Molecular characterization of Mycobacterium bovis infection in cattle and buffalo in Amazon Region, Brazil.

The aim of this study was to characterize Mycobacterium bovis from cattle and buffalo tissue samples, from two Brazilian states, and to analyse their genetic diversity by spoligotyping. Tissue samples from tuberculosis suspect animals, 57 in Amazonas State (12 cattle and 45 buffaloes) and six from Pará State (5 cattle and one buffalo) from slaughterhouses under State Veterinary Inspection, were isolated in culture medium Stonebrink. The positive cultures were confirmed by PCR and analysed by the spoligotyping technique and the patterns (spoligotypes) were identified and compared at the Mycobacterium bovis Spoligotype Database (http://www.mbovis.org/). There was bacterial growth in 44 (69.8%) of the tissues of the 63 animals, of which PCR for region of differentiation 4 identified 35/44 (79.5%) as Mycobacterium bovis. Six different spoligotypes were identified among the 35 Mycobacterium bovis isolates, of which SB0295, SB1869, SB0121 and SB1800 had already been described in Brazil, and SB0822 and SB1608 had not been described. The most frequent spoligotype in this study (SB0822) had already been described in buffaloes in Colombia, a neighbouring country of Amazonas state. The other identified spoligotypes were also described in other South American countries, such as Argentina and Venezuela, and described in the Brazilian states of Rio Grande do Sul, Santa Catarina, São Paulo, Minas Gerais, Mato Grosso do Sul, Mato Grosso and Goiás, indicating an active movement of Mycobacterium bovis strains within Brazil.

Matrix Assisted Laser Desorption Ionization-Time-of-Flight mass spectrometry identification of Mycobacterium bovis in Bovinae.

In this study, Matrix Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) mass spectrometry was used to identify Mycobacterium bovis from cattle and buffalo tissue isolates from the North and South regions of Brazil, grown in solid medium and previously identified by Polymerase Chain Reaction (PCR) based on Region of Difference 4 (RD4), sequencing and spoligotyping. For this purpose, the protein extraction protocol and the mass spectra reference database were optimized for the identification of 80 clinical isolates of mycobacteria. As a result of this optimization, it was possible to identify and differentiate M. bovis from other members of the Mycobacterium tuberculosis complex with 100% specificity, 90.91% sensitivity and 91.25% reliability. MALDI-TOF MS methodology described herein provides successful identification of M. bovis within bovine/bubaline clinical samples, demonstrating its usefulness for bovine tuberculosis diagnosis in the future.

Epidemiological Study of Mycobacterium bovis Infection in Buffalo and Cattle in Amazonas, Brazil.

Bovine Tuberculosis (BTB) is an endemic disease in about one hundred countries, affecting the economy causing a decrease in productivity, condemnation of meat, and damaging the credibility on international trade. Additionally, Mycobacterium bovis the major causative agent for BTB can also infect humans causing a variety of clinical presentations. The aim of this study was to determine BTB prevalence and the main risk factors for the Mycobacterium bovis prevalence in cattle and buffalos in Amazonas State, Brazil. Tissue samples from 151 animals (45 buffalo and 106 cattle from five herds with buffalo only, 22 herds with cattle only, and 12 herds with buffalo and cattle) were obtained from slaughterhouses under State Veterinary Inspection. M. bovis were isolated on Stonebrink medium. The positive cultures were confirmed by polymerase chain reaction (PCR) testing. The apparent herd and animal prevalence rates were 56.4 and 5.40%, respectively. Regarding animal species, the apparent prevalence rates were 3% in cattle and 11.8% in buffalo. Generalized Linear Mixed Models (GLMM) with random effect were used to assess the association with risk factors on the prevalence. Species (buffalo), herds size (>100 animals) and the presence of both species (buffalo and cattle) in the herd were the major risk factors for the infection by Mycobacterium bovis in the region. The findings reveal an urgent need for evidence-based effective intervention to reduce BTB prevalence in cattle and buffalo and prevent its spread to the human population. Studies are needed to understand why buffalo are more likely to be infected by M. bovis than cattle in Amazon. Recommendations for zoning, use of data from the inspection services to generate information regarding BTB focus, adoption of epidemiological tools, and discouragement of practices that promote the mixing of cattle and buffalo, were made.

