Effect of hyperthermia on the replication of white spot syndrome virus (WSSV) in Procambarus clarkii

The effect of hyperthermia on the development of white spot syndrome virus (WSSV) in the crayfish Procambarus clarkii was studied by competitive PCR. Crayfish were exposed to different temperatures (24 +/- 1 and 32 +/- 1 degrees C) after WSSV injection. No mortality was observed when crayfish were held at 32 +/- 1 degrees C, but mortality reached 100% when crayfish were transferred to 24 +/- 1 degrees C. Competitive PCR showed that viral levels at 32 +/- 1 degrees C remained at 10(5) copies mg(-1) tissue, while at 24 +/- 1 degrees C levels were significantly higher, rising from 10(4) to 10(10) copies mg(-1) tissue. These results suggest that hyperthermia reduces viral replication, but does not eliminate viral particles from WSSV-infected crayfish.     

Experimental infection of white spot syndrome virus in freshwater crayfish Pacifastacus leniusculus.

The signal freshwater crayfish Pacifastacus leniusculus was found to be susceptible to infection with white spot syndrome virus (WSSV). Histopathological observations of various tissues of virus-injected crayfish showed similar symptoms to those from WSSV-infected penaeid shrimp, but no appearance of white spots on the cuticle or reddish body colour were observed, although these are the prominent gross signs of white spot disease in shrimp. A gene probe for detecting WSSV was developed in order to detect the virus in affected cells and tissues using in situ hybridisation. Strong signals were observed in cells of virus-injected crayfish, but not in control-injected crayfish. The number of granular haemocytes in virus-injected crayfish was significantly higher than in sham-injected and non-injected crayfish from Days 5 to 8 (p < or = 0.05) and Days 3 to 8 (p < 0.01) post-injection, respectively. The proportion of granular haemocytes in virus-injected crayfish was also significantly higher than in sham-injected controls from Days 3 to 8 (p < 0.01). These results indicate that WSSV has a significant effect on the proportion of different haemocyte types in the freshwater crayfish.     

White spot syndrome virus (WSSV) interaction with crayfish haemocytes

WSSV particles were detected in separated granular cells (GCs) and semigranular cells (SGCs) by in situ hybridisation from WSSV-infected crayfish and the prevalence of WSSV-infected GCs was 5%, whereas it was 22% in SGCs. This indicates that SGCs are more susceptible to WSSV and that this virus replicated more rapidly in SGCs than in GCs and as a result the number of SGCs gradually decreased from the blood circulation. The effect of haemocyte lysate supernatant (HLS), containing the degranulation factor (peroxinectin), phorbol 12-myristate 13-acetate (PMA), the Ca(2+) ionophore A23187 on GCs from WSSV-infected and sham-injected crayfish was studied. The results showed that the percentage of degranulated GCs of WSSV-infected crayfish treated with HLS or PMA was significantly lower than that in the control, whereas no significant difference was observed when treated with the Ca(2+) ionophore. It was previously shown that peroxinectin and PMA have a degranulation effect via intracellular signalling involving protein kinase C (PKC), whereas the Ca(2+) ionophore uses an alternative pathway. HLS treatment of GCs and SGCs from WSSV-infected crayfish results in three different morphological types: non-spread, spread and degranulated cells. The non-spread cell group from both GCs and SGCs after treatment with HLS had more WSSV positive cells than degranulated cells, when detected by in situ hybridisation. Taken together, it is reasonable to speculate that the PKC pathway might be affected during WSSV infection. Another interesting phenomenon was that GCs from non-infected crayfish exhibited melanisation, when incubated in L-15 medium, while no melanisation was found in GCs of WSSV-infected crayfish. However, the phenoloxidase activities of both sham- and WSSV-injected crayfish in HLS were the same as well as proPO expression as detected by RT-PCR. This suggests that the WSSV inhibits the proPO system upstream of phenoloxidase or simply consumes the native substrate for the enzyme so that no activity is shown. The percentage of apoptotic haemocytes in WSSV-infected crayfish was very low, but it was significantly higher than that in the sham-injected crayfish on day 3 or 5 post-infection. The TEM observation in haematopoietic cells (hpt cells) suggests that WSSV infect specific cell types in haematopoietic tissue and non-granular hpt cells seem more favourable to WSSV infection.     

