Diagnosis of a mixed mycoplasma infection associated with a severe outbreak of bovine pinkeye in young calves.

Mycoplasma bovoculi and Mycoplasma bovis were both isolated from conjunctival swabs taken from young calves showing symptoms consistent with infectious bovine keratoconjunctivitis (pinkeye). No Moraxella spp. or other nonmycoplasma bacteria were isolated in association with this severe clinical outbreak. Based on laboratory tests and clinical observations, the first phase of the disease was likely pneumonic in nature, possibly caused by bovine respiratory syncytial virus and M. bovis. In the subsequent phase of the disease course, infection with both M. bovoculi and M. bovis resulted in ocular disease. A combination of microbiological, serological, and molecular diagnosticmethods was used to elucidate the etiology of the outbreak.     

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NM2012株致病性研究

[背景]牛支原体是牛呼吸道疾病综合征的重要病因之一,可以引起牛的多种呼吸道疾病,给养殖业带来巨大的经济损失。接种疫苗是预防牛支原体感染的最有效的手段。[目的]通过人工感染牛体试验,了解牛支原体NM2012株的致病性,为后续疫苗研究提供生产用菌种。[方法]将牛支原体NM2012株第6代、第12代、第20代菌株分别经人工气管注射感染2周龄犊牛,攻毒后连续观察27 d,记录攻毒后犊牛的临床症状,在感染后第14天,从各攻毒组分别选取3头犊牛、空白对照组选取2头犊牛进行扑杀,第27天将剩余实验犊牛处死,观察肉眼病理变化和组织学病理变化。[结果]3个代次的NM2012菌株均对犊牛具有致病性,可引起比较典型的牛支原体肺炎症状和病理变化。[结论]NM2012株6-20代仍然具有对牛的致病性,可以作为今后疫苗研究的潜在菌种。     

Isolation of mycoplasma species from the lower respiratory tract of healthy cattle and cattle with respiratory disease in Belgium

Between 1997 and 2000, a total of 150 healthy cattle and 238 animals with respiratory disease were examined for six Mycoplasma species. Attempts were made to detect Mycoplasma canis, Mycoplasma dispar and Ureaplasma diversum in calves with recurrent disease, and all three of these species were identified in calves with recurrent disease and in healthy lungs. In healthy calves, 84 per cent of bronchoalveolar lavage fluids were mycoplasma free; when cultures were positive, Mycoplasma bovirhinis was the only species isolated. Mycoplasmas were isolated from 78 per cent of animals suffering recurrent respiratory disease and from 65 per cent of acute respiratory cases. Mycoplasma bovis was isolated from bronchoalveolar lavages from 35 per cent of calves suffering recurrent respiratory disease, and from 50 per cent of acute cases, and from 20 per cent of pneumonic cases examined postmortem. M bovis was associated with other Mycoplasma species in 44 per cent of cases. M dispar was also isolated from 45.5 per cent of calves suffering recurrent respiratory disease, often in association with M bovis. M canis was identified for the first time in diseased Belgian cattle. Other mycoplasmas, including Mycoplasma arginini, Mycoplasma alkalescens and U diversum, were isolated less frequently. Associations between mycoplasmas and other pathogens were often observed. Among lungs infected with Pasteurella and/or Mannheimia species, more than 50 per cent were mixed infections with M bovis.     

Isolation of Mycoplasma bovoculi from cases of infectious bovine keratoconjunctivitis.

Five outbreaks of infectious bovine keratoconjunctivitis were examined for bacteria and mycoplasmas. Mycoplasma bovoculi was demonstrated in four of the five outbreaks. Other mycoplasmatales were represented by Ureaplasma in one sample. Moraxella bovis and Neisseria ovis were found in all the outbreaks, the former being present in the vast majority of the animals. Transmission experiments with Mycoplasma bovoculi and Moraxella bovis in combination were carried out on four young, colostrumdeprived calves. Mycoplasma bovoculi appeared to have an enhancing effect on the pathogenicity of Moraxella bovis.     

