Molecular typing of Mycobacterium bovis isolates from south-east Brazil by spoligotyping and RFLP

The identification of 163 strains of Mycobacterium bovis by polymerase chain reaction (PCR) and microbiological tests was carried out on 252 tuberculous-like lesions (TLLs) collected from slaughtered cattle in south-east Brazil. This study compared the usefulness of three genotyping techniques, IS6110-restriction fragment length polymorphism (RFLP), polymorphic guanine-cytosine-rich sequence (PGRS)-RFLP and direct repeat (DR)-spoligotyping, as applied to M. bovis isolates. Based on IS6110-RFLP genotyping we selected a group of 23 isolates containing more than one IS6110 copy, along with 16 samples containing one IS6110 copy from different geographical areas, evenly distributed among dairy (eight) and beef cattle (eight). These selected isolates were analysed by PGRS-RFLP and DR-spoligotyping genotyping. Dairy cattle (17%) display a higher frequency of multiple IS6110 copies than beef cattle (10%). A comparison between the genotype data obtained fails to show a correlation between the main clusters found by the three techniques. However, the clustering of each genotyping procedure revealed that the majority of strains are closely related. The RFLP-PGRS patterns showed a sizable group (20.5%) containing a 5.5 kb fragment and the predominant spoligotype is similar to that from the BCG vaccine strain. Unexpectedly, four strains (2.4%) showed drug resistance to 0.2 microg/ml isoniazid and 20 microg/ml ethionamide, but none of them was resistant to rifampicin or other antibiotics tested.     

Spoligotype diversity of Mycobacterium bovis and Mycobacterium caprae animal isolates

The genetic diversity of 283 Mycobacterium bovis (M. bovis) and 10 Mycobacterium caprae (M. caprae) strains, isolated between 2002 and 2007 from cattle, goat, red deer and wild boar from six different geographical regions of Portugal was investigated by spoligotyping. The technique showed a good discriminatory power (Hunter-Gaston Index, h=0.9) for the strains, revealing 29 different patterns. One pattern (SB0121) was clearly predominant, accounting for 26.3% of the isolates; ten patterns, representing 20.7% of the isolates, had never been reported previously. Multiple spoligotypes were detected in thirteen cattle and one goat herd, most of which were found in beef cattle and extensive management regions, suggesting different infection sources. With the exception of two spoligotypes, those in wildlife species were also found in domestic species.     

Evaluation of variable number tandem repeat (VNTR) loci in molecular typing of Mycobacterium bovis isolates from Ireland

Various sets of short tandem repeats such as the exact tandem repeats (ETRs), mycobacterial interspersed repetitive units (MIRUs) and variable number tandem repeat (VNTR) loci, have recently been described as effective tools in strain typing M. tuberculosis complex isolates, representative of global diversity. This study extends our previous study, evaluating the discrimination of a further 17 MIRU_VNTR loci individually and comparing the resolution of published VNTR sets and spoligotyping using a panel of 47 local M. bovis field isolates, including known epidemiologically linked isolates and 9 M. tuberculosis complex reference isolates. Individual loci differed greatly in their discrimination. The discriminatory capacity of novel combinations of the most discriminating VNTR loci was also assessed. In the panel of 47 M. bovis isolates, 17 unique profiles were resolved using VNTR set 1, whilst the MIRUs and ETRs resolved the panel into 11 and 6 profiles, respectively. A novel combination of 10 highly discriminatory VNTRs was determined, which resolved 30 unique profiles. The configuration of a multi-locus VNTR-based assay and its ability to provide a flexible, convenient and high-resolution genotyping method is discussed. We suggest a panel of VNTR markers which may be widely suitable for molecular epidemiological studies of M. bovis. However, the number and combination of informative VNTR markers selected needs to be determined empirically with reference to locally prevalent strains and will depend on the epidemiological study requirements.     

