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.     

Impact of sublethal di‐n‐butyl phthalate on the aquaculture fish species Nile tilapia (Oreochromis niloticus): histopathology and oxidative stress assessment

Phthalates such as di‐n‐butyl phthalate (DBP) and their esters are widely used plasticizers, their ubiquitous presence in daily life, inevitably leads to their restricted use due to important environmental pollution and health impacts and endocrine disruption potential. The aim of this study was to examine the effects of a sublethal concentration of 10 mg L^?1 DBP on haematocrit (HCT) values, gills and liver histology, malondialdehyde (MDA, 2‐thiobarbituric acid‐TBA reactivity) and reduced glutathione (GSH) levels in gills and liver tissue as oxidative stress biomarkers in the aquaculture fish species Nile tilapia (Oreochromis niloticus) after 24 (DBP‐24) and 96 (DBP‐96) h exposure. No differences were found between per cent HCT values in the 24 h exposure groups (P > 0.05). Response of antioxidant defence systems in liver and gill tissues of the fish were dependent on exposure duration and changed to a higher extent during 96 h. MDA levels in liver tissue increased in DBP treated fish in comparison to the control fish. However, the differences between the exposure and control groups were not significant (P > 0.05). A statistically significant decrease (P > 0.05) was recorded in gill MDA levels in the DBP‐96 group when compared to the control and DBP‐24 groups. The liver GSH levels were unchanged in the DBP treated fish. However, GSH levels were significantly lower in the gill tissue of the DBP‐96 group. Exposure to DBP caused several degenerative changes in the histology of gill and liver tissue. Gills displayed hyperaemia, epithelial lifting, oedema, talengiectasia, epithelial hyperplasia and fusion of secondary lamellae, whereas in liver several circulatory anomalies (hyperaemia, blood congestion and sinosoid dilatation) and vacuolization of hepatocytes were observed. Histopathological results demonstrated that the gills were more affected than the liver perhaps due to their direct contact with DBP.     

Effects of supplementary feeds with different protein levels on growth and economic performances of Nile tilapia (Oreochromis niloticus) cultured in a rain-fed rice–fish ecosystem

This study investigated the effects of supplementary feeds with different crude protein (CP) levels on the growth and economic performances of sex-reversed Nile tilapia (Oreochromis niloticus) reared in a rain-fed rice–fish ecosystem for a period of 120 days. There were four treatments—namely, traditional feed (21% CP), formulated feed (26% CP), commercial feed (30% CP), and control (no feed was used)—with three replicates per treatment. Fish (30.04 g) were stocked in ditches at a rate of 0.5 fish m^?2 considering the total area and fed one of the tested feeds twice daily at the rate of 2%–5% of body weight. Water-quality parameters of the ditches were monitored fortnightly, and these parameters were found suitable for fish farming. Organic matter, total nitrogen, and available phosphorous in soil were increased significantly (P < 0.05) in the feed treatments after fish cultivation. Highly significant weight gain and SGR were observed with fish fed the commercial feed as compared with other treatments. The highest benefit-cost ratio (2.15) was found in the control, followed by formulated feed (1.90), but in the case of numerical net return, it was the opposite. However, commercial feed showed the lowest benefit-cost ratio (1.67) due to higher feed cost than other supplementary feeds. Therefore, this study suggests that fish fed with a formulated feed (26% CP) produced significantly higher net economic return in the rice–fish farming system.     

Single nucleotide polymorphisms in the growth hormone gene of Oreochromis niloticus and their association with growth performance

Polymorphisms in the growth hormone (GH) gene that is associated with the growth rate of farmed fish have been the target of many breeding programmes. The present study aimed to identify single nucleotide polymorphisms (SNPs) in GH gene regions to evaluate the association of SNP variations with the growth rate of two Nile tilapia: Oreochromis niloticus (Linnaeus, 1758) strains. The targeted regions were amplified, sequenced, aligned and screened for the presence of SNPs; thereafter, performance tests were used to check for the association between SNPs and weight. Allele and genotype frequencies were estimated for each SNP and genotype. Genotype blocks or sets of SNP genotypes and frequencies were also estimated. Association between SNPs and growth rate was statistically evaluated using a univariate linear mixed model that included both fixed and random effects. A total of 10 SNPs were identified, nine in the proximal promoter and one located in the 5′ UTR, forming 10 genotype blocks. In all weight recordings, five genotype blocks were significantly associated with the highest weights. Single nucleotide polymorphisms 6‐10 were also found to be significantly associated with growth (p‐value < .05). Genotypes with higher additive genetic values for weight were identified in the Chitralada strain, suggesting a possible impact of these additive effects of the GH SNP genotype on the growth rate of Nile tilapia. These findings may be used as part of marker‐assisted selection in tilapia breeding programmes.     

