对虾的主要疾病及其诊断方法

诊断对虾病原的方法有传统的形态病理学（光镜和电镜直接观察和组织病理学等）、扩增和生物测定、微生物学和血清学方法，对皮下及造血组织坏死病毒病（IHHNV）、肝胰脏细小病毒病（HPV）、拖拉症（Taura syndrome,TS）、白斑综合症（WSSV）、斑节对虾型杆状病毒病（MBV）和杆状对虾病毒病（BP）等病原均采用非放射性的基因组探针目前已研制出了NHP、某些弧菌（Vibrio spp）和微孢子虫的传统基因探针，根据聚合酶链式反应（PCR），采用DNA扩增方法确立的检测某些病原的高敏感性方法也应用在对虾病原诊断上。     

现代生物技术检测对虾病毒在商业化应用中的限制

对虾病毒性疾病有多种,如白斑病毒、桃拉病毒、黄头病毒等。多年来,科研人员对病毒病的诊断方法进行了大量的研究,现代生物检测技术得到较多应用,如酶联免疫吸附(ELISA)、聚合酶链式反应(PCR)、核酸探针斑点杂交等．发表了较多提取病毒、改进病毒检测方     

现代生物技术检测对虾病毒病在商业化应用中的限制

对虾病毒性疾病有多种，如白斑病毒、桃拉病毒、黄头病毒等。多年来，科研人员对病毒病的诊断方法进行了大量的研究，现代生物检测技术得到较多应用，如酶联免疫吸附（ELISA）、聚合酶链式反应（PCR）、核酸探针斑点杂交等，发表了较多提取病毒、改进病毒检测方法等文章。检测方法越来越简易、灵敏，并开发有WSSV、TSV等病毒检测试剂盒。利用现代生物检测技术诊断对虾病毒病对监测养殖生产中的疾病流行趋势有一定的作用，但各种检测方法大都局限于国家机构的实验室进行，难以大规模商业化应用。是什么原因使先进的检测技术无法服务于养殖生产？现结合生产以及科研实践进行多方面探讨，供参考。     

中国对虾几个产卵场群体携带白斑综合征病毒状况调查

白斑综合征可以导致养殖对虾短时间内大面积死亡，是迄今为止对虾养殖业面临的最大挑战。本试验采用巢式PCR法对2001年采自黄渤海的中国对虾几个产卵场群体进行白斑综合征病毒检测，旨在较全面地了解黄渤海野生中国对虾携带病毒状况。各群体的阳性检出率分别为：朝鲜半岛南海岸群体55％；渤海湾群体35％；辽东湾群体94．7％；海州湾群体47．4％。结果显示，中国对虾几个产卵场群体均不同程度地携带白斑综合征病毒。辽东湾产卵场群体阳性检出率明显高于其他群体，推测人工孵化苗种放流、海湾的地理和水质条件与中国对虾的WSSV感染率相关。而中国对虾野生群体携带病毒对于对虾养殖业的影响是不容忽视的，笔者认为，只有从无特异病原(SPF)及抗特异病原(SPR)对虾养殖群体的建立着手才能从根本上避免由于对虾携带病毒而可能导致的病毒性疾病的暴发。同时，应该重视海区污染的治理，减少病毒病暴发的诱因。本试验建立了快速检测WSSV的PCR方法，1pg病毒核酸仍可检测到，为白斑综合征病毒病的防治及早期诊断提供了有效的手段。     

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。     

凡纳滨对虾传染性皮下及造血组织坏死病毒(IHHNV)可视化环介导等温扩增(LAMP)技术方法的优化与应用

传染性皮下及造血组织坏死病毒(Infectious hypodermal and hematopoietic necrosis virus, IHHNV)作为对虾疾病的主要病原之一,能够感染多种对虾,对幼虾危害尤其明显,虽然死亡率不高,但可以引起对虾生长缓慢,造成巨大的经济损失,严重影响对虾养殖业持续健康发展。本文根据IHHNV病毒基因的保守序列,采用Primer Explorer V4软件设计6条LAMP特异性引物组合,建立了一种以环介导等温核酸扩增技术(Loop-mediated isothermal amplification, LAMP)为基础的快速检测IHHNV的方法。对本研究的LAMP检测方法的敏感性和特异性进行分析,并将其灵敏度与实时荧光定量PCR、普通PCR检测方法进行比较。结果显示:LAMP检测方法在63℃恒温条件60 min内完成反应,阳性结果出现可视化的绿色,阴性结果颜色不发生变化;LAMP方法的最低检出限为10.3 copies/μL,灵敏度与荧光实时定量PCR相当,较常规PCR高。结果表明建立的LAMP方法适用于对虾IHHNV的现场快速检测。     

