EFFECT OF NEGATIVE REDOX POTENTIAL OF DRY SEDIMENT ON THE INFECTIVITY OF Vibrio parahaemolyticus, GROWTH AND HEALTH STATUS OF PACIFIC WHITELEG SHRIMP

Effect of phage therapy on survival, histopathology, and water microbiota of Penaeus vannamei challenged with Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (AHPND)

The present study aimed to select the strains of Lactic Acid Bacteria (LAB) that can antagonize Vibrio parahaemolyticus and to use these strains in preventing AHPND in culture shrimp. The research contents embraced the following four things: (1) Isolating, classifying and screening LAB strains based on determinations of morphological, physiological and bio-chemical characteristics; (2) The investigations of their antagonism against V. parahaemolyticus of LAB strains by using the agar well diffusion method; (3) The effects of dietary LAB additives on the death rate and resistance of the white-leg shrimps to AHPND; (4) Identifications of the LAB strains that have great resistance to AHPND in white-leg shrimp. By using the agar well diffusion method, the findings indicated that five out of 94 isolated LAB strains showed the highest antagonizing ability against V. parahaemolyticus strain with an inhibition zone diameter oscillating from 17.5 to 18.5 mm. Also, the death rates of the white leg shrimps in the treatments on which the shrimps were challenged with AHPND strain and fed with the LAB1, LAB2 and LAB5 supplemented feed, were significantly lower than that in the positive control treatment (54.43%), but not significantly higher than that in the negative control treatment (12.23%) in the period of 16 days after treatment. However, the histological images of shrimp hepatopancreas indicated that the infected rate by V. parahaemolyticus significantly reduced from 60.0% to 11.1% in shrimps fed with LAB-supplemented feed as compared to those in the positive control treatment (shrimps were challenged with the harmful bacteria without feeding with LAB-supplement feed). To confirm the identification of above five the best LAB strains, sequencing 16S rRNA gene was conducted. The result showed that LAB1 strain was Lactobacillus plantarum, LAB2 strain was Pediocococcus pentosaceus 1, LAB3 strain was L. garvieae, LAB4 strain was P. pentosaceus 2 and LAB5 strain was L. fermentum.

Acute hepatopancreatic necrosis disease (AHPND) has recently emerged as a serious disease of cultured shrimp. A total of 19 lactic acid bacteria (LAB) strains isolated from shrimp samples were characterized based on morphological characteristics, biochemical tests, sequencing analysis, and their ability to antagonize Vibrio parahaemolyticus, which causes AHPND in whiteleg shrimp. Results from the agar well diffusion method indicated that 3 out of 19 isolated LAB strains showed the highest antagonizing ability against AHPND V. parahaemolyticus strain with an inhibition zone diameter ranging from 18 to 20 mm. Experiments where shrimps were given feed supplemented with these LAB strains and challenged with AHPND strain showed high survival rates (approximately 80.0%), which were not significantly different as compared to those recorded in the negative control treatment (86.6%), but significantly different to those recorded in the positive control treatment (40.6%) after 16 days of the experiment. However, the histological images of shrimp hepatopancreas indicated that the infection rate significantly reduced from 60.0% to 11.1% in shrimps fed with LAB-supplemented feeds and challenged with AHPND V. parahaemolyticus strain as compared to those in the positive control treatment. A polymerase chain reaction (PCR) and 16S rRNA gene sequencing confirmed the identification of LAB strain. These results can be applied in further experiments to investigate the ability of L. plantarum in preventing AHPND in intensively cultured whiteleg shrimp.

