Computational design of novel chimeric multiepitope vaccine against bacterial and viral disease in tilapia (Oreochromis sp.).

Regarding several infectious diseases in fish, multiple vaccinations are not favorable. The chimeric multiepitope vaccine (CMEV) harboring several antigens for multi-disease prevention would enhance vaccine efficiency in terms of multiple disease prevention. Herein, the immunogens of tilapia's seven pathogens including E. tarda, F. columnare, F. noatunensis, S. iniae, S. agalactiae, A. hydrophila, and TiLV were used for CMEV design. After shuffling and annotating the B-cell epitopes, 5,040 CMEV primary protein structures were obtained. Secondary and tertiary protein structures were predicted by AlphaFold2 creating 25,200 CMEV. Proper amino acid alignment in the secondary structures was achieved by the Ramachandran plot. In silico determination of physiochemical and other properties including allergenicity, antigenicity, glycosylation, and conformational B-cell epitopes were determined. The selected CMEV (OSLM0467, OSLM2629, and OSLM4294) showed a predicted molecular weight (MW) of 70 kDa, with feasible sites of N- and O-glycosylation, and a number of potentially conformational B-cell epitope residues. Molecular docking, codon optimization, and in-silico cloning were tested to evaluate the possibility of protein expression. Those CMEVs will further elucidate in vitro and in vivo to evaluate the efficacy and specific immune response. This research will highlight the new era of vaccines designed based on in silico structural vaccine design.

Evaluation of Bacillus spp. as Potent Probiotics with Reduction in AHPND-Related Mortality and Facilitating Growth Performance of Pacific White Shrimp (Litopenaeus vannamei) Farms.

Acute hepatopancreatic necrosis disease (AHPND) is a serious bacterial disease affecting shrimp aquaculture worldwide. In this study, natural microbes were used in disease prevention and control. Probiotics derived from Bacillus spp. were isolated from the stomachs of AHPND-surviving Pacific white shrimp Litopenaeus vannamei (22 isolates) and mangrove forest soil near the shrimp farms (10 isolates). Bacillus spp. were genetically identified and characterized based on the availability of antimicrobial peptide (AMP)-related genes. The phenotypic characterization of all Bacillus spp. was determined based on their capability to inhibit AHPND-causing strains of Vibrio parahaemolyticus (VPAHPND). The results showed that Bacillus spp. without AMP-related genes were incapable of inhibiting VPAHPND in vitro, while other Bacillus spp. harboring at least two AMP-related genes exhibited diverse inhibition activities. Interestingly, K3 [B. subtilis (srfAA+ and bacA+)], isolated from shrimp, exerted remarkable inhibition against VPAHPND (80% survival) in Pacific white shrimp and maintained a reduction in shrimp mortality within different ranges of salinity (75-95% survival). Moreover, with different strains of VPAHPND, B. subtilis (K3) showed outstanding protection, and the survival rate of shrimp remained stable among the tested groups (80-95% survival). Thus, B. subtilis (K3) was further used to determine its efficiency in shrimp farms in different locations of Vietnam. Lower disease occurrences (2 ponds out of 30 ponds) and greater production efficiency were noticeable in the B. subtilis (K3)-treated farms. Taking the results of this study together, the heat-shock isolation and genotypic-phenotypic characterization of Bacillus spp. enable the selection of probiotics that control AHPND in Pacific white shrimp. Consequently, greater disease prevention and growth performance were affirmed to be beneficial in the use of these probiotics in shrimp cultivation, which will sustain shrimp aquaculture and be environmentally friendly.

Sensitive Visual Detection of AHPND Bacteria Using Loop-Mediated Isothermal Amplification Combined with DNA-Functionalized Gold Nanoparticles as Probes.

Acute hepatopancreatic necrosis disease (AHPND) is a component cause of early mortality syndrome (EMS) of shrimp. In 2013, the causative agent was found to be unique isolates of Vibrio parahaemolyticus (VPAHPND) that contained a 69 kbp plasmid (pAP1) carrying binary Pir-like toxin genes PirvpA and PirvpB. In Thailand, AHPND was first recognized in 2012, prior to knowledge of the causative agent, and it subsequently led to a precipitous drop in shrimp production. After VPAHPND was characterized, a major focus of the AHPND control strategy was to monitor broodstock shrimp and post larvae for freedom from VPAHPND by nucleic acid amplification methods, most of which required use of expensive and sophisticated equipment not readily available in a shrimp farm setting. Here, we describe a simpler but equally sensitive approach for detection of VPAHPND based on loop-mediated isothermal amplification (LAMP) combined with unaided visual reading of positive amplification products using a DNA-functionalized, ssDNA-labled nanogold probe (AuNP). The target for the special set of six LAMP primers used was the VPAHPND PirvpA gene. The LAMP reaction was carried out at 65°C for 45 min followed by addition of the red AuNP solution and further incubation at 65°C for 5 min, allowing any PirvpA gene amplicons present to hybridize with the probe. Hybridization protected the AuNP against aggregation, so that the solution color remained red upon subsequent salt addition (positive test result) while unprotected AuNP aggregated and underwent a color change from red to blue and eventually precipitated (negative result). The total assay time was approximately 50 min. The detection limit (100 CFU) was comparable to that of other commonly-used methods for nested PCR detection of VPAHPND and 100-times more sensitive than 1-step PCR detection methods (104 CFU) that used amplicon detection by electrophoresis or spectrophotometry. There was no cross reaction with DNA templates derived from non-AHPND bacteria commonly found in shrimp ponds (including other Vibrio species). The new method significantly reduced the time, difficulty and cost for molecular detection of VPAHPND in shrimp hatchery and farm settings.

