White spot syndrome virus in frozen shrimp sold at Massachusetts supermarkets.

ABSTRACT One of the most damaging viral diseases affecting the shrimp aquaculture industry is white spot disease (WSD) caused by white spot virus (WSSV), which causes high morbidity and mortality rates in penaeid shrimp and other crustaceans. The rapid spread of WSSV within wild and cultured stocks of shrimp may be caused by unregulated processing, disposal of infected imported shrimp, or the use of contaminated broodstock. The risk of introducing this virus to cultured and wild shrimp and other native species of crustaceans in the United States warrants investigation. The aim of this study is to determine the prevalence of WSSV in frozen commodity shrimp sold at four stores in the Boston area belonging to different supermarket chains. Samples from two size classes were collected in two different batches a month apart. Polymerase chain reaction was used to amplify a portion of the WSSV genome using a commercial PCR kit (ShrimpCare, DiagXotics). WSSV positive samples were visualized by electrophoresis and amplified product of selected samples was sequenced. Results showed a range of 0% to 38.7% for WSSV prevalence rate in the test populations, with an overall prevalence of 4.7%. Significant (P < 0.001) differences in WSSV prevalence were observed between shrimp from the two batches purchased a month apart, the two size classes, and the four test stores. Country of origin seemed to dominate the results. Sequence analysis confirmed the presence of WSSV genome in PCR-positive samples. Results provide preliminary evidence that an appreciable proportion of the shrimp sold in Massachusetts' supermarkets are carrying WSSV, and this constitutes a substantial risk of importation of this virus into the local environment. Further investigation is necessary to determine the risk of release of this virus into native fresh and marine water environments in Massachusetts and throughout the United States.

KEY WORDS: white spot syndrome virus, WSSV, frozen commodity shrimp

INTRODUCTION

WSD is caused by white spot virus (WSV or WSSV) a rod-shaped, double-stranded DNA nonoccluded virus that belongs to a new family of viruses (Nimaviridae) infecting crustaceans (Marks et al. 2003, van Hulten et al. 2001, Yang et al. 2001). WSSV is responsible for widespread morbidity and mortality in postlarval, juvenile, and adult stages of many species of penaeid shrimp common to aquaculture and is an Office of International Epizootics (OIE)-listed disease (Lightner 1996, Lightner et al. 1998, Lo et al. 1996, Lo et al. 1997, Wang et al. 1999, Chou et al. 1998, OIE 2004). First suspected in Taiwan in 1992 within cultured Penaeus monodon, P. japonicus, and P. penicillatus; the virus was identified in farm populations in Japan, Korea, and China in 1993 and Thailand and India in 1994 and was well established in the majority of farms throughout Indonesia and Eastern and Southern Asia by 1996 (Lightner et al. 1998, Flegel et al. 1997). WSD was first observed in the United States in P. setiferus on a farm in Texas in 1995 and has since been found in wild stocks and cultured populations in Texas, Mississippi, South Carolina, and Georgia (Lightner 1996, Lightner 1999, Lightner 1996, OIE 2004). The source of WSSV in these previously isolated North American populations was considered to be infected imported shrimp that were processed in coastal processing plants in the region (Lightner 1999, Lightner et al. 1997, Lightner 1998).

It has been demonstrated that infectious and viable WSSV can be isolated from frozen shrimp sold in some US markets (Nunan et al. 1998, Durand et al. 2000), and presence of the virus was identified by polymerase chain reaction (PCR) in one of four samples tested from a Massachusetts seafood retailer (Belak et al. 1998). The likelihood of infected shrimp reaching US markets may be enhanced by the common practice of performing emergency harvests of cultured shrimp that have succumbed to an epizootic (Lightner et al. 1997). A number of potential venues that may lead to the release of WSSV into the environment have been proposed. One such practice is the release of liquid and solid waste from processing plants, most of which are located along the coast. Another route may be the use of shrimp as bait by fishermen. Finally, disposal of by-products and carcasses of frozen shrimp in landfills and other areas, where they are accessible to gulls and various scavengers that may transport this and other viruses in their feces, may also contribute to distribution of shrimp viruses (Lightner et al. 1997, JSA 1997).

