采用假交替单胞菌(Pseudoalteromonas sp.) KL-3 2010和微小杆菌(Exiguobacterium sp.)KL-C2 2014作为益生菌,进行凡纳滨对虾(Litopenaeus vannamei)投喂实验,研究上述菌株对带毒对虾的生长与存活的影响.假交替单胞菌KL-3 2010对致急性肝胰腺坏死副溶血弧菌(Vibrio parahemolyticus) (VPAHPND 20130629002S01)有拮抗作用和胞外蛋白酶活性,微小杆菌KL-C2 2014有胞外蛋白酶活性.待试对虾经检测为白斑综合征病毒(WSSV)、致急性肝胰腺坏死病副溶血弧菌(VPAHPND)和虾肝肠胞虫(EHP)弱阳性.经过为期60d的养殖实验,结果显示,与投喂普通颗粒饲料的对照组相比,投喂假交替单胞菌KL-3 2010的对虾存活率提高了213％±43％ (P＜0.01);投喂微小杆菌KL-C2 2014的对虾平均生长率提高了105.5％±28.1％ (P＜0.05);交替投喂2株菌的对虾存活率提高了184％±52％ (P＜0.05),平均生长率提高了70.6％±32.8％.肠道可培养优势菌研究表明,2株益生菌的投喂显著影响了对虾肠道优势菌群的种类.本研究为带毒虾苗的养殖提供一种有效的病害防控和促生长的手段.

实验从山东德州市一罗非鱼(Oreochromis niloticus)养殖场循环水养殖系统的脱氮池中分离到一株具有高效脱氮特性的菌株(编号DZYC02),分别以葡萄糖、蔗糖、可溶性淀粉、丁二酸钠、乙酸钠、柠檬酸钠为碳源,研究了碳源种类对菌株DZYC02脱氨氮效果的影响;同时以蔗糖为碳源、NH4 Cl为氮源,研究了不同碳氮比、初始pH及盐度对该菌株脱氮效果的影响.结果显示:菌株DZYC02在以柠檬酸钠为碳源、C:N≥15、pH 5～7、盐度0～15的条件下具有良好的脱氮效果,24 h内对浓度为20 mg/L的NH4+的去除率达100％,48 h内对浓度为20 mg/L的NO2-去除率高达100％;将该菌采用浸泡方式感染斑马鱼(Danio rerio)进行生物安全试验,结果显示菌株DZYC02对斑马鱼表现出较好的安全特性.分别用Biolog细菌鉴定方法和16S rDNA序列分析比对法对该菌进行鉴定,结果显示菌株DZYC02为一株肺炎克雷伯氏菌(Klebsiella pneumoniae).

本研究选择1株地衣芽孢杆菌(Bacillus licheniformis) BL-9、1株枯草芽孢杆菌(Bacillus subtilis) BS-12、1株金丽假交替单胞菌(Pseudoalteromonas flavipulchra) CDM8,以1∶1∶1将其组成复合益生菌,各益生菌水体添加浓度为107 CFU/ml,进行为期30d的凡纳滨对虾(Litopenaeus vannamei)养殖实验.实验分为暂养期(7 d)、益生菌处理期(15 d)、副溶血弧菌(Vibrio parahaemolyticus)攻毒期(10 d).结果 显示,养殖水体中添加复合益生菌能显著增加水体和对虾肠道可培养细菌总数(P＜0.05),攻毒实验结束时,实验组对虾累积存活率为(73.33±6.83)％,显著高于阳性对照组(25.33±15.43)％.对虾抗病基因热激蛋白70 (Heat shock proteins 70,Hsp70)、β-1,3-葡聚糖结合蛋白-脂蛋白(Beta-1,3-glucan-binding protein-lipoprotein,βGBP-HDL)、脂多糖-β-1,3-葡聚糖结合蛋白(Lipopolysaccharide-β-1,3-glucan binding protein,LGBP)、抗菌肽Crustin在益生菌处理阶段均出现不同程度的上调,在攻毒阶段虽呈现各自不同的表达情况,但所有基因都经历了更大幅度上调.研究表明,水体中添加芽孢杆菌和假交替单胞菌组成的复合益生菌可提高凡纳滨对虾抗副溶血弧菌感染能力,对虾抗病力的提高可能与益生菌增加对虾肠道可培养细菌数量、抗病相关基因表达水平及过氧化氢酶(CAT)活性有关.

