Development and validation of a real-time TaqMan PCR assay for the detection of betanodavirus in clinical specimens

Development and validation of a real-time TaqMan PCR assay for the detection of betanodavirus in clinical specimens Panzarin V, Patarnello P, Mori A, Rampazzo E, Cappellozza E, Bovo G, Cattoli G. Betanodaviruses are the causal agents of viral encephalo-retinopathy, an infectious disease affecting more than 40 marine fish species, characterized by high morbidity and mortality. Because of its severe impact, robust diagnostic tools are required. The aim of this work was to develop and validate a real-time TaqMan PCR assay to detect betanodaviruses in clinical specimens by amplifying a conserved region of the RNA2 strand. The method proved to be specific and sensitive, being capable of detecting as low as 10 TCID(50)/ml. For clinical validation, samples from 100 marine fish were collected during a natural outbreak of disease and tested by three distinct laboratory methods, namely real-time TaqMan PCR, RT-seminested PCR and virus isolation. The results indicated optimal agreement between tests. The assay that was developed is capable of detecting members of all of the betanodavirus genetic groups currently described and can be considered a valid alternative to the time-consuming and contamination-prone nested PCR

A first attempt to produce proteins from insects by means of a circular economy

The worldwide growing consumption of proteins to feed humans and animals has drawn a considerable amount of attention to insect rearing. Insects reared on organic wastes and used as feed for monogastric animals can reduce the environmental impact and increase the sustainability of meat/fish production. In this study, we designed an environmentally closed loop for food supply in which fruit and vegetable waste from markets became rearing substrate for Hermetia illucens (BSF\u2014 black soldier fly). A vegetable and fruit-based substrate was compared to a standard diet for Diptera in terms of larval growth, waste reduction index, and overall substrate degradation. Morphological analysis of insect organs was carried out to obtain indications about insect health. Processing steps such as drying and oil extraction from BSF were investigated. Nutritional and microbiological analyses confirmed the good quality of insects and meal. The meal was then used to produce fish feed and its suitability to this purpose was assessed using trout. Earthworms were grown on leftovers of BSF rearing in comparison to a standard substrate. Chemical analyses of vermicompost were performed. The present research demonstrates that insects can be used to reduce organic waste, increasing at the same time the sustainability of aquaculture and creating interesting by-products through the linked bio-system establishment

Evaluation of direct-fed microbials on in vitro ruminal fermentation, gas production kinetic, and greenhouse gas emissions in different ruminants’ diet

IntroductionThree in vitro experiments were conducted to evaluate the effects of increasing levels of Enterococcus faecium and Saccharomyces cerevisiae (DFM1) and increasing levels of Bacillus licheniformis and Bacillus subtilis (DFM2) on in vitro ruminal fermentation parameters in three different dietary scenarios.MethodsFor Exp. 1, the basal diet consisted of 25:75 roughage:concentrate ratio (R:C) and was composed by 5 treatments: control (no additive), 2 levels of DFM1 (1X = 1.9 mg and 5X = 9.0 mg), and 2 levels of DFM2 (1X = 3.8 mg and 5X = 19 mg). The Exp. 2 consisted of a 41:59 R:C diet and was composed by 5 treatments: control (no additive) and 2 levels of DFM1 (1X = 3.8 mg and 5 X = 19 mg) and 2 levels of DFM2 (1X = 5.6 mg and 5X = 28 mg). The Exp. 3 consisted of a 100:0 R:C diet [Brachiaria (syn. Urochloa brizantha)] and was composed by the same treatments described in Exp. 1. The DFM1 contained 3.5 × 109 CFU per g of Enterococccus faecium and Saccharomyces cerevisiae, whereas the DFM2 contained Bacillus licheniformis and Bacillus subtilis at 3.2 × 109 CFU per g. In each Exp., an in vitro gas production (GP) system with 43-bottles (AnkomRF) was used in four consecutive 48 or 72-h fermentation batches to evaluate total GP (TGP), kinetics and fermentation profiles, methane (CH4), and carbon dioxide (CO2).ResultsFor Exp 1, DFM1 increased quadratically TGP at 24 and 48-h, which reflected in a greater in vitro organic matter digestibility (IVOMD). The concentrations of ammonia-N, CH4, and CO2 (mmol/g of IVOMD) reduced quadratically as DFM1 increased. For Exp. 2, DFM1 inclusion reduced butyrate concentration and acetate to propionate ratio. Regarding GHG emissions, DFM1 and DFM2 quadratically reduced CH4 and CO2 emission per IVOMD (mmol/g of IVOMD). For Exp. 3, DFM1 increased quadratically TGP at 48h with no impact on IVOMD. Otherwise, DFM2 increased linearly TGP at 24 and 48h which reflected in a greater IVOMD. The inclusion of DFM1 increased linearly iso-valerate and branched-chain volatile fatty acids (BCVFA) concentration and DFM2 addition increased BCVFA quadratically.DiscussionOverall, addition of DFM1 [Enterococccus faecium (5 × 109 CFU per g) + Saccharomyces cerevisiae (5 × 109 CFU per g)] or DFM2 [Bacillus licheniformis + Bacillus subtilis (3.2 × 109 CFU per g)] might enhance the fermentation process in the rumen and decrease greenhouse gas emissions in a dose-dependent manner, though the results are contingent on the specific type of diet

