Detection by PCR of hepatopancreatic parvovirus (HPV) and other viruses in hatchery-reared Penaeus monodon postlarvae

The prevalence of hepatopancreatic parvovirus (HPV), monodon baculovirus (MBV) and white spot syndrome virus (WSSV) in samples of Penaeus monodon postlarvae (PL10 to PL20, 10 to 20 d old postlarvae) in India was studied by PCR. Samples collected from different hatcheries, and also samples submitted by farmers from different coastal states, were analyzed. HPV was detected in 34%) of the hatchery samples and 31% of the samples submitted by farmers, using a primer set designed for detection of HPV from P. monodon in Thailand. However, none of these samples were positive using primers designed for detection of HPV from P. chinensis in Korea. This indicated that HPV from India was more closely related to HPV from P. monodon in Thailand. MBV was detected in 64% of the samples submitted by the farmers and 71% of the hatchery samples. A total of 84 % of the samples submitted by farmers, and 91% of the hatchery samples, were found positive for WSSV. Prevalence of concurrent infections by HPV, MBV and WSSV was 27% in hatchery samples and 29%, in samples submitted by farmers. Only 8% of the hatchery samples and 16% of the samples submitted by farmers were negative for all 3 viruses. This is the first report on the prevalence of HPV in P. monodon postlarvae from India.     

Diagnosis of Penaeus monodon-type baculovirus by PCR and by ELISA of occlusion bodies

The black tiger prawn Penaeus monodon is a valuable aquaculture product in Taiwan. Two specific diagnostic methods were established for P. monodon-type baculovirus, one using polymerase chain reaction (PCR) technology and the other enzyme-linked immunosorbent assay (ELISA) technology. Monodon-type baculovirus (MBV) was purified by sucrose gradient centrifugation from occlusion bodies of MBV-infected postlarvae of P. monodon. MBV DNA was subsequently purified from the occlusion bodies and its presence was confirmed by PCR using primers of the polyhedrin gene. Based on conserved sequences of the DNA polymerase genes of Autographa californica nuclear polyhedrosis virus (AcMNPV) and Lymantria dispar nuclear polyhedrosis virus (LdMNPV), primers were designed and synthesized to yield a 714 bp PCR fragment from MBV. However, the sequence of this fragment revealed low homology with that of LdMNPV and AcMNPV. From the DNA sequence of this fragment, a second set of primers was designed, and using these primers, a 511 bp DNA fragment was amplified only when MBV DNA was the template. DNA templates from AcMNPV, white spot syndrome diseased shrimp, or PMO cells (a cell line derived from the Oka organ of Penaeus monodon) did not give any amplified DNA fragment. Therefore, this primer pair was specific for the diagnosis of MBV. By using intraspleenic immunization of rabbits with purified MBV occlusion bodies, a polyclonal rabbit antiserum against MBV was obtained. This antiserum could detect nanogram levels of MBV, but did not cross react with white spot syndrome virus (WSSV), homogenates of PMO cells, postlarvae, hepatopancreatic tissue or intestinal tissue of black tiger prawns by competitive ELISA. This sensitive method could detect MBV even in tissue homogenates.     

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.     

PCR-based detection of white spot syndrome virus in cultured and captured crustaceans in India

AIMS: The occurrence and distribution of white spot syndrome virus (WSSV) among cultured and captured penaeid shrimps and crustaceans in the east coast of India was determined from November 1999 to April 2002 using PCR as a diagnostic tool. METHODS AND RESULTS: A total of 630 cultured samples consisting of 280 postlarvae collected from nine different hatcheries and 350 juvenile shrimps (40-60-day-old) collected from 18 different culture ponds were screened for WSSV. Of these cultured samples tested 53% were found to be single-step PCR positive. A total of 419 samples of captured crustaceans viz., Penaeus monodon brooders, P. indicus juveniles, Metapenaeus spp., crab Scylla serrata and Squilla mantis were also screened for WSSV by PCR, 23% of them were infected with WSSV. CONCLUSIONS: This study concluded that WSSV could be widespread in cultured and captured shrimps and other crustaceans in India. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that PCR screening of WSSV infection and rejection of infected stocks greatly assists shrimp aquaculture farmers for successful production and harvest.     

