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

False-negative reactions to the comparative intradermal tuberculin test for bovine tuberculosis

ABSTRACT: According to the Brazilian National Program for the Control and Eradication of Animal Brucellosis and Tuberculosis (PNCEBT), the routine tests for the diagnosis of bovine tuberculosis in the country are the simple intradermal tuberculin test (SITT) of the Ministry of Agriculture, Livestock and Food Supply (MAPA), the caudal fold test and the comparative intradermal tuberculin test (CITT). The latter is also used as a confirmatory test. A group of 53 animals from three dairy herds in a focal area for bovine tuberculosis, that were submitted to depopulation in the state of Rio Grande do Sul, were submitted to the CITT. Tissues were cultured and the resulting colonies were confirmed by PCR and DNA sequencing. Among the 53 animals analyzed using the CITT, 32 (60.4%) were negative, 14 (26.4%) were positive and seven (13.2%) results were inconclusive. The CITT detected 11 of the 39 animals with culture-confirmed M. bovis infection as positive. Among the total of 14 uninfected animals based on cultures, the CBT detected eight as negative. Thus, the CITT demonstrated sensitivity of 28.2% and specificity of 57.1% for the population sampled. A total of 24/32 (75.0%) of the animals with negative CITT results were culture positive (confirmed by PCR) and were considered false negatives based on the CITT. The maintenance of these false-negative animals in herds has serious implications for the control of the disease, since they can be a source of infection. The addition of complementary tests could help identify such animals and increase the odds of diagnostic success