title: Direct detection of Mycobacterium tuberculosis complex in bovine and bubaline tissues through nested-PCR.
abstract: Post-mortem bacterial culture and specific biochemical tests are currently performed to characterize the etiologic agent of bovine tuberculosis. Cultures take up to 90 days to develop. A diagnosis by molecular tests such as PCR can provide fast and reliable results while significantly decreasing the time of confirmation. In the present study, a nested-PCR system, targeting rv2807, with conventional PCR followed by real-time PCR, was developed to detect Mycobacterium tuberculosis complex (MTC) organisms directly from bovine and 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. Regarding the analytical sensitivity, DNA of the M. bovis AN5 strain was detected up to 1.5 pg by nested-PCR, whereas DNA of M. tuberculosis H37Rv strain was detected up to 6.1 pg. The nested-PCR system showed 100% analytical specificity for MTC when tested with DNA of reference strains of non-tuberculous mycobacteria and closely-related Actinomycetales. A clinical sensitivity level of 76.7% was detected with tissues samples positive for MTC by means of the culture and conventional PCR. A clinical specificity of 100% was detected with DNA from tissue samples of cattle with negative results in the comparative intradermal tuberculin test. These cattle exhibited no visible lesions and were negative in the culture for MTC. The use of the nested-PCR assay to detect M. tuberculosis complex in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.
authors.affiliation: Programa de Pós-Graduação em Ciência Animal Faculdade de Medicina Veterinária e Zootecnia Universidade Federal de Mato Grosso do Sul Campo GrandeMS Brazil Programa de Pós-Graduação em Ciência Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS, Brazil.
authors.forename: Cristina P
authors.initials: CP
authors.lastname: Araújo
authors.affiliation: Programa de Pós-Graduação em Ciência Animal Faculdade de Medicina Veterinária e Zootecnia Universidade Federal de Mato Grosso do Sul Campo GrandeMS Brazil Programa de Pós-Graduação em Ciência Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade Federal de Mato Grosso do Sul, 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 Faculdade de Medicina Veterinária e Zootecnia Universidade Federal de Mato Grosso do Sul Campo GrandeMS Brazil Programa de Pós-Graduação em Ciência Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS, Brazil.
authors.forename: Klaudia S G
authors.initials: KS
authors.lastname: Jorge
authors.affiliation: Faculdade de Medicina Veterinária e Zootecnia Universidade Federal de Mato Grosso do Sul Campo GrandeMS Brazil Faculdade de Medicina Veterinária e Zootecnia, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS, Brazil.
authors.forename: Carlos A N
authors.initials: CA
authors.lastname: Ramos
authors.affiliation: Programa de Pós-Graduação em Ciência Animal Faculdade de Medicina Veterinária e Zootecnia Universidade Federal de Mato Grosso do Sul Campo GrandeMS Brazil Programa de Pós-Graduação em Ciência Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS, Brazil.
authors.forename: Antonio F
authors.initials: AF
authors.lastname: Souza Filho
authors.affiliation: Programa de Pós-Graduação em Medicina Veterinária Universidade Federal de Santa Maria Santa MariaRS Brazil Programa de Pós-Graduação em Medicina Veterinária, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil.
authors.forename: Carlos E S
authors.initials: CE
authors.lastname: Vidal
authors.affiliation: Programa de Pós-Graduação em Medicina Veterinária Universidade Federal de Santa Maria Santa MariaRS Brazil Programa de Pós-Graduação em Medicina Veterinária, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil.
authors.forename: Agueda P C
authors.initials: AP
authors.lastname: Vargas
authors.affiliation: Instituto Biológico de São Paulo São PauloSP Brazil Instituto Biológico de São Paulo, São Paulo, SP, Brazil.
authors.forename: Eliana
authors.initials: E
authors.lastname: Roxo
authors.affiliation: Fundação Oswaldo Cruz Rio de JaneiroRJ Brazil Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil.
authors.forename: Adalgiza S
authors.initials: AS
authors.lastname: Rocha
authors.affiliation: Fundação Oswaldo Cruz Rio de JaneiroRJ Brazil Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil.
authors.forename: Philip N
authors.initials: PN
authors.lastname: Suffys
authors.affiliation: Laboratório Nacional Agropecuário Pedro LeopoldoMG Brazil Laboratório Nacional Agropecuário, Pedro Leopoldo, MG, Brazil.
authors.forename: Antônio A
authors.initials: AA
authors.lastname: Fonseca
authors.affiliation: Embrapa Gado de Leite Juiz de ForaMG Brazil Embrapa Gado de Leite, Juiz de Fora, MG, Brazil.
authors.forename: Marcio R
authors.initials: MR
authors.lastname: Silva
authors.affiliation: Universidade Federal do Pará CastanhalPA Brazil Universidade Federal do Pará, Castanhal, PA, Brazil.
authors.forename: José D
authors.initials: JD
authors.lastname: Barbosa Neto
authors.affiliation: Universidade Federal do Pará CastanhalPA Brazil Universidade Federal do Pará, Castanhal, PA, Brazil.
authors.forename: Valíria D
authors.initials: VD
authors.lastname: Cerqueira
authors.affiliation: Embrapa Gado de Corte Campo GrandeMS Brazil Embrapa Gado de Corte, Campo Grande, MS, Brazil.
authors.forename: Flábio R
authors.initials: FR
authors.lastname: Araújo
mesh_terms: D000818:Animals; D002020:Buffaloes; D002417:Cattle; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009163:Mycobacterium bovis / Q000235:genetics / Q000302:isolation & purification*; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D057089:Pathology, Molecular / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014380:Tuberculosis, Bovine / Q000175:diagnosis* / Q000382:microbiology; D014730:Veterinary Medicine / Q000379:methods*
keywords: bovine and bubaline tuberculosis; nested-PCR; real-time PCR; sanitary inspection; tissue