Susceptibility of testicular cell cultures of crab, Scylla serrata (Forskal) to white spot syndrome virus

Testicular cell culture of crab, Scylla serrata (Forskal) was used to study the effects of White spot syndrome virus (WSSV). We are showing the susceptibility of cell culture of crabs to WSSV. The proliferating cell culture of testes were maintained for more than 4 months in a medium prepared from L15 and crab saline supplemented with epidermal growth factor. The cell cultures inoculated with different concentrations of virus showed distinct cytopathic effects such as change in cell appearance, shrinkage and cell lysis. WSSV infection of cultured cells was confirmed by Nested PCR technique. The incorporation of viral DNA in cultured cells was shown by RAPD profile generated using 10-mer primers. The controls that were not exposed to WSSV did not show cytopathic effects. This work shows the usefulness of proliferating testicular cell culture for studying WSSV infection using molecular tools. Thus, this report gains significance as it opens new vistas for diagnostics and drugs for WSSV.     

WSSV对锯缘青蟹的致病性及血清酶指标影响

锯缘青蟹(Scylla serrata)俗称青蟹,是我国重要的海水养殖蟹类.近年来,浙江、福建、广东等青蟹主要养殖地区出现了严重的青蟹病害.对浙江省养殖青蟹的发病原因和流行病学调查发现,白斑综合征病毒(white spotsyndrome virus,WSSV)与青蟹发病存在较大相关性.为进一步研究WSSV对青蟹的致病性和发病机理,作者采用白斑综合征病毒的除菌过滤液,以1:10-1:10000稀释度注射感染青蟹,结果表明1:10、1:100感染组的青蟹死亡率达100%,1:1000感染组死亡率为66.7%,1:10000感染组死亡率为38.9%.根据攻毒悬液的病毒浓度计算出WSSV对青蟹的LD50为1.19×104拷贝/只(7.93×103拷贝/g组织);取WSSV感染青蟹血淋巴进行PCR检测,攻毒死亡青蟹的WSSV检出率为100%,表明WSSV对青蟹有很强的致病力.分析病毒感染濒死蟹的血清酚氧化酶(PO)、过氧化物酶(POD)、超氧化物歧化酶(SOD)、碱性磷酸酶(ALP)、谷丙转氨酶(GPT)、符草转氨酶(GOT)等主要酶指标,发现病毒感染青蟹的PO、POD和SOD活力明显低于对照组,而ALP、GPT和GOT的活力则明显高于对照组;用WSSV单克隆抗体对感染蟹进行免疫组化分析,发现WSSV主要侵染青蟹的鳃、甲壳下表皮、心脏、肠、胃等组织的上皮细胞,尤其以鳃上皮细胞损害最为严重.     

Lagenidium infection in eggs and larvae of mangrove crab (Scylla serrata) produced in Indonesia

A fungal infection occurred in the eggs and larvae of mangrove crab (Scylla serrata) in seed production in Bali, Indonesia. The causative fungus was classified as a member of the genusLagenidium (Oomycetes, Lagenidiales). After comparison of its biological and physiological characteristics with those ofL. callinectes ATCC 24973, a known parasite of various crustaceans, was concluded that the isolate is a new species ofLagenidium, L. thermophilum, because of its rapid and thermotolerant growth and unique discharge process. Fungal growth was observed on PYG agar containing 0–5.0% (w/v) NaCl and 0–2.5% (w/v) KCI. Similar pathogenicity toward the zoeae of swimming crab (Portunus trituberculatus) was demonstrated.     

