凡纳滨对虾(Litopenaeus vannamei)传染性皮下及造血组织坏死病毒(IHHNV)及虾肝肠胞虫(EHP)的荧光定量PCR检测

2013年,河北、天津等地区养殖的凡纳滨对虾(Litopenaeus vannamei)育苗期出现死苗、出苗率低的情况,生产上,仔虾个体大小差异较大,造成了严重损失。本研究采用荧光定量PCR方法(Real-time PCR)对天津大港地区采集的108尾凡纳滨对虾仔虾样品进行单尾病原检测。结果显示,传染性皮下及造血组织坏死病毒(Infectious hypodermal and hematopoietic necrosis virus,IHHNV)和虾肝肠胞虫(Enterocytozoon hepatopenaei,EHP)均有检出。IHHNV阳性检出率100%,每微克对虾组织DNA的病毒拷贝数为10~3–10~7,且个体较大的样品(1.2–2.0 cm)携带病毒拷贝数偏高;EHP阳性检出率为49.1%,每微克对虾组织DNA的拷贝数为10~3–10~5,且集中于个体较小样品(0.7–1.1 cm)。对IHHNV和EHP阳性凡纳滨对虾样品进行生物学体长与病毒载量指数相关性分析,显示IHHNV载量指数与对虾生长速率呈正相关,虾组织IHHNV平均载量达8.51×10~4 copies/μg DNA,为较高的感染水平;EHP的载量与对虾生长速率呈负相关关系,与较大个体阳性检出率较低相对应,虾组织EHP平均载量达到2.19×10~4 copies/μg DNA,为较高的感染水平。由此,该批凡纳滨对虾仔虾患病为IHHNV和EHP的混合感染所致,本研究数据为IHHNV和EHP病原混合感染流行情况及其对养殖育苗期仔虾生长的影响提供科学依据。     

粤西地区凡纳滨对虾虾肝肠胞虫、传染性皮下和造血组织坏死病毒感染情况的初步调查

针对近年来粤西地区养殖户普遍反映凡纳滨对虾长不大的情况,本实验室运用对虾EHP和IHHNV的荧光定量PCR检测方法,对该两种病原进行了流行性调查。结果显示,在87份样品中,EHP的检出率为41.38%,IHHNV的检出率为12.18%。在检测样品中,两份不同海域的海水样品有检测到EHP和IHNNV,而经处理过的养殖用水和育苗用水中未检测出上述病原;成虾的EHP检出率最高达93.75%,种虾的IHHNV检出率最高为38.10%。在本地选育的种虾和进口种虾中,均有检出EHP,检出率分别为80.00%和33.33%,进口种虾中未检测出IHHNV。在不同养殖场样品的检测结果中,EHP载量指数最高的对虾平均日增长体重最小,即载量指数为10的对虾样品日增长量为0.039 g;而EHP载量指数最低的日增量大,即载量指数为2的日增长量为0.90 g。对B养殖场不同养殖时长的对虾进行检测,结果发现3份样品的EHP载量指数分别为6、7和7;B养殖场20号池不同规格的对虾EHP载量指数均为4。     

肝胰腺细小病毒(HPV) PCR检测及流行情况调查

采用水产行业标准《对虾肝胰腺细小病毒病诊断规程第1部分:PCR检测法》(SC/T7203.1-2007)的方法,对2011-2013年期间我国沿海7个省市主要养殖对虾品种不同生长阶段的对虾样品进行该病毒携带情况的筛查。该方法的检测灵敏度为0.07 fg,相当于大约20个病毒拷贝。结果显示,639份样品的HPV阳性检出率为18.47%。其中,在中国对虾(Fenneropenaeus chinensis)、凡纳滨对虾(Litopenaeus vannamei)和日本囊对虾(Marsupenaeus japonicus)中均有阳性检出,且仔虾、幼虾、成虾各个阶段均可检出HPV,表明HPV已在我国养殖对虾中存在并流行。本研究结果为对虾养殖生产提供了疫病的科学数据,为我国养殖对虾中该病的流行情况提供了参考依据。     

