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.     

Investigation of survival time of some poultry mycoplasmas

Glucose-fermenting poultry mycoplasmas (Mycoplasma [M.] gallisepticum, M. pullorum, M. gallinaceum, M. gallopavonis) were tested in 2 experiments for their survival time at 20 degrees C and 37 degrees C on 18 different materials used on farms and in hatcheries. All mycoplasmas survived up to 16 days in egg yolk at both temperatures. On other materials, like egg shell, egg white, paper trails, feather, and others mycoplasmas generally survived 2 to 16 days at 20 degrees C. M. gallinaceum and M. gallopavonis proved more resistant to the environment than M. gallisepticum and M. pullorum.     

Mycoplasmas and acholeplasmas isolated from ducks and their possible association with pasteurellas

Two hundred and sixty-three cases of clinically diseased ducks of all ages were examined for the presence of mycoplasmas. Mycoplasmas and acholeplasmas belonging to more than eight serogroups were cultured from 68 of them, and comprised 12 M anatis, one M columbinasale, two M gallinaceum, two M gallinarum, nine M synoviae, three unidentified Mycoplasma species, 37 Acholeplasma laidlawii and one unclassified acholeplasma belonging to each of serogroups 7 and 8. They were identified by biochemical characterisation, disc growth inhibition and agar gel diffusion tests. Fifty-three (78 per cent) of the isolates occurred with species of Pasteurella: 33.8 per cent with Pasteurella anatipestifer, 32.4 per cent with P multocida and 11.8 per cent with both P anatipestifer and P multocida. Nine of the isolates (13.2 per cent) were in pure culture and six (8.8 per cent) with other agents. Of the ducks negative for mycoplasmas 33.3 per cent were infected with P anatipestifer, 25.1 per cent with P multocida and 14.4 per cent with both P anatipestifer and P multocida. There was no correlation between the infections with mycoplasmas and P anatipestifer but there was a weak association between the infections with mycoplasmas, especially M anatis and P multocida.     

Detection of mycoplasmas in eye swab specimens of cattle

In the present investigation 262 conjunctival swabs were taken from 178 cattle and examined for mycoplasmas. The isolation was possible from 111 swabs. Mycoplasmas were found in eyes with clinical symptoms of IBK (in 64 of 148 swabs investigated = 43.2%) as well as in healthy eyes (in 47 of 114 swabs investigated = 41.2%). Consequently a correlation between clinical findings and isolation of mycoplasmas could not be observed. Unfortunately 60 of 111 isolates could not be subcultivated after storage at -20 degrees C. Using the indirect immunofluorescence test 41 of the 42 surviving isolates were identified as M. bovoculi which before has not been isolated in the Federal Republic of Germany. One isolate was determined as A. laidlawii. The 17 M. bovoculi strains investigated for their biochemical reactions showed the same characteristics like the reference strain M. bovoculi M 165/69. In repeating examinations mycoplasmas could be isolated 5 times after one month and 14 times after 6 months. Cattle younger than 2 years were more often infected with mycoplasmas (62.5%) than older animals (19.4%). No difference, however, could be observed in the clinical manifestations of IBK between younger and older animals. Mycoplasmas were more frequently isolated in autumn (43.6%) than in spring (21.4%) and summer (29.3%). In most of the animals examined both eyes were colonized by mycoplasmas. No spiroplasmas could be detected in the 262 conjunctival swabs investigated.     

Specificity of oligonucleotide probes complementary to evolutionarily variable regions of 16S rRNA from Mycoplasma hyopneumoniae and Mycoplasma hyorhinis.

Mycoplasma is the common name for the smallest free-living microorganisms, the Mollicutes. Mycoplasma hyopneumoniae is of great importance in veterinary medicine, causing enzootic pneumonia in pigs. M hyorhinis can cause polyserositis and may cause pneumonia in piglets. Oligonucleotides complementary to variable regions of 16S rRNA from these mycoplasmas were designed and used as probes for detection and identification of these mycoplasmas. The probe complementary to 16S rRNA of M hyorhinis gave a very weak cross-hybridisation with M hyosynoviae in filter hybridisation experiments, but not with any of the other porcine mycoplasmas tested. Three oligonucleotide probes complementary to M hyopneumoniae 16S rRNA were tested. One of the probes (Mhp6/30) was found to be specific to M hyopneumoniae, but the other two gave cross-hybridisation with M flocculare. Using the Mhp6/30 probe in direct filter hybridisation experiments, it proved possible to detect M hyopneumoniae in lung biopsies from experimentally infected pigs.     

