Shrimp Disease Research Articles

White Spot Disease Outbreak in Confined Rain-fed Shrimp Culture Ponds of Chilka Lake: A Case Study

Incidence of large-scale mortality of cultured shrimp due to white spot disease caused by the white spot syndrome virus (WSSV) has been recorded from the confined rain-fed pond culture system in Haridas and Kusubenti villages of Chilka Lake, Orissa (India). Gross signs and symptoms of the disease and behavioural changes in shrimp were observed. Some of the soil and water quality parameters have been analysed and no adverse conditions could be observed. Detailed histopathological observations made on the moribund shrimp indicated the presence of WSSV. The probable source of the disease transmission in the confined culture system is discussed.

Gut microbiota of Pacific white shrimp (Litopenaeus vannamei) exhibits distinct responses to pathogenic and non-pathogenic Vibrio parahaemolyticus.

Acute hepatopancreatic necrosis disease (AHPND), a high-mortality-rate shrimp disease, is caused by specific Vibrio parahaemolyticus (Vp) strains with a plasmid encoding the PirABVp toxins. As a bacterial pathogen, the invasion of AHPND-causing Vp might impose pressure on commensal microbiota in the shrimp gut, while the relationship between the pathogenesis of AHPND and the dysbiosis of gut bacterial communities remains unclear. Here we explored the temporal changes of shrimp gut microbiota in response to AHPND-causing and non-AHPND-causing Vp strains, with the non-infected controls as a baseline of the shrimp gut microbiota. The diversity and composition of bacterial communities from 168 gut samples (covering three treatments at seven time points with eight individuals per set) were investigated using 16S rRNA gene metabarcoding with high-throughput sequencing. The results showed that (i) species diversity of gut bacterial communities declined in Vp-infected shrimp, independent of the strain pathogenicity; (ii) taxonomic compositions of gut bacterial communities were significantly different between shrimp infected by AHPND-causing and non-AHPND-causing Vp strains; (iii) short-term (within 6 hours) compositional shifts in the gut microbiota were found only in AHPND-causing Vp-infected shrimp; (iv) the gut microbiota of AHPND-causing Vp-infected shrimp was enriched with genera Photobacterium and Vibrio, with a decline in Candidatus Bacilliplasma; and (v) functional predictions suggested the loss of normal metabolism due to compositional shifts in the gut microbiota. Our work reveals distinct features of community dynamics in shrimp gut microbiota, associated with pathogenic versus non-pathogenic Vibrio infections, providing a new perspective of the pathogenesis of AHPND. IMPORTANCE Shrimp production is continually threatened by newly emerging diseases, such as AHPND, which is caused by specific Vp strains. Previous studies on the pathogenesis of AHPND have mainly focused on the histopathology and immune responses of the host. However, more attention needs to be paid to the gut microbiota, which acts as the first barrier to pathogen colonization. In this study, we revealed that shrimp gut microbiota responded differently to pathogenic and non-pathogenic Vp strains, with bacterial genera Photobacterium and Vibrio enriched in pathogenic Vp-infected shrimp, and Candidatus Bacilliplasma enriched in non-pathogenic Vp-infected shrimp. Moreover, functional predictions suggested that changes in taxonomic compositions would further affect normal metabolic functions, emphasizing the importance of sustaining an equilibrium in the gut microbiota. Several biomarkers associated with specific microbial taxa and functional pathways were identified in our data sets, which help predict the incidence of disease outcomes.

A recombinase polymerase amplification and Pyrococcus furiosus Argonaute combined method for ultra-sensitive detection of white spot syndrome virus in shrimp.

White spot disease (WSD) in shrimp is an acute infectious disease caused by white spot syndrome virus (WSSV). WSD has seriously threatened the security of shrimp farming, causing huge economic losses worldwide. As there is currently no effective treatment for WSD, developing early detection technologies for WSSV is of great significance for the prevention. In this study, we have established a detection method for WSSV using a combination of recombinase polymerase amplification (RPA) and Pyrococcus furiosus Argonaute (PfAgo). We have achieved a detection sensitivity of single copy per reaction, which is more sensitive than the previously reported detection methods. Additionally, we have demonstrated high specificity. The entire detection process can be completed within 75 min without the need for precise thermal cyclers, making it suitable for on-site testing. The fluorescence signal generated by the reaction can be quantified using a portable fluorescence detector or observed with the naked eye under a blue light background. This study provides an ultrasensitive on-site detection method for WSSV in shrimp aquaculture and expands the application of PfAgo in the field of aquatic disease diagnosis.