Acute hepatopancreatic necrosis disease (AHPND) is caused by PirAB toxin-producing Vibrio parahaemolyticus and has devastated the global shrimp aquaculture industry. One approach for preventing the growth of AHPND-producing Vibrio spp. is through the application of beneficial bacteria capable of inhibiting these pathogens. In this study, we focused on the inhibitory activity of Bacillus inaquosorum strain T1, which hinders V. parahaemolyticus growth in coculture experiments in a density-dependent manner; inhibition was also observed using cell-free supernatants from T1 stationary-phase cultures. Using mariner-based transposon mutagenesis, 17 mutants having a complete or partial loss of inhibitory activity were identified. Of those displaying a total loss of activity, 13 had insertions within a 42.6-kb DNA region comprising 15 genes whose deduced products were homologous to nonribosomal polypeptide synthetases (NRPSs), polyketide synthases (PKSs), and related activities, which were mapped as one transcriptional unit. Mutants with partial activity contained insertions in spo0A and oppA, indicating stationary-phase control. The levels of expression of NRPS and PKS lacZ transcriptional fusions were negligible during growth and were the highest during early stationary phase. Inactivation of sigH resulted in a loss of inhibitor activity, indicating a role for σH in transcription. Disruption of abrB resulted in NRPS and PKS gene overexpression during growth as well as enhanced growth inhibition. Our characterization of the expression and control of an NRPS-PKS gene cluster in B. inaquosorum T1 provides an understanding of the factors involved in inhibitor production, enabling this strain's development for use as a tool against AHPND-causing Vibrio pathogens in shrimp aquaculture.IMPORTANCE The shrimp aquaculture industry has been significantly impacted by acute hepatopancreatic necrosis disease (AHPND), resulting in significant financial losses annually. AHPND is caused by strains of the bacterial pathogen Vibrio parahaemolyticus, and treatment of AHPND involves the use of antibiotics, which leads to a rise in the number of antibiotic-resistant strains. Alternative treatments include the application of beneficial microorganisms having inhibitory activities against pathogens causing AHPND. In this study, we examined the ability of Bacillus inaquosorum strain T1 to inhibit the growth of an AHPND-causing Vibrio strain, and we show that this activity involves a gene cluster associated with antibacterial compound production. We found that gene expression is under stationary-phase control and that enhanced activity occurs upon inactivation of a global transition state regulator. Our approach for understanding the factors involved in producing B. inaquosorum strain T1 inhibitory activity will allow for the development of this strain as a tool for AHPND prevention and treatment.

Acute hepatopancreatic necrosis disease (AHPND) is caused by PirAB toxin-producing Vibrio parahaemolyticus and has devastated the global shrimp aquaculture industry. One approach for preventing growth of AHPND-producing Vibrio spp. is through the application of beneficial bacteria capable of inhibiting these pathogens. In this study we focus on the inhibitory activity of Bacillus inaquosorum strain T1, which hinders V. parahaemolyticus growth in co-culture experiments in a density-dependent manner; inhibition was also observed using cell-free supernatants from T1 stationary phase cultures. Using mariner-based transposon mutagenesis, 17 mutants were identified having complete or partial loss of inhibitory activity. Of those displaying total loss of activity, 13 had insertions within a 42.6 kb DNA region comprising 15 genes whose deduced products were homologous to non-ribosomal polypeptide synthetases (NRPSs), polyketide synthases (PKSs) and related activities, which were mapped as one transcriptional unit. Mutants with partial activity contained insertions in spo0A and oppA, indicating stationary phase control. Expression of NRPS and PKS lacZ transcriptional fusions were negligible during growth and highest during early stationary phase. Inactivation of sigH resulted in loss of inhibitor activity, indicating a role for σH in transcription. Disruption of abrB resulted in NRPS and PKS gene overexpression during growth as well as enhanced growth inhibition. Our characterization of the expression and control of an NRPS-PKS gene cluster in B. inaquosorum T1 provides an understanding of factors involved in inhibitor production, enabling this strain9s development for use as a tool against AHPND Vibrio pathogens in shrimp aquaculture. IMPORTANCE The shrimp aquaculture industry has been significantly impacted by acute hepatopancreatic necrosis disease (AHPND), resulting in significant financial losses annually. Caused by strains of the bacterial pathogen, Vibrio parahaemolyticus, treatment of AHPND involves the use of antibiotics, which leads to a rise in antibiotic resistant strains. Alternative treatments include the application of beneficial microorganisms having inhibitory activities against AHPND pathogens. In this study, we examine the ability of Bacillus inaquosorum strain T1 to inhibit growth of an AHPND Vibrio strain and show that activity involves a gene cluster associated with antibacterial compound production. We found that gene expression is under stationary phase control and that enhanced activity occurred upon inactivation of a global transition state regulator. Our approach for understanding the factors involved in producing B. inaquosorum strain T1 inhibitory activity will allow for development of this strain for use as a tool for AHPND prevention and treatment.

