Acute Hepatopancreatic Necrosis Disease Outbreaks Research Articles

Dietary montmorillonite clay improved Penaeus vannamei survival from acute hepatopancreatic necrosis disease and modulated stomach microbiota

Acute hepatopancreatic necrosis disease (AHPND) is causing a bacterial pandemic in shrimp farming. AHPND is caused by pathogenic strains of Vibrio parahaemolyticus (VPAHPND) that produces the bacterial toxins, PirA and PirB. Shrimp exposure to AHPND-causing bacteria is hypothesized to be through the oral route, ingested into the gut where the bacteria initially colonized the stomach and then releases the binary toxins to damage the hepatopancreas. In view of changing global regulatory controls on the use of antibiotics, there is much interest in the development of functional aquafeeds. One innovative method to mitigate AHPND is to bind the bacterial toxins by adding adsorbent clay minerals into shrimp feeds. A feeding trial using triplicate groups of Penaeus vannamei post-larvae (PL30) was conducted to evaluate the efficacy of a montmorillonite (MMT) clay [Calibrin®-Z (CL)] on growth, gut health and disease resistance to AHPND. The addition of 0.25% or 0.50% CL did not significantly (P > 0.05) impact growth and feed utilization efficiency compared to control groups fed diets with 0% CL. When challenged with VPAHPND, survival of shrimp fed 0.25% or 0.50% CL were 83.3 ± 5.5% and 93.8 ± 0.1%, respectively, and were significantly higher (P < 0.05) compared to the VPAHPND-challenged positive control group (39.6 ± 10.4%) but not significantly different compared to the unchallenged negative control group (95.8 ± 2.1%). Vibrio and total cultivable bacteria counts in the hepatopancreas of shrimp fed CL-added diets were significantly lower compared to positive control. Hepatopancreas histopathology of infected shrimp fed CL-added diets showed less damage. During AHPND infection, bacterial diversity was depressed but dietary CL tended to restore stomach bacterial richness and α-diversity index. Dietary CL modulated the stomach bacterial community possibly with beneficial impact to shrimp survival. At the phylum level, Proteobacteria and Bacteroidetes were the main groups but the relative abundance of Verrucomicrobia, Acidobacteria and Firmicutes was more prevalent in the microbiota of shrimp fed CL-added diets. Bacteria of the genus Pseudoalteromonas, Tenacibaculum, and Marinimicrobium were identified as members of the healthy microbiota and Lysobacter was observed to be relatively enriched in AHPND-infected shrimp. Demequina was identified as a potential biomarker and was significantly enriched in the stomach of the CL-added groups. This is the first report on the effectiveness of dietary clay mineral in AHPND mitigation. At least 0.25% CL is suggested to be included in commercial shrimp feeds to mitigate against production and economic losses from AHPND outbreaks in shrimp farms.

Vibrio load and percentage composition of sucrose‐fermenting vibrios in Nile tilapia ( Oreochromis niloticus ) cultured in brackish water earthen ponds and biocontrol potential of recycled tilapia‐conditioned greenwater against population growth of V. parahaemolyticus and non‐sucrose‐fermenting vibrios in the grow‐out culture of white leg shrimp ( Litopenaeus vannamei )

Vibrio are known opportunistic pathogens of pond-cultured fish and shrimp. This study determined the presumptive Vibrio count (PVC) and percentage composition of “beneficial” sucrose-fermenting vibrios (SFVs) and “pathogenic” non-SFVs in the water, sediment, gills and intestines of healthy tilapia Oreochromis niloticus collected every 2 weeks from Days 30 to 120 post-stocking in brackish water earthen ponds. PVCs in water and sediment ranged from 102–104 CFU/ml/CFU/g while in tilapias' gills and intestines, PVCs ranged from 103–106 CFU/g and 102–106 CFU/g, respectively, with SFVs dominating in water, sediment, gills (≥50%), and intestines (≥80%) of tilapia. Because use of tilapia-conditioned green water (TCGW) for shrimp's grow-out culture has been reported effective against acute hepatopancreatic necrosis disease outbreaks caused by V. parahaemolyticus, PVC, SFVs/non-SFVs (%), and V. parahaemolyticus count (VPC) in the recycled TCGW and Litopenaeus vannamei's hepatopancreas were also examined from stocking until 105 days of culture (DOC) in earthen ponds. PVCs ranging from 102–103 CFU/ml and 103–104 CFU/g were noted in water and shrimp's hepatopancreas, respectively. While non-SFVs population in water varied, a downward trend typified by an abrupt 50% reduction was noted in hepatopancreas at DOC 42, and remained or further dropped until DOC 105. VPCs in water likewise varied (undetectable–102 CFU/ml) while in hepatopancreas, counts dropped to undetectable level from DOC 70 onwards. Current data illustrate that remarkable shift of SFVs' dominance in the water and concomitantly in shrimp's hepatopancreas, plays a pivotal role in regulating non-SFVs proliferation in shrimp's hepatopancreas via competitive exclusion.

