White Spot Syndrome Virus Injection Research Articles

Resistance to white spot syndrome virus in the European shore crab is associated with suppressed virion trafficking and heightened immune responses.

All decapod crustaceans are considered potentially susceptible to White Spot Syndrome Virus (WSSV) infection, but the degree of White Spot Disease (WSD) susceptibility varies widely between species. The European shore crab Carcinus maenas can be infected with the virus for long periods of time without signs of disease. Given the high mortality rate of susceptible species, the differential susceptibility of these resistant hosts offers an opportunity to investigate mechanisms of disease resistance. Here, the temporal transcriptional responses (mRNA and miRNA) of C. maenas following WSSV injection were analysed and compared to a previously published dataset for the highly WSSV susceptible Penaeus vannamei to identify key genes, processes and pathways contributing to increased WSD resistance. We show that, in contrast to P. vannamei, the transcriptional response during the first 2 days following WSSV injection in C. maenas is limited. During the later time points (7 days onwards), two groups of crabs were identified, a recalcitrant group where no replication of the virus occurred, and a group where significant viral replication occurred, with the transcriptional profiles of the latter group resembling those of WSSV-susceptible species. We identify key differences in the molecular responses of these groups to WSSV injection. We propose that increased WSD resistance in C. maenas may result from impaired WSSV endocytosis due to the inhibition of internal vesicle budding by dynamin-1, and a delay in movement to the nucleus caused by the downregulation of cytoskeletal transcripts required for WSSV cytoskeleton docking, during early stages of the infection. This response allows resistant hosts greater time to fine-tune immune responses associated with miRNA expression, apoptosis and the melanisation cascade to defend against, and clear, invading WSSV. These findings suggest that the initial stages of infection are key to resistance to WSSV in the crab and highlight possible pathways that could be targeted in farmed crustacean to enhance resistance to WSD.

Field Experiment: Synbiotic Microcapsule Dietary Supplementation Improved the Health Status of the White Shrimp under the Challenge of Coinfection Diseases through Improvement the Beneficial Microbial Composition.

This study aimed to evaluate the effectivity of microencapsulated synbiotic (MS), Bacillus sp. NP5 and mannan oligosaccharide (MOS) dietary in different feeding frequencies in Pacific white shrimp culture field experiment. The MS was administered as a feed supplementation to enhance the immunity for prevention against co-infection with WSSV (White Spot Syndrome Virus) and Vibrio harveyi. The synbiotic was microencapsulated by the spray dryer method. Shrimps were reared in the floating net cages in the pond. Treatments included the administration of MS at different frequencies i.e, daily (A), twice a week (B), once a week (C), and without MS supplementation (consisted of negative and positive controls) with a feeding rate of 6% of shrimp biomass (5 times a day). During the challenge trial, shrimps were removed and further reared in plastic tanks, for 7 days. The shrimps (except negative control treatment) were intramuscularly injected by WSSV filtrate at the infective dosage of 10-4 copies.ml-1. Twenty four hours after WSSV injection the shrimps were immersed in the water-containing cells suspension of V. harveyi at the cell’s population dosage of 106 CFU.ml-1. Immune responses were observed for 7 days after experimental infection. The shrimps that have been treated with daily MS supplementation (A) showed better immune responses i.e., total haemocyte counts, phenoloxidase, respiratory burst, and the lower pathogenic cells abundance in the intestine compared to other treatments groups.

Effects of Synechococcus sp. PCC 7942 harboring vp19, vp28, and vp (19 + 28) on the survival and immune response of Litopenaeus vannamei infected WSSV

This study aimed to assess the effect of oral administration of Synechococcus sp. PCC 7942 harboring vp19, vp28, and vp(19 + 28)against infection by white spot syndrome virus (WSSV) on juveniles of Litopenaeus vannamei. L. vannamei was orally administrated by feeding with different mutants of Synechococcus for 10 days, and then challenged with WSSV. The cumulative mortality of vp19, vp28, vp (19 + 28) groups was lower than that of the positive control group (57.8%, 62.2%, 71.1%, respectively); vp (19 + 28) group had a better protection rate than vp19 and vp28 groups. The analysis of shrimp immunological parameters showed that, after WSSV injection, the activity of superoxide dismutase, phenol oxidase, catalase, and lysozyme in the hepatopancreas of vp19, vp28, and vp (19 + 28) groups was higher than in the positive group; at the same time, growth performances of L. vannamei of experimental groups were better than control groups. Results showed that the Synechococcus mutants harboring vp19, vp28, and vp (19 + 28) could be used both as drug and feed to also enhance the defensive ability of juvenile shrimp against WSSV infection by increasing the activity of immune related enzymes.

