Early Mortality Syndrome Research Articles

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.

Supplementation of combined mannan oligosaccharide and β-glucan immunostimulants improves immunological responses and enhances resistance of Pacific Whiteleg Shrimp, Penaeus vannamei, against Vibrio parahaemolyticus infection

A feeding trial was conducted to evaluate the combined effects of mannan oligosaccharide ( MOS) + β-glucan (BZT® PRE-GE) supplementation administered at different frequencies on the immune responses and resistance of Penaeus vannamei against Vibrio parahaemolyticus, the causative agent of Early Mortality Syndrome (EMS) disease. Four experimental treatments were run with shrimps fed with a control basal diet (TCo), fed daily with diet containing 0.2 % MOS+ β-glucan (Td), fed every 3 days with diet containing 0.2 % MOS+ β-glucan (Td3) and fed every 7 days (Td7) with diet containing 0.2 % MOS+ β-glucan. Following the 30-day feeding trial, shrimp immunological responses were quantified. Infection challenge test was also conducted to evaluate the influence of the treatments on the resistance of P. vannamei against V. parahaemolyticus infection. Results indicate that the immunostimulant application frequency did not affect shrimp survival and overall growth performance. Immunostimulant application every 3 days was found optimum to enhance the immunological responses including total hemocyte count, prophenol oxidase and serum antibacterial activities. Moreover, significant improvement in survival following the infection challenge with V. parahaemolyticus was observed in treatment fed with the immunostimulant every 3 days. Overall these findings suggest that 0.2 % MOS+ β-glucan supplementation applied every 3 days could enhance the immune responses and improve the resistance of P. vannamei against Vibrio parahaemolyticus infection. Administration of 0.2 % MOS+ β-glucan once every 3 days could be a practical immunoprophylactic strategy to manage and prevent outbreaks of mass mortalities caused by V. parahaemolyticus infection in P. vannamei farming.

ESTABLISH THE INITIAL PROCEDURE FOR DETECTING VIBRIO PARAHAEMOLYTICUS IN THE SHRIMP BY ONE STEP- PCR METHOD

Early Mortality Syndrome (EMS), also known as Acute Hepatopancreas Necrosis Disease (AHPND) in shrimps, has been identified as being caused by a strain of V. parahaemolyticus. This disease has caused enormous damage to the shrimp farming industry among countries in the world in general and Vietnam in particular. One of the main reasons for uncontrollable widespread disease is the process of identifying pathogens is slow and inaccurate, leading to no promptly preventive measurement. In order to enhance the ability to briefly detect disease and improve the current disease status, in this study the PCR process using two specific 16S rRNA and ldh primers has been developed to swiftly diagnose V. parahaemolyticus pathogen in shrimps with a detection threshold of 3.5x103 CFU/ml

Effects of salinity and shaking condition on the growth and virulence of Vibrio parahaemolyticus

Vibrio parahaemolyticus is a Gram-negative halophilic bacterium that is found in estuarine, marine and coastal environment. This organism is the major causative agent of Early Mortality Syndrome (EMS) or Acute Hepatopancreatic Necrosis Disease (AHPND) which resulted in serious damages to cultured shrimp industry. Understanding the effect of environmental factors on the growth and virulence of this potential pathogen would be beneficial for preventing its outbreak. In this study, the growth and virulence of V. parahaemolyticus was examined under different salinity and shaking condition. V. parahaemolyticus XN9 was cultured in Brain Heart Infusion (BHI) medium with different sodium chloride concentrations (2.0, 2.5 and 3.0%) and different shaking conditions (0, 120 and 240 rpm). The growth of the bacterium was recorded over 8h and six extracellular enzymes of V. parahaemolyticus XN9 including caseinase, hemolysin, lecithinase, lipase, gelatinase, chitinase were investigated using agar-based method. The growth of V. parahaemolyticus was varied among different salinity and shaking conditions. It showed the best growth at 2.0% NaCl and 240 rpm. No change in the enzymatic activity (EA) of the tested extracellular enzymes was observed while changing salinity except the significant decline of gelatinase from 3.49±0.19 to 2.77±0.17 mm following salinity increase (p < 0.05). On the other hand, regarding shaking condition, lipase was the one to increase its activity significantly following the increase of shaking speed (p < 0.05). While caseinase, lecithinase, gelatinase and lipase were well expressed in V. parahaemolyticus, no hemolytic and chitinase activity was observed in any tested conditions. In summary, our study showed that 2.0 % NaCl and 240 rpm shaking promoted the best growth of V. parahaemolyticus and resulted in highest activity of gelatinase and lipase in this bacterium.