白斑综合征病毒广泛分布于中国、东南亚、南亚、南美洲、北美洲的许多国家,并且具有较高的致病性.为探明中国不同地区白斑综合征病毒毒株的变异情况,本研究选取2017年2—6月在河北、浙江、山东、上海、福建和广东共6个省市的白斑综合征病毒病害暴发区的44份患病样本,提取其DNA,进行wsv-pol、wsv313、wsv150的PCR扩增,筛选阳性克隆并进行测序分析.分析结果显示,在2017年wsv-pol的12个阳性样本中,均在第164位出现单个甘氨酸缺失,在个别样品中也存在氨基酸的替换现象;在wsv313的PCR扩增中有9个阳性样本在第280位插入1个脯氨酸,2个样本在第64位天冬氨酸突变为甘氨酸;wsv150的阳性样本均在第587位插入碱基T.氨基酸比对结果显示,8个样本在第200位之后出现106个大片段氨基酸的缺失,同时在第196、197、198、199位发生氨基酸的替换.氨基酸比对结果表明,wsv-pol、wsv313均未出现较大程度的变异,而wsv150的氨基酸序列出现了较大程度的变异,但未表现出地区差异性.本研究将为白斑综合征病毒病原学的研究提供依据.

本研究通过对一对虾养殖场定期采样,采用分子生物学鉴定方法,分别对对虾体内及水环境中弧菌(Vibrio sp.)、虾肝肠胞虫(Enterocytozoon hepatopenaei,EHP)及主要病毒性病原进行了跟踪检测,同时,检测了水环境中的氨氮及亚硝基氮含量的变化趋势.结果 显示,该对虾养殖场中存在的主要病原为多种致病弧菌和EHP,未检测出对虾白斑综合征病毒(wssv)、传染性肌肉坏死病毒(IMNV)、偷死野田村病毒(CMNV)及传染性皮下和造血组织坏死病毒(IHHNV);该养殖场中弧菌检出种类达到16种,其中,主要的弧菌种类有副溶血弧菌(Vibrio parahaemolyticus)、溶藻弧菌(V.alginolyticus)、欧文氏弧菌(V.owensii)、创伤弧菌(V.vulnificus)、哈维氏弧菌(V.harveyi),且存在导致急性肝胰腺坏死病的副溶血弧菌(VPAHPND);养殖中期大棚养殖池水体氨氮及亚硝氮浓度分别达到(3.54-2.0)、(8.2士0.7) mg/L,显著高于室外养殖池水体氨氮及亚硝基氮浓度(P＜0.05).养殖期跟踪的7个虾池中,出现严重对虾病害的大棚养殖池比例达到100％,而室外养殖池仅出现轻度对虾病害,发病池比例为25％.根据研究结果推测,造成大棚养殖池对虾发病死亡的主要原因为养殖对虾感染弧菌及虾肝肠胞虫,同时,养殖密度大、水体氨氮及亚硝基氮浓度过高等在对虾病害发生中起到了协同作用.本研究结果可为当前对虾养殖病害防控技术提供理论支持和科学依据.

为探索应用益生菌的凡纳滨对虾零换水育苗技术,选择具有不同生态学功能的四株益生菌进行配伍形成两个益生菌组合PBE和PBN,并应用于凡纳滨对虾无节V期幼体（N5）至仔虾第五天（PL5）的育苗实验。每个实验组包含6个平行,同时设置对照组。整个实验过程不换水,PBE和PBN组的水体中连续施用益生菌组合。当幼体发育至蚤状幼体Ⅲ期（ZⅢ）、糠虾幼体Ⅲ期（MⅢ）和PL5时,检测幼体变态率、幼体体内和水体中可培养细菌总数（total culturable bacterial count,TBC）和假定弧菌总数（total presumptive Vibriobacteria,TPVB）;在实验结束时统计各实验组幼体成活率,并用Illumina测序PL5幼体体内的微生物群落。结果显示:在整个育苗周期内,未发生疫病;与对照组相比,益生菌组合显著降低了（P ＜0. 05） TPVB在幼体体内和育苗水体中的相对含量,并显著提高了（P ＜0. 05） ZⅢ幼体和仔虾PL5的变态率和成活率（各增加15%）。各实验组的PL5体内核心微生物群的多样性和结构并不存在显著性差异（P＞ 0. 05）,红杆菌科是微生物群落中最具优势的菌群,在各实验组中的相对丰度为40%～86%。研究表明,零换水条件下,益生菌组合能够有效抑制弧菌在育苗水体和凡纳滨对虾幼体体内的过度增殖,提高幼体的成活率,实现育苗的健康生产。

