A Novel Glutathione S-Transferase Gtt2 Class (VpGSTT2) Is Found in the Genome of the AHPND/EMS Vibrio parahaemolyticus Shrimp Pathogen

Glutathione S-transferases are a family of detoxifying enzymes that catalyze the conjugation of reduced glutathione (GSH) with different xenobiotic compounds using either Ser, Tyr, or Cys as a primary catalytic residue. We identified a novel GST in the genome of the shrimp pathogen V. parahaemolyticus FIM- S1708+, a bacterial strain associated with Acute Hepatopancreatic Necrosis Disease (AHPND)/Early Mortality Syndrome (EMS) in cultured shrimp. This new GST class was named Gtt2. It has an atypical catalytic mechanism in which a water molecule instead of Ser, Tyr, or Cys activates the sulfhydryl group of GSH. The biochemical properties of Gtt2 from Vibrio parahaemolyticus (VpGSTT2) were characterized using kinetic and crystallographic methods. Recombinant VpGSTT2 was enzymatically active using GSH and CDNB as substrates, with a specific activity of 5.7 units/mg. Low affinity for substrates was demonstrated using both Michaelis–Menten kinetics and isothermal titration calorimetry. The crystal structure showed a canonical two-domain structure comprising a glutathione binding G-domain and a hydrophobic ligand H domain. A water molecule was hydrogen-bonded to residues Thr9 and Ser 11, as reported for the yeast Gtt2, suggesting a primary role in the reaction. Molecular docking showed that GSH could bind at the G-site in the vicinity of Ser11. G-site mutationsT9A and S11A were analyzed. S11A retained 30% activity, while T9A/S11A showed no detectable activity. VpGSTT2 was the first bacterial Gtt2 characterized, in which residues Ser11 and Thr9 coordinated a water molecule as part of a catalytic mechanism that was characteristic of yeast GTT2. The GTT2 family has been shown to provide protection against metal toxicity; in some cases, excess heavy metals appear in shrimp ponds presenting AHPND/EMS. Further studies may address whether GTT2 in V. parahaemolyticus pathogenic strains may provide a competitive advantage as a novel detoxification mechanism.

Sensitivity Validation of EWOD Devices for Diagnosis of Early Mortality Syndrome (EMS) in Shrimp Using Colorimetric LAMP–XO Technique

Electrowetting-on-dielectric (EWOD) is a microfluidic technology used for manipulating liquid droplets at microliter to nanoliter scale. EWOD has the ability to facilitate the accurate manipulation of liquid droplets, i.e., transporting, dispensing, splitting, and mixing. In this work, EWOD fabrication with suitable and affordable materials is proposed for creating EWOD lab-on-a-chip platforms. The EWOD platforms are applied for the diagnosis of early mortality syndrome (EMS) in shrimp by utilizing the colorimetric loop-mediated isothermal amplification method with pH-sensitive xylenol orange (LAMP–XO) diagnosis technique. The qualitative sensitivity is observed by comparing the limit of detection (LOD) while performing the LAMP–XO diagnosis test on the proposed lab-on-a-chip EWOD platform, alongside standard LAMP laboratory tests. The comparison results confirm the reliability of EMS diagnosis on the EWOD platform with qualitative sensitivity for detecting the EMS DNA plasmid concentration at 102 copies in a similar manner to the common LAMP diagnosis tests.

Current status of acute hepatopancreatic necrosis disease (AHPND) and result of isolating AHPND-causing strains of farmed shrimp in Mekong Delta Region of Vietnam

Early mortality syndrome/acute hepatopancreatic necrosis (EMS/AHPND) was first detected in China in 2009. The disease spread rapidly to neighboring countries and emerged in almost major shrimp-producing regions in the world, including Vietnam. The disease has caused serious damage to the global shrimp industry and so far, there is no effective cure. In order to understand the current status of AHPND, and then to introduce effective prevention and detection measures, we collected data and shrimp samples in some provinces in the Mekong Delta to analyze and isolate the pathogenic strains. The results of our study conducted from 2014 - 2018 in four provinces (Ben Tre, Long An, Bac Lieu, Kien Giang) showed that AHPND damaged from 2.0 to 57.2% of the total shrimp farming area. In addition, we isolated 10 AHPND-positive strains via culturing and PCR. The results of representative sequencing of three strains LA1, LA5, and LA8 showed that they were 100% similarity with the previously published strain XN89. These isolated strains are used as a collection for further studies on the origin and mechanism of the disease by whole genome sequencing.

