemiluminescence (CL). Adherent M. bovis cells did not elicit a bovine neutrophil CL response over a 75 min incubation period. M. bovis inhibited the capacity of bovine neutrophils to mount a CL response. Inhibition occurred whether viable or heat-treated M. bovis cells were used and it occurred when neutrophils were stimulated with opsonized zymosan (OZ) or phorbol myristate acetate (PMA). Inhibition of the PMA stimulated neutrophil CL response required cytadherence by M. bovis cells. These findings suggest that activation of the bovine neutrophil respiratory burst was inhibited at or distal in the pathway to the activation of protein kinase C (PKC), the site of PMA stimulation, and that it was mediated by a direct interaction between the adhering M. bovis cells and the bovine neutrophil membrane.     

Molecular and antigenic characterization of a Mycoplasma bovis strain causing an outbreak of infectious keratoconjunctivitis.

An unusually high incidence of infectious keratoconjunctivitis followed by pneumonia and arthritis was observed in beef calves of a managed herd. No Moraxella spp. or bacteria other than Mycoplasma spp. were obtained from conjunctival and nasal swabs. A strategy was designed for characterization of bovine mycoplasmas at species and strain level on the basis of a combination of molecular tools and the immunoblotting method. The strategy made it possible to rapidly assign the bacterium responsible for this outbreak to one of the phylogenetic clusters of bovine mycoplasmas delineated in this study and then to identify it as Mycoplasma bovis. The strain, designated Sar 1, showed a 100% 16S rDNA sequence identity with two European strains (120/81 and MC3386) isolated in Germany and Ireland, respectively, and hosts a vsp gene analog to the vspA, vsp422-4, and vsp422-8 genes of the M. bovis reference strain PG45T and of the field strain 422. The use of a cross-reactive rabbit serum developed against the Mycoplasma agalactiae immunodominant antigen P48 confirmed the molecular findings. The immunological response of calves against M. bovis was also investigated. This is the first report on the occurrence of M. bovis on the Island of Sardinia (Italy).     

Preliminary studies on the prevalence of Mycoplasma bovis mastitis in dairy in cattle in Australia

A highly sensitive and specific PCR (MB-PCR) was used in preliminary studies to detect M. bovis in milk samples to investigate its association with high somatic cell count (SCC), an indicator of subclinical mastitis and one of the factors in down grading the quality of milk. A total of 186 and 167 herds were tested with 43% and 62% of herds positive for M. bovis in Victoria and North Queensland, respectively. The quarter milks from 52 cows with persistently high SCC were tested by MB-PCR and culture to investigate the association of M. bovis with major mastitis pathogens (MMP). M. Bovis was detected in 77% of cows of which 19% alone had M. bovis without any other bacteria, 17% had M. bovis in combination with major mastitis pathogens and 40% had M. bovis in combination with non-major mastitis pathogens. We believe that M. bovis is widespread in dairy cattle and has the potential to produce disease alone or to predispose the udder to disease caused by major mastitis and environmental pathogens. These studies have revealed a hitherto unrecognised high prevalence of M. bovis in dairy cattle in North Queensland and Victoria in Australia. These initial studies also give a clear association between M. bovis and elevated somatic cell counts.     

Coinfection with bovine viral diarrhea virus and Mycoplasma bovis in feedlot cattle with chronic pneumonia

Chronic, antibiotic-resistant pneumonia, sometimes with concurrent polyarthritis, occurs in feedlot cattle in western Canada. The prevalence of Mycoplasma bovis, bovine viral diarrhea virus, and Haemophilus somnus was determined by using immunohistochemical staining of lung and heart tissue from 2 groups of animals with this history. Mycoplasma bovis antigen was present in 44/48 cases submitted between 1995 and 1998 (retrospective group) and 15/16 of cases from 1999 (prospective group), and was associated with pulmonary necrosis. Bovine viral diarrhea virus antigen was present in association with microscopic vascular lesions in 31/48 retrospective and 9/16 of prospective cases. Types Ib and II bovine viral diarrhea virus were isolated from 4/16 prospective cases. Haemophilus somnus antigen was present in heart, lung, or both of 15/48 retrospective and 8/16 prospective cases. The results suggest that there may be synergism between bovine viral diarrhea virus and M. bovis in this pneumonia with arthritis syndrome.     

Attachment of Moraxella bovis to calf corneal cells and inhibition by antiserum

An in vitro assay was developed using calf corneal cells to assess the importance of fimbriae in the colonisation of the bovine ocular surface by Moraxella bovis, and the role of fimbrial antibodies in the bovine immune response and resistance to infectious bovine keratoconjunctivitis (IBK). Fimbriae promoted adherence of M. bovis to calf corneal cells in culture; 15 fimbriate isolates, representative of 6 fimbrial serogroups of M. bovis, adhered to the cells whereas 4 non-fimbriate isolates failed to do so. Fimbrial antibodies in hyperimmune rabbit serum inhibited attachment of all fimbriate strains of the homologous fimbrial serogroup but not those of 5 heterologous serogroups. The relevance of these results to the use of a polyvalent fimbrial vaccine in the control of IBK is discussed.