Effect of water temperature on the immune response and infectivity pattern of white spot syndrome virus (WSSV) in freshwater crayfish

The susceptibility of two species of freshwater crayfish, Pacifastacus leniusculus and Astacus astacus, to white spot syndrome virus (WSSV) by intramuscular injection was compared and the results show that both species are susceptible to WSSV. The effect of water temperature on the development of white spot disease in crayfish was also studied. Crayfish were exposed to different temperatures after WSSV injection or oral exposure and the mortalities were recorded over a period of 45 days. No mortality was observed when crayfish were held at 4+/-2 degrees C or 12+/-2 degrees C and reached 100% when these crayfish transferred to 22+/-2 degrees C. The mortalities of nearly moribund crayfish at 22+/-2 degrees C with WSSV could be delayed after transfer to temperature below 16 degrees C. These results clearly show that low temperature affects the WSSV pathogenicity in crayfish. Moreover, haemocyte counts, phenoloxidase activity, mRNA levels of prophenoloxidase (proPO) and the lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) in crayfish exposed to various water temperatures were studied. Total haemocyte and granular cell counts of crayfish held at different temperatures were not significantly (P>0.05) different, except for the total haemocyte number at 18 degrees C was significantly (P<0.05) higher than in crayfish at 4 degrees C. The percentage of granular cells in crayfish held at 4 degrees C was the highest compared to crayfish maintained at other temperatures. The phenoloxidase activities in haemocyte lysate supernatant (HLS) of crayfish at all temperature groups remained similar. The amount of proPO-mRNAs in haemocytes was much higher than the amount of LGBP-m RNAs in all the experimental groups. However, there was no change in the level of pro PO-mRNA at the tested temperatures. Interestingly, the level of LGBP-mRNA of crayfish kept at 22 degrees C was much lower than in those held at lower temperatures. Proliferation of the haematopoietic tissues was higher at high temperatures which may support replication of WSSV, and explain the high mortality of crayfish with WSSV infection at high temperature. Based on these studies it is concluded that crayfish might act as a carrier of WSSV at low water temperature and could develop white spot disease if the water temperature is increased.     

Hyperthermia reduces viral load of white spot syndrome virus in Penaeus vannamei

We have previously reported that white spot syndrome virus-infected Penaeus vannamei (also called Litopenaeus vannamei) maintained at 32 degrees C show higher survival rates and a significant increase in number of apoptotic cells when compared to infected shrimp kept at 26 degrees C. As apoptosis plays an important part in the antiviral response of invertebrates, we hypothesized that this process would reduce WSSV replication, allowing the shrimp to control the disease and survive. To test this hypothesis, shrimp were orally infected and maintained at either 26 degrees C (Group 1) or 32 degrees C (Group 2), DNA was extracted from haemolymph collected at various times from 6 to 216 h post-infection, and the number of viral units was quantified by real time PCR using SYBR Green. In parallel, histological examination was carried out to confirm the WSSV infection and to rule out concomitant diseases. Linear regression of real time PCR units (rtPCRU) of WSSV from Group 1 showed a significant increase with time post-infection (r2 = 0.7383; p < 0.001). Conversely, there was no increase in rtPCRU with time post-infection in Group 2 (r2 = 0.142), indicating that hyperthermia inhibited, either directly or indirectly, viral replication. In addition, comparison between the groups showed no difference in WSSV rtPCRU up to 48 h post-infection. After 72 h, shrimp from Group 1 had a significantly higher viral rtPCRU (ANOVA, p < 0.001). We conclude that hyperthermia-associated WSSV rtPCRU reduction could reflect either an increase in the shrimp antiviral response, or a direct negative effect on viral replication, or both.     

