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1.
White spot syndrome (WSS) is considered as a great threat to commercial farming of the tiger shrimp (Penaeus monodon). The causal agent of WSS is a DNA virus called white spot syndrome virus (WSSV). The prevalence of this dreadful virus infection
has been studied in five randomly selected hatcheries located in the Cox’s Bazar district of Bangladesh. Both one-step and
nested polymerase chain reaction (PCR) involving two pairs of primers, namely, 146F1/146R1 and 146F2/146R2, amplifying the
1447 bp and 941 bp fragments, respectively, were conducted to detect the WSSV. Out of 60 randomly collected shrimps, 12 (20%)
were found to be positive by one-step PCR, while 18 (30%) were found to be positive by nested PCR. The nested PCR was found
to be much more sensitive than the one-step PCR. The shrimp specimens showing clinical signs of WSS were positive for WSSV
by both one-step and nested PCR. Some of the apparently healthy samples were also found to be positive for WSSV by nested
PCR. Among the two primer-pairs, the inner pair amplifying the 941 bp fragment was more sensitive than the outer primer pair
amplifying the 1447 bp fragment when used in one-step PCR. 相似文献
2.
采用对虾白斑综合症病毒和传染性皮下组织坏死病毒的特异引物,在一个PCR反应中同时扩增到大小分别为271 bp和356 bp的病毒特异片段,多重PCR条件优化后可从最少100 fg级病毒DNA模板中定性检测出此两种病毒。 相似文献
3.
N Sundar Raj K S Nathiga Nambi S Abdul Majeed G Taju S Vimal M A Farook A S Sahul Hameed 《Journal of fish diseases》2012,35(12):917-925
An attempt was made to determine the replication efficiency of white spot syndrome virus (WSSV) of shrimp in different organs of freshwater rice‐field crab, Paratelphusa hydrodomous (Herbst), using bioassay, PCR, RT‐PCR, ELISA, Western blot and real‐time PCR analyses, and also to use this crab instead of penaeid shrimp for the large‐scale production of WSSV. This crab was found to be highly susceptible to WSSV by intramuscular injection. PCR and Western blot analyses confirmed the systemic WSSV infection in freshwater crab. The RT‐PCR analysis revealed the expression of VP28 gene in different organs of infected crab. The indirect ELISA was used to quantify the VP28 protein in different organs of crab. It was found that there was a high concentration of VP28 protein in gill tissue, muscle, haemolymph and heart tissue. The copy number of WSSV in different organs of infected crab was quantified by real‐time PCR, and the results revealed a steady increase in copy number in different organs of infected crab during the course of infection. The viral inoculum prepared from different organs of infected crab caused significant mortality in tiger prawn, Penaeus monodon (Fabricius). The results revealed that this crab can be used as an alternate host for WSSV replication and production. 相似文献
4.
多重RT-PCR体系检测4种虾病毒的方法 总被引：1，自引：1，他引：0
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。 相似文献
5.
Yoganandhan K Syed Musthaq S Narayanan RB Sahul Hameed AS 《Journal of fish diseases》2004,27(9):517-522
The VP28 gene of white spot syndrome virus (WSSV) was cloned into pRSET B expression vector. The VP28 protein was expressed as a protein with a 6-histidine taq in Escherichia coli GJ1158 with NaCl induction. Antiserum was raised against this recombinant-VP28 protein in rabbits and it recognized VP28 protein in naturally and experimentally WSSV-infected shrimp, marine crabs, freshwater prawns and freshwater crabs. The antiserum did not recognize any of the other known WSSV structural proteins. Various organs such as eyestalks, head muscle, gill tissue, heart tissue, haemolymph, tail tissue and appendages were found to be good materials for detection of WSSV using the antiserum and detection of WSSV was successful in experimentally infected Penaeus monodon and P. indicus at 12 and 24 h post-infection (p.i.), respectively. The antiserum was capable of detecting WSSV in 5 ng of total haemolymph protein from WSSV-infected shrimp. 相似文献
6.
