Characterization of fluoroquinolone resistance in Escherichia coli strains from ruminants.

Thirty-seven fluoroquinolone-resistant Escherichia coli strains from ruminants (according to Clinical and Laboratory Standards Institute guidelines) were screened by molecular methods for mutations in the quinolone resistance-determining region (QRDR) of the gyrA and parC genes and for the presence of the qnrA gene. One of the strains studied was an enterohemorrhagic E. coli (EHEC) strain potentially pathogenic for humans. Three E. coli strains resistant to enrofloxacin (minimal inhibitory concentration [MIC] = 2 microg/ml) but not to ciprofloxacin (MIC = 1 microg/ml) presented single mutations in the gyrA and parC genes, while 34 strains resistant to both fluoroquinolones presented double and single mutations in gyrA and parC, respectively (31 strains), or double mutations in gyrA and parC (3 strains). The EHEC strain presented a double amino acid substitution in the GyrA protein (Ser-83-->Leu and Asp-87-->Gly) and a double amino acid substitution in the ParC protein (Gly-78-->Cys and Ser-80-->Arg), one of which has not been previously described. The present study shows that most of the mutations in the QRDR of the gyrA and parC genes of fluoroquinolone-resistant E. coli strains from ruminants are the same as those seen in E. coli strains from other animal species and humans and that there are no differences in mutation patterns in the QRDR of E. coli strains from healthy ruminants and those with diarrhea. No strains carried qnrA, which indicates that this gene does not play an important role in the selection of fluoroquinolone-resistant E. coli strains from ruminants.     

Seroprevalence of Neospora caninum in non-carnivorous wildlife from Spain

Serum samples from 1034 non-carnivorous wildlife from Spain were tested for antibodies to Neospora caninum by competitive screening enzyme linked immunosorbent assay (ELISA) and confirmed by an indirect fluorescent antibody test (IFAT). High agreement was observed between results in both techniques (kappa value higher than 0.9). Prevalences of N. caninum antibodies positive by both techniques were 11.8% of 237 red deer (Cervus elaphus), 7.7% of 13 barbary sheep (Ammotragus lervia), 6.1% of 33 roe deer (Capreolus capreolus) and 0.3% of 298 wild boar (Sus scrofa). In one of 53 hares (Lepus granatensis), antibodies were found in the ELISA but could not be confirmed by IFAT due to lack of sample. Antibodies to N. caninum were not found in any of 251 wild rabbits (Oryctolagus cuniculus), 79 fallow deer (Dama dama), 27 mouflon (Ovis ammon), 40 chamois (Rupicapra pyrenaica) and three Spanish ibex (Capra pyrenaica). Statistically significant differences were observed between N. caninum seroprevalence in red deer and management of hunting estates (open versus fenced) with higher prevalence in fenced estates, and among sampling sites. Seroprevalence was particularly high in some areas (MO estate in South-Central Spain or some estates of Catalonia, North-East Spain), while no contact with N. caninum was observed in others. Results indicate that in certain areas of Spain, N. caninum is present in wildlife, especially in red deer. These results have important implications in both sylvatic cycles and may influence the prevalence of infection in cattle farms in those areas. To our knowledge, this is the first report of antibodies to N. caninum in wildlife from Spain and the first report of N. caninum antibodies in barbary sheep and wild boar.     

Progesterone supplementation during mid-gestation increases the risk of abortion in Neospora-infected dairy cows with high antibody titres

Progesterone has been reported to reduce Th1 responses during pregnancy. Thus, given that an excessive Th1 response could lead to abortion in Neospora-infected dairy cows, we hypothesized a reduction in abortion risk following progesterone supplementation during the mid-gestation period in these animals. Seropositive animals were assigned on day 120 of gestation to a Control (n=33) or Treatment (n=34) group. Treatment consisted of progesterone supplementation by fitting a progesterone releasing intravaginal device containing 1.55g of progesterone for 28 days. Mean Neospora caninum antibody titres tested by ELISA on days 40, 90 and 120 of gestation were used to classify the cows as showing high (> or =30 units) or low (<30 units) titration. Abortion rate showed a significant increase (P<0.01) in the Treatment group (35%) compared to the Control (9%). Using logistic regression procedures, there was a significant (P<0.0001) interaction between progesterone treatment and antibody titration. Progesterone-treated cows with a high antibody titre showed a likelihood of abortion 14.3 times higher than those not treated, while in cows with low antibody titres, exogenous progesterone had not effect. Our results do not support our starting hypothesis and suggest that progesterone supplementation in cows with high N. caninum antibody titres increases the risk of abortion possibly by affecting cell-mediated immune responses.     

Transmission Efficiency of <Emphasis Type="Italic">Potato Leafroll Virus</Emphasis> by Four Potato Colonizing Aphid Species in Tunisian Potato Fields

This study investigated the transmission efficiency of Potato leafroll virus (PLRV) by four potato colonizing aphid species, Myzus persicae, Macrosiphum euphorbiae, Aphis gossypii and Aphis fabae, reported from leaves and yellow water trap. Physalis floridana was used as a test plant for virus transmission. DAS-ELISA was used for virus screening of samples as well as virus detection on the test plant after transmission experiment. A 2-h period was sufficient for the tested aphids to acquire PLRV virions. However, a difference in the transmission potential occurred according to the aphid species. The highest potential was recorded for M. persicae and M. euphorbiae, at 90 and 80%, respectively. For the first time, the study revealed the PLRV transmission efficiency of A. fabae, estimated at 50%. The lowest potential rate of 30% was recorded for A. gossypii. This study highlights the PLRV transmission capacities of four potato colonizing aphids suspected to play a key role in the spread of PLRV in potato seed production sites.     

