牛分枝杆菌特异性多重PCR反应在临床样本检测中的应用

本试验应用建立的三重PCR反应检测256份血液样本,结果其中的2份样本呈阳性.应用在线blast进行同源性比较,结果发现,牛分枝杆菌特异性基因IS6110,IS1081和recA与牛分枝杆菌AF2122/97株的同源性均达到99％以上.     

水貂源牛分枝杆菌的分离与荧光PCR鉴定

目的探讨水貂源结核杆菌复合群的检测方法,并进行病原学调查。方法利用7H10培养基分离水貂结核病的病原,采用荧光探针PCR方法进行检测。结果经鉴定分离的病原菌为致病性牛分枝杆菌。结论通过荧光探针PCR能够直接对结核杆菌复合群进行亚种鉴定,该方法具有快速、敏感、特异性强的优点,能够减少动物检疫时间。     

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.     

Identification of bovine T cell stimulatory antigens using a cosmid library of Mycobacterium bovis in Mycobacterium smegmatis

Culture filtrates derived from a Mycobacterium bovis cosmid library in Mycobacterium smegmatis were screened for T cell antigens. Recognition and reactivity were measured by the levels of lymphocyte proliferation and the levels of gamma interferon (IFN-gamma) produced when the culture filtrates were incubated with peripheral blood mononuclear cells (PBMC) taken from cattle immunised with M. bovis BCG. The screening system was optimised to distinguish between M. bovis secreted antigens and normal M. smegmatis secreted proteins. From ten culture filtrates screened, two were identified that induced lymphocyte proliferation and IFN-gamma production. Analysis of the DNA inserts from the recombinant cosmids suggest that they may code for different proteins. The results demonstrate that screening recombinant M. smegmatis culture filtrates can be used to identify M. bovis T cell antigens that are recognised by immunised cattle. These antigens may be important for the development of vaccines with protective ability against bovine tuberculosis.     

Direct Detection of Mycobacterium bovis in Bovine Lymph Nodes by PCR

Thirty‐five lymph node samples were taken from animals with macroscopic lesions consistent with Mycobacterium bovis infection. The animals were identified by postmortem examination in an abattoir in the northwestern region of state of Paraná, Brazil. Twenty‐two of the animals had previously been found to be tuberculin skin test positive. Tissue samples were decontaminated by Petroff’s method and processed for acid‐fast bacilli staining, culture in Stonebrink and Lowenstein‐Jensen media and DNA extraction. Lymph node DNA samples were amplified by PCR in the absence and presence (inhibitor controls) of DNA extracted from M. bovis culture. Mycobacterium bovis was identified in 14 (42.4%) lymph node samples by both PCR and by culture. The frequency of PCR‐positive results (54.5%) was similar to that of culture‐positive results (51.5%, P > 0.05). The percentage of PCR‐positive lymph nodes increased from 39.4% (13/33) to 54.5% (18/33) when samples that were initially PCR‐negative were reanalysed using 2.5 μl DNA (two samples) and 1 : 2 diluted DNA (three samples). PCR sensitivity was affected by inhibitors and by the amount of DNA in the clinical samples. Our results indicate that direct detection of M. bovis in lymph nodes by PCR may be a fast and useful tool for bovine tuberculosis epidemic management in the region.     

Isolation of Mycoplasma bovis from bovine clinical samples in the Republic of Ireland

Mycoplasma bovis was detected in 134 (18 per cent) of 736 samples of bovine lung tissue collected from fatal pneumonia cases in the Republic of Ireland between April 1995 and December 1998. The cases occurred in 95 herds and recurred in four of them. Other respiratory pathogens were identified in 66 per cent of the M bovis-positive cases, with Pasteurella species, infectious bovine rhinotracheitis and parainfluenza 3 virus being most frequently detected. Mastitis and arthritis were less common clinical signs associated with M bovis infection; 22 cases of M bovis mastitis and five cases of M bovis arthritis were diagnosed in five herds.     

应用TaqMan荧光定量PCR检测牛分枝杆菌

为建立快速检测牛分枝杆菌(M.bovis)的TaqMan荧光定量PCR方法,本研究以GenBank登录的M.bovis特有229 bp基因为研究对象,设计并合成引物及探针。该方法具有较好的特异性,与标准质控菌株呈阳性反应,与其他微生物样品呈阴性反应;灵敏性最低检测值可达1 pg/mL;对20阳性临床样品进行荧光定量PCR检测,均为阳性;而对培养为阴性的20份临床样品进行检测,6份为阳性。该研究结果表明,建立的方法特异性强,敏感性高,稳定性好,能够用于M.bovis的鉴别检测,对牛分枝杆菌病的快速检测和早期诊断具有重要意义。     

Identification of immunodominant antigens of Mycobacterium bovis by expression library immunization

This study combines two methodologies - vector expression of a genomic library and proteomics - to identify immunogenic proteins of Mycobacterium bovis. Immunization of BALB/c mice with a plasmid DNA pool from the library, containing approximately 8000 clones, induced a humoral response that facilitated the detection of 12 antigenic proteins by Western blotting. Two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry identified four proteins (Cpn60-1, HSP70, EF-Tu, and AdoHcyase). Such genomic immunization offers the possibility of in vivo screening of potential candidate M. bovis antigens.     

