Evaluation of the bacteriological quality of raw cow's milk at various stages of the milk production chain on farms in Algeria. © OIE (World Organisation for Animal Health), 2016. / Évaluation de la qualité bactériologique du lait cru bovin à divers stades de la chaîne de production laitière dans des fermes en Algérie.

This study evaluates hygiene practices on 53 dairy farms in the Jijel and Blida regions of Algeria. A survey questionnaire was drawn up covering milking conditions and cleaning of the equipment. In parallel, bacteriological analyses were carried out to estimate the rate, source and development of bacterial contamination in raw milk produced on the farm. In addition, screening was performed to detect the presence of inhibitor residues. The results of the survey revealed poor livestock conditions and milking practices that could explain the presence of bacteria in cow's milk. The bacteriological results showed that 76.1% of milk samples taken from cow udders complied with legal standards, compared with only 35.8% of milk samples taken from storage tanks. Moreover, bacterial inhibitors were detected in 28.8% of milk samples. These results showed that the hands of milkers, udders, teat cups, utensils, the water used during milking and the milking environment were all potential sources of milk contamination by the bacteria under investigation. These results suggest that, to improve the bacteriological quality of milk, there is a need to introduce a quality policy which places a premium on milk of high bacteriological quality and aims to generalise good hygiene practices throughout the dairy production chain.

La présente étude consiste à évaluer les pratiques d'hygiène instaurées dans 53 exploitations bovines laitières réparties dans les régions de Jijel et de Blida en Algérie. Pour cela, un questionnaire d'enquête a été élaboré, portant sur les conditions de la traite et sur le nettoyage du matériel utilisé. En parallèle, des analyses bactériologiques ont été effectuées afin d'estimer le taux, l'origine et l'évolution de la contamination bactérienne du lait cru produit à la ferme. En outre, des recherches ont été effectuées pour déceler la présence de résidus d'inhibiteurs. Les résultats de l'enquête ont mis en évidence les mauvaises conditions d'élevage et des pratiques de la traite qui peuvent expliquer la présence de bactéries dans le lait de vache. Les résultats bactériologiques ont montré que 76,1 % des échantillons de lait prélevé au pis des vaches étaient conformes aux critères légaux, contre 35,8 % seulement des échantillons de lait provenant des cuves de stockage. De plus, la présence d'inhibiteurs bactériens a été décelée dans 28,8 % des échantillons de lait. Ces résultats ont permis de déterminer que les mains des trayeurs, les mamelles, les gobelets trayeurs, les ustensiles, l'eau et l'environnement de la traite étaient les sources potentielles de contamination du lait par les bactéries recherchées. À la lumière de ces résultats, l'amélioration de la qualité bactériologique du lait repose sur l'instauration d'une politique de qualité, visant à vulgariser les bonnes pratiques d'hygiène tout au long de la chaîne de production laitière et à mettre en place une prime à la qualité bactériologique du lait.

Los autores exponen un estudio destinado a evaluar las prácticas de higiene empleadas en 53 explotaciones bovinas lecheras de las regiones de Jijel y Blida (Argelia). Para ello se elaboró un cuestionario relativo a las condiciones de ordeño y a la limpieza del material utilizado. Paralelamente se realizaron análisis bacteriológicos con el fin de estimar la tasa, el origen y la evolución de la contaminación bacteriana de la leche cruda producida en cada explotación. Además, se efectuaron investigaciones para detectar la presencia de residuos de inhibidores. Los resultados de la investigación pusieron de manifiesto que las condiciones de cría y las prácticas de ordeño eran inadecuadas, lo que puede explicar la presencia de bacterias en la leche de vaca. Los resultados bacteriológicos evidenciaron que un 76,1 % de las muestras de leche tomadas en la ubre de las vacas cumplían los criterios legales, por solo un 35,8 % de las muestras procedentes de las cubas de almacenamiento. Por otro lado, se detectó la presencia de inhibidores bacterianos en un 28,8 % de las muestras de leche. Estos resultados sirvieron para determinar que las posibles fuentes de contaminación de la leche por las bacterias investigadas se encontraban en las manos de los ordeñadores, las ubres, los cubos de ordeño, los utensilios empleados, el agua y el espacio en que discurría el ordeño. A tenor de los resultados, la mejora de la calidad bacteriológica de la leche pasa por la aplicación de una política de calidad, que sirva para divulgar las prácticas idóneas de higiene en toda la cadena de producción lechera y para instituir una prima a la calidad bacteriológica de la leche.

Contribution to the study of staphylococcus contamination of cows' milk on a number of farms in Algiers: its impact on human health.

