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, VP_AHPND) 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 VP_AHPND 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.     

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 (VP_AHPND) that were later found to harbor a plasmid (pVA) encoding the Pir‐like binary toxin genes Pir ^vpA and Pir ^vpB. VP_AHPND 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 VP_AHPND 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.     

Production of acute hepatopancreatic necrosis disease toxin is affected by addition of cell‐free supernatant prepared from AI‐2‐producing Vibrio harveyi mutant

Acute hepatopancreatic necrosis disease (AHPND) causes massive mortality in shrimp ponds within the first month poststocking. The causative agent is a specific strain of Vibrio parahaemolyticus (VP_AHPND) that has acquired the capability to produce virulent binary toxins called ToxA and ToxB. This study aims to test the effect of the addition of an autoinducer‐2‐containing cell‐free supernatant (CFS) from the mutant Vibrio harveyi (VH) on growth and toxin production of VP_AHPND. The relative AI‐2‐like activity in CFS was detected by luminescence assay. The effect of CFS (5 and 9%) on growth and toxin production of VP_AHPND was evaluated. Compared to the control culture (without CFS‐VH addition), the addition of either 5 or 9% CFS‐VH affected the growth at the initial stage of VP_AHPND. Similar growth profiles of VP_AHPND were found with the addition of CFS‐VH at both concentrations. Western blot analysis suggests that the addition of CFS‐VH affected the production of both toxins. ToxA could be detected at the early hour post‐CFS‐VH inoculation, whereas the high amount of ToxB was detected when 5% CFS‐VH was added. However, interfering with the AI‐2 function with furanone, the AI‐2 antagonist resulted in a slight delay in the production of both toxins. Results from this study will help to design a novel strategy to control AHPND in shrimp culture.     

Administration of probiotic Bacillus licheniformis induces growth,immune and antioxidant enzyme activities,gut microbiota assembly and resistance to Vibrio parahaemolyticus in Litopenaeus vannamei

This study aimed to elucidate the effects of dietary Bacillus licheniformis ATCC 11946 (BL) on the growth, immune and antioxidant activities, intestinal morphology and microbiota, and susceptibility to Vibrio parahaemolyticus (VP_AHPND) in juvenile pacific white shrimp, Litopenaeus vannamei (LV). Juvenile LV (initial weight = 0.63 ± 0.001) were fed diets containing varying BL concentrations (0 (BL0), 10⁶ (BL1), 10⁷ (BL2) and 10⁸ (BL3) CFU/g feed) for 8 weeks. Growth performance, immune and antioxidant enzyme activities, and intestinal morphology significantly improved in the probiotic‐treated groups than the untreated. Regardless of the treatment group, the two dominant phyla were Proteobacteria and Bacteroidetes, whereas the two dominant genera were Ruegeria and Vibrio. Increasing inclusion of probiotics in diets led to significant increase in beneficial bacterial genera (Ruegeria, and Pseudoalteromonas) and a significant decrease in some known opportunistic pathogens (Vibrio, Tenacibaculum, Photobacterium, Kangiella and Spongiimonas) with the BL3 group witnessing the best. A 7‐day challenge study with VP_AHPND showed significantly high protection in the probiotic‐treated groups, with the BL3, BL2 and BL1 obtaining 51%, 34% and 22% relative percentage survival, respectively. In conclusion, BL at 10⁸ CFU/g feed should be used to help in shrimp production since it attained the most significant improvement.     

Quantitative genetics of disease resistance in different groups of Penaeus semisulcatus from preliminary controlled challenge test with Vibrio parahaemolyticus the aetiological agent of acute hepatopancreatic necrosis disease (AHPND)

Acute hepatopancreatic necrosis disease (AHPND) has caused losses to shrimp farmers worldwide owing to Vibrio parahaemolyticus (VP_AHPND). Selective breeding for disease resistance of shrimp was used as an alternative strategy for disease control. Five subsamples of shrimp from 20 full-sib families were subjected to a cohabitation challenge test. Juveniles of P. semisulcatus (mean weight 2.5 ± 0.17 g) were tested for survival after challenge with VP_AHPND. Mortality in the challenge test was recorded daily, and the experiment was terminated after 96 days when the mortality ceased. Moderate heritabilities were estimated in resistance to VP_AHPND based on a linear model (LM) and threshold model (TM). This indicates that disease resistance is a heritable trait, and a challenge test may serve as a basis for selection for resistance to VP_AHPND in P. semisulcatus. However, further investigation is needed to confirm and quantify the additive genetic variation for resistance to VP_AHPND in P. semisulcatus.     