WSSV has been clearly demonstrated to be highly infectious in a wide variety of crustacean species (Holthuis 1980), with more than 50 known natural and experimental hosts. It has been shown to infect the Asian shrimp species P. monodon, P. semisulcatus, P. japonicus, P. penicillatus, P. indicus, P. merguiensis, Trachypenaeus curvirostris, Metapenaeus ensis, M. monoceros, M. dobsoni, Exopalaemen orientalis, Parapenaeopsis stylifera, and Solenocera indica (Lightner 1996, Rajendran et al. 1999, Chou et al. 1998, Wang et al. 1999, Hossain et al. 2001). WSSV has also been detected or experimentally introduced in the Central and North American shrimp species P. setiferus, P. vannamei, P. stylirostris, P. aztecus, and P. duorarum (Lightner et al. 1998, Nunan et al. 1998, Wang et al. 1999). In addition to these species of shrimp, infection with WSSV has been reported in freshwater prawns and crayfish (Macrobrachium spp., Procambarus clarkii, Orconectes punctimanus, Pacifastacus leniusculus), marine crabs (Atergatis integerrimus, Doclea hybrida, Charybdis granulata, C. feriata, C. lucifera, C. cruciata, Matuta planipes, Metopograpsus messor, Macrophthalmus sulcatus, Portunus sanguinolentus, and many others), mudcrabs (Scylla serrata and S. tranquebarica), lobsters (Panulirus ornatus, P. versicolor, P. longipes, P. penicillatus, P. homarus, and P. polyphagus), with varying degrees of disease or capacity to act as asymptomatic carriers (Wang et al. 1999, Lightnet et al. 1997, Rajendran et al. 1999, Hossain et al. 2001, Otta et al. 1999, Chert et al. 2000, Jiravanichpaisal et al. 2001, Hameed et al. 2003). In view of the wide range of crustacean hosts of this virus, it is reasonable to believe that other related species may potentially be susceptible to WSSV or act as reservoirs of the virus. This could include many economically important crustacean species native to the Gulf of Maine, such as the American lobster (Homarus americanus) or the northern shrimp species (Pandalus borealis and Pleoticus robustus) (Anderson & Lindner 1971, USDC 1998). To prevent any anthropogenic exposure of our native or established species to such a virulent disease, it is imperative that the risk of importation and release of WSSV in the Massachusetts area be investigated.

It is proposed that an appreciable percentage of the imported shrimp sold in Massachusetts markets are carrying WSSV, and this would constitute a substantial risk of introduction of this exotic virus into the local fresh and marine water environments. The aim of this study is to obtain preliminary data on the proportion of commodity shrimp sold in this state that are infected with WSSV. The specific tasks were (1) to collect frozen shrimp from supermarkets in Boston, Massachusetts; (2) to determine the prevalence of WSSV in test populations of these shrimp using PCR; and (3) to sequence a subset of PCR-positive samples to confirm presence of WSSV genome.

MATERIALS AND METHODS

Test Stores

Four test stores in the Boston area were selected, one from each of four different supermarket chains. These chains were chosen based on their large market share in Massachusetts and the perceived level of their environmental awareness. This perception was based in part on the supposition that stores with a large selection of products advertised as "organic" or "earth-friendly" are more environmentally aware than stores with a small selection of such products. In that regard, two of the four chains were perceived as being environmentally "neutral," henceforth referred to as test stores 1.N and 2.N (Table 1). The remaining two chains were perceived as being environmentally "positive," and those test stores are referred to as 3.P and 4.P (Table 1).

