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.     

Genetic variation of white spot syndrome virus (WSSV) in Pacific white shrimp Litopenaeus vannamei (Boone 1931) culture of Thailand

White spot syndrome virus (WSSV) is highly pathogenic to penaeid shrimp and has caused significant economic losses in the shrimp farming industry in Thailand. Genotyping analysis was done in 124 WSSV isolates from cultured Pacific white shrimp Litopenaeus vannamei. These samples were obtained during 2007–2014 from eight provinces in Thailand. We investigated five variable loci in the virus genome: deletions in two variable regions, VR14/15 and VR23/24, and three variable number tandem repeats (VNTR) located in open reading frame (ORF) 75, 125 and 94. WSSV genotype was characterized as (X_14/15, X_23/24) (N₇₅‐N₁₂₅‐N₉₄) where X is the number of base pair deletion in the variable region and N is the number of repeat units (RUs) in a specific ORF. The deletion pattern in VR14/15 and VR23/24 regions characterized three WSSV genotypes. The most prevalent genotype was (5950_14/15, 10971_23/24), and it was found in all studied areas. At least 33 genotypes of WSSV were analysed based on 3 VNTR loci, indicating that the VNTRs of WSSV genome are highly variable. From 124 WSSV samples, two samples presented the characteristic of all five variable loci similar to WSSV collected during 2010 in Saudi Arabia (5950_14/15, 10971_23/24) (3₇₅‐6₁₂₅‐7₉₄). Many different WSSV genotypes shown in this study as compared to previously reported genotypes in Thailand suggests current status of disease epidemiology, as well as probable movements of WSSV between countries.     

壳聚糖硫酸酯提高凡纳滨对虾抗白斑综合征病毒感染力的研究

论述如下一项研究,采用壳聚糖硫酸酯添加到饲料中投喂凡纳滨对虾,4周后检测对虾血清酚氧化酶、超氧化物歧化酶和溶菌酶活性;同时进行白斑综合征病毒(WSSV)的肌肉注射感染实验,以检测壳聚糖硫酸酯对病毒的抗感染能力。实验结果显示,饲料中壳聚糖硫酸酯添加量为0.15‰和0.50‰,能显著提高凡纳滨对虾血清酚氧化酶活性;添加量为0.15‰时,能显著提高对虾血清超氧化物歧化酶活性,但添加量继续增大时反而下降;在低添加量时,对虾血清溶菌酶活性与对照组相近,添加量为0.15‰和0.50‰时,对虾血清溶菌酶活性随添加量增大而升高。凡纳滨对虾摄食添加壳聚糖硫酸酯饲料4周后,经注射WSSV攻毒感染,壳聚糖硫酸酯添加量为0.04‰、0.15‰和0.50‰试验组,对虾比成活率分别为39.3%、42.9%和53.6%,而未摄食壳聚糖硫酸酯的对照组成活率仅为17.9%。结果表明,摄食壳聚糖硫酸酯可以明显提高对虾抵御WSSV感染的能力。     

