Penaeus vannamei , the Pacific whiteleg shrimp, is a cornerstone species in global shrimp farming due to its fast growth, adaptability, and high market demand. However, disease outbreaks, particularly those caused by White Spot Syndrome Virus (WSSV) and Acute Hepatopancreatic Necrosis Disease (AHPND), continue to represent major constraints to production (Brummett et al., 2014). While controlled and standardized experimental challenge models for these diseases are critical for evaluating prophylactic and therapeutic interventions, they are still scarce. For progressing on this topic, reliable detailed data on disease pathogenesis and dy namics is paramount. Comparative data on individual versus group challenge approaches - especially across different pathogens - can bring substantial added value to the process of the development and improvement of disease challenge models . However, these data remains virtually inexistent.
In this study, we established and compared both individual and group challenge models for WSSV and AHPND in P . vannamei under controlled laboratory conditions. Individual challenge models were designed to standardize pathogen exposure and assess the efficacy of health management products with high precision. Group challenge models, meanwhile, were developed to simulate more natural disease transmission dynamics and allow for broader application of candidate treatments.
For WSSV challenges, one peroral inoculation with WSSV-infected tissue inoculum (Thai-1 strain) (Thuong et al., 2016) was used in both individually housed (1 shrimp per 10 L tank) and group housed challenges (100 shrimp per 290L tank and 10 shrimp per 10L). AHPND disease challenges were performed in the same setups but shrimp were inoculated via immersion with Vibrio parahaemolyticus TW01 (Alloul et al., 2021) . Also in these cases, one single inoculation was performed. In both cases, animals were maintained for at least 10 days post inoculation and disease and mortality were monitored several times per day.
Striking differences were observed between pathogens and challenge models. During both the individual and group WSSV challenges, the Thai-1 strain had an incubation period of 24-54 hours post infection (hpi ) and an irreversible disease progression within 78 hpi. In WSSV group challenges , a distinct pattern of reinoculation events was demonstrated, resulting in 100% final mortality in all cases (following a minimal stocking density). This progression differed markedly from the individual WSSV challenge, where mortality curves were less steep and final mortality much more limited, suggesting differences in infection dynamics and host response depending on the mode of exposure.
In contrast, AHPND challenges showed comparable disease dynamics between individual and group models. The V. parahaemolyticus TW01 strain resulted in mortal ity within 12 hours following inoculation, with an exponential mortality increase in the first 36 hours. Notably, AHPND did not result in irreversible disease progression in the early stages post-challenge. Mortality was variable depending on the trial and dosing, but final cumulative mortality stabilized between 50% and 85%. In addition, higher initial bacterial loads were required to establish consistent infections in AHPND group models in comparison with individual challenges, highlighting differences in infectious dose thresholds. This was not observed in WSSV challenge models at our facility. These findings underscore the importance of understanding pathogen-specific dynamics under different circumstances, when designing and interpreting challenge studies.
This work emphasizes that while individual challenge models offer high reproducibility and control, group challenges better reflect field-like transmission and reinfection dynamics, especially in the context of WSSV. For AHPND, the choice of model may be less critical given the similar progression observed across methods, but attention to initial dosing remains essential. This strongly suggests that the transmission of WSSV between individuals plays a very important role in field outbreaks, while this seems to be less important in AHPND, where water transmission independently from the host might be of greater importance.
In conclusion, the choice of challenge model has a significant impact on disease outcome and must be tailored to the specific pathogen and research objectives. This study contributes valuable insights for the development and refinement of disease challenge protocols in P . vannamei, ultimately supporting more effective evaluation of health management strategies in shrimp aquaculture.
This research received partial funding from Flanders Innovation and Entrepreneurship (Belgium).
References:
Brummett, R.E.; Alvial , A.; Kibenge , F.; Forster, J.; Burgos, J.M.; Ibarra, R.; St-Hilaire, S.; Chamberlain, G.C.; Lightner, D.V.; Van Khoa, L.; et al. Reducing Disease Risk In Aquaculture; The World Bank: Washington, DC, USA, 2014.
Thuong, K.; Tuan, V.; Li, W.; Sorgeloos , P.; Bossier, P.; Nauwynck , H. Per Os Infectivity of White Spot Syndrome Virus (WSSV) in White-Legged Shrimp (Litopenaeus vannamei) and Role of Peritrophic Membrane. Vet. Res. 2016, 47, 39.
Alloul , A.; Wille, M.; Lucenti, P.; Bossier, P.; Van Stappen , G.; Vlaeminck , S. Purple bacteria as added-value protein ingredient in shrimp feed: Penaeus vannamei growth performance, and tolerance against Vibrio and ammonia stress. Aquaculture. 2021. 530, 735788.