Real-time monitoring of mycobacterium genomic DNA with target-primed rolling circle amplification by a Au nanoparticle-embedded SPR biosensor.

Objective/background The present study was aimed at detecting Mycobacterium bovis in artisanal cheese using real-time quantitative polymerase chain reaction. Methods One hundred seven cheese samples (250 g) were purchased in 107 commercial establishments including neighborhood grocery stores, bakeries, and open-air markets from 19 municipalities of the state of Pernambuco, Brazil. Ten grams of each cheese sample were macerated with sterile saline solution in a sterile bag and DNA was extracted from 20 mg of the macerated material using the Wizard SV Genomic DNA Purification System. The quantitative polymerase chain reaction amplified a fragment corresponding to the region of difference 4 of M. bovis . Results Of the 107 samples analyzed, three (2.8%) were positive for M. bovis and their identities were confirmed by sequencing. This is perhaps the first report of the presence of M. bovis in artisanal cheese in the state of Pernambuco, Brazil. Conclusion The results of the present study highlight the need for improving sanitary measures during the production of artisanal cheese to prevent zoonotic tuberculosis in humans, resulting from the consumption of food contaminated with M. bovis .

Detection of Mycobacterium bovis in artisanal cheese in the state of Pernambuco, Brazil

http://www.scielo.br/pdf/bjm/v48n1/1517-8382-bjm-48-01-0139.pdf

Comparative study of Mycobacterium bovis primary isolation methods.

The present study aimed to direct detect Mycobacterium bovis in milk (n = 401) and blood (n = 401) samples collected from 401 dairy cows of 20 properties located in the state of Pernambuco, Brazil, by real-time quantitative PCR (qPCR) targeting the region of difference 4 (RD4). Risk factors possibly associated with bovine tuberculosis (BTB) were also evaluated. Of the 802 samples analyzed, one milk (0.25 %) and eight blood (2 %) samples were positive for M. bovis in the qPCR and their identities were confirmed by sequencing. Animals positive for M. bovis were found in six (30 %) of the 20 properties visited. None of the risk factors evaluated were statistically associated with BTB. M. bovis DNA was detected in one milk sample what may pose a risk to public health because raw milk is commonly consumed in Brazil.

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Molecular detection of Mycobacterium bovis in cattle herds of the state of Pernambuco, Brazil

http://www.scielo.br/pdf/bjm/v45n4/29.pdf

Validation of a real-time PCR assay for the molecular identification of Mycobacterium tuberculosis

This study aimed to develop and validate real-time PCR for the diagnosis of Mycobacterium bovis isolates. Two hundred and seventy-four M. bovis isolates and 156 M. tuberculosis isolates were tested. Both qPCRs amplified all of the 274 M. bovis samples, but none of the 156 M. tuberculosis samples. The qPCR for PE-PGRS 20 had 91% efficiency and a detection limit of 0.32 ng (sensitivity and specificity for qPCR "Mbovis.100" were 99.64 and 100%, respectively). The qPCR for RD4 had 100% efficiency, and a detection limit of 4 pg (diagnostic sensitivity and specificity were 100 and 100%. The qPCR tests were performed using 4 extraction sets, 3 qPCR kits, and with a range of equipment; yet, all combinations produced similar results in a diagnostic

/pdf/validation-of-two-real-time-pcrs-targeting-the-pe-pgrs-20-flslpn3z2c.pdf

Validation of two real-time PCRs targeting the PE-PGRS 20 gene and the region of difference 4 for the characterization of Mycobacterium bovis isolates.

Bovine tuberculosis is a worldwide spread zoonotic disease. Intradermal tuberculinizations are the most used diagnostic tests in the world. Serological tests can be an ancillary diagnosis for bovine tuberculosis. The objective of this study was to evaluate the diagnostic performance of the ELISA Mycobacterium Bovis Antibody Test Kit IDEXX ™ in infected herds, which were in different disease control stages. One hundred and twenty animals from two dairy herds of Minas Gerais state, Brazil, were subjected to the ELISA serological test and the comparative cervical tuberculin test (CCT). Diagnostic test parameters were estimated using Bayesian latent class models and concordance between tests estimated by the frequentist approach. The ELISA test presented lower sensitivity than CCT in both herds. Its sensitivity was higher in the herd in sanitation process. Specificity estimates were above 95% in both herds. Kappa index indicated low concordance or even disagreement between tests. According to the results, the ELISA IDEXX should not be used as substitution for CCT. The tests must not be associated in series. Parallel association increased diagnostic sensitivity in the herd which was in the process of sanitation.

/pdf/diagnostic-performance-of-a-commercial-elisa-used-as-a-2t1925laf2.pdf

Diagnostic performance of a commercial Elisa used as a complementary test for bovine tuberculosis in two bovine herds with different disease status.

A Brucellosis outbreak is reported in a bovine herd free from the disease for over 18 years, officially free since 2006. One heifer and one cow tested positive in serological tests for the 2008 annual recertification in a herd of almost 1000 animals. Isolation and identification by biochemical tests and molecular biology (AMOS PCR) confirmed the infection by a Brucella abortus biovar 1 field strain. It wasn't possible to find the source of the infection. However, adoption of standard procedures prescribed by the Brazilian National Brucellosis and Tuberculosis Control and Eradication Program hindered the spread of the disease. This is the first report of a Brucella infection in an officially Brucellosis-free bovine herd in Brazil.

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P. M. Soares Filho

Papers

Validation of two real-time PCRs targeting the PE-PGRS 20 gene and the region of difference 4 for the characterization of Mycobacterium bovis isolates.

Diagnostic performance of a commercial Elisa used as a complementary test for bovine tuberculosis in two bovine herds with different disease status.

Confirmação de infecção por Brucella abortus em um rebanho bovino certificado livre em Minas Gerais: relato de caso