Identification of Mycoplasmatales in pneumonic calf lungs

Lungs from 153 calves with clinical signs of pneumonia were examined post-mortem (PM) for the presence of mycoplasmas and ureaplasmas during a 38-month period. Sixty-two percent of the cases were submitted during the months when wide fluctuations in climatic conditions occur. Using indirect fluorescent antibody tests (IFAT) and culture, mycoplasmas and/or ureaplasmas were detected in 63% of the lungs examined. Mycoplasma dispar was detected in 39%, M. bovis in 36%, Ureaplasma spp. in 22% and M. bovirhinis in 8.5% of the lungs. Thirty percent of the lungs were infected with more than one species; the most frequent combination was M. bovis, M. dispar and Ureaplasma spp. (10.5%). M. arginini, M. bovigenitalium and acholeplasmas were not cultured. M. dispar was shown to remain viable for up to 15 days PM in apical and cardiac lobes held at 4 degrees C and also was detected by IFAT in the same tissues for 49 days.     

The incidence of Mycoplasma dispar, Ureaplasma and conventional Mycoplasma in the pneumonic calf lung.

This report describes the incidence of Mycoplasma dispar, ureaplasma and conventional (large colony) mycoplasma isolated from the pneumonic lungs of groups of young calves and the identification to species level of mycoplasmas in mixed populations with the aid of the indirect fluorescent antibody test. Pneumonic lung tissue yielded one or more mycoplasma species from 88% of the 153 calves cultured. The mycoplasmas identified and percent of the calves with lungs positive for each species were: M. dispar (56%), ureaplasma (44%), Mycoplasma bovis (37%), Mycoplasma arginini (33%) and Mycoplasma bovirhinis (23%). Conventional mycoplasmas isolated from two calves (1%) could not be identified using the antisera available.     

Mycoplasma species and related organisms isolated from ruminants in Britain between 1990 and 2000

Between 1990 and 2000, more than 1600 mycoplasmas and the related acholeplasmas were identified from ruminant animals by the Mycoplasma Group at the Veterinary Laboratories Agency--Weybridge. Mycoplasma bovis was the most commonly identified pathogen, mostly from pneumonic calves but occasionally from cattle with mastitis and arthritis. Mycoplasma canis was first isolated in Britain in 1995 from pneumonic calves and the number of isolates increased to 18 per cent of the total mycoplasmas isolated from cattle in 1999. The ELISA for antibodies to M. bovis detected 1971 positive samples (22 per cent) among 8959 serum samples, mainly from pneumonic cattle. Other mycoplasmas identified included Mycoplasma dispar from the lungs of cattle with respiratory disease, and Mycoplasma bovigenitalium from the reproductive tract of cows with vulvovaginitis and infertility. Mycoplasma bovirhinis and Acholeplasma species were found commonly but are thought to be more opportunistic than pathogenic. In sheep and goats, the majority of Mycoplasma species isolated were identified as Mycoplasma ovipneumoniae from pneumonic sheep, Mycoplasma conjunctivae from sheep with keratoconjunctivitis, and the ubiquitous Mycoplasma arginini.     

Etiology of respiratory disease in non-vaccinated, non-medicated calves in rearing herds

The aim of this study was to examine the occurrence of bacterial, mycoplasmal and viral pathogens in the lower respiratory tract of calves in all-in all-out calf-rearing units. According to clinical status, non-medicated calves with and without respiratory disease signs were selected of the 40 herds investigated to analyse the micro-organisms present in healthy and diseased calves. Tracheobronchial lavage (TBL) and paired serum samples were analysed for bacteria, mycoplasmas, respiratory syncytial virus (RSV), parainfluenza virus 3 (PIV3), bovine corona virus (BCV) and bovine adenovirus (BAV). Pasteurella multocida was the most common bacterial pathogen. It was isolated from 34% of the TBL samples in 28 herds and was associated with clinical respiratory disease (p < 0.05) when other pathogenic bacteria or mycoplasma were present in the sample. Mannheimia spp. and Histophilus somni were rarely found. Mycoplasma bovis was not detected at all. Ureaplasma diversum was associated with clinical respiratory disease (p < 0.05). TBL samples from healthy or suspect calves were more often negative in bacterial culture than samples from diseased calves (p < 0.05). No viral infections were detected in six herds, while 16-21 herds had RSV, BCV, BAV or PIV3. In the herds that had calves seroconverted to BCV, respiratory shedding of BCV was more frequently observed than faecal shedding. This study showed that the microbial combinations behind BRD were diverse between herds. M. bovis, an emerging pathogen in many countries, was not detected.     

