Development of a real‐time PCR assay for detection and quantification of Streptococcus iniae using the lactate permease gene

The aim of this study is the development and evaluation of a rapid and accurate quantitative PCR (qPCR)‐based protocol for detection of zoonotic pathogen Streptococcus iniae in bacterial cultures and tissues of diseased fish. For this purpose, the lactate permease‐encoding (lldY) gene was selected as a target for the design of S. iniae‐specific primers based on comparative genomic analysis using 45 sequences retrieved from NCBI genome database. Specificity and applicability of these primers were tested using 115 bacterial strains and fish tissues infected with S. iniae. Sensitivity, reproducibility and efficiency of qPCR assay were also determined. The developed qPCR assay showed 100% specificity with pure bacterial cultures or DNA extracted from S. iniae or tissues of fish infected with the bacterium. The method has high sensitivity with a detection limit of 1.12 × 10¹ amplicon copies per assay (equivalent to 2 × 10^–9 ng/µl) using bacterial DNA and of 1.44 × 10¹ gene copies in tissues of fish infected with S. iniae. In conclusion, this qPCR protocol provides an accurate and sensitive alternative for the identification of S. iniae and its detection on fish tissues that can be implemented as a routine tool in microbiological laboratories.     

White lupine as a beneficial crop in Southern Europe: I. Potential for N mineralization in lupine amended soil and yield and N2 fixation by white lupine

Two experiments were carried out at Pegões (central Portugal) to determine the potential N mineralization in a pulse amended disturbed and undisturbed soil incubated at several temperatures, and to evaluate for 2 years the yield and N₂ fixation capacity of sweet lupine (Lupinus albus L. cv. Estoril) inoculated with a mixture of rhizobia strains or nodulated by indigenous soil bacteria and submitted to conventional tillage or no-till practices. A completely randomized block design for soil mobilization with three replicates was used for the laboratory study, and completely randomized blocks for inoculation and tillage treatments with four replicates were used for the lupine yield and N₂ fixation experiment. Residue N immobilization occurred immediately after mature residue return to the soil independent of temperature, and was greater at 7 °C especially in the disturbed topsoil. Greater immobilization was also observed by doubling the amount of mature residue incorporated in the soil. This was expected since soil microorganisms would be in direct contact with the fresh organic matter and would be provided with more organic carbon under these circumstances. Nitrogen mineralization proceeded after 5 days of amendment. Potential N mineralization was higher at 25 °C than at 18 or 7 °C, for both conventional and no-till practices. At 25 °C, 42% of buried residue-¹⁵N was released over 210 days, at a smaller rate than 18 °C (49%) over 81 days. Lupine yield and N₂ fixation capacity were similar in plots that were not inoculated and those receiving the mixture of three rhizobia strains. White lupine had an efficient symbiosis with indigenous soil rhizobia at pod-filling (>99%, >100 kg N ha⁻¹ year⁻¹) which was not affected by tillage. At this stage, plant residue including visible roots and nodules accounted for a soil N input of +96 kg ha⁻¹ year⁻¹ (91% of crop N), showing the large soil N benefit by the crop at this stage. The lupine residue at pod-filling stage can be used as a green manure under the conditions of organic farming systems. The apparent N “harvest” index of the pulse at pod-filling was only 9% though at maturity phase it should result in a higher value and the legume will show a lower fertilizer N value.     

Effects of dietary exposure to brominated flame retardant BDE-47 on thyroid condition, gonadal development and growth of zebrafish

Little is known about the effects of brominated flame retardants in teleosts and some of the information currently available is inconsistent. This study examined effects of dietary exposure to 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) on thyroid condition, body mass and size, and gonadal development of zebrafish. Pubertal, 49-day-old (posthatch) fish were fed diets without BDE-47 (control) or with 1, 5 or 25 μg/g BDE-47/diet. Treatments were conducted in triplicate 30-L tanks each containing 50 zebrafish, and 15 fish per treatment (5 per tank) were sampled at days 40, 80 and 120 of exposure. Measurements were taken of body mass, standard length, head depth and head length. Sex (at 40–120 days of exposure), germ cell stage (at 40 days) and thyroid condition (at 120 days; follicular cell height, colloid depletion, angiogenesis) were histologically determined. Whole-body BDE-47 levels at study completion were within the high end of levels reported in environmentally exposed (wild) fishes. Analysis of variance was used to determine differences among treatments at each sampling time. No effects were observed on thyroid condition or germ cell stage in either sex. Reduced head length was observed in females exposed to BDE-47 at 80 days but not at 40 or 120 days. In males, no apparent effects of BDE-47 were observed at 40 and 80 days, but fish exposed to 25 μg/g had lower body mass at 120 days compared to control fish. These observations suggest that BDE-47 at environmentally relevant whole-body concentrations does not affect thyroid condition or pubertal development of zebrafish but does affect growth during the juvenile-to-adult transition, especially in males.     

