Intraspecific variation in the 16S rRNA gene sequences of Mycoplasma agalactiae and Mycoplasma bovis strains

Intraspecific variation in the 16S rRNA genes of 17 Mycoplasma agalactiae and eight Mycoplasma bovis isolates was investigated to determine the degree of sequence variation in these two species and to determine whether the polymorphisms in the 16S rRNA genes could be used for the construction of an evolutionary tree and as epidemiological markers. A high degree of variation was found within isolates (between operons) and between isolates of both species. In contrast to M. capripneumoniae no distinct evolutionary pattern could be seen, probably because there are functional systems for gene conversion in M. agalactiae and M. bovis. However, the non-European isolates of M. agalactiae shared three characteristic nucleotides and European isolates from the same or neighbouring countries were very similar. Differences within isolates included both polymorphic positions and sequence length differences between operons. The amount of variation within isolates of the respective species ranged from zero to seven polymorphisms for M. agalactiae and from zero to four polymorphisms for M. bovis. The high degree of variation suggests the potential for misdiagnosis of species in diagnostic PCR assays based on the 16S rRNA gene sequences. All isolates of both species had a thymidine in position 912 (E. coli numbering) that causes streptomycin resistance in several bacterial species and which is characteristic for the members of the hominis group. As expected, when five M. agalactiae and three M. bovis isolates were tested for streptomycin susceptibility, they all demonstrated streptomycin resistance. M. agalactiae and M. bovis were found to have high intraspecific variation in their 16S rRNA gene and the polymorphisms patterns indicate that gene conversion takes place.     

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.     

Evaluation of a PCR assay for identification and differentiation of Campylobacter fetus subspecies

Objective To evaluate a polymerase chain reaction assay for identification of Campylobacter fetus and differentiation of the defined subspecies.
Design Characterisation of bacterial strains by traditional phenotyping, polymerase chain reaction, a probabilistic identification scheme and macrorestriction profiling using pulsed field gel electrophoresis.
Procedure The results of identification of 99 bacterial strains as determined by conventional phenotyping or by poly-merase chain reaction were compared. Two of these were type strains of C fetus subsp fetus and C fetus subsp venerealis ; the remaining strains were field isolates putatively identified as C fetus . In cases where the subspecies identity was disputed, isolates were identified by means of a probabilistic identification scheme and by macrorestriction profiling.
Results The agreement between strain identities initially suggested by traditional phenotypic methods and the PCR assay was found to be 80.8%. The polymerase chain reaction proved to be a reliable technique for the species and subspecies identification of C fetus ; equivocal results were obtained in only two instances. Initial misidentifications by conventional phenotyping methods were attributed to methodological differences used in various laboratories.
Conclusion Our results indicate that misidentification of C fetus i n routine diagnostic laboratories may be relatively common. The PCR assay evaluated gave rapid and reproducible results and is thus a valuable adjunctive method for the identification of C fetus and subsequent subspecies differentiation.     

Development of real-time PCR assay for the detection of Mycoplasma bovis

Mycoplasma bovis is one of the important bovine mycoplasma involved in economically important clinical conditions like respiratory diseases, otitis media, and mastitis. The present study was undertaken with the objective of developing a SYBR Green dye-based real-time PCR assay targeting uvrC gene for the diagnosis of M. bovis. The analytical sensitivity and specificity of the assay were evaluated. The test showed 10³-fold more sensitivity than conventional PCR and detected down to 100 fg level of DNA. It was found to be specific, as no cross reactivity was shown with other related bacteria and Mycoplasma species. The developed assay was able to detect down to 40 copies of uvrC gene from spiked bovine milk samples. At present, this developed assay may be used as a valuable diagnostic tool for the detection of Mycoplasma bovis.

     

Hydrogen Peroxide Production by Mycoplasma bovis and Mycoplasma agalactiae and Effect of In Vitro Passage on a Mycoplasma bovis Strain Producing High Levels of H2O2

Hydrogen peroxide (H2O2) production and oxygen uptake during the oxidation of NADH and L-alpha-glycerophosphate (GP) by lysed cells was determined for the type and field strains of Mycoplasma bovis and M. agalactiae. NADH oxidation by all the strains showed variable production of H2O2 ranging from 0 to 1.21 mol/mol O2 taken up. All strains were unable to oxidize GP, showing absence of GP oxidase activity. Some strains were identified that produced relatively high levels of H2O2 (> 1.0 mol/ mol O2 taken up). In vitro passage of M. bovis strain 119B96 showed reduced H2O2 production: 0.52, 0.16, and 0.07 mol/mol O2 taken up after the 50th, 100th and 200th passages, respectively. SDS-PAGE analysis showed the loss of a protein band of 32 kDa after 50 passages. These preliminary studies show that not only does H2O2 production by potentially pathogenic Mycoplasma spp. vary in the field but also that similar alterations can be induced by passage in culture. In the latter case, at least in one M. bovis strain, this alteration has been shown by SDS-PAGE to be associated with a loss of specific protein production. Further study of these phenomena is essential background for the production of more efficient vaccines for mycoplasmas.     