Vibrio parahaemolyticus is one of the bacteria that can attack whiteleg shrimp and cause Acute Hepatopancreatic Necrosis Disease (AHPND) or Early Mortality Syndrome (EMS) in shrimp. One alternative that can be used is the use of seaweed-based immunostimulants, namely k-carrageenan. The purpose of this study was to evaluate the effectiveness of k-carrageenan mixed in feed with different doses on increasing the immune system of whiteleg shrimp infected with AHPND. This study was conducted for 60 days with an experimental method in the form of a Completely Randomized Design (CRD) consisting of 5 treatments and 3 replications, namely, P1 (Positive Control): Commercial Feed + Vibrio parahaemolyticus Infection; P2 (Negative Control): Commercial Feed + 0.9% NaCl Infection; P3: Commercial Feed + Kappa-carrageenan 8 g / kg + Vibrio parahaemolyticus Infection; P4: Commercial Feed + Kappa-carrageenan 10 g/kg + Vibrio parahaemolyticus Infection; P5: Commercial Feed + Kappa-carrageenan 12 g/kg + Vibrio parahaemolyticus Infection. The results of this study indicate that the addition of 12 g/kg of k-carrageenan to the feed affects the survival rate and immune system of whiteleg shrimp infected with Vibrio parahaemolyticus.

Ample evidence shows dysbiosis in the gut microbiota when comparing healthy shrimp with those affected by severe acute hepatopancreatic necrosis disease (AHPND). However, the static comparison used in available studies leads to the uncertainties regarding how and to what extent the gut microbiota responds to the progressive severity of AHPND. In addition, shrimp AHPND is featured by rapid and massive mortality, thus the initiation of AHPND must be diagnosed for preemptive therapy. For these reasons, we explored the ecological assembly of gut microbiota over shrimp AHPND progression. Increasing AHPND severity was associated with linear increase in the copies of pirAB genes, relative abundance of gut Vibrio and potentially pathogenic, and reduction in the gut bacterial diversity, stability, and relative abundance of Bdellovibrio. Negative and significant association between gut Vibrio and Bdellovibrio were noted, indicating that compromised predation exerts a role in AHPND progression. Notably, the extents of departure to the healthy shrimp gut microbiota were positively coupled with the increasing severity of AHPND. After controlling the temporal variation in the gut microbiota as healthy shrimp age, we constructed a diagnosis model that accurately diagnosed the initial, progressed or moribund stages of AHPND, with an overall accuracy of 86.5%. Shrimp AHPND induced more stochastic gut microbiotas as a consequence of the attenuated ability of diseased shrimp to select their commensals, resulting in convergent bacterial communities between gut and rearing water over AHPND progression. Collectively, our findings provide important step toward the ecological assembly of gut microbiota implicating in AHPND etiology and in diagnosing AHPND stages. KEY POINTS: • The departure of shrimp gut microbiota positively linked with AHPND severity. • The diagnosis model accurately diagnosed the stages of AHPND. • Shrimp AHPND induced more stochastic gut microbiota.

Shrimp aquaculture has been seriously affected by acute hepatopancreatic necrosis disease (AHPND), caused by a strain of Vibrio parahaemolyticus that carries the Pir toxin plasmids (V. parahaemolyticus(AHPND)). In this study, the transcription factor, Kruppel homolog 1-like of Peneaus vannamei (PvKr-h1), was significantly induced in shrimp hemocytes after V. parahaemolyticus(AHPND) challenge, suggesting that PvKr-h1 is involved in shrimp immune response. Knockdown of PvKr-h1 followed by V. parahaemolyticus(AHPND) challenge increased bacterial abundance in shrimp hemolymph coupled with high shrimp mortality. Moreover, transcriptome and immunofluorescence analyses revealed that PvKr-h1 silencing followed by V. parahaemolyticus(AHPND) challenge dysregulated the expression of several antioxidant-related enzyme genes, such as Cu-Zu SOD, GPX, and GST, and antimicrobial peptide genes, i.e., CRUs and PENs, and reduced ROS activity and nuclear translocation of Relish. These data reveal that PvKr-h1 regulates shrimps’ immune response to V. parahaemolyticus(AHPND) infection by suppressing antioxidant-related enzymes, enhancing ROS production and promoting nuclei import of PvRelish to stimulate antimicrobial peptide genes expression.

