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Phylogenetic relationships of Western Mediterranean West Nile virus strains (1996-2010) using full-length genome sequences Single or multiple introductions?

  • Sotelo, E.
  • Fernández-Pinero, J.
  • Llorente, F.
  • Vázquez, A.
  • Moreno, A.
  • Agüero, M.
  • Cordioli, P.
  • Tenorio, A.
  • Jiménez-Clavero, M. A.
In recent years, West Nile virus (WNV) has re-emerged in the Western Mediterranean region. As a result, the number of complete WNV genome sequences available from this region has increased, allowing more detailed phylogenetic analyses, which may help to understand the evolutionary history of WNV circulating in the Western Mediterranean. To this aim, the present work describes six new complete WNV sequences from recent outbreaks and surveillance in Italy in 2008-2009 and in Spain in 2008 and 2010. Comparison with other sequences from different WNV clusters within lineage 1 (clade 1a) confirmed that all Western Mediterranean WNV isolates obtained since 1996 (except one from Tunisia, collected in 1997) cluster in a single monophyletic group (here called 'WMed' subtype). The analysis differentiated two subgroups within this subtype, which appear to have evolved from earlier WMed strains, suggesting a single introduction in the area, and further dissemination and evolution. Close similarities between WNV variants circulating in consecutive years, one in Spain, between 2007 and 2008, and another in Italy between 2008 and 2009, suggest that the virus possibly overwinters in Western Mediterranean sites. The NS3249-proline genotype, recently proposed as a virulence determinant for WNV, has arisen independently at least twice in the area. Overall, these results indicate that the frequent recurrence of outbreaks caused by phylogenetically homogeneous WNV in the Western Mediterranean since 1996 is consistent with a single introduction followed by viral persistence in endemic foci in the area, rather than resulting from independent introductions from exogenous endemic foci. © 2011 SGM.
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Climatic niche and neutral genetic diversity of the six Iberian pine species A retrospective and prospective view

  • Soto, A.
  • Robledo-Arnuncio, J. J.
  • González-Martínez, S. C.
  • Smouse, P. E.
  • Alía Miranda, Ricardo
Quaternary climatic fluctuations have left contrasting historical footprints on the neutral genetic diversity patterns of existing populations of different tree species. We should expect the demography, and consequently the neutral genetic structure, of taxa less tolerant to particular climatic extremes to be more sensitive to long-term climate fluctuations. We explore this hypothesis here by sampling all six pine species found in the Iberian Peninsula (2464 individuals, 105 populations), using a common set of chloroplast microsatellite markers, and by looking at the association between neutral genetic diversity and species-specific climatic requirements. We found large variation in neutral genetic diversity and structure among Iberian pines, with cold-enduring mountain species (Pinus uncinata, P. sylvestris and P. nigra) showing substantially greater diversity than thermophilous taxa (P. pinea and P. halepensis). Within species, we observed a significant positive correlation between population genetic diversity and summer precipitation for some of the mountain pines. The observed pattern is consistent with the hypotheses that (i) more thermophilous species have been subjected to stronger demographic fluctuations in the past, as a consequence of their maladaptation to recurrent glacial cold stages; and (ii) altitudinal migrations have allowed the maintenance of large effective population sizes and genetic variation in cold-tolerant species, especially in more humid regions. In the light of these results and hypotheses, we discuss some potential genetic consequences of impending climate change. © 2010 Blackwell Publishing Ltd.
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Nitrous oxide and methane emissions from a vetch cropping season are changed by long-term tillage practices in a Mediterranean agroecosystem