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11 945 bp的缺失片段;ORF75扩增中,总RUs数目为3、4、9,其中45 bp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.

对虾白斑综合征病毒(WSSV)开放阅读框wsv112编码脱氧尿苷焦磷酸酶(dUTP pyrophosphatase,dUTPase).为研究wsvll2与宿主的互作关系,本研究采用酵母双杂交Gal4系统从凡纳滨对虾(Litopenaeus vannamei)肠道cDNA文库中筛选与wsv112互作的候选蛋白.以WSSV为模板,构建pGBKT7-112诱饵载体,转化到Y2H Gold酵母菌感受态细胞中,转化菌液涂布到不同缺陷型培养基上,检测诱饵载体的自激性和毒性.将凡纳滨对虾肠道cDNA文库与诱饵菌株pGBKT7-112接合,通过筛选力度不同的缺陷型培养基、颜色反应、PCR、测序鉴定等步骤筛选阳性克隆,将阳性菌株提取质粒,再经过回复杂交实验验证筛选出的阳性质粒与诱饵载体的作用.研究表明,诱饵载体pGBKT7-112无自激性和毒性,可用于酵母双杂交实验;经初筛和回复杂交实验最终得到2个阳性质粒,经过NCBI数据库对比,其编码的蛋白分别与日本囊对虾(Penaeus japonicus)C型凝集素(AGW27416.1)和克氏原螯虾(Procambarus clarkii) 40S核糖体蛋白S20小亚基蛋白(ALE99171.1)具有37％和98％同源性.本研究为wsv112的调控机制提供新的线索.

自健康凡纳滨对虾(Litopenaeus vannamei)分离到枯草芽孢杆菌(Bacillus subtilis)、地衣芽孢杆菌(B.licheniformis)和短小芽孢杆菌(B.pumilus),将上述芽孢杆菌以单一和3株复合的方式包裹在基础饲料表面,制成益生菌饲料;每日投喂对虾,3周后进行白斑综合征病毒(WSSV)人工感染.统计实验组和对照组的累积死亡率,测定对虾鳃组织内WSSV拷贝数,分析对虾肠道组织含半胱氨酸的天冬氨酸蛋白酶基因(Caspase)和硫氧还原蛋白基因(Trx)的相对表达量.结果 显示,感染实验结束时,A组(枯草芽孢杆菌)、B组(地衣芽孢杆菌)、C组(短小芽孢杆菌)和D组(枯草芽孢杆菌+地衣芽孢杆菌+短小芽孢杆菌复合益生菌)的对虾累积死亡率分别为(73.3±7.0)％、(63.3±5.5)％、(75.0±7.9)％和(50.0±5.3)％,显著低于对照组(PBS组)(100％);在整个感染阶段,各实验组的病毒拷贝数呈先上升后下降的趋势,但对照组呈现一直上升趋势,且显著高于实验组.抗病基因表达结果显示,WSSV感染后,各组对虾肠道Caspase相对表达量随感染时间的延长呈先上调再下调的趋势,且在18 h各组对虾肠道Caspase表达量达到最大值;益生菌摄取和WSSV感染都能刺激Trx的表达,益生菌的刺激相对平缓,且各实验组对虾肠道Trx相对表达量在WSSV感染后的18 h时陡升到最大值,极显著高于对照组,且以D组的激活能力最强.研究证实,枯草芽孢杆菌、地衣芽孢杆菌和短小芽孢杆菌均可提高对虾抗WSSV感染能力,复合芽孢杆菌抗病毒能力最突出.对虾抗病力的提高可能与芽孢杆菌减缓了病毒在靶组织的增殖速率、提高了Caspase和Trx基因表达水平相关.