Controlled bioassay systems for determination of lethal infective doses of tissue homogenates containing Taura syndrome or white spot syndrome virus

In vivo bioassay is the predominant method for evaluating the infectivity of materials potentially harboring viable shrimp pathogens and determining the relative susceptibility of shrimp species to viral infections. A controlled bioassay system for white spot syndrome virus (WSSV) and Taura syndrome virus (TSV) was developed utilizing 260 ml tissue culture flasks modified with an air exchange vent. Individual shrimp (1.00 +/- 0.25 g) were placed in separate flasks containing artificial seawater (100 to 150 ml) and held in an incubator at 27 degrees C. After a 48 h acclimation period, shrimp were either injected intramuscularly with viral inoculum or exposed to virus-laden water. Water was exchanged and shrimp were fed a commercial food pellet daily except 24 h post-infection (p.i.). Bioassays were performed with serial dilutions of stock viral preparations and shrimp mortality was recorded for 7 d p.i. Mortality rates of test animals permitted the estimation of the lethal infective doses, LD50 and LD90. The LD50 of the TSV injection preparation was estimated at viral dilutions of 1:7.692 x 10(7) (Trial 1) and 1:6.667 x 10(7) (Trial 2). The LD50s of 2 different WSSV injection preparations were estimated at 1:4.444 x 10(6) and 1:4.505 x 10(6). The LD50 for the TSV waterborne challenge was 1:9916 (Trial 1) and 1:15 710 (Trial 2) at 20 degrees C and 1:1272 at 27 degrees C. A second waterborne TSV inoculum challenge at 27 degrees C produced an LD50 of 1:2857. WSSV doses used in the waterborne challenge only reached 39% mortality, which did not allow for the estimation of effective lethal doses. Bioassay by injection proved to be a more reliable method of estimating viral infectivity compared to waterborne method. The dose-response curves developed can serve as a basis for controlled comparisons of relative levels of viral infectivity of specific tissue preparations and for controlled comparisons of relative susceptibility of shrimp species or stocks to viral pathogens.     

Characterization and diagnostic use of a monoclonal antibody for VP28 envelope protein of white spot syndrome virus

The gene encoding the VP28 envelope protein of White spot syndrome virus (WSSV) was cloned into expression vector pET-30a and transformed into the Escherichia coli strain BL21. After induction, the recombinant VP28 (rVP28) protein was purified and then used to immunize Balb/c mice for monoclonal antibody (MAb) production. It was observed by immuno-electron microscopy the MAbs specific to rVP28 could recognize native VP28 target epitopes of WSSV and dot-blot analysis was used to detect natural WSSV infection. Competitive PCR showed that the viral level was approximately 10⁴ copies/mg tissue in the dilution of gill homogenate of WSSV-infected crayfish at the detection limit of dot-blot assay. Our results suggest that dot-blot analysis with anti-rVP28 MAb could rapidly and sensitively detect WSSV at the early stages of WSSV infection.     

Characterization and Diagnostic Use of a Monoclonal Antibody for VP28 Envelope Protein of White Spot Syndrome Virus

The gene encoding the VP28 envelope protein of White spot syndrome virus (WSSV)was cloned into expression vector pET-30a and transformed into the Escherichia coli strain BL21.After induction,the recombinant VP28 (rVP28) protein was purified and then used to immunize Balb/c mice for monoclonal antibody (MAb) production.It was observed by immuno-electron microscopy the MAbs specific to rVP28 could recognize native VP28 target epitopes of WSSV and dot-blot analysis was used to detect natural WSSV infection.Competitive PCR showed that the viral level was approximately 104 copies/mg tissue in the dilution of gill homogenate of WSSV-infected crayfish at the detection limit of dot-blot assay.Our results suggest that dot-blot analysis with anti-rVP28 MAb could rapidly and sensitively detect WSSV at the early stages of WSSV infection.     

Does hyperthermia increase apoptosis in white spot syndrome virus (WSSV)-infected Litopenaeus vannamei?

