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Health & Welfare | Assessment of transmission risk in cooked, WSSV-infected shrimp

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Thursday, April 09, 2020, 07:00 (GMT + 9)

Results show shrimp meat not infectious after boiling for over 1 minute

White spot disease (WSD), caused by the white spot syndrome virus (WSSV), is one of the biggest threats to the farmed shrimp industry. WSD causes high mortalities that can reach up to 100 percent in Specific Pathogen Free (SPF) populations in grow-out conditions in a matter of days. WSSV was first reported in Taiwan in 1992. Since then, shrimp infections with WSSV have been reported in Japan, Korea, South-East Asia, South Asia, the Mediterranean, the Middle East, the Americas and Australia.

The risk of WSSV introduction is predominantly due to the international movement between producing and importing countries of infected shrimp, whether they are live or frozen – head on (HO), head on shell on (HOSO) or headless shell on (HLSO) – or as cooked products. Due to the risk of inadvertently introducing shrimp pathogens via commodity products, some countries – including Australia, the Kingdom of Saudi Arabia and others – have implemented strict protocols regulating the importation of raw shrimp and prawn products. The screening to determine the presence and/or absence of some viral etiologies is through polymerase chain reaction (PCR). Usually, if the batch is positive, the product is either destroyed or treated (cooked).

Recently, cooked shrimp infected with WSSV tested positive by polymerase chain reaction (PCR). However, there is no study to determine the infectivity of WSSV in cooked shrimp that tested positive by PCR. There have been published studies that indicated that frozen shrimp products were an avenue for WSSV introduction into the Americas in 1995, but there are only a handful of peer-reviewed publications highlighting the risk of WSSV infectivity of cooked commodity shrimp. However, these studies restricted their findings to WSSV detection by PCR and it remained unknown if cooked shrimp that contain DNA viral sequences are infectious. The purpose of our study – supported by the Aquaculture Pathology Laboratory at the University of Arizona – was to evaluate the infectivity of WSSV-infected shrimp upon cooking.

Study setup

The shrimp in the study came from a commercial shrimp facility in the United States. A total of 250 SPF Pacific white shrimp (Litopenaeus vannamei) with an average weight of 10 grams were used. This sample weight represents the typical weight of shrimp in ponds when emergency harvests are carried out at shrimp farms where WSSV is endemic and a disease outbreak is about to occur.

To produce WSSV-infected shrimp, a WSSV challenge test was conducted via oral route by feeding previously confirmed WSSV-infected, minced shrimp tissue to SPF L. vannamei. This was done at 10 percent body weight of the SPF animals divided into two rations (morning and evening) in one day. Water in the experimental tanks was kept at 25 ppt salinity and a temperature of 25 degrees-C. After the experimental challenge, mortalities were assessed every hour. SPF L. vannamei were fed healthy tissue and served as a negative control.

Once shrimp mortalities reached 50 percent, the surviving animals were removed to simulate an emergency harvest and euthanized. Gills and pleopods samples were collected and preserved for confirmation with conventional and real-time PCR assays. Five shrimp with clinical signs were fixed and preserved for histological analysis (Fig. 1).

Fig. 1: Experimental design of the study. Click to enlarge

The initial infected shrimp population was separated into six groups each with 40-shrimp; each group was assigned to a specific boiling (in freshwater) time of 0, 1, 3, 5, 10 and 30 minutes. One group, not infected with WSSV, was the negative control. Each 40-shrimp group was divided into two sub-groups with two replicates each. Sub-group 1 was boiled using the different time frames (0, 1, 3, 5, 10 and 30 minutes) before feeding to SPF L. vannamei. Sub-group 2 was also boiled for different time frames (0, 1, 3, 5, 10 and 30 minutes) but was frozen (minus-20 degrees-C) for 14 days before being fed to SPF shrimp (Fig. 2).

Fig. 2: A schematic representation of the experimental design used in this study, including the treatment of WSSV-infected tissue at different times comprising 0, 1, 3, 5, 10 and 30 minutes in boiling water, followed by either direct experimental challenge or storing samples in the freezer before conducting an experimental challenge in SPF L. vannamei.. Click to enlarge

Temperature in shrimp tissue were collected using a thermometer placed inside the tail skeletal muscle, and internal temperature data were collected before and after the cooking process. After the boiling, samples were allowed to cool down slightly and then gill and pleopod samples were collected and preserved for further analysis of both PCR and qPCR (quantitative PCR is an improved version of traditional PCR and is used to detect and quantify nucleic acids for numerous applications).

To determine the pathogenicity of the cooked shrimp, a challenge test was conducted with a SPF shrimp population. Each 90-liter tank contained 10 juveniles (2 to 3 grams). Out of each replicate from each time frame, shrimp tissues including stomach, gills and pleopods were chopped from all the shrimp and minced. The minced tissue was added to the tank in two rations (morning and evening) at 10 percent of the total body weight of the live SPF population in one day. The duration of the challenge was 11 to 12 days. Daily mortalities were recorded, and pools of gills/pleopods from moribund shrimp were collected and preserved to be used for WSSV detection by qPCR and PCR. In addition, the moribund shrimps were fixed in Davidson’s fixative for histological analysis.

The final cumulative survival was determined at the end of the challenge. Samples of pleopods of all the shrimp were pooled per tank and preserved in 95 percent ethanol for qPCR and PCR analysis. Two shrimp from each tank were fixed in Davidson’s fixative for histological analysis. Shrimp frozen for two weeks (Group 2) were used to conduct a similar WSSV infection challenge using the same methodology described above.


For detailed information on the experimental design; shrimp experimental infection, sampling and pooling, and boiling of samples; the WSSV-challenge test; DNA extraction; PCR and qPCR analyses; and histopathology, contact the corresponding author. (the article continue) |  Read full article here

Authors: Luis Fernando Aranguren Caro, Ph.D. Hung N. Mai, Ph.D. Linda Nunan, MSc Joshua Lin, BSc Brenda Noble, BSc Arun K. Dhar, Ph.D./Global Aquaculture Advocate



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