Consistent features of the gut microbiota in response to diverse shrimp Litopenaeus vannamei diseases: A meta‐analysis

AbstractDysbiosis in the gut microbiota has been intimately implicated in shrimp (Litopenaeus vannamei, Penaeidae) diseases. However, considering the variety of shrimp diseases and the variability in experimental conditions, studies addressing common features of the gut microbiota−shrimp disease relationship are limited. Through an unbiased subject‐level meta‐analysis framework, 463 shrimp gut bacterial communities from 27 studies were re‐analysed, including six lifestages and eight diseases of shrimp, with the causal agents of viral, bacterial, eukaryotic, and unknown pathogens. Shrimp lifestages and diseases were the predominant factors governing the gut microbiota. After ruling out the top lifestage‐ and disease‐specific discriminatory amplicon sequence variants (ASVs) from the gut microbiota, the top 27 disease common‐discriminatory ASVs were identified, contributing to an overall accuracy of 95.9% in diagnosing shrimp health status. By using these optimisation procedures, the accuracy of our diagnosis model was unbiased by shrimp lifestage, specific disease, sampling size, hypervariable region and sequencing platform. The shrimp eight diseases consistently and significantly increased the relative importance of stochasticity, the relative abundance of pathogenic potentials and diversified core ASVs, whereas decreased the diversity and stability of gut microbiota. Collectively, these findings illustrate the effectiveness of meta‐analysis in determining the robust and common features of the shrimp gut microbiota in response to diverse diseases. In particular, disease common‐discriminatory ASVs could accurately diagnose shrimp health status, although the data are divergent in biotic and technical variances.

The effect of alginate oligosaccharides on intestine barrier function and Vibrio parahaemolyticus infections in the white shrimp Litopenaeus vannamei

The intestine is a host-pathogen interaction site and improved intestinal barrier function help to prevent disease in shrimp. Alginate oligosaccharides (AOS) are derived from resourceful brown algae. The intestine protection properties of AOS were widely recognized, and their benefits in fish have been reported. Nevertheless, there are no reports on AOS in shrimp and other crustaceans. In the present work, we measured the effects of AOS on growth performance and disease resistance in the white shrimp Litopenaeus vannamei and investigated their effects on intestinal health. Shrimps with an initial weight of about 2 g were fed with diets supplemented with 0 (control), 0.07%, 0.2%, 0.6%, or 1.2% of AOS for 56 days and were sampled and challenged with Vibrio parahaemolyticus. Dietary AOS did not significantly influence weight gain or feed utilization (P > 0.05). However, AOS considerably decreased the seven-day cumulative mortality after the challenge at any dose (P < 0.05). Dietary AOS improved the intestinal structure, significantly boosted the intestinal villus height at 0.6% and 1.2% levels, and increased intestinal wall thickness by 0.2%, 0.6%, and 1.2%. The alkaline phosphatase and maltase activities were also increased, suggesting that AOS improved the intestinal condition. Redox homeostasis in intestinal was improved by AOS, as expressed by the enhanced total antioxidant capacity and decreased malonaldehyde content, partly due to the increased superoxide dismutase and catalase activities. Compared with the antioxidant system, AOS's stimulating effects on immunity were more significant. At any level, AOS significantly activated lysozyme activity, the expression of propo and two antimicrobial peptide genes (pen-3 and crusin). However, the lowest concentration of AOS did not stimulate the gene expression of all three assayed pattern recognition receptors (LGBP, Toll, and IMD), and only the highest concentration of AOS increased the expression of imd. These findings suggest that AOS are highly efficient immunostimulants, and various immune pathways in shrimp are differentially sensitive to AOS. Finally, our findings suggest that AOS significantly alter the gut microbiota and their relative abundance at the phylum, family, and genus levels. In conclusion, AOS significantly enhances disease resistance in L. vannamei, possibly attributed to improved intestinal development, increased intestinal immunity and altered microbiota. These findings could provide a basis for future studies on the practical use of AOS and its mechanisms of action.

