Evaluation of the discriminatory power of variable number tandem repeat (VNTR) typing of Mycobacterium bovis strains

The discriminatory power of variable number tandem repeat (VNTR) typing based on 16 known loci (12 MIRUs, 3 ETRs and VNTR 3232) was assessed for Mycobacterium bovis strains collected sequentially at the slaughterhouse of N'Djaména, Chad. Of 67 M. bovis strains analyzed, 67% were clustered. In this study, VNTR typing was highly discriminative with an overall allelic diversity (h(oa)) of 0.922. We defined five loci (ETR A, B, C and MIRU 26, 27) as highly (h>0.25), two loci (MIRU 4, and VNTR 3232) as moderately (0.11相似文献   

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.     

Comparison of 45 variable number tandem repeat (VNTR) and two direct repeat (DR) assays to restriction endonuclease analysis for typing isolates of Mycobacterium bovis

Restriction endonuclease analysis (REA), developed 25 years ago for genotyping Mycobacterium bovis strains, is an important tool for bovine tuberculosis control in New Zealand. While REA gives excellent discrimination, it is technically difficult to perform compared to PCR-based typing systems which are faster and simpler to operate. Genotyping of M. bovis by the use of variable number tandem repeat loci (VNTR) and spoligotyping, either alone or together, has now become the preferred approach for typing M. bovis. Here, we evaluated the widest range of VNTR loci yet investigated for M. bovis, including two VNTR loci not previously studied, one of which (4155) had particular utility for characterizing New Zealand isolates. VNTR typing provided substantial geographical resolution of 26 of the most commonly found REA types and this was improved by the addition of two PCR assays based on parts of the direct repeat (DR) locus. Overall, 68 REA types of M. bovis common in New Zealand were discriminated into 33 VNTR/DR groups by using a minimum of nine VNTR and two DR assays. These 11 VNTR/DR assays concorded for three isolates each of 45 of the REA types but showed some variation with at least one of the VNTR/DR assays for the remaining 23 REA types. Major differences were found in allelic variation of some VNTRs between isolates from New Zealand and other countries, emphasizing the importance of adapting M. bovis typing systems to suit individual countries.     

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.     

High Mycobacterium bovis genetic diversity in a low prevalence setting

The genetic diversity among South African Mycobacterium bovis isolates from cattle was determined by genetic fingerprinting. The restriction fragment length polymorphism (RFLP) markers IS6110 and polymorphic GC-rich sequence (PGRS) as well as spoligotyping and determination of variable number of tandem repeats (VNTR) were used to characterize sub samples of 91 M. bovis field isolates. PGRS RFLP was the single most discriminatory method and combinations of typing methods, which included IS6110 and/or PGRS had the highest discriminatory power, able to reveal 29 distinct genotypes among 35 farms with no epidemiological link. Three of the farms were co-infected with two genetically unrelated strains. In contrast to reports from European and also other colonised countries on the African continent our findings are suggestive of a high genetic diversity of M. bovis in South Africa's cattle population, implying a variety of unrelated ancestor strains. Despite effective intervention through test-and-slaughter campaigns no indication of a 'founder effect' was apparent in the panel of isolates derived from all infected provinces.     

Molecular epidemiology of human and animal tuberculosis in Ibadan, Southwestern Nigeria

From 2005 to 2007, Mycobacterium tuberculosis complex (MTC) strains were isolated from cattle, goats and pigs samples collected at the Bodija abattoir and from human samples from tuberculosis patients and livestock traders at the Akinyele cattle market in Ibadan, Southwestern Nigeria. Seventy four isolates obtained from humans (24) and livestock (50) were identified as MTC strains. Thirty two isolates were spoligotyped. Nineteen of these 32 isolates were identified as M. tuberculosis whilst 13 were identified as Mycobacterium bovis. M. bovis was isolated from two humans, whereas M. tuberculosis was isolated from a bovine, a pig and a goat. All the M. bovis isolates identified in this study belonged to the Africa 1 clonal complex. Multiple locus VNTR [variable number of tandem repeats] analysis (MLVA) was carried out on the 74 isolates. Three major clusters were defined. Group A consisted of 24 M. tuberculosis isolates (MLVA genotypes 1-18). One strain was isolated from a bovine and one from a pig. Group B consisted of 49 M. bovis strains (MLVA genotypes 19-48), mainly of cattle origin but also included four goat, nine pig and two human isolates. Group C consisted of a single M. tuberculosis isolate (MLVA genotype 49) obtained from a goat. Spoligotyping and MLVA confirmed it as clustering with the East Africa Indian clade found in humans in Sudan and the Republic of Djibouti. The isolation of three M. tuberculosis strains from livestock raises the question of their epidemiological importance as a source of infection for humans.     