Phylogenetic relationships and antibiotic resistance of Vibrio parahaemolyticus isolates related to acute hepatopancreatic necrosis disease in Korea

In this study, we performed genotype analysis based on multi-locus sequence typing (MLST) and antibiotic resistance of 27 pirAB-harboring Vibrio parahaemolyticus (VpAHPND) isolates recovered from white leg shrimp (Penaeus vannamei) from acute hepatopancreatic necrosis disease (AHPND) affected farms (in 2016, n = 22) and retail markets (in 2016, n = 5) of Korea. Most VpAHPND isolates were resistant to colistin, ampicillin, and streptomycin but susceptible to other antibiotics. The comparison of their antibiotic resistance to that of non-VpAHPND isolates revealed a significantly higher resistance of VpAHPND isolates to streptomycin. No differences, however, were observed in the minimal inhibitory concentration median (MIC50), 90th percentile (MIC90), and average multiple antibiotic resistance (MAR) index between the two groups. In MLST analysis, sequence types could not be defined for 19 isolates owing to the failure of recA gene sequencing. Seven new STs were found in five and two isolates from retail markets and AHPND affected farms, respectively. Based on the MLST-based phylogenetic tree, all isolates were divided into eight clades; 22 isolates from AHPND affected farms were grouped into three clades, and five isolates from retail markets formed singleton clades. These data suggest that most AHPND outbreaks in Korea may be related to VpAHPND isolates originating from a common clone through the acquisition of pirAB. Further, the detection of VpAHPND from retail shrimp samples suggests that the pathogen may be widely distributed in shrimp aquaculture and/or the environment of Korea.

Risk factors associated with acute hepatopancreatic necrosis disease at shrimp farm level in Bac Lieu Province, Vietnam.

Background and Aim:Acute hepatopancreatic necrosis disease (AHPND) is a severe disease in shrimp farms and adversely affected the shrimp industry of Vietnam. So far, the study on risk factors associated with AHPND outbreaks is limited. The objective of this study was to determine the potential risk factors of AHPND at the shrimp farm level in Bac Lieu Province, Vietnam.Materials and Methods:Real-time-Polymerase chain reaction was used to analyze data collected from an active surveillance program of shrimp farms in 2017 in the Vinh Tien and Vinh Lac villages, Vinh Thinh commune, Hoa Binh district in Bac Lieu Province, Vietnam. The matched case-control study selected 20 cases and 20 control farms from 134 shrimp farms. In 2018, face-to-face interviews using structured questionnaires were conducted with the farmers of these selected farms.Results:Of the 59 studied variables, seven had p≤0.2 based on bivariate analyses. The results of multivariable analysis showed that the presence of fish-eating birds on shrimp farms was a significant association with AHPND (odds ratio=8, p=0.049).Conclusion:To reduce the effect of AHPND, farmers should apply effective methods to manage wild animals such as using a grid or net to cover the pond, combined with improved biosecurity.

