Detection of gill-associated virus (GAV) by in situ hybridization during acute and chronic infections of Penaeus monodon and P. esculentus

Chronic and acute gill-associated virus (GAV) infections were examined by in situ hybridization (ISH) using a DNA probe targeting a 779 nucleotide region of the ORF1b-gene. Chronic GAV infections were observed in healthy Penaeus monodon collected from farms and healthy P. esculentus surviving experimental infection. During chronic-phase infections in both species, GAV was detected only in partitioned foci of cells with hypertrophied nuclei (spheroids) within the lymphoid organ. Acute-phase infections were observed in moribund P. monodon and P. esculentus infected experimentally with a high dose of GAV, and in moribund P. monodon collected from farms during outbreaks of disease. During acute experimental infections in P. monodon, ISH detected GAV throughout the lymphoid organ, in gills and in connective tissues throughout the cephalothorax. In moribund P. monodon collected from natural outbreaks of disease, GAV was also detected in the gills and in connective tissues of the cephalothorax, but the distribution of virus within the lymphoid organ varied. In acutely infected P. esculentus, GAV was detected in connective tissues, but was restricted to the inner stromal matrix cells and endothelial cells of intact lymphoid organ tubules. The tissue distribution of GAV identified by ISH suggests that shrimp are able to control and maintain chronic asymptomatic infection by a process involving lymphoid organ spheroids. Acute phase infections and the development of disease appear to be dose-related and involve the systemic distribution of virus in connective tissues throughout the cephalothorax.     

Differences in the susceptibility of some penaeid prawn species to gill-associated virus (GAV) infection

Four species of penaeid prawn cultured in Australia (Penaeus monodon, Penaeus esculentus, Marsupenaeus japonicus and Fenneropenaeus merguiensis) were injected with a virulent preparation of gill-associated virus (GAV). P. monodon (average weight = 8.9, 13.9 and 19.2 g), P. esculentus (average weight = 19.5 g), F. merguiensis (average weight = 10.5 g), and small (average weight = 5.8 g) M. japonicus displayed overt signs of disease and mortalities which reached 82 to 100% within 23 d post-injection. Cumulative mortalities in P. esculentus and F. merguiensis were significantly lower than for P. monodon of the same size class. Medium (average weight = 13.0 g) M. japonicus also developed overt signs of disease but cumulative mortalities were not significantly higher than uninfected controls. Large (average weight = 20.3 g) M. japoncius did not display symptoms of disease and there were no significant mortalities up to 23 d post-injection.     

In situ hybridization demonstrates that Litopenaeus vannamei, L. stylirostris and Penaeus monodon are susceptible to experimental infection with infectious myonecrosis virus (IMNV)

Infectious myonecrosis virus (IMNV) was recently found to be the cause of necrosis in the skeletal muscle of farm-reared Litopenaeus vannamei from northeastern Brazil. Nucleic acid extracted from semi-purified IMN virions showed that this virus contains a 7.5 kb RNA genome. A cDNA library was constructed, and a clone, designated as IMNV-317, was labeled with digoxigenin-11-dUTP and used as a gene probe for in situ hybridization (ISH). This probe specifically detected IMNV in infected tissues. To determine the susceptibility of 3 species of penaeid shrimp (L. vannamei, L. stylirostris, Penaeus monodon) to IMNV infection, juveniles were injected with purified virions and observed for clinical signs of infection and mortality over a 4 wk period. All L. vannamei exhibited typical lesions after 6 d, and lesions were visible in all L. stylirostris by Day 13. The clinical signs of opaque muscle were not seen in P. monodon, due to their highly pigmented exoskeleton precluding visual detection of lesions. Moderate mortality (20%) occurred in infected L. vannamei. No mortalities were observed in either L. stylirostris or P. monodon. Histological examination and ISH indicated that all 3 species are susceptible to IMNV infection. Using ISH, IMNV was detected in tissues including the skeletal muscle, lymphoid organ, hindgut, and phagocytic cells within the hepatopancreas and heart. In all 3 species, skeletal muscle cells produced the strongest ISH reactions. Based on the onset of clinical signs of infection and mortality, L. vannamei appears to be the most susceptible of these 3 species to IMNV infection.     

