Acute Hepatopancreatic Necrosis Disease Research Articles

An enzymatic recombinase amplification assay combined with CRISPR-Cas12a for the rapid detection of acute hepatopancreatic necrosis disease in shrimp Penaeus vannamei

An enzymatic recombinase amplification assay combined with CRISPR-Cas12a for the rapid detection of acute hepatopancreatic necrosis disease in shrimp Penaeus vannamei

Different Immune Responses of Hemocytes from V. parahaemolyticus-Resistant and -Susceptible Shrimp at Early Infection Stage

Vibrio parahaemolyticus is one of the main causative agents leading to acute hepatopancreatic necrosis disease, the severe bacterial disease that occurs during shrimp aquaculture. Hemocytes play important roles during Vibrio infection. Previously, we found that there were few differentially expressed genes (DEGs) between hemocytes from V. parahaemolyticus-resistant and -susceptible shrimp before infection. We considered that there should be different immune responses between them after a pathogen infection. Here, the transcriptome data of hemocytes from V. parahaemolyticus-resistant and -susceptible shrimp before and after a pathogen infection were compared. The results showed that there were 157 DEGs responsive to infection in V. parahaemolyticus-resistant shrimp, while 33 DEGs in V. parahaemolyticus-susceptible shrimp. DEGs in V. parahaemolyticus-resistant shrimp were mainly related to immune and glycolytic processes, while those in V. parahaemolyticus-susceptible shrimp were mainly related to metabolism, with only two DEGs in common. A further analysis of genes involved in glucose metabolism revealed that GLUT2, HK, FBP, and PCK1 were lowly expressed while PC were highly expressed in hemocytes of the V. parahaemolyticus-resistant shrimp, indicating that glucose metabolism in shrimp hemocytes was related to a V. parahaemolyticus infection. After the knockdown of PC, the expression of genes in Toll and IMD signaling pathways were down-regulated, indicating that glucose metabolism might function through regulating host immunity during V. parahaemolyticus infection. The results suggest that the immune responses between V. parahaemolyticus-resistant and -susceptible shrimp were apparently different, which probably contribute to their different V. parahaemolyticus resistance abilities.

Differences in shrimp pond bottom soil properties and bacterial load between acute hepatopancreatic necrosis disease (AHPND)‐infected ponds and AHPND‐free ponds and their relation to AHPND

AbstractThis study investigated the disparities in soil characteristics and pathogenic bacteria prevalence between shrimp ponds affected by acute hepatopancreatic necrosis disease (AHPND) and unaffected ponds, alongside examining the spatial distribution of soil attributes in flat‐oriented pond soil strata. Using Pearson correlation and logistic regression analyses, relationships among variables and indicators associated with AHPND prevalence were discerned, leading to the formulation of a predictive model for AHPND occurrence. Soil samples were collected from distinct locations and depths within ponds across three southern provinces of Thailand's Andaman Seaboard. The analysis revealed significantly higher concentrations of several variables, including SOD, TIC, NO2−‐N, Ca, Mn, Cu, Zn, and specified Vibrio strains, in AHPND‐afflicted ponds, especially at 0–5 cm depth. A prominent differentiation was the escalated concentration of easily decomposable organic matter (EDOM) within infected ponds, implicating potential soil and water quality deterioration alongside heightened shrimp susceptibility to AHPND. Correlational analysis showed links between bacterial densities and organic matter groupings, trace elements, exchangeable bases, and soil pH, in AHPND‐infected ponds. The logistic regression model encapsulated three soil variables (TOC, Mg, and Mn) and one pathogen variable (V. parahaemolyticus) and furnished an equation to estimate the log (odds) of AHPND occurrence, facilitating better understanding and potential forecasting of AHPND prevalence in shrimp cultivation environments.