The authors describe a survey and screening programme for staphylococcus. The study covers 14 dairy farms in the Algiers region, from which 203 samples of cows' milk were taken for bacteriological testing. The survey results show that poor husbandry conditions are the main cause of staphylococcus in cows' milk. Staphylococcus was found in the milk of 30% of the cows sampled. These results were influenced by a variety of factors, in that: the contamination rate rose with the number of pregnancies, age, and volume of milk output of the cow, as well as the bedding thickness; the milk contamination rate was greater when milking occurred outside a milking parlour and when it was performed by machine; higher rates of staphylococcus infection were found in the milk of cows at the end of lactation, in red and white breeds, and in those with cylindricalteats. Identification of the bacteria found (staphylococcus) showed that coagulase- negative staphylococci were present in 67.21% of samples, whereas coagulase- positive staphylococci were present in only 32.79%. The average count for the latter was equal to 0.54 x 10(4) colony-forming units per ml of Staphylococcus aureus. Seventy percent of the milk analysed was free from staphylococci and most of the bacteria identified were not pathogenic to consumers (coagulase- negative staphylococci); nevertheless, consuming fresh milk still presents a degree of risk.

Interaction of PTPB with the insulin receptor precursor during its biosynthesis in the endoplasmic reticulum.

PTP1B is a protein tyrosine-phosphatase predominantly located on the cystosolic surface of the endoplasmic reticulum. This tyrosine-phosphatase plays a major role in the regulation of the activity of the insulin receptor (IR). We have studied the interaction of the IR with PTP1B in living cells using bioluminescence resonance energy transfer (BRET). The IR was fused to Renilla luciferase and a substrate-trapping mutant of PTP1B was fused to the yellow variant of the green fluorescent protein (YFP). When the two partners interacted, an energy transfer occurred between the luciferase and the YFP, and a fluorescent signal, emitted by the YFP, could be detected. The interaction of the IR with PTP1B could be monitored in real time for more than 30 min. Insulin rapidly and dose-dependently stimulated this interaction. The basal (insulin-independent) interaction of IR with PTP1B was much lower with a soluble form than with the endoplasmic reticulum-targeted form of PTP1B, indicating that this basal interaction mainly occurred in the endoplasmic reticulum. In the basal state, PTP1B and the IR indeed co-localized in the endoplasmic reticulum, as demonstrated by confocal microscopy and cell fractionation experiments. Moreover, inhibition of IR processing with tunicamycin indicated that the basal interaction of PTP1B with IR occurred during biosynthesis of the IR precursor in the endoplasmic reticulum. These results strongly suggest that PTP1B not only dephosphorylates the insulin receptor that has been activated by insulin, but also regulates the insulin receptor precursor during its biosynthesis. Localisation of PTP1B to the endoplasmic reticulum may be important to prevent insulin-independent autonomous activity of the immature insulin receptor precursor.

Looking for an insulin pill? Use the BRET methodology!

Insulin exerts its biological effects through a plasma membrane receptor that possesses a tyrosine-kinase activity. This tyrosine-kinase activity depends on the autophosphorylation of the receptor on tyrosine residues and on its dephosphorylation by protein tyrosine-phosphatases. The discovery of pharmacological agents that specifically stimulate the autophosphorylation of the insulin receptor or inhibit its dephosphorylation will be of great importance for the treatment of insulin resistant or insulin deficient patients. Bioluminescence Resonance Energy Transfer (BRET) has developed in recent years as a new technique to study protein-protein interactions. In the BRET technique, one partner is fused to Renilla luciferase, whereas the other partner is fused to a fluorescent protein (e.g. YFP, Yellow Fluorescent Protein). The luciferase is excited by addition of its substrate, coelenterazine. If the two partners interact, resonance energy transfer occurs between the luciferase and the YFP, and a fluorescent signal, emitted by the YFP, can be detected. Our work indicates that this methodology could be an important tool for the search of molecules that activate insulin receptor autophosphorylation or that inhibit its dephosphorylation. Indeed, we first showed that the activation of the insulin receptor by different ligands can be monitored using a chimeric receptor with one B-subunit fused to Renilla luciferase and the other B-subunit fused to YFP. The conformational changes induced by different ligands could be detected as an energy transfer (BRET signal) between the luciferase and the YFP, that reflects the activation state of the receptor. This methodology allows for rapid analysis of the effects of agonists on insulin receptor activity and may therefore be used in high-throughput screening for the discovery of molecules with insulin-like properties. More recently, we demonstrated that the BRET methodology could also be used to monitor the interaction of the insulin receptor with protein tyrosine-phosphatase 1B, one of the main tyrosine-phosphatase that controls its activity. HEK cells were co-transfected with the insulin receptor fused to Renilla luciferase and a substrate-trapping mutant of PTP1B (PTP1B-D181A) fused to YFP. Insulin-induced BRET signal could be followed in real time for more than 30 min. Therefore, this methodology can also be used in high-throughput screening for the search of molecules that will specifically disrupt the interaction between the insulin receptor and PTP1B.

