Experimental infections reveal that common Thai crustaceans are potential carriers for spread of exotic Taura syndrome virus

Taura syndrome virus (TSV) was first reported as a serious cause of shrimp mortality limited to reared Penaeus (Litopenaeus) vannamei in the Americas, where it spread principally through regional and international transfer of live post larvae (PL) and broodstock. Subsequently, through importation of infected broodstock, TSV outbreaks spread to Asia, first to Taiwan and China and then to Thailand, Indonesia and Korea. Since its introduction to Thailand, outbreaks have occasionally been reported from rearing ponds stocked with batches of specific pathogen free (SPF) P. vannamei PL that tested negative for TSV by nested RT-PCR assay. Since it was possible that the outbreaks may have occurred via horizontal transfer of TSV from wild carrier species, we tested 5 common native crustaceans that live in and around shrimp ponds (2 palaemonid shrimp species, Palaemon styliferus and Macrobrachium lanchesteri, and 3 species of crabs, Sesarma mederi, Scylla serrata and Uca vocans) for susceptibility to TSV in experimental challenges. We found that U. vocans, S. serrata and S. mederi did not die but, respectively, gave strong RT-PCR reactions indicating heavy viral load at 5, 10 and 15 d post-injection of TSV and 10, 15 and up to 50 d after feeding with TSV-infected P. vannamei carcasses. Also after feeding, P. styliferus did not die, but a high proportion gave strong RT-PCR reactions at 5 d post-challenge and no reactions at 15 d. Similarly after feeding, M. lanchesteri showed no mortality and gave only light RT-PCR reactions at 2 d, moderate reactions at 5 d and no reaction at 15 d. By contrast, transmission experiments from the TSV-infected crabs and palaemonid shrimp via water or feeding resulted in death of all the exposed P. vannamei from 8 to 12 d post-challenge and all were positive for heavy viral load by RT-PCR assay. Despite the results of these laboratory challenge tests, natural TSV infections were not detected by nested RT-PCR in samples of these species taken from the wild. These results indicated that transmission of TSV from infected crabs and palaemonid shrimp via water or feeding might pose a potential risk to shrimp aquaculture.     

A model of Taura syndrome virus (TSV) epidemics in Litopenaeus vannamei

Taura syndrome virus (TSV) is a highly virulent pathogen of Litopenaeus vannamei, has affected shrimp aquaculture throughout the world, and threatens wild populations. Despite its importance, little work has been done on the pathogen's formal epidemiology. Therefore we developed a compartment model for epidemics of TSV in closed populations of L. vannamei. The model includes five compartments, uninfected susceptible, prepatently infected, acutely infected, chronically infected, and dead infected shrimp. The transmission coefficients, patency coefficient, virulence coefficients, and removal coefficient (disappearance of dead infected shrimp) control the dynamics of the model. We estimated the coefficients in laboratory studies and inserted the estimates in the model to characterize TSV epidemics and to estimate the basic reproduction ratio R(0) and threshold density for TSV epidemics in L. vannamei. Further we examined through computer simulation the effect of varying the coefficients on R(0). Decreases in transmission decrease R(0), decreases in virulence increase R(0), increases in patency do not affect R(0), and increases in recovery most likely increase R(0) but under some conditions might decrease it.     

Haemolymph parameters of Pacific white shrimp (Litopenaeus vannamei) infected with Taura syndrome virus

Pacific white shrimp (Litopenaeus vannamei) were injected with Taura syndrome virus (TSV) to assess shrimp immune responses and survival. TSV-infected shrimp suffered high mortality, but mock-infected and untreated shrimp experienced no mortality. Moribund shrimp were a pale, reddish colour and were lethargic and soft-shelled. Their haemolymph was clear red and coagulated poorly. In TSV-infected shrimp, the total haemocyte count (THC), hyalinocyte and granulocyte counts, and total plasma protein decreased significantly to 21%, 24%, 17% and 56% of untreated control values, respectively. Haemocyanin decreased to 67%, and clottable proteins to 80% of control values (P< 0.01). Copper and calcium ions, haemocytic transglutaminase (TGase) activity and plasma growth inhibitory activity against Vibrio harveyi also decreased significantly. Generation of intrahaemocytic superoxide anion, O(-2), in TSV-infected shrimp was significantly greater (P< 0.05) than in both control groups, no matter whether glucan stimulated or unstimulated. But the relative increase of intrahaemocytic O(-2) generation in TSV-infected shrimp response to glucan stimulation was lower in both controls. Plasma phenoloxidase (PO) activity increased significantly in TSV-infected shrimp. The plasma bacterial agglutinin titre against E. coli and V. harveyi, growth inhibition of E. coli and the concentration of magnesium ions in TSV-infected shrimp did not change significantly.In conclusion, ten of thirteen haemolymph parameters changed significantly during the host-TSV interaction. These parameters might be valuable references of shrimp health status.     