Benefits of a Biofilm System and Low Ammonia Levels to Decrees the Early Mortality Syndrome in Shrimp Larvae (Litopenaeus vannamei) Infected with (Vibrio parahaemolyticus)

The world shrimp aquaculture, has faced several problems, causing severe losses in shrimp hatcheries; between the most critical has been diseases such as early mortality syndrome (EMS) caused by (Vibrio parahaemolyticus). The EMS was initially detected in Asian countries; after, it was disseminated to Mexico and other countries. In Mexico, EMS caused severe economic losses during 2013-2016; and it has not yet been eradicated. Various causes for EMS have been reported; none is entirely accurate, but water quality is essential for successful shrimp aquaculture; therefore, the aim this work was evaluate the ammonia concentration effect on susceptibility to (EMS) on post-larvae (PL-15) shrimp (Litopenaeus vannamei) infected with (V. parahaemolyticus), using a biofilm system (water with, microalgae, dinoflagellates, protozoa and other planktonic microorganisms). So series of 5 flasks each one were arranged as following: Series S; 900 ml of filtered seawater (FSW) and 10 PL-15 shrimp per flask. Series SB; 840 ml of FSW, 60 ml of biofilm and 10 PL-15 shrimp. Series E; 900 ml of FSW, infected with 2 ml (V. parahaemolyticus) 106 CFU/ ml and 10 PL-15 shrimp. Series EN; fifteen flasks with 900 ml of FSW, 10 PL-15 shrimp, added with NH4Cl (0.535 mg/ml), to get 0.5, 1.0, 1.5, 2.0 and 2.5 mg/l final ammonia concentration in 5 sub-series of 3 flasks each one. During experiment, ammonia concentration and PL-15 shrimp mortality were evaluated in all flask. Ammonia concentration was higher in series EN than in series E; the same was observed in Series S respect to SB, but at lower values. At end of experiment, mortality in series EN was 90% Vs 60% in E. Similarly, mortality in series S was 10% Vs 0% in SB. This results confirm that the ammonia increases PL shrimp mortality, and biofilm system reduce ammonia and consequently PL-15 shrimp mortality.

Aeromonas schubertii, a novel bacterium recovered from AHPND affected farm is lethal to whiteleg shrimp, Penaeus vannamei

Retrospective diagnosis of a bacterial collection (n = 31) originated from five farms reportedly affected by early mortality syndrome (EMS) in Southeast Asia in 2016 revealed that 9/31 isolates from two farms tested positive for V. parahaemolyticus causing acute hepatopancreatic necrosis disease (VPAHPND). Molecular analysis of the 22 remaining isolates showed that 21 isolates belong to Vibrio species including VPnon-AHPND, V. vulnificus, V. cholerae, V. owensii and V. alginolyticus. One isolate from an AHPND farm was preliminarily identified as Aeromonas schubertii based on 99.43% nucleotide identity of 16S rRNA to the reference strain ATCC 43700 (X60416). Diseases caused by Vibrio bacteria have been well-studied in shrimp while pathogenic potential of non-Vibrio species has been relatively overlooked. Since the description of A. schubertii present in shrimp farms is rare, this study therefore focused on species identification and its pathogenic potential to shrimp based on a combination of multiple approaches i.e. multilocus sequence analysis (MLSA), challenge test, histopathology and in situ hybridization (ISH). Based on MLSA of 2464 bp derived from 16S rRNA (1346 bp), gyrB (568 bp) and rpoB (550 bp), this isolate was confirmed as A. schubertii. Immersion challenge using three successive 10-fold serial dilutions (2 × 104 to 2 × 106 CFU/mL) revealed that A. schubertii was pathogenic to shrimp and cumulative mortalities were dose-dependent (45–70%). The diseased shrimp exhibited gross sign of reddish body and remarkable histopathological lesion of collapsed hepatopancreatic tubules and typical encapsulation. ISH using A. schubertii-specific probe confirmed localization of bacteria in the hepatopancreas of the infected shrimp. In summary, this study reported a novel pathogenic, non-Vibrio species, A. schubertii recovered from an AHPND-affected farm causing up to 70% mortality in immersion challenge. Since A. schubertii is relatively new to shrimp, this may pose a potential risk for low salinity shrimp farming areas, active surveillance of this pathogen, therefore, should not be overlooked.