Clinical study of the disease of calves associated with Mycoplasma bovis infection

Clinical, bacteriological and serological examination of 35 calves from the age of 5 to 26 days was performed in a Holstein-Friesian dairy herd endemically infected with Mycoplasma bovis. M. bovis was isolated from 48.6% of nasal swabs taken from the calves at the age of 5 days, and from 91.4% of the same calves at the age of 26 days, indicating the gradual spread of infection. The isolation rate of Pasteurella multocida did not change much, and varied from 28.6 to 25.7%. No P. haemolytica could be detected. In addition to M. bovis and P. multocida, the herd was also infected with different viruses (including bovine viral diarrhoea virus, infectious bovine rhinotracheitis virus, bovine adenoviruses, parainfluenza-3 virus, and bovine respiratory syncytial virus) as a large proportion of the sera of newborn calves contained colostral antibodies against these viruses. In most of the newborn calves severe clinical signs (fever, depression, inappetence, hyperventilation, dyspnoea, nasal discharge and coughing) due to M. bovis infection developed. The clinical signs appeared already on the fifth day of life, and their incidence was the highest at the age of 10 to 15 days. Three calves (8.6%) died as a result of severe serofibrinous pneumonia. The surviving calves showed very poor weight gain (ranging from 1.5 to 3.5 kg) during the first two weeks of life.     

Efficacy of tulathromycin in the treatment of bovine respiratory disease associated with induced Mycoplasma bovis infections in young dairy calves

The efficacy of tulathromycin in the treatment of bovine respiratory disease (BRD) due to Mycoplasma bovis was determined following experimental infection. Two highly pathogenic strains of M. bovis (with minimum inhibitory concentration values for tulathromycin of 1 and >64 microg/ml) were inoculated into 145 calves. Four days after inoculation, calves with clinical BRD were treated subcutaneously with saline or tulathromycin (2.5 mg/kg). Compared with saline, BRD-related withdrawals, peak rectal temperatures, and lung lesion scores were significantly lower for tulathromycin-treated calves (P < .01). Tulathromycin was highly effective in the treatment of BRD due to M. bovis in calves regardless of the minimum inhibitory concentration of the challenge strain (1 or >64 microg/ml).     

Effects of age, environmental temperature and relative humidity on the colonization of the nose and trachea of calves by Mycoplasma spp

Nasal and tracheal swabs sequentially collected from three groups of eight calves between the ages of 1 and 98 days indicated that the nose and trachea were colonized by Mycoplasma spp. during the first weeks of life. Over 92% of all calves harboured Mycoplasma spp. in their noses when they were 2 weeks old, the rate of recovery falling gradually thereafter. The peak period of recovering mycoplasmas from the noses and tracheas of calves was at 6 weeks old. M. bovirhinis, M. arginini and Acholeplasma laidlawii predominated in the nose while M. dispar and M. bovirhinis predominated in the trachea. There was no association between rates of isolation and clinical signs of respiratory disease. There were no significant differences between the frequencies of isolation of Mycoplasma spp. from groups of calves kept under different environmental temperatures and relative humidities.     

Genetic analysis of virulence factors of Mannheimia (Pasteurella) haemolytica A1

Sixteen 3-week-old calves were intratracheally inoculated with Mycoplasma bovis. Follow-up consisted of regular bronchoalveolar lavages (BALs) and clinical examinations. Animals were slaughtered from 4 to 21 days after inoculation. Counts were made of the mycoplasmas and other bacteria systematically isolated from the BAL liquids and lung lobes after slaughter. On the 6th day, spectinomycin 20mg/kg was given intramuscularly in three repeated doses at 24h intervals to six randomly chosen calves. All animals had developed a persistent M. bovis infection with a maximum BAL count on the 6th day (start of treatment). Co-occuring Pasteurella multocida infection was found in most animals with a maximum rate on the 14th day. The extent of lung surface lesions varied widely (0-64%) but was greater in the later slaughtered calves. Average counts of M. bovis and P. multocida in the BAL liquids were lower in treated calves than in untreated ones but the difference was not statistically significant. However, M. bovis and P. multocida counts in the lungs of the treated group were significantly lower than in the untreated group (p=0.003 and 0.009, respectively).     