虾肝肠胞虫(Enterocytozoon hepatopenaei)实时荧光定量PCR检测方法的建立及对虾样品的检测

根据Gen Bank中公布的虾肝肠胞虫(Enterocytozoon hepatopenaei)(EHP)SSU r DNA序列设计1对特异性引物,建立并优化了EHP的SYBR Green I实时荧光定量PCR(q PCR)检测方法。结果显示,该方法在60℃的退火温度时扩增效果最好,产物的熔解曲线为1个单峰,构建的方法对8.3×101–8.3×108 copies/μl的EHP SSU r DNA片段的检测响应具有良好的线性关系,扩增产物阈值循环数(Ct)与模板起始量的对数[log(Sq)]的关系为Ct=–3.369 log(Sq)+39.364(R2=0.992),扩增效率为98.1%,检测灵敏度下限为8.3×101 copies/μl,在线性范围内具有良好的组内和组间重复性。对实际样品的检测表明该方法比已报道的套式PCR的检测灵敏度约高4倍。利用本方法对采集自江苏、海南和山东的3批凡纳滨对虾样品的肝胰腺组织DNA(Hp DNA)中的EHP SSU r DNA进行了q PCR检测,结果显示,EHP的载量指数与对虾生长速率呈负相关关系,肝胰腺中EHP载量在103 copies/(ng Hp DNA)时代表了较高的风险水平。本研究建立的q PCR方法具有特异、灵敏、快速、定量的优点,所建立的方法及检测数据可为EHP的防控提供技术参考。     

常规PCR、实时定量PCR检测传染性皮下及造血器官坏死病毒的效果分析

对虾传染性皮下及造血器官坏死病毒(IHHNV)可感染世界各地养殖对虾,给对虾养殖业造成严重经济损失。本实验首次采用实时定量PCR法对广西地区的84份凡纳滨对虾样品进行检测,同时以常规PCR检测作对照。实时定量PCR检测阳性率为79·8%,常规PCR检测阳性率为40·5%,表明广西地区养殖的凡纳滨对虾IHHNV的感染率较高。将二者检测均呈阳性的30份样品扩增产物进行序列分析测序,测序结果通过DNA STAR软件包进行分析,并通过NCBI Blast与GenBank中的序列进行比对。结果证明,测定的是IHHNV序列。30份样品的IHHNV序列很保守,可以分为4种类型,仅有两个碱基的位置发生变异。实时定量PCR检测IHHNV,快速、灵敏、准确,特异性好,可以作为检测对虾感染病毒的有效方法。     

上海主养区凡纳滨对虾8种流行病病原监测及分析

为进一步掌握上海地区养殖凡纳滨对虾(Litopenaeus vannamei)病原的流行趋势及特点,定期对上海主养区的凡纳滨对虾开展了8种流行病病原监测及分析工作。51份样品的分子检测和细菌分离鉴定结果显示,有4种病原检测出阳性,其中虹彩病毒1(DIV1)阳性检出率为29.41%,传染性皮下及造血组织坏死病毒(IHHNV)为3.92%,致急性肝胰腺坏死病副溶血弧菌(VpAHPND)为1.96%,副溶血弧菌(Vp)为19.61%；而白斑综合征病毒(WSSV)、桃拉综合征病毒(TSV)、偷死野田村病毒(CMNV)、虾肝肠胞虫(EHP)等4种病原未检出。408项病原检测结果中,4种病原的阳性总样本数量为28份,总体阳性率为6.86%。结果表明,上海地区养殖凡纳滨对虾的病原携带率处于较低水平,DIV1、副溶血弧菌(Vp)是携带的主要病原。在实际生产中应根据病原流行特点,从苗种选购、养殖过程管理、药物选用等方面进行综合防控,减少病害发生。     