虾苗中斑节对虾杆状病毒感染的调查

近年来由于对虾病毒病的流行,给对虾养殖业带来了巨大的灾难。斑节对虾杆状病毒（Penaeusmonodonbaculovirus,MBV）为肠感染病毒,主要感染肝胰腺和前中肠的上皮细胞核。其宿主范围较广,可感染多种养殖对虾,但主要感染斑节对虾（Penaeusmonodon）,其仔虾感染率和致死率均可达100％,养成期虾感染率63．6－100％,多数虾池为90％左右。由于对虾病毒病缺乏特效药治疗,放对其应采取综合防治的原则,如育好苗、管好水、科学技何及应用一些抗病毒的中草药治疗等。其中虾苗质量的优劣对养成期的对虾的生长及虾病的发生发展均有很大的关系…     

套式PCR检测WSSV出现假阳性的原因分析及控制措施

对虾白斑综合征病毒(White Spot Syndrome Virus,WSSV)是1992-1993年全国范围内对虾暴发性流行病的主要病原,到现在仍然没有能有效控制wssV疫情的技术措施。WSSV每年给我国对虾养殖业带来巨大经济损失,成为对虾养殖业可持续发展的主要障碍。目前检测WSSV病原的方法有很多种,主要有目视观察法、组织学方法、电子显微技术、生化检验法、免疫学方法和分子生物学方法等。其中分子生物学方面中PCR检测具有敏感性高、特异性高等特点,被广泛应用于对虾WSSV检测。     

2022年滨州地区养殖对虾和海水鱼苗流行病学调查和风险评估

为了解滨州地区养殖对虾与海水鱼苗流行病学现状,掌握潜在风险,为水产养殖业健康有序发展提供科学依据,2022年,开展滨州市(无棣县、沾化区、博兴县)养殖对虾和海水鱼苗种主要病害监测,对虾苗种检测白斑综合征(WSD)、传染性皮下及造血组织坏死病毒病(IHHN)、虾肝肠胞虫病(EHP)、急性肝胰腺坏死病(AHPND)、对虾虹彩病毒病(DIV-1)、桃拉综合征(TS)、传染性肌肉坏死病(IMN);海水鱼苗种检测病毒性神经坏死病(VNN)等。本次监测了65份南美白对虾样品,分别都检测8种对虾病原,共检出5份病原阳性样品,并存在有2种甚至3种病原共感染情况。建议在对虾苗种引进、繁育、养殖过程中应进行全面的多病原监测,从源头上把好苗种质量关,保障对虾健康,促进对虾养殖业发展。     

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。     

聚合酶链反应（PCR）检测养殖对虾的白斑症病毒（WSSV）感染

对虾WSSV病是亚洲对虾养殖业中的一个棘手问题。本研究采用Kimura引物 ,用PCR技术对不同生长期的中国对虾 (Penaeuschinensis)进行了WSSV的检测 ,同时也检测了对虾发病时养殖池中多见的野生厚蟹 (Helicesp .)和矛尾刺虎鱼 (Acanthogobiushasta)。检测结果表明 :分别在检测的 5尾亲虾中的 1尾 ,6尾仔虾中的 1尾 ,5尾稚虾中的 3尾及所检测的 5尾病虾和 2只厚蟹中获得到 982bp的PCR扩增产物 ,说明为WSSV感染阳性。在检测的 2尾矛尾刺虎鱼中均未获得PCR扩增产物 ,说明为WSSV感染阴性。在亲虾、虾苗以及虾池内的野生厚蟹中检测到WSSV感染的阳性结果表明 :WSSV感染的亲虾有可能是病毒的储主 ,WSSV感染的野生厚蟹有可能是病毒中间宿主或病毒的携带者 ,它们在对虾WSSV病的感染、传播中起了重要的作用     

