Oral administration of recombinant outer membrane protein A-based nanovaccine affords protection against Aeromonas hydrophila in zebrafish.

Aeromonas hydrophila, an opportunistic warm water pathogen, has always been a threat to aquaculture, leading to substantial economic losses. Vaccination of the cultured fish would effectively prevent Aeromoniasis, and recent advancements in nanotechnology show promise for efficacious vaccines. Oral delivery would be the most practical and convenient method of vaccine delivery in a grow-out pond. This study studied the immunogenicity and protective efficacy of a nanoparticle-loaded outer membrane protein A from A. hydrophila in the zebrafish model. The protein was over-expressed, purified, and encapsulated using poly lactic-co-glycolic acid (PLGA) nanoparticles via the double emulsion method. The PLGA nanoparticles loaded with recombinant OmpA (rOmpA) exhibited a size of 295 ± 15.1 nm, an encapsulation efficiency of 72.52%, and a polydispersity index of 0.292 ± 0.07. Scanning electron microscopy confirmed the spherical and isolated nature of the PLGA-rOmpA nanoparticles. The protective efficacy in A. hydrophila-infected zebrafish after oral administration of the nanovaccine resulted in relative percentage survival of 77.7. Gene expression studies showed significant upregulation of immune genes in the vaccinated fish. The results demonstrate the usefulness of oral administration of nanovaccine-loaded rOmpA as a potential vaccine since it induced a robust immune response and conferred adequate protection against A. hydrophila in zebrafish, Danio rerio.

A comparative study on tensile strength of various thermoplastic polymers sheets following thermoforming on a pre-treatment and post-treatment maxillary model of a patient: an in vitro study.

OBJECTIVES: Thermoplastic polymers show alteration in their mechanical properties after thermoforming on a dental model. The purpose of this in-vitro study was to evaluate the tensile strength of different thermoplastic polymer sheets thermoformed on a pre-treatment (moderate crowding) and post-treatment (well-aligned) maxillary model of a patient. MATERIALS AND METHODS: Forty maxillary models (Twenty Pre-treatment & twenty Post-treatment of uniform dimension) were made by duplicating them using alginate Hydrogum 5 (Zhermack). Samples were then divided into eight groups of 5 samples each. The thermoplastic sheets Imprelon® (Scheu-Dent), AVAC R® (Jaypee), Placa Crystal® (BioART), EZ-VAC® (3A Medes)-1.0 mm thick were thermoformed on these models respectively. The sample was retrieved using ceramic bur mounted on a straight hand-piece and subjected for testing using TINIUS Olsen 10ST micro universal testing machine and recorded. RESULTS: There was no statistically significant difference (P > .05) in tensile strength of thermoformed thermoplastic polymer sheets between pre-treatment and post-treatment maxillary model. Tensile strength of EZ-VAC (3A Medes) showed higher variation between pre-treatment and post-treatment maxillary model though it was found to be statistically insignificant (P > .05). Significant difference (P < .05) was seen between groups when they were compared separately among pre-treatment and post-treatment models. CONCLUSION: Placa Crystal (BioART) among the pre-treatment group, EZ - VAC (3A Medes) among the post-treatment group, showed highest tensile strength. CLINICAL RELEVANCE: Results of the study highlights the necessity to test materials in conditions which stands in accordance with the clinical scenario to a considerable extent and also emphasizes the need for further study in aligner.

In vitro determination of probiotic efficacy of Bacillus subtilis TLDK301120C24 isolated from tilapia against warm water fish pathogens and in vivo validation using gnotobiotic zebrafish model.

Eco-friendly alternatives such as probiotics are needed to prevent economically relevant infectious diseases for a successful disease-free harvest in aquaculture. The use of antibiotics has been the favored practice, but its empirical and indiscriminate use has led to antibiotic resistance in the aquatic environment and residues in the food fish. With this rationale, a probiotic was isolated from tilapia, a commercially important cultured fish worldwide. The characteristics of the probiotic were checked against common bacterial pathogens affecting aquaculture. In vitro tests demonstrated the inhibitory effects of the isolated probiotic on the growth of Aeromonas hydrophila, Edwardsiella tarda, Vibrio anguillarum, and V. alginolyticus. The candidate probiotic, referred to as TLDK301120C24, was identified as Bacillus subtilis by a battery of biochemical tests and genotypic confirmation by 16S rDNA sequencing. The in vitro results revealed the ability of the probiotic to withstand the gut conditions that included pH range of 3-9, salt concentration of 0.5-6%, and bile salt concentration of up to 6%. The isolate could hydrolyze starch (12-14 mm clearance zone), protein (20-22 mm clearance zone), and cellulose (22-24 mm clearance zone). Further, the inhibitory ability of the probiotic against aquatic pathogens was determined in vivo using gnotobiotic zebrafish by employing a novel approach that involved tagging the probiotic with a red fluorescent protein and the pathogens with a green fluorescent protein, respectively. The colonizing ability of probiotics and its inhibitory effects against the pathogens were evaluated by fluorescence microscopy, PCR, and estimation of viable counts in LBA + Amp plates. Finally, the competitive inhibition and exclusion of fish pathogens A. hydrophila and E. tarda by B. subtilis was confirmed semi-quantitatively, through challenge experiments. This study shows the potential of B. subtilis as a probiotic and its excellent ability to inhibit major fish pathogens in vivo and in vitro. It also shows promise as a potent substitute for antibiotics.

