Diagnostic platforms for shrimp white spot disease

Description

The following diagnostic protocols are intended to provide diagnosticians, pathologists, and researchers with the most current and appropriate diagnostic procedures for the WSSV. WSSV may be detected by using specific complementary DNA probes either in hybridization assays using homogenized tissue samples "blotted" and fixed onto membranes prepared from nitrocellulose or nylon, or else in situ in tissue sections. Diagnostic polymerase chain reaction (PCR) is a procedure which amplifies target amounts of small WSSV DNA. This is accomplished by using specific primers designed for the target DNA sequence. These three methods have different procedural complexity, specificity and sensitivity, and are suitable for various diagnostic purpose.

Brief protocol

WSSV detection by dot blot hybridization analysis

DNA samples are boiled for 10 min and then quench on ice. An aliquot (1 μl) of each of the DNA samples is dotted onto a sheet of positively charged nylon paper that has been presoaked with 5× SSC (1× SSC: 150 mM NaCl, 15 mM Sodium Citrate, pH 7.0) for 5 min and air dried. After cross-linking the DNA with the membrane by UV light, the blot is used for hybridization with WSSV probes which are non-radioactively labeled with digoxigenin (DIG)-dUTP (Roche). Following prehybridization at 65°C for 1 h in prehybridization solution (10 ml 5× SSC with 100 mg of blocking reagent II [Roche], 50 μl of 20% sarkosyl, 20μl of 10% sodium dodecyl sulfate [SDS]), the blot is hybridized at 65°C for 16 h with DIG-labeled probes. The detection of the DIG-labeled nucleotides in blots is accomplished by an immunological method using anti-digoxigenin antibody conjugated to alkaline phosphatase (Roche) and CSPDB, (Roche) as a cherniluminescent substrate for alkaline phosphatase. The blot is exposed to X-ray film at room temperature for 1 to 10 min to record the chemiluminescent signal. The DNA probe uses here can be directly nonradioactively labeled with DIG by PCR using primer pair pms 146F2/R2 and WSSV-TW strain as the template or indirectly labeled by a random priming method using cloned WSSV genomic DNA fragments.

WSSV detection in histological sections by In situ hybridization

Whole or sections of shrimp fix in Davidson’s AFA fixative (Humason, 1972) for 12-24 hours, the samples then can be preserved in 70% ethanol until use. Fixed tissues are embedded in Paraplast/Plus (Monoject), and histologic tissue sections of 4 to 5 μm are made. Sections are mounted onto positively charged SuperFrost*/Plus (Menzel-Glaser) microscope slides for in situ hybridization with a WSSV-specific probe labeled with DIG. Slides consisting of tissue sections of WSSV-infected uninfected shrimp are used as negative and positive controls, respectively. The in situ hybridization procedures and coloration methods follows Lightner (1996). Finally, the slides are counterstained with 0.5% aqueous Bismarck Brown Y (Sigma) for 90 seconds and then dehydrated and mounted with Permount (Fisher Scientific, Pittsburgh, Pa.) mounting media and coverslips for observation under an Microscope. The DNA probe uses here as describe previously.

