Acute Hepatopancreatic Necrosis Disease In Shrimp Research Articles

The QS regulator AphBvp promotes expression of the AHPND PirAvp and PirBvp toxins and may enhance virulence under acidic conditions.

Shrimp acute hepatopancreatic necrosis disease (AHPND) is one of the most devastating diseases to impact the global shrimp farming industry, with a mortality rate of 70 %-100 %. The key virulence factors are a pair of Photorhabdus insect-related (Pir)-like toxins, PirAvp and PirBvp. In this study, by using an in vitro transcription and translation assay, we first confirmed that the quorum sensing transcriptional regulator AphBvp could trigger the expression of its downstream genes after binding to the AphBvp binding sequence in the promoter region of the pirAvp/pirBvp operon. Next, we showed that AphBvp was essential for the expression of these toxins by using an aphBvp-deletion mutant (ΔaphBvp) derived from the AHPND-causing Vibrio parahaemolyticus. Lastly, we discovered that the expression levels of PirAvp and PirBvp were up-regulated under acidic conditions (pH 4.5), and further showed that an acidic environment promoted the binding of AphBvp to the pirABvp promoter. We speculate that this was because the acidic environment favored the formation of AphBvp tetramers, which is important for binding to DNA. Taken together, these findings improve our understanding of the gene regulatory mechanisms of pirAvp and pirBvp, and suggest that the pH value of the environment might affect the virulence of AHPND-causing V. parahaemolyticus.

Passive immunoprophylaxis with Ccombodies against Vibrio parahaemolyticus in Pacific white shrimp (Penaeus vannamei)

The Vibrio parahaemolyticus strain causing acute hepatopancreatic necrosis disease (AHPND) in shrimp secretes toxins A and B (PirAVp/PirBVp). These toxins have been implicated in pathogenesis and are targets for developing anti-AHPND therapeutics or prophylactics that include passive immunization. We have previously reported that Ccombodies (recombinant hagfish variable lymphocyte receptor B antibodies; VLRB) targeting PirBVp conferred protection against V. parahaemolyticus in shrimp when administered as a feed supplement. In this study, we screened a phage-displayed library of engineered VLRBs for PirAVp-targeting Ccombodies that were mass-produced in a bacterial expression system. We then introduced these Ccombodies into the diet of Pacific white shrimp (Penaeus vannamei) over a seven-day period. Subsequently, the shrimp were exposed to a challenge with V. parahaemolyticus. Mortality rates were then observed and recorded over the following seven days. Administering shrimp feed supplemented with Ccombodies at a high dose (100 mg per 100 g feed) reduced mortality in recipient animals (2.96-5.19%) statistically similar to mock-challenged control (1.48%), but significantly different from the Ccombody-deficient control (74.81%). This suggests that the Ccombodies provided strong protection against the bacterium. Feeding shrimp with a median dose (10 mg/100 g feed) gave statistically comparable low mortality (5.93-6.67%) as the high dose. Reducing the Ccombody dose to 1 mg/100 g feed showed variable effects. Ccombody A2 showed mortality (11.85%) significantly lower than that of the Ccombody-deficient group (74.81%), suggesting that it can effectively protect against the bacterial challenge at a low dose. Our results demonstrate the ability of the phage-displayed VLRB library to generate antigen-specific Ccombodies rapidly and simply, with the expression of high protein levels in bacteria. The protective effect provided by these Ccombodies aligns with our earlier results, strongly supporting the use of VLRB antibodies as a substitute for IgY in passive immunoprophylaxis against AHPND in shrimp.