Comparison of Restriction Pattern Polymorphism of Mycoplasma agalactiae and Mycoplasma bovis by Pulsed Field Gel Electrophoresis

Mycoplasma agalactiae and Mycoplasma bovis are closely related species in phylogenetic terms. Pulsed Field Gel Electrophoresis (PFGE) of SmaI, EclXI, Bsi WI, MluI, BssHII, SalI, XhoI, NruI and ApaI digested DNAs were used to analyse and to compare restriction fragment length polymorphism between M. agalactiae and M. bovis and to estimate their genome sizes. SmaI, EclXI and Bsi WI enzymes cleaved DNAs of both microrganisms. MluI, BssHII, SalI, XhoI and NruI digested only M. agalactiae DNA whereas ApaI cut only M. bovis DNA. The total DNA length was established to be 945 ± 8.4 Kb for M. agalactiae and 961 ± 18.9 Kb for M. bovis.     

Comparison of restriction pattern polymorphism of Mycoplasma agalactiae and Mycoplasma bovis by pulsed field gel electrophoresis.

Mycoplasma agalactiae and Mycoplasma bovis are closely related species in phylogenetic terms. Pulsed Field Gel Electrophoresis (PFGE) of SmaI, EclXI, Bsi WI, MluI, BssHII, SalI, XhoI, NruI and ApaI digested DNAs were used to analyse and to compare restriction fragment length polymorphism between M. agalactiae and M. bovis and to estimate their genome sizes. SmaI, EclXI and Bsi WI enzymes cleaved DNAs of both microrganisms. MluI, BssHII, SalI, XhoI and NruI digested only M. agalactiae DNA whereas ApaI cut only M. bovis DNA. The total DNA length was established to be 945 +/- 8.4 Kb for M. agalactiae and 961 +/- 18.9 Kb for M. bovis.     

牛支原体TaqMan探针实时荧光定量PCR检测方法的建立及应用

以牛支原体p80基因为靶基因,设计特异性引物与探针,优化反应体系,建立了牛支原体TaqMan探针实时荧光定量PCR(TaqMan real-time PCR)检测方法,并对该方法进行了特异性、敏感性、重复性评估以及临床样本检测。结果显示,特异性试验中,牛支原体2个菌株Cq值均小于18,其余13个非牛支原体菌株Cq值均大于36;敏感性试验中,最低可检3.89拷贝/μL靶DNA,是普通PCR的10~4倍(普通PCR 3.89×10~4拷贝/μL);重复性试验中,组内、组间重复的变异系数均小于1%;44份牛的临床样品利用TaqMan real time PCR检出6份阳性(阳性率为13.64%),分离培养鉴定检出7份阳性,普通PCR检测全部阴性。本研究建立了一种敏感、特异、稳定的牛支原体TaqMan real time PCR检测方法,可用于牛支原体的快速诊断和核酸定量检测。     

牛支原体免疫相关性蛋白硫锌酰胺脱氢酶的筛选与鉴定

为鉴定牛支原体(M.bovis)菌体免疫相关蛋白及其免疫原性,本实验以M.bovis湖北株为样品,通过建立M.bovis全菌蛋白的双向电泳体系,获得了分辨率及重复性良好的双向电泳(2-DE)图谱。利用western blot技术进行筛选,鉴定得到多个M.bovis蛋白,其中一个分子量为68 ku,经过质谱分析和氨基酸一级结构比对确定该蛋白为硫锌酰胺脱氢酶(LipDH)。经原核重组表达,LipDH重组蛋白与M.bovis阳性血清的western blot反应为阴性,而Dot blot及以重组表达蛋白作为包被抗原的ELISA鉴定结果均为阳性。本研究为进一步研究LipDH的功能以及采用其重组蛋白用于该蛋白缺失的M.bovis疫苗株的鉴别检测方法的建立奠定了基础。     

