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Shrimp AHPND-causing plasmids encoding the PirAB toxins as mediated by pirAB-Tn903 are prevalent in various Vibrio species.

ABSTRACT: Acute hepatopancreatic necrosis disease (AHPND) is a newly emerging shrimp disease caused by pirAB toxins encoded by a plasmid found in Vibrio parahaemolyticus. The pirAB toxins are the homologs of the Photorhabdus insect-related (Pir) toxins. Here, we report the complete sequences of the AHPND-causing plasmid isolated from V. owensii, as well as those of its 11 siblings (pVH family). In addition, we also included 13 related plasmids (pVH-r family) without the pirAB genes isolated from a variety of species within the Vibrio Harveyi clade. Furthermore, the pirAB-Tn903 composite transposon was identified in pVH, and both ends of the transposon appeared to have inserted simultaneously into the ancestor plasmid at different sites. The homologue counterparts of pirAB were also detected in a non-pVH plasmid in V. campbellii. Taken together, our results provide novel insights into the acquisition and evolution of pirAB as well as related plasmids in the Vibrio Harveyi clade.

SUBMITTER: Xiao J

PROVIDER: S-EPMC5294582 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Shrimp AHPND-causing plasmids encoding the PirAB toxins as mediated by pirAB-Tn903 are prevalent in various Vibrio species.

Xiao Jinzhou J Liu Liyuan L Ke Yiyun Y Li Xiefei X Liu Yunfei Y Pan Yingjie Y Yan Shuling S Wang Yongjie Y

Scientific reports 20170207

Acute hepatopancreatic necrosis disease (AHPND) is a newly emerging shrimp disease caused by pirAB toxins encoded by a plasmid found in Vibrio parahaemolyticus. The pirAB toxins are the homologs of the Photorhabdus insect-related (Pir) toxins. Here, we report the complete sequences of the AHPND-causing plasmid isolated from V. owensii, as well as those of its 11 siblings (pVH family). In addition, we also included 13 related plasmids (pVH-r family) without the pirAB genes isolated from a variety ...[more]

PMID: 28169338

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Project description:Unique isolates of Vibrio parahaemolyticus (VPAHPND) have previously been identified as the causative agent of acute hepatopancreatic necrosis disease (AHPND) in shrimp. AHPND is characterized by massive sloughing of tubule epithelial cells of the hepatopancreas (HP), proposed to be induced by soluble toxins released from VPAHPND that colonize the shrimp stomach. Since these toxins (produced in broth culture) have been reported to cause AHPND pathology in reverse gavage bioassays with shrimp, we used ammonium sulfate precipitation to prepare protein fractions from broth cultures of VPAHPND isolates for screening by reverse gavage assays. The dialyzed 60% ammonium sulfate fraction caused high mortality within 24-48 hours post-administration, and histological analysis of the moribund shrimp showed typical massive sloughing of hepatopancreatic tubule epithelial cells characteristic of AHPND. Analysis of the active fraction by SDS-PAGE revealed two major bands at marker levels of approximately 16 kDa (ToxA) and 50 kDa (ToxB). Mass spectrometry analysis followed by MASCOT analysis revealed that both proteins had similarity to hypothetical proteins of V. parahaemolyticus M0605 (contig034 GenBank accession no. JALL01000066.1) and similarity to known binary insecticidal toxins called 'Photorhabdus insect related' proteins A and B (Pir-A and Pir-B), respectively, produced by the symbiotic, nematode bacterium Photorhabdus luminescens. In in vivo tests, it was shown that recombinant ToxA and ToxB were both required in a dose dependent manner to cause AHPND pathology, indicating further similarity to Pir-A and -B. A single-step PCR method was designed for detection of the ToxA gene and was validated using 104 bacterial isolates consisting of 51 VPAHPND isolates, 34 non-AHPND VP isolates and 19 other isolates of bacteria commonly found in shrimp ponds (including other species of Vibrio and Photobacterium). The results showed 100% specificity and sensitivity for detection of VPAHPND isolates in the test set.