Comparative transcriptomic analysis of hepatopancreas reveals that more genes are involved in the exposure response of Vibrio parahaemolyticus PirAvp compared to PirBvp

Vibrio parahaemolyticus (VP-AHPND) is regarded as one of the main pathogens that caused acute hepatopancreatic necrosis disease (AHPND) in the Pacific white shrimp Litopenaeus vannamei. PirAvp and PirBvp toxin proteins are the main pathogenic proteins of AHPND in shrimp. Knowledge about the mechanism of shrimp response to PirAvp or PirBvp toxin is very helpful for developing new prevention and control strategy of AHPND in shrimp. In this study, the pathological sections showed that after 4 h treatment, significant pathological changes were observed in the PirBvp treated group, and no obvious pathological changes was found in PirAvp treated group. In order to learn the mechanism of shrimp response to PirAvp and PirBvp, comparative transcriptome was applied to analyze the different expressions of genes in the hepatopancreas of shrimp after treatment with PirAvp or PirBvp. A total of 9978 differentially expressed genes (DEGs) were identified between PirAvp or PirBvp-treated and PBS control shrimp, including 6616 DEGs in the PirAvp treated group and 3362 DEGs in the PirBvp treated group. There were 2263 DEGs that were commonly expressed, 4353 DEGs were only expressed in PirAvp VS PBS group and 1099 DEGs were uniquely expressed in PirBvp VS PBS group. Among these DEGs, the anti-apoptosis related pathways and immune response related genes significantly expressed in the commonly expressed DEGs of PirAvp VS PBS group and PirBvp VS PBS group, and small GTPase-mediated signaling and DNA metabolic process might relate to the host special reaction towards PirAvp and PirBvp exposure. The data suggested that the differential expression of these immune and metabolic-related genes in hepatopancreas might contribute to the pathogenicity variations of shrimp to VP-AHPND. The identified genes in this study will be useful for clarifying the response mechanism of shrimp toward different toxins of VP-AHPND and will further provide molecular basis for understanding the pathogenic mechanism of VP-AHPND.

High nutrient induces virulence in the AHPND-causing Vibrio parahaemolyticus, interpretation from the ecological assembly of shrimp gut microbiota

Shrimp diseases frequently occur during the later farming stages, when the rearing water is eutrophic. This observation provides clue that the virulence of pathogens could be induced by elevated nutrient, whereas the underlying ecological mechanism remains limited. To address this pressing knowledge, we explored how gut microbiota responded to the infection of oligotrophic (OVp) or eutrophic (EVp) pre-cultured Vibrio parahaemolyticus, a causing pathogen of shrimp acute hepatopancreatic necrosis disease (AHPND). Resulted revealed that OVp and EVp infections caused dysbiosis in the gut microbiota and compromised shrimp immunity, while the later infection led to earlier and higher mortality. Significant associations were detected between the gut microbiota and each of the measured immune activities. Neutral community model showed that the assembly of gut microbiota was more strongly governed by deterministic processes in EVp infection, followed by EVp infected and control shrimp. Additionally, there were significantly lower temporal turnover rate and average variation degree in the gut microbiota in EVp infected shrimp compared with control individuals. Notably, we identified 22 infection-discriminatory taxa after ruling out the ontogenic effect. Using profiles of the 22 indicators as independent variables, the diagnosis model accurately distinguished (an overall 85.9% accuracy) the infected status (control, OVp or EVp infected shrimp), with 81.3% accuracy at the initial infection stage. The convergent and deterministic gut microbiota in EVp infected shrimp could partially explain why it is challenge to cure APHND from an ecological viewpoint. In addition, we provided a sensitive approach for diagnosing the onset of infection, when disease symptom is unobservable.

RNA-seq analysis unveils temperature and nutrient adaptation mechanisms relevant for pathogenicity in Vibrio parahaemolyticus

Shrimp diseases frequently occur during pre-adult to adult lifestages, when the rearing water temperature and nutrient levels are high. This phenomenon provides clues that the virulence of pathogens might be induced by elevated temperature and nutrient, whereas the underlying molecular mechanism is lacking. To address this pressing knowledge, we explored how elevated temperature and nutrient stimulate the expression profiles of Vibrio parahaemolyticus, a notorious pathogen causing shrimp acute hepatopancreatic necrosis disease (AHPND). The stimulated pathogenicity was verified by pretreatment of V. parahaemolyticus under elevated temperature or nutrient condition, which caused significantly faster and higher rate of shrimp mortality. RNA-Seq analysis revealed that elevated temperature and nutrient significantly altered the transcriptional patterns of V. parahaemolyticus, resulting in 232 and 82 differential genes, respectively. Weighted Gene Co-Expression Network Analysis depicted that the two conditions induced different gene expression patterns. Specifically, elevated temperature enriched genes involved in arginine biosynthesis and flagellar assembly, whereas elevated nutrition stimulated genes facilitating citrate cycle, cysteine and methionine metabolism pathways. Based on the profiles of differential genes, we inferred that high temperature negatively regulated OpaR and ToxR, thereby activating type VI secretion system 1 (T6SS1). Concurrently, the induced expressions of adhesion Multivalent Adhesion Molecule 7 (MAM7) and flagella-related genes enable V. parahaemolyticus to actively attack host under elevated temperature. By contrast, elevated nutrient activates the T6SS1 through TfoY. These findings provide comprehensive insights into the molecular mechanism implicating in the pathogenicity of V. parahaemolyticus, and partially explain why disease frequency is increased under the scenarios of increasing temperature and eutrophication in aquaculture and natural ecosystems.