Polychaete worms--a vector for white spot syndrome virus (WSSV)

The present work provides the first evidence of polychaete worms as passive vectors of white spot syndrome virus (WSSV) in the transmission of white spot disease to Penaeus monodon broodstocks. The study was based on live polychaete worms, Marphysa spp., obtained from worm suppliers/worm fishers as well as samples collected from 8 stations on the northern coast of Tamilnadu (India). Tiger shrimp Penaeus monodon broodstock with undeveloped ovaries were experimentally infected with WSSV by feeding with polychaete worms exposed to WSSV. Fifty percent of polychaete worms obtained from worm suppliers were found to be WSSV positive by 2-step PCR, indicating high prevalence of WSSV in the live polychaetes used as broodstock feed by hatcheries in this area. Of 8 stations surveyed, 5 had WSSV positive worms with prevalence ranging from 16.7 to 75%. Polychaetes collected from areas near shrimp farms showed a higher level of contamination. Laboratory challenge experiments confirmed the field observations, and > 60% of worms exposed to WSSV inoculum were proved to be WSSV positive after a 7 d exposure. It was also confirmed that P. monodon broodstock could be infected with WSSV by feeding on WSSV contaminated polychaete worms. Though the present study indicates only a low level infectivity in wild polychaetes, laboratory experiments clearly indicated the possibility of WSSV transfer from the live feed to shrimp broodstock, suggesting that polychaete worms could play a role in the epizootiology of WSSV.     

The role of Pm-fortilin in protecting shrimp from white spot syndrome virus (WSSV) infection

Crustacean fortilin or the product of the translationally controlled tumor protein (TCTP) gene isolated from Penaeus monodon, is well conserved and has a Ca(++) binding domain. Pm-fortilin has anti-apoptotic properties and is present at high levels during the onset of viral infections in P. monodon. The possibility of using rFortilin to protect against white spot syndrome virus (WSSV) infection was tested. Injection of shrimp with rFortilin, after infection with WSSV, resulted in 80-100% survival and detection of very low levels of WSSV by PCR, whereas in moribund samples WSSV levels were very high. This result implies that injection of recombinant rFortilin decreases viral infection by an unknown mechanism, but probably by inhibiting viral replication. Using a yeast two-hybrid screen for cellular protein partners to rFortilin we identified an unknown protein that bound to fortilin. This is a novel polypeptide of 93 amino acids with a number of XPPX signature sequences that are often reported to have a function in antiviral peptides.     

Identification and agglutination properties of hemocyanin from the mud crab (Scylla serrata)

Infectious diseases have significantly delayed the growth of crab aquaculture. Identification of the immune molecules and characterization of the defense mechanisms will be pivotal to the reduction of these diseases. Hemocyanin is an important non-specific immune protein present in the hemolymph of both mollusks and arthropods. However, little is known about the hemocyanin from the mud crab Scylla serrata. In this study, we identified the S. serrata hemocyanin using affinity proteomics and investigated its agglutinative properties. The results showed that S. serrata hemocyanin consists of five subunits with molecular weights of 70, 72, 75, 76 and 80 kDa, respectively. It demonstrated agglutination activities against seven bacterial species at concentrations ranging from 7.5 to 30 μg/ml. Agglutination was inhibited by 50–200 mM of N-acetylneuraminic acid, α-d-glucose, d-galactose and d-xylose. The 76 kDa subunit was identified as the protein that primarily binds bacterial cells and we speculate that it functions as the agglutinating subunit. We showed that outer membrane proteins (Omp) of bacteria could completely inhibit agglutination and that the agglutination activities of hemocyanin against Escherichia coli ?OmpA and ?OmpX mutants were significantly decreased, suggesting that these two Omps may be important ligands of hemocyanin. Together, the data collectively suggests that the 76 kDa subunit of S. serrata hemocyanin mediates agglutination through recognition of OmpA and OmpX proteins in bacteria.     

Immunotoxic effects of nickel in the mud crab Scylla serrata

The presence of xenobiotic contaminants especially metals in coastal waters is a major concern as they are immunotoxic to aquatic animals even at low concentrations. In our present study, mud crab Scylla serrata was exposed to three sublethal concentrations (0.4, 0.6 and 0.8 mg/L) of nickel for 30 days under laboratory conditions and the alterations of hematological parameters like haemocyte count, clotting time, haemocyte viability, protein content and immunomodulatory components like phenoloxidase, phagocytosis and superoxide anion generation were measured. In addition, the accumulation patterns of nickel were measured in gills, hepatopancreas and ovary. The accumulation was more in gills when compared to hepatopancreas and ovary of crabs exposed to nickel and was not detected in the control crabs. The results revealed a significant (P < 0.05) induction of superoxide anion generation and phagocytosis activity in the haemolymph of the crabs exposed to nickel when compared to control. On the contrary, the rest of the parameters were significantly (P < 0.05) reduced in the experimental groups when compared to the control. All the studied parameters exhibited a concentration dependent response.     

