scholarly journals Metabolic Alterations in Shrimp Stomach During Acute Hepatopancreatic Necrosis Disease and Effects of Taurocholate on Vibrio parahaemolyticus

2021 ◽  
Vol 12 ◽  
Author(s):  
Ramya Kumar ◽  
Teng-Chun Tung ◽  
Tze Hann Ng ◽  
Che-Chih Chang ◽  
Yi-Lun Chen ◽  
...  
Keyword(s):  
Bile Acid ◽  
Biofilm Formation ◽  
Vibrio Parahaemolyticus ◽  
Economic Losses ◽  
Bacterial Cells ◽  
High Concentration ◽  
Metabolic Alterations ◽  
Biofilm Detachment ◽  
Acute Hepatopancreatic Necrosis Disease ◽  
Primary Bile Acid

Acute hepatopancreatic necrosis disease (AHPND), a recently emerged bacterial shrimp disease, has increased shrimp mortality and caused huge economic losses in many Asian countries. However, molecular factors underlying pathogenesis of this disease remain largely unknown. Our objective was to characterize metabolic alterations in shrimp stomach during AHPND and determine effects of taurocholate on AHPND-causing Vibrio parahaemolyticus. Based on metabolomics, pathways for lipid metabolism and for primary bile acid (BA) synthesis were majorly affected following AHPND infection. Bile acid metabolites, namely taurocholate, were downregulated in the metabolomics database. This prompted us to study effects of taurocholate on biofilm formation, PirABvp toxin release and biofilm detachment capabilities in AHPND-causing V. parahaemolyticus. Treatment of this bacterium with high concentration of taurocholate, a primary bile acid, induced biofilm formation, PirABvp toxin release and facilitated the dispersion of bacterial cells. Taken together, our findings suggest that AHPND infection can affect the lipid metabolites in shrimp stomach, and further suggest that the primary bile acid taurocholate is important for the virulence of AHPND-causing V. parahaemolyticus.

Antibiotics Application Strategies to Control Biofilm Formation in Pathogenic Bacteria

2020 ◽  
Vol 21 (4) ◽  
pp. 270-286 ◽  
Author(s):  
Fazlurrahman Khan ◽  
Dung T.N. Pham ◽  
Sandra F. Oloketuyi ◽  
Young-Mog Kim
Keyword(s):  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Extracellular Polymeric Substances ◽  
Quorum Quenching ◽  
Resistance Mechanisms ◽  
Bacterial Biofilm ◽  
Bacterial Cells ◽  
High Concentration ◽  
Biofilm Inhibition ◽  
Abiotic Surfaces

Background: The establishment of a biofilm by most pathogenic bacteria has been known as one of the resistance mechanisms against antibiotics. A biofilm is a structural component where the bacterial community adheres to the biotic or abiotic surfaces by the help of Extracellular Polymeric Substances (EPS) produced by bacterial cells. The biofilm matrix possesses the ability to resist several adverse environmental factors, including the effect of antibiotics. Therefore, the resistance of bacterial biofilm-forming cells could be increased up to 1000 times than the planktonic cells, hence requiring a significantly high concentration of antibiotics for treatment. Methods: Up to the present, several methodologies employing antibiotics as an anti-biofilm, antivirulence or quorum quenching agent have been developed for biofilm inhibition and eradication of a pre-formed mature biofilm. Results: Among the anti-biofilm strategies being tested, the sub-minimal inhibitory concentration of several antibiotics either alone or in combination has been shown to inhibit biofilm formation and down-regulate the production of virulence factors. The combinatorial strategies include (1) combination of multiple antibiotics, (2) combination of antibiotics with non-antibiotic agents and (3) loading of antibiotics onto a carrier. Conclusion: The present review paper describes the role of several antibiotics as biofilm inhibitors and also the alternative strategies adopted for applications in eradicating and inhibiting the formation of biofilm by pathogenic bacteria.

