Real-time TaqMan polymerase chain reaction detection and quantification of cow DNA in pure water buffalo mozzarella cheese: method validation and its application on commercial samples

Mozzarella cheese obtained from buffalo (Bubalus bubalis) milk is a typical Italian product certificated by means of the European Protected Designation of Origin (PDO). Mozzarella cheese can also be obtained from bovine milk or bovine/buffalo milk mixtures, but in this case, it cannot be sold as PDO product, and its label must report the actual ingredients. However, bovine milk in PDO products was frequently detected in the past, suggesting fraudulent addition or accidental contamination. Several methods based on end-point polymerase chain reaction (PCR) have been profitably applied in a large number of tests to detect the presence of undeclared ingredients, also in dairy products. In the present study we report a real-time PCR method able to quantify bovine milk addition to pure buffalo cheese products. We validated a normalized procedure based on two targets: bovine mitochondrial cytochrome b (cyt b) to detect and quantify the bovine DNA and nuclear growth hormone (GH) gene used as a universal reference marker. With the use of this real-time PCR assay, 64 commercial mozzarella di bufala cheese samples purchased at local supermarkets, dairy shops, or directly from cheese manufacturers were analyzed. The results obtained demonstrate that most of the commercial samples were contaminated with bovine milk. Therefore, this assay could be conveniently employed to carry out routine and accurate controls aimed not only to discourage any fraudulent behavior but also to reduce risks for consumer health.     

alpha(1)-Acid glycoprotein modulates apoptosis in bovine monocytes

alpha(1)-Acid glycoprotein (AGP, orosomucoid) is a normal constituent of bovine blood. AGP is an immunocalin, a binding protein that can also exert several immunomodulatory functions. In this paper we investigated the effect of bovine alpha(1)-acid glycoprotein (boAGP) on spontaneous and staurosporine-induced apoptosis of blood derived monocytes purified using magnetic cell sorting techniques. Bovine AGP was purified from blood following a chromatographic protocol. The homogeneous protein was used to stimulate the cells as well to raise a polyclonal antibody, that was used throughout all the experiments. When monocytes were incubated with high concentrations of boAGP (0.9 mg/ml), similar to those found in bovine plasma during systemic reaction to inflammation, their spontaneous apoptosis rate was suppressed, as determined by caspase-3/7 enzymatic activity assay. Similar results were obtained when apoptosis was induced by adding staurosporine, a potent protein kinase inhibitor. The apoptosis-modulating activity of boAGP was dependent on its concentration, since physiological concentrations of boAGP (0.3 mg/ml) did not exhibit a statistically significative anti-apoptotic activity. We also investigated whether this apoptosis-modulating activity was dependent on the terminal sialic acid residues exposed on the surface of the protein. Enzymatic treatment with neuraminidase, that cleaves terminal sialic acid residues, completely abolished boAGP's anti-apoptotic activity. These results suggest that the protective effect of AGP is likely mediated by its sialic acid terminal groups.     

High levels of genetic differentiation between Wolbachia-infected and non-infected populations of Folsomia candida (Collembola, Isotomidae)

The Gram-negative -Proteobacterium Wolbachia pipientis has been described as an obligate endosymbiont in many arthropod species, where it induces a variety of reproductive alterations, including parthenogenesis. Recently, this microorganism has also been detected in the parthenogenetic collembolan Folsomia candida. Here, we confirm the occurrence of the endosymbiont also in two Italian parthenogenetic populations of F. candida using ultrastructural (electron microscopy) and molecular (PCR screening on two bacterial genes) evidence. The strain isolated in the Italian populations has almost-identical gene sequences compared to that previously isolated in other populations of F. candida. In addition, we discovered a population of Folsomia cf. candida, which showed the presence of both males and females. This population is not infected by Wolbachia. A screening of two mitochondrial genes (COI and COII) showed that the bisexual population has high levels of genetic divergence in comparison with the parthenogenetic ones, even suggesting the possibility that it belongs to a different species. Furthermore, the remarkably high levels of genetic divergence between the two parthenogenetic populations suggests a possible influence of Wolbachia on inducing such differentiation, and, in the long term, speciation.     

Escherichia coli lipopolysaccharides and Staphylococcus aureus enterotoxin B differentially modulate inflammatory microRNAs in bovine monocytes

MicroRNAs (miRNAs) are a family of regulatory molecules involved in many physiological processes, including activation of cells of the immune system. This study investigated the effect of Escherichia coli lipopolysaccharide (LPS) and Staphylococcus aureus enterotoxin B (SEB) on the expression of five miRNAs involved in the inflammatory response, including miR-9, miR-125 b, miR-155, miR-146 a and miR-223, in bovine CD14(+) cells (monocytes). Incubation of monocytes with SEB induced down-regulation of miR-155, miR-223 and miR-125 b, but not the anti-inflammatory miRNA miR-146 a. Conversely, incubation with LPS upregulated both miR-155 and miR-146 a. In vitro incubation of isolated CD14(+) bovine monocytes with LPS and SEB elicited different and opposite expression of miRNAs reportedly involved in inflammatory reactions.     