Comparison of immune responses and antioxidant status of different generations of growth‐selected Portunus trituberculatus families

The purpose of this study was to investigate the effects of inbreeding on the immune responses and antioxidant status of Portunus trituberculatus juveniles. Results showed that inbreeding affected the total haemocyte count, and phagocytic, pro‐phenoloxidase (propo), phenoloxidase and antibacterial activities decreased after the seventh generation. Antioxidant status showed a similar pattern: total antioxidant capacity, superoxide dismutase (SOD) activity and GSH/GSSG in the cell‐free haemolymph and hepatopancreas decreased, while catalase activity in the cell‐free haemolymph increased (P < 0.05). There were no significant differences in α₂‐macroglobulin and bacteriolytic activities in the cell‐free haemolymph and glutathione peroxidase activity in the cell‐free haemolymph and hepatopancreas among nine inbreeding generations. Gene expression levels of proPO and crustin in haemocytes and SOD in haemocytes and hepatopancreas also decreased significantly as the inbreeding generations increased. The results suggest that a high level of inbreeding could severely reduce the physiological health of P. trituberculatus. Our obtained data would be particularly useful for P. trituberculatus breeding programmes.     

Molecular isolation and characterization of a haemocyanin of Macrobrachium rosenbergii reveal its antibacterial activities

Haemocyanin is a multi‐subunit protein complex found in the haemolymph and is involved in the immune system of crustaceans. In this study, a haemocyanin gene of Macrobrachium rosenbergii, designated MrHc, was successfully isolated. The MrHc gene contained an open reading frame (ORF) of 1,992 nucleotides, encoding a protein of 663 amino acid residues with a molecular mass of 76.5 kDa. The deduced amino acid sequence contained distinct structural motifs of the haemocyanin superfamily, including an all‐alpha domain, a copper‐containing domain and an immunoglobulin‐like domain. Based on the phylogenetic analysis, the MrHC protein demonstrated a close relationship with the haemocyanins of Palaemon carinicauda and Macrobrachium nipponense. The MrHc gene was expressed in various shrimp tissues, including the hepatopancreas, gill, haemocytes, stomach and muscle. After Macrobrachium rosenbergii nodavirus (MrNV) challenge tests, the MrHc gene was up‐regulated 237‐fold at day 2. A recombinant protein of the MrHc immunoglobulin‐like domain exhibited antibacterial activity against Vibrio vulnificus, V. parahaemolyticus, Aeromonas caviae, A. veronii, A. hydrophila and Bacillus cereus. This study suggested that MrHc may play important roles in the shrimp innate immune response to MrNV infection and bacterial infection.     

Analysis of viral load between different tissues and rate of progression of white spot syndrome virus (WSSV) in Penaeus monodon

At present the most common and most devastating disease of shrimp is caused by the white spot syndrome virus (WSSV), which has spread throughout the world mainly by different species of crustaceans carrying the virus. After experimental injection of Penaeus monodon with a known copy number of WSSV in the abdominal muscle, the rate of viral progression in different tissues at 12, 24, 36 and 48 hpi (hours post infection) was assessed using quantitative real‐time PCR. At 12 hpi the viral load was highest in haemocytes followed by pleopod, muscle and gills whereas at 48 hpi, the gills, the main target of WSSV, showed the highest viral load followed by pleopod, muscle and haemocytes. Viral copy number in the haemocytes was the lowest beyond 12 hpi indicating a remarkable reduction in the rate of viral replication in haemocytes compared with other tissues. The viral load in haemocytes, though increased again beyond 36 hpi, never surpassed the load in the other tissues. The real‐time PCR assay with its high sensitivity and wide dynamic range make it ideal for detecting low‐level WSSV infections that can occur in apparently healthy P. monodon.     

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 PirA^VP 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.     

