Evaluation of internal reference genes for quantitative expression analysis by real-time reverse transcription-PCR in somatic cells from goat milk

Reverse transcription (RT) quantitative real-time PCR (qPCR) is the most accurate and easy-to-perform technique to measure the expression level of a selected gene of interest by quantifying mRNA transcripts. The use of reference genes is commonly accepted as the most reliable approach to normalize RT-qPCR data and reduce possible errors generated in the quantification of gene expression. The optimal number and choice of reference genes are experimentally validated for specific tissues or cell types and experimental designs. To date, data on qPCR normalization in goats are scarce and the most suitable reference genes in this species have been identified for only a limited number of tissues. The aim of this study was to determine an optimal combination of stably expressed reference genes in caprine milk somatic cells (MSC) from healthy and infected mammary glands. For the purpose, we performed RT-qPCR for 10 commonly used reference genes from various functional classes and then determined their expression level in MSC from goats intramammary challenged with Staphylococcus aureus and in MSC from healthy controls, with a view to select genes whose stability would be unaffected under infection conditions. The geNorm and NormFinder algorithms were used for validating the reference genes. Furthermore, to demonstrate the importance of normalization of gene expression with appropriate reference genes, we tested the effect of using a combination of the least stable genes for expression analysis evaluation. On the basis of our evaluation, we recommend the use of a panel of reference genes that should include G6PD, YWHAZ, and ACTB for caprine MSC gene expression profiling. The expression of the 2 genes of interest, pentraxin-related protein (PTX3) and secreted phosphoprotein 1 (SPP1), was evaluated by RT-qPCR in all samples collected pre- and postinfection, and the recommended reference genes were used to normalize the data. Our study provides a validated panel of optimal reference genes for the identification of genes differentially expressed by qRT-PCR in caprine MSC. Moreover, we provided a set of intron-spanning primer sequences that could be suitable for gene expression experiments using SYBR Green chemistry on other caprine tissues and cells

Molecular characterization of Pseudomonas fluorescens isolates involved in the Italian "blue mozzarella" event.

Between June and September 2010, widespread Italian consumer reports of unusual blue spoilage on fresh dairy products were publicized, resulting in the so-called blue mozzarella event. An inordinately high number of samples from mozzarella and whey cheese products of Italian and German production subsequently tested positive for Pseudomonas fluorescens. The aim of this study was to verify whether a selected P. fluorescens strain was responsible for this apparently unusual event. Molecular characterization of 181 isolated P. fluorescens strains was conducted using a newly optimized pulsed-field gel electrophoresis protocol. Although a high number of pulsotypes was found (132), only four pulsotypes were associated with more than one production plant, and only one German isolate had the same pulsotype as was detected in two Italian plants. This is the only evidence of possible cross-contamination among cheeses from the two countries. The overall results did not support the spread of contamination from German to Italian plants or the presence of one environmental strain that spread in both countries

Highlighting priority areas for bovine viral diarrhea control in Italy : a phylogeographic approach

The prevalence and genetic diversity of bovine viral diarrhea virus (BVDV) in a geographic area are largely influenced by live animal trade and management practices. Despite control and eradication programs currently underway in several European countries, the risk of BVDV spread within and among countries is still present. BVDV-1 is the predominant type circulating in European cattle population. In this study, a phylogeographic analysis was applied to the BVDV-1 highest prevalent subtypes in Italy to reconstruct the origin and spatial-temporal distribution and to trace main viral flows between different locations to highlight priority areas for BVDV control. A comprehensive dataset of BVDV-1b (n\u202f=\u202f173) and 1e (n\u202f=\u202f172) 5' UTR sequences was analysed, including both novel and published sequences from Italy and from European countries bordering and/or with commercial cattle flows with Italy. A common phylogeographic pattern was observed for BVDV-1b and 1e subtypes: interspersion from multiple Italian areas and European countries was widespread until the end of the last century, whereas significant local clusters were observed starting from 2000. These findings support a continuous viral flow among different areas over long time scales with no evidence of significant geographical structure, while local transmission networks are limited to more recent years. Northern Italy has been confirmed as the area of origin of the main clades of both BVDV subtypes at national level, acting both as a crucial area for introduction and a maintenance source for other areas. Piedmont, Central and Southern Italian regions contributed to limited geographical distribution and local BVDV-1b and 1e persistence. On the whole, priority control measures for BVDV-1b and 1e in Italy should be focused on: i) implementation of BVDV systematic control in all Northern Italian regions to break the viral flow from larger to smaller animal populations; and ii) breaking the dynamics of infections in regions with self-maintenance of BVDV by voluntary control programs