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.     

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.     

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.     

Increasing production in Korean shrimp farms with white-spot syndrome virus PCR-negative brood stock

White-spot syndrome virus (WSSV) is a devastating, infectious virus affecting shrimp. Although sensitive techniques involving PCR have been developed to assist farmers in screening shrimp (brood stock) for WSSV prior to stocking ponds, such practices have not yet been applied in Korea. Despite the rationality of implementing screening, there has been some doubt as to whether the stocking of WSSV-PCR-negative fry epidemiologically decreases white-spot disease outbreaks. Here, we report a retrospective analysis of data from shrimp farms in the western coast of Korea where WSSV-PCR-negative brood stocks were used to stock rearing ponds. A total of 366 shrimp from Heuksan Island were sampled for WSSV with PCR. Of the tested shrimp, 7.2% (28 brood stocks) were identified as WSSV positive; only WSSV-PCR-negative shrimp were used for brood stocks. Total unit production (final shrimp production/ the area of the ponds) was higher, at 1.96, in ponds where WSSV-PCR-negative shrimp were used, as compared with 1.02 in other ponds in Korea in 2004. This retrospective analysis of WSSV in Korea may be useful to the shrimp aquaculture industry, suggesting a testable hypothesis that may contribute to the eventual control of WSSV outbreaks.     

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).     

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.     

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.     

Fenneropenaeus indicus is protected from white spot disease by oral administration of inactivated white spot syndrome virus

Fenneropenaeus indicus could be protected from white spot disease (WSD) caused by white spot syndrome virus (WSSV) using a formalin-inactivated viral preparation (IVP) derived from WSSV-infected shrimp tissue. The lowest test quantity of lyophilized IVP coated onto feed at 0.025 g(-1) (dry weight) and administered at a rate of 0.035 g feed g(-1) body weight d(-1) for 7 consecutive days was sufficient to provide protection from WSD for a short period (10 d after cessation of IVP administration). Shrimp that survived challenges on the 5th and 10th days after cessation of IVP administration survived repeated challenges although they were sometimes positive for the presence of WSSV by a polymerase chain reaction (PCR) assay specific for WSSV. These results suggest that F. indicus can be protected from WSD by simple oral administration of IVP.     

Experimental infections reveal that common Thai crustaceans are potential carriers for spread of exotic Taura syndrome virus

Taura syndrome virus (TSV) was first reported as a serious cause of shrimp mortality limited to reared Penaeus (Litopenaeus) vannamei in the Americas, where it spread principally through regional and international transfer of live post larvae (PL) and broodstock. Subsequently, through importation of infected broodstock, TSV outbreaks spread to Asia, first to Taiwan and China and then to Thailand, Indonesia and Korea. Since its introduction to Thailand, outbreaks have occasionally been reported from rearing ponds stocked with batches of specific pathogen free (SPF) P. vannamei PL that tested negative for TSV by nested RT-PCR assay. Since it was possible that the outbreaks may have occurred via horizontal transfer of TSV from wild carrier species, we tested 5 common native crustaceans that live in and around shrimp ponds (2 palaemonid shrimp species, Palaemon styliferus and Macrobrachium lanchesteri, and 3 species of crabs, Sesarma mederi, Scylla serrata and Uca vocans) for susceptibility to TSV in experimental challenges. We found that U. vocans, S. serrata and S. mederi did not die but, respectively, gave strong RT-PCR reactions indicating heavy viral load at 5, 10 and 15 d post-injection of TSV and 10, 15 and up to 50 d after feeding with TSV-infected P. vannamei carcasses. Also after feeding, P. styliferus did not die, but a high proportion gave strong RT-PCR reactions at 5 d post-challenge and no reactions at 15 d. Similarly after feeding, M. lanchesteri showed no mortality and gave only light RT-PCR reactions at 2 d, moderate reactions at 5 d and no reaction at 15 d. By contrast, transmission experiments from the TSV-infected crabs and palaemonid shrimp via water or feeding resulted in death of all the exposed P. vannamei from 8 to 12 d post-challenge and all were positive for heavy viral load by RT-PCR assay. Despite the results of these laboratory challenge tests, natural TSV infections were not detected by nested RT-PCR in samples of these species taken from the wild. These results indicated that transmission of TSV from infected crabs and palaemonid shrimp via water or feeding might pose a potential risk to shrimp aquaculture.     

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.     