致犊牛肺炎牛支原体南疆株的分离鉴定及oppF基因序列分析

本研究旨在调查新疆喀什某规模化奶牛场的犊牛死亡原因,并确定病原体.无菌采集3份因肺炎死亡的犊牛肺脏病料样品.采用牛支原体专用液体培养基和1.0%牛支原体琼脂固体筛选培养基从3份病死犊牛肺脏病料中分离得到2株牛支原体(Mycoplasma bovis,M.bovis),分别命名为M.bovis-NJ-1和M.bovis-NJ-2.通过菌落形态学观察、特异性PCR和oppF测序比对对分离株进行鉴定.结果显示,2个分离株在固体培养基上的菌落呈现典型的"煎蛋状",且Dienes染色特点符合牛支原体菌落着色特征,中心呈深蓝色;PCR能扩增出牛支原体特异的448 bp目的片段;2个分离株的oppF基因序列与牛支原体国际标准株PG45的同源性分别为96.7%和95.3%.结果表明,引起犊牛发病死亡的病原是牛支原体,本研究为犊牛支原体肺炎的快速诊断和防制提供依据.     

Prevalence of respiratory pathogens in diseased, non-vaccinated, routinely medicated veal calves

The prevalence of respiratory pathogens in diseased veal calves was determined in 24 respiratory disease outbreaks in 15 herds in Belgium. Bacteria were cultured from nasopharyngeal swabs and seroconversion against viruses and Mycoplasma bovis was determined on paired sera. At the individual calf level, Mycoplasma species, Mannheimia haemolytica and Pasteurella multocida, were isolated from 70.5 per cent, 21.5 per cent and 26.0 per cent of swabs, respectively. At the herd level, the presence of M bovis could be confirmed in 84.6 per cent of the herds examined. Seroconversion against bovine viral diarrhoea virus (BVDV) was present in 71.4 per cent of herds, parainfluenzavirus type 3 in 53.3 per cent, bovine respiratory syncytial virus in 40.0 per cent, bovine adenovirus type 3 in 46.7 per cent, bovine coronavirus in 30.0 per cent, and bovine herpesvirus type 1 in 26.7 per cent. At postmortem examination, Mycoplasma species could be cultured from 61.9 per cent of pneumonic lungs (n=21). Sixty per cent of calves tested were positive for BVDV (n=20), and 20.0 per cent were positive for bovine respiratory syncytial virus (n=16).     

Characterization studies on mycoplasmas isolated from bovine mastitis and the bovine respiratory tract.

Mycoplasmas isolated from bovine mastitis in California were classified into five distinct species. These included Mycoplasma bovis, M. bovigenitalium, M. alkalescens, M. canadenfe, and an unidentified strain, ST-6. Strains frequently recovered from the nose of young calves proved to be M. arginini, M. bovirhinis was recovered from the respiratory tract but was not a common finding.     

Isolation of Mycoplasmas from nasal swabs of calves affected with respiratory diseases and antimicrobial susceptibility of their isolates

Nasal swabs of 293 calves were examined for Mycoplasma. The samples were collected from calves affected with respiratory diseases on 71 farms in various parts of Japan between 1996 and 1997. Mycoplasma bovirhinis was isolated from 47 of 293 calves (16.0%). Mycoplasma alkalescens, M. bovis, M. arginini, M. bovigenitalium and Acholeplasma spp. were isolated from 19 (6.5%), seven (2.4%), four (1.4%), four (1.4%) and 18 (6.1%) calves, respectively. Pasteurella multocida and P. haemolytica were isolated from 60% of Mycoplasma-positive calves. However, other bacteria were not isolated from calves. To evaluate the antimicrobial susceptibility of their isolates, 68 M. bovirhinis, 21 M. alkalescens and 10 M. bovis strains were examined for 12 antimicrobial agents. All isolates showed higher susceptibility to tiamulin than to the other drugs used in the study. However, erythromycin had no effect on any of the Mycoplasma strains studied. The field isolates were less susceptible than the type strains to some drugs, such as spiramycin, oxytetracycline and tylosin.     