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.

Acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus has caused severe damage to the shrimp farming industry of Vietnam. Probiotics are chosen as a prophylactic method to mitigate the outbreak of diseases. We report in this article the isolation and evaluation of the effect of a potential probiotic, Bacillus subtilis BLD01, which enhances the survival rates and changes gut microbiota of whiteleg shrimp (Penaeus vannamei) after challenge with AHPND V. parahaemolyticus. After seven days of the challenge, the treatment where shrimps were fed with B. subtilis BLD01 strain (106 CFU/g) and challenge with AHPND V. parahaemolyticus (106 CFU/mL) showed high survival rates of 71% as compared to 33% in the treatment where shrimp were given standard feed without probiotics supplementation and challenged with AHPND V. parahaemolyticus. 16S rRNA amplicon data of the gut microbiomes of shrimps in four treatments were carried out using Illumina sequencing. A total of 231 436 reads were obtained, and a total of 14 phyla, 28 classes, 142 genera were revealed. The most abundant phyla in all subjects were Proteobacteria and Bacteroidetes. The sequence number of the Vibrio genus was the highest with 28% in treatment without B. subtillis BLD01 in-feed addition and shrimps challenged with AHPND V. parahaemolyticus. The sequence number of Bacillus genus was the highest with 3% in the treatment with B. subtillis BLD01 addition and without AHPND V. parahaemolyticus challenge. These results contribute to confirming the mechanism of action of B. subtilis against V. parahaemolyticus in the experimental model, creating a scientific basis for the development and use of probiotics products applied in shrimp farming in Vietnam.

Simple SummaryThis study aimed to evaluate the growth, survival rate, and resistance to Acute hepatopancreatic Necrosis Disease (AHPND) of white leg shrimp (Penaeus vannamei) by using Lactobacillus plantarum, Lactobacillus fermentum, and Pediococcus pentosaceus mixed with feed, and at the same time supplying CNP in a ratio of 15:1:0.1 to the water. The result showed that shrimps were fed with feed containing lactic acid bacteria (LAB), especially L. plantarum have an effective to increased shrimp growth, stimulated non-specific immune system such as total hemocyte cells, granulocyte cells, hyaline cells, and protected shrimp to ANPND. The supply of CNP to the water have increased the intensity of V. parahaemolyticus effects on shrimp health; significantly decreased non-specific immune parameters of shrimp by 30–50%, therefore increased the AHPND infected rate and mortality of shrimp compared with without CNP group. In summary, LAB has a good effect to shrimp and the supply of CNP had significantly reduced the shrimp’s immune response and increased the susceptibility of shrimp to AHPND in both cases of use with and without LAB-containing diets.This study aimed to evaluate the growth, survival rate, and resistance to acute hepatopancreatic necrosis disease (AHPND) of white leg shrimp (Penaeus vannamei) by using Lactobacillus plantarum, Lactobacillus fermentum, and Pediococcus pentosaceus mixed with feed, and at the same time supplying CNP in a ratio of 15:1:0.1 to the water. As a result, the treatments that shrimp were fed with feed containing lactic acid bacteria (LAB), especially L. plantarum, have increased shrimp growth, total hemocyte cells, granulocyte cells, and hyaline cells significantly (p < 0.05) in comparison to the control group. The supply of CNP to the water has promoted the intensity of V. parahaemolyticus effects on shrimp health and significantly decreased total hemocyte cells, granulocyte cells, and hyaline cells by 30–50% in the period after three days of the challenge, except in L. plantarum treatment, which had only a 20% decrease compared to other treatments. In CNP supplying treatments, the AHPND infected rate and mortality of shrimp were higher than those in other treatments. In summary, the supply of CNP had significantly reduced the shrimp’s immune response and promoted the susceptibility of shrimp to AHPND in both cases of use with and without LAB-containing diets.

