Protection of Penaeus monodon against white spot syndrome virus by oral vaccination

White spot syndrome virus (WSSV) occurs worldwide and causes high mortality and considerable economic damage to the shrimp farming industry. No adequate treatments against this virus are available. It is generally accepted that invertebrates such as shrimp do not have an adaptive immune response system such as that present in vertebrates. As it has been demonstrated that shrimp surviving a WSSV infection have higher survival rates upon subsequent rechallenge, we investigated the potential of oral vaccination of shrimp with subunit vaccines consisting of WSSV virion envelope proteins. Penaeus monodon shrimp were fed food pellets coated with inactivated bacteria overexpressing two WSSV envelope proteins, VP19 and VP28. Vaccination with VP28 showed a significant lower cumulative mortality compared to vaccination with bacteria expressing the empty vectors after challenge via immersion (relative survival, 61%), while vaccination with VP19 provided no protection. To determine the onset and duration of protection, challenges were subsequently performed 3, 7, and 21 days after vaccination. A significantly higher survival was observed both 3 and 7 days postvaccination (relative survival, 64% and 77%, respectively), but the protection was reduced 21 days after the vaccination (relative survival, 29%). This suggests that contrary to current assumptions that invertebrates do not have a true adaptive immune system, a specific immune response and protection can be induced in P. monodon. These experiments open up new ways to benefit the WSSV-hampered shrimp farming industry.     

Protection of Penaeus monodon against white spot syndrome virus using a WSSV subunit vaccine

Although invertebrates lack a true adaptive immune response, the potential to vaccinate Penaeus monodon shrimp against white spot syndrome virus (WSSV) using the WSSV envelope proteins VP19 and VP28 was evaluated. Both structural WSSV proteins were N-terminally fused to the maltose binding protein (MBP) and purified after expression in bacteria. Shrimp were vaccinated by intramuscular injection of the purified WSSV proteins and challenged 2 and 25 days after vaccination to assess the onset and duration of protection. As controls, purified MBP- and mock-vaccinated shrimp were included. VP19-vaccinated shrimp showed a significantly better survival (p<0.05) as compared to the MBP-vaccinated control shrimp with a relative percent survival (RPS) of 33% and 57% at 2 and 25 days after vaccination, respectively. Also, the groups vaccinated with VP28 and a mixture of VP19 and VP28 showed a significantly better survival when challenged two days after vaccination (RPS of 44% and 33%, respectively), but not after 25 days. These results show that protection can be generated in shrimp against WSSV using its structural proteins as a subunit vaccine. This suggests that the shrimp immune system is able to specifically recognize and react to proteins. This study further shows that vaccination of shrimp may be possible despite the absence of a true adaptive immune system, opening the way to new strategies to control viral diseases in shrimp and other crustaceans.     

Protection of Penaeus monodon against white spot syndrome virus by inactivated vaccine with herbal immunostimulants

To improve the immune response in tiger shrimp Penaeus monodon against WSSV infection, juveniles (350 ± 10 mg) were vaccinated with formalin-inactivated WSSV and fed with herbal immunostimulants. The methanolic extracts of herbal immunostimulants such as Acalypha indica, Cynodon dactylon, Picrorrhiza kurrooa, Withania somnifera and Zingiber officinalis were incorporated in formulated diets at different concentrations; 250 (ED(1)), 500 (ED(2)), 1000 (ED(3)) and 2000 (ED(4)) mg kg(-1) of feed and fed for 60 days after vaccination. After 30 and 60 days intervals of feeding, the shrimps were challenged with WSSV, which were isolated and propagated from the infected crustaceans. The shrimps fed with control diets (C(1)) succumbed to death within 5 days after WSSV challenge, when no vaccination and immunostimulations were given. The other control groups (C(2) and C(3)) had slight improvements in all parameters including survival. The percentage survival was significantly (P < 0.05) increased to 30, 50 and 60% in the ED(2), ED(3) and ED(4) diets respectively after 60 days challenging. The better haematological, biochemical and immunological parameters were also found in the herbal extracts supplemented diets fed vaccinated shrimps. The present study revealed that the combined effect of immunostimulation and vaccination helped to boost the immune system against WSSV infection and hence this application can be adopted for shrimp culture.     