Detection of Taura syndrome virus (TSV) strain differences using selected diagnostic methods: diagnostic implications in penaeid shrimp

Positive results were obtained with nested white spot syndrome virus (WSSV) diagnostic PCR performed on 5 commercial brands of dry-packed Artemia cysts using several WSSV genomic sequence-specific primers. In 2 brands, PCR and nucleotide sequence analysis found C-->T and C-->G point mutations in the pms 146 WSSV amplicon, but in all 5 brands, the nucleotide sequences that were successfully amplified by the rrl, rr2 and tk-tmk gene-specific primer sets were identical to those of Penaeus monodon WSSV. However, despite the inarguable presence of WSSV or WSSV-like template DNA, we were unable to detect WSSV by PCR in hatched nauplii derived from PCR-positive cysts or in P. monodon postlarvae fed Artemia nauplii hatched from such cysts. Most simply, these results suggested that the cysts were externally contaminated with WSSV or WSSV-like template material that was removed during hatching and washing of the nauplii. Given the small sequence variations found, it may also have been a variety of WSSV non-infectious for P. monodon or Artemia and derived from other crustaceans or arthropods in the Artemia environment. However, we could not establish this conclusively and a small possibility remained that the PCR template in these tests was derived from WSSV template present internally in the cysts and derived from infected Artemia adults. However small, this possibility must be vigorously tested, given the impact that a positive outcome could have on the shrimp industry.     

Development of a real-time PCR assay for detection of monodon baculovirus (MBV) in penaeid shrimp

A real-time PCR method was developed to detect monodon baculovirus (MBV) in penaeid shrimp. A pair of MBV primers to amplify a 135 bp DNA fragment and a TaqMan probe were developed. The primers and TaqMan probe were specific for MBV and did not cross react with Hepatopancreatic parvovirus (HPV), White spot syndrome virus (WSSV), Infectious hypodermal and haematopoietic virus (IHHNV) and specific pathogen free (SPF) shrimp DNA. A plasmid (pMBV) containing the target MBV sequence was constructed and used for determination of the sensitivity of the real-time PCR. This real-time PCR assay had a detection limit of one plasmid MBV DNA copy. Most significantly, this real-time PCR method can detect MBV positive samples from different geographic locations in the University of Arizona collection, including Thailand and Indonesia collected over a 13-year period.     

Occurrence, histopathology and experimental transmission of hepatopancreatic parvovirus infection in Penaeus monodon postlarvae

Hepatopancreatic parvovirus (HPV) was detected in samples of Penaeus monodon post-larvae (PL-13, PL-18, PL-19, PL-26) from 2 hatcheries in 2 provinces (Samar and Iloilo) in the Philippines. The percentage of infection was 20 to 100% in postlarvae obtained from the hatchery in Samar in August 2001. Postlarvae from the hatchery in Iloilo, sampled in October and November 2001, had 70 to 99% HPV infection. Wet mounts of squashed hepatopancreatic tissue stained with malachite green (wet-mount technique) and histopathology revealed the presence of large, usually single, basophilic intranuclear inclusion bodies in the distal tubules, which led to displacement of the nucleoli. Light microscopy showed ovoid to spherical inclusion bodies, 5 to 11 mmicrom in diameter. Transmission electron microscopy revealed that the inclusion bodies were composed of electron-dense granular material and virions. The virions appeared roughly spherical and averaged 18 to 22 nm in diameter. An experiment was undertaken to induce HPV infection by feeding P. monodon postlarvae with virus-infected postlarvae. P. monodon postlarvae (PL-16), initially determined as free from HPV, were found HPV-positive 24 h after being fed with infected material. The percentage of infection ranged from 30% at Day 1 post-infection (p.i.) to 100% at Day 7 p.i. determined by the wet-mount technique and by histopathology. This is the first report of a successful horizontal transmission of HPV in P. monodon postlarvae.     

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.     

