White spot syndrome virus (WSSV) infection in tiger shrimp <Emphasis Type="Italic">Penaeus monodon</Emphasis>: A non-lethal histopathological rapid diagnostic method using paraffin and frozen sections

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.     

High efficacy of white spot syndrome virus replication in tissues of freshwater rice‐field crab,Paratelphusa hydrodomous (Herbst)

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.     

Tissue distribution of hepatopancreatic parvo‐like virus of shrimp in freshwater rice‐field crab,Paratelphusa hydrodomous (Herbst)

An attempt was made to determine the replication efficiency of hepatopancreatic parvo‐like virus (HPV) of shrimp in different organs of freshwater rice‐field crab Paratelphusa hydrodomous (Herbst) using bioassay, PCR, RT‐PCR, ELISA, Western blot and q‐PCR analyses. Another attempt was made to use this crab as an alternative to penaeid shrimp for the large‐scale production of HPV. This crab was found to be highly susceptible to HPV by intramuscular injection. The systemic HPV infection was confirmed by PCR and Western blot analyses in freshwater crab. The expression of capsid protein gene in different organs of infected crab was revealed by RT‐PCR analysis. Indirect ELISA was used to quantify the capsid protein in different organs of the crab. The copy number of HPV in different organs of the infected crab was quantified by q‐PCR. The results revealed a steady decrease in C_T values in different organs of the infected crab during the course of infection. The viral inoculum that was prepared from different organs of the infected crab caused significant mortality in post‐larvae of tiger prawn, Penaeus monodon (Fabricius). The results revealed that this rice‐field crab could be used as an alternative host for HPV replication and also for large‐scale production of HPV.     

中国对虾(Fenneropenaeus chinensis)和凡纳滨对虾(Litopenaeus vannamei)对白斑综合征病毒的敏感性比较

本研究分别对中国对虾野生群体(Wild-Fenneropenaeus chinensis, W-Fc)、中国对虾选育群体‘黄海2号’(Selected-Fenneropenaeus chinensis, S-Fc)和凡纳滨对虾商业一代苗种(Commercial- Litopenaeus vannamei, C-Lv)采用单尾定量口饲感染白斑综合征病毒(WSSV),比较W-Fc、S-Fc及C-Lv对WSSV的敏感性差异。结果显示,感染同等含量WSSV后,W-Fc、S-Fc和C-Lv的平均存活时间分别为(124.11±39.49) h、(166.79±51.54) h和(136.90±41.99) h,3组对虾间的平均存活时间存在显著性差异(P<0.05)。3组对虾在感染期间的死亡趋势：W-Fc在96 h达到死亡高峰,并且一直持续到216 h;S-Fc和C-Lv在144 h出现死亡高峰。另外,分别在感染后的3、6、12、24、36、48、72、144 h共8个时间点对3组对虾进行活体取样,利用实时荧光定量RT-PCR技术对其进行了病毒载量检测,从对虾存活时间和体内肌肉组织病毒载量的角度比较不同对虾抗病性能的差异,结果如下：48 h时,W-Fc、S-Fc和C-Lv3对虾体内肌肉组织的病毒载量分别为(1.22×106±6.14×105)、(7.10×103±7.26×102)和(1.50×104± 4.19×103) copies/ng DNA;144 h时,3组对虾体内肌肉组织病毒载量分别为(8.44×106±1.25×106)、(3.21×106±8.21×105)和(1.49×106±6.59×105) copies/ng DNA。实验结果显示,3组对虾对WSSV敏感性从高到低依次为中国对虾野生群体、凡纳滨对虾商业苗种、中国对虾选育群体,表明中国对虾选育群体‘黄海2号’在人工感染WSSV条件下表现出了良好的抗病性能。     

Development of a monoclonal antibody‐based flow‐through immunoassay (FTA) for detection of white spot syndrome virus (WSSV) in black tiger shrimp Penaeus monodon

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.     