SpCrus3 and SpCrus4 share high similarity in mud crab (Scylla paramamosain) exhibiting different antibacterial activities

Type I crustins are crucial effectors of crustacean immune system. Various type I crustins with high sequence diversity possess different antimicrobial activities. To date, the mechanism on how the sequence diversity of type I crustins affects their antimicrobial activities is largely unclear, and how different crustins function together against bacterial invasion still remains unknown. In this study, we identified two novel type I crustins, namely, SpCrus3 and SpCrus4, from an economically important crab, Scylla paramamosain. Either SpCrus3 or SpCrus4 was highly expressed in gill. After challenges with Vibrio parahemolyticus or Staphylococcus aureus, SpCrus4 was up-regulated, whereas SpCrus3 was down-regulated. No significant expression change of SpCrus3 and SpCrus4 was observed after white spot syndrome virus injection, suggesting that these two genes may not participate in the antiviral immune responses. SpCrus3 and SpCrus4 had the common 5′ terminus and high similarity of 66.06%, but SpCrus4 exhibited stronger antimicrobial activity than that of SpCrus3. Microorganism-binding assay results revealed that both SpCrus3 and SpCrus4 exhibited binding ability to all tested microorganisms. Furthermore, the polysaccharide-binding assay showed that these two proteins exhibited strong binding activity to bacterial polysaccharides, such as lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PGN). SpCrus3 and SpCrus4 exhibited stronger binding activity to LPS or LTA than to PGN. Moreover, SpCrus4 showed stronger binding activity to LTA than that of SpCrus3, which may be responsible for the significantly distinct antimicrobial activity between these two proteins. In addition, SpCrus4 displayed stronger agglutination activity against several kinds of microorganisms than that of SpCrus3. This increased agglutination activity may also contribute to the strong antibacterial activity of SpCrus4. On the basis of all these results, a possible antibacterial mode exerted by SpCrus3 and SpCrus4 was proposed as follows. SpCrus3 was highly expressed in normal crabs to maintain low-level antibacterial activity without bacterial challenges. When crabs were challenged with bacteria, large amount of SpCrus4 was generated to exhibit strong antibacterial activity against bacterial invasion. This study provides new insights to understand the antibacterial functions and mechanisms of type I crustins.

Experimental inoculation of oriental river prawn Macrobrachium nipponense with white spot syndrome virus (WSSV).

The oriental river prawn Macrobrachium nipponense is an economically important species that is widely farmed in China. White spot syndrome virus (WSSV) is one of the most devastating pathogens of the cultured shrimp Litopenaeus vannamei, responsible for massive loss of its commercial products worldwide. We investigated the infectivity and pathogenicity of WSSV in adult M. nipponense using standardized conditions for L. vannamei. The median lethal dose of WSSV in adult M. nipponense was 103.84±0.06 copies g-1, which was about 1000-fold higher than in L. vannamei (100.59±0.22 copies g-1). WSSV was detected by 2-step PCR in the gills, hepatopancreas, muscle, stomach, heart, gut, nerve, integument, pereopod, eyestalk, testis, and ovary of experimentally infected dead M. nipponense. Lesions were observed histologically following WSSV injection, showing basophilic intranuclear inclusion bodies in the hepatopancreas and subsequently in the gills. The clearance of WSSV was observed in hepatopancreas and gills at 48 and 96 h post-inoculation, respectively. No histological lesions were detected in muscle from 0-96 h post-injection. The results show that the oriental river prawn M. nipponense can be infected by WSSV and the infections are self limiting over time; therefore, M. nipponense may serve as a useful model for studying resistance to WSSV.

