Assessing the population transmission dynamics of tilapia lake virus in farmed tilapia

A novel virus, tilapia lake virus (TiLV), has been identified as a key pathogen responsible for disease outbreak and mass mortality of farmed tilapia. We used a deterministic susceptible‐infectious‐mortality (SIM) model to derive key disease information appraised with published TiLV‐induced cumulative mortality data. The relationship between tilapia mortality and TiLV exposure dosages was described by the Hill model. Furthermore, a disease control model was proposed to determine the status of controlled TiLV infection using a parsimonious control reproduction number (R_C)‐control line criterion. Results showed that the key disease determinants of transmission rate and basic reproduction number (R₀) could be derived. The median R₀ estimate was 2.59 in a cohabitation setting with 2.6 × 10⁵ TCID50 fish^?1 TiLV. The present R_C‐control model can be employed to determine whether TiLV containment is feasible in an outbreak farm by quantifying the current level of transmission. The SIM model can then be applied to predict what additional control is required to manage R_C < 1. We offer valuable tools for aquaculture engineers and public health scientists the mechanistic‐based assessment that allows a more rigorous evaluation of different control strategies to reduce waterborne diseases in aquaculture farming systems.     

Two-year surveillance of tilapia lake virus (TiLV) reveals its wide circulation in tilapia farms and hatcheries from multiple districts of Bangladesh

Tilapia lake virus (TiLV) is an emerging pathogen in aquaculture, reportedly affecting farmed tilapia in 16 countries across multiple continents. Following an early warning in 2017 that TiLV might be widespread, we executed a surveillance programme on tilapia grow-out farms and hatcheries from 10 districts of Bangladesh in 2017 and 2019. Among farms experiencing unusual mortality, eight out of 11 farms tested positive for TiLV in 2017, and two out of seven tested positive in 2019. Investigation of asymptomatic broodstock collected from 16 tilapia hatcheries revealed that six hatcheries tested positive for TiLV. Representative samples subjected to histopathology confirmed pathognomonic lesions of syncytial hepatitis. We recovered three complete genomes of TiLV from infected fish, one from 2017 and two from 2019. Phylogenetic analyses based on both the concatenated coding sequences of 10 segments and only segment 1 consistently revealed that Bangladeshi TiLV isolates formed a unique cluster within Thai clade, suggesting a close genetic relation. In summary, this study revealed the circulation of TiLV in 10 farms and six hatcheries located in eight districts of Bangladesh. We recommend continuing TiLV-targeted surveillance efforts to identify contaminated sources to minimize the countrywide spread and severity of TiLV infection.     

Underwater video analysis as a non‐destructive alternative to electrofishing for sampling imperilled headwater stream fishes

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Detection of infectious pancreatic necrosis virus (IPNV) from asymptomatic redbanded seabream,Pagrus auriga Valenciennes,and common seabream,Pagrus pagrus (L.), using a non‐destructive procedure

A non‐destructive procedure based on nested RT‐PCR and dot‐blot hybridization has been developed for the detection of asymptomatic IPNV‐carrier fish. The pair of primers designed for RT‐PCR amplified a 599‐bp fragment of the pVP2 region within the polyprotein gene, resulting in the detection of IPNV genotype III.1. The use of a nested RT‐PCR allowed the amplification of IPNV genotypes III.1 and I.2. In addition, a 191‐bp probe was designed for hybridization studies used in combination with the nested RT‐PCR. The application of the nested RT‐PCR to analyse blood samples from asymptomatic redbanded seabream, Pagrus auriga, and common seabream, P. pagrus, specimens showed a 53.1% and 77.8% prevalence of IPNV‐carriers, respectively. The combination of nested RT‐PCR and dot‐blot hybridization increased the detection rates up to 100% for redbanded seabream and 94.4% for common seabream. Therefore, the protocol described in this study is highly sensitive and specific for the detection of IPNV in asymptomatic carrier fish, and, in addition, the results demonstrate the carrier state in two newly cultured sparid species in southern Spain.     