Apoptosis plays a critical role in development and maintenance of multicellular organisms. It has also been described as an anti-viral mechanism in both insects and vertebrates. In fact, to escape the immune system and to increase their spread, some viruses such as baculovirus produce anti-apoptotic molecules. Conversely, a recent report showing a positive correlation between the number of apoptotic cells and the severity of white spot syndrome virus (WSSV) infection in Penaeus monodon suggested that apoptosis might be the cause of death in viral-infected shrimp. Searching for the mechanisms involved in the beneficial effect of hyperthermia for WSSV-infected Litopenaeus vannamei (also called Penaeus vannamei) and considering that hyperthermia increases apoptosis in other experimental models, we investigated the presence of apoptosis by Tdt-mediated dUTP nick-end label (TUNEL), from 4 of 168 h in 3 groups of 50 L. vannamei juveniles. Group 1 consisted of experimentally infected shrimp (intramuscular injection of 3 x 10(7) viral copies) kept at 25 degrees C, Group 2 of similarly infected shrimp kept at 32 degrees C and Group 3 of uninjected shrimp kept at 32 degrees C. Apoptosis was found only in WSSV-infected individuals. Shrimp at 25 degrees C were positive for apoptotic cells in 48 (16%) of their examined tissues or organs, compared to 62 (21%) for those at 32 degrees C. Moreover, shrimp at 32 degrees C also had a significantly higher overall mean apoptotic index (AI) than shrimp at 25 degrees C (p < 0.05). Comparison of mean AI at 72, 96 and 120 h post-infection showed that individuals at 32 degrees C presented a significantly higher values than those at 25 degrees C. These results suggested that hyperthermia might facilitate apoptosis in WSSV-infected L. vannamei and might be one of the mechanisms responsible for increased survival of infected shrimp maintained at 32 degrees C.     

Hydroprene prolongs developmental time and increases mortality in wandering-phase Indianmeal moth (Lepidoptera: Pyralidae) larvae

Wandering phase Indianmeal moth, Plodia interpunctella (Hübner), larvae were exposed to the label rate of hydroprene (1.9 x 10(-3) mg [AI] /cm2) sprayed on concreted petri dishes. Larvae were exposed for 1, 3, 6, 12, 18, 24, and 30 h and maintained at 16, 20, 24, 28, and 32 degrees C and 57% RH until adult emergence. Larval developmental time and mortality were significantly influenced by temperature and exposure intervals. Maximum developmental time (47.2 +/- 1.3 d) occurred at 16 degrees C, and the minimum developmental time (7.0 +/- 0.5 d) occurred at 32 degrees C. Larval mortality generally increased at all of the five tested temperatures as exposure period increased. The greatest mortality (82.0 +/- 0.1%) occurred when larvae were exposed for 30 h at 28 degrees C, and minimum mortality (0.0 +/- 0.5%) occurred at 16 degrees C when larvae were exposed for 1 h. The relationships between temperature, exposure period, and developmental time were described by polynomial models, based on lack-of-fit tests. Hydroprene has potential to be an effective alternative to conventional insecticides in surface treatments for Indianmeal moth management. Response-surface models derived from this study can be used in simulation models to estimate the potential consequences of hydroprene on Indianmeal moth population dynamics.     

Hydroprene prolongs developmental time and increases mortality of Indianmeal moth (Lepidoptera: Pyralidae) eggs

Eggs of the Indianmeal moth, Plodia interpunctella (Hübner), were exposed to the labeled rate of hydroprene (1.9 x 10(-3) mg [AI]/cm2) sprayed on concreted petri dishes. These eggs were exposed for 1, 3, 6, 12, and 18 h and until hatching (continuous exposure) at temperatures of 16, 20, 24, 28, and 32 degrees C and 57% RH until the emergence of first instars. The developmental time and egg mortality were significantly influenced by temperature and exposure periods. At 16 degrees C, hydroprene did not cause differences in developmental time when eggs were exposed for different periods. At temperatures >16 degrees C, both exposure period and temperature influenced developmental time. The maximum developmental time (15.0 +/- 0.2 d) occurred at 16 degrees C, and the minimum developmental time (3.2 +/- 0.3 d) occurred at 32 degrees C. Mortality increased when eggs were exposed to hydroprene for longer periods at all of the five tested temperatures. The greatest mortality (81.6 +/- 2.1%) occurred when eggs were continuously exposed on treated surfaces at 32 degrees C. We used developmental time instead of rate (1/ developmental time) to fit simple linear or polynomial regression models to the development data. Appropriate models for developmental time and mortality were chosen based upon lack-of-fit tests. The regression models can be used in predictive simulation models for the population dynamics of Indianmeal moth to aid in optimizing use of hydroprene for insect management.     