Effects of Microencapsulated Synbiotic Administration at Different Dosages against heavy co-infection of White Spot Disease (WSD) and Vibrio harveyi in Pacific White Shrimp (Litopenaeus vannamei)

White spot disease (WSD) is one of infectious disease in shrimp caused by white spot syndrome virus (WSSV). This study aimed to determine the dosage immunological effects and growth performances of microencapsulated synbiotic (Bacillus NP5 and mannan oligosaccharide) at different dosages on Pacific white shrimp. The microencapsulated synbiotic was administered as feed supplementation against the co-infect ion of WSSV and Vibrio harveyi. Synbiotic was encapsulated by spray drying method, further feed supplemented to Pacific white shrimp for 30 days at a dosages of 0.5% (A), 1% (B), 2% (C) and control treatments, i.e. without any microencapsulated synbiotic administration as positive control (D) and negative control (E). The challenge test was performed on day 30 after feeding supplementation , the n the experimental shrimp s were injected by WSSV intramuscularly at the infective dosage of 10 4 copies. - ml -1 . Afterwards, 24 hours after WSSV injection the shrimps were immersed in water contained cells suspension of V. harveyi at the cells population dosage of 10 6 CFU - .ml -1 . All synbiotic treatments showed better results with the value s of T otal H aemocyte C ount (THC) , P hen oloxidase (PO) and R espiratory B urst (RB), were higher ( P <0.05) compared to positive control. The specific growth rate s (SGR) of A, B and C showed higher than both controls of D and E. The f eed conversion ratio (FCR) value of synbiotic treatments were lower (P<0.05) than both controls. However, the administration of microencapsulated synbiotic have not been able to prevent heavy impact of WSSV and V. harveyi co-infection due to lower SR and mortality pattern which continued to increase. K eywords : Synbiotic , Litopenaeus vannamei, WSSV, Vibrio harveyi, co-infection

PmVRP15, a novel viral responsive protein from the black tiger shrimp, Penaeus monodon, promoted white spot syndrome virus replication.

Suppression subtractive hybridization of Penaeus monodon hemocytes challenged with white spot syndrome virus (WSSV) has identified the viral responsive gene, PmVRP15, as the highest up-regulated gene ever reported in shrimps. Expression analysis by quantitative real time RT-PCR revealed 9410–fold up-regulated level at 48 h post WSSV injection. Tissue distribution analysis showed that PmVRP15 transcript was mainly expressed in the hemocytes of shrimp. The full-length cDNA of PmVRP15 transcript was obtained and showed no significant similarity to any known gene in the GenBank database. The predicted open reading frame of PmVRP15 encodes for a deduced 137 amino acid protein containing a putative transmembrane helix. Immunofluorescent localization of the PmVRP15 protein revealed it accumulated around the nuclear membrane in all three types of shrimp hemocytes and that the protein was highly up-regulated in WSSV-infected shrimps. Double-stranded RNA interference-mediated gene silencing of PmVRP15 in P. monodon significantly decreased WSSV propagation compared to the control shrimps (injected with GFP dsRNA). The significant decrease in cumulative mortality rate of WSSV-infected shrimp following PmVRP15 knockdown was observed. These results suggest that PmVRP15 is likely to be a nuclear membrane protein and that it acts as a part of WSSV propagation pathway.

Cloning, characterization, and expression of the macrophage migration inhibitory factor gene from the Pacific white shrimp Litopenaeus vannamei (Penaeidae)

The macrophage migration inhibitory factor (MIF) is an important proinflammatory cytokine that mediates both innate and adaptive immune responses. In this study, we identified a homolog of MIF in the Pacific white shrimp Litopenaeus vannamei. The MIF cDNA contained a 363-bp open reading frame encoding a 120-amino acid protein with a calculated molecular mass of 13.442 kDa and a theoretical isoelectric point of 6.57. The L. vannamei MIF shared high amino acid identity with MIFs of other invertebrates. Tissue distribution analysis by quantitative real-time polymerase chain reaction (qRT-PCR) revealed that the L. vannamei MIF was abundantly expressed in the blood, heart, and hepatopancreas, was moderately expressed in the gill, and was weakly expressed in the muscle and intestine. Furthermore, in order to gain a basic understanding of the role of MIF in the shrimp immune response against viral infection, its mRNA expression was determined in the hepatopancreas of L. vannamei at different stages after white spot syndrome virus (WSSV) challenge using qRT-PCR. The result indicated that the expression of MIF was significantly upregulated after WSSV injection, suggesting that MIF may be involved in the response to viral infection in shrimp.