Test Populations

Raw frozen shrimp (unknown species), were obtained from the four test stores on 2 occasions, June 25, 2001 (Week A) and July 25, 2001 (Week B, Table 1). Purchases were made a month apart to obtain samples potentially from two different shipments of shrimp to the test stores. Two different sizes of shrimp, based on count per pound, were purchased from each store when available (Table 1). Bags of shrimp selected were those that were the closest size available to either 21-25/lb (categorized as "Large size class") or 41-50/lb (categorized as "Small size class"). Enough bags of shrimp were purchased to obtain at least 100 individuals for each size class from each store (2-5 bags for large size and 1-2 bags for small size). Shrimp were kept on ice in coolers and transported to the molecular biology laboratory at Tufts University School of Veterinary Medicine in North Grafton, Massachusetts. Internal records were maintained for the country of origin and other information contained on all shrimp packages.

Selection of Test Populations and Samples to be Used for WSSV Detection

Test populations consisted of 100 individual shrimp chosen blindly from the pooled bags purchased for each combination of week, store, and size class (14 test populations total). Each individual shrimp was given a unique coded identification label. Using a generated random number chart, 75 individuals were chosen from each test population to be tested for WSSV (1,050 total, Table 1), thus providing a 95% probability of detecting the virus if the prevalence within each test population of 100 is at least 2% (Lightner 1996).

DNA Extraction 

All samples were weighed prior to tissue extraction. DNA was extracted from tissue obtained from the sixth abdominal segment of each sample using the DNeasy kit following the manufacturer's procedure (Qiagen, Valencia, CA). The concentration ([micro]g/[micro]l) of extracted DNA was determined using a spectrophotometer and corrected according to the results of quality control assays performed by electrophoresis in 1% agarose gels.

WSSV Diagnostic by PCR

Primers that amplify a 401 bp fragment of the WSSV genome, WSSV positive control template, and negative control template for PCR (ShrimpCare kit) were obtained from DiagXotics Inc. (Wilton, CT). PCR was performed in a 25-[micro]L reaction consisting of ~100 ng DNA of virus template or shrimp sample, 1.5 mM Mg[Cl.sub.2], 1.25 mM dNTP, 1 [micro]L DiagXotics' primers mix, 1x PCR buffer, and 1.5 U Taq DNA Polymerase, using a thermocycler (PTC-100 MJ Research, MA) preheated to 95[degrees]C. The PCR reaction profile consisted of a single cycle of 95[degrees]C for 2 min followed by 40 cycles of 94[degrees]C for 30 sec, 60[degrees]C for 30 sec, 72[degrees]C for 60 sec, and a final extension at 72[degrees]C for 10 min (as recommended by DiagXotics Inc.) Ten micro liters of amplified DNA product from each sample was electrophoresed in a 2% agarose gel at 90 volts for 3 h and photographed using Polaroid 677 film. To detect possible cross-contamination during PCR and gel electrophoresis, each gel contained lanes with positive and negative control templates (DiagXotics) and a control water blank. Due to use of a commercial kit and lack of access to primer sequence information for sequencing the amplified product, PCR was repeated with selected samples to confirm presence of WSSV using forward (F) and reverse (R) primers and PCR reaction mixture taken from Lo et al. (1996, 1997, 1999) and Belak et al. (1998). The PCR reaction mixtures were run in a MJ Research thermocycler as follows: 1 cycle of 4 min at 94[degrees]C, 1 min at 55[degrees]C, and 3 min at 72[degrees]C; followed by 39 cycles of 1 min at 94[degrees]C, 1 min at 55[degrees]C, 3 min at 72[degrees]C, and 5 min at 72[degrees]C for final extension before holding the samples at 4[degrees]C. PCR product was run on agarose gel as described previously. DNA was eluted from the agarose gel and purified using X-spin columns (Costar, CA) before sequencing them at Tufts University DNA Sequencing Facility in Boston, Massachusetts using 2 forward (F1, F2) and 2 reverse (R1, R2) primers. Sequence homology was performed using the programs Blastn and Blastx (http:// www.ncbi.nlm.nih.gov/blast). The sequence of one of the PCR-amplified products obtained from frozen shrimp has been deposited in the public Genbank database (accession # AY850066).