对虾白斑综合症病毒囊膜蛋白VP19的融合表达及其抗病毒感染作用

根据GenBank上WSSV囊膜蛋白基因vp19的序列,设计并合成引物,PCR扩增得到vp19基因并克隆到pGEM‐T载体中,经过BamHⅠ/HindⅢ酶切、连接并将vp19插入到pET32b表达载体中。用重组质粒pET32b-vp19转化大肠杆菌Origam(iDE3)pLysS,在IPTG诱导下,融合蛋白Trx-VP19以可溶性的形式得到表达,经SDS-PAGE和Western-blot检测显示其分子量与预期的大小相符合。目的蛋白经Ni2 柱纯化并定量后分别直接注射鳌虾和包被饲料投喂鳌虾。实验结果表明注射Trx-VP19可以提高鳌虾个体抗WSSV感染力的作用。     

Heat stress aggravates viral myocarditis in mice

While a beneficial effect of hyperthermia on viral infection has been hypothesized, there are no data on viral myocarditis in vivo. To investigate whether hyperthermia might attenuate the course or severity of viral myocarditis, we studied the pathological changes in a murine model of viral myocarditis. C3H mice were inoculated i.p. with the encephalomyocarditis virus (500 pfu). They were anesthetized and heated to a body temperature of 42.5+/-0.2 degrees C for 30 min. The latter was performed 4 hr before (n=28, HB) or 4 hr after (n=28, HA) the viral inoculation; results were compared with nonheated, infected controls (n=30, Cont). Cardiac viral titers were recorded on day 3, and the body weight (BW), heart weight (HW) and pathological changes were recorded on days 5 and 10. The incidence of spontaneous mortality on day 10 was significantly higher in the HA group (all deaths occurring by day 7 post-inoculation) as compared with the HB (35%) or Cont (18%) groups. Viral titers in the HA group (n=4) were significantly (P<0.05) higher than those in the Cont (n=7) or HB (n=7) groups (4.11+/-0.54 vs 3.01+/-0.44 and 3.23+/-0.45 LogTCID50/mg, respectively). On day 5, the HW, the BW/HW ratio, and the severity of myocardial necrosis were all significantly higher in the HA than in the Cont and HB groups. To confirm the effect of hyperthermia on the expression of heart shock protein (HSP), immunohistochemical staining was done in the virus-infected hearts. The nucleus and cytoplasm of the injured myocardium in the HA group strongly expressed HSP70, whereas the HB and Cont groups were negative for this protein. In conclusion, induction of hyperthermia after viral inoculation aggravated the viral-induced myocardial necrosis and increased the mortality rate in a murine model of viral myocarditis and induced myocardial heat shock protein 70.     

Prohibitin Interacts with Envelope Proteins of White Spot Syndrome Virus and Prevents Infection in the Red Swamp Crayfish,Procambarus clarkii