The effect of liquid smoke (Cocos nucifera) in controlling acute hepatopancreatic necrosis disease in Litopenaeus vannamei

Vibrio parahaemolyticus is found in coastal environments throughout the world and is a major pathogen that causes Acute Hepatopancreatic Necrosis Disease (AHPND) with mass mortalities in Pacific whiteleg shrimp (Penaues vannamei). The extract of coconut shell liquid smoke (Cocos nucifera) was tested for antibacterial effect against AHPND in vitro. It was further evaluated for its potential efficacy in preventing AHPND in vannamei shrimp in vivo. The water in this study has been tested and positive for Vibrio parahaemolyticus. Two experimental diets were obtained by adding 0.5% and 1% liquid smoke to a commercial shrimp diet. The dietary supplementation with liquid smoke significantly enhanced shrimp growth performance during 90 days of rearing. A liquid concentration of 0.5% showed a higher growth performance (17.56 gr) than a 1% concentration (14.4 gr). Liquid smoke added to the shrimp fed partially entered the shrimp’s digestive system, and some would dissolve into the water. This water-soluble bioactive is thought to reduce the concentration of V. parahaemolyticus in waters. This was proven by the fact that no shrimp were infected with V. parahaemolyticus during the 90-day rearing.

The effect of supplementation of Lactococcus lactis strain as probiotic on the growth and survival of Litopenaeus vannamei

Acute Hepatopancreatic Necrosis Disease (AHPND) or Early Mortality Syndrome (EMS) is a bacterial disease in shrimps caused by pathogen Vibrio parahaemolyticus. It has culminated in huge loss in global shrimp production due to mass mortality. A probiotic strain Lactococcus lactis strain FA1 was recently isolated from shark intestine, showing inhibition towards the growth of the pathogen. Due to inhibitory potential, the effect of probiotics strain on growth performance of shrimp infected with V. parahaemolyticus was evaluated. The probiotic strain was incorporated into feed for juvenile shrimps Litopanaeus vannamei for 3 weeks before which they were then challenged with pathogen Vibro parahaemolyticus. The study compares 4 shrimp groups: Control (Without any treatment); Group A (Probiotic treated, uninfected); Group B (Probiotic treated, infected) and Group C (No probiotic, infected). The survival and growth performance (weight and length gain) of shrimps were evaluated in the following 30 days. Statistical analyses (ANOVA; Post Hoc Tukey) were used to compare between shrimp groups. In general, infected shrimp demonstrated some of the key symptoms of AHPND (pale or white hepatopancreas), transparent body and erratic swimming behaviour. The supplementation of probiotics resulted in an improved survivability (65±1%) compared to infected shrimp (45±1%). The probiotic treated shrimp group showed to have better % body weight gain, in which weight gain between group B (treated/infected) and group C (untreated/infected) were significantly different (at P=0.046); and so, to group A (treated/uninfected) when compared with group C (P=0.047). There is however no significant difference in % length gain between the groups. Meanwhile, some of the infected shrimps were able to show recovery from the infection. Pre-infection probiotic treatment showed better performance compared to post-infection probiotic treatment. This indicated that the strain Lactococcus lactis is highly suitable for use as the future probiotic in shrimp aquaculture.

ZntA maintains zinc and cadmium homeostasis and promotes oxidative stress resistance and virulence in Vibrio parahaemolyticus

ABSTRACT Although metals are essential for life, they are toxic to bacteria in excessive amounts. Therefore, the maintenance of metal homeostasis is critical for bacterial physiology and pathogenesis. Vibrio parahaemolyticus is a significant food-borne pathogen that mainly causes acute gastroenteritis in humans and acute hepatopancreatic necrosis disease in shrimp. Herein, we report that ZntA functions as a zinc (Zn) and cadmium (Cd) homeostasis mechanism and contributes to oxidative stress resistance and virulence in V. parahaemolyticus. zntA is remarkably induced by Zn, copper, cobalt, nickel (Ni), and Cd, while ZntA promotes V. parahaemolyticus growth under excess Zn/Ni and Cd conditions via maintaining Zn and Cd homeostasis, respectively. The growth of ΔzntA was inhibited under iron (Fe)-restricted conditions, and the inhibition was associated with Zn homeostasis disturbance. Ferrous iron supplementation improved the growth of ΔzntA under excess Zn, Ni or Cd conditions. The resistance of ΔzntA to H2O2-induced oxidative stress also decreased, and its virulence was attenuated in zebrafish models. Quantitative real-time PCR, mutagenesis, and β-galactosidase activity assays revealed that ZntR positively regulates zntA expression by binding to its promoter. Collectively, the ZntR-regulated ZntA is crucial for Zn and Cd homeostasis and contributes to oxidative stress resistance and virulence in V. parahaemolyticus.