Dysbiosis of the shrimp ( Penaeus monodon ) gut microbiome with AHPND outbreaks revealed by 16S rRNA metagenomics analysis

Acute hepatopancreatic necrosis disease (AHPND) or early mortality syndrome (EMS) is a severe hepatopancreatic disease of shrimp, caused mainly by Vibrio parahaemolyticus. Growing evidence has shown that gut bacterial imbalance is linked to bacterial pathogenesis. AHPND disease also imposes selective pressure on the gut microbiota, which was revealed by metagenomic analysis of 16S rRNA gene. Firstly, 46 bacterial strains and 8 V. parahaemolyticus were isolated from the intestine of AHPND-infected Penaeus monodon. Secondly, PCR detection of 4 major virulence genes was carried out in 8 V. parahaemolyticus. The results showed that pirAVp could be detected in 5 strains. Thirdly, 16S rRNA gene was used to sequence the intestinal microbiota of asymptomatic and AHPND-infected shrimp. There were significant differences in gut bacteria structure between the two groups, because pirAVp toxin alone is sufficient to change the core microbiota of infected shrimp. The microbial communities of infected shrimp were enriched with Firmicutes and Bacteroidetes, but the number of Proteobacteria was reduced compared with the healthy shrimps. However, in healthy shrimp, the core microbial abundance was opposite to that of infected shrimp. AHPND infection was positively correlated with the levels of SCFAs (butyrate and propionate) producing species such as Bacteroides, Bifidobacterium, Lactobacillus and Staphylococcus. Significant differences were found in the functional gene profile category between healthy and infected shrimp gut. These findings improve the current understanding of AHPND disease's impact on the assembly of shrimp gut microbiota.

Passive Immunization with Recombinant Antibody VLRB-PirAvp/PirBvp-Enriched Feeds against Vibrio parahaemolyticus Infection in Litopenaeus vannamei Shrimp.

The causative agent of acute hepatopancreatic necrosis disease (AHPND) is the bacterium, Vibrio parahaemolyticus, which secretes toxins into the gastrointestinal tract of its host. Vibrio parahaemolyticus toxins A and B (PirAvp/PirBvp) have been implicated in the pathogenesis of this disease, and are, therefore, the focus of studies developing treatments for AHPND. We previously produced recombinant antibodies based on the hagfish variable lymphocyte receptor B (VLRB) capable of neutralizing some viruses, suggesting that this type of antibody may have a potential application for treatment of AHPND. Here, recombinant PirAvp/PirBvp, produced using a bacterial expression system, were used as antigens to screen a hagfish VLRB cDNA library to obtain PirAvp/PirBvp-specific antibodies. A cell line secreting these antibodies was established by screening and cloning the DNA extracted from hagfish B cells. Supernatants collected from cells secreting the PirAvp/PirBvp antibodies were collected and concentrated, and used to passively immunize shrimp to neutralize the toxins PirAvp or PirBvp associated with AHPND. Briefly, 10 μg of PirAvp and PirBvp antibodies, 7C12 and 9G10, respectively, were mixed with the shrimp feed, and fed to shrimp for three days consecutive days prior to experimentally infecting the shrimp with V. parahaemolyticus (containing toxins A and B), and resulting mortalities recorded for six days. Results showed significantly higher level of survival in shrimp fed with the PirBvp-9G10 antibody (60%) compared to the group fed the PirAvp-7C12 antibody (3%) and the control group (0%). This suggests that VLRB antibodies may be a suitable alternative to immunoglobulin-based antibodies, as passive immunization treatments for effective management of AHPND outbreaks within shrimp farms.

Gastrointestinal microbiota imbalance is triggered by the enrichment of Vibrio in subadult Litopenaeus vannamei with acute hepatopancreatic necrosis disease

