Increasing prevalence of Mycoplasma bovis in Danish cattle

A study on the prevalence of mycoplasmas in pneumonic bovine lungs was performed on material submitted for diagnostic purposes at the Danish Veterinary Laboratory, Copenhagen. Among the 50 examined cases 43 (86.0%) were found to be infected with mycoplasmas. The predominant mycoplasmas were Ureaplasma spp. (72.0%), M. dispar (48.0%) and M. bovis (24.0%). Other mycoplasmas were M. bovirhinis (20.0%) and M. bovigenitalium (6.0%). Among the infected lungs multiple species infections were predominant (76.7%) over single species infections (23.3%) with M. dispar-Ureaplasma (25.6%), M. bovis-Ureaplasma (18.6%) and M. dispar-M. bovirhinis-Ureaplasma (11.6%) infections being the most frequently encountered combinations. There appears to be an increasing prevalence of M. bovis (24.0%) as compared to earlier reports (0.6-2.0%), thus calling for special attention upon this mycoplasma. Pulsed field gel electrophoresis (PFGE) analysis of 11 field isolates of M. bovis from 9 different farms revealed different profiles except for 2 isolates which were recovered from the same farm. Because mycoplasmas belonging to the 'M. mycoides cluster' were not encountered during this study; it appears that the Danish cattle population is still free from this group of mycoplasma in spite of their presence in some other European countries.     

Prevalence of mycoplasmas in the respiratory tracts of pneumonic calves.

The prevalence of mycoplasmas in the respiratory tracts of 148 pneumonic calves originating from 25 herds in the Netherlands is reported. Four types of culture media were used to isolate mycoplasmas: solid modified Edward medium, 2 types of Friis media, and A7B differential agar medium. Mycoplasmas were isolated both from nasal swab specimens and lung lavage fluids collected from live calves and from nasal mucosa and lung tissue specimens collected post mortem. All of the mycoplasma strains isolated could be identified as either Ureaplasma diversum (isolated from 80% of 25 herds), Mycoplasma dispar (92%), M. bovirhinis (88%), M. bovis (20%), M. bovigenitalium (4%), M. arginini (16%), or M. canis (12%). Isolation rates of M. dispar and U. diversum were considerably higher from lung lavage fluids than from nasal swab specimens. M. bovis was detected only in fattening herds and not in dairy herds. The respiratory tracts of 75% of the calves examined contained at least 2 mycoplasma species. In total, 25 different combinations of mycoplasma species were detected in specimens collected from noses and lungs. The pathogenic species U. diversum and M. dispar had not been isolated before in the Netherlands.     

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.     

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.     

Mycoplasmas and cuffing pneumonia in a group of calves.

The mycoplasmas found in the lungs of 20 calves, housed together for six months, and the related pulmonary pathology are reported. Twelve calves had cuffing pneumonia and in this group there was a significantly higher isolation frequency of Mycoplasma dispar and Ureaplasma spp compared with the non-pneumonic group. Mycoplasma bovirhinis and Acholeplasma laidlawii were isolated from the lungs of calves in both groups. Mycoplasma arginini was not recovered from the lungs of any calf. The significance of the peribronchiolar lymphocytic accumulation in the lungs of the non-pneumonic animals and their differentiation from peribronchiolar lymphocytic cuffs is discussed.     

The nasal mycoplasmal flora of healthy calves and cows.

The nasal mycoplasmal flora of 270 healthy cows from 27 herds in the Netherlands and 35 healthy calves from 7 of these herds was examined. Various methods for isolating mycoplasmas were compared. The prevalence of the various species was as follows: Ureaplasma diversum in 3 (9%) calves; Mycoplasma dispar in 14 (40%) calves; M. bovis in 1 (3%) calf; M. bovirhinis in 23 (66%) calves and 16 (6%) cows; M. bovoculi in 8 (23%) calves and 53 (20%) cows; M. canis in 1 (3%) calf; M. equirhinis in 2 (1%) cows; M. conjunctivae in 2 (1%) cows; Acholeplasma laidlawii in 1 (3%) calf and 3 (1%) cows; and A. axanthum in 7 (3%) cows. The noses of healthy calves were less frequently colonized by the pathogenic species U. diversum and contained fewer U. diversum and M. dispar organisms than the noses of pneumonic calves. We concluded that the mycoplasmal flora of calves and healthy cows was quite different and also that cows play only a minor role in the epidemiology of pathogenic mycoplasma species of calves in the Netherlands.     

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.     