Prevalence of multi-antibiotics resistant (MAR) Vibrio parahaemolyticus in shrimp farms in Sarawak, Malaysia

The shrimp farming industry is constantly under threat due to outbreaks of infectious diseases and environmental problems. Vibrio parahaemolyticus is an important foodborne pathogen causing significant economic losses within the shrimp aquaculture industry worldwide. This research aimed to determine the prevalence of V. parahaemolyticus from different shrimp farms from the stocking to harvesting period and assess the antibiotic susceptibility of V. parahaemolyticus using the antimicrobial susceptibility test (AST). In this study, a total of 288 samples comprising twenty-four from each sample consisting of shrimp, water, effluent, and sediment samples were collected aseptically from three (n = 3) shrimp farms located at Telaga Air Farm 1 (Pond 6), Telaga Air Farm 2 (Pond 9) and Santubong Farm (Pond 7), Kuching, Sarawak. A molecular approach by polymerase chain reaction was used to confirm the presence of regulator gene, toxR, V. parahaemolyticus. A total of 14 antibiotics, including spectinomycin (SH100), imipenem (IPM10), amoxycillin/ clavulanic acid (AMC30), enrofloxacin (ENR5), bacitracin (B10), meropenem (MEM10), cephalothin (KF30), penicillin G (P10), tetracycline (TE30), kanamycin (K30), streptomycin(S25), rifampicin (RD2), erythromycin (E15), and nalidixic acid (NA30) were used. The results obtained showed that 51/288 (17.71%) of the collected samples with regulator gene, toxR V. parahaemolyticus. As a whole, this includes 31.25% (30/288) from sediment samples, 4.17% (4/288) from shrimp samples, 15.63% (15/288) from water samples, and 2.08% (2/288) from effluent water samples. A total of 54.9% (28/51) of V. parahaemolyticus acquired multiple antibiotic resistance (MAR). The resistance of antibiotics was profiled, and the multiple antibiotic resistance (MAR) indexes and classified into ten patterns. The MAR index of V. parahaemolyticus isolates ranged from 0.11 to 0.36. Vibrio parahaemolyticus isolates showed 31.38% with a MAR index &gt; 0.2, indicating that these isolates might be originated from high-risk sources. The data obtained from this study is helpful to monitor the presence of V. parahaemolyticus in the aquaculture farm management system to mitigate the hazard potentially arising from the environmental factor that causes shrimp diseases and shrimp infection.

The Effectiveness of Lactic Acid Bacteria on the Immune System of Vannamei Shrimp Infected with Bacteria Vibrio Parahaemolyticus

Diseases are a major obstacle in shrimp farming because they can cause relatively high mortality and decrease the quality of the aquaculture environment. One dangerous shrimp disease is caused by Vibrio bacteria. Today, people are turning to biological control methods by utilizing lactic acid bacteria that live as microflora in the digestive tract of aquaculture animals. This study aimed to analyze the potential of lactic acid bacteria isolates from the gut of vannamei shrimp as probiotic candidates and to analyze selected lactic acid bacteria isolates that were effective in increasing the immune response and survival of vannamei shrimp infected with Vibrio parahaemolyticus bacteria. Four BAL isolates were isolated, designated as 8A, 11B, F1, and G2. BAL isolates showed antibacterial activity against Vibrio parahaemolyticus bacteria with an inhibition zone of up to 11.5 mm, capable of living at acidic to alkaline pH (1.5-7.2), bile salts (3000 ppm) and negative catalase. The characterization of several tests such as gram-positive, inhibition test and sugar fermentation test and comparison of LAB characterization showed that LAB isolates (G2) from the intestine of vannamei shrimp were classified as Pediococcus acidilactici. The application of probiotics, namely LAB (G2) to vannamei shrimp through feed can stimulate the vaname shrimp immune system after being challenged with V. parahaemolyticus, as indicated by an increase in the number of hemocytes (THC), phagocytosis activity, and suppressing the population of V. parahaemolyticus in vannamei shrimp.