Evaluation of tryptophan biomass as an alternative to conventional crystalline tryptophan in broiler diets

Integrated overview on status, diagnosis and disease management of Acute Hepatopancreatic Necrosis Disease (AHPND) in shrimp aquaculture through metallic nanoparticles (MNPs) application – A review

Simple SummaryHeat stress is a serious issue in commercial broiler production in hot and humid countries, including Malaysia. Exposure of broilers to heat stress affects their health and productivity. In this context, antibiotics are widely used at sub-therapeutic levels as growth promoters to reduce stress and infectious diseases in order to sustain productivity in commercial broiler farms. However, the extensive use of antibiotics as growth promoters for a long time leads to the development of antibiotic-resistant bacteria and the possibility of antibiotic-resistant genes being transferred among organisms. Recently, postbiotics produced by Lactobacillus plantarum have been widely studied as a feed additive in order to replace in-feed antibiotics. However, to date, no studies have investigated the role of postbiotics in feed for broilers under heat stress.The effects of feeding different postbiotics on growth performance, carcass yield, intestinal morphology, gut microbiota, immune status, and growth hormone receptor (GHR) and insulin-like growth factor 1 (IGF-1) gene expression in broilers under heat stress were assessed in this study. A total of 252 one-day-old male broiler chicks (Cobb 500) were randomly assigned in cages in identical environmentally controlled chambers. During the starter period from 1 to 21 days, all the birds were fed the same basal diet. On day 22, the birds were weighed and randomly divided into six treatment groups and exposed to cyclic high temperature at 36 ± 1 °C for 3 h per day from 11:00 to 14:00 until the end of the experiment. From day 22 to 42 (finisher period), an equal number of birds were subjected to one of the following diets: NC (negative control) basal diet; PC (positive control) basal diet + 0.02% oxytetracycline; or AA (ascorbic acid) basal diet + 0.02% ascorbic acid. The other three groups (RI11, RS5 and UL4) were basal diet + 0.3% different postbiotics (produced from different Lactobacillus plantarum strains, and defined as RI11, RS5 and UL4, respectively). The results demonstrated that birds fed RI11 diets had significantly higher final body weight, total weight gain and average daily gain than the birds that received the NC, PC and AA treatments. The feed conversion ratio was significantly higher in the RI11 group compared with the other groups. Carcass parameters were not affected by the postbiotic-supplemented diet. Postbiotic supplementation improved villi height significantly in the duodenum, jejunum and ileum compared to the NC, PC and AA treatments. The crypt depth of the duodenum and ileum was significantly higher in NC group compared to other treatment groups except RI11 in duodenum, and UL4 in ileum was not different with NC groups. The villus height to crypt depth ratio of duodenum and ileum was significantly higher for the postbiotic treatment groups and AA than the PC and NC treatment groups. The postbiotic RI11 group recorded significantly higher caecum total bacteria and Lactobacillus count and lower Salmonella count compared to the NC and PC treatment groups. The Bifidobacterium population in the NC group was significantly lower compared to the other treatment groups. The postbiotic (RI11, RS5 and UL4) and AA treatment groups showed lower Enterobacteriaceae and E. coli counts and caecal pH than the NC and PC treatment groups. The plasma immunoglobulin M (IgM) level was significantly higher in the birds receiving postbiotic RI11 than those receiving other treatments. The plasma immunoglobulin G (IgG) level was higher in the RI11 treatment group than in the NC, AA and RS5 groups. The plasma immunoglobulin A (IgA) level was not affected by postbiotic supplements. The hepatic GHR mRNA expression level was significantly increased in birds fed postbiotics RI11, RS5 and UL4, AA and PC compared to the NC-fed birds. Postbiotic RI11 led to significantly higher hepatic IGF-1 mRNA expression level compared to the NC, PC, and AA treatments. Mortality was numerically lesser in the postbiotic treatment groups, but not significantly different among all the treatments. In conclusion, among the postbiotics applied in the current study as compared with NC, PC and AA, RI11 could be used as a potential alternative antibiotic growth promoter and anti-stress treatment in the poultry industry.

Vibrio parahaemolyticus and Vibrio harveyi causing Acute Hepatopancreatic Necrosis Disease (AHPND) in Penaeus vannamei (Boone, 1931) isolated from Malaysian shrimp ponds