Real-time qPCR is a powerful assay to estimate the 171 R/Q alleles at the PrP locus directly in a flock's raw milk: a comparison with the targeted next-generation sequencing.

The hazard to human health represented by transmissible spongiform encephalopathies in sheep is one of the major reasons for implementing the genetic selection plan to break down prion diseases. The problem is particularly important because of the risk of disease transmission from ewe to lamb via milk or colostrum. In order to establish an active and convenient monitoring of the flocks already undergone genetic selection and thus, indirectly increase consumers' security, the challenge of the work was quantifying the classical scrapie risk in bulk milk. A new quantitative real-time PCR assay for the estimation of the 171 R and Q allelic frequencies in a DNA pool representative of all the lactating ewes present in a flock was optimized and validated "in field". The repeatability range was 3.69-5.27 for R and 4.20-5.75 for Q. The ruggedness of the allele frequencies resulted 4.26 for R and 4.77 for Q. Regarding the validation "in field", none of the considered sources of variability (flock, month, number of genotyped animals and somatic cell count) showed a significant effect on flock and milk at the linear model. The targeted next-generation sequencing was also tested to evaluate its applicability in this context. Results show that the real-time PCR assay could represent a valid tool for the determination of 171 R/Q allele frequencies in bulk milk. The implementation of a service for breeder self-control along the production chain would aim to increase the production of high-security dairy products, while monitoring over time of the effects of genetic selection in the flocks.

Updating on the fungal composition in Sardinian sheep's milk by culture-independent methods.

This work applies culture-independent methods for the characterization of fungal populations (yeasts and moulds) naturally occurring in Sardinian ewe's milk sampled in the Italian areas with the largest dairy production (Sardinia and Lazio regions). Sequences of the D1/D2 variable domains at the 5' end of the 26S rRNA gene were obtained by amplification of DNA directly isolated from milk, and this allowed identification of a total of 6 genera and 15 species of fungi. Among the 6 identified genera Geotrichum spp., Candida spp., Phaeosphaeriopsis spp., Pestalotiopsis spp. and Cladosporium spp. belong to the phylum of Ascomycota, while Cryptococcus spp. is part of the phylum of Basidiomycota. In particular, two genera (Pestalotiopsis and Phaeosphaeriopsis) and two species (Plectosphaerella cucumerina and Pryceomyces carsonii) have never been reported in dairy ecosystems before. Results provide evidence that several moulds and yeasts, previously described only in ovine cheeses, are transferred directly from raw milk. The knowledge of fungal consortia inhabiting sheep raw milk is a particularly relevant issue because several species are directly involved in cheese making and ripening, determining the typical aroma. On the other hand, spoilage yeasts and moulds are involved in anomalous fermentation of cheese and may be responsible for considerable economic losses and serious risks for consumers' health.

Detection of Clostridium tyrobutyricum in milk to prevent late blowing in cheese by automated ribosomal intergenic spacer analysis.

Clostridium tyrobutyricum has been identified as the main causal agent of the late blowing defect in cheese, with major effects on quality and commercial value. In this work, for the first time, we applied automated ribosomal intergenic spacer analysis (ARISA) approach to diagnose the presence of C. tyrobutyricum in raw milk before cheesemaking. A species-specific primer set was designed and used for this original application of the ARISA. Sensitivity of detection, reproducibility of the fluorescent PCR assay, and repeatability of the capillary electrophoretic analysis of amplicons were evaluated using DNA extracted from milk added with known amounts of C. tyrobutyricum genome copies, ranging from 3 × 10(6) to 3. Results indicated that the sensitivity of the technique permits to detect the bacterium in all the samples. The reproducibility, evaluated by analyzing 3 sets of serial dilutions, resulted satisfactory, with little deviation within PCR reactions amplifying the same starting amount of template (standard deviations ≤ 0.1, coefficients of variation ≤ 3%). The peaks' fluorescence displayed an evident correspondence with the number of genome copies contained in each dilution. The capillary electrophoretic analysis, tested by running a single PCR product per dilution point in 10 repeats, resulted efficient and highly repeatable, with excellent coefficients of variation ≤ 2% and standard deviations ≤ 0.1 in all the sample sets. This application of ARISA gives good estimates of the total C. tyrobutyricum DNA content allowing a specific, fine-scale resolution of this pollutant species in a complex system as milk. A further advantage linked to the automatization of the process.

Diversity of fungal flora in raw milk from the Italian Alps in relation to pasture altitude.

The present paper explores the diversity of mycobiota inhabiting raw milk sampled at different altitudes (1400 m, 1800 m, 2200 m) from cows grazing Alpine pastures of Valle d'Aosta (North-Western Italian Alps). To this aim, multilocus sequencing was performed at barcodes commonly used for fungal identification (ITS1, D1/D2 domains of the 26S rRNA gene, and part of the ß-tubulin gene). A total of 31 species were detected, most of them yeasts, followed by moulds and by 2 sequences of macroscopic fungi. Several yeasts and moulds were well-characterized inhabitants of the dairy environment, known to positively contribute to cheesemaking. Among these, Candida was the most represented genus with a tendency to cluster at the highest altitudes (6 over 8 observations at ≥ 1800 m), and Kluyveromyces marxianus the most abundant single species, retrieved at all altitudes. The environmental ascomycetous Atrotorquata lineata, never put in relation with food nor described outside North-America, was another species among those most frequently retrieved and was detected in 6 milks at 1400 and 1800 m. The remaining fungi, in general never reported in milk, were mostly environmental. Many of them resulted associated with plants as pathogens or symbionts. Finally, the highest sampled altitude yielded a significant fungal diversity (17 species). This work enlarges the knowledge of fungal consortia inhabiting raw milk and introduces microbial ecology among the altitude-dependent factors, in the composition of Alpine pastures, with the potential of shaping the properties of milks and cheeses, together with the already described physical, chemical and botanical variables.

Identification of microbiota present on the surface of Taleggio cheese using PCR-DGGE and RAPD-PCR.

UNLABELLED: Microbial DNA from 9 batches of Taleggio PDO cheese sampled at various times during ripening, brines, swabs of wooden shelves used for cheese dry-salting, and 13 commercial cheeses were analyzed by denaturing gradient gel electrophoresis (PCR-DGGE) and/or random amplification of polymorphic DNA (RAPD-PCR). Sequencing allowed the detection of 12 genera, 27 species, and 2 unclassified bacteria. Molecular analysis allowed for the detection of microorganisms not previously associated with Taleggio such as Lactobacillus paracasei, Carnobacterium maltaromaticum, Bacillus licheniformis, Corynebacterium variabile, Psychrobacter cibarius, and Staphylococcus carnosus. For the first time Massilia spp. was detected in a dairy ecosystem. PRACTICAL APPLICATION: Indigenous species and strains of bacteria identified by this study could be used for the selection of dairy cultures to be employed routinely by manufacturers to control the Taleggio cheese production. The new cultures may give the bases for driving dairy processes and, consequently, control the typical flavor resulting from metabolic actions of environmental microorganisms.