Dietary fulvic acid effects on survival and expression of immune‐related genes in Litopenaeus vannamei challenged with Vibrio parahaemolyticus

The effects of fulvic acid (FA) on survival and immune‐related gene expression were investigated in Litopenaeus vannamei challenged with Vibrio parahaemolyticus by immersion. Shrimp were fed with different dietary FA concentrations (1, 2, 4 and 6 g/kg feed) for 20 days (first bioassay) or 8 days (second bioassay, 2 g/kg feed of FA added every 2 days) and then challenged with V. parahaemolyticus. In a third bioassay, the expression of three immune‐related genes (translationally controlled tumour protein [TCTP], superoxide dismutase [SOD] and heat‐shock protein 70 [HSP70]) in haemocytes or hepatopancreas of experimental shrimp was measured by real‐time quantitative PCR at 0, 6, 12, 24, 48, 72 and 96 hr after FA (2 g/kg feed) administration. Fulvic acid increased survival at a concentration of 2 g/kg feed supplied every two days. Interestingly, TCTP gene expression was upregulated, whereas gene expression of SOD and HSP70 was downregulated. In conclusion, dietary fulvic acid improves survival in white shrimp challenged with V. parahaemolyticus and modulates the immune response. Therefore, FA merits further evaluation as prophylactic treatment in commercial shrimp farms.     

Anti‐PirA‐like toxin immunoglobulin (IgY) in feeds passively immunizes shrimp against acute hepatopancreatic necrosis disease

Acute hepatopancreatic necrosis disease (AHPND), caused by a toxin‐producing Vibrio parahaemolyticus strain, has become a serious threat to shrimp aquaculture. The need to regulate antibiotic use prompted the development of alternative ways to treat infections in aquaculture including the use of chicken egg yolk immunoglobulin (IgY) for passive immunization. This study evaluated the protective effect of IgY against AHPND infection in Litopenaeus vannamei (Boone). IgY was isolated from eggs laid by hens immunized with recombinant PirA‐like (rPirA) and PirB‐like (rPirB) toxins. Whole‐egg powders having IgY specific to rPirA (anti‐PirA‐IgY) and rPirB (anti‐PirB‐IgY) and IgY from non‐immunized hen (control‐IgY) were mixed with basal diets at 20% concentrations and used to prefeed shrimp 3 days before the bacterial challenge test. Survival rates of the challenged shrimp fed the anti‐PirA‐IgY, anti‐PirB‐IgY and control‐IgY diets were 86%, 14% and 0%, respectively. Only the feed containing anti‐PirA‐IgY protected shrimp against AHPND. Increasing the concentration of rPirA antigen to immunize hens and lowering the amount of egg powder in feeds to 10% consistently showed higher survival rates in shrimp fed with anti‐PirA‐IgY (87%) compared with the control (12%). These results confirm that addition of anti‐PirA‐IgY in feeds could be an effective prophylactic method against AHPND infection in shrimp.     

A3α‐peptidoglycan extracted from Bifidobacterium sp. could enhance immunological ability of Apostichopus japonicus

To assess the effects of A3α‐peptidoglycan (A3α‐PG) extracted from Bifidobacterium sp. on the immune response and disease resistance of sea cucumber, different concentrations (0, 0.5, 5 and 50 mg mL^?1) of A3α‐PG suspensions were used to perform hypodermic injection on Apostichopus japonicus, followed by a Vibrio splendidus challenge. Total coelomocyte count (TCC), phagocytosis activity and activities of four immunological enzymes in both cell‐free coelomic fluid (extra‐cellular, EC) and coelomocyte lysate supernatant (intracellular, IC), including acid phosphatase (ACP), alkaline phosphatase (ALP), superoxide dismutase (SOD) and peroxidase (POD), were measured at 2, 6, 14 and 24 h post injection (hpi). The TCC was not significantly affected (P > 0.05) by A3α‐PG, ranging from 1.84 × 10⁶ to 3.53 × 10⁶ cells mL^?1. The coelomocyte phagocytosis activity was significantly activated (P < 0.05) in all the A3α‐PG treatments, whereas no significant difference was observed between them except 24 hpi (P > 0.05). The EC‐ACP activity in the 5.0 mg mL^?1 treatment increased significantly (P < 0.05) at all sampling times, while the IC‐ACP activity in the 50 mg mL^?1 treatment increased significantly (P < 0.05) at 2 hpi. Also, the 5.0 mg mL^?1 treatment had significant (P < 0.05) increase in the EC‐ALP activity within 14 hpi and the EC‐POD activity at 2 hpi, respectively, while significantly (P < 0.05) enhanced IC‐ALP and IC‐POD activities were observed in the 50 mg mL^?1 treatment within 6 hpi and at 2 hpi, respectively. Only the 5.0 mg mL^?1 treatment showed significant (P < 0.05) increase in the EC‐SOD activity at 2 hpi and IC‐SOD activity within 14 hpi, respectively. The challenge test showed that the animals treated with 50 mg mL^?1 of A3α‐PG had notably lower cumulative mortality after 14 days following V. splendidus exposure. All together, these results suggest that A3α‐PG could positively enhance immune response that effectively promotes the health status of A. japonicus against V. splendidus infection.     