Rhabdovirus infection induces public and private T cell responses in teleost fish.

Many viruses induce a strong T cell response that contributes to the elimination of infected cells presenting viral peptides by MHC molecules. The structure and expression of genes encoding molecules homologous to mammalian alphabeta TCRs have been recently characterized in rainbow trout and in several teleost species, but the alphabeta T cell response against pathogens has not been directly demonstrated. To study the modifications of the T cell repertoire during an acute viral infection in rainbow trout, we adapted the immunoscope methodology, which consists of spectratyping the complementarity-determining region 3 length of the TCRbeta chain. We showed that the naive T cell repertoire is polyclonal and highly diverse in the naive rainbow trout. Using viral hemorrhagic septicemia virus (VHSV), which provokes an acute infection in rainbow trout, we identified skewed complementarity-determining region 3 size profiles for several VbetaJbeta combinations, corresponding to T cell clonal expansions during primary and secondary response to VHSV. Both public and private T cell expansions were shown by immunoscope analysis of spleen cells from several infected individuals of a rainbow trout clone sharing the same genetic background. The public response to VHSV consisted of expansion of Vbeta4Jbeta1 T cell, which appeared early during the primary response and was strongly boosted during the secondary response.

Participation of acetaldehyde dehydrogenases in ethanol and pyruvate metabolism of the yeast Saccharomyces cerevisiae.

This work was undertaken to clarify the role of acetaldehyde dehydrogenases in Saccharomyces cerevisiae metabolism during growth on respiratory substrates. Until now, there has been little agreement concerning the ability of mutants deleted in gene ALD4, encoding mitochondrial acetaldehyde dehydrogenase, to grow on ethanol. Therefore we constructed mutants in two parental strains (YPH499 and W303-1a). Some differences appeared in the growth characteristics of mutants obtained from these two parental strains. For these experiments we used ethanol, pyruvate or lactate as substrates. Mitochondria can oxidize lactate into pyruvate using an ATP synthesis-coupled pathway. The ald4Delta mutant derived from the YPH499 strain failed to grow on ethanol, but growth was possible for the ald4Delta mutant derived from the W303-1a strain. The co-disruption of ALD4 and PDA1 (encoding subunit E1alpha of pyruvate dehydrogenase) prevented the growth on pyruvate for both strains but prevented growth on lactate only in the double mutant derived from the YPH499 strain, indicating that the mutation effects are strain-dependent. To understand these differences, we measured the enzyme content of these different strains. We found the following: (a) the activity of cytosolic acetaldehyde dehydrogenase in YPH499 was relatively low compared to the W303-1a strain; (b) it was possible to restore the growth of the mutant derived from YPH499 either by addition of acetate in the media or by introduction into this mutant of a multicopy plasmid carrying the ALD6 gene encoding cytosolic acetaldehyde dehydrogenase. Therefore, the lack of growth of the mutant derived from the YPH499 strain seemed to be related to the low activity of acetaldehyde oxidation. Therefore, when cultured on ethanol, the cytosolic acetaldehyde dehydrogenase can partially compensate for the lack of mitochondrial acetaldehyde dehydrogenase only when the activity of the cytosolic enzyme is sufficient. However, when cultured on pyruvate and in the absence of pyruvate dehydrogenase, the cytosolic acetaldehyde dehydrogenase cannot compensate for the lack of the mitochondrial enzyme because the mitochondrial form produces intramitochondrial NADH and consequently ATP through oxidative phosphorylation.

A mitochondrial pyruvate dehydrogenase bypass in the yeast Saccharomyces cerevisiae.

Spheroplasts of the yeast Saccharomyces cerevisiae oxidize pyruvate at a high respiratory rate, whereas isolated mitochondria do not unless malate is added. We show that a cytosolic factor, pyruvate decarboxylase, is required for the non-malate-dependent oxidation of pyruvate by mitochondria. In pyruvate decarboxylase-negative mutants, the oxidation of pyruvate by permeabilized spheroplasts was abolished. In contrast, deletion of the gene (PDA1) encoding the E1alpha subunit of the pyruvate dehydrogenase did not affect the spheroplast respiratory rate on pyruvate but abolished the malate-dependent respiration of isolated mitochondria. Mutants disrupted for the mitochondrial acetaldehyde dehydrogenase gene (ALD7) did not oxidize pyruvate unless malate was added. We therefore propose the existence of a mitochondrial pyruvate dehydrogenase bypass different from the cytosolic one, where pyruvate is decarboxylated to acetaldehyde in the cytosol by pyruvate decarboxylase and then oxidized by mitochondrial acetaldehyde dehydrogenase. This pathway can compensate PDA1 gene deletion for lactate or respiratory glucose growth. However, the codisruption of PDA1 and ALD7 genes prevented the growth on lactate, indicating that each of these pathways contributes to the oxidative metabolism of pyruvate.