Project description:BackgroundAcute hepatopancreatic necrosis disease (AHPND) is an important shrimp bacterial disease caused by some Vibrio species. The severity of the impact of this disease on aquaculture worldwide has made it necessary to develop alternatives to prophylactic antibiotics use, such as the application of probiotics. To assess the potential to use probiotics in order to limit the detrimental effects of AHNPD, we evaluated the effect of the ILI strain, a Vibrio sp. bacterium and efficient shrimp probiotic, using metabarcoding (16S rRNA gene) on the gastrointestinal microbiota of shrimp after being challenged with AHPND-causing V. parahaemolyticus.ResultsWe showed how the gastrointestinal microbiome of shrimp varied between healthy and infected organisms. Nevertheless, a challenge of working with AHPND-causing Vibrio pathogens and Vibrio-related bacteria as probiotics is the potential risk of the probiotic strain becoming pathogenic. Consequently, we evaluated whether ILI strain can acquire the plasmid pV-AHPND via horizontal transfer and further cause the disease in shrimp. Conjugation assays were performed resulting in a high frequency (70%) of colonies harboring the pv-AHPND. However, no shrimp mortality was observed when transconjugant colonies of the ILI strain were used in a challenge test using healthy shrimp. We sequenced the genome of the ILI strain and performed comparative genomics analyses using AHPND and non-AHPND Vibrio isolates. Using available phylogenetic and phylogenomics analyses, we reclassified the ILI strain as Vibrio diabolicus. In summary, this work represents an effort to study the role that probiotics play in the normal gastrointestinal shrimp microbiome and in AHPND-infected shrimp, showing that the ILI probiotic was able to control pathogenic bacterial populations in the host's gastrointestinal tract and stimulate the shrimp's survival. The identification of probiotic bacterial species that are effective in the host's colonization is important to promote animal health and prevent disease.ConclusionsThis study describes probiotic bacteria capable of controlling pathogenic populations of bacteria in the shrimp gastrointestinal tract. Our work provides new insights into the complex dynamics between shrimp and the changes in the microbiota. It also addresses the practical application of probiotics to solve problems with pathogens that cause high mortality-rate in shrimp farming around the world. Video Abstract.

Project description:Vibrio parahaemolyticus, the causative agent of Acute hepatopancreatic necrosis disease (AHPND), was discovered in 2013 as a unique isolate that produces toxins and kills penaeid shrimps in devasting nature in Bangladesh and causes severe economic losses. This research aimed to understand the expressions of immune genes in different stages of the host species, Penaeus monodon, against virulence and toxin genes upon being challenged with V. parahaemolyticus. Healthy post-larvae (PL) samples were collected from southwestern of Bangladesh from July 2021 to August 2022. The tryptic soy agar with 1.5% sodium chloride (NaCl) was used to inoculate the cells of V. parahaemolyticus, and the tryptic soy broth (TSB) with 1.5% NaCl was used to transfer the colonies. The spectrophotometry measured bacteria density. PCR, qPCR, SDS-PAGE, and Western blot measured gene expression and survivability after the immersion challenge. The 1 × 105CFU/mL of V. parahaemolyticus was used for 144 h.p.i (hours post-infection) challenge to six stages of post-larvae (PL) of P. monodon (PL20, PL25, PL30, PL35, PL40, and PL45), PL30 and PL35 showed 100% mortality by day 72 (h.p.i.) after exposure that indicated most vulnerable to V. parahaemolyticus. The expression of immune and toxic genes was confirmed by qPCR. The immune genes toll-like receptors (TLR), prophenoloxidase (ProPO), lysozyme (lyso), and penaeidin (PEN) of PL20 and PL25 of P. monodon were expressed robustly up-trends. PL30 and PL35 showed the lowest gene expression at the end of 72 (h.p.i.). At the end of the 144 (h.p.i.) exposure, the immune genes TLR, ProPO, lyso, and PEN expressed highest in PL45 than other post-larvae stages of P. monodon. The toxic genes (pirA, ToxR, ToxA, ToxB, tlh, tdh, and trh) in PL30 and PL35 of P. monodon after exposure of V. parahaemolyticus were expressed highest at the end of the 72 (h.p.i.). The lowest toxic genes expressions were revealed in PL20 and PL45 at the end of the 144 (h.p.i.). The SDS-PAGE analysis of proteins from the bacterium revealed identical protein profiles with toxic genes, and those toxins were further confirmed by Western blot. The 20 kDa, 78 kDa (ToxR), 20 kDa, 25 kDa (ToxA), 25 kDa (ToxB), 20 kDa, 27 kDa, 75 kDa (tdh), and 20 kDa, 27 kDa, 75 kDa, and 78 kDa (trh) proteins were strong responses in Western blot, indicating the crucial involvement of these immune-related genes in the defense and recovery of the first-line defense mechanisms during V. parahaemolyticus infection to shrimp. The all-toxic genes showed a unique homology and those derived from the common ancestor compared with V. parahaemolyticus (NCBI accession no. AP014859.1). All clades were derived with different traits with very low genetic distance, where the overall mean distance was 3.18 and showed a very uniform and homogenous pattern among the lineages. The V. parahaemolyticus infection process in different PL stages in P. monodon revealed novel insights into the immune responses. The responses may lead to the subsequent production of a DNA vaccine, enhancing shrimp health management to minimize the economic losses due to AHPND experiencing an outbreak of early mortality syndrome (EMS) toward sustainable production P. monodon (shrimp).

