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

Daily and alternate day supplementation of urea or soybean meal to ruminants consuming low-quality cool-season forage: II. Effects on ruminal fermentation

Five ruminally cannulated steers (initial BW=464 ± 26 kg) consuming low-quality forage (5% CP; 78% NDF; DM basis) were used in an incomplete 5 x 4 Latin square with four 18-d periods to determine the influence of supplemental N source and supplementation frequency (SF) on ruminal fermentation dynamics. Treatments, arranged as a 2 x 2 factorial with a negative control, consisted of urea or soybean meal (SBM) supplements offered daily (D) or alternate days (2D) plus an unsupplemented treatment (CON). Urea supplements were provided to meet 100% of the degradable intake protein requirement while SBM supplements were provided on an isonitrogenous basis. All supplemented treatments received an equal quantity of supplemental N over a 2-d period. Ruminal indigestible acid detergent fiber (IADF) passage rate was increased with supplementation (P ≤ 0.03) on the days when D and 2D supplements were provided, as well as when only D supplements were provided. In contrast, ruminal liquid fill and dilution rate were not affected by supplementation, N source, or SF on the days when D and 2D supplements were provided (P ≥ 0.24). However, when only D supplements were offered, ruminal liquid dilution rate was greater (P=0.03) for SBM supplemented steers compared with cohorts receiving supplemental urea, whereas ruminal liquid fill was greater (P=0.03) for steers fed urea supplements. Nitrogen supplementation increased (P = 0.50). In summary, providing a urea-based supplement, as infrequently as every-other-day, was an effective alternative to a SBM-based supplement in maintaining acceptable ruminal fermentation of steers consuming low-quality, cool-season forage. (C) 2013 Elsevier B.V. All rights reserved.Keywords: Non-protein N, Forage, Urea, Soybean meal, Supplementation frequencyKeywords: Non-protein N, Forage, Urea, Soybean meal, Supplementation frequenc

Characterisation of a Desmosterol Reductase Involved in Phytosterol Dealkylation in the Silkworm, Bombyx mori

Most species of invertebrate animals cannot synthesise sterols de novo and many that feed on plants dealkylate phytosterols (mostly C29 and C28) yielding cholesterol (C27). The final step of this dealkylation pathway involves desmosterol reductase (DHCR24)-catalysed reduction of desmosterol to cholesterol. We now report the molecular characterisation in the silkworm, Bombyx mori, of such a desmosterol reductase involved in production of cholesterol from phytosterol, rather than in de novo synthesis of cholesterol. Phylogenomic analysis of putative desmosterol reductases revealed the occurrence of various clades that allowed for the identification of a strong reductase candidate gene in Bombyx mori (BGIBMGA 005735). Following PCR-based cloning of the cDNA (1.6 kb) and its heterologous expression in Saccharomyces cerevisae, the recombinant protein catalysed reduction of desmosterol to cholesterol in an NADH- and FAD- dependent reaction

Susceptibility of black bullhead Ameiurus melas to a panel of ranavirus isolates

Ranaviruses are considered a serious threat to lower vertebrates, including fish, amphibians and reptiles. However, epidemiological data on these agents are lacking, and further investigations are needed to understand the role of carriers and to update the list of susceptible hosts. We carried out various experimental infections under controlled conditions to contribute to the current knowledge on the susceptibility of black bullhead Ameiurus melas to European catfish virus (ECV) and other ranaviruses. A panel of 7 ranavirus isolates was used to challenge duplicate groups of A. melas juveniles maintained in aquaria supplied with running dechlorinated tap water. The experiments were performed at 15 and 25 degrees C. The results confirmed the high susceptibility of A. melas to ECV infection. Furthermore, a significant mortality associated with the typical signs of systemic viral infections was observed in groups challenged with Epizootic haematopoietic necrosis virus (EHNV) at 25 degrees C, and to a lesser extent, at 15 degrees C. No significant mortality was recorded in fish challenged with European sheatfish virus (ESV), Frog virus 3 (FV3), Rana esculenta virus-like (REV-like), Bohle iridovirus (BIV) or short-finned eel virus (SERV)