Characterization and PCR Detection Of Binary, Pir-Like Toxins from Vibrio parahaemolyticus Isolates that Cause Acute Hepatopancreatic Necrosis Disease (AHPND) in Shrimp.

Unique isolates of Vibrio parahaemolyticus (VPAHPND) have previously been identified as the causative agent of acute hepatopancreatic necrosis disease (AHPND) in shrimp. AHPND is characterized by massive sloughing of tubule epithelial cells of the hepatopancreas (HP), proposed to be induced by soluble toxins released from VPAHPND that colonize the shrimp stomach. Since these toxins (produced in broth culture) have been reported to cause AHPND pathology in reverse gavage bioassays with shrimp, we used ammonium sulfate precipitation to prepare protein fractions from broth cultures of VPAHPND isolates for screening by reverse gavage assays. The dialyzed 60% ammonium sulfate fraction caused high mortality within 24-48 hours post-administration, and histological analysis of the moribund shrimp showed typical massive sloughing of hepatopancreatic tubule epithelial cells characteristic of AHPND. Analysis of the active fraction by SDS-PAGE revealed two major bands at marker levels of approximately 16 kDa (ToxA) and 50 kDa (ToxB). Mass spectrometry analysis followed by MASCOT analysis revealed that both proteins had similarity to hypothetical proteins of V. parahaemolyticus M0605 (contig034 GenBank accession no. JALL01000066.1) and similarity to known binary insecticidal toxins called 'Photorhabdus insect related' proteins A and B (Pir-A and Pir-B), respectively, produced by the symbiotic, nematode bacterium Photorhabdus luminescens. In in vivo tests, it was shown that recombinant ToxA and ToxB were both required in a dose dependent manner to cause AHPND pathology, indicating further similarity to Pir-A and -B. A single-step PCR method was designed for detection of the ToxA gene and was validated using 104 bacterial isolates consisting of 51 VPAHPND isolates, 34 non-AHPND VP isolates and 19 other isolates of bacteria commonly found in shrimp ponds (including other species of Vibrio and Photobacterium). The results showed 100% specificity and sensitivity for detection of VPAHPND isolates in the test set.

Draft Genome Sequences of Six Strains of Vibrio parahaemolyticus Isolated from Early Mortality Syndrome/Acute Hepatopancreatic Necrosis Disease Shrimp in Thailand.

Some strains of Vibrio parahaemolyticus cause acute hepatopancreatic necrosis disease (AHPND) in shrimp. We sequenced 3 AHPND and 3 non-AHPND strains and found that all of them lacked the pathogenicity island relevant to human infection. A unique sequence encoding a type IV pilus/type IV secretion system was found in 3 AHPND strains.

Chitinases from the black tiger shrimp Penaeus monodon: phylogenetics, expression and activities.

Chitinases are essential enzymes for crustaceans and animal alike for their molting and digestion of foods containing chitin. From the Penaeus monodon EST database, cDNA contigs and singletons for three chitinases, namely PmChi1, 2 and 3, were identified. The complete sequences for the mature PmChi1, 3 and partial PmChi2 were amplified and cloned. The reading frames of PmChi1 and 3 encoded mature proteins of 644 and 468 amino acids with calculated molecular masses of 72.4 and 51.9kDa, respectively. The amino acid sequence comparison among the penaeid chitinases revealed homology around 90%. Therefore, they were grouped together along with those of other crustaceans and insects into three groups separated from those of mammals. PmChi1, 2 and 3 were expressed mainly in hepatopancreas, gill and hepatopancreas, respectively, though small amounts were expressed in other tissues. After molting, only the expression of PmChi2 was down-regulated, while the expression of PmChi1 and 3 was relatively unchanged. The results suggested that the PmChi2 was likely involved in molting while the others might function in the digestion of chitinous foods. The recombinant PmChi1 (rPmChi1) over-produced from Escherichia coli had its optimal pH 5 but it was most stable at neutral pH. Interestingly, the optimal temperature was relatively high at 55 degrees C. Nevertheless, it was stable at lower temperature below 40 degrees C. The rPmChi1 preferentially hydrolyzed the more soluble substrates like partially N-acetylated chitin (PNAC) and colloidal chitin from shrimp shell as compared to the beta-chitin from squid pen.