Efficient control against bovine mastitis requires sensitive, rapid, and specific tests to detect and identify the main bacteria that cause heavy losses to the dairy industry. Molecular detection of pathogenic microorganisms is based on DNA amplification of the target pathogen. Therefore, efficient extraction of DNA from pathogenic bacteria is a major step. In this study, we aimed to develop a specific, sensitive, and rapid method to extract DNA directly from the main gram-positive bacteria known to cause bovine mastitis (Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis) found in milk samples. The DNA extraction method is based on the lysing and nuclease-inactivating properties of the chaotropic agent, guanidinium thiocyanate, together with the nucleic acid-binding properties of the silica particles. An efficient protocol consisting of 6 basic steps (3 of which were done twice) was developed and applied directly to milk samples. Absence of PCR inhibitors and DNA quality were evaluated by PCR amplification of the species-specific DNA sequences of the target bacteria. The level of sensitivity achieved in our experiments is applicable to milk sample analysis without sample enrichment.

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The performances and the stability of a novel subcutaneous glucose monitoring system have been evaluated. GlucoDay † (A. Menarini I.F.R. S.r.l, Florence Italy) is a portable instrument provided with a micro-pump and a biosensor coupled to a microdialysis system capable of recording the subcutaneous glucose level every 3 min. Long and short term stability of the biosensor are discussed and the results of some critical in vitro and in vivo (on rabbits) experiments are reported. A linear response up to 30 mM has been found for in vivo glucose concentration. The sensitivity referred to blood glucose is better than 0.1 mM and the zero current is typically below the equivalent of 0.1 mM. In the accuracy study a mean bias of 2.7 mg/dl and a correlation coefficient equal to 0.9697 have been found. At room temperature, an excellent membrane stability assures good performances up to 6 months from the first use. #

Aim:  To develop an easy, rapid and efficient DNA extraction procedure for Staphylococcus aureus detection with a low number of steps and removing completely the PCR inhibitors, applicable to raw milk cheese samples, and to compare phenotypical and genotypical method to detect Staph. aureus isolates and staphylococcal enterotoxins (SEs) production. Methods and Results:  A total of 33 bovine and caprine raw milk cheese samples were analysed by means of both classic microbiological and molecular techniques. All samples were positive for Staph. aureus contamination. The DNA extraction protocol optimized was found to achieve a detection limit of 100 CFU g−1 for Staph. aureus. None of the samples tested with immunological assays contained SEs but in 14 of 33 samples a mixture of se positive (sea, sec, sed, seg, sel, sej) isolates were identified. Conclusions:  Staphylococcus aureus is a food‐borne pathogen mainly detected in finished dairy products. The rapid and efficient detection of Staph. aureus isolates from dairy products is essential for consumer safety. The direct detection of pathogens from food is possible with careful attention to sample preparation and nucleic acid amplification optimization. Significance and Impact of the Study:  This study shows that raw milk cheese samples can be tested for Staph. aureus contamination with a rapid, simple and reproducible procedure.