Development of lateral flow immunoassay strip for rapid detection of acute hepatopancreatic necrosis disease-causing PirAB toxins in shrimp

Acute hepatopancreatic necrosis disease (AHPND) is currently the most affected disease of shrimp farms worlwide As rapid and specific detection methods are essential to improve disease management, a lateral flow immunoassay (LFIA) was designed for the detection of PirAvp and PirBvp toxins in shrimp. In this study, polyclonal antibody based lateral flow immunoassay (LFIA) strip has been developed. The recombinant PirAvp and PirBvp toxin‐like proteins of Vibrio parahaemolyticus (VPAHPND) was used to immunize rabbits with the highest antibody titre after the third booster. This polyclonal rabbit antiserum was purified and used to develop a LFIA strip for the detection of AHPND pathogen. This test strip could detect PirAvp and PirBvp toxins up to 125 ng/mL by naked eye within 15 min. No cross-reactivity was observed with VPnon-AHPND. Furthermore, the sensitivity and specificity of LFIA were 94.0% and 98.0%, respectively. This LFIA strip could be used as a handy tool by the farmers and technicians in situ. ® 2023 Journal of Science and Technology - NTTU

Cellular and transcriptomic response to pathogenic and non-pathogenic Vibrio parahaemolyticus strains causing acute hepatopancreatic necrosis disease (AHPND) in Litopenaeus vannamei

The shrimp industry has historically been affected by viral and bacterial diseases. One of the most recent emerging diseases is Acute Hepatopancreatic Necrosis Disease (AHPND), which causes severe mortality. Despite its significance to sanitation and economics, little is known about the molecular response of shrimp to this disease. Here, we present the cellular and transcriptomic responses of Litopenaeus vannamei exposed to two Vibrio parahaemolyticus strains for 98 h, wherein one is non-pathogenic (VpN) and the other causes AHPND (VpP). Exposure to the VpN strain resulted in minor alterations in hepatopancreas morphology, including reductions in the size of R and B cells and detachments of small epithelial cells from 72 h onwards. On the other hand, exposure to the VpP strain is characterized by acute detachment of epithelial cells from the hepatopancreatic tubules and infiltration of hemocytes in the inter-tubular spaces. At the end of exposure, RNA-Seq analysis revealed functional enrichment in biological processes, such as the toll3 receptor signaling pathway, apoptotic processes, and production of molecular mediators involved in the inflammatory response of shrimp exposed to VpN treatment. The biological processes identified in the VpP treatment include superoxide anion metabolism, innate immune response, antimicrobial humoral response, and toll3 receptor signaling pathway. Furthermore, KEGG enrichment analysis revealed metabolic pathways associated with survival, cell adhesion, and reactive oxygen species, among others, for shrimp exposed to VpP. Our study proves the differential immune responses to two strains of V. parahaemolyticus, one pathogenic and the other nonpathogenic, enlarges our knowledge on the evolution of AHPND in L. vannamei, and uncovers unique perspectives on establishing genomic resources that may function as a groundwork for detecting probable molecular markers linked to the immune system in shrimp.