口服特异性卵黄抗体对凡纳滨对虾抗WSSV感染的免疫保护效果

为探讨特异性卵黄抗体对凡纳滨对虾(Litopenaeus vannamei)抗白斑综合征病毒(white spot syndrome virus, WSSV)的免疫保护机制及效果,本研究以添加不同剂量WSSV卵黄抗体制剂(0、0.2%和0.5%)的饲料投喂凡纳滨对虾幼虾,免疫28 d后使用WSSV进行人工感染,测定感染对虾的肝胰腺免疫酶活力和免疫基因表达水平,以及感染后14 d内对虾的存活率。结果显示,WSSV感染3 d后,与未添加卵黄抗体制剂的对照组相比,0.2%免疫组对虾肝胰腺的超氧化物歧化酶(SOD)和酚氧化酶(PO)活力显著升高,酸性磷酸酶(ACP)和碱性磷酸酶(AKP)活力显著降低,热休克蛋白70基因(Hsp70)表达水平显著升高,凝集素基因(lectin)和β-1,3-葡聚糖结合蛋白–脂蛋白基因(β-GBP-HDL)表达水平显著降低;0.5%免疫组对虾肝胰腺的SOD活力显著升高,ACP和AKP活力显著降低,Hsp70基因表达水平显著升高,β-GBP-HDL基因表达水平显著降低。人工感染实验结果显示,WSSV感染14 d后,0.2%和0.5%免疫组对虾的存活率分别为48.89%和87.78%,均显著高于对照组(存活率为0),且0.5%免疫组对虾存活率显著高于0.2%免疫组。特异性卵黄抗体制剂能在一定程度上改变发病的进程,延迟对虾的发病和死亡时间,提高同期存活率。研究表明,口服特异性卵黄抗体制剂可以调节对虾肝胰腺免疫酶活力和免疫基因表达水平,显著提高凡纳滨对虾抗WSSV感染的能力。本研究为卵黄抗体抗WSSV感染机制的研究提供了参考,也为在生产上使用卵黄抗体防控WSSV感染提供了科学依据。     

Effects of acute change in salinity and moulting on the infection of white leg shrimp (Penaeus vannamei) with white spot syndrome virus upon immersion challenge

In the field, moulting and salinity drop in the water due to excessive rainfall have been mentioned to be risk factors for WSSV outbreaks. Therefore, in this study, the effect of an acute change in environmental salinity and shedding of the old cuticle shell on the susceptibility of Penaeus vannamei to WSSV was evaluated by immersion challenge. For testing the effect of abrupt salinity stress, early premoult shrimp that were acclimated to 35 g L^?1 were subjected to salinities of 50 g L^?1, 35 g L^?1, 20 g L^?1, 10 g L^?1 and 7 g L^?1 or 5 g L^?1 and simultaneously exposed to 10^5.5 SID₅₀ mL^?1 of WSSV for 5 h, after which the salinity was brought back to 35 g L^?1. Shrimp that were transferred from 35 g L^?1 to 50 g L^?1, 35 g L^?1 and 20 g L^?1 did not become infected with WSSV. Shrimp became infected with WSSV after an acute salinity drop from 35 g L^?1 to 10 g L^?1 and lower. The mortality in shrimp, subjected to a salinity change to 10 g L^?1, 7 g L^?1 and 5 g L^?1, was 6.7%, 46.7% and 53.3%, respectively (P < 0.05)_. For testing the effect of moulting, shrimp in early premoult, moulting and post‐moult were immersed in sea water containing 10^5.5 SID₅₀ mL^?1 of WSSV. The resulting mortality due to WSSV infection in shrimp inoculated during early premoult (0%), ecdysis (53.3%) and post‐moult (26.72%) demonstrated that a significant difference exists in susceptibility of shrimp during the short moulting process (P < 0.05). The findings of this study indicate that during a drop in environmental salinity lower than 10 g L^?1 and ecdysis, shrimp are at risk for a WSSV infection. These findings have important implications for WSSV control measures.     

凡纳滨对虾白斑综合征血液病理研究

对自然发病、投喂和注射感染的凡纳滨对虾白斑综合征（white spot syndrome,WSS）血液病理进行研究,结果发现：不同感染方式患病对虾的血液病理变化相似,表现为：1．患病对虾血细胞总数、透明细胞数量极显著减少,小颗粒细胞、大颗粒细胞极显著增加。2．显微病理变化主要表现为血涂片中血细胞明显减少且分布不均匀,破损或解体的细胞增多,呈典型的溶血状态。3．超微病理变化表现为,大部分血细胞坏死,少数血细胞呈不典型的凋亡。患病对虾的血细胞核中可见大量白斑综合征病毒（white spot syndrome virus,WSSV）粒子。病理变化表明血细胞是WSSV的主要靶细胞。     

Silencing Pacific white shrimp Litopenaeus vannamei LvRab7 reduces mortality in brooders challenged with white spot syndrome virus