In recent years,the fast development of aquaculture has been followed with the increased diseases such as the white spot disease to cause a great loss for the global aquaculture industry.To understand the variations in WSSV ORF14/15 and ORF23/24 and the VNTR in different regions of China in 2015,we investigated 57 samples WSSV-positive on PCR,collected in a disease outbreak area between April and October 2015.The tested samples were from Shandong,Guangdong,Hainan,Zhejiang,Tianjin and Jiangsu.Using specific primers,WSSV-positive samples were genotyped and the amplified fragments were ligated to a T-vector and transformed in Top10 cells.The positive clones were selected and sequenced.After sequencing,the fragments deleted from ORF14/15 and ORF23/24 in different samples were compared with the sequences of Th-96-Ⅱ and TW respectively,and three WSSV VNTR regions in ORF75,ORF94,and ORF125 from different samples were analyzed with the DNAMAN software.The results showed that 35 samples had the products of ORF14/15 amplification,and 14 sample had the products of ORF23/24 amplification.The lengths of the amplified fragments of ORF 14/15 were 1270 bp,1892 bp and 2075 bp,corresponding to deletions of 6530 bp,5908 bp and 5725 bp,respectively,compared with Th-96-Ⅱ.One type of ORF23/24 was confirmed,and the amplified fragment was 1140 bp,corresponding to a deletion of 12070 bp compared with the TW isolate.Ten fragments were amplified from ORF75,including 1,2,3 repeat units with 45 bp,and 1 with 102 bp.Four different ORF94 VNTRs,including 4,5,10,or 12 repeat units,were identified.The ORF 125 VNTR including 3,5 or 6 repeat units was identified.The results showed that WSSV exhibited a significant difference in some open reading frames,but a significant stability in others.

[Objective] To optimize the particle size and contained windrow conditions of soybean meal in solid fermentation laboratory trail with Bacillus licheniformis.[Method] Based on establishing the effective counting method of live bacteria in soybean,the effects of different blending methods on the bacteria count in fermented soybean meal were analyzed and the effects of different fermentation vessel,soybean meal's particle size and windrow thickness on the bacteria concentration of fermented soybean meal were studied.[Result] After fermented soybean meal was oscillated 1 min in PBS,the bacteria were released from the soybean meal for the follow-up counting.Taking 4-layer newspaper-closed 50 mL centrifuge tube as fermentation vessel,when soybean meal particles through an 80 mesh sieve and a windrow with 1.5 cm thickness were used,the best fermentation effect of laboratory trail was obtained and bacteria concentration reached 1.65 × 1010 ～ 1.76 × 1010 CFU/g.[Conclusion] This study could provide technical support for the laboratory trail method of fermented soybean meal.

[目的]了解wsv006蛋白结构变异情况,并进一步为其蛋白功能研究提供参考资料.[方法]以2016和2017年在全国18个地区采集的WSSV阳性样品为模板,采用PCR方法,用特异性引物扩增wsv006基因,经连接转化后进行测序,并对测序结果进行分析.[结果]在2016年的47份样本中有9个样本,包括河北黄骅、山东日照、河北沧州和河北唐山4个地区的样本中出现了wsv006目的条带,检出率为19％.在2017年的39份WSSV阳性样本中有16个样本,包括福建霞美镇、河北黄骅、广东湛江、山东滨州、山东东营和上海6个地区的样本中出现了wsv006的目的条带,检出率为41％.氨基酸序列比对结果显示,扩增的wsv006编码氨基酸变异包括氨基酸替换、插入、缺失和提前终止翻译,其中插入的主要是脯氨酸(P)和苏氨酸(T),插入位点在第151～166位,处于PT的交叉重复区域;终止翻译是由于核苷酸第541位插入了GAGG,在转录过程中形成了终止密码子.[结论]wsv006氨基酸序列存在明显变异.

