Results from black tiger shrimp penaeus monodon culture ponds stocked with postlarvae PCR-positive or -negative for white-spot syndrome virus (WSSV).

Commercial, intensive, earthen shrimp ponds (188) in southern Thailand were stocked with postlarvae (PL) of Penaeus monodon that had tested positive or negative for white-spot syndrome virus (WSSV) infection by polymerase chain reaction (PCR) assay. All the PL were grossly healthy. At 2 wk intervals after stocking, shrimp from each pond were examined for gross WSSV lesions and tested for WSSV by PCR. Shrimp from all the ponds stocked with WSSV-PCR-positive PL (Group 0, n = 43) eventually showed gross signs of white-spot disease (WSD) at an average of 40 d after stocking. Of the remaining ponds stocked with WSSV-PCR-negative PL (n = 145), some remained WSSV-PCR-negative throughout the study (Group 5, n = 52), while others (93) became WSSV-PCR-positive after stocking, during the first month (Group 1, n = 23), second month (Group 2, n = 40), third month (Group 3, n = 24), or fourth month (Group 4, n = 6). Crop failure was defined as a pond drain or forced harvest before 14 wk or 98 d of cultivation. For Group 0 the proportion of ponds failing was 0.953, while it was only 0.019 for Group 5. Thus, the relative risk of failure for Group 0 was approximately 50 times that of Group 5. The relative risk of failure for Group 0 was also 3 times that for ponds stocked with WSSV-PCR-negative PL. Obviously, not all WSSV outbreaks resulted in crop failure. Of the 93 ponds stocked with PCR-negative PL that later yielded WSSV-PCR-positive shrimp, 53% reached successful harvest. The study showed that PCR screening of PL and rejection of WSSV-positive batches before stocking could greatly improve the chances of a successful harvest.     

Risk factors associated with white spot syndrome virus infection in a Vietnamese rice-shrimp farming system

White spot disease (WSD) is a pandemic disease caused by a virus commonly known as white spot syndrome virus (WSSV). Several risk factors for WSD outbreaks have been suggested. However, there have been very few studies to identify risk factors for WSD outbreaks in culture systems. This paper presents and discusses the risk factors for WSSV infection identified during a longitudinal observational study conducted in a Vietnamese rice-shrimp farming system. A total of 158 variables were measured comprising location, features of the pond, management practices, pond bottom quality, shrimp health and other animals in the pond. At the end of the study period WSSV was detected in 15 of the 24 ponds followed through the production cycle (62.5%). One hundred and thirty-nine variables were used in univariate analyses. All the variables with a p-value < or = 0.10 were used in unconditional logistic regression in a forward stepwise model. An effect of location was identified in both univariate and multivariate analyses showing that ponds located in the eastern portion of the study site, closer to the sea, were more likely to test positive for WSSV by 1-step PCR at harvest. Ponds with shrimp of a smaller average size 1 mo after stocking tended to be positive for WSSV at the end of the production cycle. Average weight at 1 mo was also highlighted in multivariate analyses when considered as either a risk factor or an outcome. Other risk factors identified in univariate analyses were earlier date of stocking and use of commercial feed. A number of variables also appeared to be associated with a reduced risk of WSSV at harvest including the presence of dead post larvae in the batch sampled at stocking, presence of Hemigrapsus spp. crabs during the first month of production, feeding vitamin premix or legumes, presence of high numbers of shrimp with bacterial infection and the presence of larger mud crabs or gobies at harvest. No associations were detected with WSSV at harvest and stocking density, presence, or number or weight of wild shrimp in the pond. The multivariate model to identify outcomes associated with WSSV infection highlighted the presence of high mortality as the main variable explaining the data. The results obtained from this study are discussed in the context of WSD control and areas requiring further investigation are suggested.     

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.     