Effect of skin testing and segregation on the prevalence of bovine tuberculosis, and molecular typing of Mycobacterium bovis, in Ethiopia

In 2002, the prevalence of bovine tuberculosis (tb) among 500 cattle on Holeta Farm, near Addis Ababa, Ethiopia, was 48 per cent, and the farm was divided into positive and negative herds. After three consecutive rounds of skin testing and segregation of skin test-positive and -negative animals, the prevalence of bovine tb was reduced from 14 per cent to 1 per cent in the negative herd in a year. Spoligotyping of 41 isolates from 17 cows gave an identical and unique spoligotype pattern, which can be represented as the binary number 1100000101111110111111100010000000000100000, where 1 indicates the presence of a spacer and 0 represents a loss. This spoligotype pattern had not previously been reported on the Mycobacterium bovis spoligotype database, and it was therefore designated SB1176, Ethiopian M bovis strain 1 (EMbs1). The variable number tandem repeat (VNTR) profile of the strain was 5254(*)33.1, which differed from the VNTR profile of strains reported in Great Britain.     

Advances in molecular diagnostics for Mycobacterium bovis

The two most important molecular diagnostic techniques for bovine tuberculosis are the polymerase chain reaction (PCR) because of its rapid determination of infection, and DNA strain typing because of its ability to answer important epidemiological questions. PCR tests for Mycobacterium bovis have been improved through recent advances in PCR technology, but still lack the sensitivity of good culture methods, and in some situations are susceptible to giving both false negative and false positive results. Therefore, PCR does not usually replace the need for culture, but is used to provide fast preliminary results. DNA typing of M. bovis isolates by restriction endonuclease analysis (REA) was developed 25 years ago in New Zealand, and remains an important tool in the New Zealand control scheme, where the typing results are combined with other information to determine large and expensive possum poisoning operations. A range of other DNA typing systems developed for M. bovis in the 1990 s have assisted epidemiological investigations in some countries but are now less commonly used. Variable number tandem repeat (VNTR) typing and spoligotyping, either alone or together, have now become the preferred approaches as they are robust and amenable to electronic analysis and comparison. Spoligotyping gives only moderate discrimination but can be easily applied to large numbers of isolates, and VNTR typing provides better discrimination than all other methods except for REA. While the current typing techniques are sufficient for most epidemiological purposes, more discriminating methods are likely to become available in the near future.     

Investigations into an outbreak of tuberculosis in a flock of sheep in contact with tuberculous cattle

Tuberculosis, associated with Mycobacterium bovis infection, occurs infrequently in sheep. A sheep flock, which was potentially exposed to a high level of infection from in-contact tuberculous cattle, was examined for evidence of infection. Six sheep that had given a positive reaction to the comparative intradermal tuberculin test were examined post mortem. Tuberculous lesions were present in four of these sheep. Lesion morphology and distribution in the sheep was similar to that in cattle. M. bovis was cultured from the lesions and the isolates were strain typed by spoligotyping and variable number of tandem repeats (VNTR) typing. Tuberculin-reacting sheep also reacted positively to an assay for in vitro release of interferon-gamma. This paper describes the first report of an outbreak of tuberculosis in sheep in either Britain or Ireland. The report describes immunology and pathology findings and, using molecular typing techniques, suggests that the sheep had been infected from in-contact cattle.     

