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.     

Recent advances in our knowledge of Mycobacterium bovis: a feeling for the organism

Significant and rapid progress has been made in our knowledge and understanding of Mycobacterium bovis since the last international M. bovis conference 5 years ago. Much of this progress has been underpinned by the completion of the genome sequence. This important milestone has catalysed research into the development of a number of improved tools with which to combat bovine tuberculosis. In this article we will review recent progress made in the development of these tools and in our understanding of the organism, its evolution and spread. Comparison of the genome sequence with those of other members of the Mycobacterium tuberculosis complex has enabled insights into the evolution of M. bovis. This analysis also indicates that the M. tuberculosis complex have the propensity to adapt to new host species. The use of high throughput molecular typing methods has revealed that the recent bovine tuberculosis epidemic in Great Britain is being driven by a number of clonal expansions, which cannot be explained by random mutation and drift alone. Completion of a number of mycobacterial genome sequences has allowed the development of antigen mining techniques that rapidly identify M. bovis-specific genes. These can then be used as reagents in the gamma interferon assay to increase the specificity of the assay and also to discriminate between Bacillus of Calmette and Guérin (BCG) vaccinated animals and those infected with M. bovis. In the longer term, comparisons between the genomes of M. bovis and BCG will allow insight into how BCG became attenuated following serial passage on artificial growth media and reveal clues into how to improve the vaccine efficacy of BCG.     

牛分枝杆菌特异性PCR检测方法的建立及初步应用

根据已发表的牛分枝杆菌的pncA的基因序列，设计和合成了一对可扩增294bp目的片段的引物，建立了特异性检测牛分枝杆菌的PCR方法。对牛分枝杆菌国际参考株和国内分离株成功扩增出294bp的特异性基因片段；对人结核分枝杆菌、副结核分枝杆菌、鸟胞内分枝杆菌和草分枝杆菌DNA的PCR扩增结果均为阴性。本PCR方法检测的敏感度可达到50pg。对10份牛分枝杆菌培养阳性和10份阴性样品的DNA分别进行了PCR检测，结果10份阳性样品中有9份样品为PCR扩增阳性，阳性符合率为90％（9／10）；而10份阴性样品则PCR扩增全部为阴性，阴性符合率为100％（10／10）。本方法可做为牛分枝杆菌的快速检测和流行病学调查的工具。     

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。     

Molecular epidemiologic analysis of Mycobacterium bovis isolates from Mexico

OBJECTIVE: To assess phylogenetic relationships among Mycobacterium bovis isolates by use of random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) fingerprinting and to relate genetic profiles of isolates to epidemiologic characteristics. ANIMALS: 400 cattle with tuberculosis. PROCEDURE: Mycobacterium bovis was isolated from various organs of cattle slaughtered in 6 geographic regions of Mexico. Most cattle were adult Holsteins from large herds that did not participate in a tuberculosis control program. Four random primers and 2 selected primers were used in RAPD-PCR fingerprinting of 88 isolates. Pairwise genetic distance between isolates was obtained and subjected to cluster analysis with bootstrapping to test for levels of support. RESULTS: 98 different fragments were obtained; there was broad genetic diversity among isolates, and each isolate had a unique RAPD-genotype, including those originating from the same herd. Clustering by geographic location, affected organ, or severity of lesion was not detected. Linkage disequilibrium analysis suggested that M. bovis was highly clonal and that mutations develop at a rapid rate among isolates. CONCLUSIONS AND CLINICAL RELEVANCE: Use of RAPD-PCR could not differentiate M. bovis isolates by epidemiologic characteristics or identify common sources of infection.     