Histopathological changes in giant freshwater prawn Macrobrachium rosenbergii (de Man 1879) fed with probiotic Bacillus licheniformis upon challenge with Vibrio alginolyticus

The effect of dietary supplementation of probiotic bacterium Bacillus licheniformis on the histopathological changes in Macrobrachium rosenbergii juveniles (4.0 ± 0.02 g) challenged with known pathogenic strain of Vibrio alginolyticus are reported. Two isocaloric basal diets supplemented with probiotic bacteria B. licheniformis (1.0 × 10⁹ cfu/g feed) and other without probiotic supplementation were fed to the M. rosenbergii juveniles for 45 days. The histological observations revealed no significant changes in the hepatopancreas and gut tissues of both the experimental and the control groups which indicate that the present bacterium is a safe candidate probiont for the host. Prawns were challenged with V. alginolyticus after 45 days of feeding with probiotic diet. The histopathological studies of the hepatopancreas revealed that M. rosenbergii fed with probiotic‐supplemented diet showed less changes as compared to the prawns fed with control diet on second and fourth day of post‐experimental challenge with V. alginolyticus. Histopathological observations revealed that the gills of the prawns fed with control diet were severely affected in comparison to the prawns fed with probiotic‐supplemented diet after challenging with V. alginolyticus. Results from this study revealed the improved protection by dietary incorporation of B. licheniformis in reducing the histopathological manifestations due to V. alginolyticus infection in freshwater prawn.     

Development of a rapid immunochromatographic strip test for the detection of Vibrio parahaemolyticus toxin B that cause acute hepatopancreatic necrosis disease

Here, two monoclonal antibodies (MAbs) specific to different epitopes on ToxB, a toxin produced by Vibrio parahaemolyticus that causes acute hepatopancreatic necrosis disease (VP_AHPND), were employed to develop a rapid strip test. One MAb was conjugated to colloidal gold to bind to ToxB at the application pad, and another MAb was used to capture colloidal gold MAb–protein complexes at the test line (T) on the nitrocellulose strip. To validate test performance, a downstream control line (C) of goat anti-mouse immunoglobulin G antibody was used to capture the free colloidal gold conjugate MAb. The sample in the application buffer could be applied directly to the application well, and the test result was obtained within 15 min. The sensitivity of the kit is approximately 6.25 µg/ml of toxin, which was equivalent to the toxin produced by approximately 10⁷ cfu/ml of bacteria. This kit is convenient and easy to use since it can be used to identify VP_AHPND directly using a single colony of bacteria grown on agar culture plates. Because of its high specificity and simplicity, as well as not being reliant on sophisticated equipment or specialized skills, this strip test could be used by farmers for surveillance for ToxB-producing bacteria.     

Does Ralstonia eutropha,rich in poly‐β hydroxybutyrate (PHB), protect blue mussel larvae against pathogenic vibrios?

The natural amorphous polymer poly‐β‐hydroxybutyrate (PHB‐A: lyophilized Ralstonia eutropha containing 75% PHB) was used as a biological agent to control bacterial pathogens of blue mussel (Mytilus edulis) larvae. The larvae were supplied with PHB‐A at a concentration of 1 or 10 mg/L for 6 or 24 hr, followed by exposure to either the rifampicin‐resistant pathogen Vibrio splendidus or Vibrio coralliilyticus at a concentration of 10⁵ CFU/ml. Larvae pretreated 6 hr with PHB‐A (1 mg/L) survived a Vibrio challenge better relative to 24 hr pretreatment. After 96 hr of pathogen exposure, the survival of PHB‐A‐treated mussel larvae was 1.41‐ and 1.76‐fold higher than the non‐treated larvae when challenged with V. splendidus and V. coralliilyticus, respectively. Growth inhibition of the two pathogens at four concentrations of the monomer β‐HB (1, 5, 25 and 125 mM) was tested in vitro in LB₃₅ medium, buffered at two different pH values (pH 7 and pH 8). The highest concentration of 125 mM significantly inhibited the pathogen growth in comparison to the lower levels. The effect of β‐HB on the production of virulence factors in the tested pathogenic Vibrios revealed a variable pattern of responses.     

