Klaudia S.G. Jorge

Mycobacterium bovis identification by a molecular method from post-mortem inspected cattle obtained in abattoirs of Mato Grosso do Sul, Brazil

The presence of Mycobacterium bovis in bovine carcasses with lesions suggestive of tuberculosis was evaluated. Seventy-two carcass samples were selected during slaughter inspection procedures in abattoirs in the state of Mato Grosso do Sul, Brazil. Seventeen (23.6%) of samples showed colonies suggestive of mycobacteria that were confirmed to be acid-fast bacilli by Ziehl-Neelsen staining. Polymerase chain reaction (PCR) using primers specific for M. bovis identified M. bovis in 13 (76.5%) isolates. The PCR-restriction enzyme pattern analysis using gene encoding for the 65-kDa protein and two restriction enzymes identified the remaining four isolates that were represented by two M. tuberculosis complex and two nontuberculous mycobacteria. The results are indicative of infection of slaughter cattle by M. bovis and other mycobacteria in the state of Mato Grosso do Sul.

Detection of Mycobacterium bovis in bovine and bubaline tissues using nested-PCR for TbD1.

In the present study, a nested-PCR system, targeting the TbD1 region, involving the performance of conventional PCR followed by real-time PCR, was developed to detect Mycobacterium bovis in bovine/bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. In terms of analytical sensitivity, the DNA of M. bovis AN5 was detected up to 1.56 ng with conventional PCR, 97.6 pg with real-time PCR, and 1.53 pg with nested-PCR in the reaction mixture. The nested-PCR exhibited 100% analytical specificity for M. bovis when tested with the DNA of reference strains of environmental mycobacteria and closely-related Actinomycetales. A clinical sensitivity value of 76.0% was detected with tissue samples from animals that exhibited positive results in the comparative intradermal tuberculin test (CITT), as well as from those with lesions compatible with tuberculosis (LCT) that rendered positive cultures. A clinical specificity value of 100% was detected with tissue samples from animals with CITT- results, with no visible lesions (NVL) and negative cultures. No significant differences were found between the nested-PCR and culture in terms of detecting CITT+ animals with LCT or with NVL. No significant differences were recorded in the detection of CITT- animals with NVL. However, nested-PCR detected a significantly higher number of positive animals than the culture in the group of animals exhibiting LCT with no previous records of CITT. The use of the nested-PCR assay to detect M. bovis in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.

Direct detection of Mycobacterium tuberculosis complex in bovine and bubaline tissues through nested-PCR

Post-mortem bacterial culture and specific biochemical tests are currently performed to characterize the etiologic agent of bovine tuberculosis. Cultures take up to 90 days to develop. A diagnosis by molecular tests such as PCR can provide fast and reliable results while significantly decreasing the time of confirmation. In the present study, a nested-PCR system, targeting rv2807, with conventional PCR followed by real-time PCR, was developed to detect Mycobacterium tuberculosis complex (MTC) organisms directly from bovine and bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other Actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. Regarding the analytical sensitivity, DNA of the M. bovis AN5 strain was detected up to 1.5 pg by nested-PCR, whereas DNA of M. tuberculosis H37Rv strain was detected up to 6.1 pg. The nested-PCR system showed 100% analytical specificity for MTC when tested with DNA of reference strains of non-tuberculous mycobacteria and closely-related Actinomycetales. A clinical sensitivity level of 76.7% was detected with tissues samples positive for MTC by means of the culture and conventional PCR. A clinical specificity of 100% was detected with DNA from tissue samples of cattle with negative results in the comparative intradermal tuberculin test. These cattle exhibited no visible lesions and were negative in the culture for MTC. The use of the nested-PCR assay to detect M. tuberculosis complex in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.

Resposta imune específica de bovinos experimentalmente sensibilizados com inóculos inativados de Mycobacterium bovis e Mycobacterium avium

The presumptive diagnosis of bovine tuberculosis is based on analysis of the immune response to micobacterial antigens. This experimental simulation of sensibilization by Mycobacterium bovis and Mycobacterium avium in cattle aimed to verify the immune response by both the cervical comparative test and the evolution of the specific production of gamma-interferon, and also to identify interference of unspecified reactions by M. avium on the test results. The results support that the experimental animals started a response of delayed hypersensitivity to the inactivated bacilli, and that both diagnostic tests for bovine tuberculosis were efficient for the identification of animals sensitized with M. bovis and for discrimination of reactions generated by inoculation of cattle with M. avium.

Patterns and Processes of Mycobacterium bovis Evolution Revealed by Phylogenomic Analyses