scholarly journals The infection of Vibrio parahaemolyticus in shrimp and human

2018 ◽  
Vol 1 (1) ◽  
pp. 44 ◽  
Author(s):  
Rian Ka Praja
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Animal Health ◽  
Alternative Therapy ◽  
Penaeus Monodon ◽  
Early Mortality ◽  
Economic Losses ◽  
Early Mortality Syndrome ◽  
Thermostable Direct Hemolysin ◽  
Acute Hepatopancreatic Necrosis Disease ◽  
Very High

<p class="15" align="justify"><em>Vibrio parahaemolyticus</em> is an aquatic zoonotic agent that can threaten human and aquaculture animal health. Humans can be infected by consuming contaminated raw seafood or wound-related infections. Generally infection of <em>V. parahemolyticus</em> is orally transmitted and causes gastroenteritis in humans while in aquaculture animals especially shrimp can cause Acute Hepatopancreatic Necrosis Disease (AHPND) or Early Mortality Syndrome (EMS) with a very high mortality rate and cause economic losses. Shrimp species susceptible to infection are <em>Litopenaeus vannamei, Penaeus monodon,</em> and <em>P. chinensis</em>. <em>V. parahaemolyticus</em> produces several toxins in human disease such as thermostable direct hemolysin (TDH), TDH-related haemolysin (TRH), and thermolabile hemolysin (TLH). Meanwhile, Photorabdus insect-related (Pir) toxins consisting of PirA<sup>vp</sup> and PirB<sup>vp</sup> are the toxins associated with AHPND in shrimp. The genes that encode the toxin are used as targets to diagnose <em>V. parahaemolyticus</em> pathogens molecularly. Until now the treatment of <em>V. parahaemolyticus</em> infection is using antibiotics and fluid therapy, but there were <em>V. parahaemolyticus</em> isolates from aquaculture that have been resistant to antibiotics so that the use of antibiotics in aquaculture must be controlled and the use of alternative therapy are very important to be developed to control <em>V. parahaemolyticus</em> infection.</p><p class="15" align="justify"> </p><p>Keywords: <em>V. parahaemolyticus</em>, zoonotic, gastroenteritis, Acute Hepatopancreatic Necrosis Disease (AHPND), Early Mortality Syndrome (EMS).</p>

scholarly journals Molecular characterization of acute hepatopancreatic necrosis disease causing Vibrio parahaemolyticus strains in cultured shrimp Penaeus monodon in south‐west farming region of Bangladesh

2018 ◽  
Vol 27 (1) ◽  
pp. 57-68 ◽  
Author(s):  
Md Mostavi Enan Eshik ◽  
Nusrat Jahan Punom ◽  
Mst Khadiza Begum ◽  
Tahsin Khan ◽  
Mihir Lal Saha ◽  
...  
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Penaeus Monodon ◽  
16S Rrna Gene Sequencing ◽  
Molecular Techniques ◽  
Shrimp Farming ◽  
Economic Losses ◽  
Rrna Gene ◽  
South West ◽  
Acute Hepatopancreatic Necrosis Disease ◽  
Positive Results

Acute hepatopancreatic necrosis disease (AHPND) is an emerging shrimp disease caused by strains of Vibrio parahaemolyticus containing a unique virulent plasmid, responsible for substantial economic losses since 2009; caused up to 100% mortality in farmed shrimp Penaeus monodon. The purpose of this study was to isolate and identify the pathogenic strain of V. parahaemolyticus causing AHPND in cultured shrimp (Penaeus monodon) using classical and molecular techniques. Samples were collected from three different locations of south-west shrimp farming regions of Bangladesh viz. Sadar Upazilla of Satkhira; Mongla and Morrelganj under Bagerhat district. In this study, three selective media were used for primary isolation of V. parahaemolyticus. Among 46 primary isolates, 18 representative isolates were checked for the species-specific detection of V. parahaemolyticus using ldh primers and all of them were found to be positive. 16S rRNA gene sequencing were used to further confirm the isolates as V. parahaemolyticus. tdh primer was used to check human pathogenicity but all 18 isolates showed negative result. The isolates were further characterized to check their AHPND positivity using AP3 and AP4 primers. Ten isolates showed positive results for AP3 (55.56%) and 9 showed positive results for AP4 (50%) which indicated that the isolates were AHPND positive. This study also reported that all AHPND positive strains were resistant to the antibiotic gentamycin but sensitive to chloramphenicol, nalidixic acid, nitrofurantoin and tetracycline. The findings of this study will help the shrimp farmers and policy makers to take proper biosecurity measures to protect shrimps from AHPND and thereby sustain the shrimp production in Bangladesh. Dhaka Univ. J. Biol. Sci. 27(1): 57-68, 2018 (January)