Prevalence of Mycobacterium bovis infection in traditionally managed cattle at the wildlife-livestock interface in South Africa in the absence of control measures

Cattle are the domestic animal reservoir for Mycobacterium bovis (M. bovis) which also affects other domestic animals, several wildlife species and humans leading to tuberculosis. The study area is in a resource-poor community that is surrounded by several game parks, where M. bovis infection has been previously diagnosed in wildlife. A cross-sectional study was carried out to determine the prevalence of M. bovis infection in 659 cattle from a total of 192 traditionally managed herds using the BOVIGAM® interferon gamma assay (IFN-γ). Infection was confirmed by post mortem examination and M. bovis isolation from three test-positive cattle. Genotyping of the M. bovis isolates was done using spoligotyping and VNTR (variable number of tandem repeats typing). The apparent M. bovis prevalence rate in cattle at animal level was 12% with a true population prevalence of 6% (95% Confidence interval (C.I) 3.8 to 8.1) and a herd prevalence of 28%. Spoligotyping analysis revealed that the M. bovis isolates belonged to spoligotype SB0130 and were shared with wildlife. Three VNTR profiles were identified among the SB0130 isolates from cattle, two of which had previously been detected in buffalo in a game reserve adjacent to the study area. The apparent widespread presence of M. bovis in the cattle population raises a serious public health concern and justifies further investigation into the risk factors for M. bovis transmission to cattle and humans. Moreover, there is an urgent need for effective bTB control measures to reduce infection in the communal cattle and prevent its spread to uninfected herds.

     

Multiple strains of Mycobacterium bovis revealed by molecular typing in a herd of cattle

Mycobacterium bovis isolates from an outbreak of bovine tuberculosis in a herd of cattle in Rio de Janeiro, Brazil, were analysed by spoligotyping and variable-number tandem repeat PCR analysis of the mycobacterial interspersed repetitive unit and exact tandem repeats. Molecular typing revealed a high genetic diversity of strains in the herd. The genetic diversity could be explained by the introduction of infected animals from different sources.     

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.     

Isolation and molecular characterization of Mycobacterium bovis from Kafue lechwe (Kobus leche kafuensis) from Zambia

Bovine tuberculosis (BTB) is a chronic bacterial disease caused by Mycobacterium bovis. Infections due to M. bovis, which serves as a stable reservoir, can pose serious challenge to control and eradicate in both wildlife and livestock at the interface. This study aimed at isolating and characterizing M. bovis from Kafue lechwe (Kobus leche kafuensis) and black lechwe (Kobus leche smithemani) at the animal/human interface in Zambia. The samples with lesions compatible with BTB collected during the hunting seasons of 2009 and 2010 were cultured for isolation of mycobacteria using Stonebrink with pyruvate (BD Diagnostics, MD, USA) and Middlebrook 7H10 (BD Diagnostics) slants. Isolated mycobacteria were identified using IS6110 polymerase chain reaction and deletion analysis. Molecular characterization of the isolates was performed using spoligotyping and mycobacteria interspersed repetitive unit–variable number tandem repeat (MIRU–VNTR) with nine loci. Data was analyzed using BioNumerics software 6.1. Out of the 39 samples, acid fast bacilli were detected in 27 (69.2 %) based on smear microscopy. Seven isolates were found to belong to Mycobacterium tuberculosis complex, and all were identified as M. bovis based on deletion analysis. All seven isolates were identical on spoligotyping as belonging to the SB0120 (SIT 482). MIRU–VNTR differentiated the isolates into five different patterns. This study has confirmed that M. bovis circulates in the Kafue lechwe, and non-tuberculous mycobacteria were detected in the black lechwe in Zambia which represents a wildlife reservoir, with a potential to spillover to cattle and humans. Isolates of M. bovis from lechwe antelopes are much conserved as only one spoligotype was detected. The study has shown that three loci differentiated fairly well. This option is cheap and less laborious, and hence a better option in resource-strained country like Zambia. The study further showed that some of the loci recommended by the European Reference Laboratory are not suitable for typing M. bovis in Zambia.     