Infectious bovine keratoconjunctivitis II. Antibodies in lacrimal secretions of cattle naturally or experimentally infected with Moraxella bovis.

One or both eyes of 20 calves were inoculated one or more time with variou(s combinations of microorganism (live oor killed Moraxella bovis, infectious bovine rhinotracheitis virus, bovine adenovirus, bovine parainfluenza-3 virus and Mycoplasma bovoculi) by conjunctival instillation or direct inoculation of the conjunctivea or cornea. The eyes of all the calves received natural or artificial ultraviolet irradiation. Neither the adenovirus nor parainfluenza-3 virus became established in the eye or produced keratoconjunctivitis. Both M. bovis and infectious bovine rhinotracheitis virus became established in the bovine eye and produced disease. Subconjunctival or intracorneal inoculation of M. bovis caused a severe disease, simulating natural infectious bovine keratoconjunctivitis. Only the intracorneal inoculation of mycoplasma produced severe keratoconjunctivits. Eyes that on initial exposure to M. bovis became severly inflamed were more resistant to a second or third exposure to M. bovis, presumably by enhanced local defence mechanisms.     

Safety and efficacy testing of a novel multivalent bovine bacterial respiratory vaccine composed of five bacterins and two immunogens

Bovine bacterial respiratory diseases have been one of the most serious problems due to their high mortality and economic loss in calves. The vaccinations of bovine bacterial respiratory vaccines have been complex because of no multivalent vaccine. In this study, novel multivalent bovine bacterial respiratory vaccine (BRV) was developed and tested for its safety and efficacy. BRV was composed of two immunogens and five bacterins. These were leukotoxoid and bacterin of Mannheimia haemolytica type A, outer membrane protein and bacterin of Pasteurella multocida type A, and bacterins of Haemophilus somnus, Mycoplasma bovis, and Arcanobacterium pyogenes. ELISA antibody titers to five bacterial antigens in vaccinated guinea pigs increased, compared with those in unvaccinated ones. BRV was safe for calves and pregnant cattle in this study. In calves challenged with M. haemolytica and P. multocida, the average daily weight gain and antibody titers of vaccinated calves increased, and respiratory symptoms (P<0.05) and treatment frequency (P<0.01) of vaccinated calves significantly decreased, compared with those of unvaccinated calves. Interestingly, the antibody titers of M. haemolytica leukotoxoid and Mycoplasma bovis were closely related with the reduction of respiratory symptoms. BRV would be an ecomonical measure for the protection against bovine bacterial respiratory diseases.     

Analysis of heat shock protein 60 encoding genes of mycoplasmas and investigations concerning their role in immunity and infection

Only little is known about the heat shock proteins (Hsp) and Hsp-encoding genes of mycoplasmas. The aim of this study was to identify and sequence the hsp60 gene of Mycoplasma agalactiae, Mycoplasma arthritidis, Mycoplasma bovis, and Mycoplasma hyopneumoniae, and to investigate the immune response to Hsp60.Fragments of the hsp60 genes of M. agalactiae, M. arthritidis, M. bovis and M. hyopneumoniae representing almost the entire coding region were amplified by PCR. Two fragments of a hsp60 gene were cloned in Escherichia coli and the antibody response of pigs infected with M. hyopneumoniae against the recombinant Hsp60 fusion proteins was analysed. Within the mycoplasmas, the hsp60 genes showed sequence identities of nearly 100%, with the exception of the hsp60 gene of Mycoplasma genitalium, which was determined to be only 76.5-77.7% identical. Identities to Clostridium perfringens, Bacillus subtilis and E. coli were determined between approximately 50 and 60%. The predicted amino acid sequences of Hsp60 showed an identity of 90 to nearly 100% among mycoplasmas and 50-60% to the other bacteria indicated above. Two Hsp60 derived glutathione-S-transferase fusion proteins containing mycoplasma peptides of 28 and 35kDa were isolated. M. hyopneumoniae-ELISA positive porcine convalescent sera reacted strongly with the recombinant Hsp60 fusion proteins in Western immunoblotting indicating for the first time that mycoplasmal Hsp60 is immunogenic in natural infection.