The common shrimp disease, acute hepatopancreatic necrosis disease (AHPND), has caused extensive damage to the shrimp farming industry due to its rapid spread and high mortality rates. However, the alterations in gastrointestinal microbiota (GM) ecosystem in subadult shrimps (Litopenaeus vannamei) with AHPND occurrence are still unclear. This study identified AHPND according to clinical symptoms and the existence of the toxin gene pirAVP, analyzed the microbiome dynamics of healthy and diseased subadult shrimps, and built an ecological model for quantitative evaluation of the GM imbalance. The results showed that the GM community diversity in diseased shrimps was significantly lower than that in healthy ones. The structural composition, dysbiosis-related indices, and assembly processes of the GM community were also evidently changed in diseased shrimps. The different operational taxonomic units (OTUs) in GM of diseased shrimps were mainly composed of Vibrio spp. with a relative abundance >90%, the majority of these were neutrally distributed in the neutral model of AHPND occurrence. Of 39 biomarkers for predicting AHPND occurrence, nine belonging to Vibrio were overrepresented in diseased shrimps, whereas biomarkers such as Rhodobacteraceae OTU3427, Candiadatus aysiosphaera OTU1041, KD4-96 OTU4963, and Rhodobacter OTU3366 were enhanced in healthy shrimps. Functional prediction showed that the gene abundances of the NOD receptor signaling pathway (p = .04), Vibrio infection (p < .01), and Vibrio pathogenic cycle function (p < .01) were significantly increased in diseased compared with healthy shrimps. Disturbed GM was closely correlated with infectious diseases and pirA gene. In contrast, stable GM was maintained by multiple bacterial taxa, which may be benefit for shrimp health through regulating immune system and environmental adaptation. These results indicated that GM imbalance caused by AHPND with the characteristics of expanded variation of GM diversity, increased dysbiosis-related indices, higher abundance of potential pathogens, and lager loss of potential beneficial bacteria or ecological functions, might further accelerate the progression of AHPND outbreaks. This study may provide a basis for understanding the etiology of AHPND from a microecological perspective in subadult shrimp.

The use of machine learning to predict acute hepatopancreatic necrosis disease (AHPND) in shrimp farmed on the east coast of the Mekong Delta of Vietnam

Predicting the outbreak of disease is essential when managing shrimp farms. Acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus is a serious disease in shrimp. It is essential that shrimp farmers on the east coast of the Mekong Delta detect the disease as early as possible, because the mortality rate can reach 100%. Here, we used machine learning to predict AHPND development based on data collected since 2010 from shrimp farms in Tra Vinh, Ben Tre, Bac Lieu, and Ca Mau provinces. We initially hypothesized that the dependent variable, AHPND, was affected by 31 independent variables, but ultimately used 15 key variables to train the models. Logistic regression, artificial neural network, decision tree, and K-nearest neighbor analyses were performed, and the accuracy of the predictions was evaluated using hold-out and cross-validation tests. Logistic regression, as the most stable algorithm, was thus used to predict AHPND outbreaks in shrimp farms.

Genomic and histopathological characteristics of Vibrio parahaemolyticus isolated from an acute hepatopancreatic necrosis disease outbreak in Pacific white shrimp (Penaeus vannamei) cultured in Korea

This report describes the detailed microbial and histological characteristics of Vibrio parahaemolyticus strains associated with acute hepatopancreatic necrosis disease (VpAHPND) that infected Pacific white shrimp (Penaeus vannamei) cultured in Korea. Shrimps showing typical symptoms of AHPND, including a whitish hepatopancreas and empty gut, were reported at two private shrimp farms in Korea in 2019. In the hepatopancreas of the field samples, sloughing of undifferentiated cells in the hepatopancreatic tubule epithelium was observed upon histopathological examination, and 2 Vibrio spp.-related toxin genes (pirA and pirB) associated with AHPND were detected in the samples. Among samples obtained from the hepatopancreas, pond water, and feces (pond sediment), the copy number of the virulent AHPND plasmid was the highest in the hepatopancreas samples, followed by that in the pond water and feces (pond sediment) samples. Two VpAHPND strains were isolated from pond water and diseased shrimp. The genome of the virulent AHPND plasmid pVp_Kor-D1–2 (GenBank accession no. CP046414.1), isolated from strain 19–021-D1, was fully sequenced. We confirmed that the Korean VpAHPND strain harbored the Asian-type AHPND plasmid, which was almost identical to the AHPND-associated V. owensii plasmid pVOWZ2 previously identified in China. Furthermore, the pathogenicity of the Korean VpAHPND strains was evaluated in laboratory bioassays, and we verified that those isolates were highly pathogenic to Pacific white shrimp, causing high cumulative mortality within 3 days.

Synergistic Antimicrobial Effect of a Seaweed-Probiotic Blend Against Acute Hepatopancreatic Necrosis Disease (AHPND)-Causing Vibrio parahaemolyticus.