Project description:Vibrio parahaemolyticus is a bacterial pathogen that becomes lethal to Penaeus shrimps when acquiring the pVA1-type plasmid carrying the PirABvp genes, causing acute hepatopancreatic necrosis disease (AHPND). This disease causes significant losses across the world, with outbreaks reported in Southeast Asia, Mexico, and South America. Virulence level and mortality differences have been reported in isolates from different locations, and whether this phenomenon is caused by plasmid-related elements or genomic-related elements from the bacteria remains unclear. Here, nine genomes of South American AHPND-causing V. parahaemolyticus (VPAHPND) isolates were assembled and analyzed using a comparative genomics approach at (i) whole-genome, (ii) secretion system, and (iii) plasmid level, and then included for a phylogenomic analysis with another 86 strains. Two main results were obtained from our analyses. First, all isolates contained pVA1-type plasmids harboring the toxin coding genes, and with high similarity with the prototypical sequence of Mexican-like origin, while phylogenomic analysis showed some level of heterogeneity with discrete clusters and wide diversity compared to other available genomes. Second, although a high genomic similarity was observed, variation in virulence genes and clusters was observed, which might be relevant in the expression of the disease. Overall, our results suggest that South American pathogenic isolates are derived from various genetic lineages which appear to have acquired the plasmid through horizontal gene transfer. Furthermore, pathogenicity seems to be a multifactorial trait where the degree of virulence could be altered by the presence or variations of several virulence factors. IMPORTANCE AHPND have caused losses of over $2.6 billion to the aquaculture industry around the world due to its high mortality rate in shrimp farming. The most common etiological agent is V. parahaemolyticus strains possessing the pVA1-type plasmid carrying the PirABvp toxin. Nevertheless, complete understanding of the role of genetic elements and their impact in the virulence of this pathogen remains unclear. In this work, we analyzed nine South American AHPND-causing V. parahaemolyticus isolates at a genomic level, and assessed their evolutionary relationship with other 86 strains. We found that all our isolates were highly similar and possessed the Mexican-type plasmid, but their genomic content did not cluster with other Mexican strains, but instead were spread across all isolates. These results suggest that South American VPAHPND have different genetic backgrounds, and probably proceed from diverse geographical locations, and acquire the pVA1-type plasmid via horizontal gene transfer at different times.

Dataset Information

Shrimp AHPND-causing plasmids encoding the PirAB toxins as mediated by pirAB-Tn903 are prevalent in various Vibrio species.

Publications

Shrimp AHPND-causing plasmids encoding the PirAB toxins as mediated by pirAB-Tn903 are prevalent in various Vibrio species.

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