Adenosine 5'-monophosphate-activated protein kinase (AMPK) negatively regulates the immunity and resistance to Vibrio alginolyticus of white shrimp, Penaeus vannamei.

Shrimp immunology is vital in establishing prophylactic and therapeutic strategies for controlling pathological problems that threaten shrimp production. Apart from dietary treatments, the adenosine 5'-monophosphate-activated protein kinase (AMPK), an important regulatory enzyme that restores cellular energy balance during metabolic and physiological stress, is known to have therapeutic potential to improve shrimp's defense mechanism. Despite this, studies targeting the AMPK pathway in shrimp exposed to stressful conditions are vastly limited. In this study, AMPK was knocked down to assess the immunological changes and white shrimp, Penaeus vannamei resistance to Vibrio alginolyticus infection. Shrimps were injected individually and simultaneously with dsRNA targeting specific genes such as AMPK, Rheb, and TOR, after which the hepatopancreas was analyzed for the different gene expressions. The gene expressions of AMPK, Rheb, and TOR were effectively suppressed after being treated with dsRNAs. The Western blot analysis further confirmed a reduction in the protein concentration of AMPK and Rheb in the hepatopancreas. The suppression of AMPK gene led to a robust increase in the shrimp's resistance to V. alginolyticus, whereas the activation of AMPK by metformin decreased the shrimp's disease resistance. Among the mTOR downstream targets, the HIF-1α expression in shrimp treated with dsAMPK significantly increased at 48h but returned to normal levels when shrimp were treated with dsAMPK and either dsRheb or dsTOR. Immune responses such as respiratory burst, lysozyme activity, and phagocytic activity increased, while superoxide dismutase activity decreased following the knockdown of the AMPK gene compared to the control group. However, co-injection with dsAMPK and dsTOR or dsRheb restored immune responses to normal levels. Collectively, these results demonstrate that the inactivation of AMPK may ameliorate shrimp's innate immune response to recognize and defend against pathogens via the AMPK/mTOR1 pathway.

Transgenic Microalgae Expressing Double-Stranded RNA as Potential Feed Supplements for Controlling White Spot Syndrome in Shrimp Aquaculture.

Viral infection of farmed fish and shellfish represents a major issue within the aquaculture industry. One potential control strategy involves RNA interference of viral gene expression through the oral delivery of specific double-stranded RNA (dsRNA). In previous work, we have shown that recombinant dsRNA can be produced in the chloroplast of the edible microalga Chlamydomonas reinhardtii and used to control disease in shrimp. Here, we report a significant improvement in antiviral dsRNA production and its use to protect shrimp against white spot syndrome virus (WSSV). A new strategy for dsRNA synthesis was developed that uses two convergent copies of the endogenous rrnS promoter to drive high-level transcription of both strands of the WSSV gene element in the chloroplast. Quantitative RT-PCR indicated that ~119 ng dsRNA was produced per liter of culture of the transgenic microalga. This represents an ~10-fold increase in dsRNA relative to our previous report. The engineered alga was assessed for its ability to prevent WSSV infection when fed to shrimp larvae prior to a challenge with the virus. The survival of shrimp given feed supplemented with dried alga containing the dsRNA was significantly enhanced (~69% survival) relative to a negative control (<10% survival). The findings suggest that this new dsRNA production platform could be employed as a low-cost, low-tech control method for aquaculture.

Effect of fed dietary yeast (Rhodotorula paludigena CM33) on shrimp growth, gene expression, intestinal microbial, disease resistance, and meat composition of Litopenaeus vannamei

