Enzyme-linked immunosorbent assay (ELISA) for the detection of spring viraemia of carp virus (SVCV) in tissue homogenates of the carp, Cyprinus carpio L.

Abstract. An enzyme-linked immunosorbent assay (ELISA) for the demonstration of the virus of spring viraemia of carp (SVCV) in liver, kidney and spleen homogenates, and in infected cell cultures is described. The sensitivity of the method is 10^2·8–10^3·5 TCID₅₀ 0·lml⁻¹ of the examined fluid. The specificity has been confirmed by the ELISA inhibition test and by results of virological examinations. Contamination with bacteria or fungi of samples taken from dead fish had no effect on the results of ELISA. Specific anti-SVCV sera were used successfully for the production of conjugates for the direct immunoperoxidase and immunofluorescence detection of SVCV in infected cell cultures.     

The future of fish passage science,engineering, and practice

Much effort has been devoted to developing, constructing and refining fish passage facilities to enable target species to pass barriers on fluvial systems, and yet, fishway science, engineering and practice remain imperfect. In this review, 17 experts from different fish passage research fields (i.e., biology, ecology, physiology, ecohydraulics, engineering) and from different continents (i.e., North and South America, Europe, Africa, Australia) identified knowledge gaps and provided a roadmap for research priorities and technical developments. Once dominated by an engineering‐focused approach, fishway science today involves a wide range of disciplines from fish behaviour to socioeconomics to complex modelling of passage prioritization options in river networks. River barrier impacts on fish migration and dispersal are currently better understood than historically, but basic ecological knowledge underpinning the need for effective fish passage in many regions of the world, including in biodiversity hotspots (e.g., equatorial Africa, South‐East Asia), remains largely unknown. Designing efficient fishways, with minimal passage delay and post‐passage impacts, requires adaptive management and continued innovation. While the use of fishways in river restoration demands a transition towards fish passage at the community scale, advances in selective fishways are also needed to manage invasive fish colonization. Because of the erroneous view in some literature and communities of practice that fish passage is largely a proven technology, improved international collaboration, information sharing, method standardization and multidisciplinary training are needed. Further development of regional expertise is needed in South America, Asia and Africa where hydropower dams are currently being planned and constructed.     

Effect of repeated exposure to AQUI‐S® on the viability and growth of Neoparamoeba perurans

There have been recent efforts amongst immunologists to develop approaches for following individual fish during challenges with viral and bacterial pathogens. This study contributes to assessing the feasibility of using such approaches to study amoebic gill disease (AGD). Neoparamoeba perurans, agent of AGD, has been responsible for widespread economic and fish loss in salmonid aquaculture. With the emergence of AGD in Europe, research into infection dynamics and host response has increased. This study investigated the effect of repeat exposure to anaesthesia, a necessary requirement when following disease progression in individual fish, on N. perurans. In vitro cultures of N. perurans were exposed every 4 days over a 28‐day period to AQUI‐S^® (isoeugenol), a popular anaesthetic choice for AGD challenges, at a concentration and duration required to sedate post‐smolt salmonids. Population growth was measured by sequential counts of amoeba over the period, while viability of non‐attached amoeba in the culture was assessed with a vital stain. AQUI‐S^® was found to be a suitable choice for in vivo ectoparasitic challenges with N. perurans during which repetitive anaesthesia is required for analysis of disease progression.     

Characterization of bacteriophage HN48 and its protective effects in Nile tilapia Oreochromis niloticus against Streptococcus agalactiae infections

Streptococcus agalactiae is a causative agent responsible for massive mortalities of tilapia that has led to catastrophic losses to tilapia culture globally. Bacteriophages represent a new class of antimicrobials against bacteria. In this study, we characterized the bacteriophage HN48, which formed small and round‐transparent plaques on a double‐layer plate. With a hexagonal head and a long tail, this phage may belong to the Caudovirales according to the International Committee on Taxonomy of Viruses. HN48 was found to have a relatively wide and highly specific host range, to be sensitive to high temperature (60–80°C) and low pH (3–5), and to be relatively stable at alkaline pH (8–10). Intraperitoneal injection with HN48 had no adverse effects on tilapia and effectively inactivated the bacteria in the kidney. Fish that received phage therapy had 60% ± 3.3% survival rates and a delayed mean death time of about 3 days when compared to the control group. To the best of knowledge, this is the first study of tilapia streptococcal phage. Overall, the results indicated that phage HN48 could prevent tilapia from experimental S. agalactiae infection, suggesting it has the potential to control this disease.     

