Screening Brucella spp. in bovine raw milk by real-time quantitative PCR and conventional methods in a pilot region of vaccination, Edirne, Turkey

Abstract

Brucellosis is a worldwide zoonotic disease transmitted to humans by consumption of contaminated milk and milk products. Brucellosis is endemic in Turkey, and Edirne has a high Brucella prevalence. Brucellosis is prevented by live-attenuated vaccines for animals and the vaccination program has been in place since 1984 in Turkey. Thrace is the pilot region for this vaccination program. The gold standard diagnostic technique for brucellosis is still the isolation of suspicious bacterial colonies followed by bacteriological identification, but it is very time consuming and laborious. In many studies, Brucella has been investigated by PCR techniques. However, PCR-based methods cannot differentiate between the vaccine strain and the virulent strain; thus, the vaccine strain may interfere with the virulent strain and causes false-positive reactions. To monitor brucellosis control programs effectively, it is important to distinguish vaccine and field strains of Brucella spp. In this study, raw milk samples were collected from 99 cows at 12 different barns in 5 villages of Edirne (Turkey). Bacteriological analyses and real-time quantitative (q)PCR experiments were applied to all samples. The DNA was isolated using Biospeedy DNA-Tricky Purification Kit (Bioeksen, Istanbul, Turkey). For all reactions, Roche Light Cycler Nano (Roche Diagnostics, Mannheim, Germany) instrument and Biospeedy EvaGreen qPCR Pre-Mix (Bioeksen) were used. The data were analyzed using Roche LightCycler NanoSoftware 1.0. For samples that were negative by bacteriological analyses and positive by qPCR, we developed a novel qPCR-based method to differentiate the virulent B. abortus strains and B. abortus S19 vaccine strain. We designed qPCR primers targeting the outer membrane protein of B. abortus. The qPCR products were sequenced using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit on an ABI Prism 377 DNA sequencer (Applied Biosystems, Foster City, CA). In total, 2.02% of the samples were Brucella positive, by both bacteriological method and the novel qPCR method. We concluded that, to obtain true-positive results in Brucella spp. screening studies for milk, differentiating the virulent and vaccine strain should not be disregarded.