Statistical Analysis

The differences in the prevalence of WSSV among test populations consisting of shrimp from different test stores, supermarket environmental-awareness categories, purchase weeks, size classes, and labeled country of origin were examined by [chi square] test at a minimum level of significance of P < 0.05. Multivariate logistic regression analysis was also performed to examine the interactions between these variables in their relationships with WSSV prevalence.

RESULTS AND DISCUSSION

A 401 bp fragment for WSSV was amplified in frozen shrimp samples sold in Massachusetts' supermarkets (Fig. 1). Results showed varying prevalence rates of WSSV in each test population, ranging from 0% to 38.7% (Table 2). The overall WSSV prevalence in all samples tested was 4.7%.

[FIGURE 1 OMITTED]

The differences in WSSV prevalence between purchase weeks, size classes, and test stores were all found to be significant (P < 0.001; Table 2). The difference in prevalence between supermarket environmental-awareness categories (neutral and positive) was not significant (Table 2). However, shrimp from stores IN and 3P were significantly different to stores 2N and 4P in WSSV prevalence. The lack of a significant difference in test populations purchased from the supermarkets categorized as neutral or positive may be due to one of several factors: (1) the manner in which the supermarkets were categorized may have been invalid and did not reflect the true degree of environmental concern of the four supermarkets; (2) instead, the categorization may have been valid, but the supermarkets may not regard the risk of importation of WSSV as an issue that should affect their selection of commodity shrimp; and (3) the supermarkets may not be aware of the risks associated with importation of this or other viruses. More information regarding the policies of the four companies is necessary to determine whether they are being selective in their purchase of imported shrimp as related to the presence of viruses and other pollutants.

The presence of WSSV in batches of frozen shrimp sampled in this study coincides with the findings of similar studies conducted in this and other states (Belak et al. 1998, Nunan et al. 1998, Lo et al. 1998, Durand et al. 2000). This is the first report to investigate the percentage of infected commodity shrimp within a randomly selected lot of commercially sold shrimp from supermarkets in Massachusetts.

The variation in prevalence observed between the 14 test populations studied (0% to 38.7%; Table 2) reveals several important points regarding the analysis of the rate of importation of WSSV into the state. First of all, the significant difference in WSSV prevalence between the two batches purchased a month apart demonstrates the variability in the number of infected shrimp that may be present within the Boston market at any point in time. Here, batches purchased in Week A had a viral prevalence of 0.4% whereas those purchased in Week B had a prevalence of 9.0% (Table 2). This time sensitivity may be due to a number of factors. The countries of origin of these cultured shrimp are known to experience periodic outbreaks of WSSV within their farms that are related in part to salinity, seasonal, and climate changes (Flegel et al. 1997). Shipments of commodity shrimp to Massachusetts' supermarkets may consist of various combinations of shrimp originating from different areas of the globe. Therefore, further investigations should be carried out over a larger time scale to reveal a well fit estimation of the import rate of this and other viruses.

The second factor that introduced variation in prevalence within the test groups was the size of the shrimp studied. Samples belonging to the Large size class showed a prevalence of 8.0%, whereas those belonging to the Small size class had a prevalence of 0.2% (Table 2). This finding is in contrast to the supposition that the smaller size classes would be more likely to contain infected shrimp due to the common practice of "emergency harvest" of infected shrimp pens in aquaculture (Durand et al. 2000, Lightner et al. 1997, USEPA 1999). The study conducted by Durand et al. (2000) used small count size (>40 tails/lb) as one criterion for selection of suspected cases for testing of commodity shrimp and demonstrated presence of WSSV within three of the four lots chosen in this manner. The finding of greater viral prevalence within the larger size class in this study may be related to the possibility that our samples consisted of shrimp of different species and the fact that variability exists in the morbidity and mortality caused by WSSV within different species of shrimp (Lighther et al. 1998, Wang et al. 1999). Although a few of the bags of frozen shrimp purchased were labeled as to what species they contained, it was impossible to positively identify the species of the samples based on morphology. In light of these findings, the sampling of multiple size classes should be considered, and the species should be identified based on species-specific molecular markers in future investigations of prevalence of WSSV within commodity shrimp.

