Detection certainty of the contamination of bull sperm with mycoplasma

Tests for presence of mycoplasmas were conducted on 20 insemination bulls known as mycoplasma spreaders, with 5 sperm pellet batches of 20 pellets each being investigated for each animal. Mycoplasma contamination was positively recorded from 83 of the above 100 batches. Mycoplasma (M.) bovigenitalium, M. californicum. M. arginini, M. bovirhinis, and Acholeplasma laidlawii were typed by means of indirect immunofluorescent technique, which confirmed the presence also in the GDR of mycoplasma species described in the literature and detected in sperm samples. The solid culturing media used in the above tests, medium-B agar and cattle blood agar with staphylococcal nutrix, proved to be equally suitable for isolation of mycoplasma from sperm samples. Mycoplasma was positively identified in about one third of all pellets/batches tested. 3 pellets to one batch should be sufficient a random sample size from which to obtain information at least very close to real contamination.     

The indirect hemagglutination test for the detection of antibodies in cattle naturally infected mycoplasmas.

Stable mycoplasma antigens for the indirect hemagglutination test (IHA) were prepared employing glutaraldehyde treated sheep erythrocytes sensitized with Mycoplasma agalactiae subsp. bovis and Mycoplasma bovigenitalium antigens. Employing these antigens mycoplasma antibodies were detected in sera from cattle which had mastitic symptoms due to natural infection with either M. agalactiae subsp. bovis or M. bovigenitalium. A total of 200 cows from four herds were examined at varying intervals for the presence of M. agalactiae subsp. bovis and for the detection of antibody using growth inhibition and IHA tests. Mycoplasmas were isolated from 37 animals. Growth inhibiting antibody was detected from 56 of the 200 animals. In the IHA tests, antibody titer greater than or equal to 1:80 were detected in 148 animals, 76 of these having antibody titers greater than or equal to 1:160, while sera of 116 normal control animals had no growth inhibiting antibody and none had IHA antibody titers greater than 1:40. M. bovigenitalium was isolated from the milk of three of 26 animals in a fifth herd during an outbreak of mastitis. Growth inhibiting antibodies were demonstrated in the sera of ten of the 26 animals. However, the IHA test detected antibody titers of greater than or equal to 1:160 in 13 animals and of 1:80 in one of the 26 animals. To determine the specificity of the IHA tests, M. agalactiae subsp. bovis and M. bovigenitalium antigens were reacted with rabbit hyperimmune typing sera produced against 12 species of bovine mycoplasmatales. Homologous antisera showed IHA antibody titers of 1:1280 and 1:2560 against M. agalactiae subsp. bovis and M. bovigenitalium respectively, whereas heterologous antisera showed IHA antibody titers of less than or equal to 1:20. Also eight type-specific bovine antisera were reacted with M agalactiae subsp. bovis and M. bovigenitalium antigens in homologous and heterologous tests. Homoogous reactions showed IHA antibody titers greater than or equal to 1:320, whereas heterologous reactions showed IHA titers of less than or equal to 1:20. This IHA test promises to be useful for the detection of bovine mycoplasma antibodies in sera from cattle infected with M. agalactiae subsp. bovis or M. bovigenitalium. Thes test is sensitive, reproducible and specific and the technique is relatively simple and rapid. The antigens were stable for at least seven months.     

Prevalence of Chlamydiaceae and Mollicutes on the genital mucosa and serological findings in dairy cattle

It was the aim of this project to obtain information on the prevalence of Chlamydiaceae and Mollicutes and their potential importance for reproductive problems in cattle. Cervical or vaginal swabs were taken from 644 animals in 196 farms and blood samples were collected from 375 cattle. Out of the animals, 6.8% had aborted within the last 12 months, 2.6% showed clinical vaginitis and 11.6% clinical endometritis. Chlamydiaceae were detected and identified by PCR followed by restriction fragment length polymorphism (RFLP) analysis. For the detection and identification of Mollicutes cultivation procedures, biochemical differentiation and serological identification were used. Sera were tested for antibodies against Chlamydiaceae and Mycoplasma (M.) bovis by ELISA and against M. bovigenitalium by Western blot analysis. Chlamydophila (Cp.) abortus was found in three cervical swabs. Cp. pecorum was detected in 9% of cervical or vaginal swabs. The majority of Cp. species found was Cp. pecorum and thus fertility problems caused by Cp. abortus are limited. M. bovis was found in only one genital swab. M. bovigenitalium was rarely diagnosed (3% of cervical and 2% of vaginal swabs). M. bovigenitalium was found more often in cattle having aborted (4/32 animals) than in cattle without history of abortion (5/220, p<0.05). Ureaplasma (U.) diversum existed in 12% of cervical and 36% of vaginal swabs and was found in 8 out of 17 animals with vaginitis. Out of the animals tested, 44.9% were seropositive for Chlamydiaceae, 14.8% for M. bovis and 27.3% for M. bovigenitalium.     