  • Tellez-Rio, A.
  • García-Marco, S.
  • Navas, M.
  • López-Solanilla, E.
  • Rees, R. M.
  • Tenorio, J. L.
  • Vallejo, A.
Lower greenhouse gas (GHG) emissions from legume-based cropping systems have encouraged their use to deliver mitigation in agricultural systems. Considerable uncertainties remain about the interaction of legumes with long-term tillage systems on GHG emissions under rainfed agroecosystems. In this context, a field experiment was undertaken under a rainfed vetch crop to evaluate the effect of three long-term tillage systems (i.e. no tillage (NT), minimum tillage (MT) and conventional tillage (CT)) on nitrous oxide (N2O) and methane (CH4) emissions for 1 year. Different N2O flux patterns were observed among tillage systems during the growth period of vetch, which depended on the soil conditions favouring nitrification and denitrification. The NT system maintained a higher sink for N2O than MT and CT from January to mid-April, which significantly reduced N2O emissions at this stage. In this period, denitrification capacity and nirK gene numbers were higher for MT than NT and CT. Additionally, an increase in soil NO− 3 content and more favourable denitrification conditions in MT and NT than in CT for the last crop period increased N2O emissions in conservation tillage systems. Total annual N2O losses were significantly higher in MT (124.2 g N2O–N ha−1) than NT (51.1 g N2O–N ha−1) and CT (54 g N2O–N ha−1) in a vetch crop. Low net uptake of CH4 was observed for all tillage systems. These results suggested that long-term NT may be a better option thanMT to mitigate GHG emissions in rainfed legume-cereal rotation. © Springer-Verlag Berlin Heidelberg 2014.
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Changes induced by natural scrapie in the calretinin-immunopositive cells and fibres of the sheep cerebellar cortex

  • Toledano, A.
  • Alvarez, M. I.
  • Monleón, E.
  • Toledano-Díaz, A.
  • Badiola, J. J.
  • Monzón, M.
Calretinin (CR)-immunopositive cells and fibres in the cerebellar cortex (vermal archicerebellum and neocerebellum) of scrapie-affected, ARQ/ARQ, Rasa Aragonesa breed sheep were studied in comparison with healthy, young and aged, ARQ/ARQ, Rasa Aragonesa animals and with Manchega breed sheep. The scrapie-affected sheep showed signs of both cellular involution and hypertrophic/ hyperimmunoreactive responses in all neuronal subtypes; the distribution of the neuronal subtypes in the archi- and neocerebellum, however, did not change compared with controls. The results suggest that the different CR expression and/or CR content of cerebellar cortical neurons in scrapie-affected sheep are more related to their specific functions than any neuroprotective response. The reduction in the cell density of some CR-immunopositive neuronal subsets (i.e. unipolar brush cells) is contradictory to the supposed neuroprotective role of the calcium binding protein CR. However, the hyperimmunoreactivity of many CR-immunopositive neuronal subsets (e.g. the Purkinje cells) suggests the involvement of an over-expression of CR (transitory or restricted to selected neurons) as an adaptative mechanism to fight against the neurodegeneration caused by this prion disease. The changes in the number of immunopositive cells and the hypertrophic/ hyperimmunoreactive response seen in scrapie-affected and aged sheep suggests that some different and some similar mechanisms are at work in this disease and aging. ©Springer Science+Business Media, LLC 2011.
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Elevated non-esterified fatty acid concentrations during in vitro murine follicle growth alter follicular physiology and reduce oocyte developmental competence

  • Valckx, S. D. M.
  • Van Hoeck, V.
  • Arias-Alvarez, M.
  • Maillo, V.
  • Lopez-Cardona, A. P.
  • Gutierrez-Adan, A.
  • Berth, M.
  • Cortvrindt, R.
  • Bols, P. E. J.
  • Leroy, J. L. M. R.
Objective To study how long-term elevated non-esterified fatty acid (NEFA) concentrations, typical in metabolic disorders such as obesity or type 2 diabetes, affect murine follicular development, follicle quality, and subsequent oocyte developmental competence in vitro. Design Experimental study. Setting In vitro culture setting. Animal(s) Female and male 13-day old, B6CBAF1 mice of proven fertility were sacrificed for harvesting ovaries and epididymal sperm, respectively. Intervention(s) Early secondary murine follicles were cultured in vitro in the presence of NEFAs until the antral stage (12 days). Treatments consisted of one or a mixture of NEFAs (stearic acid [SA], palmitic acid [PA], oleic acid [OA]) in physiological (basal) or pathological (high SA, high OA, high NEFA) concentrations. Main Outcome Measure(s) Follicular development; follicle and oocyte diameters; secretion of progesterone, estradiol, and inhibin B; and luteinized granulosa cell gene expression patterns were investigated. Oocytes from NEFA-exposed follicles were fertilized in vitro, and presumptive zygotes were cultured until the blastocyst stage. Result(s) Exposure to high SA reduced follicle diameters and day-12 antrum formation. Elevated NEFA concentrations changed luteinized granulosa cell messenger-ribonucleic acid abundance of genes related to energy/fatty acid/steroid metabolism, apoptosis, and oxidative stress. High NEFA and high SA treatments increased progesterone synthesis, compared with high OA follicles. Oocyte developmental competence was substantially reduced in oocytes retrieved from high OA-, high SA-, and high NEFA-exposed follicles compared with basal-treated follicles. Conclusion(s): This study showed, for the first time, that lipolysis-linked, elevated NEFA concentrations can potentially impair fertility, by altering follicular physiology and reducing oocyte developmental competence. © 2014 by American Society for Reproductive Medicine.
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Development of a genetic indicator of biodiversity for farm animals