Effects of pH, temperature and oxygen-limited condition on the virulence of Vibrio parahaemolyticus

Introduction: Vibrio parahaemolyticus is a popular Gram-negative bacterium in the marine and estuarine regions. It can cause Early Mortality Syndrome (EMS), now named Acute Hepatopancreatic Necrosis Disease (AHPND), which resulted in severe losses to the shrimp culture. This study aimed to investigate the effect of pH, temperature, and oxygen-limited condition on the extracellular enzymatic activity of V. parahaemolyticus.
 Methods: V. parahaemolyticus XN9, an AHPND-causing strain, was cultured in Brain Heart Infusion (BHI) medium at different pHs (7.5, 8.0, 8.5 and 9.0), temperatures (25oC, 30oC, and 35oC) and different oxygen conditions (either 120rpm shaking or static with the presence of oxygen absorber packages). The activity of five extracellular enzymes, including caseinase, lecithinase, chitinase, gelatinase, and lipase, was assessed using the agar-based method with the corresponding media.
 Results: When pH was increased from 7.5 to 9.0, caseinase and lipase activity was decreased significantly by 88% and 44%. In contrast, gelatinase activity increased markedly from 0 to 1.38 ± 0.17 (+) mm, and lecithinase reached the highest activity, which was 2.96 ± 0.13 mm (++) at pH 8.5. Regarding effect of temperature, highest activity of caseinase (0.85 ± 0.13 mm (+)) and gelatinase (1.37 ± 0.25 mm (+)) was obtained at 350C, lecithinase at 30oC and lipase at 25oC. Regarding the effect of oxygen level, the activity of most tested enzymes decreased significantly following the decrease of oxygen level. The highest activity of caseinase, gelatinase, and lipase was observed when the bacteria were cultured and tested in a fully oxygenated condition while lecithinase showed the highest activity when the bacteria were cultured in oxygenated condition but tested in oxygen-limited condition. No chitinase activity was observed in any of the tested conditions.
 Conclusion: Our data suggested that extracellular enzymatic activity of V. parahaemolyticus is significantly influenced by environmental conditions. No particular testing condition resulted in the highest activity for all tested enzymes. However, warm temperature (30/ 35oC), mildly alkaline pH (pH 8.0), and fully oxygenated condition could increase the overall extracellular enzymatic activity of V. parahaemolyticus, thus increase its potential virulence.

Polyclonal antibody‐based farmer‐friendly flow‐through test for the detection of acute hepatopancreatic necrosis disease in shrimp

Early mortality syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND) is currently the most significant disease of shrimp in farms of Vietnam, Thailand, Malaysia, China and Mexico, and there is a great risk that it may spread to other shrimp farming countries. Although, an array of sophisticated detection tools for AHPND available, there is a need for a sensitive, simple and rapid detection method. In this study, a simple, sensitive, rapid and polyclonal antibody-based farmer-friendly flow-through assay (FTA) test has been developed for the detection of AHPND pathogen. The recombinant Photorhabdus insect-related (Pir) A toxin-like protein of AHPND pathogen was used to immunize rabbits at 21-day interval observed for highest antibody titre after third booster by ELISA. The raised rabbit antiserum was purified by affinity chromatography and characterized by Western blot. The antiserum showed no cross-reactivity with AHPND-free Vibrio parahaemolyticus, V. anguillarum, White Spot Virus (WSV), Aeromonashydrophila and Aphanomycesinvadans. This polyclonal rabbit antiserum was used to develop a farmer-friendly FTA test for the detection of AHPND pathogen. This simple FTA testis is more sensitive and could detect PirAVP toxin up to 0.121 µg/ml, compared with 0.242 µg/ml by immunodot assay. Furthermore, FTA test requires only 8–10 min for completion, compared with 3 hr by immunodot thus found to be more sensitive, specific and cost-effective. Collectively, sensitive FTA test would help shrimp farmers to take real-time management decisions, especially emergency harvest and finally be a better hope for the prevention of AHPND.