舟山地区大棚凡纳滨对虾生长缓慢病因的调查分析

2013年以来,浙江省舟山地区大棚养殖的凡纳滨对虾(Penaeus vannamei)普遍出现生长缓慢、养殖成功率低的现象。为了查明该原因,本研究采用分子生物学和组织病理学等方法对引起对虾生长缓慢的病因开展了调查分析。结果显示,采集的270份病虾样本中,对虾肝肠胞虫(Enterocytozoon hepatopenaei,EHP)PCR阳性检出率高达85.19%,传染性皮下及造血器官坏死病毒(infectious hypodermal and hematopoietic necrosis virus,IHHNV)检出率为0;所有采集的病虾样本中也未分离到常见的致病菌;54份正常的对虾样本中EHP和IHHNV均未检出。将病虾PCR扩增产物进行序列测定和比对分析,结果获得的序列片段与Gen Bank中已有EHP相关序列相似性高达99.55%;病虾的肝胰腺组织病理切片观察显示,在虾肝胰腺组织中可观察到处于各个生长发育阶段的EHP。通过上述研究,初步认为EHP是引起舟山地区大棚养殖对虾生长缓慢的一个重要病原。     

虾肝肠胞虫(Enterocytozoon hepatopenaei)实时荧光定量PCR检测方法的建立及对虾样品的检测

根据GenBank中公布的虾肝肠胞虫(Enterocytozoon hepatopenaei) (EHP) SSU rDNA序列设计1对特异性引物,建立并优化了EHP的SYBR Green Ⅰ实时荧光定量PCR (qPCR)检测方法.结果显示,该方法在60℃的退火温度时扩增效果最好,产物的熔解曲线为1个单峰,构建的方法对8.3×101-8.3×108 copies/μ1的EHP SSU rDNA片段的检测响应具有良好的线性关系,扩增产物阈值循环数(Ct)与模板起始量的对数[log(Sq)]的关系为Ct=-3.369 log(Sq)+39.364 (R2=0.992),扩增效率为98.1％,检测灵敏度下限为8.3× 10(1) copies/μ1,在线性范围内具有良好的组内和组间重复性.对实际样品的检测表明该方法比已报道的套式PCR的检测灵敏度约高4倍.利用本方法对采集自江苏、海南和山东的3批凡纳滨对虾样品的肝胰腺组织DNA (HpDNA)中的EHP SSU rDNA进行了qPCR检测,结果显示,EHP的载量指数与对虾生长速率呈负相关关系,肝胰腺中EHP载量在103 copies/(ng HpDNA)时代表了较高的风险水平.本研究建立的qPCR方法具有特异、灵敏、快速、定量的优点,所建立的方法及检测数据可为EHP的防控提供技术参考.     

检出虾肝肠胞虫(Enterocytozoon hepatopenaei)的凡纳滨对虾(Litopenaeus vannamei)群体的体长和体重关系

对采自天津、浙江和山东等养殖场5个凡纳滨对虾(Litopenaeus vannamei)群体的442尾个体进行虾肝肠胞虫(Enterocytozoon hepatopenaei,EHP)的Taq Man探针荧光定量PCR检测,并测量各群体每尾对虾的生物学体长和体重。引入医学上的劳累尔(Rohrer)体重指数(Ponderal index,PI,W/L3)关系建立对虾体重(W)和体长(L)关系函数。结果显示,4个凡纳滨对虾的EHP阳性群体[平均体长为(5.37±1.19)cm]的体重指数PI平均值为(5.19±0.26)×10~(–3) g/cm~3,EHP阴性群体的凡纳滨对虾群体[平均体长为(2.49±0.21)cm]为(7.96±0.51)×10~(–3) g/cm~3,根据PI=a·L~((b–3))的函数矫正EHP阴性和阳性群体的体长差异引起的PI差值后,同等体长EHP阳性群体的PI值为阴性群体的(70.5±8.7)%,表明同样大小的个体,EHP阳性群体的平均体重比阴性群体平均体重低30%;EHP阳性群体中凡纳滨对虾体长和体重的变异系数是EHP阴性群体的(2.39±0.93)和(2.05±0.86)倍,表现为对虾EHP阳性群体个体大小不均匀;EHP阳性群体体重偏差率是EHP阴性群体的2.34–3.45倍,体长相同时,EHP阳性的体重波动变大。     