Monoclonal antibodies specific to yellow-head virus (YHV) of Penaeus monodon

Monoclonal antibodies specific to 22, 67 and 135 kDa proteins of yellow-head virus (YHV) were produced from a mouse immunized with partially purified YHV isolated from the haemolymph of experimentally YHV-infected Penaeus monodon. Four groups of monoclonal antibodies were identified. One group of antibodies bound only to native protein of YHV while the others were specific to 135, 67 and 22 kDa proteins in both native and denatured forms. All antibodies could be used to detect YHV infection by means of dot blot and immunohistochemistry. However, antibodies specific to the 22 kDa protein gave the best immunohistochemistry results in terms of intensity and sharpness of staining.     

Duplex real-time PCR for detection and quantification of monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV) in Penaeus monodon

We describe a duplex real-time PCR assay using TaqMan probes for the simultaneous detection of monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV). Both MBV and HPV are shrimp enteric viruses that infect intestinal and hepatopancreatic epithelial cells. Both viruses can cause significant mortalities and depressed growth in infected larval, postlarval, and early juvenile stages of shrimp, and thus present a risk to commercial aquaculture. In this duplex assay, we combined 2 single real-time PCRs, amplifying MBV and HPV, in a one-tube PCR reaction. The 2 viruses were distinguished by specific fluorescent labels at the 5' end of TaqMan probes: the MBV probe was labeled with dichlorodimethoxyfluorescein (JOE), and the HPV probe was labeled with 6-carboxyfluorescein (FAM). The duplex real-time PCR assay was performed in a multi-channel real-time PCR detection system, and MBV and HPV amplification signals were separately detected by the JOE and FAM channels. This duplex assay was validated to be specific to the target viruses and found to have a detection limit of single copies for each virus. The dynamic range was found to be from 1 to 1 x 10(8) copies per reaction. This assay was further applied to quantify MBV and HPV in samples of infected Penaeus monodon collected from Malaysia, Indonesia, and Thailand. The specificity and sensitivity of this duplex real-time PCR assay offer a valuable tool for routine diagnosis and quantification of MBV and HPV from both wild and farmed shrimp stocks.     

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

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

Development of a monoclonal antibody specific to yellow head virus (YHV) from Penaeus monodon

A monoclonal antibody specific to yellow head virus (YHV) was produced from a mouse immunized with gill extracts prepared from laboratory-reared Penaeus monodon dually infected with YHV and white spot syndrome virus (WSSV). One clone designated V3-2B specifically bound to native and SDS-treated viral specific antigens. Immunocytochemical studies of infected gills revealed viral specific immunoreactivities in the cytoplasm of gill tissue and in haemocytes. No antibody binding was observed in gills from non-infected shrimp. In addition, immunocytochemical examination of tissues from shrimp experimentally infected with YHV gave a positive reaction, while tissues from uninfected control shrimp or shrimp experimentally infected with WSSV did not. Western blot analysis indicated that the antibody reacted with a protein of approximately 135 kD that was present only in shrimp infected with YHV. In dot-blot indirect immunoperoxidase assays, the antibody was able to detect viral associated antigen in diluted haemolymph up to 1:50 dilution and in an ammonium sulfate precipitate of haemolymph up to 1:1000 dilution. The results suggested that this antibody might be useful for development of effective diagnostic techniques for both heavy and mild YHV infections in shrimp.     