Pathological and microbiological studies on pneumonic lungs from Danish calves

During 1 year, the association between microbiological and pathological findings in 72 lungs from calves submitted to the Danish Veterinary Laboratory for diagnostic purposes was studied. All cases were evaluated pathologically and bacteriologically, whereas only 68 cases were examined for the presence of bovine respiratory syncytial virus (BRSV), parainfluenza-3 virus (PI-3 virus) and bovine coronavirus, 62 cases for bovine viral diarrhoea virus (BVD), 45 cases for bovine adenovirus and 51 cases for mycoplasmas. Based on histopathological examination, the cases were diagnosed as fibrinous and/or necrotizing bronchopneumonia, suppurative bronchopneumonia, embolic pneumonia and others. The diagnoses were based on the dominating and most severe lesions in each lung. Haemophilus somnus, Pasteurella multocida, Actinomyces pyogenes, P. haemolytica and BRSV were the most commonly found bacterial and viral lung pathogens, respectively. Pasteurella spp. and H. somnus were often associated with the more severe fibrinonecrotizing type of bronchopneumonia, whereas BRSV was primarily detected in cases of suppurative bronchopneumonia. Mycoplasma bovis was isolated from one case only, whereas M. dispar, M. bovirhinis and Ureaplasma diversum were present, often concomitantly, in the majority of cases. Aspergillus fumigatus was isolated from one case.     

Examination of cattle with respiratory diseases for Mycoplasma and bacterial bronchopneumonia agents

A total of 247 mycoplasma strains was isolated from 435 lungs, tracheobronchial secretions and nasal swabs originating from cattle with symptoms of bronchopneumonia. Mycoplasma (M.) bovis was found 89 times (36%) and was the most common mycoplasma species in the lungs. M. bovirhinis, M. bovigenitalium, M. spec. and Acholeplasma (A.) laidlawii were isolated 158 times (64%). Among these mycoplasmas M. bovirhinis was the most widespread species (114 isolations). In 55 cases (62%) M. bovis was associated with Pasteurella or Actinomyces (A.) pyogenes. The other mycoplasma species were found in 67 cases (42%) together with these bacteria. Without mycoplasmas Pasteurella and A. pyogenes occurred in 33 of the probes investigated (21%). Beside mycoplasmas Haemophilus (H.) somnus was isolated from 16 of 162 tracheobronchial secretions investigated. The results confirm earlier suppositions that in most of the cases bronchopneumonia of cattle is a multifactorial event, frequently associated with mycoplasmas--especially M. bovis.     

Examination of the role of Mycoplasma bovis in bovine pneumonia and a mathematical model for its evaluation

The authors screened 34 large cattle herds for the presence of Mycoplasma bovis infection by examining slaughtered cattle for macroscopic lung lesions, by culturing M. bovis from lung lesions and at the same time by testing sera for the presence of antibodies against M. bovis. Among the 595 cattle examined, 33.9% had pneumonic lesions, mycoplasmas were isolated from 59.9% of pneumonic lung samples, and 10.9% of sera from those animals contained antibodies to M. bovis. In 25.2% of the cases M. bovis was isolated from lungs with no macroscopic lesions. The proportion of seropositive herds was 64.7%. The average seropositivity rate of individuals was 11.3% but in certain herds it exceeded 50%. A probability model was developed for examining the relationship among the occurrence of pneumonia, the isolation of M. bovis from the lungs and the presence of M. bovis specific antibodies in sera.     

The association between serological evidence of mycoplasma infection and respiratory disease in feedlot calves.

Calves from five Ontario feedlots were bled on arrival and approximately 28 days later. Calves treated during this interval for undifferentiated respiratory disease were classified as cases and untreated calves were classified as controls. Serum was titrated blindly for antibodies to Mycoplasma bovis and Mycoplasma dispar. Indirect hemagglutination titers of 1:20 or more were assumed to reflect recent or current exposure, whereas 1:10 or less were not. The titers to M. bovis increased in all feedlots indicating active infection. The initial titers to M. dispar were higher than the titers against M. bovis, yet they increased in all feedlots except one, suggesting widespread infection with this organism. There was an increased risk (although not statistically significant) of being treated if the titer against M. bovis rose during the period. Calves with low M. dispar titers on arrival were at increased risk of being treated and titer increases were strongly associated with treatment (statistically significant). Thus, the serological results indicate high prevalence of M. bovis and M. dispar in the feedlot calves and that calves with increasing titers in particular to M. dispar are at increased risk of being treated for respiratory disease.     

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.     

Molecular and serological prevalence of Babesia bovis and Babesia bigemina in cattle from central region of Syria

A total of 207 bovine blood samples were collected from clinically healthy cattle bred in central region of Syria and examined by Giemsa-stained blood smears, nested PCR, ELISA, and IFAT to determine the molecular and serological prevalence of Babesia bovis and B. bigemina. All samples were negative to Babesia spp. by microscopic examination of blood smears. On the other hand, the overall prevalence of B. bovis and B. bigemina was 9.18% and 15.46% by nPCR, 15.46% and 18.84% by ELISA, and 18.36% and 21.74% by IFAT, respectively. Mixed infections were detected in a total of 5 samples (2.4%) by nPCR, 16 (7.73%) by ELISA and 27 (13.04%) by IFAT. Statistically significant differences in the prevalence of the two infections were observed on the basis of age and location. These data provide valuable information regarding the occurrence and epidemiology of B. bovis and B. bigemina infections in Syrian cattle, which can be employed in developing rational strategies for disease control and management.     

The effects of two Ureaplasma diversum strains on early pregnancy in heifers.