Medium Light and Medium Roast Paper-Filtered Coffee Increased Antioxidant Capacity in Healthy Volunteers: Results of a Randomized Trial

We compared the effects of medium light roast (MLR) and medium roast (MR) paper-filtered coffee on antioxidant capacity and lipid peroxidation in healthy volunteers. In a randomized crossover study, 20 volunteers consumed 482?±?61?ml/day of MLR or MR for four weeks. Plasma total antioxidant status (TAS), oxygen radical absorbance capacity (ORAC), oxidized LDL and 8-epi-prostaglandin F2α, erythrocyte superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activity were measured at baseline and after the interventions. MLR had higher chlorogenic acids-(CGA; 334?mg/150?mL) and less caffeine (231?mg/150?ml) than MR had (210 and 244?mg/150?ml, respectively). MLR also had fewer Maillard reaction products (MRP) than MR had. Compared with baseline, subjects had an increase of 21 and 26?% in TAS, 13 and 13?% in CAT, 52 and 75?% in SOD, and 62 and 49?% in GPx after MLR and MR consumption (P?相似文献   

Relationship between the incidence of latent infections caused by <Emphasis Type="Italic">Monilinia</Emphasis> spp<Emphasis Type="Italic">.</Emphasis> and the incidence of brown rot of peach fruit: factors affecting latent infection

Five field experiments were performed in commercial orchards located in Lleida (Spain) over three growing seasons, 2000–2002, in order to estimate the relationship between the incidence of latent infection caused by Monilinia spp. in peaches and the incidence of post-harvest brown rot. No latent infection was recorded at popcorn and the maximum incidence occurred pre-harvest; in some orchards a second peak was detected during the pit hardening period. Monilinia laxa is the most prevalent species isolated from peaches with brown rot. There was a positive correlation between the incidence of latent infection and that of post-harvest brown rot. The average incidence of latent infection during the crop season explained 55% of the total variation in the incidence of post-harvest brown rot. The effect of temperature (T) and duration of wetness (W) on the incidence of latent infection in peach and nectarine orchards was analysed using multiple regression. The regression analysis indicated that T and W jointly explained 83% of the total variation in the incidence of latent infection. The model predicts no latent infections when T < 8°C, and >22 h wetness are required when T = 8°C but only 5 h at 25°C are necessary for latent infection to occur. The incidence of brown rot and latent infection of peaches caused by M. laxa under controlled experimental conditions were also affected by T and W, as well as by fruit maturity and inoculum concentration. Latent infections were produced in fruit when T was not suitable for the development of brown rot symptoms. In these experiments more than 4–5 h of daily wetness were required after embryo growth in fruit sprayed to run-off with an inoculum concentration higher than 10⁴ conidia ml⁻¹ of M. laxa for brown rot and latent infections to develop. The fitted model obtained from the field data was able to predict the observed data obtained under controlled environmental conditions.     

Changes in habitat use patterns by bottlenose dolphins over a 10‐year period render static management boundaries ineffective

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Partial least squares regression (PLSR) associated with spectral response to predict soil attributes in transitional lithologies

New techniques and improvements are required to quantify soil’s chemical and physical properties on production environment, reducing environmental impacts and minimizing soil analysis time. The aim of this study is to evaluate the possibility to estimate the content of silt, sand, clay, total iron and organic matter in soils formed by different lithologies in Parana State, Brazil, using VIS-NIR spectrum associated with Partial Least Square Regression (PLSR). 200 soil samples were collected in an area formed by Lixisols, Cambisols, Ferralsols, Arenosols and Nitisols in a depths of 0–0.2 and 0.2–0.8 m. Spectral readings were obtained in laboratory by FieldSpec 3 JR sensor. The spectral curves of the samples were correlated to the attributes through PLSR. The results obtained for sand in prediction were better when compared to the other attributes, presenting R² = 0.90, r = 0.95 and RPD = 2.3. Clay and total iron presented satisfactory results, mainly for RPD values, which were above 2.4. Based on the results, it can be concluded that the PLSR technique associated with the spectral response of the soils, was able to estimate sand, clay and total iron with accuracy in a region formed by reworked materials, derived from several lithologies.     