Prohibitins (PHBs) are ubiquitously expressed conserved proteins in eukaryotes that are associated with apoptosis, cancer formation, aging, stress responses, cell proliferation, and immune regulation. However, the function of PHBs in crustacean immunity remains largely unknown. In the present study, we identified a PHB in Procambarus clarkii red swamp crayfish, which was designated PcPHB1. PcPHB1 was widely distributed in several tissues, and its expression was significantly upregulated by white spot syndrome virus (WSSV) challenge at the mRNA level and the protein level. These observations prompted us to investigate the role of PcPHB1 in the crayfish antiviral response. Recombinant PcPHB1 (rPcPHB1) significantly reduced the amount of WSSV in crayfish and the mortality of WSSV-infected crayfish. The quantity of WSSV in PcPHB1 knockdown crayfish was increased compared with that in the controls. The effects of RNA silencing were rescued by rPcPHB1 reinjection. We further confirmed the interaction of PcPHB1 with the WSSV envelope proteins VP28, VP26, and VP24 using pulldown and far-Western overlay assays. Finally, we observed that the colloidal gold-labeled PcPHB1 was located on the outer surface of the WSSV, which suggests that PcPHB1 specifically binds to the envelope proteins of WSSV. VP28, VP26, and VP24 are structural envelope proteins and are essential for attachment and entry into crayfish cells. Therefore, PcPHB1 exerts its anti-WSSV effect by binding to VP28, VP26, and VP24, preventing viral infection. This study is the first report on the antiviral function of PHB in the innate immune system of crustaceans.     

Histological alterations and immune response in the crayfish Procambarus clarkii given rVP28-incorporated diets

There is growing evidence that recombinant VP28 protein (rVP28) can significantly enhance immune response and disease resistance against white spot syndrome virus (WSSV) in shrimp, although the underlying mechanisms have not been entirely clarified yet. The aim of this study was to determine the effect of rVP28 on histological alterations and WSSV-induced apoptosis in crayfish Procambarus clarkii. Crayfish were fed commercial diets supplemented with different doses of HyNPV-VP28 infected pupae (rVP28-hp) for 4 weeks. Results showed that rVP28-hp may be used as a safe and effective source of medicinal proteins in aquaculture when supplemented in diet at low dose (10 g kg(-1) and 50 g kg(-1)), which could obviously reduce the percentage of apoptotic cells in stomach, gut and hepatopancreas tissues induced by the WSSV challenge and showed the relative percent survival (RPS) of 82.2% and 94.4%, respectively. But rVP28-hp would be detrimental to crayfish survival and decrease resistance to WSSV infection at the high dose (100 g kg(-1) and 200 g kg(-1)), with the cumulative mortality of up to 48.2% and 56.6% after WSSV challenge, respectively. During a 28-d feeding period, the survival rate of crayfish was only 54.5%-75.6%, and histopathological observation showed that one of the principal lesions was serious cell swelling, vacuolar degeneration and necrosis in hepatopancreatic epithelia and myocardial cells. These results suggested that rVP28-hp can influence the immune functions of crayfish in a dose-dependent manner, and the rVP28-hp at the dose of 50 g kg(-1) was recommended to prevent WSSV in crayfish culture.     

Time course of necrotizing hepatopancreatitis (NHP) in experimentally infected Litopenaeus vannamei and quantification of NHP-bacterium using real-time PCR