Relationship between white spot syndrome virus and indicators of quality in Penaeus monodon postlarvae in Karnataka,India

White spot disease (WSD) is a viral disease of shrimp caused by white spot syndrome virus (WSSV). Stocking WSSV-infected seed has been implicated as a major risk factor for outbreaks of WSD. In addition, the quality of postlarvae batches has been proposed as a predictor for good crops. This paper describes the relationship between indicators of quality and WSSV in postlarvae (PL) of Penaeus monodon from Karnataka, India, over the period September 1999 to January 2000. Three outcome variables were considered: the WSSV status of the PL, as determined by PCR, and 2 subjective assessments of PL quality, namely the activity of the PL and the quality of the PL as determined by research assistants and farmers, respectively. Of the 73 batches of PL, 49.3% from a random sample of farms tested positive for WSSV. After adjusting for confounding, stocking earlier in the growing season and duration of transportation were the main risk factors for the presence of WSSV. The quality assessed by farmers and the PL activity assessed by research assistants showed only fair agreement (kappa 0.252) reaffirming the subjective nature of such techniques. The only variables consistently associated with either assessment of quality in univariate analysis were PL length, number per bag and salinity of the water in the delivery bags. After adjusting for confounding, no single variable was consistently associated with PL quality and activity. The research assistants' assessment of PL activity was also associated with the hatchery and a brown-orange hepatopancreas in univariate analysis. After adjusting for confounding, a brown-orange hepatopancreas was still significant and fitted into the model together with the salinity of the water in the PL bags. The farmers' assessment of quality was associated with PL length, date of stocking and duration of transportation in both univariate and multivariable analyses. There was no relationship between quality assessment and WSSV in PCR-positive PL.     

Synthethic peptide used to develop antibodies for detection of polyhedrin from monodon baculovirus (MBV)

The use of previously published primers to amplify the monodon baculovirus (MBV) polyhedrin gene sequence by polymerase chain reaction (PCR) from post larvae (PL) of Thai Penaeus monodon resulted in failure. As a result, the putative polyhedrin protein of MBV was isolated from infected PL by homogenization, differential centrifugation and density gradient centrifugation with verification by transmission electron microscopy (TEM). By SDS-PAGE, a single major protein band at 58 kDa was obtained from the putative polyhedrin fraction and this corresponded to a previous report of the molecular weight of polyhedrin from MBV. When used for N-terminal sequence analysis, the putative polyhedrin protein yielded a sequence of 25 amino acids (M F D D S M M M E N M D D L S G D Q K M V L T L A) that did not correspond to the deduced amino acid sequence derived from a previous report of a putative MBV polyhedrin gene amplicon. Despite this, a synthetic peptide of our 25 amino acid sequence (25Pmbv) was conjugated with bovine serum albumin and used as an antigen for antiserum production in mice. Using immunohistochemistry with tissue sections of PL infected with MBV or other viruses, the mouse anti-25Pmbv antiserum showed strong immunoreactivity to occlusion bodies of MBV only. It also showed strong reactivity to the 58 kDa putative polyhedrin protein in Western blots. Altogether, the results suggest that the 58 kDa protein is Thai MBV polyhedrin and that a previously reported MBV polyhedrin gene sequence may represent another protein or polyhedrin from a different variety of MBV.     

Appendage deformity syndrome--a nutritional disease of Macrobrachium rosenbergii

Culture of the freshwater prawn Macrobrachium rosenbergii as an alternative to penaeid shrimp has recently increased in coastal areas of southern India in order to avoid numerous problems, particularly with white spot syndrome virus (WSSV). However, M. rosenbergii culture is now threatened by a new disease, appendage deformity syndrome (ADS), that also results in high mortality. Analysis of ADS prawns for viruses such as WSSV, monodon baculovirus (MBV) and infectious hypodermal and hematopoeitic necrosis virus (IHHNV) gave negative results. ADS prawns were also negative for bacterial pathogens and affected animals did not respond to antibiotic therapy. A study of potential nutritional deficiency revealed that carotenoid supplementation in the diet led to a significant decrease in ADS prawns.     

Performance of WSSV-infected and WSSV-negative Penaeus monodon postlarvae in culture ponds.

In a survey of 27 Penaeus monodon culture ponds stocked with postlarvae (approximately PL10) at medium density (approximately 40 shrimp m(-2)), single-step nested white spot syndrome virus (WSSV) PCR was used to measure the WSSV infection rates in the shrimp populations within 1 mo after stocking. Seven ponds were initially WSSV-free, and the shrimp in 5 of these were harvested successfully. In the ponds (n = 6) where detection rates were higher than 50%, mass mortality occurred during the growth period, and none of these ponds was harvested successfully. In a subsequent study, P. monodon brooders were classified into 3 groups according to their WSSV infection status before and after spawning: brooders that were WSSV-positive before spawning were assigned to group A; spawners that became WSSV-positive only after spawning were assigned to group B; and group C consisted of brooders that were still WSSV-negative after spawning. WSSV screening showed that 75, 44 and 14%, respectively, of group A, B and C brooders produced nauplii that were WSSV-positive. Most (57%; 16/28) of the brooders in group A produced nauplii in which the WSSV prevalence was high (>50%).When a pond was stocked with high-prevalence nauplii from 1 of these group A brooders, an outbreak of white spot syndrome occurred within 3 wk and only approximately 20% of the initial population survived through to harvest (after 174 d). By contrast, 2 other ponds stocked with low-prevalence and WSSV-negative nauplii (derived respectively from 2 brooders in group B), both had much higher survival rates (70 to 80%) and yielded much larger (approximately 3x by weight) total harvests. We conclude that testing the nauplii is an effective and practical screening strategy for commercially cultured P. monodon.     