White spot syndrome virus (WSSV) has caused significant losses in shrimp farms worldwide. Between 2004 and 2006, Pacific white shrimp Litopenaeus vannamei (Boone) were collected from 220 farms in Taiwan to determine the prevalence and impact of WSSV infection on the shrimp farm industry. Polymerase chain reaction (PCR) analysis detected WSSV in shrimp from 26% of farms. Juvenile shrimp farms had the highest infection levels (38%; 19/50 farms) and brooder shrimp farms had the lowest (5%; one of 20 farms). The average extent of infection at each farm was as follows for WSSV‐positive farms: post‐larvae farms, 71%; juvenile farms, 61%; subadult farms, 62%; adult farms, 49%; and brooder farms, 40%. Characteristic white spots, hypertrophied nuclei and basophilic viral inclusion bodies were found in the epithelia of gills and tail fans, appendages, cephalothorax and hepatopancreas, and virions of WSSV were observed. Of shrimp that had WSSV lesions, 100% had lesions on the cephalothorax, 96% in gills and tail fans, 91% on appendages and 17% in the hepatopancreas. WSSV was also detected in copepoda and crustaceans from the shrimp farms. Sequence comparison using the pms146 gene fragment of WSSV showed that isolates from the farms had 99.7–100% nucleotide sequence identity with four strains in the GenBank database – China ( AF332093 ), Taiwan ( AF440570 and U50923 ) and Thailand ( AF369029 ). This is the first broad study of WSSV infection in L. vannamei in Taiwan. 相似文献
7.
Melena J Bayot B Betancourt I Amano Y Panchana F Alday V Calderón J Stern S Roch P Bonami JR 《Journal of fish diseases》2006,29(10):589-600
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. 相似文献
8.
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 相似文献
9.
Omkar V. Byadgi K. M. Shankar B. T. Naveen Kumar Rajreddy Patil Iqlas Ahmed 《Aquaculture International》2014,22(2):887-900
A study was conducted on the stability of monoclonal antibody (MAb) in the hepatopancreas and hemolymph of Penaeus monodon and its effect on protection against white spot syndrome virus (WSSV) upon challenge. MAb C-5 raised against WSSV was purified and coated onto a commercial shrimp feed at dosages of 5, 10 and 15 mg/kg feed. The feed was fed to P. monodon and stability of the MAb in hepatopancreas and hemolymph was determined by immunodot and Western blot. Immunodot results indicated the presence of MAb for 2 h post-feeding in hepatopancreas and hemolymph which was dose-dependent. MAb was also detected in hemolymph by Western blot up to 1 h post-feeding. Shrimp fed with MAb were challenged with WSSV by oral and injection methods. In shrimp fed with 15 mg antibody/kg feed (0.45 μg MAb/g shrimp/day) WSSV infection significantly delayed both in oral and injection challenges with a survival of 65 and 70 % (p < 0.05), respectively, during 15 days post-challenge. MAb was stable in shrimp for passive immunization against WSSV and could be a potential tool for prophylaxis against the virus. 相似文献
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11.
白斑症病毒在日本对虾体内的感染增殖 总被引：9，自引：0，他引：9
用投喂患白斑症病毒病的虾组织人工感染日本对虾稚虾,每日取样,整虾冰冻切片,单克隆抗体的荧光抗体方法,原位观察病毒在虾体内的感染增殖,结果表明：感染后三天内,在感染虾的各组织器官内均未观察到明显的病毒感染的阳性细胞,每四天首先在鳃丝腔内的小量血细胞观察到病毒感染;第五天除血细胞外同时在血窦,鳃上皮组织,皮下组织内观察到,第六天进而在心脏,胃上皮组织内观察到：第七天进一步又在淋巴器官,中肠内观察到,八 相似文献
12.
