Detection and molecular characterization of infectious spleen and kidney necrosis virus from major ornamental fish breeding states in Peninsular Malaysia

‘Gold standard’ OIE reference PCR assay was utilized to detect the presence of infectious spleen and kidney necrosis virus (ISKNV) in freshwater ornamental fish from Malaysia. From total of 210 ornamental fish samples representing 14 species, ISKNV was detected in 36 samples representing 5 fish species. All positive cases did not show any clinical signs of ISKNV. Three restriction enzymes analyses showed that the fish were infected by identical strains of the same virus species within Megalocytivirus genus. Major capsid protein (MCP) genes of 10 ISKNV strains were sequenced and compared with 9 other reference nucleotide sequences acquired from GenBank. Sequence analysis of MCP gene showed that all strains detected in this study were closely related to the reference ISKNV with nucleotide sequence identity that was ranging from 99.8% to 100%. In addition, phylogenetic analysis of MCP gene revealed that viruses from genus Megalocytivirus can be divided into three genotypes: genotype 1 include reference ISKNV and all other strains that were detected in this study, genotype 2 include viruses closely related to red sea bream iridovirus (RSIV), and genotype 3 include viruses closely related turbot reddish body iridovirus (TRBIV).     

Production of recombinant capsid protein of Macrobrachium rosenbergii nodavirus (r‐MCP43) of giant freshwater prawn,M. rosenbergii (de Man) for immunological diagnostic methods

White tail disease (WTD) caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) is a serious problem in prawn hatcheries. The gene for capsid protein of MrNV (MCP43) was cloned into pRSET B expression vector. The MCP43 protein was expressed as a protein with a 6‐histidine tag in Escherichia coli GJ1158 with NaCl induction. This recombinant protein, which was used to raise the antiserum in rabbits, recognized capsid protein in different WTD‐infected post‐larvae and adult prawn. Various immunological methods such as Western blot, dot blot and ELISA techniques were employed to detect MrNV in infected samples using the antiserum raised against recombinant MCP43 of MrNV. The dot blot assay using anti‐rMCP43 was found to be capable of detecting MrNV in WTD‐infected post‐larvae as early as at 24 h post‐infection. The antiserum raised against r‐MCP43 could detect the MrNV in the infected samples at the level of 100 pg of total protein. The capsid protein of MrNV estimated by ELISA using anti‐rMCP43 and pure r‐MCP43 as a standard was found to increase gradually during the course of infection from 24 h p.i. to moribund stage. The results of immunological diagnostic methods employed in this study were compared with that of RT‐PCR to test the efficiency of antiserum raised against r‐MCP43 for the detection of MrNV. The Western blot, dot blot and ELISA detected all MrNV‐positive coded samples as detected by RT‐PCR.     

大鲵虹彩病毒流行株的分离及其主衣壳蛋白编码基因序列比较分析

大鲵虹彩病毒(giant salamander iridovirus, GSIV)是近年中国大陆新发现的引起人工养殖大鲵(Andrias davidianus)大规模死亡的病毒病原。为了揭示大鲵虹彩病毒流行株的基因型差异, 本研究对2010–2012年采集自全国不同大鲵养殖区域的患虹彩病毒病的大鲵样本进行了分子检测、病毒分离培养以及病毒主衣壳蛋白(major capsid protein, MCP)基因测序与比对分析。结果显示, 采自陕西、湖北、湖南、浙江、江西、福建等省的10个样本检测为阳性, 通过细胞培养获得10株病毒流行株。对该10株流行株MCP基因的测序与比对分析发现, 核苷酸序列相似性达到99.7%~100%, 其推测的氨基酸序列无明显差异, 证实中国大鲵虹彩病毒流行株属同一基因型。系统进化树分析结果表明, 所选大鲵虹彩病毒与蛙病毒分别聚为一枝, 但其亲缘关系较近。本研究结果旨为大鲵虹彩病毒病的疫苗研制及其免疫防控技术研究奠定基础。