Effect of fermented Enteromopha prolifera on the growth performance,digestive enzyme activities and serum non‐specific immunity of red tilapia (Oreochromis mossambicus × Oreochromis niloticus)

This study was conducted to evaluate the effect of dietary inclusion of different levels of fermented macroalga, Enteromorpha prolifera, on the growth performance, digestive enzyme activities and serum non‐specific immunity of red tilapia (Oreochromis mossambicus × Oreochromis niloticus). Fish were fed the control diet or the five diets supplemented with fermented E. prolifera for 7 weeks (diets 1–5 containing 10, 20, 30, 40 and 50 g kg^?1 of fermented E. prolifera respectively). The results showed that the percentage weight gain and specific growth rate (SGR) improved significantly in fish fed diets 3, 4 and 5, and regression analysis showed SGR reached the maximum in fish fed diet containing 37 g kg^?1 fermented E. prolifera, while the feed conversion ratio reduced significantly in fish fed diets 2, 3, 4 and 5, and regression analysis showed it reached its minimum in fish fed diet containing 41 g kg^?1 fermented E. prolifera. The pepsin, erepsin, gastric amylase, intestinal amylase, gastric lipase and intestinal lipase activities in fish fed diets 2, 3 and 4 were higher significantly than the control, with the exception of the pepsin activity in fish fed diet 4. The serum total superoxide dismutase, lysozyme, acid phosphatase and alkaline phosphatase activities enhanced significantly in fish fed diets 3 and 4. In conclusion, fermented E. prolifera had positive effects on the growth performance, digestive enzyme activities and serum non‐specific immunity of red tilapia and the recommended dose of fermented E. prolifera is 37–41 g kg^?1 in the diet of red tilapia.     

Effects of dietary arginine on growth performance,feed utilization,haematological parameters and non‐specific immune responses of juvenile Nile tilapia (Oreochromis niloticus L.)

An 8‐week feeding trial was conducted to evaluate the effects of dietary arginine on growth performance, feed utilization, haematological parameters and non‐specific immune responses of juvenile Nile tilapia (6.03 g). Five isonitrogenous and isoenergetic diets were formulated to contain graded levels of L‐arginine (8.5 g kg^?1, 11.5 kg^?1, 15.3 kg^?1, 18.8 kg^?1 and 22.4 kg^?1 dry diet) from dietary ingredients and crystalline arginine. Each diet was randomly assigned to triplicate groups of 25 juvenile fish (6.04 ± 0.02 g) three times daily (8:30, 12:30, and 17:00 hours) to apparent satiation. Results showed that the weight gain (WG) and special growth rate (SGR) increased with increasing dietary arginine levels up to 15.3 kg^?1 and remained nearly the same thereafter. Arginine supplementation had no impact on the survival, body composition and haematological parameters of tilapia. However, the immune responses (plasma nitric oxide content, total nitric oxide synthase (T‐NOS) and lysozyme activity) significantly (P < 0.05) increased with increasing dietary argentine level after stress. Quadratic regression analysis (y = ?0.7147x² + 25.986x + 147.53 R² = 0.8736) on weight gain against dietary arginine levels indicated that the optimal dietary arginine requirement was estimated to be 18.2 kg^?1 of the diet (corresponding to 6.24% of dietary protein on a dry weight basis). The present study indicates that arginine has an immunostimulatory potential to help juvenile Nile tilapia cope with stress conditions such as Streptococcus iniae infection.     

Effects of dietary mannanase on growth,metabolism and non‐specific immunity of Tilapia (Oreochromis niloticus)