Use of DNA amplification for the rapid identification of Mycobacterium bovis

DNA amplification using the polymerase chain reaction technique was evaluated for rapid identification of Mycobacterium bovis. Two oligonucleotide primers of 20 bases in length were constructed to target a region of the gene encoding the M. bovis secretory protein, MPB70. The amplification reaction produced a single product 372 bp in size which was readily detected by agarose gel electrophoresis. All 84 strains of M. bovis tested produced a positive signal in the amplification reaction. In addition all isolates fro the M. tuberculosis complex tested, with the exception of M. microti, gave a single band at 372 bp. No amplified product was detected when 24 other species of mycobacteria and species from four other genera were tested. The sensitivity of the test was such that a single viable cell could be detected in the reaction. This technique provides a simple and extremely sensitive method of identifying isolates of M. bovis and other pathogenic M. tuberculosis complex organisms.     

Identification of Mycobacterium bovis in bovine clinical samples by PCR species-specific primers.

Tuberculosis, caused by Mycobacterium bovis is emerging as the most important disease affecting cattle. Furthermore, it results in a major public health problem when transmitted to humans. Due to its difficult and non-specific diagnosis, M. bovis has been declared to be one of the etiologic agents causing significant economic loss in the cattle industry. Our group evaluated a more rapid and specific method, based on a new polymerase chain reaction species-specific primers, which amplifies a 470-base pair fragment of the M. bovis genome. A total of 275 milk-producing cows were studied by intradermal tuberculin test (ITT) which gave 184 positive and 91 negative cases. From them, 50 animals were taken from a cattle ranch free of tuberculosis. Three different samples were collected from each animal (blood, nasal mucus, and milk). Positive results were obtained from 26 animals by PCR (11.4%), 1 by bacteriological culturing (0.4%) and 1 by bacilloscopy (0.4%). This finding suggests, as in previous reports, that ITT, normally used for bovine tuberculosis detection, has the inconvenience of having a broad range of specificity and sensitivity, and the PCR technique is a more specific and sensitive test to detect infection associated with M. bovis. Therefore, we propose this PCR assay as a useful tool in the epidemiological characterization of infected animals in areas considered to be at high risk of transmission.     

Molecular differentiation of Mycobacterium bovis isolates. Review of main techniques and applications

Until recently, none of the Mycobacterium bovis typing techniques permitted a satisfactory differentiation of isolates. During the last 10 years, the genome of pathogenic mycobacteria has been extensively studied, and phylogenetic analyses have shown that all (except Mycobacterium avium) belong to a single genetic species: the Mycobacterium tuberculosis complex. This increase in knowledge about the genome of these bacteria has lead to the discovery of molecular markers that allow us to differentiate isolates. Because of the phylogenetic proximity of the strains, even if most of these markers have been discovered in M. tuberculosis, they could be successfully adapted to the other bacteria of the M. tuberculosis complex, especially M. bovis. The most common markers in use today are the IS6110 insertion sequence, the direct repeat (DR) region, the poly(GC) rich (PGRS) sequences and the variable number tandem repeats (VNTR) sequences. The corresponding typing techniques are briefly described, and current knowledge of polymorphism and marker stability is detailed. If molecular markers are to offer wide perspectives for field studies, these two characteristics (polymorphism and stability) must be taken into account when choosing the marker(s) used in a study. In this context, examples of the application of molecular typing techniques for M. bovis are reviewed, on the one hand with epidemiological studies for which the major problem is the comparison between isolates and, on the other, with more general studies about the population genetics of M. bovis in a given country, and about its history and its phylogeny.     

Use of a macrophage cell line for rapid detection of Mycobacterium bovis in diagnostic samples

Mycobacterium bovis isolation on bacteriological media from suspected cases of bovine tuberculosis (TB) demands laborious and time-consuming procedures. Even polymerase chain reaction (PCR) and radiometric analyses are secondary procedures and not alternatives to bacteriological procedures. Therefore, there is a need to develop new techniques aimed at rapid M. bovis detection in diagnostic samples. The human macrophage cell line THP-1 was thus investigated in experiments of M. bovis propagation and isolation from reference lymph node suspensions. THP-1 cells were shown to support a high-titered propagation within 48h of minute amounts of both M. bovis BCG and fully pathogenic M. bovis strain 503. A semi-nested PCR for TB-complex-specific insertion sequence IS6110 revealed M. bovis infection in THP-1 cells. The same was true of a flow cytometry (FC) assay for expression of M. bovis chaperonin 10 in infected cells. The reduced time for isolation and identification of M. bovis (48-72h) and the consistency of the test results make the use of macrophage cell cultures attractive and cost-effective for veterinary laboratories involved in TB surveillance.     