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.     

Prevalence of white spot syndrome virus (WSSV) in wild shrimp Penaeus monodon in the Philippines

Prevalence of white spot syndrome virus (WSSV) was determined using polymerase chain reaction (PCR) methodology on DNA extracted from the gills of wild black tiger shrimp Penaeus monodon collected from 7 sampling sites in the Philippines. These 7 sampling sites are the primary sources of spawners and broodstock for hatchery use. During the dry season, WSSV was detected in shrimp from all sites except Bohol, but during the wet season it was not detected in any site except Palawan. None of the WSSV-PCR positive shrimp showed signs of white spots in the cuticle. Prevalence of WSSV showed seasonal variations, i.e. prevalence in dry season (April to May) was higher than in the wet season (August to October). These results suggest that WSSV has already become established in the local marine environment and in wild populations of P. monodon. Thus, broodstock collected during the dry season could serve as the main source of WSSV contamination in shrimp farms due to vertical transmission of the virus in hatcheries.     

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.     

Sodium alginate from Sargassum wightii retards mortalities in Penaeus monodon postlarvae challenged with white spot syndrome virus

Sodium alginate extracted from brown seaweed Sargassum wightii (16.35 ± 1.42%, mean [±SD] yield from 5 extractions) was prepared as a powder or beads and used to enrich Artemia nauplii at concentrations of 100, 200, 300 and 400 mg l-1. The alginate-enriched nauplii were fed to Penaeus monodon shrimp postlarvae (PL) stage 15 (PL15, i.e. 15 d old) for 20 d. Mean weight gain and specific growth rate over this period were 0.24 g and 15.8%, respectively, in PL groups not fed alginate, and 0.20-0.28 g and 14.7-16.5%, respectively, in PL groups fed alginate. Amongst PL35 then challenged with white spot syndrome virus (WSSV) by immersion, all PL not fed alginate died within 9 d. However, amongst PL fed the 4 concentrations of alginate powder or beads, mortality rates reduced with increasing alginate concentration, and between 25 and 32% PL remained alive when the bioassay was terminated on Day 21. Amongst alginate-fed PL groups compared with the control group, mortality was reduced by 26.5 to 58.4%. Nested PCR detection of WSSV revealed sodium alginate concentration-dependent reductions in infection loads. The data indicate that sodium alginate extracted from brown seaweed and fed to P. monodon can retard progression of WSSV disease.     

Relationship between white spot syndrome virus and indicators of quality in Penaeus monodon postlarvae in Karnataka,India

White spot disease (WSD) is a viral disease of shrimp caused by white spot syndrome virus (WSSV). Stocking WSSV-infected seed has been implicated as a major risk factor for outbreaks of WSD. In addition, the quality of postlarvae batches has been proposed as a predictor for good crops. This paper describes the relationship between indicators of quality and WSSV in postlarvae (PL) of Penaeus monodon from Karnataka, India, over the period September 1999 to January 2000. Three outcome variables were considered: the WSSV status of the PL, as determined by PCR, and 2 subjective assessments of PL quality, namely the activity of the PL and the quality of the PL as determined by research assistants and farmers, respectively. Of the 73 batches of PL, 49.3% from a random sample of farms tested positive for WSSV. After adjusting for confounding, stocking earlier in the growing season and duration of transportation were the main risk factors for the presence of WSSV. The quality assessed by farmers and the PL activity assessed by research assistants showed only fair agreement (kappa 0.252) reaffirming the subjective nature of such techniques. The only variables consistently associated with either assessment of quality in univariate analysis were PL length, number per bag and salinity of the water in the delivery bags. After adjusting for confounding, no single variable was consistently associated with PL quality and activity. The research assistants' assessment of PL activity was also associated with the hatchery and a brown-orange hepatopancreas in univariate analysis. After adjusting for confounding, a brown-orange hepatopancreas was still significant and fitted into the model together with the salinity of the water in the PL bags. The farmers' assessment of quality was associated with PL length, date of stocking and duration of transportation in both univariate and multivariable analyses. There was no relationship between quality assessment and WSSV in PCR-positive PL.     

