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Diseases of Aquatic Organisms

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DAO 54:89-96 (2003)  -  doi:10.3354/dao054089

Controlled bioassay systems for determination of lethal infective doses of tissue homogenates containing Taura syndrome or white spot syndrome virus

Sarah Prior1,*, Craig L. Browdy1, Eleanor F. Shepard1, Rolland Laramore2, Pamela G. Parnell3

1South Carolina Department of Natural Resources, Marine Resources Research Institute, 217 Ft. Johnson Rd., Charleston, South Carolina 29422, USA
2Aquatic Animal Health Laboratory, Harbor Branch Oceanographic Institution Inc., 5600 US 1 North, Fort Pierce, Florida 34946, USA
3Clemson Veterinary Diagnostic Center, PO Box 102406, Columbia, South Carolina 29224-2406, USA

ABSTRACT: In vivo bioassay is the predominant method for evaluating the infectivity of materials potentially harboring viable shrimp pathogens and determining the relative susceptibility of shrimp species to viral infections. A controlled bioassay system for white spot syndrome virus (WSSV) and Taura syndrome virus (TSV) was developed utilizing 260 ml tissue culture flasks modified with an air exchange vent. Individual shrimp (1.00 ± 0.25 g) were placed in separate flasks containing artificial seawater (100 to 150 ml) and held in an incubator at 27°C. After a 48 h acclimation period, shrimp were either injected intramuscularly with viral inoculum or exposed to virus-laden water. Water was exchanged and shrimp were fed a commercial food pellet daily except 24 h post-infection (p.i.). Bioassays were performed with serial dilutions of stock viral preparations and shrimp mortality was recorded for 7 d p.i. Mortality rates of test animals permitted the estimation of the lethal infective doses, LD50 and LD90. The LD50 of the TSV injection preparation was estimated at viral dilutions of 1:7.692 x 107 (Trial 1) and 1:6.667 x 107 (Trial 2). The LD50s of 2 different WSSV injection preparations were estimated at 1:4.444 x 106 and 1:4.505 x 106. The LD50 for the TSV waterborne challenge was 1:9916 (Trial 1) and 1:15710 (Trial 2) at 20°C and 1:1272 at 27°C. A second waterborne TSV inoculum challenge at 27°C produced an LD50 of 1:2857. WSSV doses used in the waterborne challenge only reached 39% mortality, which did not allow for the estimation of effective lethal doses. Bioassay by injection proved to be a more reliable method of estimating viral infectivity compared to waterborne method. The dose-response curves developed can serve as a basis for controlled comparisons of relative levels of viral infectivity of specific tissue preparations and for controlled comparisons of relative susceptibility of shrimp species or stocks to viral pathogens.

KEY WORDS: TSV · WSSV · Bioassay · Shrimp · Litopenaeus vannamei

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