Pathological, Genomic and Phenotypical Characterization of Vibrio parahaemolyticus, Causative Agent of Acute Hepatopancreatic Necrosis Disease (AHPND) in Mexico (original) (raw)
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Aquaculture, 2014
The Thai Department of Fisheries (DOF), 2013 estimated that outbreaks of acute early mortality (often called early mortality syndrome or EMS) in cultivated shrimp were responsible for a 33% drop in shrimp production during the first quarter of 2013. Similar early mortality in Vietnam was ascribed to specific isolates of Vibrio parahaemolyticus that caused acute hepatopancreatic necrosis disease (AHPND) but the status of EMS/AHPND in Thailand was unclear. Here we describe the isolation and characterization of bacteria isolated from the hepatopancreas (HP) of shrimp collected from an early mortality outbreak farm in Thailand. Four independent bacterial isolates were identified as V. parahaemolyticus by BLAST analysis and by gene-specific marker detection of a lecithin dependent hemolysin (LDH) considered to be specific for the species. Immersion challenges with 3 of these and a reference isolate, obtained from China in 2010, using a previously published laboratory infection model caused very high mortality accompanied by characteristic AHPND histopathology in the shrimp HP. Tests with one of these isolates (5HP) revealed that rate of mortality was dose dependent. Using the same challenge protocol, the 4th isolate (2HP) also caused high mortality, but it was not accompanied by AHPND histopathology. Instead, it caused a different histopathology of the HP including collapsed epithelia and unique vacuolization of embryonic cells (E-cells). These results revealed the possibility of diversity in isolates of V. parahaemolyticus that may cause early mortality in shrimp cultivation ponds. Genomic and episomic DNA of these isolates and isolates of V. parahaemolyticus that cause no disease need to be compared to better understand the molecular basis of bacterial virulence in AHPND.
Applied and Environmental Microbiology, 2014
Moribund shrimp affected by acute hepatopancreatic necrosis disease (AHPND) from farms in northwestern Mexico were sampled for bacteriological and histological analysis. Bacterial isolates were molecularly identified as Vibrio parahaemolyticus by the presence of the tlh gene. The tdh-negative, trh-negative, and tlh-positive V. parahaemolyticus strains were further characterized by repetitive extragenic palindromic element-PCR (rep-PCR), and primers AP1, AP2, AP3, and AP and an ems2 IQ2000 detection kit (GeneReach, Taiwan) were used in the diagnostic tests for AHPND. The V. parahaemolyticus strains were used in immersion challenges with shrimp, and farmed and challenged shrimp presented the same clinical and pathological symptoms: lethargy, empty gut, pale and aqueous hepatopancreas, and expanded chromatophores. Using histological analysis and bacterial density count, three stages of AHNPD (initial, acute, and terminal) were identified in the affected shrimp. The pathognomonic lesions indicating severe desquamation of tubular epithelial cells of the hepatopancreas were observed in both challenged and pond-infected shrimp. The results showed that different V. parahaemolyticus strains have different virulences; some of the less virulent strains do not induce 100% mortality, and mortality rates also rise more slowly than they do for the more virulent strains. The virulence of V. parahaemolyticus strains was dose dependent, where the threshold infective density was 10 4 CFU ml ؊1 ; below that density, no mortality was observed. The AP3 primer set had the best sensitivity and specificity. Field and experimental results showed that the V. parahaemolyticus strain that causes AHPND acts as a primary pathogen for shrimp in Mexico compared with the V. parahaemolyticus strains reported to date.
Current Microbiology, 2020
Acute hepatopancreatic necrosis disease (AHPND) is a severe disease affecting recently stocked cultured shrimps. The disease is mainly caused by V. parahaemolyticus that harbors the pVA1 plasmid; this plasmid contains the pirA and pirB genes, which encode a delta-endotoxin. AHPND originated in China in 2009 and has since spread to several other Asian countries and recently to Latin America (2013). Many Asian strains have been sequenced, and their sequences are publicly accessible in scientific databases, but only four strains from Latin America have been reported. In this study, we analyzed nine pVA1-harboring V. parahaemolyticus sequences from strains isolated in Mexico along with the 38 previously available pVA1-harboring V. parahaemolyticus sequences and the reference strain RIMD 2210633. The studied sequences were clustered into three phylogenetic clades (Latin American, Malaysian, and Cosmopolitan) through pangenomic and phylogenomic analysis. The nucleotide sequence alignment of the pVA1 plasmids harbored by the Asian and Latin American strains confirmed that the main structural difference in the plasmid between the Asian and Latin American strains is the absence of the Tn3 transposon in the Asian strains; in addition, some deletions in the pirAB region were found in two of the Latin American strains. Our study represents the most robust and inclusive phylogenomic analysis of pVA1-harboring V. parahaemolyticus conducted to date and provides insight into the epidemiology of AHPND. In addition, this study highlights that disease diagnosis through the detection of the pirA and pirB genes is an inadequate approach due to the instability of these genes.