     

Comparative imaging of European eels (Anguilla anguilla) for the evaluation of swimbladder nematode (Anguillicoloides crassus) infestation

This study compares diagnostic imaging tools in detecting the parasitic swimbladder nematode Anguillicoloides crassus in Anguilla anguilla (L.) and focuses on ultrasound in an attempt to develop a non‐destructive, field diagnostic test. Ultrasound use could allow the parasite to be diagnosed without decreasing the number of critically endangered European eels through post‐mortem. In the preliminary study, eels were examined with computed radiography, computed tomography, magnetic resonance imaging, 14 MHz high‐end ultrasound and 5 MHz low‐end portable ultrasound, and the results were compared with post‐mortem findings. This ultrasound scanning technique did not produce any promising results. A second batch of eels was examined using the same high‐end and low‐end ultrasounds, but employing a different scanning technique and comparing the results with post‐mortem. This second study, scanning along the midline from below, allowed for the detection of anomalies associated with moderately infected animals. None of the eels used in this study were severely infected; thus, no conclusions can be made regarding the use of ultrasound in those animals. Overall, it was found that none of the techniques were useful in diagnosing mildly infected individuals; therefore, no single diagnostic imaging tool is sensitive enough to replace post‐mortem for definite diagnosis.     

赤点石斑鱼神经坏死病毒主衣壳重组蛋白多克隆抗体的制备

把赤点石斑鱼（Epinephelus akaara）神经坏死病毒（RGNNV）主衣壳蛋白（MCP）基因的重组表达质粒载体pRSETA-MCP转化至大肠杆菌（Estherichia coli）BL21（DE3）,经IPTG诱导表达,SDS-PAGE显示表达的重组蛋白主要以不可溶的包涵体形式存在,分子量约44.5kD。通过Ni-NTA-Agarose亲和层析柱纯化,经分析纯度达90%,之后免疫新西兰兔制备抗血清,ELISA效价达1：12800以上。Western-blot分析结果显示,该血清与表达的重组蛋白有较强反应,说明通过原核表达的重组蛋白具有良好的免疫原性。     

Development of different diagnostic techniques for Endolimax piscium (archamoebae) and their applicability in Solea senegalensis clinical samples

Systemic amoebiasis of sole is caused by Endolimax piscium, a cryptic parasitic archamoeba whose epidemiology and pathogeny are yet unknown. To establish reliable detection methods for this parasite, a battery of molecular diagnostic tools (ISH, PCR and qPCR) were developed and evaluated with a panel of clinical samples from symptomatic diseased fish and from apparently normal animals of different stocks. As there is neither enough background information on the epidemiology of the disease nor a validated reference method, comparison of tests used a composite reference method approach. The ISH technique was the most specific and sensitive in intestine samples and particularly useful as a reference confirmatory method, while the best method in muscle samples was qPCR. Application of the tests to asymptomatic fish demonstrated presence of parasites in a large proportion (>25%) of their intestines, suggesting that this is the point of entry of the amoebae and the initial stage in the development of the disease. The triggering factors that facilitate the breaching of the intestinal barrier by E. piscium, causing granulomatous lesions in other organs and systemic spreading, are not completely understood but our results point to the connective tissue as a preferential target for parasite development and migration.     

Comparative evaluation of mahua (Bassia latifolia) oil cake and castor bean (Ricinus communis) seed as fish toxicants for tilapia (Oreochromis mossambicus) and panchax (Aplocheilus panchax) with residual toxicity assessment on Labeo bata

Comparative evaluation of mahua (Bassia latifolia) oil cake (MOC) and castor bean (Ricinus communis) seed (CBS) was tested as piscicides for tilapia (Oreochromis mossambicus) and panchax (Aplocheilus panchax), and residual toxicity test was done on Labeo bata. Delayed response of CBS compared with MOC was evident as first and total mortality of tilapia was encountered at 4th and 10th hour of application, against 26 hr and 42 hr in panchax respectively. Susceptibility of the test fish to the piscicides was highly variable as tilapia was more susceptible than panchax under both the piscicides tested especially in CBS. Residual toxicity decreased polynomialy over time (R² ≥ 0.99) and reduced to below 10% on day 13–14 in both MOC and CBS treatments. Both MOC and CBS contributed positively to the nutrient pool of water in which CBS provided more nitrogen compared with MOC. N: P ratio of water in CBS treatment increased exponentially to more than 18 indicating phosphorus limitation.     