An experiment was designed to assess the effects of a commercial β‐mannanase on performance and immunity of tilapia fed plant‐based diets. A basal diet was supplemented with 0.0 (control), 0.5 and 1.0 g β‐mannase kg⁻¹ to formulate three experimental diets. Each treatment contained 4 tanks with 30 fish per tank. Trial lasted 8 weeks. Our results demonstrated that β‐mannanase addition (0.5 and 1.0 g kg⁻¹) improved significantly (P < 0.05) the final weight, specific growth rate (SGR), protein efficiency ratio (PER) and feed conversion ratio (FCR) compared with the control (0.0 g kg⁻¹). There were no significant differences in feed intake (FI) and survival rate (SR) among the 3 dietary treatments (P > 0.05). β‐mannanase supplementation also led to an increase (P < 0.05) in amylase, trypsin and Na⁺K⁺‐ATPase activities in intestine, and an decrease (P < 0.05) in aspartate transaminase (AST) and alanine transaminase (ALT) activities in serum compared with the control. However, dietary enzyme supplementation had no significant effect on the serum triacylglycerol (TG), cholesterol (CHO), high density lipoprotein cholesterol (HDL‐C), low density lipoprotein cholesterol (LDL‐C) and very low density lipoprotein cholesterol (VLDL‐C) (P > 0.05). Moreover, the dietary β‐mannanase supplementation groups exhibited an increase in the total leukocyte counts (WBC), differential leukocyte counts, respiratory burst activity, lysozyme activity and superoxide dismutase (SOD) activity compared to the controls (P < 0.05). In conclusion, β‐mannanase addition to tilapia diets improved feed utilization and non‐specific immunity resulting in improvements in growth performance.     

Differential expression of lipid metabolism‐related genes and miRNAs in Ctenopharyngodon idella liver in relation to fatty liver induced by high non‐protein energy diets

To explore the mechanism of fatty liver formation induced by high non‐protein energy diets in grass carp (Ctenopharyngodon idella), basal diet and high‐energy diets were fed to juvenile grass carp for 9 weeks. The experimental groups fed on high‐energy diets which included a high‐lipid diet (H‐LIP), a high‐carbohydrate diet (H‐CHO) and a high‐lipid and carbohydrate diet (H‐CL). The control group fed on basal diet. Growth performance, liver fat accumulation, serum biochemical indexes and the expression levels of lipid metabolism‐related genes (SREBP‐1, PPARγ, FAS, ACC1, and LPL) and miRNAs (miR‐33, miR‐122, and miR‐370) were examined at the end of the feeding trial. There were no significant differences in growth rate and feed efficiency among the four groups. However, significant increase in mesenteric and liver fat contents, and lipid droplets in the liver was induced by high‐lipid and high‐carbohydrate diets. There were significant differences in serum biochemical indicators such as AST/ALT, GLB, TG and TP, and liver fatty acid composition between the control and experimental groups. The expression levels of SREBP‐1, PPARγ, FAS, ACC1 and LPL were upregulated, while CPT‐1 was downregulated with the high‐energy treatments. Additionally, the expression levels of miR‐33, miR‐122 and miR‐370 in the liver were higher in the three high‐energy treatments than those in the control (P < 0.05). The results suggest that modifications of lipid metabolism‐related genes and miRNAs may be involved in fatty liver formation induced by high non‐protein energy diets in grass carp.     

罗非鱼湖病毒对吉富罗非鱼和E-11细胞的感染

为研究罗非鱼湖病毒在吉富罗非鱼体内和敏感细胞E-11中的感染特性,实验首先从人工感染罗非鱼湖病毒的吉富罗非鱼脾脏中获得罗非鱼湖病毒第4片段基因组,其cDNA全长1 250 bp,开放读码框长度为1 065 bp,编码354个氨基酸。通过进化树分析,该蛋白是罗非鱼湖病毒血凝素—酯酶融合蛋白(HEF)。随后通过在大肠杆菌大量表达和提纯GST融合HEF蛋白,免疫新西兰大白兔,制备了兔抗TiLV-HEF多克隆抗体。ELISA结果显示获得的抗血清效价高于1∶51 200,并且获得的抗体可以特异性识别病毒的TiLV-HEF蛋白。人工感染实验结果显示,TiLV的感染造成鱼体表面溃疡、全身性出血以及眼晶状体混浊等症状。H.E染色结果显示,肝脏形成合胞体,脾脏中含铁血黄素增加和部分细胞空泡变性。头肾出现淋巴细胞坏死,体肾蛋白质沉淀和肾小球坏死等病理症状。Western blot和免疫组织化学结果显示该病毒在所有组织中均有分布,其中脾脏、头肾和鳃中的病毒丰度高于肝脏、体肾和脑组织。通过细胞间接免疫荧光实验,发现TiLV感染E-11细胞后,HEF蛋白在细胞质中。TiLV可以通过感染吉富罗非鱼幼鱼的肝脏、脾脏...     

