Comparison of methods for uniformly challenging Black Tiger shrimp (Penaeus monodon) with gill-associated virus

Abstract

Increased disease tolerance is an important trait commonly incorporated into shrimp breeding programs. Sib-selection using experimental pathogen challenge tests are used to measure family based tolerance. It is critical for challenge tests to be standardised and repeatable for accurate genetic estimation. Accordingly, this study focused on establishing a method for uniformly challenging Black Tiger shrimp (Penaeus monodon) to an acute gill-associated virus (GAV) infection. A stock inoculum of GAV (105.27GAV RNA copiesμL−1 inoculum) was prepared from infected shrimp and used as the source of infection for the challenge methodologies investigated. Three challenge approaches compared in groups of juvenile P. monodon (2–10g) were; intramuscular injection, ingestion of fresh or frozen diced muscle tissue from GAV-infected shrimp and immersion in water containing the GAV inoculum. Intramuscular injection using a weight-standardised dose of the inoculum resulted in cumulative mortality surpassing 50% on Day 7 post-challenge. Immersion of shrimp for 2h in water comprising a 1:1000 dilution of the inoculum resulted in no mortalities over the 11day trial period. GAV RT-qPCR analysis also identified no evidence of any shrimp becoming infected by the immersion method. Feeding shrimp either fresh or frozen diced muscle tissue of moribund shrimp with an acute GAV infection generated cumulative mortality that reached/surpassed 50% on day 11 post-challenge. Compared to the survivors of the injection group (mean=107.73±0.49GAV RNA copies/μg total RNA), RT-qPCR analysis identified GAV infection loads to be less uniform and 100- to 1000-fold lower among survivors of feeding either fresh or frozen muscle tissue (mean=105.71±1.97 or 104.77±1.90GAV RNA copies/μg total RNA, respectively). Coefficient of variation (CV) values for the GAV infection load data supported survivors of injection (CV=0.06) having a more uniform infection load compared to the survivors of either fresh (CV=0.41) or frozen (CV=0.33) tissue ingestion. Of the three challenge approaches assessed for their suitability in measuring GAV tolerance, injection was clearly identified to provide the most uniform and reliable means of infecting shrimp with a defined dose of GAV and therefore is the preferred method of infection to use for GAV challenge tests.