牛分枝杆菌三重PCR检测方法的建立

为建立快速检测牛分枝杆菌(M.bovis)的三重PCR方法,本研究以M bovis ValleeⅢ株染色体DNA 为模板,分别以其recA、IS6110、IS1081基因特异性引物进行PCR扩增,建立检测M.bovis的recA-IS6110-IS1081三重PCR反应.通过PCR扩增获得大小约为860 bp、520bp和340 bp的DNA片段.BLAST序列分析表明,这3个基因片段与GenBank中登录的相关基因的核苷酸序列同源性均达到99%.同时,recA、IS6110和IS1081PCR扩增的敏感性分别达到585fg、19.5fg和19.5fg.特异性较强,只有M.bovis和人结核临床分离株扩增反应为阳性,为进一步研究recA、IS6110和IS1081基因以及其在牛结核病诊断中的应用奠定了基础.     

Patterns of antimicrobial susceptibility in Michigan wildlife and bovine isolates of Mycobacterium bovis.

The state of Michigan has recognized the presence of Mycobacterium bovis in its free-ranging white-tailed deer population since 1994. This endemic infection is primarily located in a 12-county area in the northeastern lower peninsula of Michigan. A statewide surveillance and eradication program of the disease has been in effect since 1994. Worldwide, Mycobacterium tuberculosis complex organisms have a known predilection toward development of antimicrobial resistance. The objective of this study was to investigate the antimicrobial susceptibility of M. bovis isolates from white-tailed deer in Michigan and detect any changes in susceptibility over time. M. bovis isolates from 2 fall hunting seasons (1999 and 2004) were used in this study. The fall season of 2004 marked the first documented case of direct transmission of M. bovis from a wild deer to a human in Michigan. Since M. bovis is a zoonotic disease, knowledge of susceptibility can expedite treatment options in humans. M. bovis isolates were obtained from 58 deer, 4 coyotes, 3 cattle, 2 raccoons, and 1 human case from the 2 years combined. Methods of susceptibility testing included 1% proportion agar plates and Bactec radiometric broth testing. M. bovis was found to be uniformly resistant to the antibiotic pyrazinamide; this resistance is common to all M. bovis isolates. No other antimicrobial resistance was found in any of the tested M. bovis isolates, which may be, in part, attributed to the lack of any significant treatment pressure in wildlife.     

牛分枝杆菌特异性基因的PCR扩增及二联PCR反应的建立

为了研究牛结核病的PCR诊断方法,本研究以牛分枝杆茵(M.bovis)Vallee111株染色体DNA为模板,以RecA和Ppsl基因特异性引物进行PCR扩增,获得约860 bp和430 bp的DNA片段.将PCR纯化产物进行测序,通过BLAST序列分析,与GenBank中登录的M.bovis AF2122/97 RecA基因和Ppsl基因的核苷酸序列同源性均达到99%.在同一PCR反应中同时加入RecA和Ppsl基因特异性引物建立RecA-Pps1二联PCR反应.同时,RecA和Ppsl PCR扩增的敏感性分别达到585 fg和195 fg,特异性均达到100%,为进一步研究RecA和Ppsl基因以及其在牛结核病诊断中的应用奠定了基础.     

Expression and Bioactivity Identification of eis Gene of Mycobacterium bovis in Mycobacterium smegmatis

This study was aimed to construct a shuttle expression vector of Mycobacterium bovis(M.bovis) eis gene and identify its bioactivity in recombinant Mycobacterium smegmatis (M.smegmatis). M.bovis eis gene was cloned by PCR and the shuttle expression vector pMV261-Mbeis was constructed, then it was identified by double digestion and sequencing. The recombinant plasmid was transformed into M.smegmatis mc²155 by electroporation. The expression of M.bovis eis gene in M.smegmatis was detected by SDS-PAGE and Western blotting, and the amino acids sequence of the target protein was identified by mass spectrometry. The growth curve of recombinant M.smegmatis mc²155 containing pMV261-Mbeis was successfully constructed.The results showed that pMV261-Mbeis did not affect the growth of M. smegmatis in vitro. The results of SDS-PAGE and Western blotting confirmed that the M. bovis eis gene expressed the eis protein which was about 44 ku in M. smegmatis. Mass spectrometry proved that the protein was the eis protein of M. bovis.The expression vector pMV261-Mbeis was successfully constructed and the expressed recombinant protein was proved to be have biological activities in M. smegmatis, which laid a foundation for the further study of the function of eis protein in M. bovis.     

