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Chinese (GenBank NC 003225.1), and Taiwanese WSSV (GenBank AF440570.1). The amplicon sequenced in this study also had an identity of 99% with the sequence reported in plankton collected from a shrimp farm in Guasave, Sinaloa (GenBank FJ609650.1), and an identity of 99% with the viral DNA of infected shrimp on that same farm (GenBank FJ789570.1). On April 19th, the study detected this infection at the farm selected for monitoring. The following day, mortality of shrimp reached 50%. C.gigas oysters placed as sentinels at the entrance of the water feeder into the farm detected the presence of the virus 16, 13 and 11 days before the infectious event was triggered in ponds. WSSV was not detected in oysters recovered before April 3rd (March 23rd-30th), or after April 8th (April 11th-May 25th). Also, no infectious event was recorded on those dates at the farm.
Fig. 2. Crassostrea gigas. Light microscopy images of gill tissues from an oyster cohabitated with white spot syndrome virus (WSSV)-infected Litopenaeus vannamei shrimp at 72 h that tested positive for WSSV by nested PCR. Histological tissue sections were examined either by (A−D) in situ hybridization (ISH) or (E,F) haematoxylin and eosin (H&E) staining. (A,B) ISH signal (arrows) in filaments. (B,C) Absence of ISH signal from vesicular connective tissue and basal lamina. (D) Presence of ISH signal in inter-filament spaces. (E,F) No evidence of hypertrophied cell nuclei or WSSV inclusion bodies in vesicular connective tissue and basal lamina. Bf: branchial filament; Ct: vesi cular connective tissue; m: mucocytes; Dm: demibranch of gill; Bl: basal lamina. Scale bars = (A,B,C,E,F) 100 μm, (D) 10 μm
during the period from March 23 to May 25, 2010. All samples were taken randomly from the different modules. Organisms were collected, frozen while alive, and sent to the Biochemical and Molecular Biomedicine Laboratory at the CIBNOR and the Autonomous University of Nayarit to perform analyses designed to detect WSSV using nested PCR. Control lots were also analyzed; one composed of 20 oysters gathered before the others were sent to Sinaloa, and the second collected before the sentinels were placed on the farm to begin monitoring. 6 »
Aquaculture Magazine
The diagnosis of WSSV by nested PCR only detected the presence of the viral DNA of WSSV in three of the collections conducted on April 3rd, 6th and 8th 2010, at percentages of 15%, 20% and 55%, respectively, in the oysters analyzed. An amplicon of 570 pb was obtained, which corresponds to the size expected for the product amplified by the pair of oligos PK3/PK4. The PCR products from the gills of oysters were sequenced, and an identity of 99% was found with the segment isolated from the genome of the Thai (GenBank access number AF369029.2),
Discussion Results show that sentinel oysters are capable of detecting the entrance of WSSV in ponds at least 16 days before infection is produced, thus conferring to this species of bivalve mollusk a potential value for utilization in early detection systems of this virus on shrimp farms. The possibility that the period that transpired between the alert and the outbreak of infection might depend on the status of the immune system of the
Sentinel oysters are capable of detecting the entrance of WSSV in ponds at least 16 days before infection is produced.