Necrotizing hepatopancreatitis (NHP), a severe bacterial disease affecting penaeid shrimp aquaculture, is caused by a gram-negative, pleomorphic, intracellular alpha-proteobacterium referred to as the NHP-bacterium (NHPB). The time course of NHP was investigated in experimentally infected juveniles of Kona stock Litopenaeus vannamei. Susceptible animals were individually isolated in 41 of aerated artificial seawater at salinity 30 +/- 1 ppt and maintained in a water bath at 30 +/- 1 degree C for 60 d. A total of 120 individuals were exposed per os to a 0.05 g piece of NHPB-infected hepatopancreas and 100 controls were exposed to uninfected tissue. At intervals of 3, 6, 9, 16, 23, 30, 37, 44, and 53 d post-exposure, 6 shrimp exposed to NHPB-infected tissue and 4 controls were randomly removed from the experiment; hepatopancreas samples were processed for histological and molecular analysis, and feces were processed for molecular diagnosis of NHPB infection. NHPB was first detected in the hepatopancreas through histology at 6 d post-exposure. All control shrimp were diagnosed as NHPB negative. NHPB infections classified as stage I (scattering of hepatopancreatic tubules with adjacent epithelial cells containing NHPB) and stage II (numerous infected tubules with occasional hemocyte infiltration) were observed from 6 to 37 d post-exposure. All animals that experienced NHPB-induced mortality from 16 to 51 d post-exposure were at stage III (numerous necrotic tubules, dense hemocyte infiltration, and presence of granulomas). NHPB is capable of infecting all hepatopancreatic cell types including embryonic, resorptive, fibrillar and blister-like cells. The percent of hepatopancreatic tubules containing NHPB in epithelial cells increased over time, representing bacteria multiplication and spread. Real-time PCR allowed for quantification of NHPB in hepatopancreas and feces. Over the course of infection, NHPB was present at 10(3) to 10(7) copies mg(-1) of hepatopancreas and 10(1) to 10(5) copies mg(-1) of feces. Lethal infections contained 10(6) to 10(7) copies mg(-1) of hepatopancreas and 10(3) to 10(6) copies mg(-1) of feces.     

An immersion of Gracilaria tenuistipitata extract improves the immunity and survival of white shrimp Litopenaeus vannamei challenged with white spot syndrome virus

The innate immunity and resistance against white spot syndrome virus (WSSV) in white shrimp Litopenaeus vannamei which received the Gracilaria tenuistipitata extract were examined. Shrimp immersed in seawater containing the extract at 0 (control), 400 and 600 mg L(-1) for 3 h were challenged with WSSV at 2 × 10(4) copies shrimp(-1). Shrimp not exposed to the extract and not received WSSV challenge served as unchallenged control. The survival rate of shrimp immersed in 400 mg L(-1) or 600 mg L(-1) extract was significantly higher than that of challenged control shrimp over 24-120 h. The haemocyte count, phenoloxidase activity, respiratory burst, superoxide dismutase activity, and lysozyme activity of shrimp immersed in 600 mg L(-1) extract were significantly higher than those of unchallenged control shrimp at 6, 6, 6, 6, and 6-24 h post-challenge. In another experiment, shrimp which had received 3 h immersion of 0, 400, 600 mg L(-1) extract were challenged with WSSV. The shrimp were then received a booster (3 h immersion in the same dose of the extract), and the immune parameters were examined at 12-120 h post-challenge. The immune parameters of shrimp immersed in 600 mg L(-1) extract, and then received a booster at 9, 21, and 45 h were significantly higher than those of unchallenged control shrimp at 12-48 h post-challenge. In conclusion, shrimp which had received the extract exhibited protection against WSSV as evidenced by the higher survival rate and higher values of immune parameters. Shrimp which had received the extract and infected by WSSV showed improved immunity when they received a booster at 9, 21, and 45 h post-WSSV challenge. The extract treatment caused less decrease in PO activity, and showed better performance of lysozyme activity and antioxidant response in WSSV-infected shrimp.     

Laboratory study on the effects of temperature and three ventilation rates on infestations of Varroa destructor in clusters of honey bees (Hymenoptera: Apidae)

