Sensitive and highly specific quantitative real-time PCR and ELISA for recording a potential transfer of novel DNA and Cry1Ab protein from feed into bovine milk

To address food safety concerns of the public regarding the potential transfer of recombinant DNA (cry1Ab) and protein (Cry1Ab) into the milk of cows fed genetically modified maize (MON810), a highly specific and sensitive quantitative real-time PCR (qPCR) and an ELISA were developed for monitoring suspicious presence of novel DNA and Cry1Ab protein in bovine milk. The developed assays were validated according to the assay validation criteria specified in the European Commission Decision 2002/657/EC. The detection limit and detection capability of the qPCR and ELISA were 100 copies of cry1Ab microL(-1) milk and 0.4 ng mL(-1) Cry1Ab, respectively. Recovery rates of 84.9% (DNA) and 97% (protein) and low (<15%) imprecision revealed the reliable and accurate estimations. A specific qPCR amplification and use of a specific antibody in ELISA ascertained the high specificity of the assays. Using these assays for 90 milk samples collected from cows fed either transgenic (n = 8) or non-transgenic (n = 7) rations for 6 months, neither cry1Ab nor Cry1Ab protein were detected in any analyzed sample at the assay detection limits.

Concomitant lipopolysaccharide-induced transfer of blood-derived components including immunoglobulins into milk

During a mammary immune response, the integrity of the blood-milk barrier is negatively affected and becomes leaky. The aim of the present study was to demonstrate the blood origin, and to investigate changes in the concentration, of various constituents including immunoglobulins in blood and milk during the early phase of lipopolysaccharide (LPS)-induced mastitis. Five lactating dairy cows received continuous β-hydroxybutyrate (BHBA) clamp infusions to maintain elevated BHBA blood concentrations (1.5 to 2.0 mmol/L) from 48 h before and 8h after LPS administration. One udder quarter was infused with 200 μg of Escherichia coli LPS. A second quarter served as control. Milk and blood samples were taken hourly for 8h postchallenge (PC). The somatic cell count in LPS-challenged quarters was increased from 4h PC to the end of the experiment compared with control quarters. In LPS-challenged quarters, l-lactate, BHBA, lactate dehydrogenase (LDH), IgG(1), and IgG(2) were increased at 3h PC and remained elevated until the end of experiment (8h PC) compared with control quarters. In addition, the optical density values in milk in a nonquantitative ELISA for antibodies directed against bluetongue virus (used as a measure of nonspecific antibody transfer; all animals were vaccinated) increased and, thus, indicates an increase in these antibodies in response to LPS treatment. l-Lactate concentration also increased in blood 2h PC and in the milk of control quarters during the experiment from 3h PC. A second experiment was conducted in vitro to investigate a possible contribution from destructed milk cells to l-lactate concentration and activity of LDH in milk. Aliquots of milk samples (n=8) were frozen (-20°C) or disrupted with ultrasound, respectively. Freeze thawing and ultrasound treatment increased LDH in milk samples, but had no effect on l-lactate concentrations. Results suggest that intramammary infusion of LPS induces a systemic response, as evidenced by an elevation of blood l-lactate concentration. The concomitant changes of all investigated components suggest that they were blood derived. However, the increase in blood components in the milk is not necessarily supportive of the mammary immune system, and likely a side effect of reduced blood-milk barrier integrity.

Transient Neonatal Zinc Deficiency Caused by a Heterozygous G87R Mutation in the Zinc Transporter ZnT-2 (SLC30A2) Gene in the Mother Highlighting the Importance of Zn (2+) for Normal Growth and Development

Suboptimal dietary zinc (Zn(2+)) intake is increasingly appreciated as an important public health issue. Zn(2+) is an essential mineral, and infants are particularly vulnerable to Zn(2+) deficiency, as they require large amounts of Zn(2+) for their normal growth and development. Although term infants are born with an important hepatic Zn(2+) storage, adequate Zn(2+) nutrition of infants mostly depends on breast milk or formula feeding, which contains an adequate amount of Zn(2+) to meet the infants' requirements. An exclusively breast-fed 6 months old infant suffering from Zn(2+) deficiency caused by an autosomal dominant negative G87R mutation in the Slc30a2 gene (encoding for the zinc transporter 2 (ZnT-2)) in the mother is reported. More than 20 zinc transporters characterized up to date, classified into two families (Slc30a/ZnT and Slc39a/Zip), reflect the complexity and importance of maintaining cellular Zn(2+) homeostasis and dynamics. The role of ZnTs is to reduce intracellular Zn(2+) by transporting it from the cytoplasm into various intracellular organelles and by moving Zn(2+) into extracellular space. Zips increase intracellular Zn(2+) by transporting it in the opposite direction. Thus the coordinated action of both is essential for the maintenance of Zn(2+) homeostasis in the cytoplasm, and accumulating evidence suggests that this is also true for the secretory pathway of growth hormone.