Variable number tandem repeat analysis of Mycobacterium bovis isolates from Gyeonggi-do, Korea

Bovine tuberculosis (TB) is a major zoonosis that''s caused by Mycobacterium bovis (M. bovis). Being able to detect M. bovis is important to control bovine TB. We applied a molecular technique, the variable number tandem repeat (VNTR) typing method, to identify and distinguish the M. bovis isolates from Gyeonggi-do, Korea. From 2003 to 2004, 59 M. bovis clinical strains were isolated from dairy cattle in Gyeonggi-do, Korea, and these cattle had tuberculosis-like lesions. Twenty-four published MIRU-VNTR markers were applied to the M. bovis isolates and ten of them showed allelic diversity. The most discriminatory locus for the M. bovis isolates in Korea was QUB 3336 (h = 0.64). QUB 26 and MIRU 31 also showed high discriminative power (h = 0.35). The allelic diversity by the combination of all VNTR loci was 0.86. Six loci (MIRU 31, ETR-A and QUB-18, -26, -3232, -3336) displayed valuable allelic diversity. Twelve genotypes were identified from the 59 M. bovis isolates that originated from 20 cattle farms that were dispersed throughout the region of Gyenggi-do. Two genotypes [designation index (d.i.) = e, g] showed the highest prevalence (20% of the total farms). For the multiple outbreaks on three farms, two successive outbreaks were caused by the same genotype at two farms. Interestingly, the third outbreak at one farm was caused by both a new genotype and a previous genotype. In conclusion, this study suggests that MIRU-VNTR typing is useful to identify and distinguish the M. bovis isolates from Gyeonggi-do, Korea.     

Molecular epidemiology of bovine tuberculosis in wild animals in Spain: a first approach to risk factor analysis

In human tuberculosis (Mycobacterium tuberculosis), molecular epidemiology has accurately indicated the risk factors involved in active transmission of the disease, by comparing individuals whose isolates belong to a cluster with patients whose strains are considered unique. Nevertheless, this application has not been used in bovine tuberculosis (Mycobacterium bovis). Our study describes the integration of epidemiological data into molecular classification data on M. bovis isolates. These were isolated from wild ungulates in Extremadura (western Spain) with the objective of detecting the risk factors linked to the association of strains in clades, which are indicators of the active spread of the disease. The molecular markers used were spoligotyping + VNTR typing (loci: VNTR 2165, VNTR 2461, VNTR 0577, VNTR 0580, VNTR 3192 VNTR 2163a and VNTR 2163b) on a population of 59 M. bovis strains isolated from deer (Cervus elaphus), 112 from wild boar (Sus scrofa), six from bovines, 28 from pigs and 2 from goats (n = 207). Epidemiological variables included the animal species from which the strain was isolated, pathological condition of the host (incipient lesion, early and late generalisation), date of sampling (during or after the reproductive period) and hunting season. Bivariant analysis was used to establish the risk factors connected to the association of strains and later, the variables were evaluated by means of logistic regression. Molecular typing grouped a total of 131 strains (64.21%) in 28 clusters and 76 isolates shows unique profiles. The association of strains was connected to the appearance of macroscopic lesions during the reproductive period (O.R. 4.80; 95% CI 1.09–22.99, P < 0.005), showing a possible higher transmission during the courting period. This happened mainly during the last hunting season analysed (2002–2003, O.R. 3.69; 95% CI 1.27–11.9, P < 0.05), clashing with the time of higher prevalence of the disease in wild ungulates. Active spread was not connected to any species in particular, or to any concrete pathological condition.     

Public acceptance as a determinant of management strategies for bovine tuberculosis in free-ranging U.S. wildlife

Mycobacterium marinum is a free-living bacterium that infects fish and amphibians, but is also an opportunistic pathogen in humans. Although in previous studies, the bacterium has shown a small number of Variable Number of Tandem Repeat (VNTR) polymorphic loci, the discriminatory power of the known VNTR loci is unlikely to be sufficient to distinguish such a globally ubiquitous waterborne microorganism, which has a presumably high genetic variance. The object of this study was to test M. marinum isolates from east China for strain diversity using previously described loci as well as novel VNTR loci, and to identify more discriminating VNTR loci that could enrich the genotyping tools for M. marinum. We found that 7 of the 12 tested VNTR loci, of which 5 were known loci and 2 loci were newly identified, showed good discriminatory power for the 14 M. marinum clinical isolates (Hunter-Gaston Index [HGI] value=0.990). The pathogenicity of isolates representing different VNTR clusters was also studied in a zebrafish (Danio rerio) infection model; in this model, 6 isolates representing 4 VNTR clusters induced chronic infections, whereas the American strain ATCC BAA-535 caused acute and lethal infections.     