ESTABLISH THE INITIAL PROCEDURE FOR DETECTING VIBRIO PARAHAEMOLYTICUS IN THE SHRIMP BY ONE STEP- PCR METHOD

Early Mortality Syndrome (EMS), also known as Acute Hepatopancreas Necrosis Disease (AHPND) in shrimps, has been identified as being caused by a strain of V. parahaemolyticus. This disease has caused enormous damage to the shrimp farming industry among countries in the world in general and Vietnam in particular. One of the main reasons for uncontrollable widespread disease is the process of identifying pathogens is slow and inaccurate, leading to no promptly preventive measurement. In order to enhance the ability to briefly detect disease and improve the current disease status, in this study the PCR process using two specific 16S rRNA and ldh primers has been developed to swiftly diagnose V. parahaemolyticus pathogen in shrimps with a detection threshold of 3.5x103 CFU/ml

Effects of salinity and shaking condition on the growth and virulence of Vibrio parahaemolyticus

Vibrio parahaemolyticus is a Gram-negative halophilic bacterium that is found in estuarine, marine and coastal environment. This organism is the major causative agent of Early Mortality Syndrome (EMS) or Acute Hepatopancreatic Necrosis Disease (AHPND) which resulted in serious damages to cultured shrimp industry. Understanding the effect of environmental factors on the growth and virulence of this potential pathogen would be beneficial for preventing its outbreak. In this study, the growth and virulence of V. parahaemolyticus was examined under different salinity and shaking condition. V. parahaemolyticus XN9 was cultured in Brain Heart Infusion (BHI) medium with different sodium chloride concentrations (2.0, 2.5 and 3.0%) and different shaking conditions (0, 120 and 240 rpm). The growth of the bacterium was recorded over 8h and six extracellular enzymes of V. parahaemolyticus XN9 including caseinase, hemolysin, lecithinase, lipase, gelatinase, chitinase were investigated using agar-based method. The growth of V. parahaemolyticus was varied among different salinity and shaking conditions. It showed the best growth at 2.0% NaCl and 240 rpm. No change in the enzymatic activity (EA) of the tested extracellular enzymes was observed while changing salinity except the significant decline of gelatinase from 3.49±0.19 to 2.77±0.17 mm following salinity increase (p < 0.05). On the other hand, regarding shaking condition, lipase was the one to increase its activity significantly following the increase of shaking speed (p < 0.05). While caseinase, lecithinase, gelatinase and lipase were well expressed in V. parahaemolyticus, no hemolytic and chitinase activity was observed in any tested conditions. In summary, our study showed that 2.0 % NaCl and 240 rpm shaking promoted the best growth of V. parahaemolyticus and resulted in highest activity of gelatinase and lipase in this bacterium.

Benefits of a Biofilm System and Low Ammonia Levels to Decrees the Early Mortality Syndrome in Shrimp Larvae (Litopenaeus vannamei) Infected with (Vibrio parahaemolyticus)

The world shrimp aquaculture, has faced several problems, causing severe losses in shrimp hatcheries; between the most critical has been diseases such as early mortality syndrome (EMS) caused by (Vibrio parahaemolyticus). The EMS was initially detected in Asian countries; after, it was disseminated to Mexico and other countries. In Mexico, EMS caused severe economic losses during 2013-2016; and it has not yet been eradicated. Various causes for EMS have been reported; none is entirely accurate, but water quality is essential for successful shrimp aquaculture; therefore, the aim this work was evaluate the ammonia concentration effect on susceptibility to (EMS) on post-larvae (PL-15) shrimp (Litopenaeus vannamei) infected with (V. parahaemolyticus), using a biofilm system (water with, microalgae, dinoflagellates, protozoa and other planktonic microorganisms). So series of 5 flasks each one were arranged as following: Series S; 900 ml of filtered seawater (FSW) and 10 PL-15 shrimp per flask. Series SB; 840 ml of FSW, 60 ml of biofilm and 10 PL-15 shrimp. Series E; 900 ml of FSW, infected with 2 ml (V. parahaemolyticus) 106 CFU/ ml and 10 PL-15 shrimp. Series EN; fifteen flasks with 900 ml of FSW, 10 PL-15 shrimp, added with NH4Cl (0.535 mg/ml), to get 0.5, 1.0, 1.5, 2.0 and 2.5 mg/l final ammonia concentration in 5 sub-series of 3 flasks each one. During experiment, ammonia concentration and PL-15 shrimp mortality were evaluated in all flask. Ammonia concentration was higher in series EN than in series E; the same was observed in Series S respect to SB, but at lower values. At end of experiment, mortality in series EN was 90% Vs 60% in E. Similarly, mortality in series S was 10% Vs 0% in SB. This results confirm that the ammonia increases PL shrimp mortality, and biofilm system reduce ammonia and consequently PL-15 shrimp mortality.