The response of four winter wheat (Triticum aestivum L.) varieties to field water deficit: leaf anti-oxidative protection and proteolytic activity

Introduction: Drought is one of the most significant factors that limit plant productivity. Oxidative stress is a secondary event in many unfavorable environmental conditions. Intracellular proteases have a role in the metabolism reorganisation and nutrient remobilization under stress. In order to under stand the relative significance of oxidative stress and proteolysis in the yield reduction under drought, four varieties of Triticum aestivum L. with different field drought resistance were examined. Methods: A two-year field experiment was conducted. Analyses were performed on the upper most leaf of control plants and plants under water deficitat the stages most critical for yield reduction under drought (from jointing till milk ripeness). Leaf water deficit and electrolyte leakage, malondyaldehyde level, activities and isoenzymes of superoxide dismutase, catalase and peroxidase, leaf protein content and proteolytic activity were studied. Yield components were analyzed. Results: A general trend of increasing the membrane in stability and accumulation of lipid hydroperoxides was observed with some differences among varieties, especially under drought. The anti-oxidative enzyme activities were progressively enhanced, as well as the azocaseinolytic activities. The leaf protein content decreased under drought at the last phase. Differences among varieties were observed in the parameters under study. They were compared to yield components` reduction under water deprivation.

A longitudinal study investigating the prevalence of Staphylococcus aureus genotype B in seasonally communal dairy herds

Abstract Staphylococcus aureus is a major mastitis-causing pathogen. Various genotypes have been recently identified in Switzerland but Staph. aureus genotype B (GTB) was the only genotype associated with high within-herd prevalence. The risk of introducing this Staph. aureus genotype into a herd may be increased by frequent animal movements. This may also be the case when cows from different herds of origin are commingled and share their milking equipment for a limited period of time. The aim of the present study was to determine the prevalence of Staph. aureus GTB in seasonally communal dairy herds before and after a summer period when dairy farming is characterized by mixing cows from different herds of origin in 1 communal operation. In addition, the environment was investigated to identify potential Staph. aureus GTB reservoirs relevant for transmission of the disease. A total of 829 cows from 110 herds of origin in 9 communal operations were included in the study. Composite milk samples were collected from all cows during the first or second milking after arrival at the communal operation and again shortly before the end of the season. Swab samples from the environment, involved personnel, and herding dogs present were collected before the cows arrived. At the end of the season, sampling of personnel was repeated. All samples were analyzed for the presence of Staph. aureus GTB using an established quantitative PCR. At the beginning of the season, Staph. aureus GTB-positive cows were identified in 7 out of 9 communal operations and the within-communal operation prevalence ranged from 2.2 to 38.9%. At the second sampling, all communal operations were Staph. aureus GTB positive, showing within-communal operation prevalence from 1 to 72.1%. The between-herd of origin prevalence increased from 27.3 to 56.6% and the cow-level prevalence increased from 11.2% at the beginning of the season to 29.6% at the end of the season. On 3 different communal operations, Staph. aureus GTB-positive swabs from seasonally employed personnel were identified at the end of the season. The results indicate that Staph. aureus GTB can easily spread in communal operations when cows from different herds of origin are mixed during the summer season. Effective management measures need to be designed to prevent the spread of Staph. aureus GTB in seasonally communal herds. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved. KEYWORDS: Staphylococcus aureus; biosecurity; communal herd; epidemiology

Restoring balance within cognitive unit or deservingness evaluation? The role of attitudes and deservingness in shaping emotional reactions to others’ outcomes

Based on Heider’s theory I persumed that personal attitudes are stronger predictors of emotional responses to others’ outcomes than deservingness evaluation. I manipulated outcome, attitude and deservingness experimentally, and measured specific emotions. It appeared deservingness to play a secondary role in eliciting joy and sorrow when interpersonal attitudes were involved.