Identificação por espectrometria de massa MALDI-TOF de Salmonella spp. e Escherichia coli isolados de carcaças bovinas / MALDI-TOF mass spectrometry identification of Salmonella spp. and Escherichia coli isolated from bovine carcasses

O objetivo deste trabalho foi introduzir a técnica de espectrometria de massa com fonte de ionização e dessorção a laser assistida por matriz e analisador de tempo-de-voo (MALDI-TOF) para incrementar o método tradicional microbiológico na detecção de Salmonella spp. e Escherichia coli em carcaças bovinas. Foram avaliadas 270 amostras de 90 carcaças de bovinos. Para isolamento de Salmonella spp. e E. coli, foram utilizadas, respectivamente, as metodologias descritas na ISO 6579:2002 e no Compendium of Methods for the Microbiological Examination of Foods. As análises por MALDI-TOF foram realizadas a partir de isolados cultivados em ágar nutriente ou em caldo triptona de soja, provenientes das amostras com características bioquímicas positivas (n=7), inconclusivas (n=4) e negativas (n=85) para Salmonella spp. e bioquímicas positivas (n=37) e negativas (n=85) para E. coli. Os perfis de massas foram adquiridos com o espectrômetro de massas MALDI-TOF Autoflex III SmartBeam e os espectros brutos foram processados usando o programa MALDI Biotyper (Bruker Daltonics). De acordo com a identificação preliminar, com base na morfologia das colônias e nas reações bioquímicas, sete isolados foram considerados positivos para Salmonella spp. Através do MALDI Biotyper, esses sete isolados foram classificados como pertencentes ao gênero Salmonella e, além disso, identificados como S. enterica. Quatro isolados que apresentaram características fenotípicas não usuais e resultados inconclusivos nos testes bioquímicos para Salmonella foram identificados como pertencentes aos gêneros Citrobacter e Proteus após análise por MALDI. Para E. coli, 37 amostras foram positivas pelos testes bioquímicos da espécie, o que foi confirmado por MALDI Biotyper. A metodologia MALDI-TOF permitiu a rápida confirmação da identidade de Salmonella spp. e E. coli, podendo ser utilizada para detecção desses microrganismos em isolados bacterianos de carcaças bovinas.(AU)

The aim of this study was to introduce matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry to improve the traditional microbiological method for the detection of Salmonella spp. and Escherichia coli in beef carcasses. Two hundred seventy samples from 90 beef carcasses were evaluated. The methodologies described in ISO 6579:2002 and in the Compendium of Methods for the Microbiological Examination of Foods were used for Salmonella spp. and E. coli isolation, respectively. MALDI-TOF analysis were performed on tryptone soya broth suspension isolates or directly from nutrient agar colonies, from the positive, inconclusive or negative biochemically tested samples for Salmonella and E. coli. Mass profiles were acquired on an Autoflex III SmartBeam MALDI-TOF mass spectrometer and the raw spectra were processed using the MALDI Biotyper software (Bruker Daltonics). According to the preliminary identification based on colony morphology and the biochemical reactions, seven isolates were positive for Salmonella spp. Through MALDI Biotyper these seven isolates were also classified as belonging to the genus Salmonella and further identified as S. enterica. Four isolates showing unusual phenotypic characteristics and inconclusive results in biochemical tests for Salmonella were identified as belonging to Citrobacter and Proteus genera after MALDI analysis. Regarding Escherichia coli, 37 were positive for species biochemical testing which MALDI Biotyper confirmed. MALDI-TOF methodology allowed rapid Salmonella spp. and E. coli identity confirmation and may be used to detect these microrganisms within bacterial isolates from beef carcasses.(AU)