Studies on the occurrence of infectious myonecrosis virus in pond‐reared Litopenaeus vannamei (Boone, 1931) in India

Whiteleg shrimp, Litopenaeus vannamei, with clinical sign of muscle opaqueness with reddish colour at the distal abdominal segments were observed in farms located in West Bengal State, India. The mortality of shrimp in all disease outbreak ponds ranged from 20% to 50%, and mortality increased gradually. The RT‐PCR assay of these samples using primer sets specific to infectious myonecrosis virus (IMNV) revealed its presence in the disease outbreak ponds. The IMNV infection was reproduced in healthy shrimp by intramuscular injection to satisfy River's postulates. The virus caused mortality in intramuscularly challenged shrimp, but failed to cause mortality by oral route. Tissue distribution of IMNV in infected shrimp by RT‐PCR assay revealed the presence of this virus in haemolymph, gill, hepatopancreas and muscle. This study confirms that the disease outbreak which occurred in the shrimp farms located at Purba Medinipur District, West Bengal, India, was due to IMNV.     

Development of monoclonal antibodies against VP28 of WSSV and its application to detect WSSV using immunocomb

White spot disease (WSD) is an important viral disease of penaeid shrimp caused by white spot syndrome virus (WSSV). WSSV isolated from WSD outbreaks in commercial shrimp (Penaeus monodon) farms in India were propagated in the laboratory in healthy shrimp. The virus was purified from the infected tissues by sucrose gradient centrifugation. The VP28 was electroeluted from SDS-PAGE gels and was used to immunize Balb/c mice to produce hybridomas secreting monoclonal antibodies (MAb) against WSSV. A total of five hybridoma clones secreting MAbs to VP28 were produced. The MAbs were of the isotypes IgG1, IgG2b and IgM. The MAbs reacted with VP28 of WSSV and not with any other viral or shrimp protein in western blot. The MAbs were used to develop dot immunoblot assay using an immunocomb to detect WSSV from field samples. The test developed had an analytical sensitivity of 625 pg and a diagnostic sensitivity of 100% compared to single step polymerase chain reaction (PCR). The test can be used as an alternate for first step PCR to detect WSSV from field samples.     

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.     

A synthetic antibacterial peptide from Mytilus galloprovincialis reduces mortality due to white spot syndrome virus in palaemonid shrimp

White spot syndrome virus (WSSV) isolated from Penaeus monodon was found to be highly infective for the western Mediterranean shrimp, Palaemon sp. Using polymerase chain reaction (PCR), it was demonstrated that such shrimp are not naturally carriers of WSSV. Following challenge with virus, mortality reached 100% 3.5-4 days after injection at 22 degrees C. Incubation of infected shrimp at 10 degrees C totally suppressed the mortality which rapidly developed when shrimp were returned to 18 or 22 degrees C. Preincubation of WSSV with mature synthetic mytilin significantly reduced shrimp mortality with a 50% efficient dose of about 5 microM. Survival of shrimp was not due to the development of an active mechanism of defence as re-injection of WSSV produced the same mortality pattern. Mortality was probably due to WSSV replication as dot blot failed to detect viral DNA in the injection sample but was positive 1 day post-injection. Protection by mytilin was by interaction at the virus level, preventing replication as no WSSV nucleic acid was detected by PCR even after 7 days in shrimp injected with WSSV preincubated with 10 or 50 microM mytilin.     