In this study, reduced levels of ventilation were applied to small clusters of bees under controlled conditions to determine whether lowered ventilation rates and the resulting increased levels of CO2 could increase the mortality rates of varroa. Two experiments were performed at two different temperatures (10 degrees C and 25 degrees C). Both experiments compared varroa mortality among high (360 liters/h), medium (42.5 liters/h), and low (14 liters/h) rates of ventilation. The clusters of bees (approximately 300 worker bees) in bioassay cages with 40 introduced varroa mites were placed into self-contained glass chambers and were randomly assigned to one of the three ventilation treatments within incubators set at either of the two temperatures. Bee and varroa mortality and the levels of CO2 concentration were measured in each of the experimental chambers. In both experiments, CO2 levels within the chamber increased, with a decrease in ventilation with CO2 reaching a maximum of 1.2 +/- 0.45% at 10 degrees C and 2.13 +/- 0.2% at 25 degrees C under low ventilation. At high ventilation rates, CO2 concentration in chamber air was similar at 10 degrees C (1.1 +/- 1.5%) and 25 degrees C (1.9 +/- 1.1%). Both humidity and CO2 concentration were higher at 25 degrees C than at 10 degrees C. Bee mortality was similar within all ventilation rate treatments at either 10 degrees C (11.5 +/- 2.7-19.3 +/- 3.8%) or 25 degrees C (15.2 +/- 1.9-20.7 +/- 3.5%). At 10 degrees C, varroa mortality (percentage dead) was greatest in the high ventilation treatment (12.2 +/- 2.1%), but only slightly higher than under low (3.7 +/- 1.7%) and medium ventilation (4.9 +/- 1.6%). At 25 degrees C, varroa mortality was greatest under low ventilation at 46.12 +/- 7.7% and significantly greater than at either medium (29.7 +/- 7.4%) or low ventilation (9.5 +/- 1.6.1%). This study demonstrates that at 25 degrees C, restricted ventilation, resulting in high levels of CO2 in the surrounding environment of small clusters of honey bees, has the potential to substantially increase varroa mortality.     

Changes in mortality and immunological variables of Litopenaeus vannamei parents and their filial families infected with white spot syndrome under different experimental conditions

In order to find changes in mortality and immunological variables of Litopenaeus vannamei parents and the filial WSSV-resistant and -susceptible families after infection with WSSV under different experimental conditions, the haemolymph total haemocyte count (THC), phenoloxidase (PO), and superoxide dismutase (SOD) activities were measured at days 0, 1, 3, 6, 9, 12 and 15 after challenge and shrimp mortality was also recorded. When shrimps were challenged with 10(-3) (1.29x10(6)copiesmL(-1)), 10(-4) (1.29x10(5)copiesmL(-1)) or 10(-5) (1.29x10(4)copiesmL(-1)) WSSV stock solution (0.1mLshrimp(-1)), the cumulative mortalities (mean+/-S.E.) on day 15 were 100+/-0%, 79.3+/-1.1%, and 21.7+/-2.3%, respectively. Among shrimps challenged with 10(-4) (1.29x10(5)copiesmL(-1)) WSSV dilution (0.1mLshrimp(-1)), the cumulative mortalities (mean+/-S.E.) on day 15 in high-density (100shrimpsm(-3)), middle-density (50shrimpsm(-3)), and low-density (25shrimpm(-3)) groups were 95.5+/-0%, 84.7+/-0%, and 72.3+/-0%, respectively. The immunological variables including THC, PO, and SOD were decreased significantly at the beginning of infection stage, while these immunological variables for survivors reached almost the similar levels to the non-infection control group on day 15 after challenge with 10(-4) (1.29x10(5)copiesmL(-1)) WSSV dilution (0.1mLshrimp(-1)). Cumulative mortality (mean+/-S.E.) on day 15 in 17 filial families (G(2)) ranged from 13.3+/-1.9% to 100+/-0% when shrimps were challenged with 10(-4) (1.29x10(5)copiesmL(-1)) WSSV dilution (0.1mLshrimp(-1)). Although, the PO and SOD activities for shrimps in the WSSV-resistant family were slightly higher than those in the WSSV-susceptible family at the same sampling time after infection, these differences were not significant (p<0.05).     

