Introduction
White spot syndrome virus (WSSV) can cause a cumulative mortality up to 100% within 3-10 days on shrimp farms (Dey et al., 2020). Although progress has been made in the past 30 years, a better understanding of the pathogenesis and transmission route(s) of WSSV is still necessary. This work relies heavily on the establishment of standardized in vivo experimental infection models that can be used as a tool for studying WSSV pathogenicity and for evaluating the efficacy of WSSV mitigation strategies (Prior et al., 2003). A WSSV challenge model via per os route in which shrimp are individually housed has certain advantages over a WSSV group challenge model. It allows for accurate collection of research data in a highly controlled scientific setting, while increasing the throughput in selecting and developing infection control tools. The amount of WSSV positive inoculum consumed by each shrimp can be recorded meticulously. In addition, the clinical outcomes on the level of the individual shrimp can be evaluated in detail. On the other hand, a challenge in group simulates more closely the on-farm reality of a WSSV outbreak, as it allows for disease transmission between shrimp. This might indeed generate results that can potentially be easier to extrapolate to the field, but due to the less controllable nature of this experimental setting, results may be less reproducible and accurate data collection becomes more challenging. Nevertheless, both model types can be very useful tools in WSSV research. In the past, mortality levels after oral WSSV challenges have varied between studies, in part due to a lack of knowledge on the portal of WSSV entry (Prior et al., 2003; Domínguez-Borbor et al., 2019; de Gryse et al., 2020). The objective of this study was to develop standardized and reproducible individual and group challenge models for experimental WSSV infection studies in L. vannamei whereby 100% of clinical WSSV infection is induced. By testing different experimental conditions to obtain this clinical outcome, we aim to learn more about WSSV transmission.
Materials and Methods
Specific pathogen free (SPF) Litopenaeus vannamei were imported as postlarvae (PLs) from Global Blue Technology (USA) and Miami Aqua-culture (USA). Shrimp were housed in artificial seawater at 20 ppt salinity and 27°C ± 1°C. They were injected intramuscularly with the WSSV Thai-1 strain (Escobedo-Bonilla et al., 2005). WSSV positive solid inoculum was prepared from the resulting infected carcasses. This was used to infect PL65-72 shrimp through oral route. Briefly, during the 1st experiment, in which the effect of different doses within 24h was tested, shrimp were randomly divided into four challenge groups (A1, B1, C1, D1) of 15 shrimp each and one control group (Mock1) of 10 shrimp. Shrimp were housed individually in 10L tanks. The oral infection trial followed a procedure adapted from Van Thuong, et al. (2016). Over a period of 24h, individual shrimp from groups A1, B1, C1, and D1 each received respectively 4, 5, 6, or 7 doses of WSSV positive inoculum. Mock1 shrimp received 7 doses of WSSV negative inoculum. For the 2nd experiment, in which the effect of daily repeated inoculation of 4 doses was tested, shrimp were again randomly divided into four individually housed challenge groups (A2, B2, C2, D2) of 15 shrimp each and one control group (Mock2) of 10 shrimp. In A2, B2, C2 and D2, shrimp received respectively 4 doses of WSSV positive inoculum within one day for one, two, three or four consecutive days. Mock2 shrimp received 16 doses of WSSV negative inoculum within 96h. Finally, for the 3rd experiment, where the effect of group housing was tested, shrimp were randomly divided into six groups of 100 and housed in six 250L tanks at 1.5kg/m3. Over a period of 24h, three tanks (+WSSV1, 2, 3) each received 400 doses of WSSV positive solid inoculum, while the three control tanks (-WSSV1, 2, 3) received 400 doses of WSSV negative inoculum. WSSV infection was confirmed by qPCR. The survival/mortality data were analysed statistically using the Log-rank (Mantel-Cox) test.
Results
When shrimp from the individual challenge received either 4, 5, 6 or 7 doses of WSSV positive solid inoculum within 24h, this did not result in a significantly different mortality (A1 = 16.7±4.7%; B1 = 20.0±9.4%; C1 = 20.0±9.4%, D1 = 20.0±18.9%). However, in case of daily re-inoculation, a significantly lower mortality was observed in group A2 (28.9±16.8%) compared to group D2 (57.8±3.8%). When shrimp were housed in groups and received the same number of doses as individually housed A1 and A2 shrimp, the accumulated mortality reached 100%, while in the three control tanks all shrimp survived. For each experiment, WSSV infection was confirmed by qPCR in a sample of the dead shrimp. WSSV was absent in sampled survivors and negative controls.
Discussion and conclusion
The results of the 2nd experiment showed that the probability or risk of infection in the population increased when the inoculation procedure was repeated on subsequent days. Increasing the number of doses that are given within 24h on the other hand, did not significantly raise the chances of a clinical infection in individually housed shrimp. Moreover, the uptake of inoculum by shrimp slowed down after the 5th dose, possibly because the animals were sated. In the 1st and the 2nd experiment, a clinical infection of 100% was not reached. This level could only be reached during the group challenge. This was a striking result, because the individually housed shrimp in A1, A2, and the group-housed shrimp in +WSSV1, 2, and 3 consumed the same amount of inoculum. In addition, during the group challenge cannibalization of sick shrimp was virtually absent. How WSSV transmission exactly occurs remains unknown. Proposed routes include consumption of infected tissue, water-borne transmission, and entry via the antennal gland during urination (de Gryse et al., 2020). After analysis of the results, it was hypothesized that shrimp behaviours associated with cohabitation and high stocking densities, such as increased aggression and urination, play a major role in a WSSV outbreak.
Acknowledgements
This research received funding from Flanders Innovation and Entrepreneurship (VLAIO).
References