Cloning and Expression of PirA gene of Vibrio parahaemolyticus Strain K5 Causing Acute Hepatopancreatic Necrosis Disease in Whiteleg Shrimp in E. coli Host Cell

Background: Acute hepatopancreatic necrosis disease (AHPND), is a bacterial disease of whiteleg shrimp, which has a high mortality rate (100%) and incurs economic losses. Our objective was to identify the genes which lead to cell and organ damage and investigate bioproducts to prevent and treat. Methods: Litopenaeus vannamei shrimp in Thua Thien Hue province, Vietnam were collected from an infected pond and analysed at the Institute of Biotechnology, Hue University. The PirA gene of Vibrio parahaemolyticus strain K5 was isolated and analyzed for nucleotide sequence and paired with the expression vector pQE30. The expression vector was transformed into E. coli strain M15, the PirA recombinant protein was expressed in the form of 6xHis-PirA fusion protein of about 15 kDa. PirA recombinant protein was purified and determined the PirAvp binding ratio, cloning and sequencing of PirA gene from Vibrio parahaemolyticus strain K5 causing AHPND by PCR method with specific primers and molecular weights of PirAvp and the PirAvp complex. Results: PirA gene from Vibrio parahaemolyticus strain K5 was cloned into pGEM-T easy vector (Promega, USA) and screened E. coli TOP10 colonies containing pGEM T easy/PirA recombinant plasmid on LB agar/ampicillin/IPTG/X-Gal medium. PCR showing a band of about 347 bp, matching the size of PirA gene and two nucleotide sequences (BamHI and HindIII). The results showed that PirA gene has a length of 336 bp and similar to PirA gene on GenBank (Code: KU556825.1). The results of protein extracted from E. coli M15 recombinant cells and 6xHis-PirA target protein was collected in elution fractions from EF2 to EF6, showed that the concentration of 6xHis-PirA protein and EF3 elution fraction collected a highest protein concentration (1,586.54 µg/ml). Conclusions: The purified PirA recombinant protein will provide materials for development research to create biological products to prevent and treat AHPND.

Effects of different shrimp extracts on Vibrio parahaemolyticus growth and virulence

Vibrio parahaemolyticus is the main causative agent of acute hepatopancreatic necrosis disease (AHPND) in shrimp. This study aimed to investigate how shrimp extracts affect the growth and virulence of an AHPND-causative strain known as V. parahaemolyticus XN9. To this end, the bacteria was cultured in media containing 3% extract of each of five shrimp types and their growth kinetics were compared against that from bacteria grown in brain-heart infusion (BHI) media. Eight-hour growth curves were constructed using the plate-counting method. The activity of five extracellular enzymes that contribute to bacterial virulence was examined using the agar-based method. The results showed that V. parahaemolyticus XN9’s growth was strongly enhanced in all five shrimp extract media with the highest increase (25% greater than the BHI medium) found in the giant tiger prawn extract. Additionally, all the shrimp extracts boosted the extracellular enzymatic activity of V. parahaemolyticus XN9, although to different extents. In summary, the shrimp extracts, particularly that from the prawns, not only promoted the viability and growth of V. parahaemolyticus XN9 but also its extracellular enzymatic activities.

Characteristics and Activity Anti Qourum Sensing Bacillus spp. Isolated from Penaeus vannamei Shrimp Ponds

Certain strains of V. parahaemolyticus carry a gene that encodes a toxin that causes Acute hepatopancreatic necrosis disease (AHPND) in P. vannamei. AHPND attacks shrimp post larvae within 20-30 days after stocking causing up to 100% mortality. The expression of these virulent genes is controlled by the quorum sensing system. This system is inhibited by an anti-quorum sensing (AQS) mechanism. Several Bacillus strains have AQS mechanism by producing AHL-Lactonase enzyme. Therefore, this study aimed to obtain Bacillus spp. having AQS activity for controlling AHPND. The study was conducted from isolation and selection of Bacillus isolates, as well as determination of AQS activity. From 22 samples consisting of shrimp intestines, water and pond sediment samples, a total of 151 isolates of Bacillus spp. were isolated. The screening test for AQS activity obtained 11 isolates that showed AQS activity on Cromobacterium violaceum. Determination of violacein pigment in liquid cultures of C. violaceum showed the index value of the pigment formation was between 0.025-0.166 and 0.026-0.567 at 24-hour and between 48-hour incubations, respectively. The quantitative analysis of violacein production showed that there were six isolates of Bacillus could inhibit the pigment production more than 75%. The isolates were identified as Bacillus cereus (four isolates), Bacillus thuringiensis (one isolate), and Bacillus velezensis (one isolate), respectively. The molecular analysis had confirmed that the isolates have aiiA genes encoding AHL-lactonase enzyme. These Bacillus isolates have potential application for controlling AHNPD disease.