Isolation and identification of avian mycoplasmas in Singapore.

Two hundred and forty batches of chickens with chronic respiratory syndrome were tested for mycoplasmas. One hundred and five batches (43.8%) were found to have mycoplasmosis. A total of 110 isolates of mycoplasma was cultured, of which nine isolates were identified as Mycoplasma gallisepticum, 48 avian sero-group D, 45 M. gallinarum, one M. iners and seven unclassified. 2. Identification of the mycoplasmas isolated was carried out by biochemical and serological tests (disc growth inhibition and agar gel diffusion tests). A modified agar gel diffusion test using solubilised antigens with sodium deoxycholate-glucose solution was developed for serotying of mycoplasmas.     

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.     

Mycoplasmas and mites in the ears of clinically normal goats

SUMMARY Mycoplasmas were detected in the external ear canal of goats by swabbing and culture. Up to 10⁸ colony forming units were recovered from single swabs. The resulting cultures were usually mixtures of mycoplasmas containing up to 5 species. The species present in sequential swabs varied. Pathogenic species (M.agalactiae, M.capricolum, M.mycoides subsp. capri, M.mycoides subsp. mycoides of the large colony (LC) type, M.putrefaciens) were isolated from the ears and in addition 3 untyped mycoplasmas G, U and V were often present. The same mycoplasmas were found in large numbers in the mites Psoroptes cuniculi and Raillietla caprae which were sometimes present in the external ear canal. The role of the mycoplasmas in the external ear canal as a source of infection and disease and of the mites in the spread of infection requires further elucidation.     

Microbiological and serological studies on caprine pneumonias in Oman

Eight of 10 typical cases of contagious caprine pleuro-pneumonia in Oman yielded strain F38-like mycoplasmas from the lungs in high titre, but no other mycoplasmas: both negative animals had been treated with tylosin shortly before death. Among 21 other lungs examined three of six cases of acute pneumonia yielded Mycoplasma ovipneumoniae; one also yielded M capricolum. M ovipneumoniae was also isolated from all eight cases of chronic pneumonia sampled from an abattoir, and from the lungs of three animals which died without overt signs of pneumonia. A single isolate of M arginini and three of unidentified mycoplasmas were also obtained from goats with and without pneumonia. Various bacterial species were isolated, none of which predominated. Antibodies to M mycoides subspecies capri (M m capri) and strain F38 were detected in sera from eight different sources. Assuming titres of 1 in 40 or more as positive in the indirect haemagglutination test used, 29 per cent of 422 serum samples had antibodies to M m capri alone, 2.6 per cent to strain F38 alone and 3.6 per cent to both organisms. These results confirm the presence of F38-like mycoplasmas in Oman, and indicate also widespread infection with M m capri. The role of the latter in caprine pneumonias in Oman requires elucidation.     

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.     

Examination of the 16S-23S rRNA intergenic spacer sequences of 'Candidatus Mycoplasma haemobos' and Mycoplasma haemofelis

The intergenic spacer region between the 16S and 23S rRNA genes of mycoplasmas has been used for a genetic marker for identification of the species. Here we show the intergenic spacer regions of two hemotropic mycoplasmas, Mycoplasma haemofelis and 'Candidatus Mycoplasma haemobos (synonym: 'C. M. haemobovis')' are also useful for classification of this particular group of mycoplasms. The spacer region of M. haemofelis and `C. M. haemobos' consisted of 209 and 210 base pairs, respectively, and both lacked the spacer tRNA genes. Phylogenetic analysis suggested a monophyletic relationship among hemoplasmas and M. fastidiosum. A hypothetical secondary structure predicted in the spacer regions tentatively assigned the boxA and boxB motifs peculiar to the members of the genus Mycoplasma. M. haemofelis and 'C. M. haemobos' possessed a stem-loop structure in common, despite the presence of a palindromic nucleotide substitution in the stem region.     