Effect of mannan oligosaccharides on the microbiota and productivity parameters of Litopenaeus vannamei shrimp under intensive cultivation in Ecuador

The white leg Litopenaeus vannamei shrimp is of importance to the eastern Pacific fisheries and aquaculture industry but suffer from diseases such as the recently emerged early mortality syndrome. Many bacterial pathogens have been identified but the L. vannamei microbiota is still poorly known. Using a next-generation sequencing (NGS) approach, this work evaluated the impact of the inclusion in the diet of mannan oligosaccharide, (MOS, 0.5% w/w), over the L. vannamei microbiota and production behavior of L. vannamei under intensive cultivation in Ecuador. The MOS supplementation lasted for 60 days, after which the shrimp in the ponds were harvested, and the production data were collected. MOS improved productivity outcomes by increasing shrimp survival by 30%. NGS revealed quantitative differences in the shrimp microbiota between MOS and control conditions. In the treatment with inclusion of dietary MOS, the predominant phylum was Actinobacteria (28%); while the control group was dominated by the phylum Proteobacteria (30%). MOS has also been linked to an increased prevalence of Lactococcus- and Verrucomicrobiaceae-like bacteria. Furthermore, under the treatment of MOS, the prevalence of potential opportunistic pathogens, like Vibrio, Aeromonas, Bergeyella and Shewanella, was negligible. This may be attributable to MOS blocking the adhesion of pathogens to the surfaces of the host tissues. Together, these findings point to the fact that the performance (survival) improvements of the dietary MOS may be linked to the impact on the microbiota, since bacterial lines with pathogenic potential towards shrimps were excluded in the gut.

Shewanella khirikhana sp. nov. - a shrimp pathogen isolated from a cultivation pond exhibiting early mortality syndrome.

SummaryEarly mortality syndrome (EMS) in cultivated shrimp is of complex aetiology. One of the causes is acute hepatopancreatic necrosis disease (AHPND) caused by unique Vibrio isolates that carry two Pirvp toxin genes, but other causes of EMS remain mostly unexplained. Here, we describe the discovery of a Shewanella isolate TH2012T from an EMS/AHPND outbreak pond and demonstrate its virulence for shrimp (the mean lethal concentration of 105 colony‐forming units per millilitre by immersion challenge) accompanied by distinctive histopathology, particularly of the ventral nerve cord and lymphoid organ but also including the digestive tract. On the basis of its complete genome sequence, multilocus phylogenetic trees, digital DNA–DNA hybridization analysis and differential phenotypic characteristics, we propose that Shewanella isolate TH2012T represents a novel species, separated sufficiently from the type strains S. litorisediminis and S. amazonensis to justify naming it Shewanella khirikhana sp. nov. Analysis of the TH2012T genome revealed no homologues of the Pirvp toxin genes but revealed a number of other potential virulence factors. It constitutes the first Shewanella isolate reported to be pathogenic to shrimp.

MicroRNA and mRNA interactions coordinate the immune response in non-lethal heat stressed Litopenaeus vannamei against AHPND-causing Vibrio parahaemolyticus

While Vibrio parahaemolyticus (VPAHPND) has been identified as the cause of early mortality syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND) in shrimp, mechanisms of host response remain unknown. Understanding these processes is important to improve farming practices because this understanding will help to develop methods to enhance shrimp immunity. Pre-treatment of shrimp with 5-minute chronic non-lethal heat stress (NLHS) for 7 days was found to significantly increase Litopenaeus vannamei survival against VPAHPND infection. To elucidate the mechanism involved, mRNA and miRNA expression profiles from the hemocyte of L. vannamei challenged with VPAHPND after NLHS with corresponding control conditions were determined by RNA-Seq. A total of 2,664 mRNAs and 41 miRNAs were differentially expressed after the NLHS treatment and VPAHPND challenge. A miRNA-mRNA regulatory network of differentially expressed miRNAs (DEMs) and differentially expressed genes (DEGs) was subsequently constructed and the interactions of DEMs in regulating the NLHS-induced immune-related pathways were identified. Transcriptomic data revealed that miRNA and mRNA interactions contribute to the modulation of NLHS-induced immune responses, such as the prophenoloxidase-activating system, hemocyte homeostasis, and antimicrobial peptide production, and these responses enhance VPAHPND resistance in L. vannamei.

Antibacterial Activity of Copper Nanoparticles-Chitosan Composite against Vibrio parahaemolyticus.

The Early Mortality Syndrome (EMS) caused by Vibrio parahaemolyticus has recently resulted in a serious loss in shrimp farms in the Mekong delta, Vietnam. Here, antibacterial activity of copper nanoparticles-chitosan composite (CuCS) against V. parahaemolyticus was investigated. Copper nanoparticles were synthesized using L-ascorbic acid as a green reducing agent and chitosan as a biopolymer matrix and stabilizing agent. The physical properties of CuCS were evaluated. Next, antibacterial activity of 2.5, 5.0, 10.0 ppm CuCS against V. parahaemolyticus inoculated in a sterilized shrimp-pond water was examined. CuCS at 2.5 ppm eliminated 91.47% and 95.26% of V. parahaemolyticus after 2 and 4 h of exposure, respectively. Complete elimination was attained following 2 h of 5.0 ppm CuCS exposure. A complete elimination of V. parahaemolyticus in a real EMS-infected shrimp-pond water was also described. This study is the first to report the antibacterial activity of CuCS against V. parahaemolyticus, an important pathogen in shrimp industry in the Mekong delta, Vietnam.

