The effects of feeding immunostimulant ß-glucan on the immune response of Pangasianodon hypophthalmus.

Immunostimulants are food additives used by the aquaculture industry to enhance the immune response of fish, and although ß-glucans are now commonly used for this purpose in aquaculture, little is known about their effects on the immune response of Pangasianodon hypophthalmus. Thus, a variety of immune parameters (e.g. phagocytosis, respiratory burst, lysozyme, complement, peroxidase, total protein, total anti-protease, total IgM, natural antibody titres, and specific IgM titres) was examined in this species after feeding fish with a basal control diet or diets supplemented with 0.05, 0.1, or 0.2 g/kg fungal-derived ß-glucan or 0.1% commercial yeast-derived ß-glucan, as a positive control diet, for a period of four weeks. The effect of the glucans on disease resistance was then evaluated by experimentally infecting the fish with Edwardsiella ictaluri by immersion and mortalities monitored for 14 days. Samples were collected from fish for analysis at 0, 1, 3, 7, 14, 21 and 28 days post-feeding (dpf), and also at 14 days post infection (dpi). The lowest dose of fungal-derived ß-glucan (0.05%) appeared insufficient to effectively stimulate the immune response of the fish, while those fed with the two highest levels of fungal-derived ß-glucan had enhanced immune responses compared to the control group. Significantly elevated levels of respiratory burst activity on all days examined (P < 0.05) and lysozyme activity on 7 dpf were found in the group fed 0.2% fungal-derived ß-glucan, while plasma anti-protease activity was significantly enhanced (P < 0.05) by 21 dpf, natural antibody titres by 3 dpf and complement activity by 7 dpf and also at 14 dpi in the group fed 0.1% fungal-derived ß-glucan. No statistical differences was seen in the level of mortalities between the dietary groups, although the group fed with the control diet had the highest level of mortalities and the groups fed with commercial yeast-derived ß-glucan and 0.2% fungal-derived ß-glucan the lowest.

Blue crabs Callinectes sapidus as potential biological reservoirs for white spot syndrome virus (WSSV).

White spot syndrome virus (WSSV) is a virulent pathogen of cultured shrimp and was first detected in farms in South Carolina (USA) in 1997 and subsequently in wild shrimp in 1999. We screened groups of 1808 wild Atlantic white shrimp Litopenaeus setiferus and 300 blue crabs Callinectes sapidus collected from South Carolina, Georgia, and Florida for the presence of WSSV using the Shrimple® immunoassay-strip test, with all positives and random subsets of negatives tested by TaqMan real-time PCR and in infectivity bioassays. Of 87 shrimp and 11 crabs that tested positive using the Shrimple® test, only a single C. sapidus was confirmed to be infected with WSSV by PCR and the infectivity bioassay. The data indicate that the prevalence of WSSV in these species is low in these southeastern US regions, but that C. sapidus may serve as a biological reservoir.

Effects of dietary NEXT ENHANCE®150 on growth performance and expression of immune and intestinal integrity related genes in gilthead sea bream (Sparus aurata L.).

Gilthead sea bream juveniles were fed different doses (0, 50, 100, 200, 300 ppm) of NEXT ENHANCE®150 (NE) for 9 weeks. Feed gain ratio (FGR) was improved by a 10% with all the doses, but feed intake decreased in a dose dependent manner. The optimum inclusion level to achieve maximum growth was set at 100 ppm. The hepatosomatic index did not vary and only at the highest dose, viscerosomatic and splenosomatic indexes were significantly decreased. No significant changes were found in haematological parameters, plasma biochemistry, total antioxidant capacity and respiratory burst. In a second trial, NE was given at 100 ppm alone (D1) or in combination with the prebiotic PREVIDA® (0.5%) (PRE) (D2) for 17 weeks. There were no differences in the growth rates, and FGR was equally improved for D1 and D2. No significant changes in haematology and plasma antioxidant capacity were detected. The histological examination of the liver and the intestine showed no outstanding differences in the liver, but the number of mucosal foldings appeared to be higher in D1 and D2 vs CTRL diet and the density of enterocytes and goblet cells also appeared higher, particularly in the anterior intestine. A 87-gene PCR-array was constructed based on our transcriptomic database (www.nutrigroup-iats.org/seabreamdb) and applied to samples of anterior (AI) and posterior (PI) intestine. It included 54 new gene sequences and other sequences as markers of cell differentiation and proliferation, intestinal architecture and permeability, enterocyte mass and epithelial damage, interleukins and cytokines, pattern recognition receptors (PRR), and mitochondrial function and biogenesis. More than half of the studied genes had significantly different expression between AI and PI segments. The functional significance of this differential tissue expression is discussed. The experimental diets induced significant changes in the expression of 26 genes. The intensity of these changes and the number of genes that were significantly regulated were higher at PI than at AI. At PI, both diets invoked a clear down-regulation of genes involved in cell differentiation and proliferation, some involved in cell to cell communication, cytokines and several PRR. By contrast, up-regulation was mostly found for genes related to enterocyte mass, cell epithelial damage and mitochondrial activity at AI. The changes were of the same order for D1 and D2, except for fatty acid-binding proteins 2 and 6 and the PRR fucolectin, which were higher in D2 and D1 fed fish, respectively. Thus, NE alone or in combination with PRE seems to induce an anti-inflammatory and anti-proliferative transcriptomic profile with probable improvement in the absorptive capacity of the intestine that would explain the improved FGR.