Identification and pathogenicity study of emerging fish pathogens Acinetobacter junii and Acinetobacter pittii recovered from a disease outbreak in Labeo catla (Hamilton, 1822) and Hypophthalmichthys molitrix (Valenciennes, 1844) of freshwater wetland in West Bengal,India

The disease outbreaks in aquaculture system of wetlands are the major cause of fish mortality. Among various bacterial septicaemic diseases, fish mortality caused by Acinetobacter spp. is recently reported in different fish species. Fish disease outbreak was investigated in a wetland of West Bengal, India to identify the aetiological factors involved. The moribund fish were examined and subjected to bacterial isolation. Two bacterial causative agents were identified as Acinetobacter junii and Acinetobacter pittii by biochemical characterization and 16S rRNA gene amplification. Both the isolates were oxidase‐negative, nitrate‐negative, catalase‐positive and indole‐negative. The molecular identification using 16S rRNA gene sequencing and phylogenetic tree analysis further confirmed the two Acinetobacter spp. with 97%–99% similarity. The antibiotic resistance patterns of these two bacteria revealed that both of them were resistant to β‐lactam, cefalexin, cephalothin, amoxyclav, cefuroxime, cefadroxil, clindamycin, vancomycin and penicillin. In addition, A. pittii was also resistant to other antibiotics of cephams group such as ceftazidime and cefotaxime. In the challenge experiment, both A. junii and A. pittii were found to be pathogenic with LD₅₀ of 1.24 × 10⁵ and 1.88 × 10⁷ cfu/fish respectively. Histopathological examination of gill, liver and kidney revealed prominent changes supporting bacterial septicaemia. The investigation reports for the first time on concurrent infection by A. junii and multidrug‐resistant (MDR)‐A. pittii as emerging fish pathogens to cause severe mortality in Labeo catla and Hypophthalmichthys molitrix in a freshwater wetland.     

Effects of dietary supplementation of probiotics on the growth,activities of digestive and non‐specific immune enzymes in hybrid grouper (Epinephelus lanceolatus ♂ × Epinephelus fuscoguttatus ♀)

Effects of Bacillus cereus BC‐01, Lactobacillus acidophilus LAG01, Clostridium butyricum CBG01 and their combinations as supplementation on the growth performance, digestive enzyme activities and serum non‐specific immunity of hybrid grouper (Epinephelus lanceolatus ♂×E. fuscoguttatus ♀) were assessed. Seven different diets, that is one control diet (basal feed without any probiotics, CT) and six treatment diets containing single B. cereus (Bs), L. acidophilus (Ls) and C. butyricum (Cs) at 1.0 × 10⁹ cfu/kg feed, and also their combinations in equal proportion at 1.0, 2.0 and 3.0 × 10⁹ cfu/kg feed (BLC1, BLC2 and BLC3) were prepared respectively. After 60‐day feeding trial, the final weight, specific growth rate,food consumption, food conversion efficiency and apparent digestibility coefficient of fish in Ls and BLC3 were significantly higher compared with the control (p < .05). The activities of pepsin and trypsin in the intestine of fish for Ls and BLC3 were significantly higher relative to the control (p < .05). Relative to controls, significantly enhanced amylase and lipase activities in proximal intestine except for Cs and BLC1 and lipase activities in distal intestine except for Cs were observed (p < .05). Meanwhile, activities of superoxide dismutase in the serum of fish for all treatments, lysozyme and catalase in Ls and BLC3, and glutathione peroxidase except for Cs were significantly enhanced (p < .05). Based on the above, dietary supplementation of single L. acidophilus at 1.0 × 10⁹ cfu/kg or combination of three strains at 3.0 × 10⁹ cfu/kg for hybrid grouper is recommended.