Evaluation of a high-throughput nucleic acid extraction method for the detection of Mycobacterium avium subsp. paratuberculosis in bovine fecal samples by PCR

Johne’s disease (paratuberculosis) is an economically important disease of cattle worldwide. The disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), a fastidious gram-positive bacterium. PCR is increasingly used in diagnostic laboratories for the detection of MAP in fecal samples given the rapid test turnaround time and sensitivity and specificity comparable to fecal culture. However, efficient extraction of DNA for sensitive detection of MAP by PCR is affected by the complex lipid-rich cell wall of MAP and the presence of PCR inhibitors in feces. We evaluated a high-throughput nucleic acid extraction method (MagMAX core nucleic acid purification kit with mechanical lysis module) in conjunction with an hspX gene PCR for the detection of MAP from bovine fecal samples, which resulted in correct identification of all negative (13 of 13) and positive (35 of 35) proficiency test samples obtained from the National Veterinary Services Laboratories. In addition, all 6 negative and 50 of 51 positive diagnostic specimens tested were categorized correctly.     

牛分枝杆菌eis基因在耻垢分枝杆菌中的表达及其生物学活性鉴定

试验旨在构建牛分枝杆菌eis基因的穿梭表达载体,鉴定其在重组耻垢分枝杆菌中的生物学活性。采用PCR技术扩增并克隆牛分枝杆菌eis基因,构建大肠杆菌-分枝杆菌穿梭表达载体pMV261-Mbeis,经双酶切和测序鉴定其正确性,利用电穿孔法将重组质粒转化至耻垢分枝杆菌mc²155中,采用SDS-PAGE和Western blotting技术检测牛分枝杆菌eis基因在耻垢分枝杆菌中的表达,质谱鉴定目的蛋白氨基酸序列。研究结果表明,成功构建了牛分枝杆菌eis基因穿梭表达载体pMV261-Mbeis;生长曲线表明负载重组质粒不会影响耻垢分枝杆菌的体外生长;SDS-PAGE和Western blotting检测证实了牛分枝杆菌eis基因在耻垢分枝杆菌中可表达出分子质量约44 ku的eis蛋白;质谱检测证明了该蛋白即为牛分枝杆菌eis蛋白。本研究构建的牛分枝杆菌eis基因穿梭表达质粒pMV261-Mbeis在耻垢分枝杆菌中具有生物学活性,为下一步研究表达产物eis蛋白的功能奠定了基础。     

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.     

High frequency of Mycobacterium bovis DNA in colostra from tuberculous cattle detected by nested PCR

We evaluated by nested PCR reaction, different cow secretions from a herd with 48% of prevalence of bovine tuberculosis (BTB), seeking to determine niches where Mycobacterium bovis could be found. Postmortem examination of 18 (75%) tuberculin reacting cows allowed demonstrates BTB-compatible lesions in six, all of them PCR positives in milk and four in colostra samples. Our results showed that up to 62% of the colostra analysed contained M. bovis DNA, whereas only 18% of milk gave a positive reaction. Moreover, in bronchoalveolar lavages from cattle with compatible lesions in lungs or lymph nodes, where macrophages account up to 90% of cells, we did not find evidences of M. bovis. Altogether, these results suggest that differences in the anti-bacterial capacity of bovine macrophages, dependent upon microenvironment and organ-specific factors, exist. Alternatively, we hypothesize that hypoxic conditions that are encountered in mammary glands macrophages could induce M. bovis entrance into a 'dormancy-like' state, and that the high number of colostra samples were M. bovis was detected, could be an indicator of reactivation during 'peripartum'.     

检测奶牛乳房炎主要病原菌的多重PCR方法的建立与应用

本试验针对金黄色葡萄球茵、无乳链球菌、大肠杆菌3种主要的乳房炎病原茵设计了3对特异性引物,建立了多重PCR检测方法.结果表明,本方法的特异性为100%,最低检测浓度为1.25×103cfu/mL,充分表明该方法具有快速,准确和特异的优点.     

Identification of Mycobacterium bovis isolates using a monoclonal antibody

A rapid immunoperoxidase slide assay for the identification of Mycobacterium bovis culture isolates is described. The monoclonal antibody used in this assay is specific for the M. tuberculosis complex of organisms. All M. bovis isolates tested, including 151 separate field isolates of M. bovis were positive as were 11 out of 12 M. tuberculosis strains and 4 out of 6 Bacillus Calmette Guerin (BCG) strains. One strain each of M. africanum and M. microti was negative. This assay provides a considerable improvement in both time and expense over the conventional methods of biochemical typing of M. bovis.     

Detection of Rhodococcus equi by polymerase chain reaction using species-specific nonproprietary primers.

Species-specific primers for the polymerase chain reaction (PCR) for the detection of Rhodococcus equi were developed. These primers were based on unique DNA fragments produced from R. equi reference strains and field isolates. Following random amplification of polymorphic DNA from R. equi and R. rhodochrous with a set of 40 arbitrary 10-base pair (bp) primers, a pair of species-specific primers was designed to detect a unique 700-bp fragment of R. equi chromosomal DNA. This PCR product was limited to R. equi and was not detectable in other Rhodococcus species or in a panel of additional gram-positive and gram-negative bacteria.