Improvement of degraded physical attributes of a saline-sodic soil as influenced by phytoremediation and soil conditioners

This study aimed to evaluate the use of phytoremediation and soil conditioners in the recovery of physical attributes of a saline-sodic Fluvic Neossol in Brazil Northeast. The applied treatments were: Atriplex nummularia L., as phytoremediation plant, due to its ability to extract salts from the soil; organic conditioners, such as bovine and sheep manure; gypsum and polymer, as chemical conditioners. Samples with preserved structure were collected at the time of the experiment installation and 18 months after in the layers 0–10 cm and 10–30 cm. The analyzed attributes were: water dispersed clay, dispersion index, bulk density, penetration resistance, soil porosity, and saturated hydraulic conductivity. The use of sheep manure, gypsum and polymer promoted an increase in saturated hydraulic conductivity in the 0–10 cm layer from 4.51 to 16.37 cm day^?1, 11.26 to 23.95 cm day^?1 and 7.24 to 22.77 cm day^?1, respectively. Gypsum increased the macroporosity in the superficial layer by 42.6%. Atriplex and polymer were more efficient at reducing soil penetration resistance. The polymer was more efficient at improving the physical properties. However, it is necessary to consider phytoremediation with Atriplex as a more sustainable alternative that can still be used as complementary fodder in animal feed.     

Early survival of Quercus ilex subspecies from different populations after infections and co‐infections by multiple Phytophthora species

Forests in Europe are threatened by increased diversity of Phytophthora species, but effects on trees of simultaneous infections by Phytophthora and ecological consequences of their coexistence are unknown. This study explored variation in early survival of Quercus ilex to Phytophthora infections and assessed interactions between Phytophthora species when trees were co‐infected. Three Phytophthora species (P. cinnamomi, P. gonapodyides and P. quercina), seeds from 16 populations of Q. ilex (ballota and ilex subspecies) and two infection times were used as sources of variation in two experiments. The influence of Phytophthora species, Q. ilex subspecies and populations on plant germination and survival were analysed using generalized linear mixed models and survival analysis techniques. Germination rates were not influenced by Phytophthora spp. (P = 0.194) but by the subspecies and populations of Q. ilex (P < 0.001). In Phytophthora‐infested soils, Q. ilex subsp. ilex germinated at higher rates than Q. ilex subsp. ballota. Plant survival was strongly influenced by Phytophthora species (P < 0.001), not by the subspecies and populations of Q. ilex. Seedling mortality was reduced and delayed if a less virulent Phytophthora species infected plants prior to infection by a more virulent Phytophthora species. The results help to explain oak decline syndrome and the lack of natural and artificial regeneration of Q. ilex forests. Lack of interspecific variability of early survival to Phytophthora spp. discourages direct sowing for artificial reforestation programmes. Large, thick seeds, giving plants rapid growth, are advantageous traits when soils are infested with Phytophthora spp.     

Antimicrobial Resistance Genes in Pigeons from Public Parks in Costa Rica

Antimicrobial resistance is known to be an emerging problem, but the extent of the issue remains incomplete. The aim of this study was to determine the presence or absence of nine resistance genes (bla_TEM, catI, mecA, qnrS, sulI, sulII, tet(A), tet(Q), vanA) in the faeces of 141 pigeons from four urban parks in Alajuela, Guadalupe, Tres Ríos and San José in Costa Rica. The genes were identified by real‐time PCR directly from enema samples. About 30% of the samples were positive for genes catI and sulI; between 13% and 17% were positive for qnrS, sulII, tet(A) and tet(Q); and 4% were positive for bla_TEM. The mecA and vanA genes were not detected. The average of antimicrobial resistance genes detected per pigeon was 2. Eight different patterns of resistance were identified, without differences in the sampling areas, being the most common pattern 2 (sulII positive samples). During rainy season, the genes more frequently found were sulI and tet(A). In conclusion, the urban inhabiting pigeons tested are currently carrying antimicrobial resistance genes, potentially acting as reservoirs of resistant bacteria and vectors to humans. To the authors’ knowledge, this is the first study carried out on direct detection of resistance genes in the digestive metagenomes of pigeons.