Preliminary study on haemocyte response to white spot syndrome virus infection in black tiger shrimp Penaeus monodon

White spot syndrome virus (WSSV) has been a major cause of shrimp mortality in aquaculture in the past decade. In contrast to extensive studies on the morphology and genome structure of the virus, little work has been done on the defence reaction of the host after WSSV infection. Therefore, we examined the haemocyte response to experimental WSSV infection in the black tiger shrimp Penaeus monodon. Haemolymph sampling and histology showed a significant decline in free, circulating haemocytes after WSSV infection. A combination of in situ hybridisation with a specific DNA probe for WSSV and immuno-histochemistry with a specific antibody against haemocyte granules in tissue sections indicated that haemocytes left the circulation and migrated to tissues where many virus-infected cells were present. However, no subsequent haemocyte response to the virus-infected cells was detected. The number of granular cells decreased in the haematopoietic tissue of infected shrimp. In addition, a fibrous-like immuno-reactive layer appears in the outer stromal matrix of tubule walls in the lymphoid organ of infected shrimp. The role of haemocytes in shrimp defence after viral infection is discussed.     

Changes in tissue defence system in white spot syndrome virus (WSSV) infected Penaeus monodon

The present study examined the changes occurring in the pro phenoloxidase system and antioxidant defence status in haemolymph, hepatopancreas and muscle tissue of white spot syndrome virus (WSSV) infected Penaeus monodon. Tiger shrimps (P. monodon) were infected with white spot virus by intramuscular injection of the virus inoculum. Levels of lipid peroxides and the activities of phenoloxidase, glutathione-dependent antioxidant enzymes [glutathione peroxidase (GPX), glutathione-S-transferase (GST)] and antiperoxidative enzymes [superoxide dismutase (SOD) and catalase (CAT)] were determined. WSSV infection induced a significant increase in lipid peroxidation in haemolymph, muscle and hepatopancreas of experimental P. monodon compared to normal controls. This was paralleled by significant reduction in the activities of phenol oxidase, glutathione-dependent antioxidant enzymes and antiperoxidative enzymes. The results of the present study indicate that the tissue antioxidant defence system in WSSV infected P. monodon is operating at a lower rate, which ultimately resulted in the failure of counteraction of free radicals, leading to oxidative stress as evidenced by the increased level of lipid peroxidation.     

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

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

Different responses to infectious hypodermal and hematopoietic necrosis virus (IHHNV) in Penaeus monodon and P. vannamei

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is widespread in cultured Penaeus monodon and P. vannamei in Thailand. It causes runt-deformity syndrome that is characterized by physical abnormalities and stunted growth in P. vannamei, but causes no apparent disease in P. monodon. In both species, the virus may produce Cowdry Type A inclusions in tissues of ectodermal and mesodermal origin, but these are common in P. vannamei and rare in P. monodon. The virus can be more easily detected in both species by IHHNV-specific PCR primers. By in situ hybridization (ISH) using specific IHHNV probes, fixed phagocytes associated with myocardial cells tended to show strong positive reactions in both shrimp species. Ovarian and neural tissue (neurons in the nerve ganglia and glial cells in the nerve cord) were ISH positive for IHHNV only in P. vannamei. By transmission electron microscopy, necrotic cells were found in the gills of IHHNV-infected P. vannamei, while paracrystalline arrays of virions and apoptotic cells rather than necrotic cells were found in the lymphoid organ of IHHNV-infected P. monodon. Thus, it is possible that apoptosis in P. monodon contributes to the absence of clinical disease from IHHNV. These findings reveal different responses to IHHNV infection by the 2 shrimp species. A curious feature of IHHNV infection in P. monodon was inconsistency in the comparative viral load amongst tissues of different specimens, as detected by both ISH and real-time PCR. This inconsistency in apparent tissue preference and the reasons for different cellular responses between the 2 shrimp species remain unexplained.     

