Daniela Fernandes Ramos

Molecular typing of Mycobacterium bovis isolates: a review

Mycobacterium bovis is the main causative agent of animal tuberculosis (TB) and it may cause TB in humans. Molecular typing of M. bovis isolates provides precise epidemiological data on issues of inter- or intra-herd transmission and wildlife reservoirs. Techniques used for typing M. bovis have evolved over the last 2 decades, and PCR-based methods such as spoligotyping and mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) have been extensively used. These techniques can provide epidemiological information about isolates of M. Bovis that may help control bovine TB by indicating possible links between diseased animals, detecting and sampling outbreaks, and even demonstrating cases of laboratory cross-contamination between samples. This review will focus on techniques used for the molecular typing of M. bovis and discuss their general aspects and applications.

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 typing of Mycobacterium bovis isolated in the south of Brazil

Bovine tuberculosis is a major infectious disease of the cattle. In this study, 85 M. bovis isolates from 162 lymph nodes, obtained from a herd of cattle on a farm in southern Brazil, were evaluated using spoligotyping and VNTR. The strains were grouped into five clusters and five orphans, showing a heterogenic genetic profile, what could represent diverse geographic origins of the introduced cows and/or the frequent movement of cattle between different properties.

Diagnosis of bovine tuberculosis: review of main techniques

Bovine tuberculosis (BTB) remains an important economic and zoonotic problem in Latin America. Traditionally, the fight against BTB is initiated by the implementation of routine diagnostic tests for certification of free properties. The diagnosis of BTB can be made by direct and indirect methods, in which we can mention clinical, post mortem, histopathological, immunological, bacteriological and molecular methods. The renewal of scientific interest in tuberculosis in recent year has led to develop and improve methods of diagnosis, prevention, control and eradication of BTB. The aim of this review is to present and discuss different diagnosis methods of BTB.

Anti-tuberculosis activity of oleanolic and ursolic acid isolated from the dichloromethane extract of leaves from Duroia macrophylla

Tuberculosis a major cause of death worldwide, is an infectious disease caused by Mycobacterium tuberculosis. An increase in drug-resistant tuberculosis cases and the emergence of additional resistant strains and coinfections with HIV has stimulated the search for and development of new anti-TB drugs [1]. The wide variety of natural products chemical structures plays a major role on the development of new antimycobacterial drugs generations. Duroia macrophylla is an endemic plant of the Amazon Forest [2]. To the best of our knowledge, no chemical or biological investigations other than ours [3,4] have been carried out on this species as of yet. Hence this work aims to evaluate the antimycobacterial activity of their extracts and isolate and identify the substances present in D. macrophylla active extracts.

Rational design of diagnostic and vaccination strategies for tuberculosis

The development of diagnostic tests which can readily differentiate between vaccinated and tuberculosis-infected individuals is crucial for the wider utilization of bacillus Calmette-Guérin (BCG) as vaccine in humans and animals. BCG_0092 is an antigen that elicits specific delayed type hypersensitivity reactions similar in size and morphological aspects to that elicited by purified protein derivative, in both animals and humans infected with the tubercle bacilli. We carried out bioinformatics analyses of the BCG_0092 and designed a diagnostic test by using the predicted MHC class I epitopes. In addition, we performed a knockout of this gene by homologous recombination in the BCG vaccine strain to allow differentiation of vaccinated from infected individuals. For that, the flanking sequences of the target gene (BCG_0092)were cloned into a suicide vector. Spontaneous double crossovers, which result in wild type revertants or knockouts were selected using SacB. BCG_0092 is present only in members of the Mycobacterium tuberculosis complex. Eight predicted MHC class I epitopes with potential for immunological diagnosis were defined, allowing the design of a specific diagnostic test. The strategy used to delete the (BCG_0092) gene from BCG was successful. The knockout genotype was confirmed by PCR and by Southern blot. The mutant BCG strain has the potential of inducing protection against tuberculosis without interfering with the diagnostic test based on the use of selected epitopes from BCG_0092.

Identification of Proteins from Tuberculin Purified Protein Derivative (PPD) with Potential for TB Diagnosis Using Bioinformatics Analysis

The PPD is widely used for diagnosis of bovine tuberculosis, however it lacks specificity and sensitivity, generally attributed the cross-reactions with antigens shared by other Mycobacterium species. These highlight the necessity of better diagnostic tools to detect tuberculosis in cattle. We have identified proteins present in PPD preparations (avium and bovine PPD) by LC-MS/MS (Liquid Chromatography/Mass Spectrometry/Mass Spectrometry). A total of 169 proteins were identified. From these, four PPD proteins identified in bovine PPD and absent in avium PPD (Mb1961c, Mb2898, Mb2900 and Mb2965c) were select for bioinformatics analysis to identify an antigen with potential for TB diagnosis. We identified Mb1961c, a secreted protein, which has low identity proteins found in environmental mycobacteria. This protein could be used for the development of a diagnostic test or a synthetic PPD for bovine tuberculosis.