The outbreak of acute hepatopancreatic necrosis disease (AHPND) has caused great economic losses to the shrimp culture sector. However, the use of antibiotics to fight this disease has resulted in negative impacts on human health and the environment. Thus, the use of natural alternatives to antibiotics may be a better solution. In this study, four Bacillus species obtained from the guts of shrimps (Fenneropenaeus penicillatus and Penaeus monodon) showed antimicrobial activity against the AHPND-causing Vibrio parahaemolyticus strain 3HP using the cross-streaking and agar spot methods. Two of the Bacillus isolates, B2 and BT, also showed good probiotic properties, exhibiting tolerance to bile, good adhesion to shrimp mucus, non-hemolytic, susceptibility to antibiotics and being safe towards hosts. Moreover, a seaweed-probiotic blend (a combination of Bacillus B2 and 20 mg/ml of the red seaweed Gracilaria sp.) exhibited synergistic in vitro inhibition against V. parahaemolyticus strain 3HP, with an observed inhibition zone of 5.0 mm. The broth co-culture experiment results further indicated that the seaweed-probiotic blend inhibited V. parahaemolyticus through competitive exclusion. The in vivo challenge trials also confirmed that this seaweed-probiotic blend significantly reduced the mortality of shrimps post-challenge with the AHPND-causing V. parahaemolyticus strain 3HP (p < 0.05) compared to the negative control (mortality rate = 13.88% vs 72.19%). Thus, this seaweed-probiotic blend may serve as an alternative to antibiotics in controlling the outbreak of AHPND.

Crossbreeding effects for White Spot Disease resistance in challenge tests and field pond performance in Pacific white shrimp Litopenaeus vannamei involving susceptible and resistance lines

The objective of this study was to estimate crossbreeding effects for individual survival time (STIM) in hours, and survival rate for the complete experimental period (SUEX) during challenges with White Spot Disease (WSD), as well as for individual survival rate (SURP), harvest weight at 130 days of age (HW130) and biomass production per seeded shrimp (BIOM) estimated as SURP × HW130, in commercial-like ponds with (+) or without (−) disease outbreaks in Pacific white shrimp (Litopenaeus vannamei) on the Northwest coast of Mexico. Data was obtained from 2014 to 2016 from a crossbreeding experiment involving a line of Ecuadorian shrimp with a history of WSD resistance (Resistance Line – RES) and a Mexican line with a high growth potential (Growth Line – GRO), obtained by selection for a higher growth rate in the absence of specific diseases. For challenge tests, family-marked animals were exposed per os to minced muscle tissue from WSD diseased animals and followed until mortality stabilized. Families and organisms assessed in the three years of the experimental challenges for RES, GRO and crossbreeds were 149 and 4,705, 196 and 6,186 and 144 and 4,807, respectively. Similar figures for field data were 149 and 11,490, 196 and 10,927 and 145 and 12,246, respectively. Both challenge and field data were analyzed by linear models to estimate crossbreeding effects and predicted averages for pure lines and crossbreeds. Significant direct line effects (P < 0.05) found for STIM and for SURP in all + ponds support the assumption that the RES line is more resistant to WSD than the GRO line. STIM was useful in ranking genetic groups for SURP in + ponds with an average correlation of 1.00 (P < 0.0001). The RES line had an average SURP 12.1 times that for the GRO line, and an average BIOM 16.3 times that for the GRO line in + ponds. Heterosis was positive only for HW130 (P < 0.05). The first cross between the GRO × RES lines had the highest HW130 in + ponds, while the GRO group had the highest HW130 in – ponds. As heterosis effects for BIOM were generally negative in + ponds, and loss in BIOM by using the RES rather than the GRO line is close to zero in - ponds, RES larvae could be categorized as the best alternative to minimize the risk of economic losses associated with WSD / Acute Hepatopancreatic Necrosis Disease outbreaks.

