Reduced diversity of immunoglobulin and T-cell receptor gene rearrangements in chronic inflammatory gastrointestinal diseases in dogs

Inflammatory bowel disease has a multifactorial etiology in dogs as it does in humans. Evidence has been accumulated showing an abnormal response of the immune system, mostly represented by lymphocyte infiltration in the lamina propria of the gastrointestinal tract and in the epithelium, likely driven by chronic antigenic stimulation against luminal microorganisms. A relevant role is also ascribed to the genetic predisposition typical of some canine breeds. The role of chronic antigenic stimulation is still under debate. It may be responsible for selective pressure on the lymphoid population, favouring the emergence of some lymphocyte clones. This cross-sectional study is aimed at investigating the immunoglobulin and T-cell receptor gene rearrangements in a group of dogs affected by inflammatory bowel disease. The database of a referral Veterinary Laboratory was investigated. Based upon the histological evaluation of the bioptic samples collected during endoscopy, 54 canine cases met the WSAVA criteria for diagnosing IBD and were included in the study. The histological slides were retrieved and the gDNA was purified using protocols for formalin-fixed tissue. The gDNA was PCR amplified using fluorescent-labelled primers specific for canine immunoglobulin and T-cell receptor gene rearrangements; the PCR products were analysed with fragment analysis by means of capillary electrophoresis on an automatic sequencer (GeneScanning). In 47/54 (87.3%) cases, it was possible to amplify the gDNA. Twenty-one patients out of 47 (44.7%) showed polyclonal patterns in both the immunoglobulin and the T-cell receptors, 18/47 (38.3%) showed at least one oligoclonal pattern without monoclonal ones while 8/47 (17.0%) cases showed an Ig (7/47; 14.9%) or TCR (1/47: 2.1%) monoclonal pattern. These findings indicate that reduced diversity of the immunoglobulin and T-cell receptor repertoire occurs in canine inflammatory bowel disease. The reduced diversity correlated significantly with the severity of the histological lesions and carried a significantly increased risk of death. Beside its possible role as a reliable ancillary assay, immunoglobulin and T-cell receptor GeneScanning analysis points to the possible role of aberrant chronic antigenic stimulation, leading to clonal expansion of certain lymphocyte subsets in the pathogenesis of canine IBD.     

Viability and nematophagous activity of the freeze-dried fungus Arthrobotrys robusta against Ancylostoma spp. infective larvae in dogs

Viability and in vitro and in vivo activities of freeze-dried conidia of the predatory fungus Arthrobotrys robusta (I-31) were evaluated against infective larvae (L(3)) of Ancylostoma spp. in dogs. A. robusta conidia were lyophilized and stored at 4°C for a month. Freeze-dried conidia were diluted to 1×10(3)conidia/ml and tested in vivo. The treated group consisted of a solution containing conidia (1ml) and 1000 Ancylostoma spp. (L(3)) placed on Petri dishes plated with 2% water-agar (2% WA), at 25°C, in the dark for 10 days. The control group consisted of 1000 Ancylostoma spp. L(3), plated on 2% WA. After 10 days, Ancylostoma spp. L(3) from both the treated and the control groups were recovered and counted. The in vivo test was performed on two dogs by administering a single oral dose of freeze-dried conidia (1.5×10(5)) in aqueous solution to one animal and only water to the other. Fecal samples were collected at 12, 24 and 48h after the treatments, plated 2% WA plates and incubated at 25°C for 15 days. A thousand Ancylostoma spp. L(3) larvae were spread on these plates. At day 15, infective L(3) recovered from the treated and control groups were counted. In the in vitro test, A. robusta was able to survive the freeze-drying process, grow in the plates, form traps and capture Ancylostoma spp. L(3). There was a 75.38% decrease in the number of infective larvae recovered from the treated group. The in vivo test showed that freeze-dried A. robusta conidia survived the passage through the gastrointestinal tract of the treated dog, was able to grow in the plates and capture Ancylostoma spp. L(3), reducing the number of recovered L(3) (p<0.01). Freeze-drying can be an alternative method for conservation of conidia of nematophagous fungi.     