Evaluation of dietary chitosan effects on growth performance,immunity, body composition and histopathology of Nile tilapia (Oreochromis niloticus) as well as the resistance to Streptococcus agalactiae infection

The present investigation aimed to evaluate the effect of dietary chitosan supplementation on growth performance, body composition, immune response and histopathology of Nile tilapia, and also the in vitro antibacterial activity of chitosan against Streptococcus agalactiae (S. agalactiae). About 180 fish (average body weight 39.3 ± 0.3 g) were randomly divided into three groups according to chitosan supplementation: control group (basal diet without chitosan), Ch3 group (3 g chitosan/kg diet) and Ch5 group (5 g chitosan/kg diet). Growth performance parameters and body proximate composition were measured before infection but biochemical parameters and lysozyme and antibacterial activities before and after experimental infection. Results of the present investigation showed dietary chitosan (5 g chitosan/kg diet) significantly (p < .05) improved growth performance parameters, body composition (dry matter, crude protein, ether extract, ash, and carbohydrate) and serum biochemistry (total protein, albumin, globulin, with no effect on AST, ALT, urea and creatinine) before infection in Ch5 group than the control. After infection, liver enzymes (serum AST and ALT) were maintained lower in fish fed Ch3 or Ch5 than the control. Serum lysozyme and bactericidal activities significantly increased (p < .05) in chitosan groups before and after the challenge. The mortality rate was markedly reduced in the Ch3 group and prohibited in the Ch5 group after the experimental infection. In conclusion, feeding 3 or 5 g chitosan/kg diet increased the growth rate and improved FCR of Nile tilapia. In addition, it reduced mortality by its antibacterial and immunostimulant effects.     

尼罗罗非鱼精巢支持细胞分离培养体系建立及优化

本研究分离培养尼罗罗非鱼(Oreochromis niloticus)精巢支持细胞(Sertoli cells, SCs)，建立并优化尼罗罗非鱼精巢支持细胞分离培养体系。无菌条件下，取发育至第Ⅲ期的尼罗罗非鱼精巢，PBS清洗后，剪碎精巢组织，0.25%胰蛋白酶消化，用含10%犊牛血清(Newborn bovine serum, NBS)的L-15培养液终止消化，过滤、离心，获得细胞。差速贴壁法获得SCs后，在26℃、无CO2饱和湿度恒温培养箱中培养。分别采用含10% NBS的L-15、M199、F12培养液，含5%、10%、15% NBS的L-15培养液，含1%罗非鱼血清的L-15+10% NBS培养液培养尼罗罗非鱼SCs。各组每2 d取细胞计数，绘制SCs生长曲线；甲基绿染色，倒置显微镜下观察SCs的形态。尼罗罗非鱼SCs培养1~2 d，细胞贴壁；培养3~5 d，细胞完全贴壁并迅速增殖。单个SCs呈不规则多边形，其核位于胞质中央，呈卵圆形，胞质中可见吞噬颗粒和空泡，空泡聚集于支持细胞胞质的两极或散布于核的四周。SCs在L-15培养液中贴壁生长，在F12、M199培养基中较难贴壁。与F12、M199培养液相比，L-15培养液更有助于尼罗罗非鱼SCs生长增殖(P<0.01)。与添加5%和15% NBS相比，在L-15培养基中添加10% NBS更有助于尼罗罗非鱼SCs生长增殖(P<0.05)。与未添加尼罗罗非鱼血清相比，在10% NBS+L-15培养中添加1%尼罗罗非鱼血清，能显著促进SCs生长增殖(P<0.05)。研究表明，采用胰酶消化差速贴壁法获得尼罗罗非鱼精巢支持细胞，10% NBS+1%罗非鱼血清+L-15培养液培养，能显著促进尼罗罗非鱼精巢支持细胞生长增殖。     

First serological evidence of West Nile virus infection in wild birds in Northern Algeria