Evaluation of a cocktail of phages for the control of presumptive Vibrio parahaemolyticus strains associated to acute hepatopancreatic necrosis disease

Shrimp culture is a well-established and fast-growing industry that produces economic and social benefits in many countries. However, during the last years, it was severely affected by the emergence of the Early Mortality Syndrome (EMS) or Acute Hepatopancreatic Necrosis Disease (AHPND). This disease is mainly attributed to Vibrio parahaemolyticus, and currently, there is no effective cure or treatment. In this study, the use of T2A2 and VH5e bacteriophages was evaluated to control different AHPND-positive strains (presumptively identified as V. parahaemolyticus, VPAHPND) under laboratory conditions. Lytic effect of T2A2 and VH5e bacteriophages against different strains isolated from AHPND outbreaks was corroborated. In addition, the effectiveness of the mixture of both phages was tested on a brine shrimp experimental infection model using three highly virulent VPAHPND strains. It has been found that phage-treated brine shrimp had significantly higher survival percentage compared with non-treated groups (p < .001). Also, phage cocktail was found to be harmless to the organisms. These results suggest that the phage mixture is worth considering as a possible control measure for positive AHPND strains, although it is clear that further and more extensive testing is needed.

Comparative genomic analysis unravels the transmission pattern and intra-species divergence of acute hepatopancreatic necrosis disease (AHPND)-causing Vibrio parahaemolyticus strains.

Acute hepatopancreatic necrosis disease (AHPND) is a recently discovered shrimp disease that has become a severe threat to global shrimp-farming industry. The causing agents of AHPND were identified as Vibrio parahaemolyticus and other vibrios harboring a plasmid encoding binary toxins PirAvp/PirBvp. However, the epidemiological involvement of environmental vibrios in AHPND is poorly understood. In this study, with an aim to reveal the possible transmission route of AHPND-causing V. parahaemolyticus, we sequenced and analyzed the genomes of four pairs of V. parahaemolyticus strains from four representative regions of shrimp farming in China, each including one strain isolated from diseased shrimp during an AHPND outbreak and one strain isolated from sediment before AHPND outbreaks. Our results showed that all the four shrimp-isolated and three of the sediment-isolated strains encode and secret PirAvp/PirBvp toxins and, therefore, are AHPND-causing strains. In silico multilocus sequence typing and high-resolution phylogenomic analysis based on single-nucleotide polymorphisms, as well as comparison of genomic loci in association with prophages and capsular polysaccharides (CPSs) consistently pointed to a close genetic relationship between the shrimp- and sediment-isolated strains obtained from the same region. In addition, our analyses revealed that the sequences associated with prophages, CPSs, and type VI secretion system-1 are highly divergent among strains from different regions, implying that these genes may play vital roles in environmental adaptation for AHPND-causing V. parahaemolyticus and thereby be potential targets for AHPND control. Summing up, this study provides the first direct evidence regarding the transmission route of AHPND-causing V. parahaemolyticus and underscores that V. parahaemolyticus in shrimp are most likely originated from local environment. The importance of environmental disinfection measures in shrimp farming was highlighted.

POTENTIAL GROWTH INHIBITION ACTIVITY OF FECAL MATERIALS, MUCUS AND CULTURED WATER OF TILAPIA OREOCHROMIS NILOTICUS ON ACUTE HEPATOPANCREATIC NECROSIS DISEASE (AHPND) - CAUSING PATHOGEN VIBRIO PARAHAEMOLYTICUS

Shrimp farming plays a key role in economy of many countries all over the world. Unfortunately, a disease called Acute Hepatopancreatic Necrosis Disease (AHPND) or Early Mortality Syndrome (EMS) caused by Vibrio parahaemolyticus spreading from Asia to Central America costs shrimp industry billions of dollars annually. In the past few years, scientists from multi-disciplinary field collaborated to find out a solution for this disease. Until now, there are not any effective approaches to prevent and cure this disease. However, co-culturing shrimp with tilapia was carried out to limit the outbreak of AHPND in farm scale in many countries. Some previous studies also mentioned the benefits of this farming method to prevent other pathogens. The aim of this research is to determine whether intrinsic factors or cultured water of tilapia play role in inhibition of V. parahaemolyticus – pathogen causing AHPND. These factors include: mucus on tilapia gill and skin, tilapia fecal material, and microbiota or dissolved chemicals in culturing of tilapia. Anti-V. parahaemolyticus activity of tilapia (Oreochromis niloticus) fresh and overnight incubated feces and mucus were tested using agar well diffusion method. The effectiveness of feces and mucus inhibition was not clear, both of samples generated a weak inhibition on V. parahaemolyticus. Determination of V. parahaemolyticus inhibiting factor of tilapia cultured water using challenge test showed that dissolved compounds (smaller than 0.22 µm) inhibited the growth of V. parahaemolyticus. The presence of these compounds in tilapia-cultured water reduced V. parahaemolyticus to 17 times lower than that of the negative control with the seawater alone within the first three hours post challenge.