Comprehensive cross-clade neutralization analysis of a panel of anti-human immunodeficiency virus type 1 monoclonal antibodies

Broadly neutralizing monoclonal antibodies (MAbs) are potentially important tools in human immunodeficiency virus type 1 (HIV-1) vaccine design. A few rare MAbs have been intensively studied, but we still have a limited appreciation of their neutralization breadth. Using a pseudovirus assay, we evaluated MAbs from clade B-infected donors and a clade B HIV(+) plasma against 93 viruses from diverse backgrounds. Anti-gp120 MAbs exhibited greater activity against clade B than non-B viruses, whereas anti-gp41 MAbs exhibited broad interclade activity. Unexpectedly, MAb 4E10 (directed against the C terminus of the gp41 ectodomain) neutralized all 90 viruses with moderate potency. MAb 2F5 (directed against an epitope adjacent to that of 4E10) neutralized 67% of isolates, but none from clade C. Anti-gp120 MAb b12 (directed against an epitope overlapping the CD4 binding site) neutralized 50% of viruses, including some from almost every clade. 2G12 (directed against a high-mannose epitope on gp120) neutralized 41% of the viruses, but none from clades C or E. MAbs to the gp120 V3 loop, including 447-52D, neutralized a subset of clade B viruses (up to 45%) but infrequently neutralized other clades (相似文献   

Effect of processing treatments on the white spot syndrome virus DNA in farmed shrimps (Penaeus monodon)

Aims: To investigate the effect of processing treatments on the destruction of white spot syndrome virus (WSSV) DNA in WSSV‐infected farmed shrimps (Penaeus monodon). Methods and Results: The presence of WSSV was tested by single step and nested polymerase chain reaction (PCR). The primers 1s5 & 1a16 and IK1 & IK2 were used for the single step PCR and primers IK1 & IK2–IK3 & IK4 were used for the nested PCR. Various processing treatments such as icing, freezing, cooking, cooking followed by slow freezing, cooking followed by quick freezing, canning, and cold storage were employed to destroy the WSSV DNA. Of the processing treatments given, cooking followed by quick freezing was efficient in destroying WSSV DNA in WSSV‐infected shrimp products. Canning, and cooking followed by slow freezing process had some destructive effect on the WSSV DNA, as WSSV DNA in such processed shrimp products was detected only by nested PCR. Icing, slow freezing, quick freezing, and cooking processes had no effect on the destruction of WSSV DNA. A gradual increase in the destruction of WSSV DNA was observed as the cold storage period increased. Conclusion: The results indicated that cooking followed by quick freezing process destroy the WSSV DNA. Significance and Impact of the Study: WSSV can be destroyed by cooking followed by quick freezing and this combined process can reduce the disease transmission risks from commodity shrimps to native shrimps.     

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.     

Vibrio alginolyticus associated with white spot disease of Penaeus monodon

In February 2000, white spot disease outbreaks occurred among cultured Penaeus monodon in extensive shrimp farms on the southwest coast of India. Bacteria were isolated from infected shrimp that showed reddish body coloration and white spots in the cuticle. The isolates were screened on thiosulfate citrate bile salt sucrose (TCBS) agar plates for the selection of Vibrio species. The primary isolate (QS7) was characterized as V. alginolyticus based on morphological, biochemical and physiological characteristics. Antibiotic sensitivity tests of QS7 indicated that the isolate was highly sensitive to chloramphenicol, ciprofloxacin, nalidixic acid and streptomycin. Pathogenicity tests confirmed that the isolate was virulent for P. monodon. Based on the lethal dose (LD50) value (5 x 10(6) cfu per shrimp), it was inferred that shrimp weakened by white spot syndrome virus would succumb to secondary infection by QS7.     