  • Villanueva, B.
  • Sawalha, R. M.
  • Roughsedge, T.
  • Rius-Vilarrasa, E.
  • Woolliams, J. A.
In 2002, Parties to the UN Convention on Biological Diversity made a commitment to "achieve by 2010 a significant reduction of the current rate of biodiversity loss at the global, regional and national level". In order to assess progress towards the 2010 target a limited number of indicators of biodiversity need to be developed using existing data. The objectives of this study were i) to produce an indicator of genetic diversity for livestock species; and ii) to evaluate the proposed indicator in UK sheep and cattle. The indicator proposed is the species average effective population size (Ne) for the lower tail of the distribution of Ne across breeds within the species. It is sensitive to i) genetic variation within breeds, as it is based on the Ne of individual breeds, and ii) what is happening to breeds most at risk of disappearing. A total of 31 sheep and 20 cattle UK breed societies provided the information required for estimating Ne. This represents 53% of sheep and 58% of cattle breeds native to the UK. For breeds with pedigrees available, Ne was estimated from rates of change in inbreeding. For breeds with no pedigree information, Ne was estimated from predictive equations assuming mass selection on a trait with a heritability of 0.4. For each species, the indicator was calculated by i) estimating Ne for each breed; ii) calculating the distribution of Ne; iii) finding the average Ne for the lower 20% tail of the distribution. In step iii), 20% was chosen because, given the number of breeds with available information, it provides a good compromise between giving high weight to the breeds most at risk, without being too sensitive to events surrounding a single breed. For sheep, the indicator values were 36 and 41 animals for years 2001 and 2007, respectively. Equivalent values for cattle were 26 and 34. Bootstrapping was used to assess if these changes over time would have been observed with data from all native breeds. There was no evidence that the small increase in the value of the sheep indicator, predicted from the available sample of breed data, would have been observed with all breeds. However, there was evidence (P < 0.05) to support that an increase would have been observed with data from all cattle breeds. © 2010 Elsevier B.V. All rights reserved.
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Fractionation of Miscanthus x giganteus via modification of the Formacell process

  • Villaverde, J. J.
  • Ligero, P.
  • Vega, A.
Fractionation of Miscanthus x giganteus stalks (without core) using an organosolv system with carboxylic acids in two stages (Acetosolv+Formacell) produced highly purified pulps with kappa number values of 7.3-15.6 and viscosities above 750cm3/g. The pulp yields were approximately 52-57%, and the brightness suffered significant decreases (1.1-8.2%) in the second stage.The influence of process variables in the Formacell extended stage was analysed by means of factorial designs of experiments. Optimised operating variables yielded acceptable pulp properties at reasonable chemical costs and mild energetic conditions.A comparison was performed of four organosolv processes with carboxylic acids in two stages (Acetosolv. +. Formacell. , Acetosolv. +. peroxyacetic acid, Acetosolv. +. ozone and peroxyformic acid in two stages as a modification of the Milox process), which exploit the solvent that impregnate the pulps after the first stage. © 2015 Elsevier B.V..
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Activity of goats' milk lactoperoxidase system on Pseudomonas fluorescens and Escherichia coli at refrigeration temperatures