Yeast is a health-promoting and bio-therapeutic probiotic that is commonly used in aquaculture. Rhodotorula paludigena CM33 can accumulate amounts of intracellular carotenoids and lipid, which are regarded as nutritionally beneficial compounds in various aspects. The aim of this study was to evaluate the impact of different levels of R. paludigena CM33 (RD) incorporated in a dietary composition at 0% (control), 1% (1% RD), 2% (2% RD), and 5% (5% RD) on the growth of shrimp (Litopenaeus vannamei), their immune-related gene expression, intestinal health, resistance to Vibrio parahaemolyticus (VPAHPND) infection, and meat composition. The results showed significant improvements in the specific growth rate, weight gain, and survival of shrimp fed with 1% RD, 2% RD, and 5% RD, which were higher than the control group after 4 weeks of administration. The administration of 5% RD group resulted in a decrease in cumulative mortality upon VPAHPND challenge when compared to the control group. Furthermore, the expression levels of immune-responsive genes, including proPO system (prophenoloxidase-2: PO2), antioxidant enzyme (superoxide dismutase: SOD, glutathione peroxidase: GPX, and catalase: CAT), JAK/STAT pathway (signal transducer and activator of transcription: STAT, gamma interferon inducible lysosomal thiol reductase: GILT), IMD pathway (inhibitor of nuclear factor kappa-B kinase subunit beta and epsilon: IKKb and IKKe), and Toll pathway (Lysozyme) genes, were up-regulated in the 5% RD group. In the context of microbiota, microbiome analysis revealed that the main phyla in shrimp intestines were Proteobacteria, Firmicutes, Bacteroidota, Campilobacterota, Actinobacteriota, and Verrucomicrobiota. At the genus level, Vibrio was found to be reduced in the 5% RD group, whereas the abundance of potentially beneficial bacteria Bifidobacterium was increased. The 5% RD group showed a significant increase in the levels of crude protein and crude lipid, both of which are essential nutritious components. Our results show the capability of R. paludigena CM33 as a probiotic supplement in shrimp feed in improving growth, antimicrobial responses against VPAHPND, and meat quality by increasing protein and lipid content in shrimp.

SIMULTANEOUS PATHOGEN DETECTION OF SHRIMP VIRUSES ON CULTURED TIGER SHRIMPS (Penaeus monodon) IN INDONESIA

The multiple-pathogen infection causes severe economic impact to shrimp industry in Indonesia and worldwide due to mass mortality and multiple abnormalities of the survived infected shrimps. However, multiple-pathogen detection tools in shrimp diseases have not yet widely used. The purpose in this study was to develop and applied simultaneous detection system using multiplex polymerase chain reaction (PCR) assay from natural infections caused by white spot syndrome virus (WSSV), infectious hypodermal and haematopoietic necrosis virus (IHHNV) and monodon baculovirus (MBV) in Black tiger shrimp culture. To analyze multiple-pathogen infections in the shrimp, the study designed and used three pairs of specific primers targeting DNA virus from the shrimp diseases. All amplifications used a specific master mix for multiplex PCR assay and standardized extracted nucleic acid from the samples. This mPCR assay successfully amplified the DNA of three viruses in a single tube-run by multiplex PCR for each virus. Based on the results, the study confirms that multiple-pathogen infection contributes the highest mass mortality rather than from single infection by either WSSV, IHHNV or MBV. This study also confirms that the mPCR assay is a faster, cheaper, and efficient method to detect and subsequently prevent the spreading of multi-pathogen shrimp diseases.

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

Shrimp farming production is affected severely by the presence of toxigenic strains of Vibrio parahaemolyticus, the leading etiological agent of acute hepatopancreatic necrosis disease that causes as much as 100% mortality. To date, conventional methods have not effectively controlled AHPND, and only a few studies have reported bioassays testing phage therapy against this shrimp disease, none with a metagenomic approach. Here, Penaeus vannamei juveniles infected with AHPND-causing Vibrio parahaemolyticus were treated with phage to assess shrimp survival, tissue damage, as well as water microbiota dynamics. Phage therapy reduced by 4% shrimp mortality compared to the positive control. Histopathological analysis showed a 3 h delay in mortality of shrimp in the acute stage treated with phage therapy concerning the positive control, as well as a shortening of the terminal phase. Further, surviving shrimp in the terminal stage were only found in the positive control treatment. No significant differences were observed in the diversity of water microbiota between these treatments. Interestingly, V. parahaemolyticus caused up to 65% mortality, although its density decreased rapidly, and its relative abundance in the water microbiota ranged from 0.1% to 0.4%, while the marine bacteria that predominated in the microbiota was Donghicola eburneus (25.2%–54.6%). In addition, two copies of the pVA1 plasmid per V. parahaemolyticus genome were detected during the first 12 hpi in the positive control and phage therapy treatments. Phage therapy using a single phage could not effectively control AHPND in this investigation. Therefore, methods combining different approaches to prevent or treat the AHPND, such as a phage cocktail and a toxin binder, should be considered to increase the success rate in mitigating the disease.