Detection of tilapia lake virus (TiLV) infection by PCR in farmed and wild Nile tilapia (Oreochromis niloticus) from Lake Victoria

Tilapia lake virus disease (TiLVD) has emerged to be an important viral disease of farmed Nile tilapia (Oreochromis niloticus) having the potential to impede expansion of aquaculture production. There is a need for rapid diagnostic tools to identify infected fish to limit the spread in individual farms. We report the first detection of TiLV infection by PCR in farmed and wild Nile tilapia from Lake Victoria. There was no difference in prevalence between farmed and wild fish samples (p = .65), and of the 442 samples examined from 191 fish, 28 were positive for TiLV by PCR. In terms of tissue distribution, the head kidney (7.69%, N = 65) and spleen (10.99%, N = 191), samples had the highest prevalence (p < .0028) followed by heart samples (3.45%, N = 29). Conversely, the prevalence was low in the liver (0.71%, N = 140) and absent in brain samples (0.0%, N = 17), which have previously been shown to be target organs during acute infections. Phylogenetic analysis showed homology between our sequences and those from recent outbreaks in Israel and Thailand. Given that these findings were based on nucleic acid detection by PCR, future studies should seek to isolate the virus from fish in Lake Victoria and show its ability to cause disease and virulence in susceptible fish.     

The fatty acid profiles and energetic substrates of two Nile tilapia (Oreochromis niloticus,Linnaeus) strains,Red‐Stirling and Chitralada,and their hybrid

Protein and lipid content as well as the fatty acid (FA) composition of storage tissues were analysed in two varieties of Oreochromis niloticus (Red‐Stirling and Chitralada) and their hybrid. The animals were maintained in cages for 11 months. The samples were taken when the animals weighed 10, 50, 100, 250 and 500 g. The results showed that changes in the metabolic processes occur during an increase in body mass in both varieties of tilapia and also their hybrid, but that these differences are not found in animals collected at the commercial weight. The protein content of the fillet and liver decreased with growth and the same protein content associated with growth was found for fillet lipid content. The genetic variety did not influence the FA profile of the fillet, but different genotypes had different hepatic FA compositions. Even with the same lipid content, the hepatocytes of Chitralada accumulated higher levels of polyunsaturated fatty acids (PUFA) n6 in triglycerides and increased C22:6n3 in the hepatocyte membranes. The higher n6PUFA content was compensated by a lower fraction of saturated FA in the hepatocyte triglycerides. The skin of Chitralada also had higher n6PUFA and C22:6n3 contents, suggesting a higher ability to deposit PUFA in the skin due to alterations in the liver synthetic pathway.     

饲料脂肪含量与奥尼罗非鱼幼鱼肝脏形态结构特征的相关性

研究了投喂不同脂肪含量饲料时奥尼罗非鱼幼鱼（Oreochromis niloticus×O．aureus）肝脏形态学与组织学的变化规律,旨在阐明饲料脂肪对奥尼罗非鱼幼鱼脂肪肝发生的影响。饲料脂肪水平设置为0％、2％、4％、6％和8％等5个梯度组。随着饲料脂肪水平逐渐升高,奥尼罗非鱼幼鱼的增重率与肥满度均先上升后下降,1～5组肥满度分别为1．63±0．14、1．78±0．21、1．92±0．18、1．80±0．21和1．74±0．24。脂肪水平对奥尼罗非鱼肝脏形态学有明显影响,4～5组病鱼的肝胰脏肿大,呈油腻状,颜色发黄,柔软粉糊,有的出现白色坏死病灶,部分胆囊肿大且颜色变深,肠系膜均有过量的白色脂肪沉积。脂肪水平对奥尼罗非鱼肝脏组织学亦有明显影响,1～5组试验鱼的肝细胞直径随之增大,低脂肪水平的1～3组与高脂肪水平的4～5组具有显著差异,平均值分别为8．9和17．9μm,高脂肪水平的4～5组呈现肝细胞核偏位、胞浆内脂肪滴较多、细胞透明空泡化等症状。研究认为奥尼罗非鱼幼鱼饲料中脂肪添加量超过4％时对其肝脏形态学与组织学有一定影响。     