Isolation of Bacillus strains inhibition disease resistance of Acute Hepatopancreatic Necrosis Disease on the Shrimp in Can Gio, Vietnam

Recently, the outbreak and severe damage to the shrimp farming industry is Early Mortality Syndrome (EMS) or Acute Hepatopancreatic Necrosis Disease (AHPND). The selection of Bacillus strains inhibition disease resistance of the acute hepatopancreatic necrosis disease on the shrimp is a research filed that is much attention due to Bacillus subtilis is a group of beneficial bacteria that presents in the majority of biological products for aquaculture, especially for shrimp. Competitiveness of Bacillus subtilis towards the harmful bacteria firstly is the number of bacterial cells. Beside, Bacillus has capacity to create a large amount of extracellular enzymes aid digestion, antibiotics or the inhibitor has the characteristic for resistance to the strains of Vibrio Parahaemolyticus. In this work, the inhibition ability of the Bacillus strains for the AHPND on the brackish water shrimp is studied. In which, the Bacreria is isolated and filtered from shrimp farm in Can Gio province that contain the characteristic of antagonizing Vibrio parahaemolyticus, which cause AHPND on shrimp in laboratory conditions. The results show that Bacillus subtilis produced 2,5 – Diketopiperazines capable to inbihite the growth of pandemic strain of Vibrio parahaemolyticus in Landy media for hightest activity was 200 AU/mL after 24 h of culture. This research is create the possibility of using probiotics to control the pantogenic clones Vibrio parahaemolyticus that cause AHPND for brackish water shrimp farming in Can Gio province as well as other areas in Vietnam.

Microevolution of Vibrio parahaemolyticus Isolated from Clinical, Acute Hepatopancreatic Necrosis Disease Infecting Shrimps, and Aquatic Production in China.

Vibrio parahaemolyticus is the leading cause of bacteria-associated foodborne diarrheal diseases and specifically causes early mortality syndrome (EMS), which is technically known as acute hepatopancreatic necrosis disease (AHPND), a serious threat to shrimp aquaculture. To investigate the genetic and evolutionary relationships of V. parahaemolyticus in China, 184 isolates from clinical samples (VPC, n=40), AHPND-infected shrimp (VPE, n=10), and various aquatic production sources (VPF, n=134) were collected and evaluated by a multilocus sequence analysis (MLST). Furthermore, the presence of potential virulence factors (tlh, tdh, and trh) and single nucleotide polymorphisms (SNPs) in V. parahaemolyticus isolates was assessed using genomic sequencing. Analyses of virulence factors revealed that the majority of VPC isolates (97.5%) possessed the tdh and/or trh genes, while most of the VPF isolates (83.58%) did not encode hemolysin genes. Therefore, we hypothesized that the environment is a potential reservoir that promotes horizontal DNA transfer, which drives evolutionary change that, in turn, leads to the emergence of novel, potentially pathogenic strains. Phylogenetic analyses identified VPF-112 as a non-pathogenic maternal strain isolated from aquatic products and showed that it had a relatively high evolutionary status. All VPE strains and some VPC strains were grouped into several small subgroups and evenly distributed on phylogenetic trees. Anthropogenic activities and environmental selective pressure may be important factors influencing the process of transforming strains from non-pathogenic to pathogenic bacteria.

PirABVP Toxin Binds to Epithelial Cells of the Digestive Tract and Produce Pathognomonic AHPND Lesions in Germ-Free Brine Shrimp.