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.     

Low impact of infectious hypodermal and hematopoietic necrosis virus (IHHNV) on growth and reproductive performance of Penaeus monodon

No controlled studies on the effect of infectous hypodermal and necrosis virus (IHHNV) on Penaeus monodon have been previously reported. Here we describe domesticated P. monodon that became positive for IHHNV and other viruses at variable levels of prevalence during cultivation in 16 open-air, earthen ponds. These were stocked with domesticated postlarvae (PL) that tested negative for 7 shrimp viruses including IHHNV at 6% prevalence in 3 checks using polymerase chain reaction (PCR) methods. These PL were derived from domesticated female broodstock that individually tested negative for the same viruses. At 4 mo of culture, the shrimp in some ponds without obvious mortality tested positive by PCR methods for IHHNV and 3 other viruses at variable levels of maximum estimated prevalence (MEP). Stained tissue sections showed no lesions typical of IHHNV, but in situ hybridization tests with an IHHNV-specific DNA probe were positive. There was no significant difference in mean body weight (i.e. ca. 25 g) between shrimp groups positive or negative for IHHNV. Similar results were obtained with IHHNV negative and positive adults at 1 yr. Adults that individually tested negative for all 7 viruses and some that tested lightly positive for IHHNV were bred for the next generation. There were no significant differences in the number of eggs (> 600 000) and nauplii (ca. 300,000) produced by females negative and positive for IHHNV. From these females, 11/49 (22%) IHHNV PCR-positive PL batches were obtained from PCR-negative spawners, while 8/11 (73%) were obtained from IHHNV PCR-positive spawners. The results suggested that IHHNV infection can be transmitted vertically but does not seriously retard growth of P. monodon or affect fecundity of lightly infected broodstock.     

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

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

Multiple pathogens found in growth-retarded black tiger shrimp Penaeus monodon cultivated in Thailand

In 2001-2002 throughout Thailand, black tiger shrimp Penaeus monodon farmers reported very unusual retarded growth. We have called this problem monodon slow growth syndrome (MSGS). Based on decreased national production, estimated losses due to this phenomenon were in the range of 13 000 million baht (approximately 300 million US dollars) in 2002. Since rearing practices had not changed, it was considered possible that the MSGS problem may have arisen from a new or existing pathogen. To examine this possibility, cultivated shrimp were sampled from 32 commercial rearing ponds that reported abnormally slow growth from eastern, central and southern regions of Thailand. Shrimp were randomly sampled from each pond and grouped into normal and small shrimp. Normal shrimp were defined as those with body weights (BW) of 24 g or more while small shrimp were defined as those that weighed 16.8 g or less. Pleopods were used for detection of monodon baculovirus (MBV), heptopancreatic parvovirus (HPV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) using specific polymerase chain reaction (PCR) assays. In addition, some shrimp were processed for normal histopathology and transmission electron microscopy (TEM). Most of the shrimp specimens were infected by at least 1 of these viruses but many had dual or multiple infections. Prevalence of HPV and combined HPV/MBV infections in the small shrimp was significantly higher than in the normal shrimp. In addition to the viruses, a new microsporidian species, gregarines and bacteria were also observed but were not significantly associated with the MSGS problem. Some of the small shrimp gave negative results for all these pathogens by PCR and histology and no new and unique histopathology was recognized in any of the samples. The findings suggested that HPV infection was a contributing factor but not the overriding factor responsible for MSGS. It is possible that MSGS is caused by an unknown pathogen or by some other presently unknown, non-pathogenic factor.     

Studies on effective PCR screening strategies for white spot syndrome virus (WSSV) detection in Penaeus monodon brooders.