Susceptibility to infection and pathogenicity of White Spot Disease (WSD) in non-model crustacean host taxa from temperate regions

Despite almost two decades since its discovery, White Spot Disease (WSD) caused by White Spot Syndrome Virus (WSSV) is still considered the most significant known pathogen impacting the sustainability and growth of the global penaeid shrimp farming industry. Although most commonly associated with penaeid shrimp farmed in tropical regions, the virus is also able to infect, cause disease and kill a wide range of other decapod crustacean hosts from temperate regions, including lobsters, crabs, crayfish and shrimp. For this reason, WSSV has recently been listed in European Community Council Directive 2006/88. Using principles laid down by the European Food Safety Authority (EFSA) we applied an array of diagnostic approaches to provide a definitive statement on the susceptibility to White Spot Syndrome Virus (WSSV) infection in seven ecologically or economically important crustacean species from Europe. We chose four marine species: Cancer pagurus, Homarus gammarus, Nephrops norvegicus and Carcinus maenas; one estuarine species, Eriocheir sinensis and two freshwater species, Austropotamobius pallipes and Pacifastacus leniusculus. Exposure trials based upon natural (feeding) and artificial (intra-muscular injection) routes of exposure to WSSV revealed universal susceptibility to WSSV infection in these hosts. However, the relative degree of susceptibility (measured by progression of infection to disease, and mortality) varied significantly between host species. In some instances (Type 1 hosts), pathogenesis mimicked that observed in penaeid shrimp hosts whereas in other examples (Types 2 and 3 hosts), infection did not readily progress to disease, even though hosts were considered as infected and susceptible according to accepted principles. Results arising from challenge studies are discussed in relation to the potential risk posed to non-target hosts by the inadvertent introduction of WSSV to European waters via trade. Furthermore, we highlight the potential for susceptible but relatively resistant hosts to serve as models to investigate natural mitigation strategies against WSSV in these hosts. We speculate that these non-model hosts may offer a unique insight into viral handling in crustaceans.     

White spot syndrome virus (WSSV) transmission from rotifer inoculum to crayfish

To test the possibility that shrimp pond rotifer resting eggs and hatched rotifers could transmit white spot syndrome virus (WSSV) to crayfish (Procambarus clarkii), we injected crayfish with rotifer and resting egg inocula that were WSSV-positive only by dot-blot analysis of PCR products. No crayfish became WSSV-positive after challenge with the resting egg inoculum. However, 1/15 crayfish became WSSV-positive after challenge with the rotifer inoculum. The results demonstrated that rotifers constitute a potential risk for WSSV transmission to crayfish and other cultivated crustaceans. However, the actual quantitative risk of transmission in an aquaculture setting depends on many variables that remain untested.     

Microbial Flora of Pond-Reared Brown Shrimp (Penaeus aztecus)

Agar plate counts and microbial types are reported for brown shrimp reared in 2-acre natural marshland and in 0.5-acre artificial ponds during June to October 1970. Bacterial counts of pond-reared shrimp ranged from 5 × 10⁴ to 5.5 × 10⁶ per g. At final harvest in October, bacterial counts ranged from 2 × 10⁵ to 5.5 × 10⁶ per g. In marsh ponds, bacterial counts of shrimp and pond water were lowest in August when both water temperature and salinity were high. Coryneform bacteria and to a lesser extent Vibrio were the predominant isolates from fresh pond shrimp. Shrimp stored at 3 to 5 C for 7 days were acceptable as judged by appearance and odor. Between 7 and 14 days of refrigerated storage, bacterial counts increased sharply and about 50% of the samples became unacceptable. Refrigerated storage of pond shrimp caused increases in coryneform bacteria and micrococci and decreases in Vibrio, Flavobacterium, Moraxella, and Bacillus species. Pseudomonas species were not significant in fresh or stored pond shrimp. The microbial flora of pond water usually was dominated by coryneform bacteria, Flavobacterium, Moraxella, and Bacillus species.     

Vp28 of shrimp white spot syndrome virus is involved in the attachment and penetration into shrimp cells

White spot disease (WSD) is caused by the white spot syndrome virus (WSSV), which results in devastating losses to the shrimp farming industry around the world. However, the mechanism of virus entry and spread into the shrimp cells is unknown. A binding assay in vitro demonstrated VP28-EGFP (envelope protein VP28 fused with enhanced green fluorescence protein) binding to shrimp cells. This provides direct evidence that VP28-EGFP can bind to shrimp cells at pH 6.0 within 0.5 h. However, the protein was observed to enter the cytoplasm 3 h post-adsorption. Meanwhile, the plaque inhibition test showed that the polyclonal antibody against VP28 (a major envelope protein of WSSV) could neutralize the WSSV and block an infection with the virus. The result of competition ELISA further confirmed that the envelope protein VP28 could compete with WSSV to bind to shrimp cells. Overall, VP28 of the WSSV can bind to shrimp cells as an attachment protein, and can help the virus enter the cytoplasm.     

How can we conserve cold stenotherm communities in warming Alpine ponds?