Toxicity of recombinant PirA and PirB derived from Vibrio parahaemolyticus in shrimp

Acute hepatopancreatic necrosis disease (AHPND), caused by emerging strains of Vibrio Parahaemolyticus, is of concern in shrimp aquaculture. Secreted proteins PirA and PirB, encoded by a plasmid harbored in V. parahaemolyticus, were determined to be the major virulence factors that induce AHPND. To better understand pathogenesis associated with PirA and PirB, recombinant proteins rPirA and rPirB were produced to evaluate their relative toxicities in shrimp. By challenging shrimp at concentration of 3 μM with reverse gavage method, rPirA and rPirB (approximately 0.4 and 1.5 μg per g of body weight, respectively) caused 27.8 ± 7.8% and 33.3 ± 13.6% mortality, respectively; combination of 3 μM rPirA and rPirB resulted in 88.9 ± 7.9% mortality. Analysis of protein mobility in native gel revealed that rPirB was apparently in the form of monomer while rPirA was oligomerized as an octamer-like macromolecule, suggesting that inter- and intra-molecular interactions between rPirA and rPirB enhanced the toxic effect. An attempt to block or reduce rPirA activity with a putative receptor, N-acetyl-galactosamine, was unsuccessful, implying that remodeling analysis of PirA molecule, such as the octamer observed in this study, is necessary. Results of this study provided new insight into toxic mechanism of PirA and PirB and shall help design strategic antitoxin methods against AHPND in shrimp.

Bamboo powder protects gnotobiotically-grown brine shrimp against AHPND-causing Vibrio parahaemolyticus strains by cessation of PirABVP toxin secretion

Acute hepatopancreatic necrosis disease (AHPND), caused by virulent Vibrio parahaemolyticus strains, is causing havoc in the shrimp industry. However, the culture conditions markedly influence the phenotypic status of V. parahaemolyticus. For instance, under low shaking conditions, the free-living planktonic virulent form of AHPND-causing V. parahaemolyticus switches into a non-virulent biofilm phenotype. Hence, strategies that could stimulate phenotype switching can be a novel way to control AHPND in shrimp. In this study, we evaluated the role of bamboo powder, a natural biofilm carrier, on the phenotypic switch in AHPND-causing and non-AHPND V. parahaemolyticus strains under both in vitro and in vivo conditions. The results showed that bamboo powder addition (50–100 mg/l) significantly enhanced the survival of brine shrimp larvae upon challenge with AHPND M0904 and RY strains. Furthermore, addition of bamboo powder also has profound effects on the phenotypic responses of AHPND-causing V. parahaemolyticus strains. The V. parahaemolyticus AHPND strains (M0904 and RY) grown with bamboo powder developed cellular aggregates or floccules in the culture medium and switch in the pattern of protein production and secrete alkaline phosphatase PhoX instead of PirAVP and PirBVP toxins. Based on the overall results, it can be suggested that bamboo powder induces phenotype switching, which might contribute in part to the protection of the brine shrimp larvae against AHPND-causing V. parahaemolyticus.