In vitro and in vivo evaluation of the efficacies of commercial probiotics and disinfectant against acute hepatopancreatic necrosis disease and luminescent vibriosis in Litopenaeus vannamei

The bioactivities of two commercially available probiotics and one chemical disinfectant were tested against strains of Vibrio parahaemolyticus (VPAHPND) and V. harveyi. This study aimed to determine shrimp pathogenic Vibrios' in vitro and in vivo sensitivities to commercial probiotics and a chemical disinfectant. The probiotics and disinfectant were tested first in vitro, followed by the in vivo trials. Results showed that upon administration of probiotics either through diet or adding into the tank water, the survivability of shrimp was increased during challenge with VPAHPND and V. harveyi. Also, the disinfectant was tested against the same pathogens and showed positive bactericidal effects at 2500 ppm and 5000 ppm. The present findings suggest that adding probiotics to the rearing water or the shrimp feeds effectively prevents infection by lowering the load of pathogenic bacteria. In comparison, the effectiveness of the disinfectant (PUR) depends on its appropriate concentration and timing of application. It is not only limited to rearing water but is also applicable for decontaminating pond liners, tanks, and other paraphernalia.

Alterations in the gut microbiome of whiteleg shrimp (Penaeus vannamei) postlarvae following exposure to an AHPND-causing strain of Vibrio parahaemolyticus

Acute hepatopancreatic necrosis disease (AHPND), attributed to the production of PirA/PirB toxins by certain Vibrio sp. strains, poses a significant threat to global shrimp aquaculture, causing substantial mortality and economic losses. To enhance our understanding of this disease within a closed culture system on the northern coast of Peru, we conducted a comparative analysis of the gut microbiomes between healthy and diseased postlarvae. Diseased postlarvae were obtained through exposure to an AHPND-causing strain of Vibrio parahaemolyticus. Five healthy and five diseased postlarvae were randomly sampled from experimental rearing tanks, and their medial guts were extracted. High-throughput sequencing targeting the V4 region of the 16S rRNA gene was employed for amplicon library construction, and assessments of alpha and beta diversities and taxonomic composition were conducted. Our results revealed reduced diversity and distinct compositional profiles in the gut microbiomes of diseased postlarvae. The order Rhodobacteriales was dominant in the gut microbiomes of healthy postlarvae. In contrast, the order Vibrionales (including an unassigned genus within Vibrionales, Vibrio, and Pseudoalteromonas) exhibited the highest abundance in diseased postlarvae. In conclusion, exposure to an AHPND-causing strain of V. parahaemolyticus induces significant dysbiosis in the gut microbiome of whiteleg shrimp postlarvae.

Species Richness and Diversity of Gut Microbiota may Reduce AHPND in the Whiteleg Shrimp, Litopenaeus vannamei

Acute hepatopancreatic necrosis disease (AHPND) causes massive economic loss in shrimp culture sector. Eco-friendly feed additives, which can reduce antibiotic resistance in bacteria and antibiotic residues present in aquaculture products, have become popular in shrimp farming. These additives have proven to maintain a balance host gut microbiota in preventing pathogen infection. In the present study, gut microbiota of Litopenaeus vannamei, which had been administrated with different diets (probiotic: Bacillus B2; prebiotic: Gracilaria changii, G; and a combination of Bacillus B2 and G. changii, B2+G) and post-challenged with AHPND-causing Vibrio parahaemolyticus strain 3HP, was determined using the PCR-DGGE method. The results showed that the gut microbiota of L. vannamei was dominated by the phylum Proteobacteria. The abundance of Proteobacteria was higher in the B2+G and G treatment groups. High abundance of native gut microbiota, such as Proteobacteria might prevent the adhesion of virulent bacteria by competitive exclusion, and thus, reducing the chance of AHPND infection. Moreover, the shrimp from the B2+G treatment group with higher survival in the challenge tests possessed a higher diversity and species richness of gut microbiota. This suggests that higher diversity and species richness of beneficial gut microbiota probably interfering bacterial communication among the pathogens by either destructing or neutralising their quorum sensing signals. Consequently, this will prevent the colonisation of pathogens in the gut and resulted in a higher survival of the shrimp challenged with V. parahaemolyticus.