Nutritional Contribution of Biofloc within the Diet of Growout and Broodstock of Litopenaeus vannamei,Determined by Stable Isotopes and Fatty Acids

The relative contributions of feed sources were determined through the isotopic signal (δ¹³C and δ¹⁵N) and fatty acid profile of feed items, shrimp muscle, and eggs of Litopenaeus vannamei reared in a biofloc system. In the growout phase, the isotope analysis showed the biofloc particle size class ≥250 μm contributed 55–100%; size ≥50 < 250 μm contributed 0–22%; and artificial feed contributed 0–45%. Principal component analysis applied to fatty acid profiles showed that biofloc ≥250 μm and artificial feed were the most important items in shrimp growout. For the egg production, isotope analysis suggested that the most important feed sources according to their relative contributions were polychaetes (0–100%), followed by artemia biomass (0–86%) and semi‐moist feed (0–66%), with lower contributions from squid, mussel, and the muscle of L. vannamei broodstock that had been cultured in biofloc. In terms of fatty acids, the most important items were artemia, polychaetes, and semi‐moist feeds. This work clarified the importance of feed sources for shrimp during culture in biofloc systems and during reproduction. Analysis of stable isotopes and fatty acids can be successfully used to trace the assimilation of nutrients during the nutrition of shrimp.     

Dietary calcium requirement and effects on growth and tissue calcium content of juvenile grass carp (Ctenopharyngodon idella)

A growth trial was conducted to estimate the optimum concentration of dietary calcium (Ca) for grass carp (Ctenopharyngodon idella). Triplicate groups of grass carp (4.52 ± 0.02 g) were fed diets containing graded levels (2.75, 4.51, 6.24, 7.99, 9.66 and 11.5 g kg^?1) of Ca for 8 weeks. Weight gain, feed efficiency and protein efficiency ratio were linearly increased up to the 7.99 g kg^?1 dietary Ca and then maintained stable beyond this level (P < 0.05). Dietary Ca levels higher than 7.99 g kg^?1 significantly increased the ash contents of whole body, vertebrae and scales. Ca contents in whole body, vertebrae and scales were linearly increased up to the 7.99 g kg^?1 dietary Ca and then maintained stable beyond this level (P < 0.05). In contrast, dietary Ca levels higher than 9.66 g kg^?1 significantly decreased Mg contents in whole body, vertebrae and scales. Dietary Ca levels higher than 7.99 g kg^?1 significantly increased plasma alkaline phosphatase activity. However, plasma Ca, P and Mg contents were not significantly affected by dietary Ca supplements (P > 0.05). Polynomial regression analysis indicated that 10.4 g kg^?1 dietary Ca was required for maximal tissue storage and mineralization as well as optimal growth.     

The evaluation of practical diets on a basis of digestible crude protein,lysine and methionine for Litopenaeus vannamei

A feeding trial was conducted to evaluate the efficacy of replacing fish meal (FM) with blood meal (BM), poultry by‐product meal (PBM), meat and bone meal (MBM) and shrimp head meal (SHM), rapeseed meal (RM) and peanut meal (PM) on a digestible basis of crude protein and lysine and methionine in five practical diets for the Pacific white shrimp at the FM levels of 300, 250, 200, 150 and 100 g kg^?1 under laboratory conditions. Each of the five experimental diets was hand‐fed to four replicate tanks of shrimp with an average weight of 0.33 ± 0.03 g to satiation at each meal. The shrimp were fed three times a day over a six‐week period. The per cent weight gain of initial body weight (WG%) was significantly lower in shrimp fed 100 g kg^?1 FM diet, but the value for hepatosomatic index (HSI) and the level of blood urea nitrogen (BUN) tended to be higher in shrimp fed 100 g kg^?1 FM diet than those in shrimp fed other diets. The lowest value for feeding rate (FR) occurred for shrimp fed the basal diet and was significantly lower than that in shrimp fed the FM diets at 100–150 g kg^?1. Shrimp fed diets containing 200 g kg^?1 or lower FM had significantly lower feed utilization than those fed the 250 g kg^?1 FM diet and the basal diet. The protein efficiency ratio (PER) in the shrimp fed the basal diet was significantly higher than in the other FM diets. Decreasing the FM replacement level significantly reduced nutrient digestibility except in the cases of ash and gross energy, but it did not affect the survival, condition factor (CF), body composition, digestive enzyme activity or plasma transaminase activity. The results of the study indicate that feeding a diet formulated on a digestible basis and involving FM replacement with other protein sources at a greater replacement proportion will not produce a level of shrimp growth equal to that achieved by feeding the basal diet.     