Virulence and genotypes of white spot syndrome virus infecting Pacific white shrimp Litopenaeus vannamei in north‐western Mexico

White spot syndrome virus (WSSV) has caused substantial global economic impact on aquaculture, and it has been determined that strains can vary in virulence. In this study, the effect of viral load was evaluated by infecting Litopenaeus vannamei with 10‐fold serial dilution of tissue infected with strain WSSV Mx‐H, and the virulence of four WSSV strains from north‐western Mexico was assessed along with their variable number of tandem repeat (VNTR) genotypes in ORF75, ORF94 and ORF125. The LD₅₀ of the Mx‐H strain was a dilution dose of 10^?7.5; the mortality titre was 10^9.2 LD₅₀ per gram. In shrimp injected with 10^2.5 to 10^6.5 LD₅₀, no significant virulence differences were evident. Using mortality data, the four WSSV strains grouped into three virulence levels. The Mx‐F strain (intermediate virulence) and the Mx‐C strain (high virulence) showed more genetic differences than those observed between the Mx‐G (low‐virulence) and Mx‐H (high‐virulence) strains, in ORF94 and ORF125. The application of high‐viral‐load inocula proved useful in determining the different virulence phenotypes of the WSSV strains from the Eastern Pacific.     

White spot syndrome virus epizootic in cultured Pacific white shrimp Litopenaeus vannamei (Boone) in Taiwan

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.     

Comparison of white spot syndrome virus quantification of fleshy shrimp Fenneropenaeus chinensis in outdoor ponds between different growing seasons by TaqMan real‐time polymerase chain reaction

In the present study, we used TaqMan real‐time polymerase chain reaction to quantify and compare infection of white spot syndrome virus (WSSV) with shrimp production of Fenneropenaeus chinensis cultured in outdoor ponds along the west coast of the South Korea. In 2007, a total of 60 specimens in summer and 116 specimens in autumn were collected from 12 growing‐out ponds and 12 harvest ponds respectively. Pond harvest data were obtained from farmers. Of the summer samples, all specimens were WSSV positive, with a wide range of 12.4–7.0 × 10⁷ (mean 7.5 × 10⁶) copies ng^?1 DNA; shrimp production was 1.7 metric tonnes per hectare (mt ha^?1). Of the 116 autumn‐sample specimens, 81 (69.8%) were WSSV positive; WSSV infection had been decreased dramatically, to 0–7.2 (mean 3.5) copies ng^?1 DNA. Shrimp production of autumn ponds was 2.1 mt ha^?1. Statistical analysis indicated that the difference in WSSV infections detected in summer and autumn was highly significant (P<0.01). In summer, seven ponds (58.3%) with low‐WSSV infection loads (0–1000 WSSV copies ng^?1 DNA) had shrimp production of 2.7 mt ha^?1; the others had shrimp production of only 0.2 mt ha^?1. The mean shrimp production between the two infection levels showed a highly statistically significant difference (P<0.01).     

Assessment of transmission risk in WSSV-infected shrimp Litopenaeus vannamei upon cooking

White spot syndrome virus has been a threat to the global shrimp industry since it was discovered in Taiwan in 1992. Thus, shrimp-producing countries have launched regulations to prevent import of WSSV-infected commodity shrimp from endemic areas. Recently, cooked shrimp that is infected with WSSV tested positive by PCR. However, there is no study to determine the infectivity of WSSV in cooked shrimp that tested positive by PCR. In the present study, WSSV-infected shrimp were cooked at boiling temperature for different times including 0, 1, 3, 5, 10 and 30 min. Upon exposure to boiling temperature, WSSV-infected shrimp were fed to SPF shrimp (Litopenaeus vannamei). The result showed experimentally challenged shrimp from 0-min treatment (positive control) indeed got infected with WSSV. However, experimentally challenged shrimp that were fed tissues boiled at 1, 3, 5, 10 and 30 min were not infected with WSSV. Mortality data showed that only the positive control (0-min) treatment displayed high mortality, whereas no mortality was observed in any other treatment category. These findings suggest that cooking shrimp at boiling temperature for at least 1 min might prevent any potential spread of WSSV from endemic countries to other geographical areas where WSSV has not yet been reported.     