Serine protease inhibitor 3 (Serpin3) from Penaeus vannamei selectively interacts with Vibrio parahaemolyticus PirAvp.

Acute Hepatopancreatic Necrosis Disease (AHPND) represents a significant challenge in the field of shrimp aquaculture. This disease is primarily caused by Vibrio parahaemolyticus strains harbouring the pVA1 plasmid encoding the PirAvp and PirBvp toxins. To combat this epidemic and mitigate its devastating consequences, it is crucial to identify and characterize the receptors responsible for the binding of these pathogenic toxins. Our studied discovered that Penaeus vannamei's Serine protease inhibitor 3 (PvSerpin3) derived from shrimp hepatopancreatic tissues could bind to recombinant PirAvp , confirming its role as a novel PirAvp -binding protein (PA BP). Through comprehensive computational methods, we revealed two truncated PirAvp -binding proteins derived from PvSerpin3 called Serpin3(13) and Serpin3(22), which had higher affinity to PirAvp than the full-length PvSerpin3. The PA BP genes were amplified from a cDNA library that was reversed from total RNA extracted from shrimp, cloned and expressed in Escherichia coli. Three PA BP inclusion bodies were refolded to obtain the soluble form, and the recovery efficacy was found to be 100% for Serpin3 and Serpin3(13), while Serpin3(22) had a recovery efficacy of roundly 50%. Co-Immunoprecipitation (co-IP) and dot blot assays substantiated the interaction of these recombinant PA BPs with both recombinant PirAvp and VPAHPND (XN89)-producing natural toxins.

RNA interference confirmed a close association of CYP, ApD, and UCH with the function of CpG ODNs in Litopenaeus vannamei

CpG oligonucleotides (CpG ODNs) have been demonstrated to enhance the innate immune response in various organisms, including Litopenaeus vannamei, a species of significant economic importance. However, the precise mechanism or the genetic factors of CpG ODNs underlying function in L. vannamei remained elusive. Our previous experiment has identified five potential marker genes, including cytochrome P450 (CYP), gamma-butyrobetaine dioxygenase (γ-BDO), apolipoprotein D (ApD), prostaglandin reductase 1 (PGR1), and ubiquitin carboxyl-terminal hydrolase (UCH). In this study, RNA interference (RNAi) was employed to validate the relevance of these five genes in responding the stimulation of CpG ODNs. Briefly, shrimps were fed CpG ODNs and subsequently injected dsRNA targeting these five genes for interference. Finally, the non-specific immune parameters of shrimps, including the expression of immune-related genes and antioxidant capacity of hepatopancreas, as well as the mortality of shrimps after pathogen attack were assessed. The tissue distribution analysis revealed a high abundance of CYP in the hepatopancreas, γ-BDO in the hemocytes, ApD in the eyestalk, PGR1 in the hemocytes, and UCH in the gonad. After blocking CYP, ApD, and UCH through RNAi, a significant down-regulation of the mRNA expression levels of immune-related genes, including ALF, TNF-α, Toll, and TOR, was observed. Additionally, a notable decline in the activity of antioxidant enzymes, such as CAT, GPX, GST, and SOD, was also observed. Moreover, the mortality rates of shrimps in the CYP, ApD, and UCH inhibited groups significantly increased compared to other treatment groups following exposure to Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (VpAHPND) and white spot syndrome virus (WSSV). These results collectively suggest a close association of CYP, ApD, and UCH with the function of CpG ODNs in shrimps which will provide significant theoretical insights into comprehending the mechanism of CpG ODNs and optimizing their practical application in shrimp's aquaculture.

