White spot syndrome is a viral epizootic that affects most commercially cultivated marine shrimp species. In Asia alone, white spot syndrome virus (WSSV) has caused losses valued at several billion US dollars within a few years. The disease first emerged in East Asia in the early 1990s and has since spread throughout most shrimp farming regions of Asia and the Americas. Disease outbreaks usually result in high mortalities in affected ponds. However, shrimp may also be infected chronically with no signs of disease and often obtain the infection in hatcheries from infected broodstock. A wide range of wild and cultured crustaceans can also act as asymptomatic carriers of infection.
In this study, a semi-quantitative nested-PCR test was used to determine the prevalence of WSSV infection in 245 batches of Penaeus monodon postlarvae collected from hatcheries in the Mekong Delta region of Vietnam between December 2001 and June 2002. The overall prevalence of infection was 9.0% with variations from 1.7% to 31.3% in postlarvae batches sampled from different provinces. The peak of infection prevalence occurred in postlarval batches collected between February and April 2002. Based on the collection information of these postlarvae batches, the prevalence range of original broodstocks used to produce these postlarvae batches was similar in provinces of the orth-East Southern region (3.3-21.7%), the South Central Coast region (10.825.0%) and the Mekong Delta region (4.2-33.3%).
During the same season, juvenile P. monodon were sampled from 4 healthy and 15 diseased grow-out ponds in three provinces of the Mekong Delta and tested for WSSV infection using a ·commercial semi-quantitative PCR test. Of 511 juveniles sampled, 170 (33.3%) were WSSV-positive using the WSSV IQ-2000 PCR kit. Of the positive samples, 55.9% were graded as high-level, 16.5% as medium-level and 27.6% as low-level infections. Only 9 of the 243 juveniles (3.7%) sampled from 4 healthy ponds were WSSV-positive and each was graded as a low-level infection. In the 14 diseased ponds, the proportion of WSSV-infected juveniles varied from 11.8% to 100.0%. In one diseased pond, no WSSV-positive samples were detected amongst 18 juveniles sampled, suggesting the disease was not due to WSSV.
Genotyping PCR protocols were developed by targeting tandem repeat sequences at two variable loci in the WSSV genome and were used to examine the extent of variation among WSSV isolates from P. monodon and other crustaceans in different regions of Vietnam. The first genotyping PCR detected variations in the number of a 54 nucleotide (nt) tandem repeat sequence (TRSI) located between genes encoding the large (RR1) and small (RR2) subunits of ribonucleotide reductase. Analysis of 162 WSSV isolates showed common variations in the number of 54 ot repeats and that some broodstock harboured more than one genotype. In healthy broodstock, postlarvae, and juvenile shrimp from ponds, WSSV genotypes containing 4-, 5-, 6- 7-, 8- and 9-TRSI elements were detected with no evidence of any predominant genotype. However, amongst shrimp sampled from disease outbreak ponds, the 7-TRSI genotype predominated (86%). In other crustaceans collected from shrimp ponds, WSSV genotypes containing higher numbers of repeats (i.e. 9-, 14- and 23-TRSl) were found. In several ponds containing diseased shrimp infected with the 7-TRSI genotype, other WSSV genotypes were detected in co-inhabitant healthy wild crustaceans.
Sixty-two WSSV samples that were genotyped at the TRSI site were selected for analysis using the TRS2 site genotyping PCR. As in the TRS1 peR, broodstock commonly contained more than one TRS2 genotype. In healthy broodstock, postlarvae and juveniles, WSSV genotypes containing 3-, 4-, 5-, 6-, 7- and 8-TRS2 elements were detected with no evidence of any predominant genotype. In contrast, amongst juveniles from diseased ponds, the 3-TRS2 (88%) was predominant and all samples of shrimp from the same diseased pond contained the same TRS2 genotype. The predominant 3TRS2 genotype paralleled the predominance of 7-TRSI genotype in diseased juveniles while there was no evidence of linkage between two genotypes in healthy shrimp. A WSSV isolate from wild shrimp with the 14-TRSI genotype also displayed an unusuaJly high number of repeats at site TRS2 (I1-TRS2 genotype ).
These findings indicate that genotype analysis can identify individual WSSV isolates and trace potential sources of infection and disease. Therefore, this approach will provide a useful tool for future detailed investigations of the epidemiology of white spot infection in shrimp and other crustaceans.