Identification of immunogenic proteins of Flavobacterium columnare by two‐dimensional electrophoresis immunoblotting with antibacterial sera from grass carp,Ctenopharyngodon idella (Valenciennes)

Flavobacterium columnare is a Gram‐negative bacterium causing columnaris disease of freshwater fish worldwide, and development of efficacious vaccines has been a continuous challenge in aquaculture. In this study, 14 proteins were identified from cellular components of F. columnare using an immunoblotting approach in two‐dimensional electrophoresis map gels with antibacterial sera from grass carp, Ctenopharyngodon idella (Valenciennes), and then anti‐grass carp‐recombinant Ig (rIg) polyclonal antibodies. These proteins were characterized conclusively by matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF/TOF MS). The 14 proteins are immunogenic molecules of F. columnare, including chaperonins DnaK, GroEL and trigger factor, and translation elongation factor G, translation elongation factor Tu, 30S ribosomal subunit protein S1, dihydrolipoamide succinyltransferase, succinyl‐CoA synthetase, SpoOJ regulator protein, alcohol dehydrogenase, fructose‐bisphosphate aldolase, 3‐hydroxybutyryl‐CoA dehydrogenase and two conserved hypothetical proteins. These identified immunogenic proteins may provide candidate molecules for the development of vaccines against columnaris disease.     

Supplementation of heat‐inactivated Bacillus clausii DE5 in diets for grouper,Epinephelus coioides,improves feed utilization,intestinal and systemic immune responses and not growth performance

In recent years, more and more attentions have been paid to the development and application of probiotics in aquaculture, and viable probiotics have been extensively studied, while rare information was available about inactivated probiotics in aquaculture. Therefore, in this study, a feeding trial was designed to investigate the effect of dietary supplementation of heat‐inactivated probiotic Bacillus clausii DE5 on growth performance, immune response and key immune genes expression in head kidney and intestine in grouper Epinephelus coioides. Fish were fed for 60 days with control diet (C) and two experimental diets containing 1.0 × 10⁸ CFU/g live (T1) and heat‐inactivated (T2) B. clausii DE5, respectively. The probiotic treatments did not affect the final weight, weight gain (WG) and specific growth rate (SGR) of E. coioides at days 30 and 60 (p > .05), while both heat‐inactivated and live B. clausii DE5 significantly decreased the feed intake and feed conversion ratio (FCR) at day 60 (p < .05). Serum lysozyme activity and complement C3 level in the two probiotic treatments were significantly higher than those in the control (p < .05). The lysozyme activity and complement C3 level at day 60 were significantly higher than those at day 30 (p < .05), while no significant interaction effect between diet and administration date was observed. Moreover, the heat‐inactivated B. clausii DE5 significantly improved the expression of TLR5, pro‐inflammatory cytokines (IL‐8 and IL‐1β) and TGF‐β1 in head kidney and intestine (p < .05), while the live probiotic did not show any significant effect on the expression of these key immune‐related genes in head kidney and intestine. These results indicate that dietary supplementation of heat‐inactivated B. clausii DE5 effectively improved feed utilization and both the local and systemic immune responses of E. coioides.     

Dietary nucleotide‐rich yeast supplementation improves growth,innate immunity and intestinal morphology of Pacific white shrimp (Litopenaeus vannamei)

An 8‐week feeding trial was conducted to evaluate the effects of dietary nucleotide (NT)‐rich yeast supplementation on growth, innate immunity and intestinal morphology in Pacific white shrimp (Litopenaeus vannamei). Four isonitrogenous and isolipidic practical diets were formulated to contain 0 (control), 10, 30 and 50 g/kg of NT‐rich yeast, respectively. A total of 480 shrimp with an average initial body weight of 1.86 ± 0.02 g were randomly allocated into four groups, with four replicates per group and 30 shrimp each replicate. The results indicated that shrimp fed the diet containing 50 g/kg NT‐rich yeast had significantly higher weight gain (WG), specific growth rate (SGR) and protein efficiency ratio (PER) than those fed the control diet, and the lowest feed conversion ratio (FCR) was observed in the shrimp fed the 50 g/kg NT‐rich yeast supplemental diet. However, there was no significant difference in survival among all treatments. The crude protein of whole shrimp in the 50 g/kg NT‐rich yeast group was higher than that in the control group. Total protein, triglyceride concentrations, the activities of aspartate aminotransferase and alanine aminotransferase in serum were significantly influenced by the dietary NT‐rich yeast supplementation. The activities of serum phenoloxidase (PO) and lysozyme (LZM) of shrimp fed the diet containing 50 g/kg NT‐rich yeast were higher than those in shrimp fed the other diets. Relative expressions of alp and lzm significantly upregulated in the 30 g/kg NT‐rich yeast group compared to the control group. The intestinal fold height and fold width in the 30 g/kg NT‐rich yeast group were significantly higher than those fed the control diet; and the highest microvillus height occurred in the shrimp fed the 50 g/kg NT‐rich yeast diet. In summary, dietary 30–50 g/kg NT‐rich yeast supplementation promotes growth performance, enhances innate immunity and improves intestinal morphology of Litopenaeus vannamei.