White spot syndrome virus (WSSV) in cultured Penaeus monodon in the Philippines

The prevalence and geographic distribution of white spot syndrome virus (WSSV) infection among cultured penaeid shrimp in the Philippines was determined from January to May, 1999, using PCR (polymerase chain reaction) protocol and Western blot assays. A total of 71 samples consisting of 18 post-larvae (PL) and 53 juvenile/adult shrimp samples (56 to 150 days-of-culture, DOC) were screened for WSSV. Of the 71 samples tested, 51 (72%) were found positive for WSSV by PCR: 61% (31/51) after 1-step PCR and 39% (20/51) after 2-step, non-nested PCR. Of the PL and juvenile/adult shrimp samples tested, 50 and 79% were positive for WSSV, respectively. By Western blot, only 6 of the 51 (12%) PCR-positive samples tested positive for WSSV. Of the 20 samples negative for WSSV by PCR, all tested negative for WSSV by Western blot assay. This is the first report of the occurrence of WSSV in the Philippines.     

Studies on effective PCR screening strategies for white spot syndrome virus (WSSV) detection in Penaeus monodon brooders.

We re-tested stored (frozen) DNA samples in 5 independent polymerase chain reaction (PCR) replicates and confirmed that equivocal test results from a previous study on white spot syndrome virus (WSSV) in brooders and their offspring arose because amounts of WSSV DNA in the test samples were near the sensitivity limits of the detection method. Since spawning stress may trigger WSSV replication, we also captured a fresh batch of 45 brooders for WSSV PCR testing before and after spawning. Replicates of their spawned egg batches were also WSSV PCR tested. For these 45 brooders, WSSV prevalence before spawning was 67% (15/45 1-step PCR positive, 15/45 2-step PCR positive and 15/45 2-step PCR negative). Only 27 (60%) spawned successfully. Of the successful spawners, 56% were WSSV PCR positive before spawning and 74% after. Brooders (15) that were heavily infected (i.e. 1-step PCR positive) when captured mostly died within 1 to 4 d, but 3 (20%) did manage to spawn. All their egg batch sub-samples were 1-step PCR positive and many failed to hatch. The remaining 30 shrimp were divided into a lightly infected group (21) and a 2-step PCR negative group (9) based on replicate PCR tests. The spawning rates for these 2 groups were high (81 and 78%, respectively). None of the negative spawners (7) became WSSV positive after spawning and none gave egg samples positive for WSSV. In the lightly infected group (21), 6 brooders were 2-step WSSV PCR negative and 15 were 2-step WSSV PCR positive upon capture. However, all of them were WSSV PCR positive in replicate tests and after spawning or death. Four died without spawning. The remaining 17 spawned but only 2 gave egg samples PCR negative for WSSV. The other 15 gave PCR positive egg samples, but they could be divided into 2 spawner groups: those (7) that became heavily infected (i.e. 1-step PCR positive) after spawning and those (8) that remained lightly infected (i.e. became or remained 2-step PCR positive only). Of the brooders that became heavily infected after spawning, almost all egg sample replicates (91 %) tested 2-step PCR positive. One brooder even gave heavily infected (i.e. 1-step PCR positive) egg samples. For the brooders that remained lightly infected after spawning, only 27% of the egg sample replicates were 2-step PCR positive. Based on these results, we recommend that to avoid false negatives in WSSV PCR brooder tests screening tests should be delayed until after spawning. We also recommend, with our PCR detection system, discarding all egg batches from brooders that are 1-step PCR positive after spawning. On the other hand, it may be possible with appropriate monitoring to use eggs from 2-step PCR positive brooders for production of WSSV-free or lightly infected postlarvae. These may be used to stock shrimp ponds under low-stress rearing conditions.     