Outbreak of pneumonia and arthritis in beef calves associated with Mycoplasma bovis and Mycoplasma californicum

During an outbreak of pneumonia and arthritis in beef calves and young cattle on a large farm in north-west Germany, Mycoplasma bovis and Mycoplasma californicum were isolated from tracheobronchial lavage fluids and synovial fluids. The microbiological findings in dead and living animals and the immunohistochemical demonstration of M californicum antigen in lung and arthritic joint tissue, indicated that under poor housing conditions and possibly other predisposing factors, this mycoplasma, like M bovis, can colonise the respiratory tract and may be able to cross the respiratory mucosal barrier to spread through an infected animal and cause systemic infections that may contribute to severe arthritis.     

Experimental infections of bulls with Mycoplasma (M.) bovis and M. bovigenitalium

M. bovis or M. bovigenitalium species experimentally used in intrapreputial infection were re-isolated from 7 of 8 bulls. Clinically manifest diseases did not develop at all, though slight inflammatory lesions were recorded from the genital tract of 2 animals. Mycoplasma findings are discussed together with results obtained from spermatological and hematological investigations.     

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.     

牛支原体pdhc基因的克隆、表达及其亚细胞定位

试验旨在研究牛支原体（Mycoplasma bovis,M.bovis）武威株二氢硫辛酰胺转乙酰酶（PDHc-E2）基因序列特征及其在牛支原体细胞中的位置。参照GenBank中牛支原体HB0801株pdhc基因（登录号：CP002058.1）设计引物,应用PCR扩增获得牛支原体武威株pdhc基因,在测序及序列分析的基础上,应用Overlap PCR完成点突变后将其克隆至pET-28a（+）中,构建原核表达载体pET-pdhc。pET-pdhc转化大肠杆菌Rosetta（DE3）感受态细胞后经IPTG诱导获得融合蛋白,将纯化蛋白免疫新西兰兔制备多抗血清,应用iELISA和Western blotting对牛支原体武威株PDHc-E2在细胞内的分布进行初步研究。结果显示,牛支原体武威株pdhc基因CDS全长735 bp,编码244个氨基酸,与国内牛支原体分离株HB0801、Hubei-1、CQ-W70、NM2012等基因序列完全一致,与国际标准株PG45同源性为99.2%,与无乳支原体（M.agalactiae）同源性为90.9%~91.2%,与加利福尼亚支原体（M.californicum）ST6株的同源性仅为78.4%,基因序列非常保守;通过Overlap PCR将该基因中4个编码色氨酸的TGA密码子突变为TGG,且完成点突变后的基因在大肠杆菌中成功表达,重组蛋白大小约为29 ku,主要以可溶性形式存在,iELISA结果显示,重组蛋白PDHc-E2具有较高的免疫原性,可刺激新西兰兔产生高水平的抗体,血清效价高达1：100 000;亚细胞定位结果表明,制备的多抗血清与重组蛋白PDHc-E2、牛支原体全菌蛋白、牛支原体膜蛋白、牛支原体胞浆蛋白均能发生特异性结合,说明该蛋白在牛支原体细胞膜和细胞质中均有分布,为膜相关蛋白,但在细胞质中的分布多于细胞膜。本研究结果为进一步研究牛支原体的生物学功能提供了理论依据。     

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.     

The "in vitro" effect of antibiotics on mycoplasmas under conditions of the preparation of bull sperm

Bull sperm was experimentally contaminated with Mycoplasma (M.) bovis, M. bovigenitalium, and Acholeplasma laidlawii (10(5) colony-forming units/ml sperm). Antibiotics were tested for their effectiveness under conditions of preparation of sperm for artificial insemination, primarily for their capability of killing all mycoplasma by the end of sperm equilibration (cooling to +4 degrees C within 5 h). Mycoplasma decontamination was achievable only by means of nourseothricin and lincospectin, in a concentration of 0.8 mg/ml sperm diluent which did not yet impair bull sperm vitality.     

牛支原体肺炎流行RT-PCR方法的初步诊断

设计5对引物,对山东某一牛交易市场采集的64份鼻腔棉拭子样品进行了病原学检测。对所有样品进行差异脲原体、牛支原体、牛生殖道支原体、牛眼支原体和牛传染性胸膜肺炎(CBPP)PCR病原学检测,PCR产物连接T载体测序。PCR结果显示,牛支原体阳性样品7份,牛生殖道支原体、牛眼支原体和牛丝状支原体全部为阴性;测序结果显示,所测样品核酸序列与牛支原体和无乳支原体核酸序列都具有很高的同源性。对核酸序列进行遗传进化树分析表明,相对于无乳支原体而言,所测核酸序列与牛支原体物种具有更近的遗传距离。     

Studies of bovine mycoplasma mastitis. 1. Review of literature on the occurrence of bovine mastitis with Mycoplasma involvement]

The clinical pattern as well as the pathologico-anatomic or histological changes due to mycoplasma mastitis are neither specific nor pathognomic. Mastitis pathogens so far described included M. bovis, M. bovigenitalium, A. laidlawii, A. axanthum, M. alkalescens, M. canadense, M. dispar, M. bovirhinis, strains of Group 7 according to Leach, strain ST 6, and ureaplasma strains. In the GDR, enzootic mastitis has been confined to A. laidlawii and A. axanthum.     

