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 of fspring arose because amounts of WSSV DNA in the test samples were near the sensitivity limits of the detection method. Since spawning stress may trig ger WSSV replication, we also captured a fresh batch of 45 brooders for WSS V PCR testing before and after spawning. Replicates of their spawned egg ba tches were also WSSV PCR tested. For these 45 brooders, WSSV prevalence bef ore 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 grou p (21) and a 2-step PCR negative group (9) based on replicate PCR tests. Th e spawning rates for these 2 groups were high (81 and 78 %, respectively). None of the negative spawners (7) became WSSV positive after spawning and n one 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 posit ive upon capture. However, all of them were WSSV PCR positive in replicate tests and after spawning or death. Four died without spawning. The remainin g 17 spawned but only 2 gave egg samples PCR negative for WSSV. The other 1 5 gave PCR positive egg samples, but they could be divided into 2 spawner g roups: those (7) that became heavily infected (i.e. 1-step PCR positive) af ter spawning and those (8) that remained lightly infected (i.e. became or r emained 2-step PCR positive only). Of the brooders that became heavily infe cted after spawning, almost all egg sample replicates (91 %) tested 2-step PCR positive. One brooder even gave heavily infected (i.e. 1-step PCR posit ive) egg samples. For the brooders that remained lightly infected after spa wning, only 27 % of the egg sample replicates were 2-step PCR positive. Bas ed on these results, we recommend that to avoid false negatives in WSSV PCR brooder tests screening tests should be delayed until after spawning. We a lso recommend, with our PCR detection system, discarding all egg batches fr om 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 postlarv ae. These may be used to stock shrimp ponds under low-stress rearing condit ions.