Epidemiology of Mycobacterium bovis infections of pigs and wild boars using a molecular approach

A molecular epidemiological approach was applied to establishing a possible role for the wild boar as a natural reservoir of Mycobacterium bovis in Sierra de Villuercas, Western Spain; an area free of farmed cattle and wild deer populations. Spoligo and VNTR typing were used over a three year period to study the epidemiological relationship between the occurrence of bovine tuberculosis (TB) in extensively bred Iberian pigs and indigenous wild boar. The 37 sampled wild boar showed different degree of calcified granulomatous lesions in retropharyngeal, mediastinal and pulmonary lymph nodes. The 25 sampled Iberian pigs showed calcified lesions, mainly in the respiratory tract. Lesions located in the mesenteric lymph nodes appeared secondarily. M. bovis was isolated from all affected animals. Twenty-five and 37 isolates of M. bovis were obtained from domestic pigs and wild boar, respectively. Our findings provide evidence that supports the possibility of cross infection between wild boar and domestic pig populations. This is contrary to the generally held belief that swine represent an epidemiological dead end host and play no role in the epidemiology of M. bovis.     

MIRU-VNTR typing of Mycobacterium avium in animals and humans: heterogeneity of Mycobacterium avium subsp. hominissuis versus homogeneity of Mycobacterium avium subsp. avium strains

Epidemiological studies on Mycobacterium avium are requisite for revealing infection sources and disease transmission. They are based upon genotyping methods like RFLP and MIRU–VNTR. In our study, MIRU–VNTR typing was applied to 121 previously RFLP typed M. avium field isolates to compare the discriminatory power of both methods. The applicability of MIRU–VNTR typing was studied for isolates from a limited geographic area, namely 41 M. avium subsp. avium and 80 M. avium subsp. hominissuis isolates. Among the former, exhibiting 12 IS901 RFLP types, five MIRU–VNTR types were found with discriminatory index (DI) of 0.716. Among the latter, exhibiting 56 IS1245 RFLP types, 18 MIRU–VNTR types were found with DI of 0.866. Concomitant use of both methods increased DI to 0.981 and 0.995, respectively. MIRU–VNTR typing employing the selected markers provided discernible discrimination among M. avium subsp. hominissuis isolates, but more discriminative markers are needed for M. avium subsp. avium isolates.     

Persistence and molecular evolution of Mycobacterium bovis population from cattle and wildlife in Doñana National Park revealed by genotype variation

The role of wildlife in tuberculosis epidemiology is being widely studied since it can affect the effectiveness of eradication campaigns in cattle. The health problem is enhanced when it concerns also wildlife welfare and biodiversity conservation. This study was performed to understand the epidemiology of Mycobacterium bovis population affecting livestock and wild animals in the Do?ana National Park using bacteriology and molecular characterisation techniques. Tuberculosis research was performed on 1209 cattle and wild animals (artiodactyla and carnivore) collected over 6 years in the Park. One hundred and sixty-three animals were found to be infected with M. bovis, comprising 7.96% of the cattle and 20.53% of the wild animals tested. Spoligotyping revealed nine patterns, being SB1232 and SB1230 the most prevalent (77.30% and 15.34% of infected animals, respectively). MIRU-VNTR analysis of a selected panel of 92 isolates showed eight different profiles, including several spoligotypes within the same MIRU-VNTR profile. The discriminatory capacity of both techniques in this panel was similar. The results obtained by combination of both techniques corroborate that wildlife species are infected with the M. bovis strains which are more prevalent in cattle and reveal their persistence. Genotype variation between isolates strongly suggests micro-evolutionary events in the M. bovis population in the same area. This study in the Do?ana National Park exposes the risk of introduction of domestic animals into wildlife areas when there is not a warranty of disease freedom, appropriate diagnostic techniques and control measures.