Update on early mortality syndrome/acute hepatopancreatic necrosis disease by April 2018

Outbreaks of acute hepatopancreatic necrosis disease (AHPND) have caused great economic losses to many shrimp‐producing countries in Asia since its first appearance in 2009. The causative agent was reported in 2013 as specific isolates of Vibrio parahaemolyticus (VPAHPND) that were later found to harbor a plasmid (pVA) encoding the Pir‐like binary toxin genes Pir vpA and Pir vpB. VPAHPND isolates colonize the shrimp stomach and release the binary toxins that cause massive sloughing of tubule epithelial cells followed by shrimp mortality. More recent information indicates that pVA plasmid and variants occur in many V. parahaemolyticus serotypes and also in other Vibrio species such as Vibrio campbellii, Vibrio harveyi, and Vibrio owensii. Information on such genomic and proteomic studies of different VPAHPND isolates from different countries are reviewed. A cohort study carried out in Thailand in 2014 indicated that AHPND outbreaks account for only a portion of the disease outbreaks reported by shrimp farmers as outbreaks of early mortality syndrome (EMS). It is recommended that a regional research network and surveillance program for newly emerging or re‐emerging pathogens be established to speed up the process of diagnosis and the implementation of coordinated control measures and to avoid a repeat of the EMS/AHPND scenario.

PirA & B toxins discovered in archived shrimp pathogenic Vibrio campbellii isolated long before EMS/AHPND outbreaks

Toxins PirvpA and PirvpB were first described in 2014 from Vibrio parahaemolyticus (VP) isolates discovered in 2013 as the cause of acute hepatopancreatic necrosis disease (AHPND) that emerged as a new disease of Chinese cultivated shrimp in 2009. It was subsequently reported from Thailand in 2012. By using PirvpA and PirvpB specific monoclonal antibodies (MAbs) together with reference VPAHPND isolates to screen archived bacterial cultures, we discovered 3 isolates of Vibrio (previously identified as V. harveyi) including VH 639, VH 1526 and VH Surat that were positive for both PirA and PirB toxins, even though the isolates were collected in Thailand over 10 years prior to reported Thai AHPND outbreaks. Western blot analysis revealed that the PirA/B toxins from these Vibrio isolates matched the molecular masses of the PirvpA/B toxins of previously described VPAHPND isolates. PCR analysis using VPAHPND detection methods that target the PirvpA and PirvpB toxin genes (AP4 and TUMSAT-Vp3) gave amplicons of the expected size from the 3 Vibrio isolates, and sequence analysis of the amplicons revealed 99% identity with the sequences of the PirvpA and PirvpB genes of previously described VPAHPND isolates. Pathogenicity tests performed by reverse gavage and immersion challenge tests demonstrated the pattern of AHPND pathology. Species confirmation of these 3 Vibrio isolates by multilocus sequence analysis (MLSA) of 16S rRNA, rpoD, rctB, and toxR genes revealed that they belong to V. campbellii clade. The results revealed that V. campbellii isolates capable of producing PirA and PirB toxins were present in Thailand up to 7 years before AHPND was reported from China and up to a decade before it was reported from Thailand. The plasmid or chromosomal location of the PirA and PirB toxin genes in these archived V. campbellii isolates and their phylogenetic relationship to current VPAHPND isolates are being determined.

PirA- and pirB-like gene identification in Micrococcus luteus strains in Mexico.

