三疣梭子蟹14-3-3基因的克隆及其在低盐和病原胁迫后的表达分析

本研究利用cDNA末端快速扩增技术(RACE)克隆了三疣梭子蟹(Portunus trituberculatus)14-3-3基因(Pt14-3-3)。Pt14-3-3基因cDNA序列全长为2510 bp,包含开放阅读框741 bp,编码由247个氨基酸组成的蛋白,分子量为27.98 kDa。序列比对结果显示,Pt14-3-3基因与中华绒螯蟹14-3-3基因同源性最高(100%)。系统进化树分析表明,Pt14-3-3氨基酸序列与中华绒螯蟹(Eriocheir sinensis)紧密聚为一支。组织表达分析显示,Pt14-3-3基因在肝胰腺、肌肉、鳃、心脏、眼柄、血淋巴中均有表达,其中,在肝胰腺中的表达量最高。低盐胁迫后,Pt14-3-3基因在鳃和肝胰腺中的表达量分别于48 h和12 h显著上调并达到最大值,分别为对照组的1.34倍(P<0.05)和7.54倍(P<0.05)。人工感染副溶血弧菌(Vibrio parahaemolyticus)和白斑综合征病毒(WSSV)后,Pt14-3-3基因在肝胰腺和血细胞中整体均呈显著上调表达,最高上调17.52倍(P<0.05)。本研究结果表明,Pt14-3-3基因可能在三疣梭子蟹低盐适应和免疫反应中发挥重要作用。     

口服特异性卵黄抗体对凡纳滨对虾抗WSSV感染的免疫保护效果

为探讨特异性卵黄抗体对凡纳滨对虾(Litopenaeus vannamei)抗白斑综合征病毒(white spot syndrome virus, WSSV)的免疫保护机制及效果,本研究以添加不同剂量WSSV卵黄抗体制剂(0、0.2%和0.5%)的饲料投喂凡纳滨对虾幼虾,免疫28 d后使用WSSV进行人工感染,测定感染对虾的肝胰腺免疫酶活力和免疫基因表达水平,以及感染后14 d内对虾的存活率。结果显示,WSSV感染3 d后,与未添加卵黄抗体制剂的对照组相比,0.2%免疫组对虾肝胰腺的超氧化物歧化酶(SOD)和酚氧化酶(PO)活力显著升高,酸性磷酸酶(ACP)和碱性磷酸酶(AKP)活力显著降低,热休克蛋白70基因(Hsp70)表达水平显著升高,凝集素基因(lectin)和β-1,3-葡聚糖结合蛋白–脂蛋白基因(β-GBP-HDL)表达水平显著降低;0.5%免疫组对虾肝胰腺的SOD活力显著升高,ACP和AKP活力显著降低,Hsp70基因表达水平显著升高,β-GBP-HDL基因表达水平显著降低。人工感染实验结果显示,WSSV感染14 d后,0.2%和0.5%免疫组对虾的存活率分别为48.89%和87.78%,均显著高于对照组(存活率为0),且0.5%免疫组对虾存活率显著高于0.2%免疫组。特异性卵黄抗体制剂能在一定程度上改变发病的进程,延迟对虾的发病和死亡时间,提高同期存活率。研究表明,口服特异性卵黄抗体制剂可以调节对虾肝胰腺免疫酶活力和免疫基因表达水平,显著提高凡纳滨对虾抗WSSV感染的能力。本研究为卵黄抗体抗WSSV感染机制的研究提供了参考,也为在生产上使用卵黄抗体防控WSSV感染提供了科学依据。     

白斑综合征病毒2014年中国毒株变异区的序列比较

为研究白斑综合征病毒(White spot syndrome virus,WSSV)在中国不同地区的分子流行病学变异特征,对2014年1-8月期间在病害暴发区采集到的48份PCR检测阳性样本,用ORF75、ORF94和ORF125引物扩增目的片段,连接转化已克隆目的片段,测序分析不同样本ORF75、ORF94和ORF125重复序列数目的差异.结果显示,不同地区毒株ORF75的重复单元数目有4、10、11、12、13不等,ORF94的重复单元数目有4和14,而ORF125的重复单元数目有0、3、5、6、7不等.结果表明,流行在中国大部分地区的WSSV存在一定程度变异,毒株间的变异在ORF75、ORF94和ORF125上比较明显.     