Bovine tuberculosis in South Darfur State, Sudan: an abattoir study based on microscopy and molecular detection methods

Bovine tuberculosis (BTB) is a widespread zoonosis in developing countries but has received little attention in many sub-Saharan African countries including Sudan and particularly in some parts such as Darfur states. This study aimed to detect bovine tuberculosis among caseous materials of cattle slaughtered in abattoirs in South Darfur State, Sudan by using microscopic and PCR-based methods. The study was a cross-sectional abattoir-based study which examined a total of 6,680 bovine carcasses for caseous lesions in South Darfur State between 2007 and 2009. Collected specimens were examined for the presence of acid-fast bacilli (AFB) by using microscopic and culture techniques. Isolated mycobacteria were identified by selected conventional cultural and biochemical tests in comparison to a single tube multiplex PCR (m-PCR) assay which detect Mycobacterium bovis-specific 168-bp amplicons. Of the total 6,680 slaughtered cattle examined in South Darfur, 400 (6 %) showed caseations restricted to lymph nodes (86.8 %) or generalized (13.2 %). Bovine tuberculosis was diagnosed in 12 (0.18 %), bovine farcy in 59 (0.88 %), unidentified mycobacteria in 6 (0.09 %), and missed or contaminated cultures in 7 (0.1 %). Out of 18 cultures with nonbranching acid-fast rods, 12 amplified unique 168-bp sequence specific for M. bovis and subsequently confirmed as M. bovis. With the exception of the reference M. tuberculosis strains, none of the remaining AFB amplified the 337-bp amplicon specific for M. tuberculosis. It could be concluded that bovine tuberculosis is prevalent among cattle in South Darfur representing 4.5 % from all slaughtered cattle with caseous lesions. The study sustains microscopy as a useful and accessible technique for detecting AFB. m-PCR assay proved to be valuable for confirmation of BTB and its differentiation from other related mycobacteriosis, notably bovine farcy.     

Mycobacterium nonchromogenicum in nasal mucus from cattle in a herd infected with bovine tuberculosis

Skin test negative cattle from a herd containing an unusually high proportion (194/382) of tuberculin skin test positive cattle were investigated for remaining Mycobacterium bovis infected animals. Blood samples from the skin test negative cattle, analysed by an antibody ELISA and an interferon-gamma assay, were mostly test negative for M. bovis. Radiometric culture of nasal mucus samples from 48 of the cattle yielded 22 culture positives with acid-fast bacilli and cording in 6 of these. Subculture on solid media was successful for 7, including 2 with cording of the 22 radiometric culture positives. Mycobacterium tuberculosis complex DNA probe testing using the Accuprobe (Gen-Probe, Inc.) and M. tuberculosis complex-specific PCR amplification, performed on the solid media subcultures, were negative. 16S rRNA PCR and sequence analysis were successful for 6 of the 7 solid media subcultures obtained and revealed the presence of Mycobacterium nonchromogenicum in all 6 subcultures. This is the first report of M. nonchromogenicum in nasal mucus of cattle. The observation highlights the importance of integrating definitive tests such as the PCR for diagnosis of bovine tuberculosis and indicates a possible zoonotic risk.     

