Evaluation of Extraction Methods to Detect Noroviruses in Ready-to-Eat Raw Milk Minas Artisanal Cheese.

This study aimed to assess two homogenization methods to recover norovirus from Minas artisanal cheese (MAC) made with raw bovine milk obtained from four microregions of the Minas Gerais state, Brazil, with different ripening times and geographical and abiotic characteristics. For this purpose, 33 fiscal samples were artificially contaminated with norovirus GI and GII, and Mengovirus (MgV), used as an internal process control (IPC). TRIzol® reagent and Proteinase K homogenization methods were evaluated for all samples were then subjected to RNA extraction using viral magnetic beads and RT-qPCR Taqman® for viral detection/quantification. Proteinase K method showed better efficiency results for both norovirus GI and GII, with means recovery efficiency of 45.7% (95% CI 34.3-57.2%) and 41.4% (95% CI 29.1-53.6%), respectively, when compared to TRIzol method (16.6% GI, 95% CI 8.4-24.9%, and 12.3% GII, 95% CI 7.0-17.6%). The limits of detection for norovirus GI and GII for this method were 101GC/g and 103GC/g, respectively, independent of cheese origin. MgV was detected and revealed in 100% success rate in all types of cheese, with mean recovery efficiency of 25.6% for Proteinase K, and 3.8% for the TRIzol method. According to cheese origin, Triangulo Mineiro MAC had the highest mean recovery rates for the three viral targets surveyed (89% GI, 87% GII, and 51% MgV), while Serro MAC showed the lowest rates (p < 0.001). Those results indicate that the proteinase K adapted method is suitable for norovirus GI and GII detection in MAC and corroborated MgV as an applicable IPC to be used during the process.

Recovery of Brucella in raw milk Minas artisanal cheese approved for consumption by official inspection agency in Brazil: assessment of prevalence and risk factors through One Health integrated approaches.

BACKGROUND: Minas artisanal cheese (MAC) from the Serro region is a Brazilian intangible cultural heritage. Produced from raw milk, it may carry zoonotic pathogens such as Brucella. This study included a randomized survey for the prevalence of Brucella-positive MAC and its associated factors. METHODS: MAC samples (n=55), each one from a different rural family-based cheese-processing agroindustry, were analysed for Brucella by direct polymerase chain reaction (PCR) species-specific DNA detection and cultivation-based approaches. RESULTS: Among 55 MACs that were analysed, we found 17 Brucella DNA-positive samples (30.9% [95% confidence interval {CI} 18.7 to 43.1]) by PCR and, for the first time, from one MAC (1.8% [95% CI 0.5 to 9.7]), viable Brucella abortus was recovered by cultivation. Higher values for two variables, the number of lactating cows per herd (p=0.043) and daily milk production per herd (p=0.043), were each associated with Brucella-positive MAC, which concentrated in three high-risk and one low-risk spatial clusters. CONCLUSIONS: MAC may be a source of Brucella for humans, since the positive samples were from batches that were sold by cheesemakers. This should be of concern and encourage cooperation between the health and agriculture sectors in order to mitigate this public health risk through One Health integrated approaches.