Detection of tet(M) gene from raw milk by rapid DNA extraction followed by a two-step PCR with nested primers.

The likelihood that milk and milk products may act as a vehicle for antibiotic-resistant bacterial genes has become a concern to the food industry and a public health issue, and the demand for rapid tests has increased. The purity of DNA extracted from food samples is a key issue in the sensitivity and usefulness of biological analyses, such as PCR for pathogens and nonpathogens. A rapid, phenol-chloroform free method based on a modification of a sodium iodide DNA extraction, followed by a two-step PCR was developed for direct detection of the tet(M) gene in milk samples within a single working day. This study compares the proposed method with a traditional phenol solvent extraction method and with a commercial kit (QIAamp DNA blood mini kit, Qiagen). The three DNA extraction methods were used to ensure access to the tet(M) gene from 1 ml of raw milk, inoculated with a strain of Enterococcus faecalis, which carries the tet(M) gene. The proposed method, followed by a two-step PCR with nested primers specific for the tet(M) gene, was able to reach a detection limit below 10 CFU/ml in less than 4 h, including the two amplification cycles, thus outperforming in sensitivity and rapidity both the traditional and the commercial method

The relationship between S. aureus and branched-chain amino acids content in composite cow milk

The early diagnosis of mastitis is an essential factor for the prompt detection of the animal for further actions. In fact, if not culled, infected cows must be segregated from the milking herd and milked last, or milked with separate milking units. Besides microbiological analysis, the somatic cell count (SCC) commonly used as predictor of intramammary infection, frequently lead to a misclassification of milk samples. To overcome these limitations, more specific biomarkers are continuously evaluated. The total amino acid content increases significantly in mastitic milk compared to normal milk. S. aureus requires branched-chain amino acids (BCAAs—isoleucine, leucine, and valine) for protein synthesis, branched-chain fatty acids synthesis, and environmental adaptation by responding to their availability via transcriptional regulators. The increase of BCAAs in composite milk has been postulated to be linked to mammary infection by S. aureus. The aim of this work is to demonstrate, by a direct ion-pairing reversed-phase method, based on the use of the evaporative light-scattering detector (IP-RP-HPLC-ELSD), applied to 65 composite cow milk samples, a correlation between the concentration of isoleucine and leucine, and S. aureus load. The correlation coe cient, r, was found to be 0.102 for SCC (p = 0.096), 0.622 for isoleucine (p < 0.0001), 0.586 for leucine (p < 0.0001), 0.013 for valine (p = 0.381), and 0.07 for tyrosine (p = 0.034), standing for a positive correlation between S. aureus and isoleucine and leucine concentration. The link between the content of BCAAs, isoleucine and leucine, and udder infection by S. aureus demonstrated with our study has an important clinical value for the rapid diagnosis of S. aureus mastitis in cows.http://www.mdpi.com/journal/animalsam2020Paraclinical Science

Pelt removal and lamb carcass contamination

This research characterised the effect of air inflation for pelt removal in lambs. Two preliminary trials, each consisting of two groups of two lambs were carried out. One group of animals was depelted with air inflation and the other without. Immediately after pelt removal, each carcass was subjected to swab sampling at multiple sites and the microbiological contamination of the carcasses, including total mesophilic count, coliform organisms and Staphylococcus spp., assessed. The slight differences between the two groups were non-significant. Mean levels of microbiological contamination of all the carcasses after pelting (the total mesophilic count was 1.98 (on those depelted without air inflation and 2.35 on those where air inflation had been applied. The numbers of coliform organisms were 0.02 and 0.31, and Staphylococcus spp. were 1.12 and 1.43, respectively, for non-air inflated and air inflated carcasses

The effect of depelting with air inflation on the appearance and microbiology of lamb carcasses

Air inflation is currently used in Italy for depelting lambs under 15kg live weight. The lamb carcasses have a better appearance and there is a lower incidence of cuts to the subcutaneous fat or muscles. However, the prevention of physical and microbiological hazards should be secured. Hand depelting with air inflation resulted in carcasses having a better appearance in terms of shape and L* value, and surface microbial load similar to carcasses depelted without air inflation