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.Instituto de BiotecnologíaFil: Bacanelli, Gisele. Federal University of Mato Grosso do Sul. Biotechnology and Biodiversity of the Central Western Region Postgraduate Program; BrasilFil: Olarte, Larissa C. Federal University of Mato Grosso do Sul. Biochemistry and Molecular Biology Multicentric Postgraduate Program; BrasilFil: Silva, Marcio Roberto. Empresa Brasileira de Pesquisa Agropecuária (Embrapa). Gado de Leite; BrasilFil: Rodrigues, Rudielle A. Federal University of Mato Grosso do Sul. Faculty of Veterinary Medicine. Veterinary Sciences Postgraduate Program; BrasilFil: Carneiro, Paulo A. M. Michigan State University. Center for Comparative Epidemiology; Estados UnidosFil: Kannene, John B. Michigan State University. Center for Comparative Epidemiology; Estados UnidosFil: Pasquatti, Taynara N. Dom Bosco Catholic University; BrasilFil: Takatani, Haruo. Agricultural Defense Agency of Amazonas; BrasilFil: Zumarraga, Martin Jose. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Etges, Rodrigo N. Secretary of Agriculture, Livestock and Irrigation; BrasilFil: Araujo, Flabio Ribeiro de. Empresa Brasileira de Pesquisa Agropecuária (Embrapa). Gado de Corte; BrasilFil: Verbisck, Newton V. Empresa Brasileira de Pesquisa Agropecuária (Embrapa). Gado de Corte; Brasi

Analysis of membrane protein genes in a Brazilian isolate of Anaplasma marginale

The sequencing of the complete genome of Anaplasma marginale has enabled the identification of several genes that encode membrane proteins, thereby increasing the chances of identifying candidate immunogens. Little is known regarding the genetic variability of genes that encode membrane proteins in A. marginale isolates. The aim of the present study was to determine the degree of conservation of the predicted amino acid sequences of OMP1, OMP4, OMP5, OMP7, OMP8, OMP10, OMP14, OMP15, SODb, OPAG1, OPAG3, VirB3, VirB9-1, PepA, EF-Tu and AM854 proteins in a Brazilian isolate of A. marginale compared to other isolates. Hence, primers were used to amplify these genes: omp1, omp4, omp5, omp7, omp8, omp10, omp14, omp15, sodb, opag1, opag3, virb3, VirB9-1, pepA, ef-tu and am854. After polimerase chain reaction amplification, the products were cloned and sequenced using the Sanger method and the predicted amino acid sequence were multi-aligned using the CLUSTALW and MEGA 4 programs, comparing the predicted sequences between the Brazilian, Saint Maries, Florida and A. marginale centrale isolates. With the exception of outer membrane protein (OMP) 7, all proteins exhibited 92-100% homology to the other A. marginale isolates. However, only OMP1, OMP5, EF-Tu, VirB3, SODb and VirB9-1 were selected as potential immunogens capable of promoting cross-protection between isolates due to the high degree of homology (over 72%) also found with A. (centrale) marginale

Development of enzyme-linked immunosorbent assays based on recombinant MSP1a and MSP2 of Anaplasma marginale

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant MSP1a and MSP2 from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. The high sensitivities (99% for both tests) and specificities (100% for both tests) were confirmed with sera from cattle positive or negative for A. marginale antibodies, respectively, by immunofluorescent antibody test. By the analysis of 583 sera from cattle of three regions of the state of Pernambuco, Brazil, the agreement between both tests was high, with a kappa index of 0.89. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle