Evidence of potential vertical transmission of tilapia lake virus

Tilapia lake virus disease (TiLVD) is an emerging viral disease in tilapia with worldwide distribution. Although the horizontal transmission of TiLV has been demonstrated through the cohabitation of infected fish with susceptible fish, no direct experiment showed the potential of vertical transmission from broodstock to progeny. In this study, natural outbreaks of TiLV in broodstock and fry in two tilapia hatcheries were confirmed. The TiLV genomic RNA was detected in liver and reproductive organs of infected broodstock, while infective virus was isolated in susceptible cell line. In situ hybridization assay confirmed the presence of TiLV in the ovary and testis of naturally infected fish and experimentally challenged fish. Moreover, early detection of TiLV in 2‐day‐old fry and the presence of TiLV genomic RNA and viable virus in the testis and ovary suggested the possible transfer of this virus from infected broodstock to progenies. As infective virus was present in gonads and fry in natural outbreak and experimental fish, the importance of biosecurity and prevention of the virus to establish in the hatchery should be emphasized. Hence, the development of TiLV‐free broodstock and the maintenance of high biosecurity standards in the hatcheries are essential for any attempt of virus eradication.     

Tilapia Lake Virus was not detected in non-tilapine species within tilapia polyculture systems of Bangladesh

Sixteen countries, including Bangladesh, have reported the presence of tilapia lake virus (TiLV), an emerging tilapia pathogen. Fish polyculture is a common farming practice in Bangladesh. Some unusual mortalities reported in species co-cultivated with TiLV-infected tilapia led us to investigate whether any of the co-cultivated species would also test positive for TiLV and whether they were susceptible to TiLV infection under controlled laboratory experiments. Using 183 samples obtained from 15 farms in six districts across Bangladesh, we determined that 20% of the farms tested positive for TiLV in tilapia, while 15 co-cultivated fish species and seven other invertebrates (e.g. insects and crustaceans) considered potential carriers all tested negative. Of the six representative fish species experimentally infected with TiLV, only Nile tilapia showed the typical clinical signs of the disease, with 70% mortality within 12 days. By contrast, four carp species and one catfish species challenged with TiLV showed no signs of TiLV infection. Challenged tilapia were confirmed as TiLV-positive by RT-qPCR, while challenged carp and walking catfish all tested negative. Overall, our field and laboratory findings indicate that species used in polycultures are not susceptible to TiLV. Although current evidence suggests that TiLV is likely host-specific to tilapia, targeted surveillance for TiLV in other fish species in polyculture systems should continue, in order to prepare for a possible future scenario where TiLV mutates and/or adapts to new host(s).     

Blood and liver biopsy for the non‐destructive screening of tilapia lake virus

Detection of tilapia lake virus (TiLV) in tilapines is mainly from visceral organs of killed fish. However, lethal sampling might not be viable to broodstock and economically important ornamental cichlids. To contribute towards screening of the virus in asymptomatic infected fish, a subclinically infected population of Nile tilapia adults obtained from a local farm was preliminarily tested to compare different non‐lethal sampling methods, for example liver biopsy, gill biopsy, fin clip, mucus, faeces and blood for detection of TiLV. Only liver and blood samples gave positive results by PCR. Since blood sampling is relatively simpler, it was further used for five naturally co‐cultured juvenile fish species from above‐mentioned farm including 40 red tilapia broodstock and 20 Nile tilapia adults from two other different farms. The results showed that from the tested fish, 4 of 5 Nile tilapia, 2 of 5 hybrid red tilapia and 3 of 5 giant gourami blood samples tested positive, while 38 of 40 blood samples of red tilapia tested positive for TiLV in second‐step PCR. Sequencing representative PCR amplicons of positive samples confirmed sequence identity to TiLV. In conclusion, both blood and liver biopsy are practical non‐destructive sampling platforms for TiLV screening in cichlids with blood being more convenient, especially for tilapia broodstock.     

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.     

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.     

冻罗非鱼片的复配冰衣护色效果研究

针对冷冻罗非鱼片在贮运过程中色泽品质退化的问题开展试验,寻求一种防止冻鱼片贮藏期间变色的新方法。采用响应面试验设计,在发色罗非鱼片和不发色罗非鱼片的冰衣中加入不同添加剂,测定鱼片中高铁肌红蛋白含量,比较组氨酸、抗坏血酸钠、异抗坏血酸钠、山梨酸钾及烟酰胺对鱼片的冰衣护色效果。结果显示,山梨酸钾与异抗坏血酸钠为较优的添加剂组合。以高铁肌红蛋白含量拟合公式中变色系数为试验指标,得到未发色鱼片护色的优化配方为:异抗坏血酸钠0.32‰,山梨酸钾0.68‰,此时变色系数为13.79;发色罗非鱼片的最适护色组合为:异抗坏血酸钠0.32‰,山梨酸钾0.68‰,变色系数为10.42。研究表明,冰衣护色法对防止冷冻罗非鱼片的褪色及褐变是有效果的,可作为罗非鱼片出口加工和贮运的实用技术,能有效减缓冷冻罗非鱼片的褐变速度,延长其鲜红色或原色的保持时间。     

速冻温度对罗非鱼片品质的影响

通过测定不同冷冻方式冻罗非鱼片的理化指标,试图找到最优处理方式,并为冻罗非鱼片工厂化生产提供参考。通过设置双螺旋冷冻机不同的冷冻温度(-35℃、-38℃、-41℃、)冻结新鲜罗非鱼片,将冻结后的鱼片置于-18℃的冷库贮藏,每隔7 d对鱼片样品的pH、水分活度(Aw)、质构参数、挥发性盐基氮(TVB-N)等物理及化学指标进行检测并分析结果。数据表明,冷冻温度对鱼片色差值、失水率、硬度有较大影响,且冷冻温度越低,鱼片品质越好,对咀嚼性、弹性、TVB-N影响不大,对pH则无明显影响。综上,在现有加工工艺的冷冻温度波动范围内,选择较低冷冻温度,提高冻结速率可延缓冻罗非鱼片品质劣变速度,故建议企业加工时,将冷冻温度控制在-40℃,冷冻时间控制在40 min以尽可能保持鱼片品质。     

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.     

Nutrient composition and sensory evaluation of fillets from wild-type Nile tilapia (Oreochromis niloticus, Linnaeus) and a red hybrid (Florida red tilapia × red O. niloticus)

A comparison of composition and sensory evaluation were performed on fillets of Oreochromis niloticus (wild type) and red hybrid tilapia (RHT) (Florida red tilapia ♂× red O. niloticus♀). Fifty male tilapia, 25 O. niloticus (initial weight 159.3 g) and 25 red hybrid (initial weight 132.4 g), were placed in each of three 2.0 m³ volume tanks. The fish were fed a commercial feed containing 35.9% crude protein during the 120‐day experimental period and then killed in cold water (3°C). All the fish were filleted. Twelve fillets from each treatment were used for proximate analyses, five fillets from each treatment were used for fatty acids analyses and the remaining fillets were used for sensory evaluation. The compositional analyses showed similar moisture, true protein and ash content in both genetic groups, but a lower crude fat content was measured in the red hybrid fillets (0.70%) compared with that of O. niloticus fillets (0.97%). No differences between O. niloticus and the red hybrid were observed in their fillet profile of fatty acids. In the sensory evaluation test, a difference in fillet flavour between both genetic groups was perceived. Of the 112 consumers in the preference test, 81.2% perceived a difference in fillet flavour between the two tilapias, with a general preference for the red hybrid over the wild‐type O. niloticus. The benefits of cultivating a RHT with a low lipid content are discussed.     

Tilapia lake virus: The story so far

Tilapia lake virus (TiLV) is a highly contagious pathogen that has detrimental effects on tilapia farming. This virus was discovered in 2014 and has received tremendous global attention from the aquaculture sector due to its association with high fish mortalities and its strong economic impact on the tilapia aquaculture industry. Currently, TiLV has been reported in 16 countries, and this number is continuing to rise due to improved diagnostic assays and surveillance activities around the world. In this review, we summarize the up-to-date knowledge of TiLV with regard to TiLV host species, the clinical signs of a TiLV infection, the affected tissues, pathogenesis and potential disease risk factors. We also describe the reported information concerning the virus itself: its morphology, genetic make-up and transmission pathways. We review the current methods for virus detection and potential control measures. We close the review of the TiLV story so far, by offering a commentary on the major TiLV research gaps, why these are delaying future TiLV research and why the TiLV field needs to come together and proceed as a more collaborative scientific community if there is any hope limiting the impact of this serious virus.     

利用热泵装置干燥罗非鱼片试验研究

为了充分利用热泵干燥装置的优势,以罗非鱼片为试验材料,研究了干燥温度、风速、物料厚度及形状等因素对罗非鱼片干燥特性的影响,获得了不同风速、不同厚度、不同温度条件下鱼片干燥曲线。结果表明,热泵干燥过程中各因素的合理匹配能有效提高罗非鱼片的干燥速度,强化热泵干燥的良好效果。不同厚度的鱼片只有找到匹配的温度和风速条件,才能使干燥阶段达到传热传质的平衡状态,在降低临界湿含量的同时维持后期的干燥速度,避免局部干缩而影响物料品质和外观。越薄的鱼片越易找到匹配的条件,厚3 mm鱼片更容易找到匹配的干燥条件。     

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1044 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∶51200,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)和免疫组化(IHC)对人工感染TiLV的吉富罗非鱼不同组织进行检测,结果显示,NP蛋白在肝脏、脑、体肾和头肾等组织中均有表达,以肝脏组织中表达量最高。为了解吉富罗非鱼对TiLV的免疫反应,通过实时荧光定量PCR(qRT-PCR)测定免疫因子TNF-α和TGF-β在主要免疫器官脾脏和头肾组织中的表达。研究表明,在感染早期(感染后12~24 h),病毒可显著抑制TNF-α和TGF-β在脾脏和头肾中的表达,可能通过抑制宿主这些免疫因子来促进病毒自身早期的复制。本研究将为进一步解读TiLV的致病机理及其高效防控提供理论基础。     

Virucidal effects of common disinfectants against tilapia lake virus

Tilapia lake virus (TiLV) is an emerging virus associated with high fish mortality and economic losses. This study investigates the virucidal effects of the following disinfectants (active ingredients) on TiLV: 2.5 ppm iodine, 10 ppm sodium hypochlorite (NaOCl), 300 ppm hydrogen peroxide (H₂O₂), 80 ppm formalin and 5,000 ppm (0.5%) Virkon^®. Factors that affect the disinfectants’ efficacy, including temperature, contact time and soiling (organic matter) interference, were examined under conditions mimicking natural aquaculture practices. TiLV inactivation of higher than 5 log₁₀ TCID₅₀ ml^?1 was achieved after 10 min and at 28°C for all disinfectants except formalin; similar inactivation levels were reached by NaOCl and Virkon^® at 10 min and 4°C. Extended exposure to formalin from 10 to 60 min at 28°C rendered more than 5 log₁₀ inactivation. Increasing synthetic organic matter in the water to mimic soiling interference reduced the efficacy of NaOCl, iodine and H₂O₂ when tested at 10 min and 28°C; however, Virkon^® still achieved more than 5 log₁₀ inactivation. This study demonstrates that most common disinfectants effectively reduced viral loads to minimum levels. To limit the spread of TiLV in aquaculture farms and related facilities, the appropriate use of such disinfectants should therefore be promoted and implemented.     