Nanovaccines to Combat Aeromonas hydrophila Infections in Warm-Water Aquaculture: Opportunities and Challenges.

The application of nanotechnology in aquaculture for developing efficient vaccines has shown great potential in recent years. Nanovaccination, which involves encapsulating antigens of fish pathogens in various polymeric materials and nanoparticles, can afford protection to the antigens and a sustained release of the molecule. Oral administration of nanoparticles would be a convenient and cost-effective method for delivering vaccines in aquaculture while eliminating the need for stressful, labour-intensive injectables. The small size of nanoparticles allows them to overcome the degradative digestive enzymes and help deliver antigens to the target site of the fish more effectively. This targeted-delivery approach would help trigger cellular and humoral immune responses more efficiently, thereby enhancing the protective efficacy of vaccines. This is particularly relevant for combating diseases caused by pathogens like Aeromonas hydrophila, a major fish pathogen responsible for significant morbidity and mortality in the aquaculture sector. While the use of nanoparticle-based vaccines in aquaculture has shown promise, concerns exist about the potential toxicity associated with certain types of nanoparticles. Some nanoparticles have been found to exhibit varying degrees of toxicity, and their safety profiles need to be thoroughly assessed before widespread application. The introduction of nanovaccines has opened new vistas for improving aquaculture healthcare, but must be evaluated for potential toxicity before aquaculture applications. Details of nanovaccines and their mode of action, with a focus on protecting fish from infections and outbreaks caused by the ubiquitous opportunistic pathogen A. hydrophila, are reviewed here.

Evaluation of Probiotic Efficacy of Bacillus subtilis RODK28110C3 Against Pathogenic Aeromonas hydrophila and Edwardsiella tarda Using In Vitro Studies and In Vivo Gnotobiotic Zebrafish Gut Model System.

The indiscriminate use of antibiotics in aquaculture has led to the emergence of resistance; hence, eco-friendly, host-specific alternatives to mitigate bacterial infections have become imminent. In this study, bacteria that could possibly serve as probiotics were isolated and evaluated for their efficacy with in vitro experiments and in vivo zebrafish gut model. One isolate from each of the 23 rohu fish (Labeo rohita) was shortlisted after preliminary screening of several isolates and tested for their ability to inhibit two important warm water bacterial fish pathogens, Aeromonas hydrophila, and Edwardsiella tarda. An isolate (RODK28110C3) that showed broad-spectrum inhibitory activity against a battery of different isolates of the two fish pathogens included in this study and maintained in our repository was selected for further characterization. The culture was identified phenotypically as Bacillus subtilis and confirmed by 16S rDNA sequencing. The isolate was able to hydrolyze fish feed constituents that include starch, protein, and cellulose. Further in vitro tests ensured that the potential isolate with probiotic attributes could tolerate different gut conditions, which included a range of pH, salinity, and varying concentrations of bile salt. Exposure of 4 days post fertilization zebrafish embryos to the RFP-tagged isolate confirmed the colonization of B. subtilis in the gut of the zebrafish embryo, which is an important attribute of a probiotic. The isolate was able to inhibit both A. hydrophila and E. tarda in gnotobiotic zebrafish embryo in triplicate. The study demonstrates the probiotic characteristics of the B. subtilis isolated from L. rohita and its ability to inhibit A. hydrophila and E. tarda using in vitro conditions and in the zebrafish gut and could serve as an effective alternative to antibiotics in aquaculture.

Evaluation of loop-mediated isothermal amplification assay for visual detection of Acinetobacter baumannii directly from soil and water sample from Mangalore.