Acute hepatopancreatic necrosis disease (AHPND) was first reported in China in 2009 and afterwards in Mexico in 2013. AHPND is caused by Vibrio parahaemolyticus and affects Penaeus monodon and Litopenaeus vannamei shrimp cultures. The bacterium contains the pirA- and pirB-like genes in 69- to 70-Kb plasmids, which encode the toxins that produce the disease. The aim of this study was to determine whether pirA- and pirB-like genes existed in bacterial genera distinct from Vibrio before the first cases of AHPND were documented in Mexico. Two bacterial isolates were selected from shrimp farms in Nayarit in 2006 and analysed by nested-PCR to determine the presence of pirA- and pirB-like genes. The two isolates chosen did indeed show the presence of these genes, and those findings were confirmed by sequencing. Both strains matched to the bacterial species Micrococcus luteus. Results revealed two important situations: (a) the pirA- and pirB-like genes were present in a bacterial species that has not been reported previously (Micrococcus luteus); and (b) pirA- and pirB-like bacterial genes were present in Mexico before the first AHPND outbreak was reported in China.

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.

Delineating the Origins of Vibrio parahaemolyticus Isolated from Outbreaks of Acute Hepatopancreatic Necrosis Disease in Asia by the Use of Whole Genome Sequencing.

Acute hepatopancreatic necrosis disease (AHPND) is an emerging penaeid shrimp disease caused by Vibrio parahaemolyticus. Although V. parahaemolyticus has been isolated and sequenced from several Asia countries, the epidemiological links among the AHPND outbreaks in different locations remain unclear. In this study, we sequenced the genomes of nine strains isolated in China between 2014 and 2016 from four sampling sites in three provinces. Analysis of single nucleotide polymorphisms (SNPs) among the nine isolates yielded an average of 35,519 SNPs per isolate, ranging from 35,001 SNPs to 35,889 SNPs relative to the reference genome FDA_R31. To capture the genetic diversity of V. parahaemolyticus in Asia and Mexico, 93 published genomes were included in the analysis. Phylogenetic analysis divided the 102 isolates into 5 clades from I to V, with the majority belonging to Clade I and Clade II. There were at least 12 independent AHPND related clones. The results indicated that the clones recovered from AHPND affected shrimps in Asia were genomically distinct in various locations and there are no epidemiological links between Asian and Mexico outbreaks. Core genome analysis of pVA-1-like plasmid sequences from V. parahaemolyticus revealed that the AHPND-associated plasmids were also genetically diverse. Homology analysis of the publicly available microbial genomes showed that the conjugative transfer gene clusters of the plasmids in AHPND-causing strains were found in 27 V. parahaemolyticus strains and several other Vibrio sp. from 10 countries including five strains isolated prior to the first identification of AHPND outbreak, indicating that the backbone of AHPND- associated plasmid was widely distributed around the globe. In conclusion, at least 11 origins of AHPND outbreaks were identified; as AHPND-causing plasmid is widely distributed globally, prevention strategies for AHPND need to focus on microbial management in the aquaculture system and establishing ecological friendly aquaculture practices instead of detection of plasmid alone. However, more strains from other Asia countries as well as Mexico need to be included for whole genome sequencing (WGS) for reconstruction of the global transmission and the spread patterns of AHPND.

Genetic diversity of Vibrio parahaemolyticus strains isolated from farmed Pacific white shrimp and ambient pond water affected by acute hepatopancreatic necrosis disease outbreak in Thailand.

Acute hepatopancreatic necrosis disease (AHPND) is an emerging shrimp disease that causes massive die-offs in farmed shrimps. Recent outbreaks of AHPND in Asia have been causing great losses for shrimp culture and have become a serious socioeconomic problem. The causative agent of AHPND is Vibrio parahaemolyticus, which is typically known to cause food-borne gastroenteritis in humans. However, there have been few reports of the epidemiology of V. parahaemolyticus AHPND strains, and the genetic relationship among AHPND strains is unclear. Here, we report the genetic characterization of V. parahaemolyticus strains isolated from AHPND outbreaks in Thailand. We found eight isolates from AHPND-suspected shrimps and pond water that were positive for AHPND markers AP1 and AP2. PCR analysis confirmed that none of these eight AP-positive AHPND strains possesses the genes for the conventional virulence factors affecting to humans, such as thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH) and type III secretion system 2. Phylogenetic analysis by multilocus sequence typing showed that the AHPND strains are genetically diverse, suggesting that AHPND strains were not derived from a single genetic lineage. Our study represents the first report of molecular epidemiology of AHPND-causing V. parahaemolyticus strains using multilocus sequence typing, and provides an insight into their evolutionary mechanisms.