罗非鱼湖病毒核蛋白的克隆表达、抗体制备及其组织分布

近年来罗非鱼湖病毒(TiLV)在多个国家流行,对世界罗非鱼养殖业造成严重威胁。中国是罗非鱼第一养殖大国,尽管我国大陆还没有TiLV的正式报道,鉴于吉富罗非鱼是我国重要的罗非鱼养殖品种,其对TiLV的感染特性研究具有重要意义。本实验采用TiLV对吉富罗非鱼进行人工感染,随后在肝脏组织中克隆和测定了TiLV第6片段基因。罗非鱼湖病毒第6片段基因cDNA全长1 044 bp,开放读码框(ORF)为954 bp,编码317个氨基酸,预测分子量为36.38 ku;5′非编码区(NCR)为19 bp,3′非编码区(NCR)为972 bp。系统进化树分析表明该蛋白属于TiLV核蛋白(NP)。随后在大肠杆菌中表达和提纯了GST融合NP蛋白,在新西兰大白兔上制备了多克隆抗体。通过酶联免疫吸附实验(ELISA)检测抗体效价为1∶51 200,且抗体可特异性识别感染组织中的病毒NP蛋白。对吉富罗非鱼不同组织进行苏木精—伊红(H.E)染色观察,发现肝脏组织坏死并形成合胞体,脾脏部分细胞出现空泡、坏死,含铁血黄素增多,头肾细胞坏死,鳃丝上皮细胞明显解离脱落,鳃小片黏连,脑组织细胞肿大。通过蛋白印迹法(WB)和免...     

Virulence assay of rhizoid and non‐rhizoid morphotypes of Flavobacterium columnare in red tilapia,Oreochromis sp., fry

Numerous isolates of Flavobacterium columnare were previously recovered from red tilapia, Oreochromis sp., exhibiting columnaris‐like disease in Thai farms, and the phenotypic and genetic characteristics were described. The objective of this study was to determine the virulence of two morphotypes (rhizoid and non‐rhizoid colonies) of F. columnare and to determine their ability to adhere to and persist in red tilapia fry. The results showed that the typical rhizoid isolate (CUVET1214) was a highly virulent isolate and caused 100% mortality within 24 h following bath challenge of red tilapia with three different doses. The non‐rhizoid isolate (CUVET1201) was avirulent to red tilapia fry. Both morphotypes adhered to and persisted in tilapia similarly at 0.5 and 6 h post‐challenge as determined by whole fish bacterial loads. At 24 and 48 h post‐challenge, fry challenged with the rhizoid morphotype exhibited significantly higher bacterial loads than the non‐rhizoid morphotype. The results suggested that an inability of the non‐rhizoid morphotype to persist in tilapia fry may explain lack of virulence.     

A comparative study on the apparent digestibility of selected feedstuffs in hybrid catfish (Clarias macrocephalus × Clarias gariepinus) and Nile tilapia (Oreochromis niloticus)

The apparent digestibility (AD) of dry matter (DM), organic matter (OM), crude protein (CP) and essential amino acids (EAA) in five selected feedstuffs was evaluated in hybrid catfish and Nile tilapia. The AD of DM, OM and CP in cassava leaf meal (CLM) was lower (P < 0.05) than in groundnut meal (GNM), soybean meal (SBM), sesame husk meal (SSH) and shrimp head meal (SHM). The AD of DM, OM and CP among GNM, SBM, SSH and SHM was not different (P > 0.05). The AD of most EAA was higher in SSH (P < 0.05) than in CLM, GNM, SBM and SHM. The AD of most EAA among CLM, GNM, SBM and SHM was not different (P > 0.05). The AD of DM and OM in CLM was higher (P < 0.05) in hybrid catfish than in Nile tilapia, while there was no difference in the AD of CP in CLM between fish species. The AD of DM, OM, CP and EAA among GNM, SBM, SSH and SHM did not differ between hybrid catfish and Nile tilapia. In conclusion, there were only small differences in the nutritional properties between the selected feedstuffs in both fish species. Most EAA in the selected feedstuffs were equally well utilized by hybrid catfish and Nile tilapia.