Up-regulation of ribophorin I after yellow head virus (YHV) challenge in black tiger shrimp Penaeus monodon

This work constitutes the second report from a continuing investigation of shrimp genes that may be involved in apoptosis associated death resulting from yellow head virus (YHV) infection. Here, we describe from the black tiger shrimp Penaeus monodon, a ribophorin I-like gene that is probably a subunit of the oligosaccharyltransferase complex (OST), a key enzyme in N-linked glycosylation that occurs in the endoplasmic reticulum. The OST complex also contains DAD1 (defender against apoptotic death 1) that has been reported to control apoptosis and that we have previously reported from P. monodon. The full length ribophorin I of P. monodon comprised 2157 bp with the ORF of 1806 bp corresponding to 601 deduced amino acids and three putative N-linked glycosylation sites. Analysis revealed hydrophobic properties implying that it could be a membrane protein. Tissue distribution analysis using real-time RT-PCR with SYBR Green revealed that ribophorin I was endogenously expressed in all examined tissues of normal shrimp. However, unlike DAD1 that was down-regulated after YHV challenge, ribophorin I expression was up-regulated and remained high until the moribund stage.     

A syntenin-like protein with postsynaptic density protein (PDZ) domains produced by black tiger shrimp Penaeus monodon in response to white spot syndrome virus infection

We report the isolation and characterization of products from a subtractive cDNA library from the haemolymph of Penaeus monodon experimentally infected with white spot syndrome virus (WSSV). One cDNA derived from up-regulated mRNA was identified. A homology search indicated similarity to the putative protein syntenin (TE8). The nearly complete nucleotide sequence of TE8 was obtained by rapid amplification of cDNA (RACE). Its putative protein product contained a tandem repeat of PDZ domains (postsynaptic density protein or PSD-95, DlgA and ZO-1). We propose that TE8 may function as an adapter that couples PDZ-binding protein(s) in a signaling pathway involved in the shrimp response to WSSV.     

Experimental infection of Penaeus vannamei by a rickettsia-like bacterium (RLB) originating from P. monodon

A rickettsia-like bacterium (RLB), which caused severe mortalities of commercially farmed Penaeus monodon in the southwest region of Madagascar, was investigated to determine whether the organism would produce the same disease in P. vannamei. Two series of bioassays were performed to determine whether this RLB could be transmitted to P. vannamei through injection and per os exposure. The first series of challenge bioassays used frozen, RLB-infected P. monodon tissue from Madagascar as the inoculum and feed for the injection, and per os bioassays with specific pathogen free (SPF) P. vannamei. In the second series of bioassays, frozen RLB-infected P. vannamei tissue derived from the first series of injection bioassays was used as the inoculum to challenge by injection and per os SPF P. vannamei. Disease status was determined through standard histological techniques and by in situ hybridization assays with a digoxigenin-labeled probe specific for this RLB. The results indicated that P. vannamei did develop the RLB infection when injected with either RLB infected P. monodon or P. vannamei tissue homogenates. This contrasts with results from the per os exposure to the RLB in which the disease could not be reproduced.     

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

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

Generation of monoclonal antibodies specific to Hepatopancreatic parvovirus (HPV) from Penaeus monodon

Hepatopancreatic parvovirus (HPV) was isolated from the hepatopancreas (HP) of slow growth Penaeus monodon by urografin gradient centrifugation. The presence of HPV in the fraction was monitored by PCR and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Only 1 major 54 kDa protein band was observed in the strong PCR-positive fractions used to immunize mice for monoclonal antibody production. After cell fusion, the first step in selecting specific antibodies was performed by dot-blot assay with purified HPV viral particles. The second screening step was carried out using Western blots of purified HPV proteins and immunohistochemistry of HPV-infected HP tissue. Four monoclonal antibodies were isolated; these bound to the 54 kDa protein in Western blots and to intranuclear inclusion bodies in tubule epithelial cells of HPV-infected prawn tissue by immunohistochemistry. None of the antibodies showed cross-reactivity either to uninfected shrimp tissue or to other shrimp viruses tested. These reagents have potential for use in developing a highly sensitive immunoassay such as sandwich ELISA or a convenient kit for detection of HPV infection.