Development of an in vitro model to assess protein bioavailability in diets for common octopus (Octopus vulgaris)

An in vitro model designed to assess protein bioavailability in diets for growing Octopus was developed. The model required a previous assessment of some functional features of protein digestion in this species like the main producing organs, optimum pH for activity and total production per g tissue. The main producing organs identified were the salivary glands and the hepatopancreas (HP), being optimum pH for protease activity quite different in both organs (mostly alkaline in the posterior salivary glands and acid in the HP). In spite of the high‐specific protease activity measured in the salivary glands, a major role of the HP in protein hydrolysis is suggested due to the much bigger size of this viscera. All this information was used as a basis to develop an in vitro two‐step hydrolysis process, which simulated protein hydrolysis performed by these two organs using the Octopus enzymes. The assay was used to evaluate differences in amino acid bioavailability from several protein sources (casein, gelatin, fish meal, squid meal and krill meal) that could be used as feed ingredients for this species. As significant differences were detected both in total amount and in rate of release of the amino acids from such proteins, the model is proposed as a complementary tool in the selection and nutritional evaluation of protein ingredients to be used in diets for Octopus.     

Comparative effects of dietary supplementation with maggot meal and soybean meal in gibel carp (Carassius auratus gibelio) and darkbarbel catfish (Pelteobagrus vachelli): growth performance and antioxidant responses

A 6‐week growth trial was conducted to investigate the effect of dietary supplementation with maggot meal (MGM) and soybean meal (SBM) on the growth performance and antioxidant responses of gibel carp (GC) and darkbarbel catfish (DC). The basal diet was formulated to contain 114 g kg⁻¹ fish meal (FM) and 200 g kg⁻¹ SBM. The basal diet was supplemented with either 280 g kg⁻¹ FM (Control), 390 g kg⁻¹ MGM or 450 g kg⁻¹ SBM to obtain three isonitrogenous (crude protein: 380 g kg⁻¹) and isocaloric (gross energy: 16 kJ g⁻¹) diets. For GC, a significant decrease in specific growth rate (SGR) was only observed in fish fed the SBM diet compared with the control (P < 0.05). Principal components analysis (PCA) of GC showed a higher similarity in antioxidant response to dietary supplementation with MGM and SBM proteins between liver and intestine, but the DC did not. The present results suggest that supplementing 390 g kg⁻¹ MGM protein to basal diet cause an enhancement of the antioxidant capacity in GC, but supplementing 390 g kg⁻¹ MGM and 450 g kg⁻¹ SBM proteins to basal diets resulted in a significant attenuation of the antioxidant capacity in DC.     