Development of a semi-nested PCR for the improved detection of Mycoplasma bovis from bovine milk and mucosal samples

A new forward primer, Mb-F, was designed to improve the sensitivity and reproducibility of the Mycoplasma bovis-specific PCR developed by Ghadersohi et al. [Vet. Microbiol. 56 (1997) 87] for testing clinical samples. A semi-nested (SN) PCR configuration was developed and this provided enhanced sensitivity and reproducibility. The detection limit of the SN PCR was in the range of 10-100cfu/ml and the correct amplicon was amplified from 9.15pg/microliter of total extracted DNA (mixture of M. bovis and bovine cellular DNA). A dot blot assay was also developed and compared with the SN PCR on a number of randomly selected milk and mucosal samples. The dot blot had the same level of detection as the SN PCR. The specificity of the SN configuration was confirmed by Southern blot analysis and automated sequencing of the PCR product. The results from the tests on the samples from cattle, together with those from sheep, provided evidence that M. bovis is host-specific and that most cattle are colonised. The assay was shown to be specific, sensitive and reproducible and could be used successfully to detect M. bovis directly from clinical material without pre-enrichment.     

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.     

牛支原体TaqMan实时荧光定量PCR检测方法的建立

本研究旨在建立一种快速检测牛支原体(Mycoplasma bovis)的实时荧光定量PCR检测方法。根据GenBank中收录的牛支原体OPPD/F基因序列(登录号:AF130119),利用Primer 5.0软件设计特异性引物与TaqMan探针,以重组质粒作为绝对定量模板,构建检测牛支原体的TaqMan实时荧光定量PCR检测方法。结果显示该检测方法线性关系良好,标准曲线的相关系数R2=0.994,扩增效率E=1.280。荧光定量PCR法检测的敏感性是常规PCR的10倍;特异性良好,检测9种相关细菌和病毒均为阴性;重复性好,组内及组间样本检测Ct值均小于2%。牛支原体TaqMan实时荧光定量PCR检测方法较之于常规PCR方法有快速、特异性强、敏感性高、稳定性好的优点。     

内蒙古地区乳样中牛支原体的分离及PCR鉴定

为检测内蒙古地区某牛场患乳房炎的奶牛乳样中是否存在牛支原体,同时建立直接提取乳样中牛支原体DNA进行PCR检测的方法,本研究无菌采集乳房炎乳样,通过支原体的分离培养、形态学观察和生化试验,初步鉴定分离株为牛支原体,然后提取液体培养基菌体DNA进行牛支原体特异性PCR鉴定,同时,采用Chelex-100法直接提取原乳样中菌体DNA进行PCR鉴定,并测序分析扩增片段序列。液体培养物提取DNA与Chlex-100法直接提取原乳样菌体DNA进行特异性PCR均扩增出目的条带,该片段序列与GenBank中牛支原体oppD/oppF基因的同源性达到99.8%,证实分离株为牛支原体。说明Chlex-100法可直接提取乳样中牛支原体DNA进行快速PCR鉴定。     

牛支原体和牛病毒性腹泻病毒二重二温式PCR检测方法的建立

为建立一种牛支原体(Mycoplasma bovis,MB)和牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)的快速鉴别诊断方法,针对MB的uvr C基因和BVDV的5'端非编码区(5'-UTR)保守基因序列,分别设计两对特异引物,并将三温式PCR扩增程序简化为二个温度梯度,建立了鉴别MB和BVDV的二重二温式PCR方法。该方法能同时扩增MB和BVDV,扩增产物大小分别为412和170 bp。特异性试验结果显示,该方法对参试的所有毒株只扩增MB和BVDV基因组,对其它牛病原体无扩增;敏感性试验结果显示,该方法最低能同时检测到104拷贝的两种目的核酸;干扰性试验结果显示,该方法能同时检测两个模板不同浓度的组合,试验结果不受模板影响。综上,本研究所建立的二重二温式PCR方法特异、敏感、快速、简便,可应用于MB和BVDV临床鉴别诊断和流行病学调查。     

Mycoplasma (M.) bovis antiserum "Dessau"--a diagnostic material for the identification of M. bovis

Mycoplasma bovis antiserum "Dessau" was used to investigate 4,553 mycoplasma strains, between 1985 and 1987, using a method described elsewhere. This diagnostic Agent is successfully used at present at all Regional Institutes of Veterinary Services throughout the GDR.