Proton Spin-Lattice Relaxation in Silkworm Cocoons: Physisorbed Water and Serine Side-Chain Motions

The molecular dynamic behavior of silkworm cocoons produced by a single Bombyx mori strain was investigated by means of high- and low-resolution solid-state NMR experiments. Cocoons with different moisture content were prepared to study the effects of physisorbed water on their molecular dynamics in the MHz regime, which was probed through the measurement of 1H T1 relaxation times at 25 MHz in the 25-95 °C temperature range. The water content of the different samples was determined from the analysis of 1H free-induction decays. In addition to the rotation of methyl groups, mostly from alanine, and to the reorientation of physisorbed water molecules, already identified in previous works as relaxation sinks, the reorientation of serine side-chains was here found to contribute to 1H T1 above room temperature. The analysis of the trends of 1H T1 versus temperature was carried out in terms of semiempirical models describing the three main motional processes, and indicated that methyl rotation, water reorientation and serine side-chain motions are the most efficient relaxation mechanisms below 0 °C, between 0 and 60 °C, and above 60 °C, respectively. The activation energies were found to decrease passing from serine to water to methyl motions

Post-translational control of human hemoglobin synthesis The role of the differential affinity between globin chains in the control of mutated globin gene expression

The interactions between β-thalassemia and the humen hemoglobin (Hb) α-chain variants, Hb Hasharon, Hb O Indonesia and Hb J Paris, and between α-thalassemia and the β-chain variants, Hb S, Hb C and Hb G San José, which are characterized by preferential decrease of the abnormal Hb level in peripheral blood, have been studied. Both biosynthesis studies in reticulocytes and determination of the relative affinity of abnormal chains for normal complementary chains by in vivo recombination experiments, involving globin chains previously isolated in their native form, have been carried out in order to provide insights on the molecular events following the synthesis of the mutant chains under conditions of complementary chain deficiency. Furthermore, we have measured the relative affinity for complementary chain of βD Los Angeles- and αJ Rovigo-chains, the level of which does not decay in thalassemic carriers, and of αLegnano- and βOsu Christiansborg-chains, which have not yet been observed in association with thalassemias. Our experiments indicated that the differential affinity for β-chains is not always the major post-translational control mechanism which regulates the level of certain α-chain variants in β-thalassemic heterozygotes, and that preferential removal of abnormal chains by proteolytic enzymes is likely to play an important role in most cases. On the other hand, the low affinity of certain variant β-chains for α-chains may offer an explanation for the low level of certain β-chain variants in peripheral blood of non-thalassemic carriers, as well as to their decrease under conditions of relative α-chain deficiency (α-thalassemias). © 1980