大黄鱼肿大细胞病毒不同毒株的细胞培养及主要衣壳蛋白基因比较

为建立大黄鱼肿大细胞病毒的培养方法,明确其分类地位,用肿大细胞病毒检测呈阳性的大黄鱼幼鱼病料 (FD201807和SA201808)肾组织匀浆液感染鳜仔鱼细胞系 (mandarin fish fry cell line-1,MFF-1)并连续传代,从病料组织匀浆液和细胞冻融液中提取病毒DNA,克隆病毒主要衣壳蛋白基因 (mcp),测序后与NCBI GenBank中的虹彩病毒科肿大细胞病毒属病毒mcp以及2018—2020年所检出的15株大黄鱼肿大细胞病毒mcp进行比对分析。结果显示,病毒传至第4代才可引起MFF-1细胞病变,细胞病变的主要特征为细胞脱壁、变圆、折光度增强;感染时间越长脱壁细胞越多,同时培养液中的颗粒增加;透射电镜下可见感染细胞的细胞质散在大小为130~150 nm的六边形病毒粒子和空壳。感染细胞的病变周期随传代代次的增加而缩短,第15代次的FD201807株感染细胞80%细胞病变的时间为3 d,第15代次的SA201808株感染细胞80%细胞病变的时间为7~8 d。mcp序列比对和聚类分析发现,SA201808株与FD201807株的mcp序列存在21个碱基差异,二者的mcp序列分别与大黄鱼虹彩病毒(large yellow croaker iridovirus, LYCIV) LYCIV-Zhoushan (GenBank: MW139932.1)和花鲈虹彩病毒 (Lateolabrax maculatus iridovirus, LMIV) (GenBank: MH577517.1)相近。15株从大黄鱼病料检出的肿大细胞病毒中,12株的mcp序列与SA201808株聚类;3株与FD201807聚类。本研究利用MFF-1细胞系分离培养了大黄鱼肿大细胞病毒,揭示了大黄鱼肿大细胞病毒存在差异,为更好地了解大黄鱼肿大细胞病毒提供了数据参考。     

Effects of temperature and dietary protein on gene expression of Hsp70 and Wap65 and immunity of juvenile mirror carp (Cyprinus carpio)

Juvenile mirror carp were fed diets containing 303.4, 321.7, 341.2, 361.0 and 379.1 g kg^?1 proteins, respectively, and reared at different water temperatures (18, 23 and 28°C) for 60 days. Gene expression of heat shock protein gene (Hsp70) and the warm temperature acclimation‐related 65 kDa protein gene (Wap65), immunity and antioxidant status in the carp were investigated. Results indicated that the contents of serum complement 3 (C3), complement 4 (C4) and immunoglobulin M (IgM), as well as activities of liver superoxide dismutase (SOD) and lysozyme (LSZ) were significantly enhanced with increasing dietary protein (P < 0.05), while content of malondiadehyde (MDA) decreased. Gene expression level of Wap65 in the liver significantly increased with dietary protein, while gene expression of Hsp70 decreased. The contents of C3, C4 and IgM, the activities of SOD and LSZ and gene expression level of Wap65 in the liver significantly increased with temperature. These results suggest that: Serum immune parameter, antioxidant enzymes and Hsp70 and Wap65 expression interact in fish to improve ability to adapt to the environment; and the optimal conditions for the immunity of carp are 348.1?354.5 g kg^?1 protein at 18°C, 352.3?364.9 g kg^?1 at 23°C and 360.2?364.3 g kg^?1 at 28°C, and the optimum temperature for carp is 23°C.     

Comparative genomic analysis of different virulence strains reveals reasons for the increased virulence of Aeromonas veronii

Aeromonas veronii is an important zoonotic and aquatic agent. More and more cases have shown that it has caused huge economic losses in the aquaculture industry in addition to threatening human health. But the reasons for the increasing virulence of A. veronii are still unclear. In order to further understand the reasons for the increased virulence of A. veronii, we conducted a comparative analysis of the genomes of A. veronii with different virulence. The analysis revealed that there are multiple virulence factors, such as those related to fimbriae, flagella, toxins, iron ion uptake systems and type II, type III and type VI secretion systems in the virulent strain TH0426 genome. And comparative analysis showed that there were two complete type III secretion systems (API1 and API2), of which the API2 and iron ion transport system were unique to the TH0426 strain. In addition, TH0426 strain also has unique functional gene clusters, which may play important roles in terms of resisting infection, adapting to different environments and genetic evolution. These particular virulence factors and gene clusters may be the important reasons for the increased virulence. These insights will provide a reference for the study of the pathogenesis of A. veronii.     