White spot syndrome virus (WSSV) is a major threat for farmed shrimp worldwide. RNA interference (RNAi) is the most recent tool against viral diseases. Rab7 silencing effectively inhibited virus infections in juvenile shrimp, but the antiviral effect in brooders remains unknown. This study found a homologue Penaeus monodon Rab7 gene in Litopenaeus vannamei brooders from Mexico. Sequence identity was >99% to a Thai LvRab7 sequence and >94% to Rab7 sequences from P. monodon or Marsupenaeus japonicus. Animals treated with a partial (494 bp) or a complete (618 bp) LvRab7 dsRNA sequences and challenged 48 h post treatment (hpt) with a high WSSV dose showed 80–88% mortality respectively. Shrimp treated with 4 or 20 μg LvRab7 dsRNA and challenged with a WSSV high dose had 80% mortality each, but it was reduced to 33% and 40%, respectively, with a low dose. Efficacy of dsRNA to reduce shrimp mortality was dependent on virus dose used regardless of dsRNA concentration. A significant reduction in LvRab7 mRNA levels was observed at 120 hpt. In conclusion, silencing LvRab7 in brooders showed a mild antiviral effect against a WSSV challenge at 48 hpt.     

Trypsin enzyme activity during larval development of Litopenaeus vannamei (Boone) fed on live feeds

Larval stages of the Pacific white shrimp, Litopenaeus vannamei (Boone) were fed standard live diets of mixed microalgae from the first to the third protozoea (PZ1 to PZ3), followed by Artemia nauplii until post‐larvae 1 (PL1). Trypsin enzyme activity for each larval stage was determined using N‐α‐p‐toluenesulphonyl‐l ‐arginine methyl ester (TAME) as a substrate. Results were expressed as enzyme content to assess ontogenetic changes during larval development. Tissue trypsin content (IU µg^?1 DW for each larval stage) was significantly highest at the PZ1 stage and declined through subsequent stages to PL1. This contrasts with previously observed patterns of trypsin development in Litopenaeus setiferus (Linnaeus) and other penaeid genera, which exhibit a peak in trypsin activity at the third protozoea/first mysis (PZ3/M1) larval stage. Litopenaeus vannamei larvae transferred to a diet of Artemia at the beginning of the second protozoea (PZ2) stage were significantly heavier on reaching the first mysis stage (M1) than those fed algae, while survival was not significantly different between treatments. At both PZ2 and PZ3 stages, trypsin content in larvae feeding on Artemia was significantly lower than in those feeding on algae. The rapid decline in trypsin content from PZ1 and the flexible enzyme response from PZ2 suggest that L. vannamei is physiologically adapted to transfer to a more carnivorous diet during the mid‐protozoeal stages.     

Competition of infectious hypodermal and haematopoietic necrosis virus (IHHNV) with white spot syndrome virus (WSSV) for binding to shrimp cellular membrane

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) and white spot syndrome virus (WSSV) are two widespread shrimp viruses. The interference of IHHNV on WSSV was the first reported case of viral interference that involved crustacean viruses and has been subsequently confirmed. However, the mechanisms underlying the induction of WSSV resistance through IHHNV infection are practically unknown. In this study, the interference mechanisms between IHHNV and WSSV were studied using a competitive ELISA. The binding of WSSV and IHHNV to cellular membrane of Litopenaeus vannamei was examined. The results suggested that there existed a mutual competition between IHHNV and WSSV for binding to receptors present on cellular membrane of L. vannamei and that the inhibitory effects of WSSV towards IHHNV were more distinct than those of IHHNV towards WSSV.     