Integration between molecular and morphological markers for the exploitation of olive germoplasm (Olea europaea)

Three olive cultivars (Oliva Nera di Colletorto, Noccioluta, and a probably a new local genotype) from two strictly related areas of Molise region (south-centre of Italy) were characterized by combining molecular data (eight SSRs analyzed on leaves) and morphological features (thirty-one parameters from leaves, drupes and pits). Both molecular and morphological analyses have shown a very good separation of the three endemic cultivars. A high correlation between morphological and molecular data was found using Mantel's test. The morphological traits of pits were less influenced by environmental pressure than the leaves and drupes; therefore, the pits are more affected by genetic control and might be considered a helpful tool for cultivar characterization and identification. Potential and limitations of three statistical models computed to perform cultivar identification by morphological measures is also discussed. We demonstrated that molecular and morphological analyses are useful for distinguishing new accessions and studying local varieties to preserve genetic diversity, even at small geographical scale in such an unequivocal way; hence the methodology could be proposed as a tool to discriminate widespread cultivars, with long genetic distances.     

Influence of grape density and harvest date on changes in phenolic composition, phenol extractability indices, and instrumental texture properties during ripening

Changes in the phenolic composition, phenol extractability indices, and mechanical properties occur in grape berries during the ripening process, but the heterogeneity of the grapes harvested at different ripening stages affects the reliability of the results obtained. In this work, these changes were studied in Nebbiolo grapes harvested during five consecutive weeks and then separated according to three density classes. The changes observed in chemical and mechanical parameters through the ripening process are more related to berry density than harvest date. Therefore, the winemaker has to select the flotation density according to the objective quality properties of the wine to be elaborated. On the other hand, the stiffer grapes were associated with a higher accumulation of proanthocyanidins. The harder grapes provided the higher concentration and extractability of flavanols reactive to vanillin, whereas the thicker ones facilitated the extraction of proanthocyanidins.     

Impact of glutathione on the formation of methylmethine- and carboxymethine-bridged (+)-catechin dimers in a model wine system

This study was performed to assess the impact of glutathione on the reaction between (+)-catechin and carbonyl compounds in wine-related conditions. (+)-Catechin (0.50 mM) and either glyoxylic acid (0.25 mM) or acetaldehyde (0.25 mM) were added to a model wine system with 0.0, 0.25, and 2.5 mM of glutathione added. UPLC-DAD and LC-MS analysis showed that the formation of carbonyl-bridged (+)-catechin dimers was inhibited in the samples with a glutathione to carbonyl ratio of 10:1 compared to the samples without glutathione. At a ratio of 1:1, glutathione inhibited the acetaldehyde-bridged dimers but only had a minor impact on the glyoxylic acid-bridged dimers. Further investigations showed that this trend of inhibition by glutathione on the glyoxylic acid-derived dimer was independent of temperatures, 20 °C vs 45 °C, or the presence of metal ions, 0.2 mg/L copper(II) and 5 mg/L iron(II). (1)H NMR analysis and LC-MS analysis provided evidence that glutathione inhibited dimer formation via different mechanisms depending on the carbonyl compound. For acetaldehyde-derived dimers, the main mode of inhibition was the ability of glutathione to form a (methyl-glutathionyl-methine)-(+)-catechin complex. Alternatively, the formation of a glutathione-glyoxylic acid addition product impeded the reaction between glyoxylic acid with (+)-catechin. These results demonstrate that glutathione, at sufficient concentration, can have a substantial impact on carbonyl-derived polymerization reactions in wine-like conditions.     

