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

ABSTRACT: BACKGROUND:Due 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. RESULTS:Two 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 ?-lactam, fluoroquinolone, tetracycline and macrolide groups in both MSR16 and MSR17 strains. CONCLUSIONS:The 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.

SUBMITTER: Ahmmed S

PROVIDER: S-EPMC6889531 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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

Ahmmed Shawon S Khan Md Abdullah-Al-Kamran MA Eshik Md Mostavi Enan MME Punom Nusrat Jahan NJ Islam Abul Bashar Mir Md Khademul ABMMK Rahman Mohammad Shamsur MS

BMC microbiology 20191203 1

<h4>Background</h4>Due 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-w ...[more]

PMID: 31796006

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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

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

Publications

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

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