Clearance of white spot syndrome virus (WSSV) and immunological changes in experimentally WSSV-injected Macrobrachium rosenbergii

A time course experimental challenge of WSSV was carried out to examine the clearance of WSSV in Macrobrachium rosenbergii and the consequent immunological changes. The experimental animals were injected with WSSV and the samples of gills, pleopods, head soft tissue and hemolymph were collected at different intervals of 1, 3, 5, 10, 25, 50, 75 and 100days post infection (p.i.). WSSV infection and clearing were confirmed by single step PCR, nested PCR and bioassay. At 3days p.i., M. rosenbergii became lethargic and stopped feeding in contrast to the control prawns that behaved and fed normally. However, the WSSV-injected prawns suffered no mortality during the experimental period and recovered without any further gross signs of disease or any mortality over a period of 100days p.i. The single step PCR analysis showed positive at 1, 3 and 5days p.i. in gills, head soft tissue, pleopods and hemolymph, and all the organs showed negative at 10days p.i. onwards. The nested PCR results showed that all organs were positive for WSSV from 3days p.i. and extended up to 25days p.i. At 50days p.i, head soft tissue sample alone showed WSSV-positive while all other organs were negative by nested PCR. All the organs at 75 and 100days p.i. showed nested PCR negative for WSSV as observed in the control prawn. The hemolymph collected from experimentally infected M. rosenbergii at 1, 3 and 5days p.i. caused 100% mortality at 40h p.i., 55h p.i. and 72h p.i, respectively in Penaeus monodon whereas hemolymph collected at 10, 25, 50, 75 and 100days p.i. failed to cause mortality in shrimp. The moribund shrimp showed WSSV-positive and surviving shrimp showed negative by PCR. Immunological parameters such as proPO, O(2)(-) and clotting time in WSSV-injected M. rosenbergii were found to be significantly higher than those of the control groups, whereas THC and superoxide dismutase were significantly lower when compared to control groups.     

Fenneropenaeus indicus is protected from white spot disease by oral administration of inactivated white spot syndrome virus

Fenneropenaeus indicus could be protected from white spot disease (WSD) caused by white spot syndrome virus (WSSV) using a formalin-inactivated viral preparation (IVP) derived from WSSV-infected shrimp tissue. The lowest test quantity of lyophilized IVP coated onto feed at 0.025 g(-1) (dry weight) and administered at a rate of 0.035 g feed g(-1) body weight d(-1) for 7 consecutive days was sufficient to provide protection from WSD for a short period (10 d after cessation of IVP administration). Shrimp that survived challenges on the 5th and 10th days after cessation of IVP administration survived repeated challenges although they were sometimes positive for the presence of WSSV by a polymerase chain reaction (PCR) assay specific for WSSV. These results suggest that F. indicus can be protected from WSD by simple oral administration of IVP.     

Bioassay evidence for the transmission of WSSV by the harpacticoid copepod Nitocra sp

White Spot Syndrome Virus (WSSV) is now one of the most devastating and virulent viral agents threatening the penaeid shrimp culture industry and has been responsible for serious economic losses for shrimp farms worldwide. One remarkable characteristic of WSSV is its wide reservoir range, which contributes to its wide geographical distribution. Among epizootiological surveys, there is substantial evidence for WSSV-positive copepods found in shrimp farming ponds. Therefore, copepods are suspected to be the vector of WSSV. In the present study, nested-PCR analysis showed positive results in the harpacticoid copepod Nitocra sp. exposed to WSSV by virus-phytoplankton adhesion route. Oral route and intramuscular injection were used to test the pathogenicity of WSSV isolated from the WSSV-positive Nitocra sp. For the oral route of infection, Marsupenaeus japonicus postlarvae were fed with WSSV-positive copepods. The shrimp postlarvae in the infected treatment became WSSV-positive and occurred 52.50+/-5.00% mortality which was significant higher (P <0.05) than that in the control treatment (20.00+/-0.00%) when postlarvae were fed with WSSV free copepods. In the intramuscular injection challenge, M. japonicus juveniles were injected with the copepods inoculum extracted from the WSSV-positive Nitocra sp., and showed 72.50+/-9.57% mortality which was also significant higher (P <0.05) than that in the control treatment (22.50+/-5.00%) when juveniles were received mock injection of a tissue homogenate prepared from WSSV-negative Nitocra sp. Based on these laboratory challenge studies, it was confirmed that the copepods can serve as a vector in WSSV transmission.