Multifunctional carbonized nanogels to treat lethal acute hepatopancreatic necrosis disease

Background Shrimp aquaculture has suffered huge economic losses over the past decade due to the outbreak of acute hepatopancreatic necrosis disease (AHPND), which is mainly caused by the bacteria Vibrio parahaemolyticus (V. parahaemolyticus) with the virulence pVA1 plasmid, which encodes a secretory photorhabdus insect-related (Pir) toxin composed of PirA and PirB proteins. The Pir toxin mainly attacks the hepatopancreas, a major metabolic organ in shrimp, thereby causing necrosis and loss of function. The pandemic of antibiotic-resistant strains makes the impact worse. Methods Mild pyrolysis of a mixture of polysaccharide dextran 70 and the crosslinker 1,8-diaminooctane at 180 ℃ for 3 h to form carbonized nanogels (DAO/DEX-CNGs) through controlled cross-linking and carbonization. The multifunctional therapeutic CNGs inherit nanogel-like structures and functional groups from their precursor molecules. Results DAO/DEX-CNGs manifest broad-spectrum antibacterial activity against Vibrio parahaemolyticus responsible for AHPND and even multiple drug-resistant strains. The polymer-like structures and functional groups on graphitic-carbon within the CNGs exhibit multiple treatment effects, including disruption of bacterial membranes, elevating bacterial oxidative stress, and neutralization of PirAB toxins. The inhibition of Vibrio in the midgut of infected shrimp, protection of hepatopancreas tissue from Pir toxin, and suppressing overstimulation of the immune system in severe V. parahaemolyticus infection, revealing that CNGs can effectively guard shrimp from Vibrio invasion. Moreover, shrimps fed with DAO/DEX-CNGs were carefully examined, such as the expression of the immune-related genes, hepatopancreas biopsy, and intestinal microbiota. Few adverse effects on shrimps were observed. Conclusion Our work proposes brand-new applications of multifunctional carbon-based nanomaterials as efficient anti-Vibrio agents in the aquatic industry that hold great potential as feed additives to reduce antibiotic overuse in aquaculture. Graphical Abstract

Effects of a Single Cell Protein (Methylococcus capsulatus, Bath) in Pacific White Shrimp (Penaeus vannamei) Diet on Growth Performance, Survival Rate and Resistance to Vibrio parahaemolyticus, the Causative Agent of Acute Hepatopancreatic Necrosis Disease

The efficacy of a single cell protein (SCP) methanotroph (Methylococcus capsulatus, Bath) bacteria meal (FeedKind®, Calysta, Menlo Park, CA, United States), in Pacific white shrimp (Penaeus vannamei) diets was studied to determine growth performance, survival rate and disease resistance against Vibrio parahaemolyticus causing Acute Hepatopancreatic Necrosis Disease (AHPND). The growth trial was assigned in a completely randomized design (CRD) with four treatments and 5 replicates of each, T1: a fishmeal-based control containing 15% fish meal and 3 diets with graded levels of methanotroph bacteria meal, namely T2: 5% methanotroph bacteria meal, T3: 10% methanotroph bacteria meal, and T4: 15% methanotroph bacteria meal. Shrimp were fed ad libitum for 6 weeks on trial diets to assess growth. Subsequent to the growth trial, three replicates of the same groups were exposed to V. parahaemolyticus by a single bath challenge and held for a further 15 days on the same diets as the growth study to assess survival and resistance. No significant differences (p > 0.05) in survival or in growth performance, including final weight, weight gain, specific growth rate, feed consumption or feed conversion ratio of white shrimp fed feeds containing methanotroph bacteria meal or control diets for 6 weeks. Immune markers such as hemocyte counts, phenoloxidase, superoxide dismutase and lysozyme activity were similar across all groups after the 6-week feeding trial. In a V. parahaemolyticus challenge, methanotroph bacteria meal in the diet significantly promoted the survival rate, and the reduction of Vibrio sp. in the hepatopancreas of white shrimp. Hemocyte count and phenoloxidase activity showed no significant differences (p > 0.05) between diet treatment groups, but hemolymph protein was significantly higher (p < 0.05) in shrimp fed diets containing 15% methanotroph bacteria meal after challenge. The Vibrio colony counts from hepatopancreas in the treatment groups were all significantly lower than the control (p < 0.05). The findings show that methanotroph bacteria meal can entirely replace fishmeal in white shrimp diets and the 15% inclusion of methanotroph bacteria meal in shrimp diet shows no adverse effects on growth performance, feed utilization and survival rate. In addition, shrimp fed methanotroph bacteria meal diets exhibited improved survival rates to an AHPND challenge.