title: Detection of Mycobacterium bovis in bovine and bubaline tissues using nested-PCR for TbD1.
abstract: 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.
authors.affiliation: Programa de Pós-graduação em Ciência Animal FAMEZ, UFMS, Campo Grande, MS, Brazil.
authors.forename: Cristina P
authors.initials: CP
authors.lastname: Araújo
authors.affiliation: Programa de Pós-graduação em Ciência Animal FAMEZ, UFMS, Campo Grande, MS, Brazil.
authors.forename: Ana Luiza A R
authors.initials: AL
authors.lastname: Osório
authors.affiliation: Programa de Pós-graduação em Ciência Animal FAMEZ, UFMS, Campo Grande, MS, Brazil.
authors.forename: Kláudia S G
authors.initials: KS
authors.lastname: Jorge
authors.affiliation: Bolsista DTI, CNPq, Campo Grande, MS, Brazil.
authors.forename: Carlos Alberto N
authors.initials: CA
authors.lastname: Ramos
authors.affiliation: Programa de Pós-graduação em Ciência Animal FAMEZ, UFMS, Campo Grande, MS, Brazil.
authors.forename: Antonio Francisco S
authors.initials: AF
authors.lastname: Filho
authors.affiliation: PPGMV, UFSM, Santa Maria, RS, Brazil.
authors.forename: Carlos Eugênio S
authors.initials: CE
authors.lastname: Vidal
authors.affiliation: Instituto Biológico de São Paulo, São Paulo, SP, Brazil.
authors.forename: Eliana
authors.initials: E
authors.lastname: Roxo
authors.affiliation: Faculdade de Computação, UFMS, Campo Grande, MS, Brazil.
authors.forename: Christiane
authors.initials: C
authors.lastname: Nishibe
authors.affiliation: Faculdade de Computação, UFMS, Campo Grande, MS, Brazil.
authors.forename: Nalvo F
authors.initials: NF
authors.lastname: Almeida
authors.affiliation: Laboratório Nacional Agropecuário, Pedro Leopoldo, MG, Brazil.
authors.forename: Antônio A F
authors.initials: AA
authors.lastname: Júnior
authors.affiliation: Embrapa Gado de Leite, Juiz de Fora, MG, Brazil.
authors.forename: Marcio R
authors.initials: MR
authors.lastname: Silva
authors.affiliation: UFPA, Castanhal, PA, Brazil.
authors.forename: José Diomedes B
authors.initials: JD
authors.lastname: Neto
authors.affiliation: UFPA, Castanhal, PA, Brazil.
authors.forename: Valíria D
authors.initials: VD
authors.lastname: Cerqueira
authors.affiliation: Instituto de Biotecnología, Instituto Nacional de Tecnología Agropecuaria, Buenos Aires, Argentina.
authors.forename: Martín J
authors.initials: MJ
authors.lastname: Zumárraga
authors.affiliation: Embrapa Gado de Corte, Campo Grande, MS, Brazil.
authors.forename: Flábio R
authors.initials: FR
authors.lastname: Araújo
mesh_terms: D000818:Animals; D002417:Cattle; D004269:DNA, Bacterial / Q000235:genetics*; D009163:Mycobacterium bovis / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D014380:Tuberculosis, Bovine / Q000175:diagnosis* / Q000382:microbiology*
chemical_list: D004269:DNA, Bacterial