Adaptation of Sherpas to high altitude has been studied and compared with that of Caucasians acclimatized to high altitude. Sherpas living permanently at 4000 m above sea level do not have increased hematological parameters (i.e., red cell number, hematocrit, hemoglobin content, and 2,3-diphosphoglycerate/hemoglobin ratio) and have a higher affinity of blood for oxygen as compared with acclimatized Caucasians. Sherpas permanently living at low altitude, on the contrary, have lower affinity of blood for oxygen than do Caucasians living at comparable altitude and are mildly "anemic." Various other red cell biochemical parameters (possibly related to adaptation to altitude) have also been studied in the same population.We suggest that Sherpas are genetically better adapted to high altitude than are Amerindians living on the Peruvian highlands, possibly as a consequence of a much more prolonged exposure to such an ecological factor of selection as high altitude.The populations living for thousands of years at high altitudes may adapt to low oxygen tension with a variety of mechanisms (for a review see ref. 1). In a previous paper (2), we have shown that Amerindians living on the Peruvian highlands show an increased Bohr effect (determined on hemolysates diluted in 0.1 M phosphate buffer) when they are compared with acclimatized Europeans. This fact has been interpreted as an evolutionary adaptation to low oxygen tension that results in an increase in the release of oxygen to the tissues. In contrast, analysis of a Sherpa population, living at the same altitude in the Himalayas, failed to show any difference in the oxygen affinity of diluted hemolysates from that of acclimatized Europeans (3).In order to understand how Sherpas have achieved their remarkable adaptation to altitude, we have studied in the present research, among a Sherpa population living permanently at 3800-3900 m above sea level, (i) some hematological parameters, i.e., red cell count, hematocrit value, and hemoglobin (Hb) content; (ii) oxygen dissociation curves determined on whole blood; (iii) concentration of some metabolites possibly involved in the adaptation to altitude, i.e., 2,3-diphosphoglycerate (2,3 P2G), ATP, ADP, lactate, and reduced glutathione; (iv) rate of synthesis of 2,3-P2G in presence and absence of oxygen; and (v) activity of some enzymes of the erythrocyte. Most of these parameters were determined also on some Sherpas living permanently at 1200 m above sea level.This Hb concentration was determined as cyanmethemoglobin (4). The histograms in Fig. 1 The oxygen dissociation curves were determined in whole blood 5-6 days after sampling, at constant pH and C02 pressure (6). The data (Fig. 2

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The aim of this study was to evaluate the reproducibility, the accuracy and the reliability of a continuous subcutaneous glucose measuring system. The GlucoDay system (A. Menarini I.F.R. S.r.l.-Florence, Italy) is a portable instrument provided with a micro-pump and a biosensor, coupled to a microdialysis system (see part 1). This instrument has demonstrated high reliability coupled with a low degree of invasivity. The profiles of glucose monitoring allow to achieve an excellent knowledge of the real variation of glucose in diabetic patients. The reproducibility study showed a bias lower than 10% between instruments. The accuracy study showed a difference from the reference method lower than 15%.

-A differential pH method for the routine determination of urea in milk is presented. The measure is based upon a single enzymatic reaction, the urea hydrolysis by urease, which causes a pH variation directly proportional to the urea content in the milk sample. No sample pretreatment is required. Repeatability expressed as a coefficient of variation was 0.85 % at a level of 416 mg-urea-L-1. Recovery of added urea averaged 99.4 %. The preservatives Bronopol and sodium azide at a ten times higher level than recommended did not affect the results. Comparison with the manual enzymatic French reference method showed a very good agreement. The method is very simple, accurate and rapid. © InralElsevier, Paris urea 1 differential pH 1 milk 1 enzymatic assay Résumé -Dosage de l'urée dans le lait avec la technique de pHmétrie différentielle. Une méthode de pHmétrie différentielle pour la détermination de l'urée dans le lait est présentée. La mesure est basée sur une seule réaction enzymatique, l'hydrolyse de l'urée par l'enzyme urease, qui provoque une variation de pH directement proportionnelle au niveau de l'urée dans l'échantillon de lait. Aucun prétraitement de l'échantillon n'est nécessaire. Le coefficient de variation de la répétabilité vaut 0,85 % au niveau de 416 rng-urée-L -1. La détermination de l'urée ajoutée était en moyenne de 99,4 %. L'addition de conservateurs tels que le Bronopol ou l'azide de sodium à une concentration dix fois supérieure à la quantité conseillée n'avait aucune influence sur les résultats. La comparaison avec la méthode manuelle enzymatique française de référence donne une très bonne corrélation. La méthode est très simple, précise et rapide. © Inra!Elsevier, Paris urée 1 pHmétrie différentielle liait 1 essai enzymatique * Correspondence and reprints. 262 M. Luzzana, R. Giardino