Comparative studies of the persistence of animal mycoplasmas under different environmental conditions

A comparison of the persistence of mycoplasmas in animals was carried out. When inoculated into liquid media, strains of Mycoplasma bovis, M. arginini, Acholeplasma laidlawii, and A. axanthum persisted for 59-185 days post-inoculation. The survival periods were not significantly influenced by temperature (4, 30, 37 degrees C, and room temperature). The survival periods for M. bovigenitalium, M. gallisepticum, M. bovirhinis, and M. gateae ranged from <7 to 185 days depending on medium components and temperature. Further, it was determined that strains of M. bovigenitalium, M. bovis, M. bovirhinis, M. arginini, and A. laidlawii persisted in a dry paper disc for at most 28, 126, 154, 56 and over >168 days at 4 degrees C, respectively. At 4 degrees C, strains of M. gallisepticum, M. columborale, M. edwardii, M. felis, and M. gateae survived for at most 28, 21, 42, 28, 28 and 70 days, respectively. At 30 degrees C, strains of M. bovis, M. bovirhinis, M. arginini, A. laidlawii, and M. gallisepticum persisted for at most 28, 84, 56, >168 and 14 days, respectively, but strains of M. gallisepticum, M. columborale, M. edwardii, M. felis, M. gateae, and U. diversum did not survive for more than 14 days. In an outdoor environment, strains of M. bovirhinis and A. laidlawii survived for at most 28 and 14 days, respectively. Finally, it was found that 14 isolates of M. gallisepticum persisted for periods similar to those of the reference strains. The results under dry conditions at a variety of temperatures presented contribute to understanding the epizootiology of mycoplasmal infections in the field.     

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.     

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.     

Incidence of mycoplasma mastitis in Czechoslovakia

By the mycoplasmic examination of 143 dairy cows exhibiting clinical signs of mastitis, Mycoplasma californicum and M. bovigenitalium were detected as the causal agents of the disease at two localities. The occurrence of specific antibodies in milk, and/or blood serum, was characteristic of the course of the disease, as documented by the results of the examination of more than 300 samples. In some milk samples taken from cows suffering from mastitis of obscure etiology and obtained from State Veterinary Institutes, antibodies to some of the five species of bovine Mycoplasma involved in the disease were found at 22 localities. The antibodies were detected by means of the indirect haemagglutination test and ELISA method. Both these tests are suitable for such an examination, as indicated by the comparison of the results.     

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.     

The course of mastitides in cows infected into the mammary gland with strains of Mycoplasma bovigenitalium and M. bovis]

Six cows were inoculated into the mammary gland with eight mycoplasma strains isolated from the genital tract of bulls and two type strains. The milk of cows infected with Mycoplasma bovigenitalium strains isolated from the genital tracts of bulls showed a change in the appearance and contained large quantities of mycoplasmas and specific antibodies. The mastitis was most intense in about 9 days and began to subside in 17 days infection. The type strain of M. bovigenitalium PG11 failed to produce mastitis. On the other hand, the type strain of M. bovis PG45 produced severe mastitis after a 14-day latency period, with the infection spreading to the uninoculated quarters, causing atrophy of the mammary gland, and persisting till slaughter. The sera of all cows that developed mastitis after experimental infection contained high titres of specific antibodies. The two infecting mycoplasma species were recovered from the inner organs and mammary glands of these cows after slaughter.     

Mycoplasmas: use of polyvalent antisera for identification by indirect immunofluorescence.

It would be an advantage, under many circumstances, to be able to make use of polyvalent antisera in the process of identifying mycoplasmas. As the indirect immunofluorescence test is sufficiently sensitive and also generally accepted as being rather specific, this technique was chosen to investigate whether polyvalent antisera are applicable in routine identification of mycoplasmas. Three polyvalent sera were used, each consisting of 9 or 10 rabbit antisera raised against 29 of the more common species of the genus Mycoplasma. Twenty-six field strains were examined. One strain did not react with any of the 3 polyvalent antisera although it was later identified as M. bovigenitalium. The remaining 25 strains reacted with 1 and only 1 of the polyvalent antisera and were subsequently identified by immunofluorescence utilizing monospecific antisera. Strains of the following species were identified: M. arginini, M. bovigenitalium, M. bovis, M. bovoculi, M. canis, M. capricolum, M. cynos, M. edwardii, M. hominis, M. hyorhinis, M. molare, M. mycoides subsp. mycoides and M. opalescens. It is concluded that polyvalent antisera may be used in identification procedures and thereby permit the use of a limited number of monospecific antisera without preceding biochemical testing.     

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.     

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.