Identification of Bacillus strains for biological control of catfish pathogens.

Bacillus strains isolated from soil or channel catfish intestine were screened for their antagonism against Edwardsiella ictaluri and Aeromonas hydrophila, the causative agents of enteric septicemia of catfish (ESC) and motile aeromonad septicaemia (MAS), respectively. Twenty one strains were selected and their antagonistic activity against other aquatic pathogens was also tested. Each of the top 21 strains expressed antagonistic activity against multiple aquatic bacterial pathogens including Edwardsiella tarda, Streptococcus iniae, Yersinia ruckeri, Flavobacterium columnare, and/or the oomycete Saprolegnia ferax. Survival of the 21 Bacillus strains in the intestine of catfish was determined as Bacillus CFU/g of intestinal tissue of catfish after feeding Bacillus spore-supplemented feed for seven days followed by normal feed for three days. Five Bacillus strains that showed good antimicrobial activity and intestinal survival were incorporated into feed in spore form at a dose of 8×10(7) CFU/g and fed to channel catfish for 14 days before they were challenged by E. ictaluri in replicate. Two Bacillus subtilis strains conferred significant benefit in reducing catfish mortality (P<0.05). A similar challenge experiment conducted in Vietnam with four of the five Bacillus strains also showed protective effects against E. ictaluri in striped catfish. Safety of the four strains exhibiting the strongest biological control in vivo was also investigated in terms of whether the strains contain plasmids or express resistance to clinically important antibiotics. The Bacillus strains identified from this study have good potential to mediate disease control as probiotic feed additives for catfish aquaculture.

Effects of endosulfan exposure and Taura Syndrome Virus infection on the survival and molting of the marine penaeid shrimp, Litopenaeus vannamei.

Molting in crustaceans is an important endocrine-controlled biological process that plays a critical role in growth and reproduction. Many factors can affect this physiological cycle in crustaceans including environmental stressors and disease agents. For example the pathology of Taura Syndrome Virus (TSV) of shrimp is closely related to molting cycle. Similarly, endosulfan, a commonly used pesticide is a potential endocrine disruptor. This study explores interrelationships between pesticide exposure, virus infection and their interactions with physiology and susceptibility of the shrimp. Litopenaeus vannamei (Pacific white shrimp) were challenged with increasing doses of endosulfan and TSV (TSV-C, a Belize reference strain) to determine the respective median lethal concentrations (LC(50)s). The 96-h endosulfan LC(50) was 5.32 µg L(-1), while the 7-d TSV LC(50) was 54.74 mg L(-1). Subsequently, based on their respective LC(50) values, a 20-d interaction experiment with sublethal concentrations of endosulfan (2 µg L(-1)) and TSV (30 mg L(-1)) confirmed a significant interaction (p<0.05, χ(2)=5.29), and thereby the susceptibility of the shrimp. Concurrently, molt-stage of animals, both at the time of exposure and death, was compared with mortality. For animals challenged with TSV, no strong correlation between molt-stage and mortality was observed (p>0.05). For animals exposed to endosulfan, animals in the postmolt stage were shown to be more susceptible to acute toxicity (p<0.05). For animals exposed to both TSV and endosulfan, interference of endosulfan-associated stress lead to increasingly higher susceptibility at postmolt (p<0.05) during the acute phase of the TSV disease cycle.