Microbiome analysis of Litopenaeus vannamei reveals Vibrio as main risk factor of white faeces syndrome

The diversity of the gut microbiota substantially influences the health of the host, including shrimps. White faeces syndrome (WFS) is a multifactorial shrimp disease that can cause severe financial losses in Litopenaeus vannamei aquaculture industry. WFS in shrimps is caused not only by the microsporidian Enterocytozoon hepatopenaei infection but also by gut microbial dysbiosis, specifically the Vibrio species. The objectives of this study are to explore the gut microbiota composition in L. vannamei shrimps and to gain insights into the genetic factors associated with the development of WFS. For this, PacBio Sequel II platform was utilized to obtain full-length sequencing of the 16S rRNA gene (V1-V9). Significant variations in the intestinal bacterial abundance were observed between healthy and WFS-affected shrimps. At the genus level, Candidatus Bacilloplasma, Photobacterium, Pirellula, Rhodobacter and Lactococcus are abundantly present in the healthy shrimps, whereas, Cyanobium PCC-6307, Staphylococcus, and Vibrio are overrepresented in WFS shrimp. V. parahaemolyticus was identified as the predominant species in WFS samples. Function prediction analysis revealed that shrimp samples affected by WFS exhibited decreased lipid metabolism pathways, while showing elevated nucleotide metabolism and glycan biosynthesis pathways. Additionally, exosome and bacterial toxin pathways are more commonly found in diseased samples than in healthy ones, suggesting the potential involvement of unfavourable gut flora colonization. Our study provides clear understanding of the gut microbial community, specifically at the species level, distinguishing between WFS and healthy shrimps. Additionally, it emphasizes the role of Vibrio in the development of WFS and underscores the significance of analysing the core genes related to pathogens in studying the etiology of the disease.

Shrimp in labs: Biosecurity and hydro-social life

Since the 1980s, modern shrimp aquaculture has been seriously affected by shrimp diseases worldwide. Shrimp aquaculture cooperates with scientists in the lab to develop biosecurity strategies and biotechnology products, such as specific-pathogen-free shrimp, vaccines and probiotics, to tackle the risk of shrimp disease. Securing shrimp health needs to manage the breeding environment, particularly water quality and water ecologies. Shrimp and water travel between the lab and the field for monitoring, experimentation and disease prevention. This article proposes the notion of hydro-social life to analyse how biosecurity strategies and biotechnology products are developed in the lab and deployed in the field by visiting private, governmental and university laboratories in the Vietnamese Mekong Delta, Hô` Chí Minh City, and Taiwan. I argue that scientists innovate biotechnology products to improve biosecurity strategies by reconfiguring hydro-social lives, like managing shrimp health and water quality. The development and deployment of biosecurity from the lab to the field are influenced by capitalist forms of life and social relations.

Pyrococcus furiosus Argonaute with isothermal amplification for fast and ultra-sensitive diagnosis of acute hepatopancreatic necrosis disease in shrimp

The acute hepatopancreas necrosis disease (AHPND) caused by Vibrio species containing the virulence Photorhabdus insecticidal-related genes, PirAvp and PirBvp, is a high-mortality-rate disease in shrimp farming with strong transmission, quick onset, and severe symptoms. It is vital to control the disease transmission with frequent screening and early diagnosis; hence, developing fast diagnostic methods for AHPND with point-of-care testing (POCT) capacity is particularly important. In this study, we have established a method for fast and ultra-sensitive diagnosis of AHPND by combining a nuclease, Pyrococcus furiosus Argonaute (PfAgo), with recombinase polymerase amplification (RPA). Through systematic screening and optimizations, a strategy of using two guide DNA (gDNA) molecules to program PfAgo is realized for precisely tailored nuclease cleavage. The fabricated procedure can be completed in 75 min with a sensitivity of one copy per reaction, which is better than all the currently available molecular diagnosis methods for AHPND. The procedure requires only simple device support, which is suitable for POCT. This study exhibits the advantages of the PfAgo cleavage system concerning flexibility and sensitivity. The established RPA-PfAgo method is valuable for the prevention of AHPND in shrimp farms, presenting a good example of utilizing PfAgo for highly sensitive molecular diagnosis.