Romina and Cristina: Two New Strawberry Cultivars with High Sensorial and Nutritional Values

ABSTRACT

The two cultivars (Romina and Cristina) recently released from the Marche Polytechnic University (Ancona, Italy) strawberry (Fragaria × ananassa) breeding program, show high adaptability to non-fumigated soil. Furthermore, ‘Romina’ showed very early ripening, conic or bi-conic fruit shape, good taste with high sweetness, high firmness and shelf life, and high vitamin C and folate contents. ‘Cristina’ showed very late ripening, high productivity, large fruit of conical shape, good taste, and high vitamin C and folate contents. The European Union patents of ‘Romina’ and ‘Cristina’ have been transferred to the United States, and nursery production has begun following United States rules to determine future market interest.     

Guava (<Emphasis Type="Italic">Psidium guajava L</Emphasis>. cv. Red Suprema) Crude Extract Protect Human Dermal Fibroblasts against Cytotoxic Damage Mediated by Oxidative Stress

We analyzed guava fruits (Psidium guajava L. cv. Red Suprema) from Cuba to determine their chemical composition, total antioxidant capacity, as well as their protective effect against oxidative damage using an in vitro model of human dermal fibroblasts. The guava fruit is a natural source of bioactive compounds, such as polyphenols, vitamin C, folates and beta carotenes with proven health benefits. Human dermal fibroblasts were pre-incubated with different concentrations of guava crude extract and then subjected to oxidative stress using the AAPH stressor. The number of apoptotic and dead cells, as well as the markers of oxidative damage such as lipid and protein oxidation significantly decreased when cells were pre-incubated with guava crude extract and then exposed to the stressor. The activity of antioxidant enzymes also improved when cells were pre-incubated with guava crude extract in comparison to cells subjected to stress without prior pre-incubation with the guava extract. The results obtained in this study highlight the health benefits of guava regarding oxidative stress, proving it to be an important source of bioactive compounds associated with important biological properties.     

A tryptophan-enriched diet improves feed intake and growth performance of susceptible weanling pigs orally challenged with Escherichia coli K88

We tested the effect of Trp addition to a standard weaning diet and oral challenge with enterotoxigenic Escherichia coli K88 (ETEC) on growth and health of piglets susceptible or nonsusceptible to the intestinal adhesion of ETEC. Sixty-four pigs weaned at 21 d of age were divided into 3 groups based on their ancestry and BW: a control group of 8 pigs fed a basal diet (B), the first challenged group of 28 pigs fed B diet (BCh), and the second challenged group of 28 pigs fed a diet with Trp (TrpCh). The Trp diet was produced by the addition of 1 g of l-Trp/kg to the basal diet. On d 5, pigs were orally challenged with 1.5 mL suspension containing 10(10) cfu ETEC/mL or placebo, and killed on d 9 or 23. Based on in vitro villus adhesion assay, the pigs (except the B group) were classified as susceptible (s(+)) or nonsusceptible (s(-)) to the intestinal ETEC adhesion. Thus, after the challenge, treatments were B, BChs(-), BChs(+), TrpChs(-), and TrpChs(+). Pigs susceptible to ETEC were 50.0% in the BChs(+) group (3 pigs lost included) and 46.4% in the TrpChs (+) group (1 pig lost included). During the first 4 d after challenge, the challenge reduced ADG (P < 0.05), and this reduction was greater in susceptible pigs (P < 0.05) than nonsusceptible ones. Tryptophan increased ADG and feed intake in susceptible pigs (P < 0.05) from challenge to d 4, but not thereafter. Tryptophan supplementation did not improve the fecal consistency and did not reduce the number of pigs positive for ETEC in feces on d 4 after the challenge. The K88-specific immunoglobulin A activity in blood serum tended to be greater in challenged pigs (P = 0.102) and was not affected by the addition of Trp. Villous height was affected by the addition of Trp and challenge in different ways, depending on the site of small intestine. The need to consider the phenotype for the adhesion of the ETEC in studies with different supply of Trp was clearly evident. When compared with practical weaning standard diets, Trp supplementation allowed susceptible pigs to partially compensate for the effects of ETEC challenge by increasing feed intake and maintaining an adequate BW growth. This is of practical importance for the formulation of diets for pigs selected for lean growth because of the presence of an association between this trait and the susceptibility to the intestinal adhesion of ETEC.