Multifunctional Carbonized Nanogels to Treat Lethal Acute Hepatopancreatic Necrosis Disease

2021 ◽  
Author(s):  
Shao-Chieh Yen ◽  
Ju-Yi Mao ◽  
Hung-Yun Lin ◽  
Huai-Ting Huang ◽  
Scott G. Harroun ◽  
...  
Keyword(s):  
Functional Groups ◽  
Vibrio Parahaemolyticus ◽  
Feed Additives ◽  
Economic Losses ◽  
Multiple Drug ◽  
Loss Of Function ◽  
Bacterial Membranes ◽  
Resistant Strains ◽  
Acute Hepatopancreatic Necrosis Disease ◽  
The Impact

Abstract 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 neuralization 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.

scholarly journals QseC Inhibition as a Novel Antivirulence Strategy for the Prevention of Acute Hepatopancreatic Necrosis Disease (AHPND)-Causing Vibrio parahaemolyticus

2021 ◽  
Vol 10 ◽  
Author(s):  
Qian Yang ◽  
Peizhuo Zou ◽  
Zhi Cao ◽  
Qingyao Wang ◽  
Songzhe Fu ◽  
...  
Keyword(s):  
Broad Spectrum ◽  
Vibrio Parahaemolyticus ◽  
Pathogenic Bacteria ◽  
White Shrimp ◽  
Economic Losses ◽  
Resistant Bacteria ◽  
Flagellar Motility ◽  
Type Vi Secretion System ◽  
Global Regulation ◽  
Acute Hepatopancreatic Necrosis Disease

Acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus resulted in great economic losses in global shrimp aquaculture. There is an urgent need for development of novel strategies to combat AHPND-causing V. parahaemolyticus (VpAHPND), given that one of the greatest challenges currently is the widespread use of antibiotics and subsequent emergence of multidrug-resistant bacteria. Here, we proposed a broad-spectrum antivirulence approach targeting a conserved histidine kinase, QseC, which has been demonstrated to activate virulence expression in several Gram-negative pathogens. Our results showed that QseC mediated the catecholamine stimulated effects on growth and flagellar motility of VpAHPND. Transcriptome analysis revealed that QseC was involved in the global regulation of the virulence of VpAHPND as the ΔqseC mutant exhibited a decreased expression of genes related to type IV pilin, flagellar motility, and biofilm formation, while an overexpression of type VI secretion system and cell wall biosynthesis. Subsequently, the bacterial catecholamine receptor antagonist LED209 not only neutralized the stimulatory effects of host catecholamines on the growth and motility of VpAHPNDin vitro, but also attenuated the virulence of VpAHPND towards brine shrimp larvae and white shrimp in vivo. Additionally, LED209 presented no interference with pathogen growth, nor the toxicity to the experimental animals. These results suggest that QseC can be an attractive antivirulence therapy target, and LED209 is a promising candidate for development of broad-spectrum antivirulence agents. This is the first study that demonstrated the role of QseC in the global regulation of VpAHPND infection and demonstrated the antivirulence potential of LED209, which provides insight into the use of an antivirulence approach for targeting not only VpAHPND, but also a much larger collection of pathogenic bacteria.

scholarly journals Vibrio alginolyticus influences quorum sensing-controlled phenotypes of acute hepatopancreatic necrosis disease-causing Vibrio parahaemolyticus