Can widely used cell type markers predict the suitability of immortalized or primary mammary epithelial cell models?

BACKGROUND Mammary cell cultures are convenient tools for in vitro studies of mammary gland biology. However, the heterogeneity of mammary cell types, e.g., glandular milk secretory epithelial or myoepithelial cells, often complicates the interpretation of cell-based data. The present study was undertaken to determine the relevance of bovine primary mammary epithelial cells isolated from American Holstein (bMECUS) or Swiss Holstein-Friesian (bMECCH) cows, and of primary bovine mammary alveolar epithelial cells stably transfected with simian virus-40 (SV-40) large T-antigen (MAC-T) for in vitro analyses. This was evaluated by testing their expression pattern of cytokeratin (CK) 7, 18, 19, vimentin, and α-smooth muscle actin (α-SMA). RESULTS The expression of the listed markers was assessed using real-time quantitative PCR, flow cytometry and immunofluorescence microscopy. Characteristic markers of the mesenchymal (vimentin), myoepithelial (α-SMA) and glandular secretory cells (CKs) showed differential expression among the studied cell cultures, partly depending on the analytical method used. The relative mRNA expression of vimentin, CK7 and CK19, respectively, was lower (P < 0.05) in immortalized than in primary mammary cell cultures. The stain index (based on flow cytometry) of CK7 and CK19 protein was lower (P < 0.05) in MAC-T than in bMECs, while the expression of α-SMA and CK18 showed an inverse pattern. Immunofluorescence microscopy analysis mostly confirmed the mRNA data, while partly disagreed with flow cytometry data (e.g., vimentin level in MAC-T). The differential expression of CK7 and CK19 allowed discriminating between immortal and primary mammary cultures. CONCLUSIONS The expression of the selected widely used cell type markers in primary and immortalized MEC cells did not allow a clear preference between these two cell models for in vitro analyses studying aspects of milk composition. All tested cell models exhibited to a variable degree epithelial and mesenchymal features. Thus, based on their characterization with widely used cell markers, none of these cultures represent an unequivocal alveolar mammary epithelial cell model. For choosing the appropriate in vitro model additional properties such as the expression profile of specific proteins of interest (e.g., transporter proteins) should equally be taken into account.

The influence of the rearing period on intramammary infections in Swiss dairy heifers: A cross-sectional study

Healthy replacement heifers are one of the foundations of a healthy dairy herd. Farm management andrearing systems in Switzerland provide a wide variety of factors that could potentially be associated withintramammary infections (IMI) in early lactating dairy heifers. In this study, IMI with minor mastitispathogens such as coagulase-negative staphylococci (CNS), contagious pathogens, and environmentalmajor pathogens were identified. Fifty-four dairy farms were enrolled in the study. A questionnaire wasused to collect herd level data on housing, management and welfare of young stock during farm isitsand interviews with the farmers. Cow-level data such as breed, age at first calving, udder condition andswelling, and calving ease were also recorded. Data was also collected about young stock that spent aperiod of at least 3 months on an external rearing farm or on a seasonal alpine farm. At the quarterlevel, teat conditions such as teat lesions, teat dysfunction, presence of a papilloma and teat lengthwere recorded. Within 24 h after parturition, samples of colostral milk from 1564 quarters (391 heifers)were collected aseptically for bacterial culture. Positive bacteriological culture results were found in 49%of quarter samples. Potential risk factors for IMI were identified at the quarter, animal and herd levelusing multivariable and multilevel logistic regression analysis. At the herd level tie-stalls, and at cow-level the breed category “Brown cattle” were risk factors for IMI caused by contagious major pathogenssuch as Staphylococcus aureus (S. aureus). At the quarter-level, teat swelling and teat lesions were highlyassociated with IMI caused by environmental major pathogens. At the herd level heifer rearing at externalfarms was associated with less IMI caused by major environmental pathogens. Keeping pregnant heifersin a separate group was negatively associated with IMI caused by CNS. The odds of IMI with coagulase-negative staphylococci increased if weaning age was less than 4 months and if concentrates were fed tocalves younger than 2 weeks. This study identified herd, cow- and quarter-level risk factors that may beimportant for IMI prevention in the future.