Investigation on extraction methods of garlic (Allium sativum L.) with activity against three strains of Vibrio causing disease in shrimp

Garlic (Allium sativum L.) is a plant with potent antibacterial activity that has been known for a long time. In this study, we extracted different extracts from garlic such as essential oil, polysaccharide, 5% acetic acid extract, ethanol extract, fractional ethyl acetate extract, fractional ethanol extract and fractional water extract to find the garlic extract with the best antibacterial activity against Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholera, all species causing white feces disease and acute hepatopancreatic necrosis disease in shrimp. The results showed that ethanol extract and essential oil had antibacterial activity in all three strains and garlic essential oil inhibited all 3 strains of Vibrio with the highest sterile ring diameter of 80 ± 0 mm by fumigation method. The MIC and MBC values of both ethanol extract and essential oil showed better resistance to V. vulnificus than to V. parahaemolyticus and V. cholera. The ethanol extract had MIC = 8.33 mg/ml and MBC = 12.50 mg/ml and the essential oil had MIC = 0.03 μl/100 μl and MBC = 0.05 μl/100 μl. The results of the time-kill curve showed that both the ethanol extract and the essential oil were able to kill all three strains of Vibrio only after 24 hours when added to the bacterial medium, while tetracycline only inhibited V. cholera, V. parahaemolyticus during the first 8 hours and 24 hours for V. vulnificus.

Scavenger receptor B2, a type III membrane pattern recognition receptor, senses LPS and activates the IMD pathway in crustaceans

The immune deficiency (IMD) pathway is critical for elevating host immunity in both insects and crustaceans. The IMD pathway activation in insects is mediated by peptidoglycan recognition proteins, which do not exist in crustaceans, suggesting a previously unidentified mechanism involved in crustacean IMD pathway activation. In this study, we identified a Marsupenaeus japonicus B class type III scavenger receptor, SRB2, as a receptor for activation of the IMD pathway. SRB2 is up-regulated upon bacterial challenge, while its depletion exacerbates bacterial proliferation and shrimp mortality via abolishing the expression of antimicrobial peptides. The extracellular domain of SRB2 recognizes bacterial lipopolysaccharide (LPS), while its C-terminal intracellular region containing a cryptic RHIM-like motif interacts with IMD, and activates the pathway by promoting nuclear translocation of RELISH. Overexpressing shrimp SRB2 in Drosophila melanogaster S2 cells potentiates LPS-induced IMD pathway activation and diptericin expression. These results unveil a previously unrecognized SRB2-IMD axis responsible for antimicrobial peptide induction and restriction of bacterial infection in crustaceans and provide evidence of biological diversity of IMD signaling in animals. A better understanding of the innate immunity of crustaceans will permit the optimization of prevention and treatment strategies against the arising shrimp diseases.

Characterization of vB_ValM_PVA8, a broad-host-range bacteriophage infecting Vibrio alginolyticus and Vibrio parahaemolyticus.

Phage therapy was taken as an alternative strategy to antibiotics in shrimp farming for the control of Vibrio species of Vibrio parahaemolyticus and Vibrio alginolyticus, which cause substantial mortality and significant economic losses. In this study, a new Vibrio phage vB_ValM_PVA8 (PVA8), which could efficiently infect pathogenic isolates of V. alginolyticus and V. parahaemolyticus, was isolated from sewage water and characterized by microbiological and in silico genomic analyses. The phage was characterized to be a member of the Straboviridae family with elongated head and contractile tail by transmission electron microscopy. Genome sequencing showed that PVA8 had a 246,348-bp double-stranded DNA genome with a G + C content of 42.6%. It harbored totally 388 putative open reading frames (ORFs), among them 92 (23.71%) assigned to functional genes. Up to 27 transfer RNA (tRNA) genes were found in the genome, and the genes for virulence, antibiotic resistance, and lysogeny were not detected. NCBI genomic blasting results and the phylogenetic analysis based on the sequences of the large terminase subunits and the DNA polymerase indicated that PVA8 shared considerable similarity with Vibrio phage V09 and bacteriophage KVP40. The phage had a latent period of 20 min and a burst size of 309 PFUs/infected cell with the host V. alginolyticus, and it was stable over a broad pH range (4.0-11.0) and a wide temperature span (-80°C to 60°C), respectively, which may benefit its feasibility for phage therapy. In addition, it had the minimum multiplicity of infection (MOI) of 0.0000001, which revealed its strong multiplication capacity. The shrimp cultivation lab trials demonstrated that PVA8 could be applied in treating pathogenic V. parahaemolyticus infection disease of shrimp with a survival rate of 88.89% comparing to that of 34.43% in the infected group, and the pond application trails confirmed that the implementation of PVA8 could rapidly yet effectively reduce the level of the Vibrio. Taken together, PVA8 may be potential to be explored as a promising biological agent for Vibrio control in aquaculture farming industry.