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.     

A Kazal type serine proteinase SPIPm2 from the black tiger shrimp Penaeus monodon is capable of neutralization and protection of hemocytes from the white spot syndrome virus

A Kazal type serine proteinase SPIPm2 is abundantly expressed in the hemocytes and shown to be involved in innate immune response against white spot syndrome virus (WSSV) in Penaeus monodon. The SPIPm2 is expressed and stored in the granules in the cytoplasm of semigranular and granular but not the hyaline hemocytes. Upon WSSV challenge and progression of infection, the SPIPm2 was secreted readily from the semigranular and granular hemocytes. The more they secreted the SPIPm2, the less they were distinguishable from the hyaline cells. The WSSV-infected cells were either semigranular or granular hemocytes or both and depleted of SPIPm2. The rSPIPm2 was able to temporarily and dose-dependently neutralize the WSSV and protect the hemocytes from viral infection judging from the substantially less expression of WSSV late gene VP28. The antiviral activity was very likely due to the binding of SPIPm2 to the components of viral particle and hemocyte cell membrane.     

Protein profiling in the gut of Penaeus monodon gavaged with oral WSSV‐vaccines and live white spot syndrome virus

White spot syndrome virus (WSSV) is a pathogen that causes considerable mortality of the farmed shrimp, Penaeus monodon. Candidate ‘vaccines’, WSSV envelope protein VP28 and formalin‐inactivated WSSV, can provide short‐lived protection against the virus. In this study, P. monodon was orally intubated with the aforementioned vaccine candidates, and protein expression in the gut of immunised shrimps was profiled. The alterations in protein profiles in shrimps infected orally with live‐WSSV were also examined. Seventeen of the identified proteins in the vaccine and WSSV‐intubated shrimps varied significantly compared to those in the control shrimps. These proteins, classified under exoskeletal, cytoskeletal, immune‐related, intracellular organelle part, intracellular calcium‐binding or energy metabolism, are thought to directly or indirectly affect shrimp's immunity. The changes in the expression levels of crustacyanin, serine proteases, myosin light chain, and ER protein 57 observed in orally vaccinated shrimp may probably be linked to immunoprotective responses. On the other hand, altered expression of proteins linked to exoskeleton, calcium regulation and energy metabolism in WSSV‐intubated shrimps is likely to symbolise disturbances in calcium homeostasis and energy metabolism.     

Seasonal variation in white spot syndrome virus-positive samples in broodstock and post-larvae of Penaeus monodon in Thailand

Records of PCR test results for white spot syndrome virus (WSSV) were reviewed in Thailand from 1998 to 2000 for wild Penaeus monodon broodstock purchased by hatcheries and for post-larvae (PL) brought by farmers to diagnostic laboratories for testing. Samples for PCR comprised DNA extracts from the last pleopod dissected from broodstock females after the first spawning and DNA extracts from whole, homogenized PL. There was a consistent pattern of fluctuation in percentage of WSSV-positive broodstock and PL. In broodstock, the fluctuation pattern was similar each year, with a low percentage (0 to 6%) from January to May and a higher percentage (6 to 18%) for the rest of the year, with a peak from September to November. The fluctuation pattern for PL was similar but offset with peaks and troughs occurring approximately 2 mo after those for the broodstock. The peak percentages of broodstock-positive samples were roughly constant from year to year, but those for PL decreased progressively in magnitude from 1998 to 2000. Examination of a small number of hatcheries in 2000 revealed that the percentage of WSSV-positive PL samples was significantly lower for hatcheries that routinely discarded WSSV-PCR-positive wild broodstock when compared to hatcheries that did not.