Evaluation of Spoligotyping and MIRU-VNTR for Mycobacterium bovis in Xinjiang, China

Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB) in cattle, is also a pathogen for human and other mammals. In this study, 406 cows were screened for bTB by both single intradermal comparative cervical tuberculin (SICCT) test and IFN-γ assay. 135 M. bovis were isolated from 31 SICCT and IFN-γ double-positive cows in Xinjiang Uygur Autonomous Region of China. Spoligotyping and MIRU-VNTR were evaluated for genotyping, and 4 and 7 genotypes were identified, respectively. A new combination of nine MIRU-VNTR loci was most discriminative for M. bovis clones from Xinjiang. Interestingly, two new spoligotypes (SB1903 and SB1904) and special repeat numbers of three loci (ETR-D, QUB 1895 and QUB 3336) were discovered in this study. These results indicated a specific epidemic conservation in Xinjiang, China. M. bovis strains with the unique genotypes were isolated from the herds maintaining parent cows imported from the bTB-free countries, suggesting a possible transmission from the local breed of Xinjiang brown cattle.     

结核杆菌多位点环介导等温扩增检测方法的建立

为建立一种快速、高敏感、高特异、鉴别结核分枝杆菌和牛分枝杆菌的方法,本研究根据大多数致病性结核分枝杆菌共有序列esat-6,结核分枝杆菌和牛分枝杆菌gyrB共有特异性序列,以及结核分枝杆菌种特异序列mtp40设计6对特异性引物对痰液中的结核分枝杆菌进行检测,并与鉴别培养基分离培养结果以及常规PCR鉴定结果进行比较。实验结果表明,建立的LAMP检测方法具有很高的特异性。可区分致病性结核分枝杆菌和非致病结核分枝杆菌,也可鉴别结核分枝杆菌和牛分枝杆菌。LAMP检测技术的灵敏度比经典PCR技术高100倍左右,可检测到7拷贝/反应。另外,用3种方法同时检测样品发现,LAMP与细菌培养的符合率为90.91%,LAMP与常规PCR检测结果的符合率为100%。LAMP检测方法可以在流行病学调查及现场快速诊断方面广泛应用,并为临床治疗提供依据。     

Cloning and characterization of a Moraxella bovis cytotoxin gene

OBJECTIVE: To identify the Moraxella bovis cytotoxin gene. PROCEDURE: Hemolytic and nonhemolytic strains of M. bovis were compared by use of western blotting to identify proteins unique to hemolytic strains. Oligonucleotide primers, designed on the basis of amino acid sequences of 2 tryptic peptides derived from 1 such protein and conserved regions of the C and B genes from members of the repeats in the structural toxin (RTX) family of bacterial toxins, were used to amplify cytotoxin-specific genes from M. bovis genomic DNA. Recombinant proteins were expressed, and antisera against these proteins were produced in rabbits. RESULTS: Several proteins ranging in molecular mass from 55 to 75 kd were unique to the hemolytic strain. An open reading frame encoding a 927-amino acid protein with a predicted molecular mass of 98.8 kd was amplified from M. bovis genomic DNA. The deduced amino acid sequence encoded by this open reading frame was homologous to RTX toxins. Antisera against the recombinant carboxy terminus encoded by this open reading frame neutralized hemolytic and cytolytic activities of native M. bovis cytotoxin. CONCLUSIONS AND CLINICAL RELEVANCE: A gene was identified in M bovis that encodes a protein with sequence homology to other RTX toxins. Results of cytotoxin neutralization assays support the hypothesis that M. bovis cytotoxin is encoded by this gene and belongs in the RTX family of bacterial exoproteins. Identification of this gene and expression of recombinant cytotoxin could facilitate the development of improved vaccines against infectious bovine keratoconjunctivitis.     