Intermediary metabolic response and gene transcription modulation on the Sub-Antarctic notothenioid Eleginops maclovinus (Valenciennes, 1930) injected with two strains of Piscirickettsia salmonis

Pathogen interactions with cultured fish populations are well studied, but their effects on native fishes have not been characterized. In Chile, the disease caused by bacterial species Piscirickettsia salmonis represents one of the main issues and is considered to be one of the important pathogens in the field of aquaculture. They have been found to infect native fish. Therefore, it is necessary to understand the impact of P. salmonis on native species of local commercial value, as well as the potential impact associated with the emergence of antibiotic-resistant strains of P. salmonis. Due to this purpose, the native fish Eleginops maclovinus was used in our study. Fish were randomly distributed in tanks and intraperitoneally inoculated with two strains of P. salmonis. No mortality was recorded during the experiment. Cortisol, glucose and total α-amino acid levels increased in fish injected with AUSTRAL-005 strain compared to sham-injected and LF-89-inoculated fish. Moreover, results showed an increase in the activity of carbohydrates and lipids metabolism in liver; and an increase in the carbohydrates, lipids and total α-amino acid metabolism in muscle after injection with AUSTRAL-005. Our results suggest that P. salmonis modulates the physiology of E. maclovinus and the physiological impact increase in the presence of the antibiotic-resistant strain AUSTRAL-005.     

Establishing bacterial infectivity models in striped Catfish Pangasianodon hypophthalmus (Sauvage) with Edwardsiella ictaluri

A bacterial infectivity challenge model of Edwardsiella ictaluri in striped catfish was developed. All experiments were conducted using a bacterial isolate of E. ictaluri that had been recovered during a natural outbreak of bacillary necrosis of Pangasianodon (BNP) in farmed striped catfish Pangasianodon hypophthalmus in Vietnam. Time of immersion in 10⁷ CFU.ml⁻¹ had a significant effect on mortality. The immersion bacterial dose of 10⁷ CFU/ml for 30 s resulted in a cumulative percentage mortality of 63%. Three to four days post-bacterial challenge, fish showed gross clinical signs of natural BNP and E. ictaluri was recovered and identified from these fish. Moreover, a cohabitation challenge was evaluated as an alternative challenge method, although the mortalities among the infected fish were lower at around 15%–40%. This study confirmed the horizontal transmission of E. ictaluri in striped catfish and elucidated that cohabitation challenge could be used in reproducing the disease under controlled conditions.     

Stability comparison of three candidate internal reference genes in Chinese giant salamander (Andrias davidianus)

The Chinese giant salamander (Andrias davidianus) as food and medicinal product has been an important aquaculture object in China. Study of gene function in the Chinese giant salamander requires accurate normalization though the use of appropriate reference genes. In this study, the expression levels of three candidate reference genes including β‐actin, GAPDH and cytb of different tissues, different developmental stages and different challenges in Chinese giant salamander were evaluated by qPCR. The stabilities of these three reference genes were analysed by geNorm, NormFinder and BestKeeper software. The results showed that the expression of GAPDH was more stable than that of β‐actin and cytb in four tissues and at two developmental stages of Chinese giant salamander. Compared with GAPDH and cytb, β‐actin was the most stable in spleen of Chinese giant salamander treated with LPS or GSIV. Therefore, the result showed that GAPDH was the suitable reference gene in different tissues and at different developmental stages of Chinese giant salamander. The β‐actin could be used as a reference gene in spleen of Chinese giant salamander challenged with LPS and GSIV. This study provides convincing information for the GAPDH and β‐actin as suitable reference gene in Chinese giant salamander of different tissues, different developmental stages and different challenges respectively.     