Acute hepatopancreatic necrosis disease (AHPND) is an emerging shrimp disease causing great losses for the shrimp culture industry worldwide since 2010. In the present study, a bacterial strain 20160303005-1 was isolated from the hepatopancreas tissue of Litopenaeus vannamei with early mortality syndrome (EMS), and was identified as Vibrio parahaemolyticus based on its physiological and biochemical characteristics; and the analysis of both 16S rRNA and groEL gene sequence. The serotype of the bacterium is O1:KUT (K untypeable). It revealed positive amplification of the genes pirAVP and pirBVP which is related to cause AHPND in a virulence plasmid harboring this strain. However, the isolate examined showed negative amplification results for the virulent clinical V. parahaemolyticus strain markers—thermostable direct hemolysin gene tdh and TDH-related hemolysin gene trh. The immersion challenge test with L. vannamei was also employed to study pathogenicity and histopathology. The results showed that the isolate was highly virulent, with a median lethal dose (LD50) value of 7.96×103 CFU/ml. The empty gut in shrimp was observed at 6 h post-challenged. The hepatopancreas appeared pale and atrophic at 9 h. More than half of the shrimps died at 12 h, and up to 100％ died at 24 h. Subsequent histological analyses showed that the hepatopancreas tubules collapsed with massive sloughing of hepatopancreas epithelial cells, which was the typical pathological characteristics of AHPND. Among 21 antibiotics tested, the isolate was resistant to amoxicillin, cefalotin, ticarcillin, cefuroxime, and cotrimoxazol; however, it was sensitive to gentamicin, ciprofloxacin, and other 14 antibiotics tested. These results provide basic data for epidemiology and drug control research on V. parahaemolyticus in aquaculture.

为探究中国白斑综合征病毒(white spot syndrome virus,WSSV)的分子流行病学变异情况,选取2016年1-7月从我国3省9市采集到的47份WSSV阳性样本,提取WSSV核酸后,使用特定引物对目的片段进行扩增,继而进行测序并分析比较不同地区样本的ORF14/15和ORF23/24序列缺失情况,ORF75、ORF94和ORF125的VNTR及SNPs变化情况.结果显示,在ORF14/15的扩增中,有4种缺失片段,分别是6 540 bp、6 530 bp、5 950 bp和5 140 bp,而在ORF23/24扩增中则有3种大片段缺失,分别是12 073 bp、12 070bp和11 945 bp,在所有样本中ORF75的RUs数目分别为11、8、10、3,ORF94的RUs数目分别为5、7、8、11、14,ORF125的RUs数目为4、5、6.SNPs分析结果表明,ORF94片段中含有5个和8个RUs的在48位的碱基均为T,含有7个RUs的在48位的碱基为T、G、G、G、G、T、T,含有11个RUs的在48位的碱基为T、T、T、T、T、T、G、T、G、T、T,含有14个RUs的在48位的碱基为T、T、T、T、G、G、T、G、G、T、T、T、T、T,含有所有RUs数目的ORF125片段在8、18、25、66和69位置的碱基均为G、G、G、G和A,,而在9、50、53、61和63位的碱基则出现了4种变异.研究结果表明,2016年的样本中,WSSV毒株存在一定程度的变异情况.主要表现在ORF14/15和ORF23/24均出现了新的缺失片段,ORF75、ORF94和ORF125的VNTR以及SNPs变异情况也存在明显差异.

