Isolation and identification of mycobacteria from livestock specimens and milk obtained in Brazil

Leite, Clarice Queico Fujimura; Anno, Ivone Shizuko; Leite, Sérgio Roberto de Andrade; Roxo, Eliana; Morlock, Glenn P.; Cooksey, Robert C.

doi:10.1590/s0074-02762003000300005

Cited by 75 publications

(72 citation statements)

References 18 publications

(11 reference statements)

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“…In accordance with the report of Hermans et al (1990), no amplification was observed for M. avium, M. fortuitum, M. terrae, M. vacae, M. xenopi, M. flavescens, and M. scrofulaceum, which are non-MTC species commonly isolated from cattle in Brazil (PARDO et al, 2001;LEITE et al, 2003).…”

Section: Discussionsupporting

confidence: 89%

“…In those animals, the occurrence of visible gross lesions in the mammary gland (MG) or supramammary lymph nodes (LN) is less than 0.5% (SERRANO-MORENO et al, 2008). M. bovis has been isolated from milk from storage tanks, inadequately pasteurized milk, and milk from tuberculin non-reacting cattle (PARDO et al, 2001;LEITE et al, 2003). This situation stresses the need for sensitive and accurate procedures for the rapid identification of the bacteria in milk, to assist in the control of this zoonosis.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Use of PCR for detection of bovine tuberculosis bacillus in milk of positive skin test cows

Carvalho

Castro

Fernandes

et al. 2014

Braz. J. Vet. Res. Anim. Sci.

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The causative agent of bovine tuberculosis (BTB) is Mycobacterium bovis, a bacterium belonging to the M. tuberculosis complex (MTC). The definitive diagnosis is achieved through isolation and identification of M. bovis from clinical samples, using a combination of traditional culture and biochemical methods, which is considered the "gold standard". This procedure is cumbersome and time-consuming. We evaluated a PCR assay for the direct detection of MTC DNA in milk of positive skin test cows, using primers that were previously tested and proven reliable to target the IS6110 element. Milk previously seeded with M. bovis was used as the starting material, for standardization of the technique. The procedure involved extracting the DNA by enzymatic lysis (proteinase K and lysozyme), phenol, chloroform, isoamyl alcohol, followed by ethanol precipitation and PCR. The PCR assay allowed us to detect BTB in artificially contaminated milk, with a detection limit of 100 CFU/mL, and was also able to detect the bacillus in 50% (75/150) of the milk samples tested. This procedure could be used to assist the in vivo diagnosis of BTB, complementing sorological or microbiological tests, becoming an alternative option for epidemiological studies of BTB transmission and preventing contaminated milk from entering the food supply.Keywords: Bovine tuberculosis. Molecular biology. Mycobacterium tuberculosis complex. Milk. ResumoO agente causador da tuberculose bovina (BTB) é o Mycobacterium bovis, uma bactéria pertencente ao complexo M. tuberculosis (CMT). O diagnóstico definitivo é realizado através do isolamento e identificação de M. bovis em amostras clínicas, utilizando uma combinação de cultura bacteriológica e métodos bioquímicos, que são considerados como "padrão de ouro". Entretanto, esses procedimentos são trabalhosos e demorados. No presente estudo, foi avaliado um ensaio de PCR para a detecção direta de DNA de CMT em leite de vacas positivas ao teste cutâneo, utilizando primers previamente testados e comprovadamente confiáveis, para amplificação da região de interesse IS6110. Leite previamente contaminado com M. bovis foi utilizado como material de partida para a padronização da técnica. O procedimento envolveu a extração do DNA por lise enzimática (proteinase K e lisozima), fenol, clorofórmio, álcool isoamílico, seguido de precipitação com etanol e PCR. O ensaio de PCR detectou BTB em leite artificialmente contaminado, com um limite de detecção de 100 UFC/mL, e também foi capaz de detectar o bacilo em 50% (75/150) das amostras de leite testadas. A técnica de PCR pode ser utilizada para auxiliar o diagnóstico in vivo da BTB, além de complementar os testes sorológicos ou microbiológicos, tornando-se uma alternativa para estudos epidemiológicos da transmissão de BTB, prevenindo que o leite contaminado entre na cadeia de alimentos.Palavras-chave: Tuberculose bovina. Biologia molecular. Complexo Mycobacterium tuberculosis. Leite.