Dietary protein level and natural food management in the culture of blue (Litopenaeus stylirostris) and white shrimp (Litopenaeus vannamei) in microcosms

The effect of dietary protein level and natural food management on the production parameters of blue and white shrimp, as well as on water quality, was evaluated in a microcosms system (plastic pools simulating aquaculture ponds). Two experimental trials were carried out in the facilities of DICTUS, University of Sonora, Northwest México. Treatment with low protein diet (LP) consisted of a low protein input (diet with 250 g kg^?1 crude protein) through the culture period; treatment with high protein diet (HP) consisted of a high protein input (diet with 400 g kg^?1 crude protein) through the trial, and finally treatment VP consisted of an adjustment of protein input (diets with 250, 350 or 400 g kg^?1 crude protein), depending on the abundance of biota (zooplankton and benthos) in the system. Each species responded differently to the treatments. For blue shrimp, low protein input resulted in the lowest final body weight (12.9 ± 0.6 g) and biomass (696.0 g pool^?1). Survival and feed conversion ratio were similar in the three treatments. For white shrimp, the best growth, biomass and food conversion ratio were obtained in the low protein input treatment. Water quality parameters such as nitrate, ammonia and organic matter during the two trials, were better for LP and VP treatments. White shrimp seems to have lower protein requirements than blue shrimp. For the blue shrimp culture, adjusting protein input according to natural food abundance (zooplankton and benthos) in the system, seems to be advantageous because of the possibility of getting a production similar to that obtained with a high protein input through the farming period, but at lower feed cost, and with a lower environmental impact. It is concluded that a high protein input through the whole farming period is not the best feeding strategy for any of the two species.     

The effects of sediment redox potential and stocking density on Pacific white shrimp Litopenaeus vannamei production performance and white spot syndrome virus resistance

Redox potential represents the intensity of anaerobic condition in the pond sediment, which may affect the dominant microbial transformations of substances, the toxins production, mineral solubility, as well as the water quality in the sediment–water interface inhabited by the shrimp. This study evaluates the effect of sediment redox potential in conjunction with stocking density on shrimp production performance, immune response and resistance against white spot syndrome virus (WSSV) infection. A completely randomized two factors experimental design was applied with three different sediment redox potential, i.e. ?65, ?108 and ?06 mV, and two shrimp densities, i.e. low (60 shrimp m^?2) and high (120 shrimp m^?2). Shrimp juveniles with an initial mean body weight of 5.32 ± 0.22 g were maintained in semi‐outdoor fibre tanks (270 L in capacity) for 35 days of experimental periods. At the bottom of each tank, 5‐cm deep soil substrate with different redox potential was added according to the treatments. The survival and biomass production were significantly reduced at ?206 mV sediment redox potential, regardless of stocking density. Highly negative sediment redox potential (?206 mV) and higher stocking density significantly reduced total haemocyte counts and phenoloxydase activity, and shrimp resistance to WSSV infection. We recommend to maintain the redox potential of pond sediment at a level of more than ?206 mV.     

凡纳滨对虾不同世代生长性状的变异

对从美国进口的选育凡纳滨对虾(Litopenaeus vannamei)海南群体(进口亲虾繁育的第1世代,G1)、山东和饶平群体(G2)、湛江2和湛江3群体(G3)、湛江1和上海群体(G4)共7个养殖群体4个世代1150个个体的生长性状体长和体重进行了分析。7个群体的平均体长(范围)分别为14.76(13.25~15.99)、8.46(6.28~10.48)、9.24(4.28~10.70)、7.75(5.13~9.36)、11.38(8.13~14.12)、5.25(3.47~6.83)和7.14(4.14~9.00),变异系数分别为0.04、0.08、0.08、0.09、0.12、0.14、0.14,平均体重(范围)分别为33.41(24.33~39.74)、5.19(1.80~9.68)、6.95(3.18~11.34)、4.62(1.52~9.87)、15.03(6.00~26.96)、1.47(0.48~3.42)、3.29(0.49~6.20),变异系数分别为0.10、0.23、0.21、0.27、0.32、0.39、0.36。体长和体重的变异系数随着繁育世代的增加而增加,其中体重的变异系数每繁殖1代增加10%,其第1代的变异系数与美国选育的亲本群体相同。体长、体重相关与回归分析表明,体长与体重相关极显著(P<0.01),体长和体重的回归方程为W=0.01L2.93。表明随着繁育世代的增加,生长性状逐代分化。     