Acinetobacter baumannii is a well-known nosocomial pathogen that commonly inhabits soil and water and has been implicated in numerous hospital-acquired infections. The existing methods for detecting A. baumannii have several drawbacks, such as being time-consuming, expensive, labor-intensive, and unable to distinguish between closely related Acinetobacter species. Thus, it is important to have a simple, rapid, sensitive, and specific method for its detection. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay using hydroxynaphthol blue dye to visualize A. baumannii by targeting its pgaD gene. The LAMP assay was performed using a simple dry bath and was shown to be specific and highly sensitive, as it could detect up to 10 pg/µl of A. baumannii DNA. Further, the optimized assay was used to detect A. baumannii in soil and water samples by culture-medium enrichment. Out of 27 samples tested, 14 (51.85%) samples were positive for A. baumannii through LAMP assay, while only 5 (18.51%) samples were found to be positive through conventional methods. Thus, the LAMP assay has been found to be a simple, rapid, sensitive, and specific method that can be used as a point-of-care diagnostic tool for detecting A. baumannii.

Evaluation of reverse transcriptase-polymerase spiral reaction assay for rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2.

BACKGROUND AND AIM: Existing real-time reverse transcriptase PCR (RT-qPCR) has certain limitations for the point-of-care detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since it requires sophisticated instruments, reagents and skilled laboratory personnel. In this study, we evaluated an assay termed the reverse transcriptase-polymerase spiral reaction (RT-PSR) for rapid and visual detection of SARS-CoV-2. METHODS: The RT-PSR assay was optimized using RdRp gene and evaluated for the detection of SARS-CoV-2. The time of 60min and a temperature of 63°C was optimized for targeting the RNA-dependent RNA polymerase gene of SARS-CoV-2. The sensitivity of the assay was evaluated by diluting the in-vitro transcribed RNA, which amplifies as low as ten copies. RESULTS: The specific primers designed for this assay showed 100% specificity and did not react when tested with other lung infection-causing viruses and bacteria. The optimized assay was validated with 190 clinical samples in two phases, using automated RTPCR based TrueNat test, and the results were comparable. CONCLUSIONS: The RT-PSR assay can be considered for rapid and sensitive detection of SARS-CoV-2, particularly in resource-limited settings. To our knowledge, there is as yet no RT-PSR-based kit developed for SARS-CoV-2.

Evaluation of loop-mediated isothermal amplification assay along with conventional and real-time PCR assay for sensitive detection of pathogenic Vibrio parahaemolyticus from seafood sample without enrichment.

The primary reason for foodborne illness is improper seafood safety testing, and hence, an appropriate tool for testing is the key to control the outbreaks. The current study aimed to develop a loop-mediated isothermal amplification (LAMP) assay to detect pathogenic Vibrio parahaemolyticus, important foodborne pathogen, targeting tdh, and trh genes. The specificity of the LAMP assay was good without any false-positive and false-negative results. The assay was highly sensitive and could detect the pathogenic V. parahaemolyticus as low as 1 CFU/reaction in spiked seafood samples and 1 pg of extracted DNA. Out of 62 seafood samples from India's southwest coastal region tested with LAMP assay, eight (12.9%) were positive for trh, and seven (11.29%) samples were positive tdh gene. LAMP-based on tdh and trh was found to be significantly more sensitive (p < 0.05) than conventional PCR and nearly equal sensitive as real-time PCR (RT-PCR) for the detection of pathogenic V. parahaemolyticus. Our study shows that LAMP assay can be a better approach as a point-of-care (POC) diagnostic tool and could detect pathogenic V. parahaemolyticus on seafood samples directly without enrichment and isolation. The high sensitivity and simplicity make LAMP assay a better alternative method than the conventional method and RT-PCR for the detection of pathogens. LAMP assay can be considered as a good alternative to PCR for the routine detection of pathogenic V. parahaemolyticus in seafood.

Rapid visual detection of Vibrio parahaemolyticus in seafood samples by loop-mediated isothermal amplification with hydroxynaphthol blue dye.

The detection and monitoring of Vibrio parahaemolyticus pathogen in aquatic foods have become essential for preventing outbreaks. In this study, loop-mediated isothermal amplification (LAMP) assay with the azo dye, hydroxynaphthol blue (HNB) was developed targeting species-specific tlh gene. The assay was carried out on 62 seafood samples that included clam and shrimp and compared with conventional LAMP assay performed with the commonly used fluorescent dye, conventional PCR, and real-time PCR (RT-PCR). Of 62 samples studied for tlh gene, 32 (51.61%) gave positive by HNB-LAMP, which comprised 22 (70.96%) clam samples and 10 (32.25%) shrimp samples. The HNB-LAMP assay was found to be highly sensitive, specific, and superior to conventional PCR (p > 0.05). RT-PCR presented higher sensitivity than HNB-LAMP; however, it has the limitation of being cost-intensive and requiring technical expertise to perform. HNB-LAMP is affordable, rapid, simple, and easy to perform, allowing naked eye visualization.