Reactive oxygen system plays an important role in shrimp Litopenaeus vannamei defense against ­Vibrio parahaemolyticus and WSSV infection

The present study investigated the in vivo hemocytic and hepatopancreatic response to Vibrio parahaemolyticus and white spot syndrome virus (WSSV) injection in shrimp Litopenaeus vannamei. The proliferation of bacteria and virus in shrimp, animal mortality, total hemocyte counts (THCs), phenoloxidase (PO) activity, respiratory burst, and gene expression of immune factors associated with immune recognition (lectin), prophenoloxidase (proPO) activation, and the anti-microorganism (lysozyme) and active oxygen defense response (including respiratory burst, cytosolic manganese superoxide dismutase [C-MnSOD], and catalase [CAT]) were quantified. Shrimp death rate increased significantly and was time-dependent after V. parahaemolyticus or WSSV injection. The production of superoxide anion, and the gene expression including lectin in hemocytes, proPO in the hepatopancreas, lysozyme, C-MnSOD and CAT could be induced by injection with V parahaemolyticus and WSSV. The highest value of lysozyme was in the hemocytes with 66.59 times (at 3 h) greater expression than in the control group after WSSV injection and 3.69 times (24 h) greater than in the control group after V parahaemolyticus injection. In the hepatopancreas, CAT expression showed a significant increase, with up to 16 times greater expression than in the control group at 6 h postinjection with WSSV and 7.02 times greater expression than in the control group at 48 h postinjection with V parahaemolyticus (p < 0.05). However, significant decreases in PO activity and proPO transcripts in hemocytes and lectin transcripts in the hepatopancreas were detected after V parahaemolyticus and WSSV injection (p < 0.05). The results suggest that lysozyme, the antioxidase system, and reactive oxygen species might play a crucial role in shrimp defense against bacterial and viral infection.

A Kazal-type serine proteinase inhibitor SPI Pm2 from the black tiger shrimp Penaeus monodon is involved in antiviral responses

A five-domain Kazal-type serine proteinase inhibitor, SPI Pm2, from Penaeus monodon has recently been implicated in antiviral responses for it is up-regulated upon viral infection and needs further studies. The SPIPm2 genomic gene was composed of seven exons and six introns. The genomic DNA segments coding for each Kazal domain were separated by introns of variable lengths supporting the hypothesis of gene duplication in the Kazal-type gene family. RT-PCR and Western blot analysis revealed that the SPIPm2 transcript and its five-domain protein product were expressed mainly in the hemocytes and less in gill, heart and antennal gland. Upon white spot syndrome virus (WSSV) infection, the SPI Pm2 was only detected in the hemocytes and plasma. Immunocytochemical study of P. monodon hemocytes showed that the percentage of SPI Pm2-producing hemocytes was reduced by about half after WSSV infection. Quantitative RT-PCR revealed further that the SPI Pm2 was up-regulated early in the hemocytes of WSSV-infected shrimp and gradually reduced as the infection progressed. Injection of the recombinant SPI Pm2 (rSPI Pm2) prior to WSSV injection resulted in a significant inhibition of WSSV replication. The rSPI Pm2 injection also prolonged the mortality rate of WSSV-infected shrimp. Therefore, the SPI Pm2 was involved in the innate immunity against WSSV infection in shrimp.

A single WAP domain (SWD)-containing protein with antipathogenic relevance in red swamp crayfish, Procambarus clarkii

Single WAP domain-containing proteins (SWDs) are small proteins possessing a whey acidic protein (WAP) domain at the C-terminal region. In this study, a complementary deoxyribonucleic acid (cDNA) of SWD-containing protein was isolated from the hemocytes of red swamp crayfish, Procambarus clarkii called Pc-SWD. The full-length cDNA sequence is 998 bp. The deduced amino acid sequence consists of 74 residues with a signal peptide of 20 residues. The mature peptide has a single WAP domain which contains eight conserved cysteine residues forming a “four-disulphide core” (4-DSC). The predicted molecular mass of the mature protein is 5.97 kDa, with an estimated pI of 7.71. Tissue distribution analysis by reverse-transcribed polymerase chain reaction (RT-PCR) revealed that Pc-SWD transcripts were primarily found in the hemocytes, heart, hepatopancreas, gills, and intestine. The results of time course analysis demonstrated that expression of Pc-SWD was decreased at 6 h followed by a significant upregulation from 48 h to 72 h in hemocytes after white spot syndrome virus (WSSV) injection. A similar expression pattern was found in the hepatopancreas after WSSV injection. In addition, Pc-SWD expression was visibly upregulated in the gills from 6 h to 72 h after WSSV injection. The results of Western bolt revealed that Pc-SWD was constitutively expressed in the heart, hepatopancreas, gills, and intestine of unchallenged crayfish. A weak band was detected in the hemocytes and hemolymph of unchallenged P. clarkii. The Pc-SWD expression was upregulated in the hemocytes and gills after challenging with WSSV; however, no obvious change occurred in the heart and intestine. rPc-SWD could bind to both Gram-negative and -positive bacteria strongly. Moreover, rPc-SWD exhibited specific proteinase inhibitory activity against the secretory proteinase(s) from B. subtilis and P. aeruginosa. All these findings suggest that Pc-SWD possibly functions as an immunity effector in defense against the invasion of crayfish pathogens.