Antibacterial spectrum of synthetic herbal-based polyphenols against Vibrio parahaemolyticus isolated from diseased Pacific whiteleg shrimp (Penaeus vannamei) in Thailand

Acute hepatopancreatic necrosis disease (AHPND) was first reported as a new shrimp disease in 2009, which caused serious disease in shrimp culture with global economic losses and high rates of mortality. Vibrio parahaemolyticus (VP) was recently identified as the leading causative agent of AHPND that contains Pir toxin bearing plasmid coding toxins (PirA and PirB). Classic antibiotic treatments have been used for the control of infections, however, the frequent use of antibiotics has led to the emergence of antibiotic-resistant bacterial strains of public health concern. The present study aimed to investigate the potential use of synthetic herbal-based polyphenols compounds as natural, eco-friendly antimicrobial agents for disease control. Polyphenols and their active ingredients were investigated against 96 VP isolates retrieved from Pacific whiteleg shrimp (Penaeus vannamei) in Thailand and compared with various commercial antibiotics using a broth microdilution method, time-kill assay, and scanning electron microscope (SEM). All VP isolates were identified using conventional bacteriology and molecular-based tools. Bacteriological examination revealed that all isolates harbored toxR gene with specific amplicons of 368 bp. Only 56 isolates were positive to AP3, a toxin-encoding gene of AHPND-VP with a fragment size of 336 bp. In terms of susceptibility, all isolates were entirely resistant to ampicillin and amoxicillin, whereas they showed different susceptibility patterns to the other tested antibiotics. Among the polyphenols, there was no apparent activity of syringic acid and rutin, while vanillic acid showed higher bactericidal activity against VP isolates at MICs and MBCs of 1024–2048 μg mL−1. Pyrogallol exhibited the strongest antibacterial activity even at very low concentrations with a MIC and MBC of 32–256 μg mL−1, and the majority of cells showed disruption and an indistinguishable cell wall structure by SEM analysis. The present study offers pyrogallol as a potential synthetic herbal-based antimicrobial agent for the adoption of inventory management practices and control of V. parahemolyticus causing AHPND in shrimp producing sectors.

Comparative genomic provides insight into the virulence and genetic diversity of Vibrio parahaemolyticus associated with shrimp acute hepatopancreatic necrosis disease

Acute hepatopancreatic necrosis disease (AHPND) is an important shrimp disease of economic importance which causes mass mortality of cultivated penaeid shrimps in Southeast Asian countries, Mexico and South America. This disease was originally caused by Vibrio parahaemolyticus (VPAHPND) which is reported to harbour a transferable plasmid carrying the virulent PirAB-like toxin genes (pirABvp). However, little is known about the pathogenicity of VPAHPND. To extend our understanding, comparative genomic analyses was performed in this study to identify the genetic differences and to understand the phylogenetic relationship of VPAHPND strains. Seven Vibrio parahaemolyticus strains (five VPAHPND strains and two non-VPAHPND strains) were sequenced and 31 draft genomes of V. parahaemolyticus were retrieved from NCBI database and incorporated into the genomic comparison to elucidate their genomic diversity. The study showed that the genome sizes of the VPAHPND strains were approximately 5 Mbp. Ten sequence types (STs) were identified among the VPAHPND strains using in silico-Multilocus Sequence Typing analysis (MLST) and ST 970 was the predominant ST. Phylogenetic analysis based on MLST and single nucleotide polymorphisms (SNP) showed that the VPAHPND strains were genetically diverse. Based on the comparative genomic analysis, several functional proteins were identified from diiferent categories associated with virulence-related proteins, secretory proteins, conserved domain proteins, transporter proteins, and phage proteins. The CRISPR analysis showed that VPAHPND strains contained less number of CRISPRs elements than non-VPAHPND strains while six prophages regions were identified in the genomes, suggested the lack of CRISPR might promote prophage insertion. The genomic information in this study provide improved understanding of the virulence of these VPAHPND strains.

Horizontal Plasmid Transfer Promotes the Dissemination of Asian Acute Hepatopancreatic Necrosis Disease and Provides a Novel Mechanism for Genetic Exchange and Environmental Adaptation.