Population structure of Mycobacterium bovis isolates from cattle in Mexico

The molecular fingerprints of 878 isolates of Mycobacterium bovis collected from cattle between 2009 and 2010 in different regions of Mexico were used in this study. One hundred and ninety-four spoligotypes were observed in total with a high degree of heterogeneity. Sixty-four percent of the isolates grouped into just nine spoligotypes, and 27% fell into only two spoligotypes: SB0673 and SB0669; 149 were orphan spoligotypes. The two predominant spoligotypes were found in almost all states in Mexico, especially in central Mexico, where there is a high concentration of dairy cattle; however, some spoligotypes were closely associated with restricted geographical areas. The hypothetical evolutionary relationship among spoligotypes was estimated using the spoligoforest program in the spolTools webpage. Four trees with connected components and nine unconnected nodes were found. The biggest tree had SB0140 strain as a root, suggesting this as the oldest strain in the tree. However, the relationship of this spoligotype with SB0673 and SB0669 was weak. The discriminatory power of spoligotyping for this M. bovis sample of isolates was 0.94, and the recent transmission index (RTI) 0.83, suggesting a high rate of recent transmission of some strains of M. bovis in the population. This parameter indicates that new measures are required to stop the dissemination of tuberculosis in cattle.     

Tuberculosis due to Mycobacterium bovis and Mycobacterium caprae in sheep

Tuberculosis was diagnosed in three flocks of sheep in Galicia, Spain, in 2009 and 2010. Two flocks were infected with Mycobacterium bovis and one flock was infected with Mycobacterium caprae. Infection was confirmed by the comparative intradermal tuberculin test, bacteriology, molecular analysis and histopathology. Sheep have the potential to act as a reservoir for tuberculosis.     

Genotypic diversity in field isolates of Babesia bovis from cattle with babesiosis after vaccination

Objective To determine whether particular genotypes of Babesia bovis were common to field isolates obtained from cattle properties in Queensland where the B bovis vaccine had apparently failed.
Design A comparative study of polymerase chain reaction genotypes in different populations of B bovis .
Procedure Two polymerase chain reaction assays were applied to analyse DNA extracts of B bovis vaccine (K, T and Dixie strains) and 27 field isolates from 24 properties where disease outbreaks had occurred despite the use of the vaccine. To evaluate the stability of the genotypes identified, 11 of the field isolates were inoculated into experimental cattle that had either been previously vaccinated with T strain or not vaccinated.
Results No particular genotype of B bovis was responsible for the problems observed in previously vaccinated herds. None of the isolates had genotypes identical to the vaccine strains used. No geographic trends among the genotypes were observed. Isolates that originated from the same property also had different genotypes. Blood passage of the 11 field isolates in either previously vaccinated or nonvaccinated cattle did not alter the original genotype.
Conclusion No particular genotypes identified by the Bv80 and BvVA1 polymerase chain reaction assays could be associated with vaccine failures.     

Simultaneous detection and strain differentiation of Mycobacterium bovis directly from bovine tissue specimens by spoligotyping

Culture of Mycobacterium bovis is used routinely to support field diagnosis of bovine tuberculosis; however, this method is slow. Rapid detection and strain-typing of M. bovis directly from 37 lesioned bovine lymph node specimens was performed by the polymerase chain reaction (PCR) based method, spoligotyping. Mycobacterial DNA was extracted from the specimens using a nucleic acid sequence capture technique. Two sets of specimens were tested, the first set comprising 16 decontaminated tissue homogenates from lesioned lymph node specimens which had been processed for BACTEC culture and a second set of 21 non-decontaminated lesioned lymph node specimens. Both sets of specimens had been frozen before analysis. Sequence capture PCR enabled detection and strain-typing of M. bovis directly from 15 of the 16 decontaminated homogenates and all 21 of the non-decontaminated tissues. Four spoligotype (ST) patterns were obtained from each set; ST1, ST2, ST3 and ST16 were detected in the decontaminated specimens and ST1, ST2, ST11 and ST14 in the non-decontaminated specimens. For both sets of specimens, ST1 was the predominant strain type detected. ST patterns obtained from the BACTEC cultures of the decontaminated specimens were in agreement with those obtained directly from the tissue. The sensitivity of detection by sequence capture-PCR compared very favourably with that of BACTEC culture. ST patterns were obtained directly from tissues of 34 of the 35 culture positive specimens and the two culture negative specimens. DNA extraction from the 21 non-decontaminated specimens involved an initial stomaching treatment. An assessment of sequence capture on both liquid alone and liquid and tissue homogenate combined, following stomaching, indicated that PCR was less successful on the liquid component alone.     

