Detecção de Brucella spp. por nested PCR e fatores associados à sua ocorrência em queijos artesanais produzidos com leite cru

Abstract

Although raw milk artisanal cheeses (RMAC) represent an important segment of the food production chain in Brazil, their consumption may pose a public health risk, as they are made from unpasteurized milk and may therefore harbor zoonotic agents. Brucellosis, caused by bacteria of the genus Brucella, is particularly relevant in this context, although investigation of this agent is not required in foods in Brazil according to the microbiological standards established by regulatory acts. Polymerase chain reaction (PCR) represents a promising tool for the detection of Brucella spp. in foods; however, its application depends on obtaining high-quality DNA and controlling interfering substances present in dairy products. The aim of this study was to determine the frequency of Brucella spp. in RMAC from nine producing regions in the states of Minas Gerais and Piauí, as well as to evaluate epidemiological factors potentially associated with the occurrence of the agent. A total of 222 RMAC samples were evaluated using different DNA extraction methodologies, from which a guanidinium thiocyanate-based protocol was selected and standardized. The detection of Brucella spp. was performed by nested PCR targeting the eryD gene, with samples considered positive when the expected 363-bp fragment was detected in the second reaction. Associated factors were evaluated using univariate and spatial statistical analyses, considering the production site, counts of total and fecal coliforms and coagulasepositive Staphylococcus (CPS), the presence of Coxiella burnetii, the rainy season, ripening period, moisture content, and pH. Fragments of the eryD gene were detected in 24 samples (10.8%; 95% CI: 7.1–15.7%), distributed across four producing regions (Serro, Araxá, Canastra, and Piauí). In nine samples, only the 469-bp fragment was observed, even after the second reaction, and these results were considered inconclusive regarding the presence of the agent. The production site was significantly associated with the occurrence of Brucella spp. (p < 0.0001), as were CPS counts (p = 0.03). Odds Ratio (OR) analysis indicated a higher likelihood of positivity among samples with elevated CPS counts (p = 0.04). The frequency of positive samples decreased with increasing time elapsed since the official recognition of the regions as traditional cheese-producing areas by the state animal health authority (p < 0.0001). Spatial analyses identified two high-density clusters of positive samples, one in Minas Gerais and the other in Piauí. The results demonstrated the occurrence of Brucella spp. DNA in RMAC from different producing regions and reinforce the importance of investigating this agent in foods produced with raw milk, contributing to the understanding of epidemiological aspects related to the production chain, strengthening surveillance and control actions, and implementing continuous educational initiatives with producers, under the One Health perspective.