Lastly, test store of origin proved to be a significant source of variation in percentage of infected shrimp within the test populations. Test stores 2.N and 4.P had the lowest overall prevalence of WSSV (0.3% and 1.3%, respectively) whereas test stores 1.N and 3.P had the highest overall prevalence (9.7% and 10%, respectively) (Table 2). This variation may be due in part to the countries from which the shrimp sold in each store originated and when the shrimp were imported. As stated previously, these factors may affect the relative number of infected shrimp within the shipments to each store. Therefore, future studies need to consider sampling from different sources within the Massachusetts market.

There seems to be a connection between shrimp originating from Thailand and higher prevalence of WSSV. A multivariate logistic regression analysis showed that indeed most of the positive samples were from Thailand (P < 0.0001). Within the shrimp from Thailand, positive samples were significantly those of large shrimp (P < 0.0001), week B (P < 0.0001), and stores 1.N and 3.P (P < 0.0001), with these two stores 1.N and 3.P not differing among themselves (P = 0.91). To address food safety and food security issues, future epidemiologic studies should be performed using state-of-the-art technology to identify species, confirm country of origin, and acquire information on cultured versus wild origin rather than based on consumer product labeling only. Sensitive DNA fingerprinting technologies have been developed (Meehan et al. 2003, Alcivar-Warren et al. unpublished) and should be used in the future to trace species and country of origin of imported shrimp.

DNA sequence analysis confirmed that the PCR-positive product amplified from frozen shrimp sold in Massachusetts' supermarkets corresponded to a portion of the WSSV genome (Fig. 2). The partial WSSV sequences obtained from seven PCR fragments were similar in all samples (not shown). The 896 nucleotides sequence of amplified product TURW.WSSVcom562.F2 was deposited in GenBank (accession # AY850066). Blastx homology searches revealed that TURW.WSSVcom562.F2 had 99% identity in 292 (4984-5275) amino acids of WSSV ORF167 (AAK77836.1) whereas the 363 bp of TURW.WSSVcom872.R2 sequence showed 84% similarity to ORF167. These sequences were 99% to 100% similar to the China strain of WSSV (Alcivar-Warren et al. unpublished). Based on prior knowledge of infective material in commodity shrimp and the presented sequence data, results support the supposition that this virus is being imported into Massachusetts and may constitute a substantial risk of importation of this exotic virus into the local fresh and marine water environments. Potential environmental contamination with this virus may have existed in this state since 1998 (Belak et al. 1998). However, because most of the shrimp sold in Massachusetts' supermarkets are from tails-only (no heads-on), the possibility of the virus to be homogeneously distributed throughout an infected shrimp may be lower and not equally likely to result in transmission (Soto et al. 2001). Further investigation is necessary to determine the risk of release of this virus into the marine environment in Massachusetts. Epidemiologic models are needed to predict the threshold density of potential susceptible native species below which an outbreak of WSSV will not occur (Soto et al. 2001, Lotz & Soto 2002). Regulations of shrimp imports need to be developed and implemented to preserve our native resources and to protect domestic aquaculture enterprises.

Figure 2. DNA sequence of PCR-positive fragment obtained from a
Dozen shrimp purchased at a supermarket in Boston, Massachusetts
(TURW.WSSVcom562F2; GenBank accession # AY850066), aligned with
the sequence obtained from a shrimp injected experimentally
(TURW.WSSVini226F2) with the China strain of the virus
(Alcivar-Warren et al. unpublished). Only 348 bp out of the 896 bp
of the TURW.WSSVcom562F2 sequence is shown. Template DNA isolation
and sequencing methodology is described in Materials and Methods
section.