Role of the cultivar in choosing Clementine fruits with a high level of health-promoting compounds

Thirteen cultivars and two hybrids of Clementine fruits (Citrus clementina Hort. Ex. Tan) cultivated in Italy were characterized according to pH, titratable acidity, total soluble solids, total polyphenols, carotenoids, vitamin C, hesperidin, rutin, narirutin and naringin and radical scavenging activity. The presence of rutin in Clementine fruit juice is reported for the first time here. The results indicated that all chemical parameters statistically differentiated each cultivar (P < 0.001). In particular, principal component analysis showed a clear discrimination of five cultivars from all the other varieties based on vitamin C and total polyphenols for the Caffin cultivar, which showed also the highest antioxidant activity; narirutin for the Etna hybrid cultivar; hesperidin, rutin and total soluble solids for the SRA 89 cultivar; and naringin, hesperidin and rutin for the Esbal cultivar. Moreover, the Mandalate hybrid cultivar showed the lowest antioxidant activity as well as vitamin C and total polyphenols content, while titratable acidity and naringin level were the highest. The antioxidant activity assessed in all the fruits was closely correlated with vitamin C and total polyphenols content, rather than with the flavonoid compounds.     

Inflow and outflow water quality control in coastal aquaculture systems: a case study

Coastal water bodies are a particularly heterogeneous resource, typified by high spatial and temporal variability that could influence the aquaculture in coastal zones. However, the development of coastal aquaculture may produce negative impacts on the coastal area by the potential release of nutrients and organic matter that can be a source of pollution in receiving waters. The aim of this paper was to evaluate the performance of constructed wetland in controlling the dynamics of deoxygenating matter (organic matter and ammonia) and eutrophicating matter [organic matter and soluble reactive phosphorus (SRP)] in the waters entering (inflow) and flowing out (outflow) from a coastal aquaculture fish farm. We observed that constructed wetland systems are effective in removing fractions of total suspended solids, COD, total ammonia nitrogen and SRP contained in the inflow water with higher efficiency in the spring period (60.37%, 14.89%, 65.38% and 17.6% respectively) than in the summer period (45.10%, 8.06%, 32.43% and 8.00% respectively). Similar pattern was recorded for the treatment of the outflow waters, showing that the wetland system reduced most of the deoxygenating and eutrophicating matter produced as a consequence of feeding and fish metabolic activity. During the summer season, high algae mortality can reduce the performance of the wetland system in the outflow water control; this lower efficiency could be improved by controlling the biomass of algae by vegetation harvesting.     

Feeding,growth, and blood chemistry of the tambaqui (Colossoma macropomum) held under self-feeding and time-restricted automatic feeding conditions

This work evaluated the effects of food frequencies and self-feeding on the behavior, performance, and blood biochemistry of Colossoma macropomum. Juveniles weighing 3.63?±?0.05 g were distributed in 27 aquariums (40 L), with six fish per aquarium, to evaluate nine treatments in three replications in a 12L:12D photoperiod. The treatments were 24-h self-feeding (SF); daytime self-feeding at will (SFD); nighttime self-feeding at will (SFN); and feeding at fixed frequencies of two (2D), four (4D), and six times (6D) daytime and twice (2 N), four (4 N), and six times (6 N) nightly. The treatments were offered in automatic feeders, with fixed frequencies programmed. Extruded commercial feed, with 46% protein, was used. The experimental period was 40 days. In the SF treatment, the animals exhibited 95.7% nocturnal and 4.3% diurnal feeding activity (p?<?0.05). In the treatment SFD and SFN, the animals showed feeding activity throughout the day and night. Final weight, weight gain, and total feed intake were higher for animals at fixed daytime and nighttime frequencies (p?<?0.05). Feed conversion was better for animals in SF, SFD, 4D, SFN, and 4 N (0.89, 0.76, 0.91, 0.85, and 0.88, respectively) (p?<?0.05). Cholesterol and high-density lipoproteins were lower for fish in SF, SFN, 2 N, 4 N, and 6 N (p?<?0.05). SF treatment animals had lower triglyceride content, 154.20 mg dL^?1 (p?<?0.05). Colossoma macropomum has nocturnal food preferences, however, with remarkable plasticity. Juveniles of this species fed four times during the day or night show good performance. Dietary patterns influenced the blood metabolic indicators of this species and the periods studied.