Usefulness of dead shrimp specimens in studying the epidemiology of white spot syndrome virus (WSSV) and chronic bacterial infection

This paper describes the utility of dead shrimp samples in epidemiological investigations of the white spot syndrome virus (WSSV) and chronic bacterial infections. A longitudinal observational study was undertaken in shrimp farms in Kundapur, Karnataka, India, from September 1999 to April 2000 to identify risk factors associated with outbreaks of white spot disease (WSD) in cultured Penaeus monodon. As a part of the larger study, farmers were trained to collect and preserve dead and moribund shrimp (when observed) during the production cycle. At the end of the production cycle, 73 samples from 50 ponds had been collected for histopathology and 55 samples from 44 ponds for PCR. Intranuclear viral inclusion bodies diagnostic of WSSV infection were detected in dead samples from 32 ponds (64 %). Samples of dead shrimp from 18 ponds (36%) showed no histopathological evidence of WSSV infection. However, of these, samples from 13 ponds (26%) showed clear evidence of shell, oral, enteric and systemic chronic inflammatory lesions (CIL) in the form of haemocytic nodules, typical of bacterial infection. Samples from 5 ponds (10%) were negative for both WSSV and CIL. Samples from 8 ponds had dual WSSV and CIL, although both WSSV and CIL were only observed in the same shrimp from 1 pond. Useful information was obtained from these shrimp despite the presence of post-mortem changes. Samples from 19 ponds (43%) tested positive for WSSV by 1-step PCR and samples from an additional 10 ponds (22.7%) were positive by 2-step nested PCR. Samples from 15 ponds (34.1%) were negative for WSSV by 2-step nested PCR. There was moderate to substantial agreement between PCR and histopathology in the diagnosis of WSSV infection in dead shrimp. WSSV infection in dead shrimp was significantly associated with crop failures as defined by a shorter length of the production cycle (<90 d) and lower average weight at harvest (<22 g). WSSV infection was also associated with lower survival (<50%), but this was not significant. Ponds with CIL did not experience any crop failures, and the presence of CIL was significantly associated with successful crops. The study demonstrates that samples of dead shrimp can provide useful information for disease surveillance and epidemiological investigations of WSSV and chronic bacterial infections.     

High variation in repetitive DNA fragment length for white spot syndrome virus (WSSV) isolates in Thailand

White spot syndrome virus (WSSV) presently causes the most serious losses to shrimp farmers worldwide. Earlier reports of high DNA sequence homology among isolates from widely separated geographical regions suggested that a single virus was the cause. However, we have found surprisingly high variation in the number of 54 bp DNA repeats in ORF94 (GenBank AF369029) from 55 shrimp ponds (65 shrimp samples) experiencing WSSV outbreaks in Thailand in 2000 and 2002. These were detected by PCR amplification using primers ORF94-F and ORF94-R flanking the repeat region. Altogether, 12 different repeat groups were found (from 6 to 20 repeats) with 8 repeats being most frequent (about 32%). Extracts prepared from individual shrimp in the same outbreak pond belonged to the same repeat group while those collected at the same time from separate WSSV outbreak ponds, or from the same ponds at different times, usually belonged to different repeat groups. This suggested that different outbreaks were caused by different WSSV isolates. In contrast to the highly variable numbers of repeats, sequence variation within the repeat region was confined to either T or G at Position 36. These variations may be useful for epidemiological studies on the local and global movement of WSSV, since there is high variation in the number of repeats (good for local studies) but little sequence change (good for global studies).     

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.     

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.     

Evaluation of an immunodot test to manage white spot syndrome virus (WSSV) during cultivation of the giant tiger shrimp Penaeus monodon

A monoclonal antibody-based immunodot test was compared to a polymerase chain reaction (PCR) assay for managing white spot syndrome virus (WSSV) on shrimp farms at Kundapur and Kumta situated in Udupi and Uttar Kannada Districts, respectively, of Karnataka on the west coast of India. Of 12 grow-out farms in Kundapur, 6 (F1 to F6) yielded shrimp samples that were negative for WSSV by both immunodot test and 1-step PCR from stocking to successful harvest. Samples from the other 6 farms (F7 to F12) were positive for WSSV by both immunodot test and 1-step PCR at various times post stocking, and their crops failed. In the 2 farms at Kumta (F13, F14), immunodot and 1-step PCR results were both negative, and harvests were successful. In contrast to 1-step PCR results, farms F5, F6, F13, and F14 gave positive results for WSSV by 2-step PCR, and they were successfully harvested at 105 d post stocking. Our results indicate that an inexpensive immunodot assay can be used to replace the more expensive 1-step PCR assay for disease monitoring.     