Pathogenicity of Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (AHPND) in shrimp (Litopanaeus vannamei) in Serang, Banten, Indonesia

Abstract. Saputra A, Maftuch, Andayani S, Yanuhar U. 2023. Pathogenicity of Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (AHPND) in shrimp (Litopanaeus vannamei) in Serang, Banten, Indonesia. Biodiversitas 24: 2365-2373. Vannamei shrimp target EMS (Early Mortality Syndrome)/AHPND (Acute Hepotopancreatic Necrosis Disease) cases in several Asian nations, including China, Vietnam, Malaysia, Thailand, India, and Indonesia. AHPND cases on shrimp farms have been reported in several Indonesian regions. The aim of this study was to evaluate the histopathological pathogenicity of Vibrio parahaemolyticus in different shrimp tissues exposed to different doses and durations of exposure. PCR amplification of V. parahaemolyticus was performed using Vp.flaE-79F (5?-GCA GCT GAT CAA AAC GTT GAG T-3?) and Vp.flaE-934R (5?-GATACCTTGTTACGACTT -3?) primers for molecular identification of bacteria isolated from shrimp exhibiting AHPND symptoms. V. parahaemolyticus positive target band at 897 bp. Bacterial isolates with green colonies on TCBS agar media were chosen for other test. Challenge tests with healthy shrimp were conducted using the V. parahaemolyticus strain. The challenge test on shrimp demonstrated the same clinical and pathological symptoms as the signs of infected shrimp in rivers, including swimming into the container’s corner and changes in the carapace’s pale color and empty midgut. The results showed that challenged shrimp displayed necrosis, atrophy, granulomas, cell sloughing, and had significantly considerable hemocyte infiltration that led to melanization. The highest percentage (63.33%) of damage was recorded in the treatment with a concentration of 108 CFU mL-1. It was also observed that virulence of V. parahaemolyticus strain varied with different concentrations. At concentrations of 108 CFU mL-1 and 106 CFU mL-1, virulence of V. parahaemolyticus strain caused 100% mortality in 73 hour post-infection (p.i). The lowest concentration limit for pathogenicity was 104 CFU mL-1 with a mortality of 48% at 73 p.i. The challenge test results showed that V. parahaemolyticus strain that causes AHPND was the main pathogen affecting vannamei shrimp in Serang, Banten, Indonesia.

A bacterial binary toxin system that kills both insects and aquatic crustaceans: Photorhabdus insect-related toxins A and B.

Photorhabdus insect-related toxins A and B (PirA and PirB) were first recognized as insecticidal toxins from Photorhabdus luminescens. However, subsequent studies showed that their homologs from Vibrio parahaemolyticus also play critical roles in the pathogenesis of acute hepatopancreatic necrosis disease (AHPND) in shrimps. Based on the structural features of the PirA/PirB toxins, it was suggested that they might function in the same way as a Bacillus thuringiensis Cry pore-forming toxin. However, unlike Cry toxins, studies on the PirA/PirB toxins are still scarce, and their cytotoxic mechanism remains to be clarified. In this review, based on our studies of V. parahaemolyticus PirAvp/PirBvp, we summarize the current understanding of the gene locations, expression control, activation, and cytotoxic mechanism of this type of toxin. Given the important role these toxins play in aquatic disease and their potential use in pest control applications, we also suggest further topics for research. We hope the information presented here will be helpful for future PirA/PirB studies.