采用OIE标准检测养殖对虾中传染性皮下及造血组织坏死病毒(IHHNV)的PCR检出类型

利用世界动物卫生组织(OIE)推荐的4对引物(389F/R、392F/R、77012F/77353R 和309F/R)，通过普通 PCR 方法，对本实验室2011?2012年采集于国内不同地区的对虾样品进行 IHHNV(Infectious hypodermal and hematopoietic necrosis virus)检测，并对国内存在的 IHHNV 检出类型进行初步分析。检测结果显示，在凡纳滨对虾、斑节对虾、中国对虾、宽沟对虾中均检测出了 IHHNV，而在脊尾白对虾中未检出。其中凡纳滨对虾阳性率最高，中国对虾阳性检测率最低。对虾样品2011年阳性率高于2012年，华东地区高于华北、华南两地。此外，根据4对引物的检测结果，得到国内 IHHNV 的4种 PCR 检出类型。     

Multiple infections caused by white spot syndrome virus and Enterocytozoon hepatopenaei in pond‐reared Penaeus vannamei in India and multiplex PCR for their simultaneous detection

White leg shrimp, Penaeus vannamei, were collected on a monthly basis from grow‐out ponds located at Tamil Nadu and Andhra Pradesh states along the east coast of India for screening of viral and other pathogens. Totally 240 shrimp samples randomly collected from 92 farms were screened for white spot syndrome virus (WSSV), infectious hypodermal and haematopoietic necrosis virus (IHHNV), infectious myonecrosis virus (IMNV) and Enterocytozoon hepatopenaei (EHP). The number of shrimp collected from shrimp farms ranged from 6 to 20 based on the body weight of the shrimp. All the shrimp collected from one farm were pooled together for screening for pathogens by PCR assay. Among the samples screened, 28 samples were WSSV‐positive, one positive for IHHNV and 30 samples positive for EHP. Among the positive samples, four samples were found to be positive for both WSSV and EHP, which indicated that the shrimp had multiple infections with WSSV and EHP. This is the first report on the occurrence of multiple infections caused by WSSV and EHP. Multiplex PCR (m‐PCR) protocol was standardized to detect both pathogens simultaneously in single reaction instead of carrying out separate PCR for both pathogens. Using m‐PCR assay, naturally infected shrimp samples collected from field showed two prominent bands of 615 and 510 bp for WSSV and EHP, respectively.     

Pre-exposure to infectious hypodermal and haematopoietic necrosis virus or to inactivated white spot syndrome virus (WSSV) confers protection against WSSV in Penaeus vannamei (Boone) post-larvae

Larvae and post-larvae of Penaeus vannamei (Boone) were submitted to primary challenge with infectious hypodermal and haematopoietic necrosis virus (IHHNV) or formalin-inactivated white spot syndrome virus (WSSV). Survival rate and viral load were evaluated after secondary per os challenge with WSSV at post-larval stage 45 (PL45). Only shrimp treated with inactivated WSSV at PL35 or with IHHNV infection at nauplius 5, zoea 1 and PL22 were alive (4.7% and 4%, respectively) at 10 days post-infection (p.i.). Moreover, at 9 days p.i. there was 100% mortality in all remaining treatments, while there was 94% mortality in shrimp treated with inactivated WSSV at PL35 and 95% mortality in shrimp previously treated with IHHNV at N5, Z1 and PL22. Based on viral genome copy quantification by real-time PCR, surviving shrimp previously challenged with IHHNV at PL22 contained the lowest load of WSSV (0-1x10(3) copies microg-1 of DNA). In addition, surviving shrimp previously exposed to inactivated WSSV at PL35 also contained few WSSV (0-2x10(3) copies microg-1 of DNA). Consequently, pre-exposure to either IHHNV or inactivated WSSV resulted in slower WSSV replication and delayed mortality. This evidence suggests a protective role of IHHNV as an interfering virus, while protection obtained by inactivated WSSV might result from non-specific antiviral immune response.     