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.     

Detection of mycoplasma in mastitic milk by PCR analysis and culture method.

Mycoplasma alkalescens, M. bovigenitalium, M. bovirhinis and M. bovis were directly detected from milk specimens by a polymerase chain reaction (PCR) when milk specimens were centrifuged and treated with mycoplasmal lysis buffer. The sensitivity of this PCR method was 110 to 1,400 colony forming units (CFU). This method was useful for the detection of mycoplasmas in milk specimens from cows at an early stage of mycoplasmal mastitis since a small amount of mycoplasma could be detect in milk without culture. The results were available within 12 hr, which is faster than conventional culture techniques. M. bovirhinis was detected in more than 70% of mastitic milk specimens when mycoplasmas were detected in milk specimens from 30 cows with mastitis by this PCR method.     

抗猪支原体共同抗原单克隆抗体的制备与鉴定

以猪肺炎支原体（Mycoplasma hyopneumoniae,Mhp)168菌株F332作为抗原，免疫8周龄BALB/c小鼠，利用淋巴细胞杂交瘤技术，获得两株能稳定分泌特异单克隆抗体的杂交瘤细胞株。两株单抗与Mhp和猪鼻支原体（M.hyorhinis,Mh)等反应，而不与鸡支原体，对照血清等反应。结果表明两株单抗可能是针对猪支原体共同抗原的单抗，用SDS-PAGE电泳和Western-blot分析猪支原体的膜蛋白成分。结果显示，猪支原体的共同抗原是80kd和30kd蛋白，两株单抗均与Mhp和Mh的30kd蛋白条带反应，表明这两株单抗特异地针对猪支原体的共同抗原成分30kd蛋白，可以看出，这两株单抗在Mhp的抗原分析，血清学诊断和疫苗质量监测以及猪源支原体污染细胞检测等方面有重要应用价值。     

Comparison of Sampling Procedures for Isolating Pulmonary Mycoplasmas in Cattle

Three sampling procedures were compared to determine the optimal technique for isolating mycoplasmas in cattle with respiratory diseases. The prevalence of mycoplasmas isolated from these animals is also reported. In the first group, bronchoalveolar lavage (BAL) and nasal swab cultures were compared with the corresponding lung cultures from cattle necropsied for fatal respiratory diseases (n = 20). In a second group, nasal swabs were compared with corresponding BAL cultures in living animals with recurrent respiratory pathologies (n = 49). There was complete agreement between the paired BAL and lung cultures. In contrast, nasal cultures were not representative of the mycoplasmas present in the lower respiratory airways. The relative sensitivity and specificity of the nasal swab technique compared to BAL in living animals confirmed that the nasal swab cultures were not predictive of lower respiratory airway pathogens, such as Mycoplasma bovis. BAL is considered to be the best method for isolating M. bovis in cattle with respiratory diseases as it combines reliability and feasibility under field sampling conditions. In the present study, Mycoplasma dispar (43%) and M. bovis (29%) were mainly isolated in mixed infections. This confirms the need to search for mycoplasmas in routine examinations and to take them into account in therapeutic strategies for respiratory diseases in cattle.     