Kinetic analysis of internalization of white spot syndrome virus by haemocyte subpopulations of penaeid shrimp,Litopenaeus vannamei (Boone), and the outcome for virus and cell

Little is known about the innate antiviral defence of shrimp haemocytes. In this context, the haemocytes of penaeid shrimp Litopenaeus vannamei (Boone) were separated by iodixanol density gradient centrifugation into five subpopulations (sub): sub 1 (hyalinocytes), sub 2 and 3 (prohyalinocytes), sub 4 (semigranulocytes) and sub 5 (granulocytes) and exposed to beads, white spot syndrome virus (WSSV) and ultraviolet (UV)‐killed WSSV. In a first experiment, the uptake of beads, white spot syndrome virus (WSSV) and UV‐killed WSSV by these different haemocyte subpopulations was investigated using confocal microscopy. Only haemocytes of sub 1, 4 and 5 were internalizing beads, WSSV and UV‐killed WSSV. Beads were engulfed by a much larger percentage of cells (91.2 in sub 1; 84.1 in sub 4 and 58.1 in sub 5) compared to WSSV (9.6 in sub 1; 10.5 in sub 4 and 7.9 in sub 5) and UV‐killed WSSV (12.9 in sub 1; 13.3 in sub 4; and 11.8 in sub 5). In a second experiment, it was shown that upon internalization, WSS virions lost their envelope most probably by fusion with the cellular membrane of the endosome (starting between 30 and 60 min post‐inoculation) and that afterwards the capsid started to become disintegrated (from 360 min post‐inoculation). Expression of new viral proteins was not observed. Incubation of haemocyte subpopulations with WSSV but not with UV‐killed WSSV and polystyrene beads resulted in a significant drop in haemocyte viability. To find the underlying mechanism, a third experiment was performed in which haemocyte subpopulations were exposed to a short WSSV DNA fragment (VP19) and CpG ODNs. These small DNA fragments induced cell death. In conclusion, WSSV is efficiently internalized by hyalinocytes, semigranulocytes and granulocytes, after which the virus loses its envelope; as soon as the capsids start to disintegrate, cell death is activated, which in part may be explained by the exposure of viral DNA to cellular‐sensing molecules.     

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.     

Effect of culture system on the nutrition and growth performance of Pacific white shrimp Litopenaeus vannamei (Boone) fed different diets

Two 8-week feeding trials were conducted with juvenile Pacific white shrimp, Litopenaeus vannamei (Boone) to compare the growth and performance of animals fed a series of experimental and commercial pelleted shrimp and fish feeds and dietary feeding regimes within an indoor running-water culture system and an outdoor zero-water-exchange culture system. The best overall shrimp growth performance was observed for animals fed the experimental shrimp diet and all-day feeding regime under outdoor zero-water-exchange culture conditions. Final body weight and average weekly growth rate under these conditions were 2.8 and 3.4 times greater, respectively, than animals of similar size fed with the same diet under indoor running-water culture conditions. Although direct comparison between indoor and outdoor culture systems is difficult because of the lower indoor water temperatures, and consequently lower mean daily feed intake of animals, it is believed that the higher growth and feed performance of animals reared under outdoor `green-water' culture conditions was primarily due to their ability to obtain additional nutrients from food organisms endogenously produced within the zero-water-exchange culture system. The most promising features of zero-water-exchange culture systems are that they offer increased biosecurity, reduced feed costs and water use for the farmer, and by doing so provide a potential avenue of moving the shrimp culture industry along a path of greater sustainability and environmental compatibility.     

The effects of variation in feed protein level on the culture of white shrimp, Litopenaeus vannamei (Boone) in low-water exchange experimental ponds

The present study was conducted to evaluate the effect of varying dietary protein level on pond water quality and production parameters of white shrimp Litopenaeus vannamei (Boone). Experimental units consisted of nine 400‐m² earthen ponds with a low water exchange. Two treatments were tested: treatment HP consisted of shrimp fed a high‐protein diet (40%) during the whole grow‐out, and treatment LP consisted of the use of a low‐protein diet for the complete farming period. No differences on any of the water quality parameters were observed among treatments. Excellent survival (over 85%) and feed conversion ratios (around 1.6), and acceptable growth (over 12 g) and biomass (from 1721 to 1793 kg ha^?1) were recorded in all experimental ponds. No significant differences in any of the production parameters were found among treatment groups.     