Vibrio parahaemolyticus is an important foodborne pathogen and has recently gained particular notoriety because it causes acute hepatopancreatic necrosis disease (AHPND) in shrimp, which has caused significant economic loss in the shrimp industry. Here, we report a whole-genome analysis of 233 V. parahaemolyticus strains isolated from humans, diseased shrimp, and environmental samples collected between 2008 and 2017, providing unprecedented insight into the historical spread of AHPND. The results show that V. parahaemolyticus is genetically diverse and can be divided into 84 sequence types (STs). However, genomic analysis of three STs of V. parahaemolyticus identified seven transmission routes in Asia since 1996, which promoted the transfer of an AHPND-associated plasmid. Notably, the insertion sequence (ISVal1) from the plasmid subsequently mediated the genetic exchange among V. parahaemolyticus STs and resulted in the deletion of an 11-kb region regulating cell mobility and the production of capsular polysaccharides. Phenotype assays confirmed that this deletion enhanced biofilm formation, providing a novel mechanism for environmental adaptation. We conclude that the transmission mode of AHPND consists of two steps, the transmission of V. parahaemolyticus and the subsequent horizontal transfer of the AHPND-associated plasmid. This plasmid allows ISVal1 to mediate genetic exchange and improve pathogen fitness in shrimp ponds. Current shrimp farming practices promoted such genetic exchanges, which highlighted a risk of the emergence of new virulent populations, with potentially devastating consequences for both aquaculture and human health. This study addressed the basic questions regarding the transmission mechanism of AHPND and provided novel insights into shrimp and human disease management.IMPORTANCE Global outbreaks of shrimp acute hepatopancreatic necrosis disease (AHPND) caused by V. parahaemolyticus represent an urgent issue for the shrimp industry. This study revealed that the transmission mode of AHPND consists of two steps, the transregional dissemination of V. parahaemolyticus and the horizontal transfer of an AHPND-associated plasmid. Surprisingly, the introduction of the AHPND-associated plasmid also offers a novel mechanism of genetic exchange mediated by insertion sequences, and it improved the fitness of V. parahaemolyticus in a harsh environment. The results presented herein suggest that current shrimp farming practices promote genetic mixture between endemic and oceanic V. parahaemolyticus populations, which introduced the plasmid and accelerated bacterial adaptation by the acquisition of ecologically important functions. This entails a risk of the emergence of new virulent populations both for shrimp and humans. This study improves our understanding of the global dissemination of the AHPND-associated plasmid and highlights the urgent need to improve biosecurity for shrimp farming.

Complete genome sequence analysis of the Vibrio owensii strain SH-14 isolated from shrimp with acute hepatopancreatic necrosis disease.

Vibrios are a group of very important bacterial pathogens in marine aquaculture industry and cause serious aquatic animal diseases, such as shrimp acute hepatopancreatic necrosis disease (AHPND). A new AHPND pathogen, the Vibrio owensii strain SH-14, was isolated from diseased shrimp in Shanghai, China. In this study, to better understand the pathogenesis of AHPND at the genomic level, the genome of the strain SH-14 was completely sequenced and analyzed. The SH-14 consists of two circular chromosomes of 3,689,702bp and 2,430,445bp, and of two plasmids named as pVHvo (69,148bp) and pVHvo-R (78,918bp), respectively. The pVHvo encodes the bi-toxic genes of pirAB, responsible for shrimp AHPND. The whole genomes contain a total of 5703 predicted open reading frames (ORFs), 129 tRNA genes and 37 rRNA genes. The average nucleotide identities (ANIs) between the SH-14 and the other V. owensii strains are all greater than 95%, confirming a new V. owensii strain of the SH-14. The taxonomic affiliation of the SH-14 is also supported by whole-genome alignment and nucleotide identity dotplot analyses. These results pave the way for further study of spread and epidemic of shrimp AHPND.