Isolation and identification of mycoplasmas from the nasal cavity of sheep

Mycoplasmas isolated from the nasal cavity of sheep in a ram test station were examined to determine their identity and prevalence. Specimens were obtained for mycoplasmal culture in 1980, 1982, and 1983 from 558 sheep, and mycoplasmas were isolated from 630 specimens from 320 sheep (57.3%). The isolates were characterized and differentiated into groups on the basis of sensitivity to digitonin, fermentation of glucose, and hydrolysis of arginine. Isolates in some groups were further characterized by use of additional diagnostic media, and their identity was confirmed by agglutination or growth inhibition with antiserum prepared from reference mycoplasmas. Of the 320 sheep with mycoplasmas, 293 had Mycoplasma ovipneumoniae, 12 had M arginini, and 1 had M capricolum. Two sheep had Acholeplasma spp, and 3 sheep had unidentified Mycoplasma spp. The remaining 9 sheep had M ovipneumoniae in combination with Acholeplasma spp (n = 3), M arginini (n = 3), M capricolum (n = 2), and an unidentified Mycoplasma spp (n = 1). The biochemical reactions of the M ovipneumoniae from the 293 sheep were similar, but varied in the degree of growth and fermentation in the basal medium containing glucose. The high prevalence of M ovipneumoniae indicated that it may be commensal in the upper respiratory tract of healthy sheep.     

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.     

In vitro activity of tiamulin against porcine mycoplasmas

The activity of tiamulin against Mycoplasma hyopneumoniae, M flocculare, M hyorhinis and M hyosynoviae grown in liquid medium was assessed in vitro. With the first three of these mycoplasmas, the activity of tylosin and oxytetracycline was observed in parallel. Tiamulin was more active against M hyopneumoniae and M flocculare, but there was less disparity between the three antibiotics with the strain of M hyorhinis tested. Tiamulin was notably more active against M hyosynoviae than against M hyopneumoniae. It was more difficult to suppress M hyopneumoniae than the other mycoplasmas with tiamulin. This persistence of M hyopneumoniae was more striking when M hyopneumoniae and M hyosynoviae were tested in parallel.     

Anatomic location of Mycoplasma mycoides subsp. capri and Mycoplasma agalactiae in naturally infected goat male auricular carriers

This study sought to determine whether male goat auricular carriers of mycoplasmas known to cause contagious agalactia could harbour these microorganisms at anatomical sites other than the ears. A microbiological study was conducted in 6 naturally infected bucks that had been diagnosed as chronic auricular asymptomatic carriers of Mycoplasma (M.) mycoides subsp. capri (Mmc) more than one year previously. To detect mycoplasmas, cultures and PCR were performed on 46 samples taken from each goat from the cardio-respiratory, digestive, nervous, lymph and genitourinary systems and several joints. Of a total of 274 samples analyzed, 28 were positive for mycoplasmas (10.1%): Mmc was detected in 17 (6.1%), Mycoplasma (M.) agalactiae in 12 (4.3%) and both microorganisms were identified in one of the samples. In all 6 goats, mixed infection was observed despite none being auricular carriers of M. agalactiae. Mycoplasma spp. were identified at 15 different sites; the most frequent sites being the joints (31.2%, 5 positive samples), lymph nodes (25%, 4 positive samples) and respiratory tract (25%, 4 positive samples). Positive results were also obtained in three brain tissue (18.7%), two cardiac tissue (12.5%) and one ileum, urethra, testicle and bulbourethral gland (6.25%) samples. The histopathological findings may suggest the presence of mild chronic conditions in some of the organs where the bacteria were found. Our findings reveal for the first time the capacity of Mmc and M. agalactiae to colonize several other organ systems in chronically naturally infected auricular carriers, possibly representing an added risk factor for the spread of these microorganisms. In the case of M. agalactiae, colonization seemed to be independent of the animal's auricular carrier state.     

The inactivation of mycoplasmas and bacteria in calf serum by 60Co gamma rays

Reported in this paper is the use of 60Co gamma radiation to inactivate mycoplasmas in calf serum, newborn calf serum, and fetal calf serum. A dose of 3 kGy, independent of dose rate, was found to be sufficient for inactivation in the above sera of several mycoplasmas, including Acholeplasma laidlawii, Mycoplasma (M.) orale, M. arginini, M. hyorhinis, and M. bovis. The critical dose proved to be at 2 kGy. No difference was found to exist between the above species in susceptibility to irradiation in diluted sera (50%) and 10% in Eagle MEM). Sensibility of wild mycoplasma strains was found to be identical with that of laboratory strains. Hence, 60Co gamma irradiation of sera appears to be a safe method by which to make sera mycoplasma-free. Bacillus subtilis in calf serum was inactivated by doses above 18 kGy, with the critical dose being 15 kGy.