Model of white spot syndrome virus (WSSV) epidemics in Litopenaeus vannamei

White spot syndrome virus (WSSV) is devastating shrimp aquaculture throughout the world, but despite its economic importance no work has been done on modeling epidemics of this pathogen. Therefore we developed a Reed-Frost epidemic model for WSSV in Litopenaeus vannamei. The model includes uninfected susceptible, latently infected, acutely infected, and dead infected shrimp. The source of new infections during an outbreak is considered to be dead infected shrimp. The transmission coefficient, patency coefficient, virulence coefficient, and removal coefficient (disappearance of dead infected shrimp) control the dynamics of the model. In addition, an explicit area parameter is included to help to clarify the distinction between density and absolute shrimp population size. An analysis of the model finds that as number of shrimp, initial dose, transmission coefficient, patency coefficient, virulence coefficient, or removal coefficient changes, the speed of the epidemic changes. The model predicts that a threshold density of susceptible shrimp exists below which an outbreak of WSSV will not occur. Only initial dose, transmission coefficient, removal coefficient, and area coefficient affect the predicted threshold density. Increases in the transmission coefficient reduce the threshold value, whereas increases in the other factors cause the threshold value to increase. Epidemic models may prove useful to the shrimp aquaculture industry by suggesting testable hypotheses, some of which may contribute to the eventual control of WSSV outbreaks.     

Experimental infection of twenty species of Indian marine crabs with white spot syndrome virus (WSSV)

Twenty species of Indian marine crabs were experimentally infected with white spot syndrome virus (WSSV), via the oral route and intramuscular injection, to determine their viral susceptibility. We determined that 16 species (Calappa philargius, Charybdis annulata, C. lucifera, Doclea hybrida, Grapsus albolineatus, Halimede ochtodes, Liagore rubronaculata, Lithodes maja, Matuta miersi, Paradorippe granulata, Parthenope prensor, Philyra syndactyla, Podophthalmus vigil, Portunus sanquinolentus, Scylla serrata and Thalamita danae) were susceptible and 4 (Atergatis integerrimus, Charybdis natator, Demania splendida or Menippe rumphii) were refractive at 50 d post-infection (p.i.). The presence of WSSV in these crabs was confirmed by PCR tests, histology and bioassay. WSSV was found in the gill, heart, eyestalks, striated muscle and cephalothoraxic tissue. The 4 WSSV-refractive species represent potential reservoirs or carriers of WSSV.     

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.     

Identification of polymorphic microsatellite loci in the mud crab Scylla serrata (Brachyura: Portunidae)

Five microsatellite loci are identified and characterized from the genome of Scylla serrata, a widespread and commercially important species of coastal marine crab. The loci were detected by randomly screening for di‐ and tri‐nucleotide repeat units within a partial genomic library developed for the species. The five loci consist of dinucleotide repeats and are both co‐dominant and polymorphic within the species. A sample (N = 36) of S. serrata from one Australian population has an average observed heterozygosity of 0.875 and provides no evidence of either linkage among loci or significant deviation from random mating expectations across loci. PCR products for each of the five loci were also observed from a small sample of three other species within the Scylla genus. These markers may provide genetic information that will be useful for both aquaculture and studies of natural populations of the genus.     