Prevalence of Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) and White Spot Syndrome Virus (WSSV) in Litopenaeus vannamei in the Pacific Ocean off the Coast of Panama

Abstract.— In March 2000, 104 wild caught Litopenaeus wannamei broodstock, captured off the Pacific coast of Panama, were screened for the following penaeid viruses: infectious hypodermal and hematopoietic necrosis virus (IHHNV) and white spot syndrome virus (WSSV). The purpose of this study was to determine the prevalence of IHHNV and WSSV in wild shrimp in this area of the Western Hemisphere and to acquire specific pathogen free (SPF) L. vannamei for inclusion into the Oceanic Institute's genetic breeding program. The prevalence of the viruses was determined using the dot blot hybridization format, which is a commercially available molecular method for detecting these viruses. Dot blot hybridization assays can be used as an initial screening method to detect moderately to highly infected shrimp. The results from the dot blot assays indicated the prevalence of IHHNV in 28% and WSSV in 2% of the 104 hemolyrnph samples tested. Results from this study were used to establish the initial candidate SPF status of the animals that were assessed and to determine the prevalence of two serious pathogens of penaeid shrimp captured from the wild of the Pacific Ocean in the Central American region off the coast of Panama.     

Simultaneous presence of infectious hypodermal and hematopoietic necrosis virus (IHHNV) and Type A virus-related sequence in Penaeus monodon from India

Detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimp is complicated by the fact that certain virus-related sequences are integrated into the genome of Penaeus monodon in some parts of the world, which has been reported so far from Africa and Australia. In this study, we evaluated the highly specific and sensitive diagnostic primer sets for detection of infectious IHHNV and integrated virus-related sequence in 177 samples of P. monodon from India. A nested primer set, IHHNV648F/R and IHHNV309F/R was used to specifically detect infectious IHHNV and not the virus-related sequences. IHHNV was detected in 67.4% postlarvae (PL) and 34% adult samples using this primer set. The OIE recommended primers IHHNV392F/R and IHHNV389F/R gave positive reaction for 86.7% PL and 67% adult samples, while the primer pair 77012F and 77353R gave positive reaction with 46.7% PL and 20% adult samples. These primers were found to detect virus-related sequence integrated into the shrimp genome. The analysis of virus-related sequence by MG831F/R primers showed that 33.7% PL and 31.7% adult shrimp possessed Type A virus-related sequence. 22.8% PL and 10.5% adults had both IHHNV and Type A virus-related sequence. Cloning and sequencing 832 bp virus-related sequence from P. monodon from India revealed presence of five shrimp DNA markers between 439 and 825 bp. This study is the first conclusive report on the presence of Type A virus-related sequence in P. monodon from India.     

Effect of Low Salinity on Growth and Survival of Postlarvae and Juvenile Litopenaeus vannamei

The effect of low salinity on survival and growth of the Pacific white shrimp Litopenaeus vannamei was examined in the laboratory due to the interest of raising shrimp inland at low salinities. In three separate experiments, individual L. vannamei postlarvae (∼ 0.1 g) were cultured at salinities of either 0.5, 1, 1.5, 2, or 3 ppt ( N = 5 or 10/treatment) for 18 to 40 d at 30 C in individual 360-mL containers. In each experiment controls of 0 and 30 ppt were run. There was no postlarval survival at salinities < 2 ppt. Survival was significantly different ( P < 0.01) at 2 ppt (20%) compared to 30 ppt (80%). Growth was also significantly different ( P < 0.01) at 2 and 3 ppt compared to 30 ppt (416%, 475%, and 670%, respectively). A fourth experiment compared juveniles (∼ 8 g) and postlarvae (∼ 0.05 and 0.35 g). Shrimp were cultured at salinities of 0, 2, 4, and 30 ppt for 40 d at 25 C, in individual 360-mL and 6-L containers ( N = 7/treatment). There was no postlarval survival at < 2 ppt. Postlarval survival at 4 ppt (86%) was not significantly different (P > 0.05) from 30 ppt (100%). Juveniles exhibited better survival at lower salinities (100% at 2 ppt) than 0.05 and 0.35 g postlarvae (29% and 14% respectively, at 2 ppt). The effects of salinity on growth varied with sizdage. Final growth of 0.05 g postlarvae at 2 ppt (693%) was significantly less ( P < 0.01) than at 4 ppt (1085%) and 30 ppt (1064%). Growth of 0.35 g postlarvae was significantly less ( P < 0.01) for 4 ppt (175%) than for 30 ppt (264%). There was no growth data for juveniles (8 g). It appears from these experiments that the culture of L. vannamei poses risks when performed in salinities less than 2 ppt.     