Thermoregulatory behavior of the crayfish Procambarus clarki in a burrow environment

The behavioral thermoregulation of the red swamp crayfish, Procambarus clarki, was investigated in its burrow environment. In the field, air and water temperatures within crayfish burrows fluctuated less compared with surface temperatures in the Mojave Desert. However, crayfish could still experience sub-optimal temperature regimes inside burrows. In the laboratory, P. clarki heated and cooled more rapidly in water than in air. In a thermal gradient, the crayfish selected a water temperature of 22 degrees C and avoided water temperatures above 31 degrees C and below 12 degrees C. Observations of behavior in an artificial burrow showed that P. clarki displayed three main shuttling behaviors between water and air in response to temperature. The number of bilateral emersions and emigrations, as well as the amount of time spent in air (in a 24 h period), were significantly greater at 34 degrees C than at 12, 16, 22 or 28 degrees C. This reflected an increased use of the behavioral thermoregulation at temperatures approaching the critical thermal maximum of this species. Upon migrating from 34 degrees C water into 38 degrees C air, crayfish body temperature decreased significantly. These periods of emersion were interspersed with frequent dipping in the water, allowing the crayfish to gain the benefits of evaporative cooling, without the physiological costs incurred by long-term exposure to air.     

The effect of different acclimation temperatures on the prophenoloxidase system and other defence parameters in Litopenaeus vannamei

Water temperature changes (higher and lower than 24 degrees C) were shown to have a significant effect on dopamine (DA) concentration, haemocyte count and the proPO system in the white shrimp Litopenaeus vannamei. No significant difference in any of the parameters was observed in the control group. DA concentration in haemolymph in the experimental groups increased to a peak value at 0.5 days; meanwhile serine protease (SP) activity and proteinase inhibitor (PI) activity decreased. Total haemocyte count (THC), differential haemocyte count (DHC) and PO activity were lowest at 1 day. All defence parameters became stable after 1-3 days, while the total haemocyte and large granular cell count stabilized after 6 days. After these stabilized, there was no significant difference in DA concentration and PI activity between the control and experimental groups, as was the case for the THC, DHC, PO and SP activities of shrimp held at higher temperatures. However these latter four parameters in the lower temperature groups were distinctly lower than the control group. alpha(2)-Macroglobulin activity in the experimental groups increased to a peak value after 1 day compared with the control and then stabilized after 6 days when the activity levels in higher temperature groups were higher than the control, while the lower temperature groups had no significant difference from the control. It was therefore concluded that water temperature changes modulated the immune system of L. vannamei.     

Effects of white spot syndrome virus infection on immuno-enzyme activities and ultrastructure in gills of Cherax quadricarinatus

In this study, we explored the pathogenic mechanism of white spot syndrome virus (WSSV) in crayfish, Cherax quadricarinatus, by investigating activities of enzymes related to innate immune function during infection. After 6-12 h of exposure to WSSV, the activities of four enzymes, phenoloxidase (PO), peroxidase (POD), superoxide dismutase (SOD) and lysozyme (LSZ), increased in the gills of C. quadricarinatus but then sharply decreased during longer infection times. Except for PO, the activities of other enzymes in the WSSV-infected crayfish (Group II) were significantly lower than those of the controls at 72 h post-exposure (P < 0.01). Interestingly, the enzyme activities in the group treated with polysaccharides before challenge with WSSV (Group III) were higher than those in Group II. This phenomenon demonstrated that the polysaccharides could improve the immuno-enzyme activities and enhance the organism's antiviral defenses. Morphological examination by transmission electron microscopy revealed abundant WSSV particles and significant damage in the gills of infected crayfish. WSSV infection caused parts of the gill epithelium and microvilli to be reduced in number and size or damaged; meanwhile, the mitochondria morphology changed, with parts of the cristae diminished leaving large vacuoles. Moreover, electron dense deposits appeared and heterochromatinized nuclei could be seen in blood cells with ruptured nuclear membranes and outflow of nucleoplasm. The findings of this study furthers our understanding of the biochemical alterations induced by viral infections, including changes in the antioxidant status, oxidative stress and lysozyme activity, which could help to advance strategies for control of WSSV in crayfish.     