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。 相似文献
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高位池养殖过程凡纳滨对虾携带WSSV情况的动态变化 总被引：1，自引：0，他引：1
为了更好地预防对虾白斑综合征(WSS)的暴发,探讨该病毒病的流行规律,笔者针对养殖过程中对虾的携带WSSV情况展开调查。调查于2010年7月-2010年11月广东省汕尾市红海湾养殖场进行,从10口凡纳滨对虾高位养殖池中随机抽取6口进行跟踪采样。收集指标包括对虾生长状况、基本环境指标、浮游微藻种群结构和对虾病毒携带量等。本文重点报道利用实时定量PCR-TaqMan探针法检测6口精养池塘对虾体内WSSV的携带量变化情况,检测结果显示:①1-3号虾池苗种携带WSSV,其波动范围在1.3×103~1.7×104copy/g之间;②对虾在养殖过程中均带毒,鳃组织中的平均病毒携带量(2.3×109copy/g)多于肌肉组织中的平均病毒携带量(3.2×108copy/g),且变化趋势一致,但没有显著性差异(P>0.05);③在整个养殖过程中对虾WSSV携带量总体呈现波动上升的趋势,期间各池出现过数次高值。前期WSSV拷贝数的波动范围在1.3×103~3.0×107copy/g之间,后期上升到1.5×106~1.2×1011copy/g,使得某些池塘养殖对虾WSS暴发。调查结果说明:1)对虾携带WSSV可以进行养殖生长;2)WSSV在对虾体内的含量是变化的,且其变化存在着一定的规律性;3)这种变化规律主要体现在带毒量随着养殖时间的进行及外界水环境中某些主要因子的变化而变化,如:养殖时间越长,带毒量越高;养殖环境中某些关键环境因子的改变,如:温差较大,不良藻相转换,天气骤变等均可引起对虾体内病毒含量较大的波动。鉴此,作者提出,构建并维持良好的浮游微藻的群落结构,注意有害藻相改变时保持养殖水体环境稳定,对环境突变前后都做好应对对虾应激的措施等可以极大程度地减少WSS暴发的可能。本研究通过对WSSV的密切跟踪,旨在更好的反映其在养殖环境下的动态变化规律,以及受各种环境因子影响的情况,从而为预防对虾WSS提供依据和参考。 相似文献
15.
R Sudhakaran T Mekata T Kono K Supamattaya N T H Linh Y Suzuki M Sakai T Itami 《Journal of fish diseases》2009,32(7):611-617
White spot syndrome virus (WSSV) is an important shrimp pathogen responsible for large economic losses for the shrimp culture industry worldwide. The nucleic acids of the virus must be adequately preserved and transported from the field to the laboratory before molecular diagnostic analysis is performed. Here, we developed a new method to isolate WSSV-DNA using Flinders Technology Associates filter paper (FTA matrix card; Whatman) without centrifugation or hazardous steps involved. FTA technology is a new method allowing the simple collection, shipment and archiving of nucleic acids from haemolymph samples providing DNA protection against nucleases, oxidation, UV damage, microbial and fungal attack. DNA samples prepared from 10-fold dilutions of moribund shrimp haemolymph using FTA matrix cards were analysed using semi-quantitative and quantitative polymerase chain reaction (PCR) and were compared with two commercially available DNA isolation methods, the blood GenomicPrep Mini Spin Kit (GE Healthcare) and the DNAzol (Invitrogen). Sequence analysis was performed for the DNA samples prepared using the various isolation procedures and no differences in the sequence among these methods were identified. Results based on the initial copy number of DNA prepared from the GenomicPrep Mini Spin Kit are a little more sensitive than the DNA prepared from FTA matrix cards, whereas the DNAzol method is not suitable for blood samples. Our data shows the efficiency of retention capacity of WSSV-DNA samples from impregnated FTA matrix cards. Matrix cards were easy to store and ship for long periods of time. They provide ease of handling and are a reliable alternative for sample collection and for molecular detection and characterization of WSSV isolates. 相似文献
16.
Experimental infection of European crustaceans with white spot syndrome virus (WSSV) 总被引：3，自引：1，他引：3
V Corbel Z Zuprizal C Shi Huang Sumartono J-M Arcier & J-R Bonami 《Journal of fish diseases》2001,24(7):377-382
Eight European marine and freshwater crustaceans were experimentally infected with diluted shrimp haemolymph infected with white spot syndrome virus (WSSV). Clinical signs of infection and mortalities of the animals were routinely recorded. Diagnosis was by direct transmission electron microscopy (TEM), DNA hybridization (dot-blot and in situ hybridization) using WSSV probes and by PCR using WSSV specific primers. High mortality rates were noted between 7 to 21 days post-infection for Liocarcinus depurator , Liocarcinus puber , Cancer pagurus , Astacus leptodactylus , Orconectes limosus , Palaemon adspersus and Scyllarus arctus . Mortality reached 100%, 1 week post-infection in P. adspersus . When infection was successful, direct TEM observation of haemolymph revealed characteristic viral particles of WSSV, some observed as complete virions (enveloped), others as nucleocapsids associated with envelope debris. WSSV probes showed strong positive reactions in dot-blots and by in situ hybridization in sections and specific virus DNA fragments were amplified successfully with WSSV primers. White spot syndrome virus was pathogenic for the majority of the crustaceans tested. This underlines the epizootic potential of this virus in European crustaceans. 相似文献
17.