对来自河北黄骅(HH)、山东平度(PD)、江苏吴江(WJ)和山东日照(RZ)的4个凡纳滨对虾(Litopenaeus vannamei)群体进行了对虾生长参数测量,用TaqMan qPCR检测了凡纳滨对虾各群体的肝胰腺组织中和RZ群体多种组织中的虾肝肠胞虫数量(Amount of Enterocytozoon hepatopenaei,EHP).结果显示,在主要生长相关参数中,RZ群体最优,该群体EHP载量也最低.不同群体的样本数EHP对数直方图的模式存在差异,HH和PD群体的EHP对数呈双峰分布,而WJ和RZ群体的EHP对数呈单峰分布,代表EHP在不同群体中可能存在不同的传播模式.EHP对数呈单峰分布的群体或从多峰分布的群体中分离出的高EHP对数子群体的对虾体长或体重与EHP对数呈显著的负相关.RZ群体中,各个体不同组织中EHP从高到低的顺序依次是肝胰腺＞中肠＞血淋巴＞鳃＞肌肉.肝胰腺、中肠和鳃3个组织中EHP对数相互间的相关性为99.9％的极显著水平(P＜0.001);除了中肠与血淋巴和肝胰腺与血淋巴以外,其余组织间EHP对数的相关性也达到极显著(P＜0.01)或显著(P＜0.05)水平.用DIG标记的EHP探针对肝胰腺、肌肉、鳃、肠道组织的原位杂交显示,肝胰腺是主要的EHP感染组织,其他组织中杂交信号较弱,但各组织中有少数细胞的EHP易感.

对虾急性肝胰腺坏死病（Acute hepatopancreatic necrosis disease，AHPND）是由致AHPND副溶血弧菌（AHPND-causing Vibrio parahaemolyticus，VpAHPND）携带的pVA1-like质粒所表达的PirAVp和PirBVp毒力蛋白对对虾肝胰腺的急性毒性所致。本研究用2.19×105 CFU/ml VpAHPND分离株20130629002S01对凡纳滨对虾（Litopenaeus vannamei）进行浸泡感染，于感染后2~9 d采集对虾的肝胰腺、鳃、肠道、肌肉组织，采用实时荧光定量PCR方法，检测各组织中的pirAVp拷贝数。结果显示，感染后凡纳滨对虾各组织均能检测到pirAVp，其中，肝胰腺在感染后第4天达到峰值，为8.71×104 copies/mg，而鳃、肌肉、肠道分别在第3、4、5天达到峰值，分别为9.08×103、2.59×104、5.76×104 copies/mg。早期感染鳃组织中先出现VpAHPND的富集，在高死亡发生期，VpAHPND数量在肝胰腺和肠道出现高峰，在死亡数量逐渐下降的后期，各组织的VpAHPND均快速下降，肠道、肝胰腺和肌肉中的VpAHPND水平趋于接近。对虾肝胰腺组织病理切片显示，同一时间有临床症状的病虾和濒死对虾相比，濒死对虾表现出更严重的AHPND病理特征，且二者的组织病理特征均随着感染时间的延长变得更为严重，但检测到的VpAHPND数量呈下降趋势。研究表明，在VpAHPND感染过程中，组织中的pirAVp基因数量不能代表对虾的发病程度，发病程度及组织病理严重的AHPND样品中VpAHPND的数量不一定处于高水平状态。

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the pathogens causing the significant diseases of crustaceans listed by the OIE (Office intemaptional despizooties)Manual of Diagnostic Tests for Aquatic Animals.Infection with IHHNV does not produce any pathognomonic gross clinical signs,although runt-deformity syndrome (RDS) has been noted in infected juvenile Litopenaeus vannamei and Penaeus monodon.Enterocytozoon hepatopenaei (EHP) was first found and isolated in Thailand in'2009,which caused slow growth of P.monodon.IHHNV and EHP were found in the samples of L.vannamei postlarvae which were collected from hatchery farms in Tianjin and Hebei Province in 2013.The diseased shrimps showed some clinical signs with high mortality,slow growth and difference of individual growth rate.The IHHNV and EHP individual detection was conducted on these samples using the real-time PCR technique.In total,108 out of 108 (100％) L.vannamei postlarvae samples were IHHNV positive,and 53 out of 108 (49.1％) L.vannamei postlarvae samples were EHP positive.Positive samples contained approximately 10-10 copies/(μg DNA) of IHHNV and big size of individual shrimp carried higher number of virus copies.Positive samples contained approximately 103-105 copies/(μg DNA) of EHP and mainly the small sized individual shrimp.The analysis showed that the IHHNV loads were positively correlated with the shrimp growth rate.IHHNV loads above 8.51× 104 copies/μg DNA represented a high risk level.EHP loads were negatively correlated with the shrimp growth rate.EHP loads above 2.19× 104 copies/μg DNA represented a high risk level.Our study showed that the diseased L.vannamei postlarvae samples occurred in the hatchery farms of Tianjin and Hebai Province infected with shrimp pathogens of IHHNV and EHP.Data provides scientific basis on the effects of pathogens infection on the growth of cultured penaeid shrimp.