Vertical transmission and concurrent infection of multiple bacterial pathogens in naturally infected red tilapia (Oreochromis spp.)

Horizontal transmission of various bacterial pathogens in tilapia is well described, but there is scant information regarding their vertical transmission. This study aimed to determine the possibility of vertical transmission of two commonly reported bacterial pathogens (Francisella noatunensis subsp. orientalis and Shewanella putrefaciens) in natural stocks of red tilapia (Oreochromis spp.). Vertical transmission of these pathogens via gametes was evaluated using in vitro fertilization from 10 different families and analysing for the presence of bacteria in milt, unfertilized eggs, fertilized eggs and offspring at various ages (1‐day‐old larvae, 10‐day‐old fry and 30‐day‐old fingerlings), as well as water samples using colorimetric LAMP assay. The study revealed that both F. n. orientalis (6/10) and S. putrefaciens (4/10) was transmitted vertically to the fertilized eggs. Analysis of the water samples from different water sources (brood stock tanks, hatching chamber and larval rearing tanks) showed that both the pathogens were present in water samples with highest prevalence for F. n. orientalis followed by S. putrefaciens. Analyses for the presence of two pathogens in various organs (gonads, gill, liver, spleen, kidney and brain) of the healthy tilapia broodstock without any clinical symptoms of disease demonstrated they were carriers of S. putrefaciens and F. n. orientalis. This is the first documented evidence that vertical transmission via the broodstock of tilapia may also play an important role in transmitting these problematic pathogens to their progeny and underlines the necessity to modify the current disease management strategies in tilapia aquaculture.     

一氧化碳发色处理对罗非鱼鱼片暗色肉贮藏过程色泽影响分析

以罗非鱼（Orochromis niloticus♀×0．aureus♂）为研究对象,分析了一氧化碳（CO）发色处理对鱼片背部暗色肉贮藏过程中色泽的影响。结果表明,CO发色处理可以明显提高鱼片的红绿色值（a^＊）,并影响贮藏过程中色泽变化,特别是表征鱼片色泽的a^＊。冷藏初期发色鱼片a^＊略有上升,而后缓慢下降,但未发色鱼片则始终呈缓慢下降的趋势,冻藏期间3种处理鱼片a^＊均缓慢下降;发色鱼片冷藏期间保持鲜红色泽达13d,冻藏达半年。其他2项色泽指标亮度（L^＊）和黄蓝色值（b^＊）贮藏过程中变化趋势基本一致。贮藏过程中a^＊的变化情况与肌肉组织对CO的结合程度及稳定性密切相关,一般经CO发色处理的鱼片贮藏1～2d后鱼片的a^＊会进一步提高,这在冷藏过程中表现尤为明显。CO发色处理对鱼肉中的色素成分肌红蛋白（Mb）起到一定的保护作用,在某种程度上抑制或减缓了Mb氧化反应的发生,阻止高铁肌红蛋白（met—Mb）的生成。     

Experimental early pathogenesis of Streptococcus agalactiae infection in red tilapia Oreochromis spp.

Streptococcus agalactiae causes a severe systemic disease in fish, and the routes of entry are still ill‐defined. To address this issue, two groups of 33 red tilapia Oreochromis spp. each of 10 g were orally infected with S. agalactiae (n = 30), and by immersion (n = 30), six individuals were control‐uninfected fish. Three tilapias were killed at each time point from 30 min to 96 h post‐inoculation (pi); controls were killed at 96 h. Samples from most tissues were examined by haematoxylin–eosin (H&E), indirect immunoperoxidase (IPI) and periodic acid‐Schiff; only intestine from fish infected by gavage was evaluated by transmission electron microscopy. The results of both experiments suggest that the main entry site of S. agalactiae in tilapia is the gastrointestinal epithelium; mucus seems to play an important defensive role, and environmental conditions may be an important predisposing factor for the infection.