NGHIÊN CỨU KHẢ NĂNG KHÁNG VI KHUẨN Vibrio spp. GÂY BỆNH HOẠI TỬ GAN TỤY CẤP (AHPND) TRÊN TÔM CHÂN TRẮNG (Litopenaeus vannamei) CỦA DỊCH CHIẾT TỪ CÂY CHÓ ĐẺ THÂN XANH (Phyllanthus amarus Schum)

Kết quả nghiên cứu nhằm xác định khả năng kháng khuẩn của dịch chiết từ cây chó đẻ thân xanh (Phyllanthus amarus); nồng độ ức chế tối thiểu (MIC) và nồng độ tiêu diệt tối thiểu (MBC) trên 2 chủng vi khuẩn Vibrio parahaemolitycus (K15) và Vibrio sp. (VT33) ở mật độ 106 CFU/mL gây bệnh hoại tử gan tụy cấp (Acute Hepatopancreatic Necrosis Disease –AHPND) còn được gọi là hội chứng tôm chết sớm (Early Mortality Syndrome - EMS) trên tôm chân trắng (Litopenaeus vannamei) nuôi tại Thừa Thiên Huế, kết quả ban đầu ghi nhận: dịch chiết được chiết xuất dạng cao từ cây chó đẻ thân xanh (P. amarus) có khả năng kháng cả 2 chủng Vibrio parahaemolitycus (K15) và Vibrio sp. (VT33) thử nghiệm từ nồng độ 250 đến 1.000 mg/mL, thể hiện ở đường kính vòng kháng khuẩn dao động từ 16,60–21,40 mm đối với chủng Vibrio parahaemolitycus (K15) và từ 17,60 – 23,60 mm đối với Vibrio sp. (VT33). Kết quả cũng xác định hiệu quả của dịch chiết trên 2 chủng Vibrio parahaemolyticus (K15) và Vibrio sp. (VT123) thể hiện giá trị MIC và MBC tương ứng đạt 125 mg/mL và 500 mg/mL; 62,5 và 500 mg/mL.

Silver Nanoparticles Synthesized with Rumex hymenosepalus: A Strategy to Combat Early Mortality Syndrome (EMS) in a Cultivated White Shrimp

Early Mortality Syndrome (EMS) or Acute Hepatopancreatic Necrosis Syndrome (AHPNS) is a disease produced by gram-negative bacteria Vibrio parahaemolyticus (V. parahaemolyticus), which has caused declines in worldwide production of a white shrimp Litopenaeus vannamei (L. vannamei). In this work, we propose the implementation of silver nanoparticles (AgNPs) synthesized with Rumex hymenosepalus (Rh) extract as an alternative on V. parahaemolyticus control. AgNPs were characterized by UV-Vis spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and selected area electron diffraction (SAED). AgNP mean sizes by DLS were 80.82±1.16 nm and sizes between 2 and 10 nm by TEM, with a zeta potential of −47.72±1.05 mV. This study evaluated AgNPs and Rh antimicrobial capacity on V. parahaemolyticus at different concentrations; the minimum inhibitory concentration (MIC) found was 25 μg/mL for AgNPs and 220 μg/mL for Rh. Additionally, were carried out time-kill curves and reactive oxygen species (ROS) generation for 1 and 4 MIC. Both concentrations (MIC) were tested for toxicity on Artemia nauplii from Artemia franciscana (A. franciscana), because nauplii were used as biocarriers for AgNPs and Rh extract on L. vannamei. Once the shrimp were treated, they were challenged with Vibrio infection and it was found that those who were treated with both agents showed greater survival than the control. V. parahaemolyticus and postlarval samples were taken from the bioassay and fixed and prepared for TEM and SEM in order to search NPs in internal structure of bacteria and the hepatopancreatic area of shrimps; AgNPs were detected in both cases. AgNPs and Rh extract show antibacterial properties on the infected shrimp with V. parahaemolyticus. The action mechanisms are interaction with the bacterial membrane and ROS generation; these effects are produced by both agents.