While the epidemiology of Flaviviruses has been extensively studied in most of the Mediterranean basin, little is known about the current situation in Algeria. In order to detect the circulation of West Nile (WNV) and Usutu viruses (USUV) in Kabylia, 165 sera were collected from two wild birds species, namely the long distance migrant Turdus philomelos (song thrush) (n = 92) and the resident Passer domesticus (house sparrow) (n = 73). A total of 154 sera were first analyzed by commercial competition ELISA. WNV and USUV micro-neutralization tests were performed on all c-ELISA positive sera and all samples with poor volume. Overall, 7.8 % (CI95 %: 3.5–11.9) were positive by c-ELISA. Positive results were detected in 12.5 % (CI95 %:5.6–19.4) of song thrushes and 1.5 % (CI95 %: 0.0–4.5) for sparrow.Micro-neutralization tests revealed an overall seroprevalence of 6.7 % for WNV (CI95 %: 2.9–10.3), Neutralizing antibodies were found in 8.7 % (CI95 %: 3.0–14.4) for song thrushes and in 4.1 % (CI95 %: 0.0–8.7) of sparrows. The current study demonstrates significant seroprevalence of WNV antibodies in wild birds in Algeria.     

5味中药醇提物对尼罗罗非鱼白细胞免疫活性的作用

将超微五倍子、白头翁、地榆、柯子、虎杖粉碎后过160目筛,按照料液比1∶10将药粉与60%的乙醇水溶液混合,再用超声波粉碎机超声1 h,用滤纸过滤收集滤液,置于多样品平行蒸发仪中浓缩后制得中药提取物。用吸取0.2 mL抗凝剂的无菌注射器自尼罗罗非鱼尾静脉采血0.5 mL,与L-15细胞培养基按体积比1∶9的比例混合成血浆,与中草药提取液单用、联用共孵育,利用氮蓝四唑还原法和流式细胞仪技术分别测定中草药提取液对尼罗罗非鱼外周血白细胞呼吸爆发活性和白细胞吞噬活性的影响。试验结果显示,五倍子、白头翁、地榆、柯子、虎杖单用均能显著提高白细胞氧呼吸爆发活性,呼吸爆发增加率分别为128.68%、123.96%、28.37%、44.44%和78.93%;试验组白细胞吞噬率分别较对照组增加44.04%、44.66%、95.00%、10.35%和64.93%。在两味中药双联用试验中,对白细胞氧呼吸爆发活性呈协同作用的组合为白头翁+地榆,呼吸爆发联用指数为1.28;对白细胞吞噬活性呈协同作用的组合为白头翁+虎杖、柯子+虎杖、五倍子+虎杖和白头翁+柯子,吞噬联用指数分别为11.45、6.20、3.03和1.06。     

Surveillance and Early Warning of West Nile Virus Lineage 2 Using Backyard Chickens and Correlation to Human Neuroinvasive Cases

In 2010, a West Nile virus (WNV) epidemic was reported in Central Macedonia, Northern Greece, with 197 neuroinvasive disease (WNND) cases in humans. The following 3 years, WNV spreads to new areas of Greece and human cases reoccurred during the transmission periods. After the initial outbreak, a WNV surveillance system using juvenile backyard chickens was established in Central Macedonia (after the 2011 outbreak) and Eastern Macedonia‐Thrace (after the 2012 outbreak). Sera were screened for the presence of antibodies against WNV using cELISA and serum neutralization test, to monitor the spread of WNV and to assess the correlation between the WNV point seroprevalence in chickens and the incidence rates of human WNND cases in the aforementioned areas. WNV seroprevalence in chickens was 10.4% (95% CI: 7–15) in Central Macedonia (2011) and 18.1% (95% CI: 14–23) in Eastern Macedonia‐Thrace (2012). Seroprevalence in chickens and incidence rates of human WNND cases in Eastern Macedonia‐Thrace were strongly positively correlated (ρ = 0.98, P = 0.005) at the regional unit level, with the incidence of WNND in humans increasing with increasing WNV point seroprevalence in chickens. In Central Macedonia, the correlation was weaker (ρ = 0.68, P = 0.20), apparently due to small number of reported human WNND cases. Another study was also conducted using juvenile backyard chickens in Central Macedonia, aiming to detect early WNV enzootic circulation, before the onset of human cases during 2011 and 2013. The first seroconverted chickens were detected about 1.5 months before the laboratory diagnosis of any human WNND cases in Central Macedonia, for both years. WNV surveillance, using juvenile backyard chickens, was reliable for the identification of areas with WNV enzootic and silent transmission, and for early warning. Timely diffusion of information to public health authorities facilitated the successful implementation of preparedness plans to protect public health.