检出虾肝肠胞虫(Enterocytozoon hepatopenaei)的凡纳滨对虾(Litopenaeus vannamei)群体的体长和体重关系

对采自天津、浙江和山东等养殖场5个凡纳滨对虾Litopenaeus vannamei)群体的442尾个体进行虾肝肠胞虫(Enterocytozoon hepatopenaei,EHP)的TaqMan探针荧光定量PCR检测,并测量各群体每尾对虾的生物学体长和体重.引入医学上的劳累尔(Rohrer)体重指数(Ponderal index,PI,W/L3)关系建立对虾体重(W)和体长(L)关系函数.结果显示,4个凡纳滨对虾的EHP阳性群体[平均体长为(5.37±1.19) cm]的体重指数PI平均值为(5.19±0.26)×10-3 g/cm3,EHP阴性群体的凡纳滨对虾群体[平均体长为(2.49±0.21)cm]为(7.96±0.51)×10-3 g/cm3,根据PI=a·L(b-3)的函数矫正EHP阴性和阳性群体的体长差异引起的PI差值后,同等体长EHP阳性群体的PI值为阴性群体的(70.5±8.7)％,表明同样大小的个体,EHP阳性群体的平均体重比阴性群体平均体重低30％;EHP阳性群体中凡纳滨对虾体长和体重的变异系数是EHP阴性群体的(2.39±0.93)和(2.05±0.86)倍,表现为对虾EHP阳性群体个体大小不均匀;EHP阳性群体体重偏差率是EHP阴性群体的2.34-3.45倍,体长相同时,EHP阳性的体重波动变大.     

Histopathological Studies on Wild Broodstock of White Shrimp Penaeus vannamei in the Platanitos Area, Adjacent to San Blas, Nayarit, Mexico

In Mexico, the capture of wild broodstock of Penaeus vannamei or P. stylirostris for hatchery production of postlarvae is a common practice. During September 1995 and March-April 1996, high mortalities were observed in the wild broodstock captured in the Platanitos area, on the coast of San Blas, Nayarit, Mexico. As a result of these mortalities, many hatcheries closed down their facilities. Because early histological analysis had shown that some of these animals were infected with different pathological agents, such as bacteria and viruses, the present study was carried out to determine the prevalence and severity grade of infectious diseases affecting these shrimp. During March 1996, a total of 28 female and 28 male shrimp were randomly selected from a recently captured stock of 600 breeders of P. vannamei . A second sampling (28 females and 28 males) was performed from a stock of 800 breeders captured in April 1996. On this occasion, the shrimp were sampled after 10 d of acclimation in a hatchery. The animals selected for examination were those with necrosis in the cuticule, red coloration in the telson and uropods, soft cuticle, empty gut, and erratic swimming. All of the specimens were subjected to histological analysis and dot blot hybridization with a gene probe specific for Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV). The results showed that both stocks were infected with at least three different viruses (Infectious Hypodermal and Hematopoitetic Virus, Taura Syndrome Virus and Hepatopancreatic Parvo-Like Virus), gram-negative bacteria and other parasites such as gregarines. It is likely that the high mortalities observed are due to the recent introduction of TSV in the wild population of P. vannamei .     

A single-step multiplex PCR for simultaneous detection of white spot syndrome virus and infectious hypodermal and haematopoietic necrosis virus in penaeid shrimp

White spot syndrome virus (WSSV) and infectious hypodermal and haematopoietic necrosis virus (IHHNV) are the major viral pathogens of penaeid shrimp worldwide (Lightner & Redman 1998). Litopenaeus vannamei was introduced into China from the Americas, and quickly became widely cultured. Following its introduction, both IHHNV and WSSV have become important pathogens of cultured penaeid shrimp and have had a huge impact on the culture industry in China in recent years.