白斑综合征病毒感染克氏原螯虾后的PCR检测及组织病理学研究 总被引：3，自引：0，他引：3
采用投喂感染白斑综合征病毒(White Spot Syndrome Virus,WSSV)对虾肌肉的方式,对养殖克氏原螯虾(Procambarus clarkii)进行人工感染,以确定WSSV对养殖克氏原螯虾的易感性。结果发现,投喂病虾感染组螯虾的死亡率达到90%,而对照组未出现死亡。采用PCR对试验组螯虾的肌肉进行WSSV检测,发现投喂感染组的阳性检出率为100%,对照组的阳性检出率均为0。PCR检测结果发现,濒死螯虾的肝胰腺、中肠、肌肉、鳃、性腺、心脏六种组织的PCR结果均为WSSV阳性,而对照组的各组织检测结果均为阴性。组织切片的光镜观察也证实,濒死螯虾的肝胰腺、中肠、肌肉、鳃、性腺、心脏及血淋巴等组织均发生了不同程度的病变。 相似文献
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19.
White spot syndrome virus (WSSV) infection was induced in tiger shrimp, Penaeus monodon, under laboratory conditions, and histopathological changes in subcuticular epithelial cells of the eye stalk and pleopod were studied sequentially at different time post-challenge. Routine histological techniques using paraffin embedded tissues, as well as frozen tissues, were used to document WSSV infection. Histological manifestations such as cellular hypertrophy in the subcuticular epithelial cells of the eyestalk and pleopod could be detected as early as 18 h post-infection (p.i.) before the manifestation of clinical signs of the disease. However, no histopathological changes could be detected before 18 h p.i.. Hypertrophy of the nuclei in the epithelial cells was pronounced after 24 h p.i. Marked necrosis, and eosinophilic intranuclear inclusions, characteristic of early stages of WSSV infection were observed between 24–36 h p.i. Clinical signs of the disease appeared at 48 h p.i. The presence of WSSV at early asymptomatic stages of p.i. has been tested in parallel samples using polymerase chain reaction, for further confirmation of WSSV. This paper discusses the potential of a non-lethal and rapid histopathological diagnostic method to document WSSV infection, using the eyestalk or pleopod, when expensive DNA based diagnostics are not available or affordable. 相似文献
20.
R Patil K M Shankar B T N Kumar A Kulkarni P Patil N Moger 《Journal of fish diseases》2013,36(9):753-762
A flow‐through immunoassay (FTA), an improved version of immunodot, was developed using a nitrocellulose membrane baked onto adsorbent pads enclosed in a plastic cassette to detect white spot syndrome virus (WSSV) in shrimp. Sharp purple dots developed with WSSV against the white background of the nitrocellulose membrane. The detection limits of WSSV by the FTA and immunodot were 0.312 and 1.2 μg mL?1 crude WSSV protein, respectively. The FTA could be completed in 8–10 min compared with 90 min for immunodot. The FTA was 100 times more sensitive than 1‐step polymerase chain reaction (PCR) and in between that of the 1‐ and 2‐step PCR protocol recommended by the Office of International Epizootics (OIE). In experimental, orally infected shrimp post‐larvae, WSSV was first detected 14, 16 and 18 h post‐infection (hpi) by FTA, immunodot and one‐step PCR, respectively. The FTA detected WSSV 2 and 4 h earlier than immunodot and one‐step PCR, respectively. The FTA was more sensitive (25/27) than one‐step PCR (23/27) and immunodot (23/27) for the detection of WSSV from white spot disease outbreak ponds. The reagent components of the FTA were stable giving expected results for 6 m at 4–8 °C. The FTA is available as a rapid test kit called ‘RapiDot’ for the early detection of WSSV under field conditions. 相似文献