The transcriptomic response to viral infection of two strains of shrimp (Litopenaeus vannamei).

The extent to which data-intensive studies of the transcriptome can provide insight into biological responses is not well defined, especially in the case of species (such as shrimp) where much physiological and biochemical knowledge is missing. In this study we took a transcriptomic approach to gain insight into the response to viral infection of two strains of the Pacific whiteleg shrimp (Litopenaeus vannamei) that differ in their resistance to Taura Syndrome Virus (TSV). Changes in gene expression in the hepatopancreas following infection with TSV and Yellow Head Virus (YHV) were assessed using a cDNA microarray containing 2469 putative unigenes. The null hypothesis tested was that significant differences between the transcriptomic responses to viral infection of resistant and sensitive strains would not be detected. This hypothesis was broadly rejected, with the most surprising observation being that the baseline (control, unchallenged) sensitive and resistant strains expressed distinguishable transcriptomic signatures. The resistant line was pre-disposed to lower expression of genes encoding viral (and host) proteins. Many of the genes differentiating resistant and sensitive lines are involved in protein metabolism, cellular trafficking, immune defense and stress response, although it was not possible to clearly identify candidate genes responsible for TSV resistance. In contrast to TSV challenge, YSV either failed to perturb the host transcriptome or created a "confused" response that was difficult to interpret.

Non-specific activation of antiviral immunity and induction of RNA interference may engage the same pathway in the Pacific white leg shrimp Litopenaeus vannamei.

Many questions remain unanswered regarding RNAi-based mechanisms and dsRNA-induced antiviral immune responses in penaeid shrimp. In this study, we report the characterization in the white leg shrimp Litopenaeus vannamei of RNAi pathway associated proteins Lv-Ago 1 and Lv-Ago 2, two members of the Argonaute family of proteins, as well as Lv-sid 1, the first shrimp homologue of Sid-1, a membrane channel-forming protein implicated in the cellular import of dsRNA. To decipher their functional implication in RNAi-related phenomena, we monitored their relative expression following stimulation by specific and non-specific RNA duplexes of diverse length. The findings show that the length of small RNA duplexes plays a critical role in the activation of both RNAi-related and innate antiviral responses. They also suggest that these two mechanisms of antiviral response may activate the same pathway, requiring Lv-Sid 1 and Lv-Ago 2 induction.

Knocking down a Taura syndrome virus (TSV) binding protein Lamr is lethal for the whiteleg shrimp Penaeus vannamei.

A cDNA encoding a laminin receptor protein (Lamr) has been isolated from hemocytes of the Pacific white shrimp Penaeus (Litopenaeus) vannamei (Pv), based on primers designed from a previously published Lamr sequence of a Taura syndrome virus (TSV) binding protein of the black tiger shrimp Penaeus monodon (Pm). The deduced amino acid sequence of PvLamr shares 97% identity with PmLamr and has significant homology to laminin receptors and ribosomal protein p40 from various organisms. Tissue distribution analysis by RT-PCR revealed that Lamr transcripts were widely expressed in all tested tissues of P. monodon and Penaeus vannamei. PmLamr was constructed and expressed in Escherichia coli, and the recombinant protein was purified and used to raise a polyclonal antibody. The antiserum reacted with purified recombinant PmLamr and crude muscle tissue proteins from both P. monodon and P. vannamei, but not with hemocyte-free shrimp hemolymph. Examination of protein localization by immunohistochemical analysis revealed the presence of Lamr positive cytoplasm in subcuticular epithelial cells, hematopoietic tissues, epithelial cells of the stomach, epithelial cells of the anterior midgut cecum, antennal gland epithelial cells, F cells of the hepatopancreas, cells in the ovarian zone of proliferation and spheroid cells in the lymphoid organ. RNA interference-mediated silencing of the messenger from Lamr in P. vannamei led to shrimp mortality and indicated an essential function of Lamr for shrimp viability. A negative consequence was that the effect of Lamr knockdown on shrimp infection by Taura syndrome virus could not be assessed.

Lack of evidence for Litopenaeus vannamei Toll receptor (lToll) involvement in activation of sequence-independent antiviral immunity in shrimp.