Freshwater biodiversity has shown to be highly vulnerable to climate warming, alpine cold stenotherm populations being especially at risk of getting extinct. This paper aims at identifying the environmental factors favouring cold stenotherm species in alpine ponds. This information is required to provide management recommendations for habitats restoration or creation, needed for the mitigation of the effects of climate warming on alpine freshwater biodiversity. Cold stenotherm species richness as well as total (i.e. stenotherm and eurytherm) richness were analyzed for aquatic plants, Coleoptera and Odonata in 26 subalpine and alpine ponds from Switzerland and were related to environmental factors ecologically relevant for pond biodiversity. Our results confirmed that the set of environmental variables governing pond biodiversity in alpine or subalpine ponds is specific to altitude. Altitude and macrophyte presence were important drivers of cold stenotherm and total species richness, whereas connectivity did not show any significant relation. Therefore, the management of pond biodiversity has to be ‘altitude-specific’. Nevertheless, cold stenotherm species from the investigated alpine ponds do not show some specific requirements if compared to the other species inhabiting these ponds. Therefore, both total and cold stenotherm species richness could be favoured by the same management measures.     

Fatal,virus-associated peripheral neuropathy and retinopathy in farmed Penaeus monodon in eastern Australia. II. Outbreak descriptions

Outbreaks of 'peripheral neuropathy and retinopathy' (PNR) occurring during 2 consecutive growout periods (typically October-April) are described for an intensive Penaeus monodon farm in eastern Australia. In the 1998/99 growout period, outbreaks graded minor to severe occurred in 22 of 25 ponds, 12 to 25 wk post-stocking. In the severely affected index pond, harvested 8 wk after outbreak recognition in mid-January, estimated survival for the period late December to harvest was 50%. Minor to moderate losses could be attributed to PNR in the other ponds. Mean survival over the same period for the 14 ponds harvested within 5 wk of outbreak recognition was 93% (83 to 100%); for the 7 ponds harvested 5 to 8 wk after outbreak recognition was 79% (67 to 92%) and for the 3 unaffected ponds was 90% (86 to 95%). Analysis indicated a significantly lower risk (Fisher's exact p = 0.016) of an outbreak in the 2 ponds stocked only with postlarvae from one hatchery (D) versus the 18 ponds stocked only with postlarvae from 3 other hatcheries (A, B and C). In the 1999/2000 growout period, minor to severe PNR outbreaks occurred in all 26 ponds, each stocked with postlarvae from the same hatchery (E), 19 to 21 wk post-stocking. Stocking date in 1999/2000 appeared to influence PNR outbreak severity; for ponds stocked on 2 of the 7 stocking dates versus those stocked on remaining dates, the crude relative risks (CRR) of a severe outbreak, or either a moderate or severe outbreak, were 11.25 (1.55 < CRR < 81.40) and 2.63 (1.30 < CRR < 5.31), respectively. Although inconclusive, study findings are consistent with the hypothesis that 'gill-associated virus' (GAV), the putative causal pathogen identified in a separate pathological study, entered ponds via postlarvae, and that prevalence and/or severity of infection within postlarval batches influenced outbreak severity. The generally high survival in ponds harvested soon after outbreak recognition, together with PNR prevalence of approximately 50% in prawns collected from 4 ponds 7 wk before those ponds were recognised as affected, also suggest that GAV is highly infectious and that PNR has a relatively long incubation period and/or clinical course.     

Genotyping of white spot syndrome virus prevalent in shrimp farms of India

DNA extracts from white spot syndrome virus (WSSV) that had infected post-larvae and juveniles of cultured shrimp, wild shrimp and crabs, which had been collected from different hatcheries and farms located along both the east and west coasts of India, revealed considerable variation in several previously identified WSSV DNA repeat regions. These include the 54 bp repeat in ORF 94, the 69 bp repeat in ORF 125 and the compound 45 and 57 bp repeat region in ORF 75. In ORF 94, 13 genotypes were observed with the number of repeats ranging from 2 to 16 units. While 7 repeat units were commonly observed (11.3%), no samples with 11 or 15 repeat units were found. In ORF 125, 11 types were found, with repeats ranging from 2 to 14 units. The most prevalent genotype displayed 4 repeat units (47.1%); no samples with 6 or 13 repeats were observed. The compound repeat region of ORF 75 displayed 6 different patterns of repeats. Samples with the same repeat pattern in one ORF did not always show identical repeat patterns in one or both of the other repeat regions. These data suggest that combined analysis of all 3 variable loci could be used to differentiate and characterize specific WSSV strains. For general epidemiological studies, the best marker with maximum variation is ORF 94, followed by ORF 125 and ORF 75. The 3 repeat regions above were used to compare WSSV genotypes from disease outbreaks on 3 sets of farms from different locations in the state of Andhra Pradesh. The genotypes within each farm set were almost identical, but differed between farm sets, suggesting that WSSV transmission occurred directly through virus carriers or water exchange between adjacent farms at each location. These findings show that genotyping can be a useful epidemiological tool for tracing the movement of WSSV within infected populations.