Nanopore long reads enable the first complete genome assembly of a Malaysian Vibrio parahaemolyticus isolate bearing the pVa plasmid associated with acute hepatopancreatic necrosis disease

Background: The genome of Vibrio parahaemolyticus MVP1, isolated from a Malaysian aquaculture farm with shrimp acute hepatopancreatic necrosis disease (AHPND), was previously sequenced using Illumina MiSeq and assembled de novo, producing a relatively fragmented assembly. Despite identifying the binary toxin genes in the MVP1 draft genome that were linked to AHPND, the toxin genes were localized on a very small contig precluding proper analysis of gene neighbourhood. Methods: The genome of MVP1 was sequenced on Nanopore MinION to obtain long reads to improve genome contiguity. De novo genome assembly was performed using long-read only assembler followed by genome polishing and hybrid assembler. Results: Long-read assembly produced three complete circular MVP1 contigs: chromosome 1, chromosome 2 and the pVa plasmid encoding pirABvp binary toxin genes. Polishing of the long-read assembly with Illumina short reads was necessary to remove indel errors. Complete assembly of the pVa plasmid could not be achieved using Illumina reads due to identical repetitive elements flanking the binary toxin genes leading to multiple contigs. These regions were fully spanned by the Nanopore long-reads resulting in a single contig. Alignment of Illumina reads to the complete genome assembly indicated there is sequencing bias as read depth was lowest in low-GC genomic regions. Comparative genomic analysis revealed a gene cluster coding for additional insecticidal toxins in chromosome 2 of MVP1 that may further contribute to host pathogenesis pending functional validation. Scanning of publicly available V. parahaemolyticus genomes revealed the presence of a single AinS-family quorum-sensing system that can be targeted for future microbial management. Conclusions: We generated the first chromosome-scale genome assembly of a Malaysian pirABVp-bearing V. parahaemolyticus isolate. Structural variations identified from comparative genomic analysis provide new insights into the genomic features of V. parahaemolyticus MVP1 that may be associated with host colonization and pathogenicity.

Genomic and evolutionary features of two AHPND positive Vibrio parahaemolyticus strains isolated from shrimp (Penaeus monodon) of south-west Bangladesh

BackgroundDue to its rapid lethal effect in the early development stage of shrimp, acute hepatopancreatic necrosis disease (AHPND) has been causing great economic losses, since its first outbreak in southeast China in 2009. Vibrio parahaemolyticus, carrying the pirA and pirB toxin genes is known to cause AHPND in shrimp. The overall objective of this study was to sequence the whole genome of AHPND positive V. parahaemolyticus strains isolated from shrimp (Peneaus monodon) of the south-west region of Bangladesh in 2016 and 2017 and characterize the genomic features and emergence pattern of this marine pathogen.ResultsTwo targeted AHPND positive V. parahaemolyticus strains were confirmed using PCR with 16S rRNA, ldh, AP3 and AP4 primers. The assembled genomes of strain MSR16 and MSR17 were comprised of a total of 5,393,740 bp and 5,241,592 bp, respectively. From annotation, several virulence genes involved in chemotaxis and motility, EPS type II secretion system, Type III secretion system-1 (T3SS-1) and its secreted effectors, thermolabile hemolysin were found in both strains. Importantly, the ~ 69 kb plasmid was identified in both MSR16 and MSR17 strains containing the two toxin genes pirA and pirB. Antibiotic resistance genes were predicted against β-lactam, fluoroquinolone, tetracycline and macrolide groups in both MSR16 and MSR17 strains.ConclusionsThe findings of this research may facilitate the tracking of pathogenic and/or antibiotic-resistant V. parahaemolyticus isolates between production sites, and the identification of candidate strains for the production of vaccines as an aid to control of this devastating disease. Also, the emergence pattern of this pathogen can be highlighted to determine the characteristic differences of other strains found all over the world.

Efficacy of Rose Myrtle Rhodomyrtus tomentosa Seed Extract against Acute Hepatopancreatic Necrosis Disease in Pacific Whiteleg Shrimp Penaeus vannamei.