南美白对虾亲虾暴发性传染病病原调查

对福建、广东沿海对虾育苗场的南美白对虾亲虾暴发性传染病进行病原学研究,结果表明大部分亲虾感染了白斑综合症病毒,还有一些亲虾同时感染桃拉综合症病毒。发病对虾体色发红,部分对虾体表可见大小不一的白色斑点。对这些病毒的PCR扩增产物进行测序,并进行同源性比较,结果表明导致本次对虾传染病的WSSV基因片段与基因库已报告的WSSV具有很高的同源性。结合流行病学特点,可确定这些亲虾主要因感染了WSSV和TSV而发病死亡。     

Development of a polymerase chain reaction (PCR) procedure for the detection of baculovirus associated with white spot syndrome (WSBV) in penaeid shrimp

The causative viral agent was purified from diseased shrimp Penaeus japonicus with white spot syndrome (WSBV). Several hundred clones were obtained from libraries of the purified viral genomic DNA. According to the results of nucleotide sequence analysis, none of the WSBV clones showed considerable sequence homology with those of other known viruses, indicating that WSBV is a new virus causing a serious disease in shrimp. Based on the sequence data of WSBV genomic DNA, a pair of polymerase chain reaction (PCR) primers was designed. After 30 cycles of PCR amplification of viral genomic DNA extracted from WSBV, a single product of the expected size was detected. Southern blot hybridization confirmed that the amplified product was specific to the DNA of WSBV. The PCR system was able to detect 1 pg of WSBV DNA after 30 cycles, and efficiently amplify the target region of WSBV gene in the total nucleic acids extracted either from the diseased shrimp or hatchery shrimp with no signs of viral infection.     

多重RT-PCR体系检测4种虾病毒的方法

根据多重RT-PCR的技术原理,利用对虾传染性表皮与造血组织坏死症病毒、白斑综合征病毒、黄头病毒和桃拉综合征病毒的基因序列分别设计了4对特异引物,建立多重RT-PCR体系用于虾4种病毒的检测。多重RT-PCR体系能特异地扩增出IHHNV、WSSV、YHV和TSV的目的片段:TSV特异性扩增片段508 bp,WSSV 特异性扩增片段435 bp,IHHNV 特异性扩增片段301 bp 和YHV。特异性扩增片段614 bp。结果表明,多重PCR虾病毒检测系统具有较高的特异性和敏感性,并对其它对虾病原呈阴性。IHHNV、TSV、WSSV和YHV模板在多重PCR虾病毒检测体系中的检测下限分别为0.1,1,0.02和0.2 pg。病毒感染病料检测试验中,该检测体系的检测结果与单纯PCR的检测结果呈现出较好的吻合度。     

Detailed monitoring of white spot syndrome virus (WSSV) in shrimp commercial ponds in Sinaloa, Mexico by nested PCR

WSSV has caused great losses to the global shrimp industry in recent years. This virus can infect shrimps asymptomatically. However, once the clinical signs are developed, mortalities can reach 100% in 3-10 days. PCR has been extensively used to detect WSSV in a specific and sensitive manner. Nested PCR is even more sensitive than single-step PCR and had been used for the detection of WSSV in asymptomatic populations. In this work, a detailed monitoring of WSSV by nested PCR in shrimp commercial ponds in Guasave County, State of Sinaloa, Mexico, is presented. Five ponds from two different farms were monitored for growth and presence of WSSV. At the beginning of the culture, ponds from both farms showed no or very slight WSSV presence. A 3-day period of rain occurred at both farms 10 and 14 weeks of culture for farms 1 and 2, respectively. At this time, WSSV was widely distributed in the shrimp populations of farm 1 according to nested-PCR data, although no visual symptoms were observed. In ponds of farm 2, WSSV was present at low level. However, the number of PCR-positive groups was drastically increased in both farms by nested and single-step PCR. Abrupt fluctuations in temperature and salinity were documented in farm 2 after the rain, which may have contributed to the increasing of viral load in the pond's shrimp populations. Twelve days after the rain period, estimated mortalities of 80% occurred in farm 1. Nevertheless, the study ponds at farm 2 culture continued normally for three more weeks and were harvested successfully (52% and 67% of survival for ponds 1 and 2, respectively). The removal of 40% and 50% of shrimp population 2-4 days after the raining period may have contributed to the thriving of the cultures. Analyses of the presence of WSSV in individuals of both sexes indicated that there is no preference for this virus to infect male or female shrimp. Also, no differences in weight were found between WSSV infected and non-infected individual shrimps, as well as nested-PCR positive against single-step PCR positive organisms. Nested PCR is more useful to monitor shrimp cultures than single-step PCR since it allows knowing how widely distributed the virus is in asymptomatically populations.