Ultrastructure of the replication site in Taura syndrome virus (TSV)-infected cells

Taura syndrome virus (TSV) is a member of the family Dicistroviridae that infects Pacific white shrimp Litopenaeus vannamei (also called Penaeus vannamei), and its replication strategy is largely unknown. To identify the viral replication site within infected shrimp cells, the viral RNA was located in correlation with virus-induced membrane rearrangement. Ultrastructural changes in the infected cells, analyzed by transmission electron microscopy (TEM), included the induction and proliferation of intracellular vesicle-like membranes, while the intracytoplasmic inclusion bodies and pyknotic nuclei indicative of TSV infection were frequently seen. TSV plus-strand RNA, localized by electron microscopic in situ hybridization (EM-ISH) using TSV-specific cDNA probes, was found to be associated with the membranous structures. Moreover, TSV particles were observed in infected cells by TEM, and following EM-ISH, they were also seen in close association with the proliferating membranes. Taken together, our results suggest that the membranous vesicle-like structures carry the TSV RNA replication complex and that they are the site of nascent viral RNA synthesis. Further investigations on cellular origins and biochemical compositions of these membranous structures will elucidate the morphogenesis and propagation strategy of TSV.     

Branchial carbonic anhydrase activity and ninhydrin positive substances in the Pacific white shrimp, Litopenaeus vannamei, acclimated to low and high salinities

The Pacific white shrimp, Litopenaeus vannamei, acclimated to 30 ppt salinity, was transferred to either low (15 and 5 ppt), or high (45 ppt) salinity for 7 days. Hemolymph osmolality, branchial carbonic anhydrase activity, and total ninhydrin-positive substances (TNPS) in abdominal muscle were then measured for each condition. Hemolymph osmotic concentration was regulated slightly below ambient water osmolality in shrimp acclimated to 30 ppt. At 15 and 5 ppt, shrimp were strong hyper-osmotic regulators, maintaining hemolymph osmolality between 200 and 400 mOsm above ambient. Shrimp acclimated to 30 ppt and transferred to 45 ppt salinity were strong hypo-osmotic and hypo-ionic regulators, maintaining hemolymph osmolality over 400 mOsm below ambient. Branchial carbonic anhydrase (CA) activity was low (approximately 100 micromol CO(2) mg protein(-1) min(-1)) and uniform across all 8 gills in shrimp acclimated to 30 ppt, but CA activity increased in all gills after exposure to both low and high salinities. Anterior gills had the largest increases in CA activity, and levels of increase were approximately the same for low and high salinity exposure. Branchial CA induction appears to be functionally important in both hyper- and hypo-osmotic regulations of hemolymph osmotic concentrations. Abdominal muscle TNPS made up between 19 and 38% of the total intracellular osmotic concentration in shrimp acclimated to 5, 15, and 30 ppt. TNPS levels did not change across this salinity range, over which hemolymph osmotic concentrations were tightly regulated. At 45 ppt, hemolymph osmolality increased, and muscle TNPS also increased, presumably to counteract intracellular water loss and restore cell volume. L. vannamei appears to employ mechanisms of both extracellular osmoregulation and intracellular volume regulation as the basis of its euryhalinity.     

Taura syndrome virus loads in Litopenaeus vannamei hemolymph following infection are related to differential mortality

Taura syndrome is an economically important disease that can cause catastrophic losses of farmed shrimp. Without effective treatments for Taura syndrome virus (TSV), one approach to managing the problem is to selectively breed shrimp populations with increased disease resistance. To better understand why some shrimp can survive exposure to TSV, information is needed on how viral loads progress and persist following infection. Data reported here show that mortalities occurring mostly within 1 wk of infection are associated with high viral titers, and titers as high as 10(8.7) genome copies per microl hemolymph can persist for up to 3 wk in survivors. Thereafter, and up to approximately 9 wk post-exposure, most surviving shrimp remain chronically infected with TSV loads ranging from 10(4) to 10(8) genome copies per microl hemolymph. Challenging shrimp from families with varying TSV resistance showed that in shrimp from less resistant families, the TSV load in hemolymph increased earlier and reached higher peaks than in shrimp from more resistant families. Although TSV loads in moribund shrimp from families differing in resistance did not differ significantly, infection loads among survivors were lower in shrimp from more resistant families. Taken together, the data suggest that lethal infection loads can occur in both more and less susceptible shrimp and that survivors represent shrimp in which viral expansion is better contained.     