Acute hepatopancreatic necrosis disease (AHPND), a newly emergent farmed penaeid shrimp bacterial disease originally known as early mortality syndrome (EMS), is causing havoc in the shrimp industry. The causative agent of AHPND was found to be a specific strain of bacteria, e.g., Vibrio and Shewanella sps., that contains pVA1 plasmid (63–70 kb) encoding the binary PirAVP and PirBVP toxins. The PirABVP and toxins are the primary virulence factors of AHPND-causing bacteria that mediates AHPND and mortality in shrimp. Hence, in this study using a germ-free brine shrimp model system, we evaluated the PirABVP toxin-mediated infection process at cellular level, including toxin attachment and subsequent toxin-induced damage to the digestive tract. The results showed that, PirABVP toxin binds to epithelial cells of the digestive tract of brine shrimp larvae and produces characteristic symptoms of AHPND. In the PirABVP-challenged brine shrimp larvae, shedding or sloughing of enterocytes in the midgut and hindgut regions was regularly visualized, and the intestinal lumen was filled with moderately electron-dense cells of variable shapes and sizes. In addition, the observed cellular debris in the intestinal lumen of the digestive tract was found to be of epithelial cell origin. The detailed morphology of the digestive tract demonstrates further that the PirABVP toxin challenge produces focal to extensive necrosis and damages epithelial cells in the midgut and hindgut regions, resulting in pyknosis, cell vacuolisation, and mitochondrial and rough endoplasmic reticulum (RER) damage to different degrees. Taken together, our study provides substantial evidence that PirABVP toxins bind to the digestive tract of brine shrimp larvae and seem to be responsible for generating characteristic AHPND lesions and damaging enterocytes in the midgut and hindgut regions.

Silver Nanoparticles Synthesized with Rumex hymenosepalus: A Strategy to Combat Early Mortality Syndrome (EMS) in a Cultivated White Shrimp

Early Mortality Syndrome (EMS) or Acute Hepatopancreatic Necrosis Syndrome (AHPNS) is a disease produced by gram-negative bacteria Vibrio parahaemolyticus (V. parahaemolyticus), which has caused declines in worldwide production of a white shrimp Litopenaeus vannamei (L. vannamei). In this work, we propose the implementation of silver nanoparticles (AgNPs) synthesized with Rumex hymenosepalus (Rh) extract as an alternative on V. parahaemolyticus control. AgNPs were characterized by UV-Vis spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and selected area electron diffraction (SAED). AgNP mean sizes by DLS were 80.82±1.16 nm and sizes between 2 and 10 nm by TEM, with a zeta potential of −47.72±1.05 mV. This study evaluated AgNPs and Rh antimicrobial capacity on V. parahaemolyticus at different concentrations; the minimum inhibitory concentration (MIC) found was 25 μg/mL for AgNPs and 220 μg/mL for Rh. Additionally, were carried out time-kill curves and reactive oxygen species (ROS) generation for 1 and 4 MIC. Both concentrations (MIC) were tested for toxicity on Artemia nauplii from Artemia franciscana (A. franciscana), because nauplii were used as biocarriers for AgNPs and Rh extract on L. vannamei. Once the shrimp were treated, they were challenged with Vibrio infection and it was found that those who were treated with both agents showed greater survival than the control. V. parahaemolyticus and postlarval samples were taken from the bioassay and fixed and prepared for TEM and SEM in order to search NPs in internal structure of bacteria and the hepatopancreatic area of shrimps; AgNPs were detected in both cases. AgNPs and Rh extract show antibacterial properties on the infected shrimp with V. parahaemolyticus. The action mechanisms are interaction with the bacterial membrane and ROS generation; these effects are produced by both agents.

Diagnosis and potential treatments for acute hepatopancreatic necrosis disease (AHPND): a review.

Acute hepatopancreatic necrosis disease (AHPND) or formerly known as early mortality syndrome (EMS) is an emerging disease that has caused significant economic losses to the aquaculture industry. The primary causative agent of AHPND is Vibrio parahaemolyticus, a Gram-negative rod-shaped bacterium that has gained plasmids encoding the fatal binary toxins Pir A/Pir B that cause rapid death of the infected shrimp. In this review, the current research studies and information about AHPND in shrimps have been presented. Molecular diagnostic tools and potential treatments regarding AHPND were also included. This review also includes relevant findings which may serve as guidelines that can help for further investigation and studies on AHPND or other shrimp diseases.

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.

Complete Genome Sequence of Shewanella sp. Strain TH2012, Isolated from Shrimp in a Cultivation Pond Exhibiting Early Mortality Syndrome.

Here, we present the complete genome sequence of a Shewanella isolate, TH2012, from a shrimp pond in which shrimp exhibited early mortality syndrome (EMS)/acute hepatopancreatic necrosis disease (AHPND). The complete genome of TH2012 has a prophage-like element and a number of potential virulence factors, making TH2012 a possible contributing factor to EMS outbreaks.

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.