Penaeus monodon chitin-binding protein (PmCBP) is involved in white spot syndrome virus (WSSV) infection

White spot syndrome virus (WSSV) can cause the most serious viral disease of shrimp and has a wide host range among crustaceans. Although researches show a lot about its genome and structure, information concerning the mechanism of how WSSV infects' cells is lacking. In this study, some experiments were applied to confirm the biological meaning of the protein–protein interaction between WSSV envelope protein, VP53A, and Penaeus monodon chitin-binding protein (PmCBP). Immunofluorescent study indicated that PmCBP is located on the cell surface of host cells. PmCBP amounts of about 34 kDa can be detected in both P. monodon and Litopenaeus vannamei tissues by Western blotting. In the in vivo neutralization experiment, both rVP53A and rPmCBP that were produced by Esherichia coli can promote resp. a 40% and 20% survival rate of the shrimp which were challenged by WSSV. Furthermore, a yeast-two-hybrid result revealed that PmCBP could interact with at least 11 WSSV envelope proteins. Those findings suggest that PmCBP may be involved in WSSV infection.     

Study of epileptiform activity in cerebral ganglion of mud crab Scylla serrata

An attempt is made to induce in mud crab (Scylla serrata) epileptiform activities that resemble the generalized epileptic seizures. Cerebral ganglion of crab was exposed in situ, to a convulsant drug pentylenetetrazole (PTZ) 100 mM, for induction of seizures. Also, crabs were pretreated with antiepileptic drug viz sodium valproate (120 μmol/l) to inhibit epileptiform activities. The surface electrical discharges of cerebral ganglion were recorded using Unkelscope (MIT, USA) in control as well as experimental animals. The cerebral ganglion of crab showed a pattern of high cerebral electrical discharges after PTZ treatment compared to control. The sodium valproate promoted sedative action in control and prevented PTZ-mediated epileptiform discharges. Glutamate and GABA contents in cerebral ganglion were assayed. Glutamate level increased (31.45%) during PTZ treatment with concomitant decrease (43.93%) in GABA. Sodium valproate had no effect on glutamate concentration, but it decreased GABA by 24.75%. The present study shows that epileptiform activities can be induced in crabs.     

Usefulness of dead shrimp specimens in studying the epidemiology of white spot syndrome virus (WSSV) and chronic bacterial infection

This paper describes the utility of dead shrimp samples in epidemiological investigations of the white spot syndrome virus (WSSV) and chronic bacterial infections. A longitudinal observational study was undertaken in shrimp farms in Kundapur, Karnataka, India, from September 1999 to April 2000 to identify risk factors associated with outbreaks of white spot disease (WSD) in cultured Penaeus monodon. As a part of the larger study, farmers were trained to collect and preserve dead and moribund shrimp (when observed) during the production cycle. At the end of the production cycle, 73 samples from 50 ponds had been collected for histopathology and 55 samples from 44 ponds for PCR. Intranuclear viral inclusion bodies diagnostic of WSSV infection were detected in dead samples from 32 ponds (64 %). Samples of dead shrimp from 18 ponds (36%) showed no histopathological evidence of WSSV infection. However, of these, samples from 13 ponds (26%) showed clear evidence of shell, oral, enteric and systemic chronic inflammatory lesions (CIL) in the form of haemocytic nodules, typical of bacterial infection. Samples from 5 ponds (10%) were negative for both WSSV and CIL. Samples from 8 ponds had dual WSSV and CIL, although both WSSV and CIL were only observed in the same shrimp from 1 pond. Useful information was obtained from these shrimp despite the presence of post-mortem changes. Samples from 19 ponds (43%) tested positive for WSSV by 1-step PCR and samples from an additional 10 ponds (22.7%) were positive by 2-step nested PCR. Samples from 15 ponds (34.1%) were negative for WSSV by 2-step nested PCR. There was moderate to substantial agreement between PCR and histopathology in the diagnosis of WSSV infection in dead shrimp. WSSV infection in dead shrimp was significantly associated with crop failures as defined by a shorter length of the production cycle (<90 d) and lower average weight at harvest (<22 g). WSSV infection was also associated with lower survival (<50%), but this was not significant. Ponds with CIL did not experience any crop failures, and the presence of CIL was significantly associated with successful crops. The study demonstrates that samples of dead shrimp can provide useful information for disease surveillance and epidemiological investigations of WSSV and chronic bacterial infections.     