Acute hepatopancreatic necrosis disease (AHPND) is a new emerging bacterial disease that has been recently reported to cause mass mortalities in Pacific whiteleg shrimp Penaeus vannamei. Antibiotics have been used to treat bacterial diseases in shrimp, but most of them have been ineffective and have resulted in drug residues in the harvested shrimp products. In this study, an alternative approach was tested for its efficacy in controlling AHPND. The extract of rose myrtle Rhodomyrtus tomentosa seed, a traditional Vietnamese medicine, was tested for antibacterial effect against three AHPND bacterial strains invitro (Vibrio parahaemolyticus [VPAHPND ] KC12.020, VPAHPND KC13.14.2, and V.harveyi KC13.17.5) and was further evaluated for its potential efficacy in prevention of AHPND in shrimp invivo. The invitro studies showed that the antibacterial activity of the R.tomentosa extract was dose dependent, with the strongest bacterial susceptibility (≥18.0mm) at a concentration of around 3,500μg/disc. The invivo studies showed that after challenge with VPAHPND KC12.020, the survival rates for shrimp in the groups that received feed pellets supplemented with extract at 3.5% or 7.0% (survival ~48.9% and 52.2%, respectively) were significantly higher than the zero survival rate in the positive control group, which received feed without the extract. These results indicate that the use of the R.tomentosa extract as an alternative therapy for control of AHPND in shrimp could help to minimize disease outbreaks. As a result, the extract is further expected to reduce drug/chemical residues in shrimp products.

Multiplex SYBR Green and duplex TaqMan real-time PCR assays for the detection of Photorhabdus Insect-Related (Pir) toxin genes pirA and pirB

Acute hepatopancreatic necrosis disease (AHPND), also known as Early mortality syndrome (EMS), is a recently emerged lethal disease that has caused major economic losses in shrimp aquaculture. The etiologic agents are Vibrio spp. that carry Photorhabdus Insect-Related (Pir) toxin genes pirA and pirB. A multiplex SYBR Green real-time PCR was developed that detects pirA, pirB, and two internal control genes, the shrimp 18S rRNA and the bacterial 16S rRNA genes in a single reaction. The pirB primers amplify the 3'-end of the pirB gene allowing the detection of Vibrio spp. mutants that contain a complete deletion of pirA and the partial deletion of pirB. The assay also detects mutants that contain the entire pirA gene and the deletion of the pirB gene. Since both toxin genes are needed for disease development, this assays can distinguish between pathogenic strains of Vibrio spp. that cause AHPND in shrimp and mutants that do not cause disease. The amplicons for pirA, pirB, 18S rRNA and 16S rRNA showed easily distinguishable melting temperatures of 78.21 ± 0.18, 75.20 ± 0.20, 82.28 ± 0.34 and 85.41 ± 0.21 °C respectively. Additionally, a duplex real-time PCR assay was carried out by designing TaqMan probes for the pirA and pirB primers. The diagnostic sensitivity and specificity was compared between the SYBR Green and TaqMan assays. Both assays showed similar sensitivity with a limit of detection being 10 copies for pirA and pirB, and neither assays showed any cross reaction with other known bacterial and viral pathogens in shrimp. The high sensitivity of both assays make them suitable for the detection of low copies of the pirA and pirB genes in AHPND causing Vibrio spp. as well as for detecting non-pathogenic mutants.

PirVP genes causing AHPND identified in a new Vibrio species (Vibrio punensis) within the commensal Orientalis clade

Acute hepatopancreatic necrosis disease (AHPND) has extended rapidly, causing alarming shrimp mortalities. Initially, the only known causative agent was Vibrio parahaemolyticus carrying a plasmid coding for the mortal toxins PirVP. Recently, it has been found that the plasmid and hence the disease, could be transferred among members of the Harveyi clade. The current study performs a genomic characterization of an isolate capable of developing AHPND in shrimp. Mortality studies and molecular and histopathological analyses showed the infection capacity of the strain. Multilocus sequence analysis placed the bacteria as a member of the Orientalis clade, well known for containing commensal and even probiotic bacteria used in the shrimp industry. Further whole genome comparative analyses, including Vibrio species from the Orientalis clade, and phylogenomic metrics (TETRA, ANI and DDH) showed that the isolate belongs to a previously unidentified species, now named Vibrio punensis sp. nov. strain BA55. Our findings show that the gene transfer capacity of Vibrio species goes beyond the clade classification, demonstrating a new pathogenic capacity to a previously known commensal clade. The presence of these genes in a different Vibrio clade may contribute to the knowledge of the Vibrio pathogenesis and has major implications for the spread of emerging diseases.