Viruses,biosecurity and specific pathogen-free stocks in shrimp aquaculture

The greatest threat to the future of world shrimp aquaculture is disease, in particular the virulent untreatable viruses, infectious hypodermal and haematopoietic necrosis virus (IHHNV), taura syndrome virus (TSV), yellow head virus (YHV), and white spot syndrome virus (WSSV). To overcome these hazards, the industry of the future must be based on: (i) specific pathogen-free and genetically improved shrimp stocks; (ii) biosecure systems including enclosed, reduced water-exchange/increased water-reuse culture systems; (iii) biosecure management practices; and (iv) co-operative industry-wide disease control strategies. Specific pathogen-free shrimp are those that are known to be free of specified pathogens and such stocks will ensure that seed shrimp are not the conduit for introduction of pathogens and that if pathogens are encountered the stocks will not be severely affected. Commercially acceptable biosecure culture systems that are under cover and use recirculated sea water will need to be developed for shrimp production. Adherence to operating protocols that incorporate strict biosecurity practices, including restricted access and disinfection strategies, will need to become standard. Co-operative efforts will include: early warning surveillance; co-ordination of harvest and water exchange schedules of contaminated ponds; processor co-operation to ensure that processing wastes are not threats; quick response to outbreaks.     

White spot syndrome virus (WSSV) in Litopenaeus vannamei captured from the Gulf of California near an area of extensive aquaculture activity

For the shrimp farming industry of Mexico, disease outbreaks caused by white spot syndrome virus (WSSV) are relatively recent. Efforts to control the virus are assisted by monitoring for its prevalence in aquaculture systems, but few attempts have been made to search for it in carriers from coastal waters. To search for WSSV carriers in the Gulf of California, we made surveys off the coast of Sinaloa, Mexico, in March 2001, November 2001, and September 2003 using polymerase chain reaction (PCR) assays and histopathology. WSSV-positive shrimp were detected only in November 2001, after hurricane Julliete. This suggested possible dispersal of WSSV to the marine environment from infected shrimp farms.     

Feeding hermit crabs to shrimp broodstock increases their risk of WSSV infection

White spot syndrome virus (WSSV) is a serious shrimp pathogen that has spread globally to all major shrimp farming areas, causing enormous economic losses. Here we investigate the role of hermit crabs in transmitting WSSV to Penaeus monodon brooders used in hatcheries in Vietnam. WSSV-free brooders became PCR-positive for WSSV within 2 to 14 d, and the source of infection was traced to hermit crabs being used as live feed. Challenging hermit crabs with WSSV confirmed their susceptibility to infection, but they remained tolerant to disease even at virus loads equivalent to those causing acute disease in shrimp. As PCR screening also suggests that WSSV infection occurs commonly in hermit crab populations in both Vietnam and Taiwan, their use as live feed for shrimp brooders is not recommended.     

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.     

DNA fragmentation, an indicator of apoptosis, in cultured black tiger shrimp Penaeus monodon infected with white spot syndrome virus (WSSV)