Biofilm inhibitor Con A lectin feed additive protects shrimp against pathogenic Vibrio harveyi and V. parahaemolyticus

Extracellular matrix (ECM) is a major component of all bacterial biofilms that protect the bacterial cells living within. It consists mainly of carbohydrates, proteins, lipids, and nucleic acids. We characterized the ECM of an acute hepatopancreatic necrosis disease (AHPND) causing Vibrio parahaemolyticus isolate 3HP and two luminescent shrimp disease-causing V. harveyi isolates (1114GL or VH0 and its Siphoviridae phage-lysogenized isolate, VH1) using 11 FITC-labeled plant lectins with different terminal sugar specificities. Canavalia ensiformis lectin (Con A), wheat germ lectin (Triticum vulgaris, WGA), Griffonia simplicifolia lectin II (GSL-II), and Maclura pomifera Lectin (MPL) were found to react with the biofilms of all three isolates. Based on these results and availability, Con A and WGA and their respective specific sugars (D-mannose or glucose and N-acetyl-D-glucosamine) were tested for effects on V. harveyi and V. parahaemolyticus biofilm formations. Biofilms of these bacteria were evaluated by thickness after growth with and without the two lectins and with and without their respective sugar targets at different concentrations. Only Con A at 0.1 mg/ml and 0.5 mg/ml (with and without D-mannose or d-glucose) were found to inhibit biofilms of all three Vibrio isolates in a dose-dependent manner. Con A was then tested for efficacy in protecting shrimp against V. harveyi (VH1) and V. parahaemolyticus 3-HP by its addition to shrimp feed at 0.05 mg/g and 0.5 mg/g and fed for seven days before they were immersion challenged with either VH1 or 3HP on day 8. After the challenge, the shrimp continued to be fed with their respective feed, and their health and survival were monitored for another 11 and 12 days for the VH1 and 3HP experiments, respectively. From both experiments, the groups fed with Con A-supplemented feed gave significantly higher survival rates [51.9% and 53.6% (VH1) and 52.8% and 62.9% (3HP)] than those fed with un-supplemented feed [38.3% (VH1) and 21.2% (3HP)] (p < 0.05). These results suggested that D-mannose/d-glucose plays an essential role in V. harveyi and V. parahaemolyticus biofilm formations, and Con A lectin might be helpful in monitoring and controlling biofilms to prevent Vibrio diseases.

Application of Bacillus Coagulans as Paraprobiotic Against Acute Hepatopancreatic Necrosis Disease in Shrimp.

Paraprobiotics, known as non-viable or ghost probiotics, have attracted attention for their benefits over live microbial cells. This study was designed to investigate the paraprobiotic effects of heat-killed Bacillus coagulans on the white leg shrimp Litopenaeus vannamei. The paraprobiotic formulation was prepared in three different concentrations including B. coagulans 1 (107 cells g-1 diet), B. coagulans 2 (108 cells g-1 diet), and B. coagulans 3 (109 cells g-1 diet) through heat inactivation method. Preliminary toxicity assessments revealed that post-larvae shrimps (mean weight ± SE: 0.025 ± 0.007g) treated with B. coagulans 1, 2 and 3 paraprobiotic formulations exhibited no mortality, confirming the non-toxic nature of the formulated diet. In a 90-day feeding trial involving juvenile shrimps (mean weight ± SE: 0.64 ± 0.05g), growth parameters and feed conversion ratios improved in all experimental groups. Subsequently, these shrimps were challenged with Vibrio parahaemolyticus, revealing that paraprobiotic-fed shrimps exhibited significant survival rate improvements. Oxidative stress-related enzyme activities, such as superoxide dismutase and catalase, increased in paraprobiotic-fed shrimps post-Vibrio challenge, while the challenged control group showed decreased activity (p < 0.001). Nitric oxide levels are also increased in paraprobiotic-treated shrimp, with B. coagulans 3 showing a significant rise in nitric oxide activity (p < 0.001). This study further demonstrated the positive impact of paraprobiotic treatment on digestive enzymes, immune-related parameters (e.g., total hemocyte count, prophenoloxidase, and respiratory burst activity), and overall disease resistance. These findings suggest that B. coagulans paraprobiotics have the potential to enhance antioxidant, antibacterial, and immune-related responses in L. vannamei, making them a valuable addition to shrimp aquaculture.