                  1                                        50
TURW.WSSVinj226F2 TTACTCCTcA gtAACGAGCA TCtGGTATCC TCTTTCGCAT
  TCGCCCGCCC
TURW.WSSVcom562F2 TTACTCCTCA gaAACGAGCA T.tgGTATCc TCTTTCGCAT
  TCGCCCGCCC

                  51                                      100
TURW.WSSVinj226F2 AGAAGTCTCC ATGGAAGAAA TTAGAGCCAC ACCCTATCAG
  GCCAACAAGC
TURW.WSSVcom562F2 AGAAGTCTCC ATGGAAGAAA TTAGAGCCAC ACCCTATCAG
  GCCAACAAGC

                  101                                     150
TURW.WSSVinj226F2 TTATTAGTGA CAAACATTAC GTGATGAACA TGTCCAAGAT
  CGATTCTAGA
TURW.WSSVcom562F2 TTATTAGTGA CAAACATTAC GTGATGAACA TGTCCAAGAT
  CGATTCTAGA

                  151                                     200
TURW.WSSVinj226F2 GTAACAGGAT CTTCCCTCCT TAAGAAGGTT AGCGAATGGA
  CTGAAATGAG
TURW.WSSVcom562F2 GTAACAGGAT CTTCCCTCCT TAAGAAGGTT AGCGAATGGA
  CTGAAATGAG

                  201                                     250
TURW.WSSVinj226F2 AATGAACTCC AACTTTAATG GAACATTTGA ACCATCAAGA
  CTCGCCCTCT
TURW.WSSVcom562F2 AATGAACTCC AACTTTAATG GAACATTTGA ACCATCAAGA
  CTCGCCCTCT

                  251                                     300
TURW.WSSVinj226F2 CCAACTCTGG CATGACAACG GCAGGAGTCA ACNCTCGACG
  TTATTGTCAA
TURW.WSSVcom562F2 CCAACTCTGG CATGACAACG GCAGGAGTCA A.CCTCGACG
  TTATTGTCAA

                  301                                     350
TURW.WSSVinj226F2 ACCAAATAA TGCAAGAAGT GTACTAGGAA TATTGGAAT
  G~~~~~~~~~
TURW.WSSVcom562F2 ACCAAATAA TGCAAGAAGT GTACTAGGAA TATTGGAAT
  GTCATCGCCA

TABLE 1.
Summary of frozen shrimp obtained from different supermarkets
in Massachusetts.

                                       Week A

        Supermarket (a)      Large size        Small size
                              class (a)         class (a)

1.N     # of samples              75                75
        Count/lb                21-25             51-60
        Mean weight [+         18.81 [+          8.05 [+
          or -] SD, g         or -] 1.92        or -] 0.98
        Country of origin      Thailand          Thailand
        Species                   nd                nd
2.N (b) Number of samples         75                75
        Count/lb                21-25             41-50
        Mean weight [+        18.84 [+           9.39 [+
          or -] SD, g         or -] 2.06        or -] 1.43
        Country of origin       India            Honduras
        Species              Black tiger            nd
                              shrimp (d)
3.P     Number of samples         75              na (c)
        Count/lb               26-30/lb
        Mean weight [+        15.57 [+
          or -] SD, g         or -] 1.15
        Country of origin      Thailand
        Species                   nd
4.P     Number of samples         75                75
        Count/lb                26-30             41-50
        Mean weight [+        15.59 [+           9.43 [+
          or -] SD, g         or -] 2.71        or -] 0.64
        Country of origin      Thailand           Belize
        Species                   nd         White shrimp (e)
Total   # of samples             300               225

                                        Week B

        Supermarket (a)      Large size        Small size        Totals
                              class (a)         class (a)

1.N     # of samples              75                75              300
        Count/lb                21-25             51-60
        Mean weight [+        20.36 [+           7.71 [+
          or -] SD, g         or -] 2.50        or -] 1.20
        Country of origin      Thailand          Thailand
        Species                   nd                nd
2.N (b) Number of samples         75                75              300
        Count/lb                21-25             41-50
        Mean weight [+        18.77 [+           9.68 [+
          or -] SD, g         or -] 1.97        or -] 1.00
        Country of origin      Thailand          Honduras
        Species              Black tiger            nd
                              shrimp (d)
3.P     Number of samples         75              na (c)            150
        Count/lb               26-30/lb
        Mean weight [+        14.38 [+
          or -] SD, g         or -] 1.26
        Country of origin      Thailand
        Species                   nd
4.P     Number of samples         75                75              300
        Count/lb                26-30             41-50
        Mean weight [+        15.66 [+           9.29 [+
          or -] SD, g         or -] 1.23       or -] 0.96 g
        Country of origin      Thailand           Belize
        Species                   nd         White shrimp (e)
Total   # of samples             300               225             1050

