Aquaculture Europe 2023

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Add To Calendar 19/09/2023 12:00:0019/09/2023 12:15:00Europe/ViennaAquaculture Europe 2023EFFECT OF SALINITY DROP ON SUSCEPTIBILITY TO WSSV INFECTION IN Litopenaeus vannamei SHRIMP USING A PER OS CHALLENGE MODELSchubert 5The European Aquaculture Societywebmaster@aquaeas.orgfalseDD/MM/YYYYaaVZHLXMfzTRLzDrHmAi181982


Natasja Cox1,2*, Evelien De Swaef1, Mathias Corteel1, Wim Van Den Broeck4, Peter Bossier3 ,  João  J. Dantas-Lima1 , Hans  J. Nauwynck2


1IMAQUA, 908 0 Lochristi, Belgium

2Laboratory of Virology, D epartment of Translational Physiology, Infectiology and Public Health, Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, Belgium

3 Laboratory of Aquaculture & Artemia Reference Center, Department of Animal Sciences and Aquatic Ecology, Faculty of Bioscience Engineering, Ghent University, 9000 Ghent, Belgium

4 Department of Morphology,  Medical Imaging, Orthopedics , Physiotherapy and Nutrition, Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, Belgium

*Correspondence :



 In shrimp farms, salinity drop in the water due to excessive rainfall has been mentioned to be  a risk factor for WSSV outbreaks (Thuong et al., 2016a). It was hypothesized that when WSSV is introduced into the rearing water and a sudden lowering of the salinity occurs, this could lead to an uptake of water through the nephropores into the antennal gland, as shrimp attempt to regulate their haemolymph osmolarity and urinary ion excretion .  Once the cells of the antennal gland become infected, the virus infection can spread further into the body. An experimental WSSV immersion challenge  mimics a natural water-borne WSSV transmission. Thuong et al. (2016a), performed an experiment in which shrimp were immersed in sea water containing 10 5.5 SID50 mL-1 of WSSV . Subsequently, these shrimp were subjected to a salinity change from 35 to 5 g l-1. After 5 hours, the salinity was restored to 35 g l-1 . The  mortality  due to WSSV infection was 53%. There was no mortality in the control group  without  a salinity drop . This suggested an important role of a salinity drop in the WSSV infectivity during an immersion challenge . However, WSSV is also reported to be transmitted through consumption of infected tissues (Wang et al., 1999). In the current study, we examined the effect of a salinity change  on  infection and  mortality during a  per os  WSSV challenge, because it simulates natural WSSV infections  through cannibalism.  By testing  these  conditions, we aim  to investigate if salinity change is also a risk factor for WSSV infection during an oral WSSV challenge in L. vannamei . These results could then be used in future work to further elucidate WSSV transmission dynamics.

Materials and Methods

Virus stock production: specific pathogen free (SPF) Litopenaeus vannamei were imported as postlarvae (PLs) from the United States of America (USA) .  Shrimp were housed in artificial seawater at 20 ppt salinity and 27°C ± 1°C. They were injected intramuscularly with t he WSSV Thai-1 strain (Escobedo-Bonilla et al., 2005). WSSV positive solid inoculum was prepared from the resulting infected carcasses .

WSSV challenge and salinity drop: The inoculum was used  to infect PL76  shrimp through oral route.  Briefly, during the experiment,  shrimp were randomly divided into three challenge groups (A, B, C)  consisting  of  three replicates of 10 shrimp . Ten shrimp were assigned to a first  negative control (Mock1- 15 ppt drop ). Another  group of  ten shrimp served as the second  negative control (Mock2 – 30 ppt drop ). Shrimp were housed individually in 10L tanks. Shrimp from groups A, B, and Mock1 were acclimatised to 20 ppt salinity, while shrimp from groups C and Mock2 were acclimatised to 35 ppt salinity.  The oral infection trial followed a procedure adapted from Van Thuong , et al. (2016b).  Group A remained at a salinity of 20ppt during and after the oral WSSV challenge. I ndividual shrimp from groups  B, C, Mock1, and Mock2, were transferred into seawater with a 5ppt salinity. Groups A, B, and C received WSSV positive inoculum, while Mock1 and Mock 2 received negative solid inoculum. After a period of 5 hours, the salinity in the individual tanks from groups B, and Mock1 was restored to 20ppt, while the salinity in the tanks of groups C and Mock2 was restored to 35ppt. The animals were observed in the following days and the experiment ended when no mortality was observed for 48hours. WSSV infection presence or absence  in the tissues of collected shrimp  was confirmed by qPCR.  The survival/mortality data were analysed  statistically  using the Log-rank (Mantel-Cox) test.


 At the end of the challenge trial, cumulative mortality  rates in the WSSV-challenged groups A, B, and C were respectively 21%, 33% and 40%. The differences in mortality rates showed a trend between group A, that was not subjected to a drop in salinity, and group C, that was subjected to a 30ppt drop (from 35 to 5 ppt) (p-value = 0.0952). In the t wo  control  groups, Mock1 and Mock2, that were subjected to a salinity drop  of respectively 15 and 30ppt, all shrimp survived.  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 experiment showed that the probability or risk of infection in the population  increased  when the  animals were  subjected to a salinity drop during an oral WSSV challenge.  This  result was similar to the results obtained by Thuong et al. (2016a) during their WSSV immersion experiments with the same change in salinity. It suggests that salinity change could indeed be a risk factor for WSSV infection in the field, where natural WSSV transmission  occurs both by  water-borne or cannibalism routes .  De Gryse et al. (2020) argued that this could be explained, because  sudden salinity drop during, e.g., heavy monsoon rains, aggression, establishment of social dominance, and feed intake (cannibalism) are conditions where frequent urination ,  and thus frequent opening of the nephropore, takes place. Subsequently,  this  could  create a window of opportunity for WSSV invasion, making  entry via the antennal gland  possible  (de Gryse et al., 2020).


 This research received funding  from  Flanders Innovation  and Entrepreneurship (VLAIO, Belgium).


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