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.

Discovery of the Genes in Response to White Spot Syndrome Virus (WSSV) Infection in Fenneropenaeus chinensis Through cDNA Microarray

We used microarray technology to study differentially expressed genes in white spot syndrome virus (WSSV)-infected shrimp. A total of 3136 cDNA targets, including 1578 unique genes from a cephalothorax cDNA library and 1536 cDNA clones from reverse and forward suppression subtractive hybridization (SSH) libraries of Fenneropenaeus chinensis, plus 14 negative and 8 blank control clones, were spotted onto a 18 x 18 mm area of NH(2)-modified glass slides. Gene expression patterns in the cephalothorax of shrimp at 6 h after WSSV injection and moribund shrimp naturally infected by WSSV were analyzed. A total of 105 elements on the arrays showed a similar regulation pattern in artificially infected shrimp and naturally infected moribund shrimp; parts of the results were confirmed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The up-regulated expression of immune-related genes, including heat shock proteins (HSP70 and HSP90), trehalose-phosphate synthase (TPS), ubiquitin C, and so forth, were observed when shrimp were challenged with WSSV. Genes including myosin LC2, ATP synthase A chain, and arginine kinase were found to be down-regulated after WSSV infection. The expression of housekeeping genes such as actin, elongation factor, and tubulin is not stable, and so these genes are not suitable as internal standards for semiquantitative RT-PCR when shrimp are challenged by WSSV. As a substitute, we found that triosephosphate isomerase (TPI) was an ideal candidate of interstandards in this situation.

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

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

Time-course and levels of apoptosis in various tissues of black tiger shrimp Penaeus monodon infected with white-spot syndrome virus.

This study focused on apoptosis in various tissues of the black tiger shrimp Penaeus monodon following white spot syndrome virus (WSSV) injection. The study included: (1) light microscopy (LM) and transmission electron microscopy (TEM) of various tissues; (2) fluorescent LM of nuclear DNA by staining with 4, 6-diamidine-2-phenyl indole dihydrochloride (DAPI) and TdT-mediated dUTP nick-end labelling (TUNEL) techniques; and (3) determination of caspase-3 activity. Juvenile P. monodon were injected with WSSV, and several tissues of ectodermal and mesodermal origin were studied at different intervals after injection. The total haemocyte count had decreased to one-tenth of its original level 60 h after WSSV injection. By LM, extensive destruction by WSSV was observed in the stomach epithelium, gills, hematopoietic tissue, hemocytes and the heart, but the most severely affected tissue was the subcuticular epithelium. TEM revealed that at 6 h post-injection (p.i.) the chromatin of infected nuclei was marginated, and by 24 h p.i. the nuclei were filled with enveloped and non-enveloped WSSV virions. At later stages of the infection, the nucleus extruded WSSV particles. Chromatin margination and nuclear condensation and fragmentation (i.e. signs of apoptosis) were observed as early as 6 h p.i. in all affected tissues, but occurred in cells without WSSV virions rather than in cells with virions. The occurrence of apoptosis was supported by data obtained using TUNEL and by DAPI-staining and progressed from 6 to 60 h p.i. In addition, caspase-3 activity in WSSV-infected shrimp was about 6-fold higher than that in uninfected shrimp. The data strongly suggests that apoptosis occurs following WSSV infection in P. monodon, but the extent to which it contributes to shrimp mortality requires further investigation.

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.