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ARS Home » Southeast Area » Auburn, Alabama » Aquatic Animal Health Research » Research » Publications at this Location » Publication #421216

Research Project: Integrated Research to Improve Aquatic Animal Health in Warmwater Aquaculture

Location: Aquatic Animal Health Research

Title: Characterization and molecular identification of Vibrio parahaemolyticus isolated from Litopenaeus vannamei following an outbreak in a low salinity recirculating system

Author
item SIMPSON, KELLY - Auburn University
item Garcia, Julio
item MARTIN, BRITTANY - Auburn University
item KELLY, ANITA - Auburn University
item BRUCE, TIMOTHY - Auburn University
item DAVIS, ALLEN - Auburn University
item Beck, Benjamin
item ROY, LUKE - Auburn University

Submitted to: Aquaculture
Publication Type: Abstract Only
Publication Acceptance Date: 12/10/2024
Publication Date: 3/6/2025
Citation: Simpson, K., Garcia, J.C., Martin, B., Kelly, A.M., Bruce, T.J., Davis, A.D., Beck, B.H., Roy, L.A. 2025. Characterization and molecular identification of Vibrio parahaemolyticus isolated from Litopenaeus vannamei following an outbreak in a low salinity recirculating system [ABSTRACT]. Aquaculture 2025, New Orleans, Louisiana. March 6-10, 2025.

Interpretive Summary: Vibrio parahaemolyticus (Vp) is a gram-negative bacterium commonly found in estuarine, marine, and coastal environments and is known to induce disease throughout the global shrimp aquaculture industry. This pathogen is the causative agent of acute hepatopancreatic necrosis disease (AHPND) or early mortality syndrome (EMS) in Pacific white shrimp, Litopenaeus vannamei. This study describes the characterization of a Vp isolate (ARS-2-2024-GL) obtained from a naturally occurring outbreak in shrimp reared within a low salinity recirculating system (RAS; 363 L system). The RAS comprised 16 tanks (23L) maintained at 29 °C, each stocked with 10 shrimp (average weight of 2.63 g). Salinity at stocking was 9.5 ppt and was decreased approximately 2 ppt/day. During acclimation, a disease outbreak occurred. Affected shrimp displayed clinical signs of pale and watery hepatopancreas, alongside lethargy. The moribund and deceased shrimp were collected and necropsied, and microbial samples were aseptically collected from the eyes, hepatopancreas, and gastrointestinal tract. The samples were initially cultured on marine agar for the general isolation of bacterial samples and CHROMagar™ Vibrio for the isolation and detection of Vibrio spp. and results were positive. Biochemical and phenotypic profiles were developed using the API 20 E strip system, Biolog Gen. III ID microplate identification system, and Fatty Acid Methyl Analysis (FAME). ARS-2-2024-GL API 20E strip results yielded a profile code 534612, correctly identifying the isolate to the genus level as Vibrio spp. Then, the phenotypic profile of ARS-2-2024-GL was generated with the Biolog Gen. III ID microplate system, which classified the sample as V. parahaemolyticus. Additionally, FAME analysis of the isolate identified a core group of fatty acids (12:0, 13:0 iso, 14:0, 16:0 iso, 16:0, 17:0 iso, 17:1 '8c, 17:1 '6c, 17:0, 18:1 '9c, 18:0, Summed Feature 3, Summed Feature 5, and Summed Feature 8) yielding a sim index ID of .357 V. parahaemolyticus. 16S rRNA sequencing confirmed the isolate identification to be Vp. Preliminary bacterial challenges were carried out in L. vannamei using ARS-2-2024-GL to identify an appropriate challenge method and dosage. Cumulative percent mortality (CPM) from the immersion challenge was 11%, (low dose), 0% (intermediate), 13.5% (medium) and 15 % (high). Reverse gavage injection CPM was 4% (low), 8% (medium) and 39% (high). These findings suggest the outbreak in the RAS was most likely caused by Vp. We are currently investigating additional Vp isolates obtained from the outbreak and establish an effective L. vannamei challenge model for these specific isolates.

Technical Abstract: Vibrio parahaemolyticus (Vp) is a gram-negative bacterium commonly found in estuarine, marine, and coastal environments and is known to induce disease throughout the global shrimp aquaculture industry. This pathogen is the causative agent of acute hepatopancreatic necrosis disease (AHPND) or early mortality syndrome (EMS) in Pacific white shrimp, Litopenaeus vannamei. This study describes the characterization of a Vp isolate (ARS-2-2024-GL) obtained from a naturally occurring outbreak in shrimp reared within a low salinity recirculating system (RAS; 363 L system). The RAS comprised 16 tanks (23L) maintained at 29 °C, each stocked with 10 shrimp (average weight of 2.63 g). Salinity at stocking was 9.5 ppt and was decreased approximately 2 ppt/day. During acclimation, a disease outbreak occurred. Affected shrimp displayed clinical signs of pale and watery hepatopancreas, alongside lethargy. The moribund and deceased shrimp were collected and necropsied, and microbial samples were aseptically collected from the eyes, hepatopancreas, and gastrointestinal tract. The samples were initially cultured on marine agar for the general isolation of bacterial samples and CHROMagar™ Vibrio for the isolation and detection of Vibrio spp. and results were positive. Biochemical and phenotypic profiles were developed using the API 20 E strip system, Biolog Gen. III ID microplate identification system, and Fatty Acid Methyl Analysis (FAME). ARS-2-2024-GL API 20E strip results yielded a profile code 534612, correctly identifying the isolate to the genus level as Vibrio spp. Then, the phenotypic profile of ARS-2-2024-GL was generated with the Biolog Gen. III ID microplate system, which classified the sample as V. parahaemolyticus. Additionally, FAME analysis of the isolate identified a core group of fatty acids (12:0, 13:0 iso, 14:0, 16:0 iso, 16:0, 17:0 iso, 17:1 '8c, 17:1 '6c, 17:0, 18:1 '9c, 18:0, Summed Feature 3, Summed Feature 5, and Summed Feature 8) yielding a sim index ID of .357 V. parahaemolyticus. 16S rRNA sequencing confirmed the isolate identification to be Vp. Preliminary bacterial challenges were carried out in L. vannamei using ARS-2-2024-GL to identify an appropriate challenge method and dosage. Cumulative percent mortality (CPM) from the immersion challenge was 11%, (low dose), 0% (intermediate), 13.5% (medium) and 15 % (high). Reverse gavage injection CPM was 4% (low), 8% (medium) and 39% (high). These findings suggest the outbreak in the RAS was most likely caused by Vp. We are currently investigating additional Vp isolates obtained from the outbreak and establish an effective L. vannamei challenge model for these specific isolates.