Effects of high temperatures on functional responses of haemocytes in the clam Chamelea gallina

The effects of high temperatures on the clam, Chamelea gallina, generally recognised as a low tolerant bivalve species, were studied by evaluating some functional responses of the haemocytes. The animals were kept for 7days at 20, 25 and 30 degrees C and total haemocyte count (THC), phagocytosis, lysozyme activity (in both haemocyte lysate and cell-free haemolymph), activity and expression of the antioxidant enzyme superoxide dismutase (SOD) (in both haemocyte lysate and cell-free haemolymph) were chosen as biomarkers of exposure to high temperatures. The survival-in-air test was also performed. During the experiment, the clams showed differing burrowing behaviour: the animals kept at 20 and 25 degrees C burrowed completely, whereas at 30 degrees C the clams progressively emerged from the sediment and then remained on the surface. The highest temperature significantly increased THC, whereas it decreased the phagocytic activity of haemocytes. The haemocyte size frequency distribution in clams kept at 30 degrees C showed that the cell population of about 8-10microm was markedly reduced compared to clams kept at 20 and 25 degrees C. In clams maintained at 25 degrees C, lysozyme activity was significantly increased in haemocyte lysate, whereas it was markedly decreased in cell-free haemolymph. Total SOD activity significantly decreased in haemocytes from clams held at 30 degrees C whereas it increased in cell-free haemolymph from clams held at 25 degrees C and 30 degrees C. A significant decrease in haemocyte Mn-SOD and Cu/Zn-SOD activities was found with increasing temperature. In cell-free haemolymph, the highest Mn-SOD activity was recorded at 30 degrees C, whereas the Cu/Zn-SOD activity showed no significant changes in clams maintained at different temperatures. SOD isoform expression exhibited different patterns in haemocyte lysate and cell-free haemolymph. The resistance to air exposure of clams kept at 30 degrees C was shown to decrease significantly, LT(50) values fell from 6days in clams kept at 20 degrees C and 25 degrees C to 4days in those kept at 30 degrees C.     

Modulation of the innate immune system in white shrimp Litopenaeus vannamei following long-term low salinity exposure

Immune parameters, haemocyte lifespan, and gene expressions of lipopolysaccharide and β-glucan-binding protein (LGBP), peroxinectin (PX), integrin β, and α2-macroglobulin (α2-M) were examined in white shrimp Litopenaeus vannamei juveniles (0.48 ± 0.05 g) which had been reared at different salinity levels of 2.5‰, 5‰, 15‰, 25‰, and 35‰ for 24 weeks. All shrimp survived during the first 6 weeks. The survival rate of shrimp reared at 2.5‰ and 5‰ was much lower (30%) than that of shrimp reared at 15‰, 25‰, and 35‰ (76%~86%) after 24 weeks. Shrimp reared at 25% grew faster. Shrimp reared at 2.5‰ and 5‰ showed lower hyaline cells (HCs), granular cells (GCs), phenoloxidase activity (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity, and lysozyme activity, but showed a longer haemocyte lifespan, and higher expressions of LGBP, PX, integrin β, and α2-M. In another experiment, shrimp which had been reared at different salinity levels for 24 weeks were challenged with Vibrio alginolyticus (6 × 10(6) cfu shrimp(-1)), and WSSV (10(3) copies shrimp(-1)) and then released to their respective seawater. At 96-144 h, cumulative mortalities of shrimp reared at 2.5‰ and 5‰ were significantly higher than those of shrimp reared at 15‰, 25‰, and 35‰. It was concluded that following long-term exposure to 2.5‰ and 5‰ seawater, white shrimp juveniles exhibited decreased resistance against a pathogen due to reductions in immune parameters. Increases in the haemocyte lifespan and gene expressions of LGBP, integrin β, PX, and α2-M indicated that shrimp had the ability to expend extra energy to modulate the innate immune system to prevent further perturbations at low salinity levels.     

Change in water temperature on the immune response of Taiwan abalone Haliotis diversicolor supertexta and its susceptibility to Vibrio parahaemolyticus

Taiwan abalones, Haliotis diversicolor supertexta, held in 30 parts/per thousand seawater at 28 degrees C, were injected with TSB-grown Vibrio parahaemolyticus (1.6x10(5) cfu abalone(-1)) and then transferred to 20, 24, 28 and 32 degrees C. All abalones transferred to 32 degrees C died by 72 h. The mortality of V. parahaemolyticus-injected abalone held at 20 and 24 degrees C was significantly lower over 24-96 h, compared to animals held at 28 and 32 degrees C. In a separate experiment designed to measure immune function, abalones held in 30 per thousand seawater at 28 degrees C and then transferred to 20, 24, 28 and 32 degrees C were examined for total haemocyte count, phenoloxidase activity, respiratory burst, and phagocytic activity to V. parahaemolyticus after 24, 72 and 120 h. The phenoloxidase activity and phagocytic activity decreased significantly, whereas respiratory burst increased significantly in abalone transferred to 32 degrees C. It is concluded that transfer of abalone from 28 degrees C to 32 degrees C reduced their innate immunity and resistance against V. parahaemolyticus infection.     