Fifty black tiger shrimp Penaeus monodon from commercial cultivation ponds in Malaysia were examined by Tdt-mediated dUTP nick-end labeling (TUNEL) fluorescence assay and agarose gel electrophoresis of DNA extracts for evidence of DNA fragmentation as an indicator of apoptosis. From these specimens, 30 were grossly normal and 20 showed gross signs of white spot syndrome virus (WSSV) infection. Of the 30 grossly normal shrimp, 5 specimens were found to be positive for WSSV infection by normal histology and by nested polymerase chain reaction (PCR) analysis. All of the specimens showing gross signs of WSSV infection were positive for WSSV by normal histology, while 5 were positive by nested PCR only (indicating light infections) and 15 were positive by 1-step PCR (indicating heavy infections). Typical histological signs of WSSV infection included nuclear hypertrophy, chromatin condensation and margination. None of the 25 grossly normal shrimp negative for WSSV by 1-step PCR showed any signs of DNA fragmentation by TUNEL assay or agarose gel electrophoresis of DNA extracts. The 10 specimens that gave PCR-positive results for WSSV by nested PCR only (i.e., 5 grossly normal shrimp and 5 grossly positive for WSSV) gave mean counts of 16 +/- 8% TUNEL-positive cells, while the 25 specimens PCR positive by 1-step PCR gave mean counts of 40 +/- 7% TUNEL-positive cells. Thus, the number of TUNEL positive cells present in tissues increased with increasing severity of infection, as determined by gross signs of white spots on the cuticle, the number of intranuclear inclusions in histological sections, and results from single and nested PCR assays. DNA extracts of PCR-positive specimens tested by agarose gel electrophoresis showed indications of DNA fragmentation either as smears or as 200 bp ladders. Given that DNA fragmentation is generally considered to be a hallmark of apoptosis, the results suggested that apoptosis might be implicated in shrimp death caused by WSSV.     

In vivo titration of white spot syndrome virus (WSSV) in specific pathogen-free Litopenaeus vannamei by intramuscular and oral routes

White spot syndrome virus (WSSV) is a devastating pathogen in shrimp aquaculture. Standardized challenge procedures using a known amount of infectious virus would assist in evaluating strategies to reduce its impact. In this study, the shrimp infectious dose 50% endpoint (SID50 ml(-1)) of a Thai isolate of WSSV was determined by intramuscular inoculation (i.m.) in 60 and 135 d old specific pathogen-free (SPF) Litopenaeus vannamei using indirect immunofluorescence (IIF) and 1-step polymerase chain reaction (PCR). Also, the lethal dose 50% endpoint (LD50 ml(-1)) was determined from the proportion of dead shrimp. The median virus infection titers in 60 and 135 d old juveniles were 10(6.8) and 10(6.5) SID50 ml(-1), respectively. These titers were not significantly different (p > or = 0.05). The titration of the WSSV stock by oral intubation in 80 d old juveniles resulted in approximately 10-fold reduction in virus titer compared to i.m. inoculation. This lower titer is probably the result of physical and chemical barriers in the digestive tract of shrimp that hinder WSSV infectivity. The titers determined by infection were identical to the titers determined by mortality in all experiments using both i.m. and oral routes at 120 h post inoculation (hpi), indicating that every infected shrimp died. The determination of WSSV titers for dilutions administered by i.m. and oral routes constitutes the first step towards the standardization of challenge procedures to evaluate strategies to reduce WSSV infection.     

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.     

Development of a Rapid Method for Identifying Carryover Contamination of Positive Control DNA,Using a Chimeric Positive Control and Restriction Enzyme for the Diagnosis of White Spot Syndrome Virus by Nested PCR

Chimeric positive plasmids have been developed to minimize false-positive reactions caused by polymerase chain reaction (PCR) contamination. Here, we developed a rapid method for identifying false-positive results while detecting white spot syndrome virus (WSSV) by nested PCR, using chimeric positive plasmids. The results of PCRs using WSSV diagnostic primer sets showed PCR products of a similar size (WSSV 1st PCR product, 1,447 bp; WSSV 2nd PCR product, 941 bp) using WSSV chimeric plasmids or DNA from shrimp infected with WSSV. The PCR products were digested with DraI for 1 h at 37 °C. The digested chimeric DNA separated into two DNA bands; however, the WSSV-infected shrimp DNA did not separate. Thus, chimeric plasmid DNA may be used as positive control DNA instead of DNA from WSSV-infected shrimp, in order to prevent PCR contamination. Thus, the use of restriction enzyme digestion allowed us to rapidly distinguish between WSSV DNA and WSSV chimeric plasmid DNA.