Utilization of Caridina nilotica (Roux) meal as a protein ingredient in feeds for Nile tilapia (Oreochromis niloticus)

The effects of replacing fish meal with Caridina nilotica as a protein ingredient on growth performance, nutrient utilization, carcass, proximate composition and economic benefits in Nile tilapia (Oreochromis niloticus) culture was evaluated. Replacement of the FM with C. nilotica was done at 25%, 50%, 75% and 100% (D25, D50, D75 and D100) and the substitution effects was compared with the control diet (D0, 0% C. nilotica). After 140 days of culture, the best growth performance, nutrient utilization and economic benefits occurred in fish groups fed diets with 25% C. nilotica inclusion. However, growth performance in fish fed diets D50 and D75 were comparable with the control (P > 0.05). At 100% substitution level of FM with C. nilotica, the growth performance and fish survival was lower than control. Protein and lipid contents in the fish and their digestibilities were highest in diet D25 and decreased with increasing levels of substitution of FM with C. nilotica. This study demonstrate that utilization of local protein sources (C. nilotica) can be effectively used to replace up to 75% of FM in the diets without compromising growth performance, survival, nutrient utilization and economic benefits in O. niloticus culture.     

Comparative studies on dietary protein requirements of juvenile and on‐growing gibel carp (Carassius auratus gibelio) based on fishmeal‐free diets

Two trials were conducted to investigate protein requirements of juvenile (3.18 g in Trial 1) and on‐growing (87.1 g in Trial 2) gibel carp, Carassius auratus gibelio var. CAS III. Six isoenergetic diets containing 250–500 g kg^?1 dietary protein were formulated using soy protein concentrate (SPC) and casein as protein sources. The results showed that weight gain (WG) increased when dietary protein increased from 250 to 400 g kg^?1 and decreased at 400 to 500 g kg^?1 CP in Trial 1, while WG increased when dietary protein increased from 250 to 350 g kg^?1 and kept constant at 350 to 500 g kg^?1 CP in Trial 2. With increasing dietary protein, feed conversion ratio (FCR) decreased, while protein retention efficiency (PRE) decreased in Trial 1 and was not affected in Trial 2. Apparent digestibility coefficient of protein (ADC_p) increased with increasing dietary protein in two trails. Trypsin activity increased with dietary protein in the juveniles and was not affected in on‐growing fish. Hepatic alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities increased with dietary protein. Broken‐line and quadratic regression of WG estimated that dietary protein requirements for maximum growth were about 402–427 g kg^?1 for the juvenile and 337–418 g kg^?1 for on‐growing gibel carp.     

Isolation and evaluation of putative probiotic strains from different teleost to prevent Pseudomonas aeruginosa infection in Cyprinus carpio

Probiotics renowned as valuable microbes serve as a potential alternative to control diseases in aquaculture and are considered as an efficient and environment‐friendly approach to reduce the use of antibiotics. The present study aims at the isolation of putative probiotic bacteria from the intestinal tract of different fish species from the Doaba region of Punjab, India. In this study, isolated bacterial strains were characterized based on their morphological, biochemical and molecular characterization by 16S rRNA gene sequencing, followed by in vitro evaluation of different selection parameters described in FAO/WHO guidelines. A total of 169 different bacterial strains were isolated from the gastrointestinal tract of 52 different fish species. After in vitro evaluation, out of 169 bacterial strains only five bacteria (S3, S7, BDK2', BDK7 and BDK9) identified as Enterococcus and Bacillus species showed antagonistic activity against the fish pathogen Pseudomonas aeruginosa (MTCC 4 673). These isolates were screened based on their response towards bile tolerance, pH tolerance, adhesion and drug susceptibility to different antibiotic discs. And, the in vivo results indicated improved growth and survival against the infection (P. aeruginosa) after oral administration of the probiotics. The observations of in vitro and in vivo evaluation indicate that these isolated probiotic strains serve as effective probiotics and can be used as a novel and safe treatment to cure current issues in aquaculture.     