Phloroglucinol-Mediated Hsp70 Production in Crustaceans: Protection against Vibrio parahaemolyticus in Artemia franciscana and Macrobrachium rosenbergii

The halophilic aquatic bacterium, Vibrio parahaemolyticus, is an important aquatic pathogen, also capable of causing acute hepatopancreatic necrosis disease (AHPND) in shrimp resulting in significant economic losses. Therefore, there is an urgent need to develop anti-infective strategies to control AHPND. The gnotobiotic Artemia model is used to establish whether a phenolic compound phloroglucinol is effective against the AHPND strain V. parahaemolyticus MO904. We found that pretreatment with phloroglucinol, at an optimum concentration (30 µM), protects axenic brine shrimp larvae against V. parahaemolyticus infection and induced heat shock protein 70 (Hsp70) production (twofolds or more) as compared with the control. We further demonstrated that the Vibrio-protective effect of phloroglucinol was caused by its prooxidant effect and is linked to the induction of Hsp70. In addition, RNA interference confirms that phloroglucinol-induced Hsp70 mediates the survival of brine shrimp larvae against V. parahaemolyticus infection. The study was validated in xenic Artemia model and in a Macrobrachium rosenbergii system. Pretreatment of xenic brine shrimp larvae (30 µM) and Macrobrachium larvae (5 µM) with phloroglucinol increases the survival of xenic brine shrimp and Macrobrachium larvae against subsequent V. parahaemolyticus challenge. Taken together, our study provides substantial evidence that the prooxidant activity of phloroglucinol induces Hsp70 production protecting brine shrimp, A. franciscana, and freshwater shrimp, M. rosenbergii, against the AHPND V. parahaemolyticus strain MO904. Probably, phloroglucinol treatment might become part of a holistic strategy to control AHPND in shrimp.

Pathogenicity and genomic characterization of Vibrio parahaemolyticus strain PB1937 causing shrimp acute hepatopancreatic necrosis disease in China

Acute hepatopancreatic necrosis disease (AHPND) outbreaks in cultured shrimps were identified in Zhangpu, China. One Vibrio parahaemolyticus strain PB1937 was isolated from the cultured shrimps and was confirmed as a causative agent of the AHPND outbreak by employing Koch’s four postulates. Challenge tests with 106 cells ml−1 of strain PB1937 caused 100% mortality of shrimps, indicating it had sufficient virulence to cause the outbreak. Phylogenomic analysis revealed a clear divergence between PB1937 and 14 publicly available V. parahaemolyticus strains and divided 11 AHPND-causing strains into six genomic clusters. Prophage profiling of above strains revealed strong correlations with their genomic relationship, while Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) were almost absent in the genomes. The binary toxin gene pirABvp directly related to the development of AHPND was found in a 70-kb plasmid p1937-1 in PB1937 but was absent in a 78-kb novel plasmid p1937-2, which shared 46% sequence similarity with p1937-1. Comparative genomic analysis revealed that PB1937 has a novel truncated type VI secretion system (T6SS1) which possibly affects its antibacterial activity. In addition, three novel genomic islands were reported. The analysis of the genomes gave some clues regarding the correlation of virulence with its genomic trait for the AHPND strains.

Draft Genome Sequence of Vibrio parahaemolyticus Strain M1-1, Which Causes Acute Hepatopancreatic Necrosis Disease in Shrimp in Vietnam.

ABSTRACTWe report here the genome sequence of Vibrio parahaemolyticus strain M1-1, which causes a mild form of shrimp acute hepatopancreatic necrosis disease (AHPND). Compared to other virulent strains, the M1-1 genome appeared to express several additional genes, while some genes were missing. These instabilities may be related to the reduced virulence of M1-1.

The Infection of Vibrio parahaemolyticus in Shrimp and Human

Vibrio parahaemolyticus 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 V. parahemolyticus 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 Litopenaeus vannamei, Penaeus monodon, and P. chinensis. V. parahaemolyticus 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 PirAvp and PirBvp are the toxins associated with AHPND in shrimp. The genes that encode the toxin are used as targets to diagnose V. parahaemolyticuspathogens molecularly. Until now the treatment of V. parahaemolyticus infection is using antibiotics and fluid therapy, but there were V. parahaemolyticus 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 V. parahaemolyticus infection.