Apparent digestibility of selected feed ingredients for white shrimp Litopenaeus vannamei,Boone

Apparent digestibility coefficients of dry matter (DM), crude protein, crude lipid, gross energy, phosphorus and amino acids in Peruvian fish meal (FM), fermented soybean meal, extruded soybean meal, soybean meal, peanut meal, wheat gluten meal, corn gluten meal, shrimp byproduct meal, meat and bone meal (MBM), poultry meat meal and plasma protein meal (PPM) were determined for white shrimp (Litopenaeus vannamei). A reference diet (RF) and test diets (consisting of 70% RF diet and 30% of the feedstuff) were used with 0.5% chromic oxide as an external indicator. A total of 1440 shrimp (initial mean body weight 1.05 ± 0.01 g) were randomly stocked into thirty‐six 500‐L fibreglass tanks with 40 shrimp per tank and three tanks per diet. Faeces were collected from triplicate groups of shrimp by a faecal collection vessel attached to the shrimp‐rearing tank. The shrimp were fed to apparent satiation four times a day and the feeding experiment lasted for 6 weeks. Statistics indicate that apparent DM digestibilities for white shrimp (L. vannamei) were the highest for FM, ranged 52.83–71.23% for other animal products and 69.98–77.10% for plant products. The protein and lipid from plant and animal sources were well digested by white shrimp. Apparent protein and lipid digestibility were in the range 87.89–93.18% and 91.57–95.28%, respectively, in plant products, and 75.00–92.34% and 83.72–92.79%, respectively, for animal products. The white shrimp demonstrated a high capacity to utilize phosphorus in the ingredients. The apparent phosphorus digestibility ranges of animal feedstuffs and plant feedstuffs were 58.90–71.61% and 75.77–82.30% respectively. Amino acid availability reflected protein digestibility, except that in MBM, for which the availability of some amino acid was lower, possibly due to protein damage during processing. Digestibility information could promote the use of ingredient substitution in least‐cost formulated diets for white shrimp.     

白斑综合征病毒(WSSV)3种PCR检测方法的灵敏度比较

为了探讨不同PCR检测方法的灵敏度,分别利用TaqMan实时定量PCR、世界动物卫生组织(OIE)公布的巢式PCR引物(简称OIE)、黄海水产研究所种质资源与工程育种研究室(GB)设计的引物(简称GB)及2种巢式PCR对应的一步法PCR,对具有不同白斑综合征病毒(White Spot Syndrome Virus,WSSV)含量的中国明对虾(Fenneropenaeus chinensis)样品进行检测.结果显示,当使用已知病毒含量的标准品进行检测时,TaqMan实时定量PCR方法可以检测到l0个WSSV拷贝;OIE巢式PCR与GB巢式PCR方法分别可检测到104和103个WSSV拷贝;单独使用OIE巢式PCR的外引物和内引物扩增时,分别可检测到5×104和2.5×104个WSSV拷贝;单独使用GB巢式PCR的外引物和内引物进行一步法PCR扩增时,分别可检测到104和5×103个WSSV拷贝.使用上述PCR方法分别对44份未知WSSV含量的样品进行验证,定量PCR方法检测阳性率为84.09%,OIE巢式PCR与GB巢式PCR方法检测的阳性率分别为18.18%和27.27%;单独使用OIE巢式PCR的外引物和内引物扩增检测的阳性率均为15.91%;单独使用GB巢式PCR的外引物和内引物扩增检测的阳性率分别为18.18%和20.45%.根据以上结果,PCR方法检测WSSV的灵敏度由高到低依次为:定量PCR、巢式PCR、一步法PCR.     

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.