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11567
Author(s):  
Panida Paopradit ◽  
Natta Tansila ◽  
Komwit Surachat ◽  
Pimonsri Mittraparp-arthorn
Keyword(s):  
Quorum Sensing ◽  
Biofilm Formation ◽  
Vibrio Parahaemolyticus ◽  
Extracellular Polymeric Substances ◽  
Capsular Polysaccharide ◽  
Regulatory Gene ◽  
Challenge Test ◽  
Great Ability ◽  
Acute Hepatopancreatic Necrosis Disease ◽  
Reporter Bacteria

Background Acute hepatopancreatic necrosis syndrome (AHPND) caused by Vibrio parahaemolyticus strain (VPAHPND) impacts the shrimp industry worldwide. With the increasing problem of antibiotic abuse, studies on quorum sensing (QS) system and anti-QS compounds bring potential breakthroughs for disease prevention and treatment. Methods In this study, the cell-free culture supernatant (CFCS) and its extract of V. alginolyticus BC25 were investigated for anti-QS activity against a reporter bacteria, Chromobacterium violaceum DMST46846. The effects of CFCS and/ or extract on motility, biofilm formation and extracellular polymeric substances (EPSs) of VPAHPND PSU5591 were evaluated. Moreover, the effects of V. alginolyticus BC25 on virulence of VPAHPND PSU5591 were investigated by shrimp challenge test. The potentially active anti-QS compounds presented in the extract and effect on gene expression of VPAHPND PSU5591 were identified. Results The CFCS of V. alginolyticus BC25 and its extract showed a significant anti-QS activity against the reporter bacteria as well as swimming and swarming motilities, biofilms, and EPSs production by VPAHPND PSU5591. Transcriptome analysis revealed that V. alginolyticus BC25 extract significantly reduced the flagella genes involved in biofilm formation and iron-controlled virulence regulatory gene of VPAHPND PSU5591. Whereas, the LuxR family transcriptional regulator gene, c-factor, a cell-cell signaling gene, and capsular polysaccharide were up-regulated. The potentially active anti-QS compounds identified in extract were Cyclo-(L-Leu-L-Pro), and Cyclo-(L-Phe-L-Pro). Furthermore, V. alginolyticus BC25 enhanced disease resistance against VPAHPND PSU5591 in tested shrimp larvae. Conclusion These findings suggest that V. alginolyticus BC25 could provide natural anti-QS and anti-biofilms compounds and has great ability to be used as biocontrol agent against VPAHPND infection in shrimp aquaculture.

scholarly journals Isolation and characterization of infectious Vibrio parahaemolyticus, the causative agent of AHPND, from the whiteleg shrimp (Litopenaeus vannamei)

2017 ◽  
Vol 44 (3) ◽  
pp. 470-479
Author(s):  
Patricia López-León ◽  
Antonio Luna-González ◽  
Ruth Escamilla-Montes ◽  
María del Carmen Flores-Miranda ◽  
Jesús A. Fierro-Coronado ◽  
...  
Keyword(s):  
Causative Agent ◽  
Biofilm Formation ◽  
Vibrio Parahaemolyticus ◽  
Bacterial Strains ◽  
Isolation And Characterization ◽  
Commercial Farms ◽  
Whiteleg Shrimp ◽  
Acute Hepatopancreatic Necrosis Disease ◽  
Selection Of

Vibrio parahaemolyticus, the causative agent of acute hepatopancreatic necrosis disease (AHPND), was isolated from the hepatopancreas of moribund whiteleg shrimp of commercial farms from Guasave, Sinaloa, Mexico. The isolates were screened on thiosulfate citrate bile salt sucrose agar plates for the selection of green colonies and further characterized through PCR with AP3 primers, 89F/R primers, hemolysin genes, hemolytic and enzymatic activity, hydrophobicity, autoaggregation, and biofilm formation. Bioassays by immersion challenge were conducted to confirm the pathogenicity of selected bacterial strains. In addition, the LC50 was calculated for each isolate. All isolates (35) belonged to V. parahaemolyticus, but three isolates did not correspond to strains that cause AHPND since they were negative with 89F/R primers. All isolates were αhemolytic and showed biofilm formation (from moderate to strong). Isolates were hydrophobic or hydrophilic and showed high autoaggregation capacity. Eight strains did not kill shrimp and eleven were pathogenic, but differences in virulence were found among them perhaps due to α-hemolysis and differences in biofilm formation and hydrophobicity. Therefore, performed characterization may help to understand the pathogenicity of V. parahaemolyticus. Finally, results showed that smaller shrimp are less resistant to V. parahaemolyticus infection.