Further evaluation of the efficacy of emamectin benzoate for treating Pseudocapillaria tomentosa (Dujardin 1843) in zebrafish Danio rerio (Hamilton 1822)

Pseudocapillaria tomentosa is a pathogenic nematode parasite, causing emaciation and severe inflammatory lesions in the intestines in zebrafish Danio rerio (Hamilton 1822). Emamectin benzoate is commercially available analogue of ivermectin used for treating salmon for sea lice, under the brand name SLICE^®, and we have used this for treating zebrafish with the P. tomentosa. Here, SLICE^®, 0.2 per cent active emamectin benzoate, was used for oral treatments at 0.35 mg emamectin benzoate/kg fish/day for 14 days starting at 7 days post‐exposure (dpe). Another experiment entailed initiating treatment during clinical disease (starting at 28 dpe). Early treatment was very effective, but delaying treatment was less so, presumably due to inappetence in clinically affected fish. We evaluated emamectin benzoate delivered in water, using Lice‐Solve? (mectinsol; 1.4% active emamectin benzoate) in two experiments. Application of four 24‐hr treatments, space over 7 days was initiated at 28 dpe at either 0.168 or 0.56 mg emamectin benzoate/L/bath, and both treatments completely eradicated infections. This was 3 or 10 times manufacture's recommended dose, but was not associated with clinical or histological side effects.     

Efficacy of calcein as a chemical marker of green‐lipped mussel (Perna canaliculus) larvae and its potential use for tracking larval dispersal

An optimal chemical shell marking protocol was developed for the New Zealand green‐lipped mussel, Perna canaliculus with a view to its future use in larval tracking experiments. Larval P. canaliculus aged either 10, 15 or 19 days post fertilization were immersed in treatments of 50, 100 and 200 mg L^?1 of calcein for a period of 24 h before measurements of shell mark brightness were taken. There was 100% marking success in all calcein treatments for all age classes, with 19‐day larvae immersed in 200 mg L^?1 calcein producing the brightest mark. Growth was not affected by calcein immersion; however, 10‐day larvae exhibited significantly higher levels of mortality compared with 15‐ and 19‐day larvae suggesting a reduced resilience to the marking protocols in younger larvae. In a mass staining experiment, a solution of 100 mg L^?1 calcein was used to successfully stain15.6 million hatchery reared P. canaliculus larvae. Calcein, therefore, offers a low impact method with which to stain the sensitive early life stages of this species thus providing a rapid method for identifying individuals of interest, i.e. individuals released in the wild or specific family lines within a hatchery environment.     

Flavobacteria colonizing the early life stages of hatchery‐incubated Chinook salmon Oncorhynchus tshawytscha (Walbaum 1792) are markedly diverse

Flavobacterial diseases are significant impediments to hatchery‐based fishery conservation and aquaculture productivity worldwide. Recent studies revealed a multitude of novel flavobacteria within the reproductive fluids and unfertilized eggs of feral Chinook salmon Oncorhynchus tshawytscha broodstock, some of which were associated with systemic disease. Herein, embryonated eggs/fry from these broodstock were assayed for flavobacteria while in incubator stacks in three hatcheries over 2 years, as was the water entering hatchery incubators. Overall, >65% of sampled eggs and 38% of fry were colonized by flavobacteria. One hundred and ninety‐one egg and fry‐associated flavobacterial isolates were characterized phenotypically and via 16S rRNA gene sequencing and phylogenetic analyses, revealing that the majority fell into 22 clades (i.e., 15 Flavobacterium spp. groups and seven Chryseobacterium spp. groups) that varied in presence by facility. Although some matched previously described fish‐pathogenic species, the majority were distinct from all described flavobacteria and likely represent novel species. Of concern, iodophor disinfection at the commonly utilized dose/duration for egg‐surface disinfection did not eliminate flavobacteria. Results also implicated maternal routes of infection and source water for some flavobacteria. In total, study findings underscore the complexity of flavobacterial ecology within hatchery environments and highlight the need for improved hatchery biosecurity practices.