Nested WSSV diagnostic PCR

200 mg of tested organisms are rapidly frozen in liquid nitrogen and crushed to a fine powder. The processed tissue is placed in 2.4 ml digestion buffer (100 mM NaCl, 10 mM Tris-HC1, pH 8, 25 mM EDTA, pH 8, 0.5% SDS, 0.1 mg ml-1proteinase K) and incubates at 65°C for 12 to 18 h. The digest is deproteinized by successive phenol/chloroform/isoamyl alcohol extractions, recover by ethanol precipitation, and dry and resuspend in 0.1 X TE buffer at 65°C for 30 min, and then store at 4°C until use for PCR. The primer pairs,146F1/146R1 (146F1: 5'-ACTACTAACTTCAGCCTATCTAG-3'; 146R1: 5'-TAATGCGGGTGTAATGTTCTTACG) is used for the 1-step PCR. The DNA samples use for amplification is total 0.1 μg in a 100 μl reaction mixture containing, 200 μM of each dNTP, 100 pmol of each primer, 2 units of thermo stable DNA polymerase. The amplification is performed for 1 cycle of 94°C for 4 min, 55°C for 1 min, 72°C for 2 min; 39 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 2 min, plus a final 5 min extension at 72 °C after 40 cycles. Control reaction containing no template DNA is run for PCR reactions. A portion (10 μl) from each of the completed PCR reactions is mixed with 1 μl loading buffer and subject to electrophoresis on 1 % agarose gels containing ethidium bromide at a concentration of 0.5 μg ml-1, and visualize by ultraviolet transillurnination. At the nested PCR amplification, 10 μl of the first step PCR product is added to 90 μl of PCR cocktail containing the nested primer pair, 146F2 and 146R2 (146F2, 5'-GTAACTGCCCCTTCCATCTCCA-3'; 146R2, 5'-TACGGCAGCTGCTGCACCTTGT-3’), and this is then subjected to a second step of amplification over the same 40 cycles. For the analysis of PCR products, 10 μl of the final reaction mixture is used for gel electrophoresis.

References

1. Lightner, D.V. (ed.) (1996) A Handbook of Pathology and Diagnostic Procedures for Diseases of Penaeid Shrimp. Baton Rouge, La.: World Aquaculture Society.
2. Humason, G.L. (ed.). (1972) Animal Tissue Technique. 3rd Edition. W.H. Freeman and Co., San Francisco, CA.

Some of our publications that have used this platform

1. Hsu, H.C., Lo, C.F., Lin, S.C., Liu, K.F., Peng, S.E., Chang, Y.S., Chen, L.L., Liu, W.J., and Kou, G.H. (1999) Studies on effective PCR screening strategies for white spot syndrome virus (WSSV) detection in Penaeus monodon brooders. Dis Aquat Org 39:13-19.
2. Kou, G.H., Peng, S.E., Chiu, Y.L., and Lo, C.F. (1998) Tissue distribution of white spot syndrome virus (WSSV) in shrimp and crabs. In: Advances in Shrimp Biotechnology, Flegel, T.W. (ed.). Bangkok, Thailand: National Center for Genetic Engineering and Biotechnology, 267-271.
3. Lo, C.F., Leu, J.H., Ho, C.H., Chen, C.H., Peng. S.E., Chen, Y.T., Chou, C.M., Yeh, P.Y., Huang, C.J., Chou, H.Y., Wang, C.H., and Kou, G.H. (1996a) Detection of baculovirus associated with white spot syndrome (WSBV) in penaeid shrimps using polymerase chain reaction. Dis Aquat Org 25:133-141.
4. Lo, C.F., Ho, C.H., Peng, S.E., Chen, C.H., Hsu, H.C., Chiu, Y.L., Chang, C.F., Liu, K.F., Su, M.S., Wang, C.H., and Kou, G.H. (1996b) White spot syndrome baculovirus (WSBV) detected in cultured and captured shrimp, crab and other arthropods. Dis Aquat Org 27:215-225.
5. Lo, C.F., Ho, C.H., Chen, C.H., Liu, K.F., Chiu, Y.L., Yeh, P.Y., Peng, S.E., Hsu, H.C., Liu, H.C., Chang, C.F., Su, M.S., Wang, C.H., and Kou, G.H. (1997a) Detection and tissue tropism of white spot syndrome baculovirus (WSBV) in captured brooders of Penaeus monodon with a special emphasis on reproductive organs. Dis Aquat Org 30:53-72.
6. Lo, C.F., Wang, C.H., and Kou, G.H. (1997b) White spot syndrome (WSS): pathology, hosts and prevalence in captured shrimp and crabs in Taiwan. In: New Approaches to Viral Diseases of Aquatic Animals: NRIA International Workshop Proceedings, Inui,Y. (ed.). Mie, Japan: National Research Institute of Aquaculture, 206-217.
7. Lo, C.F., Hsu, H.C., Tsai, M.F., Ho, C.H., Peng, S.E., Kou, G.H., and Lightner, D.V. (1999). Specific genomic DNA fragment analysis of different geographical clinical samples of shrimp white spot syndrome virus. Dis Aquat Org 35:175–185.