Identification of Brucella sp. isolated in Brazil from 1976 to 2013 by Bruce-Ladder PCR

Abstract

Background: Brucella sp. are the causative agents of brucellosis, an infectious disease that affects various species of animals and can be transmitted to humans through direct contact with infected animals, indirectly by the ingestion of rawmilk products, and during the handling of strains or infected material in the laboratory. Being a zoonosis, the detection of Brucella species in animals is essential for the prevention of the disease in humans and to perform a good program of control in infected herds. This study aimed at identifying Brucella field strains isolated from 1976 to 2013 in Brazil, using the modified Bruce-Ladder method, to evaluate the performance of this technique. Materials, Methods & Results: Eighty-three strains of Brucella sp. were included in the study, i.e. 21 reference strains (nine B. abortus, one B. canis, four B. melitensis, two B. ovis and five B. suis) and 62 field strains (six B. canis, one B. suis and 55 B. abortus). For the identification of the genus and/or species of Brucella, biochemical and physiological tests, including MacConkey-agar growth, glucose fermentation, haemolysis, catalase, oxidase and urease tests, nitrate reduction, citrate utilization, H2 S production and CO2 requirement, were performed. Genomic DNA was extracted from pure cultures through heat-lysis of bacterial cultures and the genus was confirmed by a genus-specific PCR (bcsp31 target gene), before performing the modified Bruce-Ladder PCR for the confirmation of the Brucella species. No problems of specificity were observed with the Bruce-Ladder PCR. However, the 1,682 bp fragment was not systematically amplified, even after several modifications such as the concentration of mix components, annealing temperatures and time. Therefore, an individual PCR using primers specific to this fragment was needed for complete identification of some strains. Also, only one kind of Polymerase gave the best results. All Brucella reference strains and negative controls gave the expected results. All field strains previously identified as B. abortus, B. canis and B. suis by biochemical and physiological tests were confirmed by the modified Bruce-Ladder PCR. All isolated Brucella abortus presented a Bruce-Ladder PCR profile expected for field strains, excluding the vaccine strains. Discussion: The modified Bruce-Ladder PCR identified properly all Brucella species (reference and field strains) and proved to be a reliable technique, thus facilitating the identification of the species in the laboratory, reducing the manipulation of these bacteria and the associated danger. Albeit the difficulties of amplification of one fragment for some strains, when using the multiplex technique, this method is fast and without risks after inactivation of the strains. Most studies on animal brucellosis in Brazil were only based on serological tests without identification of the pathogen; while the knowledge of the particular species and/or biovars that occur in Brazil, as well as their distribution, is important to monitor the spread of Brucella among sensitive species and among farms. Our results showed also that B. abortus is still the predominant species isolated in cattle in Brazil. The knowledge of the species that occur in Brazil can help to identify the source of infection and the measures of control to be applied, while it is also very important to trace the dispersion of strains among farms.