Susceptibility of the Australian freshwater crayfish Cherax destructor albidus to white spot syndrome virus (WSSV)

Cherax destructor occurs naturally and/or is farmed in all Australian mainland states and territories and is of major cultural, economical and conservation significance. The aim of this study was to determine susceptibility of the commercially important subspecies C. destructor albidus to white spot syndrome virus (WSSV), a hazard to crustaceans and currently considered to be exotic to Australia. In challenge tests by intramuscular injection, C. destructor albidus displayed a similar level of susceptibility to white spot disease (WSD) as Penaeus monodon (i.e. 100% mortality in 3 d). In one oral challenge test where C. destructor albidus was subjected to significant temperature stress, over 50% died of severe WSD within 14 d post challenge. All dead and moribund crayfish displayed histopathological lesions typical for WSD and gave positive results for WSSV in DNA dot blot hybridization tests. Survivors to 30 d (n = 3) showed no lesions and gave negative dot blot test results. In a second oral challenge test without temperature stress, mortality was delayed but reached 75% by 30 d. However, no typical WSD lesions were observed in the dead, dying or surviving crayfish and dot blot test results were negative. The results suggested that C. destructor albidus would be less susceptible than P. monodon to WSSV exposure via natural routes of infection in farms and in the wild. This information may be useful for disease import risk analysis for WSSV.     

Preliminary study on haemocyte response to white spot syndrome virus infection in black tiger shrimp Penaeus monodon

White spot syndrome virus (WSSV) has been a major cause of shrimp mortality in aquaculture in the past decade. In contrast to extensive studies on the morphology and genome structure of the virus, little work has been done on the defence reaction of the host after WSSV infection. Therefore, we examined the haemocyte response to experimental WSSV infection in the black tiger shrimp Penaeus monodon. Haemolymph sampling and histology showed a significant decline in free, circulating haemocytes after WSSV infection. A combination of in situ hybridisation with a specific DNA probe for WSSV and immuno-histochemistry with a specific antibody against haemocyte granules in tissue sections indicated that haemocytes left the circulation and migrated to tissues where many virus-infected cells were present. However, no subsequent haemocyte response to the virus-infected cells was detected. The number of granular cells decreased in the haematopoietic tissue of infected shrimp. In addition, a fibrous-like immuno-reactive layer appears in the outer stromal matrix of tubule walls in the lymphoid organ of infected shrimp. The role of haemocytes in shrimp defence after viral infection is discussed.     

Changes in tissue defence system in white spot syndrome virus (WSSV) infected Penaeus monodon

The present study examined the changes occurring in the pro phenoloxidase system and antioxidant defence status in haemolymph, hepatopancreas and muscle tissue of white spot syndrome virus (WSSV) infected Penaeus monodon. Tiger shrimps (P. monodon) were infected with white spot virus by intramuscular injection of the virus inoculum. Levels of lipid peroxides and the activities of phenoloxidase, glutathione-dependent antioxidant enzymes [glutathione peroxidase (GPX), glutathione-S-transferase (GST)] and antiperoxidative enzymes [superoxide dismutase (SOD) and catalase (CAT)] were determined. WSSV infection induced a significant increase in lipid peroxidation in haemolymph, muscle and hepatopancreas of experimental P. monodon compared to normal controls. This was paralleled by significant reduction in the activities of phenol oxidase, glutathione-dependent antioxidant enzymes and antiperoxidative enzymes. The results of the present study indicate that the tissue antioxidant defence system in WSSV infected P. monodon is operating at a lower rate, which ultimately resulted in the failure of counteraction of free radicals, leading to oxidative stress as evidenced by the increased level of lipid peroxidation.     

Effects of oral recombinant VP28 expressed in silkworm (Bombyx mori) pupa on immune response and disease resistance of Procambarus clarkii

The envelope protein VP28 of white spot syndrome virus (WSSV) was overexpressed in the silkworm Bombyx mori, which was achieved by using a baculovirus (HyNPV) expression system and by making silkworm pupa as an alternative host, and then it was directly supplemented in diet at a dose of 20 g kg⁻¹ without purification. During a 30 day feeding period, the levels of phenoloxidase (PO) and superoxide dismutase (SOD) in the haemolymph of the tested Procambarus clarkii increased greatly (P < 0.05) when compared to the control crayfish fed with wild-type HyNPV baculovirus-infected silkworms or normal silkworms. Compared with two controls, the crayfish which had been infected for 20 days showed a significantly lower (P < 0.05) mean cumulative mortality (15.6%), which respectively, resulted in relative percent survivals (RPS) of 83.7 and 84.4%. The efficacy to inhibition of viral infection was further studied by in situ hybridization with a WSSV-specific DNA probe. The high levels of PO and SOD might be important for developing resistance against WSSV in these crayfish.     