Typing of Mycobacterium bovis isolates from cattle and other animals in the same locality

The effects of high doses of the beta-2 agonists iso-prenaline, salbutamol and fenoterol on the myocardium were studied experimentally in sheep. Each drug was given intravenously in progressively increasing doses to four sedated animals and four controls. The experiments were repeated during hypoxaemia and animals were necropsied 3 days later.     

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.     

Excretion of Mycobacterium bovis by experimentally infected cattle

Three groups, each of five calves, four to seven months old, were inoculated intranasally with different numbers of Mycobacterium bovis. Infection was established readily in the calves which received an inoculum containing either 10(6) or 10(4) colony forming units (cfu). After every infection there was a lag period during which the organisms could not be isolated from specimens of nasal mucus. All the animals excreted M bovis and the time of commencement, quantity and duration of excretion appeared to be related to the inoculation dose. Excretion continued for many weeks, and for two calves excretion became intermittent over many months. All the calves which were given inocula of 92 cfu failed to develop the disease and no immunological responses were detected; however, M bovis was isolated from nasal secretions from one of these animals 100 days after inoculation.     

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   

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.     

High-throughput multiplex MIRU-VNTR typing of Mycobacterium bovis

Spoligotyping is the most widely used method for genotyping Mycobacterium bovis (M. bovis). However, its discriminatory power varies widely between countries. MIRU-VNTR typing could be a promising alternative, although it generally requires the time consuming and laborious simplex PCR assays using standard agarose gel electrophoresis. The accuracy of this approach depends on good standardization and a certain degree of expertise. This study presents a version of MIRU-VNTR based on three triplex PCRs with automatic allelic assignation of the products analyzed in capillary electrophoresis. The technique was prospectively applied to 44 M. bovis and two Mycobacterium caprae (M. caprae) isolates, and 22 different MIRU-VNTRtypes were obtained; with spoligotyping, only 14 different types were obtained. The proposal makes it possible to shorten response times, automate procedures, and increase accuracy, thus minimizing errors in assigning genotypes. It would enable the switch from a standard limited method of genotyping M. bovis to a high-throughput discriminatory fingerprinting approach.     

Mycobacterium bovis infection and tuberculosis in cattle

This review considers the possible events that can occur when cattle are exposed to Mycobacterium bovis and, where appropriate, draws on principles accepted for tuberculosis infection in humans and laboratory animal models. Consideration is given to the many complex factors which influence the outcome of challenge with tubercle bacilli. These include features inherent to the mycobacterium, the host and the environment. It is apparent that clinical disease probably occurs only in a relatively small, but undetermined, proportion of cattle that are exposed to Al. bovis. The majority of animals may clear infection or control the bacilli, possibly in a condition of latency. It is concluded that a better understanding of the dynamics of the events following M. bovis exposure and subsequent infection in cattle would be of significant benefit in developing new tools appropriate for disease control and to designing optimal approaches for their application.     

Genotypic diversity in Babesia bovis field isolates and vaccine strains from South Africa

Genotypic diversity in Babesia bovis (cause of Asiatic redwater in cattle) vaccine strains and field isolates from South Africa were investigated using the Bv80 gene as well as microsatellites. The S11 vaccine strain possessed both A and B alleles of the Bv80 gene, as well as genotypic diversity within each allele type as defined by repeat variation resulting in different amplicon sizes. Rapid serial passage of vaccine strain from passage S10 to S24 resulted in loss of genotypic diversity that yielded a single allele A genotype with an amplicon size of 558 bp. This suggested that clonal selection occurred during rapid passaging. Extensive genotypic diversity exists in 44 field isolates characterized with both Bv80 A and B alleles, but can be readily distinguished from the S24 vaccine strain using either the Bv80 allele specific PCR assays or using multi-locus micro-satellite typing. This indicated that no recent documented clinical cases of Asiatic redwater were caused by the reversion to virulence of the current vaccine strain.     

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.     

A mathematical model for Mycobacterium bovis excretion from tuberculous cattle

An analysis was carried out of available information from a series of experiments on the excretion of M. bovis from infected cattle. The analysis indicated that an inverse exponential relationship exists between 'dose' of organisms given and the delay before excretion commences. This relationship was represented mathematically. Available field data supported the relationship and indicated that in natural bovine tuberculosis excretion of M. bovis begins around 87 days after infection occurs. It is also suggested that the data supports the concept of single nuclei infections in cattle.