Taura syndrome virus (TSV) in Thailand and its relationship to TSV in China and the Americas

The cultivation of exotic Penaeus vannamei in Thailand began on a very limited scale in the late 1990s, but a Thai government ban on the cultivation of P. monodon in freshwater areas in 2000 led many Thai shrimp farmers to shift to cultivation of P. vannamei. Alarmed by the possibility of Taura syndrome virus (TSV) introduction, the Thai Department of Fisheries required that imported stocks of P. vannamei be certified free of TSV by RT-PCR (Reverse Trasciption Polymerase Chain Reaction) testing. During the interval of allowed importation, over 150,000 broodstock shrimp were imported, 67% of these from China and Taiwan. Despite the safeguards, TSV outbreaks occurred and we confirmed the first outbreak by RT-PCR in early 2003. This resulted in a governmental ban on all shrimp broodstock imports from February 2003, but TSV outbreaks have continued, possibly due to original introductions or to the continued illegal importation of stocks. To determine the origin of the TSV in Thailand, the viral coat protein gene VP1 was amplified by RT-PCR from several shrimp specimens found positive for TSV by RT-PCR from January to November 2003. These included 7 samples from P. vannamei disease outbreaks in Thailand, 3 other non-diseased shrimp samples from Thailand and Burma and 6 samples including P. vannamei and P. japonicus from China. Comparison revealed that the Thai, Burmese and Chinese TSV types formed a clade distinct from a clade of TSV types from the Americas.     

Specific monoclonal antibodies raised against Taura syndrome virus (TSV) capsid protein VP3 detect TSV in single and dual infections with white spot syndrome virus (WSSV)

The gene sequence encoding VP3 capsid protein of Taura syndrome virus (TSV) was cloned into pGEX-6P-1 expression vector and transformed into Escherichia coli BL21. After induction, recombinant GST-VP3 (rVP3) fusion protein was obtained and further purified by electro-elution before use in immunizing Swiss mice for production of monoclonal antibodies (MAb). One MAb specific to glutathione-S-transferase (GST) and 6 MAb specific to VP3 were selected using dot blotting and Western blotting. MAb specific to VP3 could be used to detect natural TSV infections in farmed whiteleg shrimp Penaeus vannamei by dot blotting and Western blotting, without cross reaction to shrimp tissues or other shrimp viruses, such as white spot syndrome virus (WSSV), yellow head virus (YHV), monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV). These MAb were also used together with those specific for WSSV to successfully detect TSV and WSSV in dual infections in farmed P. vannamei.     

Molecular Epidemiological Investigation of Infectious Hypodermal and Hematopoietic Necrosis Virus and Taura Syndrome Virus in Penaeus Vannamei Cultured in China

The Infectious hypodermal and hematopoietic necrosis virus(IHHNV) and Taura syndrome virus(TSV) are two important shrimp viruses in cultured shrimp in America.These two viruses were transmitted to China at the beginning of the 21st century.In this study,214 shrimp samples of Penaeus vannamei were collected from seven different areas of China and tested by PCR for IHHNV and TSV infection.The results showed that there were a high prevalence of IHHNV(65.42%) and low prevalence of TSV(3.27%) in the tested samples.Several samples were found to be co-infected with these two viruses.A 3 kb fragment of 7 positive IHHNV samples and a structure protein region(ORF2) of three TSV positive samples were amplified and sequenced.The sequence comparison indicated that both IHHNV and TSV sequenced in China have a low genetic variations compared with the prototype IHHNV and TSV from Hawaii.Phylogenetic analysis showed that TSV isolates were clustered into two groups,Asia and America group,which was genetically correlated to geographic distribution.     

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.     

Review of salinity effects on abundance,growth, and survival of nearshore life stages of pink shrimp (Farfantepenaeus duorarum)

The pink shrimp (Farfantepenaeus duorarum) has been selected as an ecological indicator to assess ecological effects on estuaries of implementation of the Comprehensive Everglades Restoration Plan that seeks to restore historical freshwater flows and nearshore salinity regimes in southern Florida. Concern over altered freshwater delivery impacts on pink shrimp productivity was expressed as early as the 1960s. The present review assessed pink shrimp scientific literature of the past 75+ years (>500 publications) to glean information relevant to understanding potential influence of freshwater management on pink shrimp productivity. The review was organized around “Essential Fish Habitat” metrics concerning abundance, growth, survival, distribution, productivity, and behavior. It summarizes previous pink shrimp field, laboratory, and modeling studies. Where possible, statistical analyses and meta-analyses of previously published data were performed to investigate consistency among independent findings. Pink shrimp occur in a wide range of salinities (0.5–67 ppt). A majority of studies (53.3%) reported maximal abundance between ∼20 to 35 ppt salinities. One laboratory study reported maximal growth at 30 ppt. Meta-analysis of reported growth rates did not yield results due to non-convergence of regression models. Reported survival was maximal at ∼30 ppt and remained high (>80% survival) across salinities of ∼15 to 40 ppt. A regression model that combined survival data across studies confirmed a previously reported parabolic relationship between salinity and survival; in this regression, 35 ppt maximized survival. Productivity, conditional upon survival and growth, was maximized at polyhaline (18–30 ppt) conditions. Inshore hypersalinity (>40 ppt) may elicit young pink shrimp behavioral cues counterproductive to settlement in nearshore areas. Virtually no information exists regarding postlarval pink shrimp movement or preference relative to salinity gradients. Realization and preservation of nearshore polyhaline conditions and elimination of hypersalinity should maximize growth, survival, and density, thus improving pink shrimp productivity. New and updated statistical models predicting pink shrimp distribution, abundance, growth, survival, and productivity relative to salinity conditions are needed to better guide freshwater management decisions.