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).     

Dietary beta-1,3-glucan effectively improves immunity and survival of Penaeus monodon challenged with white spot syndrome virus

The effectiveness of dietary beta-1,3-glucan (BG), derived from Schizophyllum commune, in modulating the non-specific immunity of the grass prawn Penaeus monodon and its resistance to white spot syndrome virus (WSSV) were investigated. Juvenile P. monodon (6.5+/-0.4 g) were fed for 20 days on a series of test diets containing graded levels of BG (0, 1, 2, 10, 20 g kg(-1)diet) and were then challenged by injection of WSSV. The haemolymph total haemocyte count (THC), phagocytosis (PI), phenoloxidase (PO), superoxide anion (O(2)(-)) and superoxide dismutase (SOD) production were measured at days 0, 1, 3, 6, 9, 12 and 24 after challenge, and shrimp survival rate was also recorded. All the shrimps fed on diets containing BG no more than 1 g kg(-1)died by day 12. Conversely, the survival rate of shrimp fed with the diet containing 10 g kg(-1)BG was significantly higher (P<0.05) by day 9 than that of the other groups. When screened by the WSSV PCR diagnostic procedure, the percentages of surviving juveniles of the BG 2, 10, 20 g kg(-1)groups that were 2-step WSSV negative, were 55, 65 and 65%, respectively. The haemolymph THC, PO, O(2)(-)and SOD production of the 2, 10 and 20 g kg(-1)BG diet groups dropped drastically immediately after the WSSV challenge but subsequently returned to normal. Therefore, oral administration of BG at an optimal level of 10 g kg(-1)diet for 20 days effectively enhanced the immune system and improved the survival of WSSV-infected P. monodon.     

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.     

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.     

White spot syndrome virus (WSSV) transmission from rotifer inoculum to crayfish

To test the possibility that shrimp pond rotifer resting eggs and hatched rotifers could transmit white spot syndrome virus (WSSV) to crayfish (Procambarus clarkii), we injected crayfish with rotifer and resting egg inocula that were WSSV-positive only by dot-blot analysis of PCR products. No crayfish became WSSV-positive after challenge with the resting egg inoculum. However, 1/15 crayfish became WSSV-positive after challenge with the rotifer inoculum. The results demonstrated that rotifers constitute a potential risk for WSSV transmission to crayfish and other cultivated crustaceans. However, the actual quantitative risk of transmission in an aquaculture setting depends on many variables that remain untested.     

Vaccination of shrimp (Penaeus chinensis) against white spot syndrome virus (WSSV)

Two structural protein genes, VP19 and VP466, of white spot syndrome virus (WSSV) were cloned and expressed in Sf21 insect cells using a baculovirus expression system for the development of injection and oral feeding vaccines against WSSV for shrimps. The cumulative mortalities of the shrimps vaccinated by the injection of rVP19 and rVP466 at 15 days after the challenge with WSSV were 50.2% and 51.8%, respectively. For the vaccination by oral feeding of rVP19 and rVP466, the cumulative mortalities were 49.2% and 89.2%, respectively. These results show that protection against WSSV can be generated in the shrimp, using the viral structural protein as a protein vaccine.     

红螯光壳螯虾白斑综合症血液病理学研究

采用Wright-Geimsa染色法和电镜技术对人工感染的红螯光壳螯虾(Cherax quadricarinatus)白斑综合症(White spot syndrome,WSS)血液病理学进行了研究。结果显示:患病螯虾血细胞总数、透明细胞(AH)数量极显著减少(P<0.01),大颗粒细胞(LGH)极显著增加(P<0.01);病毒感染后3种血细胞大小均有增加趋势,透明细胞和大颗粒细胞的核质比(NP)较感染病毒前极显著下降(P<0.01)。显微病理学变化主要表现为血涂片中血细胞明显减少,病变、破损或解体的细胞增多,至濒死期螯虾血液呈典型的溶血状态。超微病理学变化表现为血细胞受到了损伤。高尔基体变形、线粒体结构模糊破损;核膜变形核固缩、细胞核高度异染色质化;濒临死亡的螯虾血细胞细胞器和染色质溶解,胞浆水肿,细胞溶解坏死。在患病螯虾的血细胞核中清晰可见WSSV粒子。