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Standardized white spot syndrome virus (WSSV) inoculation procedures for intramuscular or oral routes
C M Escobedo-Bonilla1, L Audoorn, M Wille
1Laboratory of Aquaculture & Artemia Reference Center, Faculty of Bioscience Engineering, Ghent University, Rozier 44, 9000 Ghent, Belgium.
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.
Insights
Standardized White Spot Syndrome Virus (WSSV) inoculation methods using defined doses improve experimental reproducibility. A 30 shrimp infectious dose 50% endpoint (SID50) is recommended for WSSV challenges.
Area of Science:
- Aquaculture
- Virology
- Animal Health
Background:
- White Spot Syndrome Virus (WSSV) causes significant losses in shrimp aquaculture.
- Previous WSSV infectivity studies lacked standardized challenge doses, hindering result comparability.
Purpose of the Study:
- To determine the reproducibility of defined WSSV doses (10, 30, 90 SID50) via intramuscular (i.m.) and oral inoculation.
- To establish standardized WSSV challenge protocols for Litopenaeus vannamei.
Main Methods:
- Three experiments were conducted using specific pathogen-free (SPF) Litopenaeus vannamei.
- WSSV challenge was performed using i.m. or oral routes with three defined doses (10, 30, 90 SID50).
- Disease onset, cumulative mortality, and median lethal time (LT50) were recorded to assess reproducibility.
Main Results:
- Both i.m. and oral WSSV inoculation routes induced disease within 24-36 hpi.
- Intramuscular inoculation showed non-significant differences in LT50 across doses, while oral inoculation revealed significant differences (p < 0.05).
- A dose of 30 SID50 was identified as a reliable standard for both WSSV challenge routes.
Conclusions:
- Standardized WSSV inoculation using defined doses enhances experimental reproducibility.
- The proposed methods and 30 SID50 dose are suitable for future WSSV challenge studies in shrimp.
- These protocols can be adapted for other crustacean species and WSSV strains.