Injection of non-specific dsRNA initiates a broad-spectrum innate antiviral immune response in the Pacific white shrimp, Litopenaeus vannamei, however, the receptor involved in recognition of this by-product of viral infections remains unknown. In vertebrates, dsRNA sensing is mediated by a class of Toll-like receptors (TLRs) and results in activation of the interferon system. Because a TLR (lToll) was recently characterized in L. vannamei, we investigated its potential role in dsRNA recognition. We showed that injection of non-specific RNA duplexes did not modify lToll gene expression. A reverse genetic approach was therefore implemented to study its role in vivo. Silencing of lToll did not impair the ability of non-specific dsRNA to trigger protection from white spot syndrome virus and did not increase the shrimp susceptibility to viral infection, when compared to controls. In contrast, gene-specific dsRNA injected to specifically silence lToll expression activated an antiviral response. These data strongly suggest that shrimp lToll plays no role in dsRNA-induced antiviral immunity.

The role of crustins in Litopenaeus vannamei in response to infection with shrimp pathogens: An in vivo approach.

Crustin antimicrobial peptides, identified in crustaceans, are hypothesized to have both antimicrobial and protease inhibitor activity based on their primary structure and in vitro assays. In this study, a reverse genetic approach was utilized to test the hypothesis that crustins are antimicrobial in vivo in response to bacterial and fungal challenge. Injection of double-stranded RNA specific to a 120-bp region of LvABP1, one of the most prominent crustin isoforms, yielded a significant reduction in the expression of both crustin mRNA and protein within the hemocytes. To test the role of crustins in the shrimp immune response, RNAi was first used to suppress crustin expression and animals were subsequently injected with low pathogenic doses of either Vibrio penaeicida or Fusarium oxysporum. A significant increase in mortality in crustin-depleted animals was observed in animals infected with V. penaeicida as compared to controls, whereas no significant change in shrimp mortality was observed following infection with F. oxysporum.

Contributions of functional genomics and proteomics to the study of immune responses in the Pacific white leg shrimp Litopenaeus vannamei.

The need for better control of infectious diseases in shrimp aquaculture and the ecological importance of crustacea in marine ecosystems have prompted interest in the study of crustacean immune systems, particularly those of shrimp. As shrimp and other crustacea are poorly understood from the immunological point of view, functional genomic and proteomic approaches have been applied as a means of quickly obtaining molecular information regarding immune responses in these organisms. In this article, a series of results derived from transcriptomic and proteomic studies in shrimp (Litopenaeus vannamei) are discussed. Expressed Sequence Tag analysis, differential expression cloning through Suppression Subtractive Hybridization, expression profiling using microarrays, and proteomic studies using mass spectrometry, have provided a wealth of useful data and opportunities for new avenues of research. Examples of new research directions arising from these studies in shrimp include the molecular diversity of antimicrobial effectors, the role of double stranded RNA as an inducer of antiviral immunity, and the possible overlap between antibacterial and antiviral responses in the shrimp.

Knocking down caspase-3 by RNAi reduces mortality in Pacific white shrimp Penaeus (Litopenaeus) vannamei challenged with a low dose of white-spot syndrome virus.

Apoptosis has long been observed in viral target organs of white-spot syndrome virus (WSSV)-infected shrimp and whether the phenomenon helps the shrimp to survive the infection or is a factor leading to mortality is still controversial. If the shrimp mortality is a result of triggered apoptosis, then inactivation of caspase-3, a key protein in the induction of apoptosis, should improve shrimp survival upon challenge with WSSV. To test this prediction, we identified and characterized a caspase-3 homologue (cap-3) from the Pacific white shrimp Penaeus (Litopenaeus) vannamei and used this information to silence cap-3 expression by RNA interference prior to WSSV challenge. After confirming the efficacy of cap-3 silencing, its effects on mortality at high and low doses of WSSV were evaluated. In a high-dose WSSV challenge, cap-3 silencing had no significant effect on WSSV-induced mortality, except for a delay in mean time to death. However, at a low-dose WSSV challenge, cap-3 silencing correlated with a lower level of cumulative mortality, relative to silencing of a control gene, suggesting that apoptosis may exacerbate rather than decrease mortality in WSSV-challenged shrimp.

Anti-lipopolysaccharide factor in Litopenaeus vannamei (LvALF): a broad spectrum antimicrobial peptide essential for shrimp immunity against bacterial and fungal infection.