Discrimination of isolates of Mycobacterium bovis in Northern Ireland on the basis of variable numbers of tandem repeats (VNTRs)

The ability to reproducibly discriminate Mycobacterium bovis isolates and trace their transmission has the potential to clarify sources of infection and major routes of transmission for bovine tuberculosis (TB). A PCR-based genotyping assay has been developed to discriminate between strains of M bovis by examining multiple sites in its genome that consist of variable numbers of tandem repeats (VNTRS). The discriminatory power and reproducibility of this VNTR typing has been compared with that of the established PCR-based spoligotyping technique by using a panel of 461 isolates of M bovis prevalent in Northern Ireland. The VNTR assay discriminated 40 different profiles, the most prevalent of which constituted 21 per cent of the total, compared with 14 profiles discriminated by spoligotyping, the most prevalent of which constituted 65 per cent. No significant differences were observed between the prevalences of the VNTR profiles in the years from 1999 to 2003. A preliminary evaluation indicated that most genotypes predominated in particular areas of the country. This VTNR typing assay was found to be highly discriminating, with the performance characteristics to support its systematic application to the molecular epidemiology of bovine TB.     

Polymerase chain reaction detection of Mycobacterium tuberculosis complex and Mycobacterium avium organisms in formalin-fixed tissues from culture-negative ruminants

In the US eradication program for bovine tuberculosis, a definitive diagnosis depends on the isolation of Mycobacterium bovis. However, in some cases bacterial culture is unsuccessful, even though the tissue is considered suspicious by histopathology because granulomatous lesions and acid-fast organisms are present. The purpose of this study was to determine if polymerase chain reaction (PCR) tests on formalin-fixed tissue would successfully identify the organisms observed in suspect lesions from culture-negative animals. Diagnostic laboratory records were used to select paraffin blocks of tissue from 102 ruminants that had suspect microscopic lesions but no bacterial isolation. Sections from these blocks were examined with PCR primers for IS6110 to detect Mycobacterium tuberculosis complex infection, or with 16S ribosomal RNA and IS900 primers for detection of Mycobacterium avium. The PCR tests successfully identified a mycobacterial infection in 58 of 102 tissues, including 41 M. tuberculosis complex and 17 M. avium (11 subspecies paratuberculosis). These results demonstrate that PCR testing of formalin-fixed tissue, in combination with bacterial culture, may increase the effectiveness of laboratory diagnostic efforts to detect and identify the most common mycobacterial diseases of ruminants.     

Rapid detection of Mycobacterium bovis on its lipid profile by thin layer chromatography

Sixteen Mycobacterium bovis (M. bovis) strains isolated from bovine tissues and one standard reference strain of M. bovis AN5 alongwith other species of mycobacteria for comparison were investigated for the presence of phenolic glycolipid (PGL) and phthiocerol dimycocerosate (PDIM) for rapid identification of M. bovis by thin-layer chromatography (TLC). The study indicated presence of PGL with an Rf value of 0.75 in chloroform-methanol solvent in all 17 M. bovis strains. The dimycocerostate A corresponding to spot A was the major constituent among all the three spots in M. bovis strains. TLC appeared to be a promising alternative to conventional biochemical methods for identification of M. bovis taking into consideration both PGL and PDIM lipids.     

Antibodies to mycobacteria in cattle not infected with Mycobacterium bovis

An indirect anti-IgG enzyme-linked immunosorbent assay (ELISA) using a whole cell sonicate of Mycobacterium bovis as the coating antigen, was used to detect anti-mycobacterial antibodies in cattle not infected with M. bovis. False positive M. bovis ELISA scores were produced in 6 cattle experimentally inoculated with Mycobacterium avium-intracellulare-scrofulaceum (MAIS) serovars 2, 8, 9, 14 and 18 and Mycobacterium flavescens, respectively. False positive ELISA results were also found in 39.5% of cattle from which other mycobacteria were cultured and in 56.4% of necropsied cattle with other pathological conditions. No M. bovis was cultured from these animals. Other groups of animals, with no pathological conditions, which had been tuberculin-tested negative, tuberculin-tested positive and never tuberculin tested showed positive ELISA results in 15.4%, 73.6% and 42.4% of the respective groups. The variation of these non-specific responses in uninfected cattle highlights the need for careful selection of negative controls in evaluating ELISAs for the diagnosis of bovine tuberculosis.