Outbreak of viral haemorrhagic septicaemia (VHS) in lumpfish (Cyclopterus lumpus) in Iceland caused by VHS virus genotype IV

A novel viral haemorrhagic septicaemia virus (VHSV) of genotype IV was isolated from wild lumpfish (Cyclopterus lumpus), brought to a land‐based farm in Iceland, to serve as broodfish. Two groups of lumpfish juveniles, kept in tanks in the same facility, got infected. The virus isolated was identified as VHSV by ELISA and real‐time RT‐PCR. Phylogenetic analysis, based on the glycoprotein (G) gene sequences, may indicate a novel subgroup of VHSV genotype IV. In controlled laboratory exposure studies with this new isolate, there was 3% survival in the I.P. injection challenged group while there was 90% survival in the immersion group. VHSV was not re‐isolated from fish challenged by immersion. In a cohabitation trial, lumpfish infected I.P. (shedders) were placed in tanks with naïve lumpfish as well as naïve Atlantic salmon (Salmo salar L.). 10% of the lumpfish shedders and 43%–50% of the cohabiting lumpfish survived after 4 weeks. 80%–92% of the Atlantic salmon survived, but no viral RNA was detected by real‐time RT‐PCR nor VHSV was isolated from Atlantic salmon. This is the first isolation of a notifiable virus in Iceland and the first report of VHSV of genotype IV in European waters.     

Development of piscirickettsiosis in Atlantic salmon (Salmo salar L.) smolts after intraperitoneal and cohabitant challenge using an EM90‐like isolate: A comparative study

Piscirickettsiosis, caused by the intracellular Gram‐negative bacteria Piscirickettsia salmonis, is at present the most devastating disease in the Chilean salmon industry. The aim of this study was to analyse disease development after challenge with a P. salmonis strain (EM90‐like) under a controlled environment by comparing intraperitoneal challenge with cohabitation challenge. The P. salmonis EM90‐like isolate was cultured in a liquid medium for the challenge of 400 Atlantic salmon (Salmo salar) smolts. Cumulative mortality was registered, necropsy was performed, and bacterial distribution in the tissues and histopathological changes were analysed. The results revealed a similar progression of the disease for the two different challenge models. Pathological and histopathological changes became more visible during the development of the clinical phase of the disease. Bacterial DNA was identified in all the analysed tissues indicating a systemic infection. Bacterial tropism to visceral organs was demonstrated by real‐time quantitative PCR and immunohistochemistry. Better knowledge of disease development during P. salmonis infection may contribute to further development of challenge models that mimic the field situation during piscirickettsiosis outbreaks. The models can be used to develop and test future preventive measures against the disease.     

2017年中国部分地区白斑综合征病毒高变异区的序列分析

白斑综合征病毒(WSSV)自暴发以来给全球范围内的对虾养殖产业带来了巨大的损失。为了解我国凡纳滨对虾(Litopenaeus vannamei)养殖区WSSV的流行变异情况,选取2017年中国部分地区的42个WSSV阳性样本,对ORF14/15、ORF23/24、ORF75、ORF94和ORF125共5个可变区进行PCR特异性扩增,分析其序列的缺失变异和重复单元(Ru)中单核苷酸多态性的变化。研究结果显示,在ORF14/15的扩增中共出现4种缺失片段;ORF23/24只出现11945bp的缺失片段;ORF75扩增中,总RUs数目为3、4、9,其中45bp的RUs在12、27、80位点发生多核苷酸多态性;ORF94的RUs数目为6,其各重复单元在48位发生单核苷酸多态性;ORF125的RUs数目为4、6、7不等,其各重复单元分别在20、27、50、53、61位发生碱基突变。研究结果表明,2017年样本中,WSSV在中国大部分地区均出现一定程度的缺失变异,其中部分可变区表现出缺失情况的稳定性,某些可变区的重复单元数目及SNP表现出地区的差异性以及不稳定性。