Two field isolates of Ureaplasma diversum spp. were used to infect heifers at the time of insemination in a preliminary study to observe the effect of infection on early pregnancy. M84-14c-1 was a field isolate from a bull's prepuce typed by immunofluorescence to be similar to U. diversum strain T-44 (Group C). M84-477c-4 was a field isolate from bovine semen typed by immunofluorescence to be similar to U. diversum strain T-288 (Group A). All three heifers infected with M84-477c-4 had a mild granular vulvitis at some time during the trial. None was pregnant when slaughtered 27 days after infection. The result of infection with M84-14c-1, a preputial isolate, was not consistent. One heifer had no infection and a normal pregnancy, one heifer was infected with an abnormal pregnancy, and one heifer was open but ureaplasmas were not detected until day 17 of the trial.     

Efficiency of a recombinant MSA-2c-based ELISA to establish the persistence of antibodies in cattle vaccinated with Babesia bovis

Bovine babesiosis is caused by Babesia bovis and B. bigemina in Argentina. These protozoans are prevalent north of parallel 30 degrees S, where their natural vector Rhipicephalus (Boophilus) microplus is widespread. To prevent babesiosis outbreaks in endemic areas, an increasing population of 4-10-month-old calves are vaccinated with low virulence B. bovis R1A (BboR1A) and B. bigemina S1A (BbiS1A) strains. In non-endemic areas, an additional calf population is also vaccinated and boostered as adults, before they are relocated to R. microplus-endemic areas of the country. Serological tests are currently utilized not only to determine the status of natural Babesia spp. infections, but also to confirm the infection caused by vaccine strains. For this purpose, an indirect enzyme immunoassay (ELISA) based on the recombinant major surface antigen-2c (rMSA-2c) of B. bovis expressed in Escherichia coli, was standardized using sera from Babesia spp. experimentally infected cattle. ELISA(rMSA-2c) was validated using sera obtained weekly during 336 days from steers primed and boostered with BboR1A and/or BbiS1A on days 0 and 154, then compared with the immunofluorescent-antibody test (IFAT). Western blot (WB) protein analysis was used to confirm the specificity of the immune response to rMSA-2c. The sensitivity and specificity for ELISA(rMSA-2c) were 92 and 96% after the Babesia spp. priming and 88 and 73% after the boostering immunization, respectively. The sensitivity and specificity for IFAT were 99 and 90% after priming and 92 and 98% after boostering, respectively. Unlike IFAT, ELISA(rMSA-2c) detected a remarkable delayed booster response and a significant drop in specificity between 35 and 84 days after the booster immunization. Simultaneously, 87.5% of cattle boostered with B. bigemina showed cross-reactions in the ELISA(rMSA-2c), particularly between 63 and 77 days after the inoculation. A reaction against E. coli was observed, since bands of approximately 40 and/or 42kDa were detected using sera from cattle before and after Babesia spp. inoculations. ELISA(rMSA-2c) showed to be useful between 42 and 98 days after priming with Babesia spp. live vaccine to evaluate the success of infecting cattle. However, after boostering the test showed low specificity.     

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.     

ECOLOGY OF MYCOPLASMAS IN CLINICALLY HEALTHY CATS

Mycoplasmas were isolated from various sites and organs of a series of 319 clinically healthy cats. They include M. felis, M. gateae, M. Arginini, A. laidlawii, feline ureaplasmas, M. pulmonis, M. arthritidis, and M. gallisepticum. In addition, there were 10 strains of mycoplasmas which could not be identified with the specific antiserums by growth inhibition tests. Antibody against M. felis was demonstrated by haemagglutination-inhibition and complement-fixation tests in cats which were over 6 months of age. However, no such antibody against M. felis was detected in animals which were less than 6 months old. No antibody against A. laidlawii, M. gateae, M. arginini and feline ureaplasmas was demonstrated by the same serological methods. The significance of these mycoplasmas in cats is described.     

Cross-reactive proteins among eight bovine mycoplasmas detected by monoclonal antibodies

Whole cell proteins of eight bovine mycoplasmas (M. bovoculi, M. bovis, M. dispar, M. bovirhinis, M. arginini, M. verecundum, M. canadense, M. alkalescens) were separated by SDS-PAGE and transferred to nitrocellulose paper. Rabbit anti-M. bovoculi serum was found to react with immunoblots of all mycoplasma species tested. These cross-reactive proteins were in the range of 35,000-100,000 molecular weight. Monoclonal antibody MA25.5 developed against a M. bovoculi 94 kDa surface protein cross-reacted with a band of 62 kDa from M. dispar and three bands of 89, 85 and 74 kDa from M. arginini only while MA18.13 that recognized a band of 57 kDa from M. bovoculi did not react with the other species. The role of MA25.5 monoclonal antibody in inhibiting the growth of M. bovoculi, M. dispar and M. arginini was tested using the metabolic-inhibition (MI) test. Monoclonal antibody MA25.5 inhibited the growth of M. bovoculi and also inhibited M. dispar growth but at lower MI titers, while it showed no effect on the growth of M. 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.     

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).