Microsporidia Enterocytozoon hepatopenaei (EHP) was detected using probe based real-time PCR from 442 shrimp coming from 5 farmed Litopenaeus vannamei populations in Tianjin,Zhejiang,and Shandong Province.The biological length and body weight of shrimp were measured individually.The Rohrer's ponderal index (PI,W/L3) originally used in medical science was introduced in this study to establish the fuction of body weight (W) and biological length (L) of shrimp.The results showed that the PI of 4 EHP positive populations with an average biological length at (5.37±1.19) cm was calculated as (5.19±0.26)× 10-3 g/cm3,while that of the EHP negative population with an average biological length was (7.96±0.51)×10-3 g/cm3.After adjusting of the difference of PI caused by biological length according to the function PI=a.L(b-3),the PI of EHP positive populations was about (70.5±8.7)％ of that of the EHP negative populations at the same biological length.It indicated that the average body weight of shrimp in an EHP positive population was about 30％ smaller than that of shrimp with same size in biological length in an EHP negative population.The coefficient of variation (CV) of biological length and body weight in EHP positive populations was (2.39±0.93) times and (2.05±0.86) times of those in the EHP population,respectively,suggesting significant size variation in the EHP positive populations.The deviation ratio of actual body weight to body weight estimated on biological length in the EHP positive populations was 2.34 to 3.45 times of that in the EHP negative population,indicating the shrimp with same size in the EHP positive populations had greater fluctuation in body weight.

This paper firstly introduces the framework of global biosecurity strategy, and then illustrates the recommendations from Food and Agriculture Organization (FAO), World Organization for Animal Health (OIE) and the International Aquatic Veterinary Biosecurity Consortium for the global biosecurity strategy. Lastly, it gives some examples for the practices of shrimp breeding industries biosecurity in the United States, Thailand and Indonesia, which provides enlightenments for the healthy and sustainable development of Chinese aquaculture.

为查明黄头病毒(Yellow Head Virus,YHV)在我国的存在和变异情况,本研究采用世界动物卫生组织(OIE)《水生动物诊断手册》中YHV套式RT-PCR检测方法对2012-2014年采集的299份样品进行了YHV监测,并对部分YHV阳性样品基因进行了克隆测序及系统发育分析.流行病学调查显示,299份样品中YHV的阳性率为11％,我国养殖的中国明对虾、凡纳滨对虾、日本囊对虾以及罗氏沼虾的部分样品中均检出了YHV,中国明对虾和罗氏沼虾是本次调查中新发现的YHV自然宿主,而且YHV在中国明对虾中的检出率最高.对6份较强阳性样品YHV基因组ORF1b内1002 bp的分型片段进行克隆测序和序列分析,序列比对结果显示,阳性样品的YHV与国外报道的YHV的6个基因型相似度为81.0％-90.5％;系统发育分析显示,6份阳性样品归于同一分支,但与已知YHV的6个基因型均不在同一分支内,其与YHV基因1型(YHV-1)亲缘关系较近.对阳性样品YHV基因组ORF3内编码gp116蛋白的一段序列进行克隆测序,得出其序列长度为509 bp,与YHV-1a的545 bp、YHV-1b的383 bp和YHV-2(即鳃联病毒GAV)的476 bp均不同;依据该片段构建的系统发育树显示,6份阳性样品归于同一分支,与YHV已知的6个基因型不同,与YHV-1亲缘关系较近,且与YHV-1a相似度大于YHV-1b.对其中两份样品的ORF2序列进行比对显示,两份样品序列相似性为99.8％,蛋白序列完全相同,与YHV-1的序列相似度为85.9％,与YHV-2相似性为80.9％.样品调查结果对增补YHV的宿主范围有重要意义;YHV核酸检测和序列比对结果表明,感染我国养殖对虾的YHV为一种新的致病株型.