Defining desired genetic gains for Pacific white shrimp (Litopenaeus vannamei) breeding objective using participatory approaches.

The objective of this study was to define desired genetic gains from economically important traits of Pacific white shrimp (Litopenaeus vannamei) using participatory approaches. Two questionnaires were sent out to 100 Pacific white shrimp farmers in all five Iranian shrimp farming provinces. Questionnaire A (Q-A) includes management factors and farming environments. Moreover, in this questionnaire, farmers were asked to rank the fourth most important traits in shrimp among 10 economic traits in the list for genetic improvement. In questionnaire B (Q-B), priorities of the four traits with the highest value were obtained using pairwise comparison. The results showed that the four most important traits were white spot syndrome virus resistance (WSSV), growth rate before 4 months (GR), acute hepatopancreatic necrosis disease resistance (AHPND), and female total weight at ablation (FTW). Medians of the best individual preference values were WSSV (0.222), GR (0.173), AHPND (0.157), and FTW (0.053). Most disagreements were found between the social group preference values in the commercial products and water salinity categories. Desired genetic gains were 1.71%, 1.57%, 0.53% and 0.31% for GR, AHPND, WSSV and FTW, respectively. This study highlighted that despite environmental and management differences, participatory approaches can achieve desired genetic results for Pacific white shrimp breeding programme.

Development of a multiplex real-time enzymatic recombinase amplification assay for differentiation of yellow head virus genotype 1 and 2 in Penaeus vannamei

At present, eight genotypes of yellow head virus (YHV) have been identified, and the main pathogenic genotypes are yellow head virus 1 (YHV1) and gill-associated virus (GAV), which have caused mass death and serious economic losses of shrimp industry. The prevention of YHV complex infection is highly dependent on early and rapid diagnostic, however, the available molecular technologies require complex procedures or expensive equipment. In this study, a multiplex rapid detection method of YHV1 and GAV based on real-time enzymatic recombinase amplification (YHV1/GAV-ERA) was established. Primers and probes were designed to detect the conserved ORF1b gene of YHV1 and GAV. Best primers and probes were selected to effectively amplify the target gene, and the sensitivity was reached 102 copies/μL within 6.17 ± 0.49 min. While multi-nested PCR could reach the detection limit of 1.9 copies/μL and 2.1 copies/μL for YHV1 and GAV by consuming higher time and instrument cost. The YHV1/GAV-ERA can effectively amplify the target gene at 32–45 °C within 30 min, and the optimum reaction temperature is 42 °C. No cross reaction with white spot syndrome virus (WSSV), Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (VpAHPND), Enterocytozoon hepatopenaei (EHP), infectious hypodermal and hematopoietic necrosis virus (IHHNV) and healthy shrimp DNA were observed. Additionally, the test results of YHV1/GAV-ERA were 100% consistent with the industrial standard multi-nested PCR in shrimp samples. This method can distinguish the two genotypes of YHV and get rid of the dependence on the thermal cycle instrument, and provides a feasible rapid detection method for on-site screening of pathogens.

Development of lateral flow immunoassay strip for rapid detection of acute hepatopancreatic necrosis disease-causing PirAB toxins in shrimp

Acute hepatopancreatic necrosis disease (AHPND) is currently the most affected disease of shrimp farms worlwide As rapid and specific detection methods are essential to improve disease management, a lateral flow immunoassay (LFIA) was designed for the detection of PirAvp and PirBvp toxins in shrimp. In this study, polyclonal antibody based lateral flow immunoassay (LFIA) strip has been developed. The recombinant PirAvp and PirBvp toxin‐like proteins of Vibrio parahaemolyticus (VPAHPND) was used to immunize rabbits with the highest antibody titre after the third booster. This polyclonal rabbit antiserum was purified and used to develop a LFIA strip for the detection of AHPND pathogen. This test strip could detect PirAvp and PirBvp toxins up to 125 ng/mL by naked eye within 15 min. No cross-reactivity was observed with VPnon-AHPND. Furthermore, the sensitivity and specificity of LFIA were 94.0% and 98.0%, respectively. This LFIA strip could be used as a handy tool by the farmers and technicians in situ.&#x0D; ® 2023 Journal of Science and Technology - NTTU