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。     

A simple method for purifying the White Spot Syndrome Virus using ultrafiltration

A very simple and efficient method was developed for isolating intact White Spot Syndrome Virus (WSSV) particles from infected Litopenaeus vannamei tissue. No density gradient centrifugation, ultracentrifugation or protease inhibitors were required for the purification of intact WSSV virions using microfilters (100 kDa cut-off) combined with several steps of conventional centrifugation procedures. A mortality assay was run using healthy shrimp to prove that the virions obtained were infective. The concentrated viral preparations were further studied using polyacrylamide gel electrophoresis (PAGE). At least five distinct protein bands were detected when intact purified WSSV virions were found by sodium dodecyl sulphate-PAGE, followed by Coomassie Brilliant R-250 staining. The estimated molecular weights of these proteins were 23, 24, 29, 32 and 42-kDa, which could correspond to viral protein. Using this method, the virus does not lose its ability to infect healthy shrimp.     

Highly efficient double‐stranded RNA transfection of penaeid shrimp using cationic liposomes

Yellow head virus (YHV) is a viral pathogen of shrimp that has caused high economic loss in the Thai shrimp farming industry, and the RNA interference mechanism has potential as a virus control strategy, as previous studies have shown that injection of virus specific double‐stranded RNA (dsRNA) can inhibit viral replication in shrimp. However, to date, complete inhibition viral replication has not been achieved. This study sought to determine whether cationic liposomes mixed with an antiviral dsRNA (YHV‐Pro‐dsRNA) were able to enhance protection against YHV. The results showed that injection of a YHV‐Pro‐dsRNA/cationic liposome complex markedly increased protection against YHV as compared with naked YHV‐Pro‐dsRNA, suggesting that cationic liposomes enhance the uptake of protective dsRNA into shrimp cells.     

Ex‐post facto analysis of diseases of the Gulf of Mexico′s white shrimp Litopenaeus setiferus

This study presents an Ex‐Post Facto analysis of diseases of wild juvenile and adults of Litopenaeus setiferus collected from a field survey at the Natural Protected Area of Terminos lagoon, southern Mexico. The objective of the present approach was to determine if sampling site and/or shrimp age were contributing risk factors for disease between juvenile and adult shrimp; if there was a determined period of time in a year cycle when diseases were more critical, and if the analysis would help to decision‐ making considering what population would pose less risk of disease‐carrying when withdrawn for experimental purposes; all under an after‐the‐fact (ex‐post facto) approach. We identified that juvenile shrimp were at more risk of contracting some diseases in the estuarine environment and June, July and August months, were found to be a critical period when colonizing and parasitic diseases maintained a significant high prevalence in the shrimp population. These assumptions may help for decision‐making when wild shrimp have to be withdrawn from their natural environment for research purposes.     

A rapid non-enzymatic method of DNA extraction for PCR detection of white spot syndrome virus in shrimp

The present study describes a simple method of extraction of white spot syndrome viral DNA (WSSV) from infected shrimp for the polymerase chain reaction (PCR) detection of WSSV. The DNA preparation using this method was found to be free from the host DNA, RNA and protein, and is suitable for different PCR protocols such as single‐step PCR, nested PCR and single‐tube semi‐nested PCR. This method of extraction has worked successfully for extracting the WSSV‐DNA from different organs (haemolymph, eyestalk, carapace, head muscle, heart, gills, appendages, heptopancreas, stomach, intestine, abdominal muscle and tail muscle) of WSSV‐infected adult shrimp, and WSSV‐infected larvae and postlarvae.