DAO 68:181-188 (2006)  -  doi:10.3354/dao068181

Standardized white spot syndrome virus (WSSV) inoculation procedures for intramuscular or oral routes

C. M. Escobedo-Bonilla1,2, L. Audoorn3, M. Wille1, V. Alday-Sanz4, P. Sorgeloos1, M. B. Pensaert2, H. J. Nauwynck2,*

1Laboratory of Aquaculture & Artemia Reference Center, Faculty of Bioscience Engineering, Ghent University, Rozier 44, 9000 Ghent, Belgium
2Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium
3Biomath, Department of Applied Mathematics, Biometrics and Process Control, Ghent University, Coupure Links 653, 9000 Ghent, Belgium
4INVE technologies, Hoogveld 93, 9200 Dendermonde, Belgium
*Corresponding author. Email:

ABSTRACT: In the past, strategies to control white spot syndrome virus (WSSV) were mostly tested by infectivity trials in vivo using immersion or per os inoculation of undefined WSSV infectious doses, which complicated comparisons between experiments. In this study, the reproducibility of 3 defined doses (10, 30 and 90 shrimp infectious doses 50% endpoint [SID50]) of WSSV was determined in 3 experiments using intramuscular (i.m.) or oral inoculation in specific pathogen-free (SPF) Litopenaeus vannamei. Reproducibility was determined by the time of onset of disease, cumulative mortality, and median lethal time (LT50). By i.m. route, the 3 doses induced disease between 24 and 36 h post inoculation (hpi). Cumulative mortality was 100% at 84 hpi with doses of 30 and 90 SID50 and 108 hpi with a dose of 10 SID50. The LT50 of the doses 10, 30 and 90 SID50 were 52, 51 and 49 hpi and were not significantly different (p > 0.05). Shrimp orally inoculated with 10, 30 or 90 SID50 developed disease between 24 and 36 hpi. Cumulative mortality was 100% at 108 hpi with doses of 30 and 90 SID50 and 120 hpi with a dose of 10 SID50. The LT50 of 10, 30 and 90 SID50 were 65, 57 and 50 hpi; these were significantly different from each other (p < 0.05). A dose of 30 SID50 was selected as the standard for further WSSV challenges by i.m. or oral routes. These standardized inoculation procedures may be applied to other crustacea and WSSV strains in order to achieve comparable results among experiments.

KEY WORDS: Litopenaeus vannamei · WSSV · Experimental inoculation · Intramuscular route · Oral route · LT50 · Probit analysis

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