Effect of Temperature, pH, and Water Activity on Biofilm Formation by Salmonella enterica Enteritidis PT4 on Stainless Steel Surfaces as Indicated by the Bead Vortexing Method and Conductance Measurements

2005 ◽  
Vol 68 (10) ◽  
pp. 2149-2154 ◽  
Author(s):  
E. GIAOURIS ◽  
N. CHORIANOPOULOS ◽  
G.-J. E. NYCHAS
Keyword(s):  
Stainless Steel ◽  
Water Activity ◽  
Metabolic Activity ◽  
Biofilm Formation ◽  
Salmonella Enteritidis ◽  
Ph Value ◽  
Effect Of Temperature ◽  
Bacterial Cells ◽  
High Concentration ◽  
Steel Surfaces

An assay was developed in an effort to elucidate the effect of important environmental parameters (temperature, pH, and water activity [aw]) on Salmonella Enteritidis biofilm formation on stainless steel surfaces. To achieve this, a modified micro-biological technique used for biofilm studying (the bead vortexing method) and a rapid method based on conductivity measurements were used. The ability of the microorganism to generate biofilm on the stainless surfaces was studied at three temperatures (5, 20, and 37°C), four pH values (4.5, 5.5, 6.5, and 7.4), and four aw values (0.5, 1.5, 5.5, and 10.5% NaCl). Results obtained by the bead vortexing method show that maximum numbers of adherent bacteria per square centimeter (106 CFU/cm2) were attained in 6 days at 20°C. Biofilm formation after 7 days of incubation at 20°C was found to be independent of the pH value. In addition, the high concentration of sodium chloride (10.5% NaCl, aw = 0.94) clearly inhibited the adherence of cells to the coupons. Conductance measurements were used as a supplementary tool to measure indirectly the attachment and biofilm formation of bacterial cells on stainless steel surfaces via their metabolic activity (i.e., changes in the conductance of the growth medium due to microbial growth or metabolism). Results obtained by conductance measurements corresponded well to those of the bead vortexing method. Furthermore, we were able to detect cells that remained attached on the metal surfaces even after vortexing via their metabolic activity. The results, except for demonstrating environmental-dependent Salmonella Enteritidis biofilm formation, indicated that traditional vortexing with beads did not remove completely biofilm cells from stainless steel; hence, conductance measurements seem to provide a more sensitive test capable to detect down to one single viable organism.

scholarly journals Multifunctional carbonized nanogels to treat lethal acute hepatopancreatic necrosis disease

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Shao-Chieh Yen ◽  
Ju-Yi Mao ◽  
Hung-Yun Lin ◽  
Huai-Ting Huang ◽  
Scott G. Harroun ◽  
...  
Keyword(s):  
Functional Groups ◽  
Vibrio Parahaemolyticus ◽  
Feed Additives ◽  
Economic Losses ◽  
Multiple Drug ◽  
Loss Of Function ◽  
Bacterial Membranes ◽  
Resistant Strains ◽  
Acute Hepatopancreatic Necrosis Disease ◽  
The Impact

Abstract 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

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

2022 ◽  
Author(s):  
Nguyen Quang Linh ◽  
Khanh Van Nguyen ◽  
Dung Quoc Tran ◽  
Van Khanh Tran Quang
Keyword(s):  
Recombinant Protein ◽  
Vibrio Parahaemolyticus ◽  
Expression Vector ◽  
Cloning And Expression ◽  
Economic Losses ◽  
Bacterial Disease ◽  
Parahaemolyticus Strain ◽  
E Coli ◽  
Whiteleg Shrimp ◽  
Acute Hepatopancreatic Necrosis Disease

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 &micro;g/ml). Conclusions: The purified PirA recombinant protein will provide materials for development research to create biological products to prevent and treat AHPND.

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