Aquaculture, 2019
Acute hepatopancreatic necrosis disease (AHPND) first emerged as a new shrimp disease in 2009 that heavily affected shrimp industry leading to global economic losses. The etiological agent was previously identified as Vibrio parahaemolyticus that carries a plasmid containing toxins (PirA and PirB). However, recent researches revealed that V. parahaemolyticus is not the only bacterial species capable of causing AHPND, thus this study screened on bacterial strains with AHPND toxins from Penaeus vannamei shrimps in Malaysia. Out of the 86 isolated total strains, 12 AHPND positive strains were arbitrarily selected and were evaluated in in vivo assay using Artemia franciscana as a model organism. All the 12 AHPND positive strains with PirA and PirB genes demonstrated significant mortalities (P < 0.05) of A. franciscana compared to the negative control. The 12 AHPND positive strains were identified using molecular methods of 16S rRNA, RctB and RpoD region amplifications to belonged to the Harveyi clade and were closely related to V. parahaemolyticus and Vibrio harveyi. Further test showed that the yellow colony V. harveyi strain BpShHep24 was found to be more virulent than the green colony V. parahaemolyticus strain BpShHep31 in shrimp P. vannamei challenge test. Histological
Dhaka University Journal of Biological Sciences, 2018
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 isol...
Aquaculture International
Acute hepatopancreatic necrosis disease (AHPND) is an emerging shrimp (Penaeus monodon) disease caused by Vibrio parahaemolyticus (VP) since 2013 in Bangladesh. The aim of this work was to evaluate a PCR and RT-PCR techniques as rapid methods for detecting V. parahaemolyticus AHPND-positive P. monodon using genetic markers. Healthy and diseased shrimp (P. monodon) samples were collected from three monitoring stations. The samples were enriched in TCBS plates and DNA extraction from the cultured bacteria. DNA quantifications, PCR amplification, RT-PCR, and gene sequencing were done for the detection of V. parahaemolyticus AHPND-positive P. monodon. The sequence of PCR amplicons showed 100% identity and significant alignment with V. parahaemolyticus. The primers used provided high specificity for V. parahaemolyticus in PCR detection compared with another Vibrio species. In the PCR, amplification resulted positive amplicons, whereas, non-AHPND isolates showed no amplicons. Neighbor-joining methods indicated that all genes evolved from a common ancestor and clades have different traits with very low genetic distance and low variability. The pairwise alignment scores of atpA, tox, blaCARB, 16S rRNA, and pirA genes were 100.0, 98.90, 98.89, 95.53, and 41.42, respectively. The RT-qPCR exposed variable expression levels for all genes in the AHPND-positive strain. Homology analysis and distance matrix exhibited all genes to have the lowest similarity and most divergence, offering the highest specificity. In this study, the expression and variability of target genes confirmed the presence of V. parahaemolyticus in all sampling sites. The results suggested that PCR amplification, RT-qPCR, and gene sequencing can be used for the rapid detection of V. parahaemolyticus in AHPND-positive P. monodon that may lead to subsequent prevention and treatment research in the future for managing this disease.
International Journal of Food Microbiology, 2007
Nine hundred cases of seafood related diarrhea were reported in the region of Puerto Montt, Chile during the austral summer of 2006. This is the continuation of the large outbreaks associated with the consumption of seafood containing the Vibrio parahaemolyticus serovar O3:K6 pandemic clonal group that arose last decade in Chile. The initial outbreaks occurred during the summer of 1998 in Antofagasta (23°39′S 70°24′W). Subsequently, outbreaks there were rare, but since 2004 outbreaks have been frequent farther south in Puerto Montt (41°29′S 72°24′W). The large outbreaks in Puerto Montt and their rarity in Antofagasta is atypical because the seawater temperature at Puerto Montt is 5°C lower than at Antofagasta and the presence of V. parahaemolyticus in seafood has been associated with higher water temperatures. To better understand the role of seafood in outbreak occurrences in these regions, we analyzed the V. parahaemolyticus populations in clinical cases and shellfish from Puerto Montt during diarrhea outbreaks in 2006 and in shellfish from Antofagasta, where no cases were observed. Enrichment culture from shellfish yielded no V. parahaemolyticus from samples from the north, but its presence was detected in 80% of the samples from the south. Grouping of the V. parahaemolyticus isolates by the fragment restriction pattern of their DNA showed that all pathogenic (tdh+) isolates obtained from Puerto Montt shellfish corresponded to the serovar O3:K6 South East Asian pandemic clone, while the non-pathogenic (tdh−) isolates corresponded to at least six discrete groups. The possible causes for the disappearance of the pandemic strain from the north and its persistence in the south are discussed.
2016
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 LC 50 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 capa...
Pertanika Journal of Tropical Agricultural Science, 2022
The acute hepatopancreatic necrosis disease (AHPND) epidemic from 2010 to 2013 significantly affected white shrimp (Penaeus vannamei) production in Malaysia. This study aims to determine the status of AHPND in P. vannamei culture from detecting PirA/B toxin genes in hepatopancreas tissues and isolation of Vibrio parahaemolyticus for identification of pathogenic strain from major white shrimp producing farms in Malaysia. Bacteria from the hepatopancreas organ were cultured on tryptic soy agar and identified using API® 20 NE (bioMérieux, France) for Vibrio species. Confirmation of PirA/B toxin genes in hepatopancreas and V. parahaemolyticus isolates were determined by polymerase chain reaction (PCR). Twenty-three V. parahaemolyticus isolates were identified from 7.7% of the analysed samples. Fourteen (14) (4.7%) were detected with PirA/B toxin genes from districts of Johor such as Batu Pahat (1) and Kota Tinggi (8), Alor Setar, Kedah (3), Tawau, Sabah (1), and Kuching, Sarawak (1). In...
Background Due to its rapid lethal effect in the early stage of shrimp, acute hepatopancreatic necrosis disease (AHPND) 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 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 it...