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Section: Discussionsupporting

confidence: 89%

Section: Introductionmentioning

confidence: 99%

Use of PCR for detection of bovine tuberculosis bacillus in milk of positive skin test cows

Carvalho

Castro

Fernandes

et al. 2014

Braz. J. Vet. Res. Anim. Sci.

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“…According to a tuberculin testing survey conducted in 1998, an average of 7.1% of herds was infected, ranging from 2.8% in the Central West to 58.3% in the Northern regions. M. bovis has been isolated from livestock pathological specimens (bovine, buffalo and swine), and from raw milk, confirming the risk for humans ingesting raw or un-pasteurized milk, as well as under-cooked animal products (Leite et al, 2003).…”

Section: Introductionmentioning

confidence: 89%

Diagnosis of bovine tuberculosis: review of main techniques

2015

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Bovine tuberculosis (BTB) remains an important economic and zoonotic problem in Latin America. Traditionally, the fight against BTB is initiated by the implementation of routine diagnostic tests for certification of free properties. The diagnosis of BTB can be made by direct and indirect methods, in which we can mention clinical, post mortem, histopathological, immunological, bacteriological and molecular methods. The renewal of scientific interest in tuberculosis in recent year has led to develop and improve methods of diagnosis, prevention, control and eradication of BTB. The aim of this review is to present and discuss different diagnosis methods of BTB.Keywords: tuberculosis bovine, diagnosis, Mycobacterium bovis, new techniques. Diagnóstico de tuberculose bovina: revisão das principais técnicas ResumoA tuberculose bovina (BTB) continua sendo um importante problema econômico na América Latina, com potenciais consequências zoonóticas. Tradicionalmente, a luta contra a tuberculose bovina tem sido iniciada pela execução de testes de diagnóstico de rotina para a certificação de propriedades livres da doença. O diagnóstico de BTB pode ser feito através de métodos diretos e indiretos, nos quais podemos citar os métodos clínicos, post mortem, histopatológicos, imunológicos, bacteriológicos e moleculares. A renovação do interesse científico em tuberculose nos últimos anos tem levado à necessidade de desenvolver e melhorar os métodos de diagnóstico, prevenção, controle e erradicação da BTB. O objetivo deste artigo é analisar e discutir sobre os diferentes métodos de diagnóstico de BTB.Palavras-chave: tuberculose bovina, diagnóstico, Mycobacterium bovis, novas técnicas.

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“…The use of a single molecular technique can produce false negative results, hence the need for applying more than one type of technique. Leite et al (2003), using culture and identification by PCR and RFLP-PCR, found 68.2% of positivity for M. bovis in pathological bovine and bubaline samples. The efficiency of the culture used as a first criterion for M. bovis identification was low (23.6%).…”

mentioning

confidence: 99%

Mycobacterium bovis identification by a molecular method from post-mortem inspected cattle obtained in abattoirs of Mato Grosso do Sul, Brazil

Araújo

Leite

Prince

et al. 2005

Mem. Inst. Oswaldo Cruz

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Isolation and identification of mycobacteria from livestock specimens and milk obtained in Brazil

Cited by 75 publications

References 18 publications

Use of PCR for detection of bovine tuberculosis bacillus in milk of positive skin test cows

Use of PCR for detection of bovine tuberculosis bacillus in milk of positive skin test cows

Diagnosis of bovine tuberculosis: review of main techniques

Mycobacterium bovis identification by a molecular method from post-mortem inspected cattle obtained in abattoirs of Mato Grosso do Sul, Brazil

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