  • Zapico, P.
  • Gaya, P.
  • Nuñez, M.
  • Medina, M.
Bactericidal activity of the lactoperoxidase (LP) system against Pseudomonas fluorescens was observed in refrigerated raw goats' milk. Mean decreases in the levels of P. fluorescens of 1.69 log units at 4°C and 1.85 log units at 8°C were achieved during the first 24 h by LP-system activation. Inhibitory activity depended on temperature and length of incubation. P. fluorescens counts lower than the initial level were recorded in activated LP-system milk for 5 days at 4°C and 3 days at 8°C. Escherichia coli did not grow in raw goats' milk at 4°C, and the influence of LP-system activation at this temperature on E. coli counts was negligible. At 8°C, E. coli was able to grow in control milk with no apparent lag phase. In contrast, a lag phase of 2 days was observed in activated LP-system milk at 8°C, resulting in lower E. coli counts than those of control milk during the first 5 days. Copyright ©, International Association of Milk, Food and Environmental Sanitarians.
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Studies on the presence and absence of glycerol in unfrozen and frozen ram semen Fertility trials and the effect of dilution methods on freezing ram semen in the absence of glycerol

  • Abdelhakeam, A. A.
  • Graham, E. F.
  • Vazquez, I. A.
Trials were conducted to study fertility of ram semen under various breeding conditions. In trial 1, 60 cycling ewes were randomly divided into five treatment groups of 12 ewes. Group 1 was bred naturally. Groups 2 through 5 were artificially inseminated with pooled, diluted semen group 2 with fresh, diluted semen; group 3 with semen stored at 5 °C for 6 h; group 4 with semen containing 3% glycerol ( v v) stored at 5 °C for 6 h; and group 5 with frozen-thawed glycerolated (3%) semen. Lambing rates were 83, 91, 83, 41, and 33%, respectively. Trial 2 studied the site of semen deposition and the effect of dialysis on frozen-thawed glycerolated ram semen fertility. Seventy-eight ewes were divided into three treatment groups. Two groups of 33 ewes each were inseminated in the cervix; group 1 with nondialyzed glycerolated thawed semen and group 2 with glycerolated thawed semen dialyzed (110) against TEST-yolk-glucose extender for 30 min at 5 °C. In group 3, 12 ewes received intrauterine inseminations of glycerolated frozen-thawed semen. Percentage lambing rates were 33, 48, and 67%, respectively. Dialysis of frozen-thawed semen did not improve lambing rate significantly (P > 0.05). However, intrauterine insemination of glycerolated frozen-thawed semen yielded a significantly higher lambing rate (P < 0.05). The effect of different dilution methods on survival of ram sperm frozen in the absence of glycerol is described. Ram semen diluted by the cold dilution method and frozen in the absence of glycerol was inseminated into 46 cycling ewes. A lambing rate of 52% was obtained. © 1991.
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Short communication Inactivation of microbial contaminants in raw milk La Serena cheese by high-pressure treatments

  • Arqués, J. L.
  • Garde, S.
  • Gaya, P.
  • Medina, M.
  • Nuñez, M.
La Serena cheese, a Spanish variety made from Merino ewes' raw milk, has a high pH value, low salt content, and high moisture, conditions that are all favorable for growth and survival of contaminating microorganisms, including pathogens. To improve its microbiological quality and safety, high-pressure treatments at 300 or 400 MPa for 10 min at 10°C were applied to 2 batches of La Serena cheese on d 2 or 50 of ripening. Cheese treated on d 2 at 300 MPa showed viable aerobic counts that were 0.99 log units lower than those for control cheese on d 3 and showed counts of enterococci, coagulase-positive staphylococci, gram-negative bacteria, and coliforms that were 2.05, 0.49, 3.14, and 4.13 log units lower, respectively, than control cheese. For cheese treated on d 2 at 400 MPa, the respective reductions in counts were 2.02, 2.68, 1.45, 3.96, and 5.50 log units. On d 60, viable aerobic counts in cheese treated on d 50 at 300 MPa were 0.50 log units lower than those in control cheese, and counts of enterococci, gram-negative bacteria, and coliforms were 1.37, 2.30, and 4.85 log units lower, respectively. For cheese treated on d 50 at 400 MPa, the respective reductions in counts were 1.29, 1.98, 4.47, and >5 log units. High-pressure treatments at 300 or 400 MPa on d 2 or 50 reduced significantly the counts of undesirable microorganisms, improving the microbiological quality and safety of La Serena cheese immediately after treatment and at the end of the ripening period. © American Dairy Science Association, 2006.
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