Controlling Properties and Cytotoxicity of Chitosan Nanocapsules by Chemical Grafting

The tunability of the properties of chitosan-based carriers opens new ways for the application of drugs with low water-stability or high adverse effects. In this work, the combination of a nanoemulsion with a chitosan hydrogel coating and the following poly (ethylene glycol) (PEG) grafting is proven to be a promising strategy to obtain a flexible and versatile nanocarrier with an improved stability. Thanks to chitosan amino groups, a new easy and reproducible method to obtain nanocapsule grafting with PEG has been developed in this work, allowing a very good control and tunability of the properties of nanocapsule surface. Two different PEG densities of coverage are studied and the nanocapsule systems obtained are characterized at all steps of the optimization in terms of diameter, Z potential and surface charge (amino group analysis). Results obtained are compatible with a conformation of PEG molecules laying adsorbed on nanoparticle surface after covalent linking through their amino terminal moiety. An improvement in nanocapsule stability in physiological medium is observed with the highest PEG coverage density obtained. Cytotoxicity tests also demonstrate that grafting with PEG is an effective strategy to modulate the cytotoxicity of developed nanocapsules. Such results indicate the suitability of chitosan as protective coating for future studies oriented toward drug delivery.     

Interacting effects of soil degradation and precipitation on plant productivity in NE Patagonia,Argentina

Our objective was to examine the effects of inter-annual variation of precipitation on productivity of two dominant species (Chuquiraga avellanedae, an evergreen shrub, and Nassella tenuis, a perennial grass) in two communities of contrasting soil degradation: a herbaceous steppe with shrubs (HSS) and a degraded shrub steppe (SS). Data were collected during two consecutive years with different annual precipitation. Aboveground productivity was determined nondestructively using a double sampling approach. The number of inflorescences per plant was recorded too. Perennial grass productivity was lower in SS than in HSS in both years, while shrub productivity was lower in SS only during the year of below average precipitation. With rising precipitation the perennial grass increased the number of inflorescences while the evergreen shrub augmented vegetative biomass. In summary, the effects of precipitation on plant productivity are community dependent; abiotic factors, such as superficial and sub-superficial soil characteristics, and biotic factors, such as leaf area index (LAI) or tussock sizes, may interact to influence the responses of species to precipitation. Our results suggest that if precipitation increased, this would favor the dominance of shrubs over grasses.     

Methods for Cercospora coffeicola protoplast isolation and genetic transformation with the green fluorescent protein

Cercospora coffeicola is the causal agent of brown eye spot on coffee leaves. Although the disease has significant importance, few molecular studies have been done with C. coffeicola. Here we report a protocol for isolating protoplasts as well as development of a genetic transformation system using Green Fluorescent Protein. High yields of protoplasts (≈10⁸/ml) were obtained from mycelial cultures from five isolates of C. coffeicola. One isolate was transformed with a vector encoding hygromycin resistance and Green Fluorescent Protein. Out of 43 hygromycin-resistant transformants obtained, Green Fluorescent Protein was highly expressed in one (2.3 %).     

Estimating nocturnal ecosystem respiration from the vertical turbulent flux and change in storage of CO2

Micrometeorological measurements of nighttime ecosystem respiration can be systematically biased when stable atmospheric conditions lead to drainage flows associated with decoupling of air flow above and within plant canopies. The associated horizontal and vertical advective fluxes cannot be measured using instrumentation on the single towers typically used at micrometeorological sites. A common approach to minimize bias is to use a threshold in friction velocity, u_*, to exclude periods when advection is assumed to be important, but this is problematic in situations when in-canopy flows are decoupled from the flow above. Using data from 25 flux stations in a wide variety of forest ecosystems globally, we examine the generality of a novel approach to estimating nocturnal respiration developed by van Gorsel et al. (van Gorsel, E., Leuning, R., Cleugh, H.A., Keith, H., Suni, T., 2007. Nocturnal carbon efflux: reconciliation of eddy covariance and chamber measurements using an alternative to the u_*-threshold filtering technique. Tellus 59B, 397–403, Tellus, 59B, 307-403). The approach is based on the assumption that advection is small relative to the vertical turbulent flux (F_C) and change in storage (F_S) of CO₂ in the few hours after sundown. The sum of F_C and F_S reach a maximum during this period which is used to derive a temperature response function for ecosystem respiration. Measured hourly soil temperatures are then used with this function to estimate respiration R_Rmax. The new approach yielded excellent agreement with (1) independent measurements using respiration chambers, (2) with estimates using ecosystem light-response curves of F_c + F_s extrapolated to zero light, R_LRC, and (3) with a detailed process-based forest ecosystem model, R_cast. At most sites respiration rates estimated using the u_*-filter, R_ust, were smaller than R_Rmax and R_LRC. Agreement of our approach with independent measurements indicates that R_Rmax provides an excellent estimate of nighttime ecosystem respiration.