We re-tested stored (frozen) DNA samples in 5 independent polymerase chain reaction (PCR) replicates and confirmed that equivocal test results from a previous study on white spot syndrome virus (WSSV) in brooders and their offspring arose because amounts of WSSV DNA in the test samples were near the sensitivity limits of the detection method. Since spawning stress may trigger WSSV replication, we also captured a fresh batch of 45 brooders for WSSV PCR testing before and after spawning. Replicates of their spawned egg batches were also WSSV PCR tested. For these 45 brooders, WSSV prevalence before spawning was 67% (15/45 1-step PCR positive, 15/45 2-step PCR positive and 15/45 2-step PCR negative). Only 27 (60%) spawned successfully. Of the successful spawners, 56% were WSSV PCR positive before spawning and 74% after. Brooders (15) that were heavily infected (i.e. 1-step PCR positive) when captured mostly died within 1 to 4 d, but 3 (20%) did manage to spawn. All their egg batch sub-samples were 1-step PCR positive and many failed to hatch. The remaining 30 shrimp were divided into a lightly infected group (21) and a 2-step PCR negative group (9) based on replicate PCR tests. The spawning rates for these 2 groups were high (81 and 78%, respectively). None of the negative spawners (7) became WSSV positive after spawning and none gave egg samples positive for WSSV. In the lightly infected group (21), 6 brooders were 2-step WSSV PCR negative and 15 were 2-step WSSV PCR positive upon capture. However, all of them were WSSV PCR positive in replicate tests and after spawning or death. Four died without spawning. The remaining 17 spawned but only 2 gave egg samples PCR negative for WSSV. The other 15 gave PCR positive egg samples, but they could be divided into 2 spawner groups: those (7) that became heavily infected (i.e. 1-step PCR positive) after spawning and those (8) that remained lightly infected (i.e. became or remained 2-step PCR positive only). Of the brooders that became heavily infected after spawning, almost all egg sample replicates (91 %) tested 2-step PCR positive. One brooder even gave heavily infected (i.e. 1-step PCR positive) egg samples. For the brooders that remained lightly infected after spawning, only 27% of the egg sample replicates were 2-step PCR positive. Based on these results, we recommend that to avoid false negatives in WSSV PCR brooder tests screening tests should be delayed until after spawning. We also recommend, with our PCR detection system, discarding all egg batches from brooders that are 1-step PCR positive after spawning. On the other hand, it may be possible with appropriate monitoring to use eggs from 2-step PCR positive brooders for production of WSSV-free or lightly infected postlarvae. These may be used to stock shrimp ponds under low-stress rearing conditions.     

Prevalence and effect of spawner-isolated mortality virus on the hatchery phases of Penaeus monodon and P. merguiensis in Australia

Spawner-isolated mortality virus (SMV) has been associated with mortalities in broodstock of Penaeus monodon and with mid-crop mortality syndrome on grow-out farms. Epidemiological evidence suggested an association between the SMV status of broodstock and subsequent survival of their progeny, and this paper describes investigations into that association. The faeces of 909 broodstock in 9 different groups were tested by a polymerase chain reaction (PCR) for SMV and positive results were confirmed by an internal dot-blot. Seventy-seven spawners (8.5%) were positive for SMV with prevalence ranging from 0 to 24% among groups. The prevalence in spawners of P. monodon was higher (24%) than in P. merguiensis (4%). Three longitudinal studies were undertaken to compare the survival of progeny from broodstock that were positive to SMV with those that were not. Survival in hatchery tanks of progeny from SMV-positive spawners was lower than those from SMV-negative spawners with reductions of 23% (p = 0.01), 7.3% (p = 0.214) and 18.9% (p = 0.129) in the 3 studies. The conclusions were less consistent when examined during each of the later stages of growth in hatchery pools, nursery and grow-out ponds, with progeny from SMV-postive spawners sometimes having better survival rates. However, survival was better overall in progeny from SMV-negative spawners. Simple linear regression showed survival was negatively related to the proportion of postlarvae from SMV-positive spawners, with a decrease in survival of 5.6% for each 10% increase in the proportion of postlarvae coming from SMV-positive spawners (p = 0.006). Data from 38 ponds showed 6.71% of losses were due to SMV. If these losses were consistent across the entire industry, the annual loss due to SMV would have been approximately AUD 3 million in 1999/2000.