(a) Bags of frozen shrimp were selected for purchase at each store
based on the count per pound label on the packages. Because all four
stores did not stock shrimp of the same count per pound, the product
that was the nearest to the desired count per pound value for each
size class was chosen for purchase (21-25/lb for Large and 41-50/lb
for Small). The same brand of shrimp were purchased on Weeks A and B
from each store, with the exception of store 2.N.

(b) The same brand purchased on Week A for the Large size class was
not available on Week B. A comparable brand was chosen.

(c) This store did not carry a comparable sized raw shrimp.

(d) Black Tiger shrimp is a common name for Penaeus monodon.

(e) White shrimp is a common name for Penaeus (Litopenaeus) vannamei.

na = not applicable; nd = not stated in shrimp package.

TABLE 2.
Prevalence of White Spot Syndrome Virus (WSSV) in frozen shrimp
obtained from different supermarkets in Massachusetts. (1)

                                   Week A

  Supermarket           Large size            Small size

1.N                   0/75 (0%)             0/75 (5%)
2.N                   1/74 (2) (1.4%)       0/75 (0%)
3.P                   1/75 (1.3%)           N/A (4)
4.P                   0/75 (0%)             0/75 (0%)
Total # of samples    2/299 (0.7%)          0/225(0%)
Week totals                      2/524 (0.4%)
                                 Large size
Size class totals              48/599 (8.1%)

                                   Week B

  Supermarket           Large size            Small size

1.N                   29/75 (38.7%)         0/75(0%)
2.N                   0/75 (0%)             0/74 (3) (0%)
3.P                   14/75 (18.7%)         N/A (4)
4.P                   3/75 (4%)             1/75 (1.3%)
Total # of samples    46/300 (15.3%)        1/224 (0.4%)
Week totals                      47/524 (9.0%)
                                  Small size
Size class totals                1/449 (0.2%)

                                            Environmental
Supermarket           Supermarket totals    class totals

1.N                   29/300 (9.7%)         30/598 (5.0%)
2.N                   1/298 (0.3%)
3.P                   15/150 (10%n)         19/450 (4.2%)
4.P                   4/300 (1.3%)
Total # of samples
Week totals                      49/1048 (5) 4.7%)

Size class totals

(1) Prevalence = percentage of samples testing positive to WSSV.

(2) One sample deleted due to failure to extract any
measurable amount of DNA.

(3) Results were not obtained for one sample in this group
due to loss of PCR amplified sample.

(4) This store does not carry a comparable sized raw shrimp.

(5) 47 of the 49 positive samples were in shrimp labeled
as from Thailand.

ACKNOWLEDGMENTS

This work was partially supported by NIH short-term training grant T35DK07635 (C.R.), USDA/CSREES #99-38808-7431 (A. A-W.) and the Department of Environmental and Population Health at TUSVM (A. A-W.)

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CARA REVILLE, (1) JENNIFER AL-BEIK, (1) DAWN MEEHAN-MEOLA, (1) ZHENKANG XU, (1,2) MICHELE L. GOLDSMITH, (3) WILLIAM RAND, (4) AND ACACIA ALCIVAR-WARREN (1,2,*)

(1) Environmental and Comparative Genomics Section, (2) International Marine Shrimp Environmental Genomics Initiative (IMSEGI) and (3) Center for Animals and Public Policy, Department of Environmental and Population Health, Tufts University School of Veterinary Medicine, North Grafton, Massachusetts, 01536 and (4) Department of Community Health, Tufts University School of Medicine, Boston, Massachusetts, 02111

* Correspondence author: E-mail: [e-mail]