Antimicrobial peptides are an essential component of the innate immune system of most organisms. Expressed sequence tag analysis from various shrimp (Litopenaeus vannamei) tissues revealed transcripts corresponding to two distinct sequences (LvALF1 and LvALF2) with strong sequence similarity to anti-lipopolysaccharide factor (ALF), an antimicrobial peptide originally isolated from the horseshoe crab Limulus polyphemus. Full-length clones contained a 528bp transcript with a predicted open reading frame coding for 120 amino acids in LvALF1, and a 623bp transcript with a predicted open reading frame coding for 93 amino acids in LvALF2. A reverse genetic approach was implemented to study the in vivo role of LvALF1 in protecting shrimp from bacterial, fungal and viral infections. Injection of double-stranded RNA (dsRNA) corresponding to the LvALF1 message resulted in a significant reduction of LvALF1 mRNA transcript abundance as determined by qPCR. Following knockdown, shrimp were challenged with low pathogenic doses of Vibrio penaeicida, Fusarium oxysporum or white spot syndrome virus (WSSV) and the resulting mortality curves were compared with controls. A significant increase of mortality in the LvALF1 knockdown shrimp was observed in the V. penaeicida and F. oxysporum infections when compared to controls, showing that this gene has a role in protecting shrimp from both bacterial and fungal infections. In contrast, LvALF1 dsRNA activated the sequence-independent innate anti-viral immune response giving increased protection from WSSV infection.

Insights into the immune transcriptome of the shrimp Litopenaeus vannamei: tissue-specific expression profiles and transcriptomic responses to immune challenge.

Infectious disease constitutes a major obstacle to the sustainability of shrimp aquaculture worldwide and a significant threat to natural populations of shrimp and other crustacea. The study of the shrimp immune system, including the response to viral infection, has been hampered by a relative lack of molecular genetic information and of tools suitable for high-throughput assessment of gene expression. In this report, the generation of a cDNA microarray encompassing 2,469 putative unigenes expressed in gills, circulating hemocytes, and hepatopancreas of Litopenaeus vannamei is described. The unigenes printed on the microarray were derived from the analyses of 7,021 expressed sequence tags obtained from standard cDNA libraries as well as from libraries generated by suppression subtractive hybridization, after challenging shrimp with a variety of immune stimuli. The general utility of the cDNA microarray was demonstrated by interrogating the array with labeled RNA from four different shrimp tissues (gills, hemocytes, hepatopancreas, and muscle) and by analyzing the transcriptomic response of shrimp to a lethal challenge with white spot syndrome virus. Our results indicate that white spot syndrome virus infection upregulates (in the hepatopancreas) genes encoding known and potential antimicrobial effectors, while some genes involved in protection from oxidative stress were found to be downregulated by the virus.

Double-stranded RNA and antiviral immunity in marine shrimp: inducible host mechanisms and evidence for the evolution of viral counter-responses.

Double-stranded RNA (dsRNA) is a common virus-associated molecular pattern and a potent inducer of antiviral responses in many organisms. While it is clear that the specific RNA interference (RNAi) response, a phenomenon triggered by dsRNA, serves antiviral functions in invertebrates, innate (non-specific) antiviral immune reactions induced by dsRNA (e.g. the Interferon response) have long been thought to be restricted to vertebrates. Recent work in an underappreciated experimental model, the penaeid shrimp, is challenging these traditional distinctions, by demonstrating the existence of both innate (non sequence-specific) and RNAi-related (sequence-specific) antiviral phenomena in crustacea. Here we discuss the evidence for this bivalent role of dsRNA in the initiation of antiviral responses in shrimp, and present new data that suggest that the antiviral functions of the shrimp RNAi machinery have imposed selective pressures on an evolving viral pathogen. These findings open the door for the discovery of novel mechanisms of innate immunity, and provide a basis for the future development of strategies to control viral diseases in the commercially important penaeid shrimp.

Inactivation of White Spot Syndrome Virus (WSSV) by normal rabbit serum: implications for the role of the envelope protein VP28 in WSSV infection of shrimp.