Evolutionary trajectory of white spot syndrome virus (WSSV) genome shrinkage during spread in Asia

Background

White spot syndrome virus (WSSV) is the sole member of the novel Nimaviridae family, and the source of major economic problems in shrimp aquaculture. WSSV appears to have rapidly spread worldwide after the first reported outbreak in the early 1990s. Genomic deletions of various sizes occur at two loci in the WSSV genome, the ORF14/15 and ORF23/24 variable regions, and these have been used as molecular markers to study patterns of viral spread over space and time. We describe the dynamics underlying the process of WSSV genome shrinkage using empirical data and a simple mathematical model.

Methodology/Principal Findings

We genotyped new WSSV isolates from five Asian countries, and analyzed this information together with published data. Genome size appears to stabilize over time, and deletion size in the ORF23/24 variable region was significantly related to the time of the first WSSV outbreak in a particular country. Parameter estimates derived from fitting a simple mathematical model of genome shrinkage to the data support a geometric progression (k<1) of the genomic deletions, with k = 0.371±0.150.

Conclusions/Significance

The data suggest that the rate of genome shrinkage decreases over time before attenuating. Bioassay data provided support for a link between genome size and WSSV fitness in an aquaculture setting. Differences in genomic deletions between geographic WSSV isolates suggest that WSSV spread did not follow a smooth pattern of geographic radiation, suggesting spread of WSSV over long distances by commercial activities. We discuss two hypotheses for genome shrinkage, an adaptive and a neutral one. We argue in favor of the adaptive hypothesis, given that there is support for a link between WSSV genome size and fitness.     

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.     

Standardized white spot syndrome virus (WSSV) inoculation procedures for intramuscular or oral routes

In the past, strategies to control white spot syndrome virus (WSSV) were mostly tested by infectivity trials in vivo using immersion or per os inoculation of undefined WSSV infectious doses, which complicated comparisons between experiments. In this study, the reproducibility of 3 defined doses (10, 30 and 90 shrimp infectious doses 50% endpoint [SID50]) of WSSV was determined in 3 experiments using intramuscular (i.m.) or oral inoculation in specific pathogen-free (SPF) Litopenaeus vannamei. Reproducibility was determined by the time of onset of disease, cumulative mortality, and median lethal time (LT50). By i.m. route, the 3 doses induced disease between 24 and 36 h post inoculation (hpi). Cumulative mortality was 100% at 84 hpi with doses of 30 and 90 SID50 and 108 hpi with a dose of 10 SID50. The LT50 of the doses 10, 30 and 90 SID50 were 52, 51 and 49 hpi and were not significantly different (p > 0.05). Shrimp orally inoculated with 10, 30 or 90 SID50 developed disease between 24 and 36 hpi. Cumulative mortality was 100% at 108 hpi with doses of 30 and 90 SID50 and 120 hpi with a dose of 10 SID50. The LT50 of 10, 30 and 90 SID50 were 65, 57 and 50 hpi; these were significantly different from each other (p < 0.05). A dose of 30 SID50 was selected as the standard for further WSSV challenges by i.m. or oral routes. These standardized inoculation procedures may be applied to other crustacea and WSSV strains in order to achieve comparable results among experiments.     

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.     

A new shell disease in the mud crab Scylla serrata from Port Curtis, Queensland (Australia)

In 1994 a previously unreported rust spot shell disease was seen in mud crabs Scylla serrata - Forskal from Port Curtis, central Queensland, Australia. Of 673 crabs surveyed, 21.7% had shell lesions. Of these, 82.9% had rust spot lesions on the carapace. The majority of rust spot-affected crabs (78.8%) were female. Rust spot lesions were predominantly non-perforated (89.4%) and 54.8% were bilaterally symmetrical. There was also a gender difference in the areas of the carapace most commonly affected. The main histological features of the rust spot lesion included: a cavity in the upper endocuticle; indentation of the endocuticle below the cavity; remains of muscle attachment adhesive epithelium within the cavity; fibrous connective tissue between the damaged carapace and the attached muscle; and islands of endocuticle in this fibrous connective tissue. Histopathology of the internal organs failed to find evidence of an infectious or parasitic cause of the rust spot lesions. The cause(s) of the syndrome appear(s) to be non-infectious.