Mycobacterium bovis is an important animal pathogen worldwide that parasitizes wild and domesticated vertebrate livestock as well as humans. A comparison of the five M. bovis complete genomes from the United Kingdom, South Korea, Brazil, and the United States revealed four novel large-scale structural variations of at least 2,000 bp. A comparative phylogenomic study including 2,483 core genes of 38 taxa from eight countries showed conflicting phylogenetic signal among sites. By minimizing this effect, we obtained a tree that better agrees with sampling locality. Results supported a relatively basal position of African strains (all isolated from Homo sapiens), confirming that Africa was an important region for early diversification and that humans were one of the earliest hosts. Selection analyses revealed that functional categories such as “Lipid transport and metabolism,” “Cell cycle control, cell division, chromosome partitioning” and “Cell motility” were significant for the evolution of the group, besides other categories previously described, showing importance of genes associated with virulence and cholesterol metabolism in the evolution of M. bovis. PE/PPE genes, many of which are known to be associated with virulence, were major targets for large-scale polymorphisms, homologous recombination, and positive selection, evincing for the first time a plethora of evolutionary forces possibly contributing to differential adaptability in M. bovis. By assuming different priors, US strains originated and started to diversify around 150–5,210 ya. By further analyzing the largest set of US genomes to date (76 in total), obtained from 14 host species, we detected that hosts were not clustered in clades (except for a few cases), with some faster-evolving strains being detected, suggesting fast and ongoing reinfections across host species, and therefore, the possibility of new bovine tuberculosis outbreaks.

ELISA BASEADO EM MPB70 E P27 RECOMBINANTES PARA DETECÇÃO DE ANTICORPOS CONTRA Mycobacterium bovis

Bovine tuberculosis is a major disease caused by the bacterium Mycobacterium bovis. Skin tests and slaughter policies have reduced the incidence of bovine tuberculosis in many countries. However, more practical and efficient tools with high sensitivity and specificity values are needed. The aim of the present study was to develop an ELISA using the recombinant proteins MPB70 and p27 from M. bovis in order to detect antibodies against this bacterium in cattle. Sensitivity and specificity were respectively 88.7% and 94.6% for ELISA-MPB70 and 98.1% and 91.9% for ELISA-p27. The use of a serological test such as ELISA with recombinant MPB70 and p27, together with cell tests, may solve some of the problems regarding the diagnosis of bovine tuberculosis, such as inconclusive results and the lack of detection in anergic animals in advanced stages of the disease.

Identificação e genotipagem de Mycobacterium bovis em bovinos positivos no teste intradérmico para tuberculose em Mato Grosso do Sul

Spoligotyping was performed in the present study to genotype Mycobacterium bovis isolates obtained from tissues of cattle that were positive in the comparative intradermal tuberculin test (CITT) in the state of Mato Grosso do Sul (Brazil). Tissue samples from 13 positive cattle from different municipalities of the state were cultured using a Stonebrink medium. The resulting colonies were subjected to Ziehl-Neelsen staining and all isolates exhibited the staining characteristics of AFB. The 13 isolates of AFB were identified by means of a multiplex PCR (mPCR) assay. The hsp65 gene was targeted for the identification of Mycobacterium spp., whereas the IS6110 insertion sequence was targeted for the identification of the Mycobacterium tuberculosis complex (MTC) and the rvd1rv2031c region was explored for the detection of Mycobacterium bovis. The spoligotyping assay was performed to genotype mycobacterial isolates. Of the 13 cattle, seven had at least one lesion suggestive of tuberculosis in the retropharyngeal, parotid and lung lymph nodes or lung. The remaining six exhibited no lesions suggestive of the disease. In the mPCR, 11 of the 13 isolates (84.6%) were positive for Mycobacterium spp., 8/13 (61.5%) were positive for the MTC and 7/13 (53.8%) were positive for M. bovis. Based on the spoligotyping, eight isolates were grouped into three different groups of genotypes and one isolate exhibited an orphan type. Four isolates exhibited spoligotype pattern SB0121, while two isolates were associated with the pattern SB1145, another two were associated with pattern SB0881 and one was associated with pattern SB0140. Spoligotyping confirmed the genetic diversity present among isolates found in the state of Mato Grosso do Sul. In addition, SB0121 was confirmed as the predominant profile.

ELISA using a recombinant chimera of ESAT-6/MPB70/MPB83 for Mycobacterium bovis diagnosis in naturally infected cattle

Bovine tuberculosis (bTB) control programs generally rely on intradermal tuberculin tests for the antemortem diagnosis of Mycobacterium bovis infection in cattle, but these tests detect only a portion of the infected animals. The aim of the present study was to evaluate the diagnostic coverage of a combination of the bTB antemortem techniques known as the comparative intradermal tuberculin test (CITT) and an ELISA based on a recombinant chimera of ESAT-6/MPB70/MPB83 as the antigen in cattle. The results were compared to postmortem findings based on M. bovis culturing and PCR. Paired comparisons of all data (n=92) demonstrated that ELISA and LST results compared to the culturing results did not present significant differences (P=0.27 on McNemar’s test and P=0.12 on Fisher’s exact test, respectively). Using culturing as the gold standard, the sensitivity and specificity of ELISA were 79.5% (95% CI: 64.5–89.2%) and 75.5% (95% CI: 62.4–85.1%), respectively, whereas LST demonstrated 100% sensitivity (95% CI: 91.03–100%) and 92.5% specificity (95% CI: 82.1–97.0%). The ELISA results did not reveal significant differences in relation to the LST results (P>0.99 on Fisher’s exact test). Using the latter as the gold standard, the sensitivity and specificity of ELISA were 79.1% (95% CI: 64.8–88.6%) and 79.6% (95% CI: 66.4–88.5%), respectively. The use of ELISA with the recombinant chimera of ESAT-6/MPB70/MPB83 as the antigen complements the diagnostic coverage provided by CITT and increases the removal of infected animals from herds.