White Spot Syndrome Virus (WSSV) is a highly pathogenic and prevalent virus affecting crustacea. A number of WSSV envelope proteins, including vp28, have been proposed to be involved in viral infectivity based on the ability of specific antibodies to attenuate WSSV-induced mortality in vivo. In the present study, a series of monoclonal and polyclonal antibodies targeting vp28 were tested for their ability to neutralize WSSV infectivity, with the purpose of identifying epitopes potentially involved in vp28-mediated infection of shrimp. Surprisingly, when used as protein A-purified immunoglobulin, none of the antibodies tested were capable of inhibiting WSSV infectivity. This included one polyclonal preparation that has been previously shown to inactivate WSSV, when used as whole rabbit serum. Moreover, strong inactivation of WSSV by some rabbit sera was observed, in a manner independent of anti-vp28 antibodies. These results underscore the problems associated with using heterogeneous reagents (e.g. whole rabbit antiserum) in viral neutralization experiments aimed at defining proteins involved in infection by WSSV. In light of this, the potential of anti-vp28 antibodies to specifically neutralize WSSV should be reconsidered.

Double-stranded RNA induces sequence-specific antiviral silencing in addition to nonspecific immunity in a marine shrimp: convergence of RNA interference and innate immunity in the invertebrate antiviral response?

Double-stranded RNA (dsRNA) is a common by-product of viral infections and a potent inducer of innate antiviral immune responses in vertebrates. In the marine shrimp Litopenaeus vannamei, innate antiviral immunity is also induced by dsRNA in a sequence-independent manner. In this study, the hypothesis that dsRNA can evoke not only innate antiviral immunity but also a sequence-specific antiviral response in shrimp was tested. It was found that viral sequence-specific dsRNA affords potent antiviral immunity in vivo, implying the involvement of RNA interference (RNAi)-like mechanisms in the antiviral response of the shrimp. Consistent with the activation of RNAi by virus-specific dsRNA, endogenous shrimp genes could be silenced in a systemic fashion by the administration of cognate long dsRNA. While innate antiviral immunity, sequence-dependent antiviral protection, and gene silencing could all be induced by injection of long dsRNA molecules, injection of short interfering RNAs failed to induce similar responses, suggesting a size requirement for extracellular dsRNA to engage antiviral mechanisms and gene silencing. We propose a model of antiviral immunity in shrimp by which viral dsRNA engages not only innate immune pathways but also an RNAi-like mechanism to induce potent antiviral responses in vivo.

Induction of antiviral immunity by double-stranded RNA in a marine invertebrate.

Vertebrates mount a strong innate immune response against viruses, largely by activating the interferon system. Double-stranded RNA (dsRNA), a common intermediate formed during the life cycle of many viruses, is a potent trigger of this response. In contrast, no general inducible antiviral defense mechanism has been reported in any invertebrate. Here we show that dsRNA induces antiviral protection in the marine crustacean Litopenaeus vannamei. When treated with dsRNA, shrimp showed increased resistance to infection by two unrelated viruses, white spot syndrome virus and Taura syndrome virus. Induction of this antiviral state is independent of the sequence of the dsRNA used and therefore distinct from the sequence-specific dsRNA-mediated genetic interference phenomenon. This demonstrates for the first time that an invertebrate immune system, like its vertebrate counterparts, can recognize dsRNA as a virus-associated molecular pattern, resulting in the activation of an innate antiviral response.

Sampling and evaluation of white spot syndrome virus in commercially important Atlantic penaeid shrimp stocks.

In 1997, white spot syndrome virus (WSSV) was discovered in shrimp culture facilities in South Carolina, USA. This disease was known to cause devastating mortalities in cultured populations in Southeast Asia and prompted concern for the health of wild populations in the USA. Our study surveyed wild shrimp populations for the presence of WSSV by utilizing molecular diagnostics and bioassay techniques. A total of 1150 individuals (586 Litopenaeus setiferus, 477 Farfantepenaeus aztecus and 87 F. dourarum) were examined for the presence of WSSV DNA by PCR. A total of 32 individuals tested positive and were used in a bioassay to examine the transmission of disease to healthy individuals of the culture species L. vannamei. DNA sequencing of PCR products from a positive individual confirmed that the positive individuals carried WSSV DNA. Significant mortalities were seen in test shrimp injected with tissue extracts from heavily infected wild shrimp. These data confirm the existence of WSSV in wild shrimp stocks along the Atlantic Coast and that the virus can cause mortalities in cultured stocks.

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.