The synergistic Lactobacillus plantarum L20 and Sargassum polycystum -added diet for improvement of Black tiger shrimp, Penaeus monodon ‘s growth, immune responses, bacterial profiles, and resistance against Vibrio parahaemolyticus associated Acute hepatopancreatic necrosis disease (AHPND) infection

Acute hepatopancreatic necrosis disease (AHPND) has inflicted massive economic losses and posed a considerable threat to the development of penaeid shrimp aquaculture in Malaysia. The restriction on antibiotic uses have necessitated exploring alternatives preventive measures against AHPND outbreak, such as the use beneficial bacteria and nutritional additives. In this study, diets which were fed to Penaeus monodon postlarvae (PL15) were fed with diets containing probiotic (L. plantarum at 108 CFU/mL), prebiotic (S. polycystum at 2%) and synbiotic diets (combination of probiotic and prebiotic) formulations. These diets were administered in triplicate for 35 days, followed by an immersion challenge with 6 × 105 CFU/mL AHPND-causing V. parahaemolyticus S2–4. Sampling of shrimp’s cephalothorax and abdomen from the feeding trial and the immersion challenge were conducted for gene expression and histopathology analyses. Results indicated that the synbiotic-fed group displayed the most significant weight gain, specific growth rate, and protein conversion ratio among the tested groups. In addition, shrimp cephalothorax from this group displayed a significant immune response, with expression of LGBP, peroxinectin, prophenoloxidase during post-feeding trial, and expression of prophenoloxidase, toll-like receptor, penaeidin, during post-challenge trial. Furthermore, the highest L. plantarum concentration in abdomen, lowest V. parahaemolyticus S2–4 concentration in cephalothorax and highest percent survival of shrimp during post-challenge were observed in this group. Therefore, this study highlighted the positive effect of synbiotic-supplemented diet on growth, immune response, and disease resistance of shrimp, offering a promising and sustainable solution to alleviate substantial production losses in shrimp farming.

Dietary administration of yeast (Saccharomyces cerevisiae) hydrolysate from sugar byproducts promotes the growth, survival, immunity, microbial community and disease resistance to VP (AHPND) in Pacific white shrimp (Litopenaeus vannamei)

This study investigated the effects of yeast hydrolysate (YH) from sugar byproducts on various parameters in Pacific white shrimp (Litopenaeus vannamei). The study found no significant differences in water quality parameters across all treatment tanks, ensuring that the observed effects were not due to environmental variations. There were no significant differences in growth parameters between the control group and groups receiving YH at different dosages. However, the group given YH at 10.0 g/kg feed exhibited a notably higher survival rate and higher expression of growth-related genes (IGF-2 and RAP-2A) in various shrimp tissues. YH was associated with enhanced immune responses, including lysozyme activity, NBT dye reduction, bactericidal activity, and phagocytic activity. Notably, the 10.0 g/kg feed group displayed the highest phagocytic index, indicating a dose-dependent immune response. Expression of immune-related genes (ALF, LYZ, ProPO, and SOD) was upregulated in various shrimp tissues. This upregulation was particularly significant in the gills, hepatopancreas, intestine, and hemocytes. While total Vibrio counts remained consistent, a reduction in green Vibrio colonies was observed in the intestine of shrimp treated with YH. YH, especially at 5.0 and 10.0 g/kg feed dosages, significantly increased survival rates and RPS values in response to AHPND infection. The findings of this study suggest that incorporating additives derived from yeast byproducts with possible prebiotic properties obtained from sugar byproducts can lead to positive results in terms of enhancing growth performance, immunity, histological improvements, and resistance to V. parahaemolyticus, the causative agent of acute hepatopancreatic necrosis disease (AHPND).