One Step Rapid Sensitive Method for the Diagnosis of Hemolysin Gene of Aeromonas hydrophila by Polymerase Chain Reaction.

Aeromonas hydrophila is a Gram-negative bacterium that has been linked to serious illnesses in both humans and animals. The presence of hemolysin, a virulence factor, is critical in the development of A. hydrophila-related illnesses. As a result, precise and timely detection of the hemolysin gene is critical for efficient diagnosis and prevention of many illnesses. The PCR is used in this study to detect the hemolysin gene of A. hydrophila in a novel, fast, and highly sensitive one-step technique. Specific primers were constructed to amplify a conserved area within the hemolysin gene to achieve both specificity as well as sensitivity. The PCR assay was rigorously optimized, taking temperature, primer concentration, and reaction time into account, in order to maximize the efficiency and reliability of this method. In conclusion, this method's simplicity, sensitivity, and specificity make it highly promising for regular diagnostic applications. Its application would allow for the early detection of A. hydrophila infections, allowing for more effective treatment and control methods.

Facile Synthesis of Benzimidazoles via Oxidative Cyclization of Acyclic Monoterpene Aldehyde with Diamines: Studies on Antimicrobial and in Vivo Evaluation of Zebrafish.

Citral (1a), a bioactive component of Cymbopogon citratus (lemongrass) could be isolated and semi-synthetic analogs synthesized with improved therapeutic properties. Herein we first report describes citral (1a) as a primary material for the synthesis of benzimidazole derivatives between various o-phenylenediamines (2a-l) in the presence of Diisopropylethylamine (DIPEA) as a commercially available environmentally benign base, ethanol as a green solvent and the yield of all benzimidazole derivatives (3a-l) was between 68-76 %; The semi-synthetically prepared benzimidazole derivatives (3a-l) were assessed for their anti-bacterial and anti-fungal properties. The benzimidazole compounds (3a-b, and 3g-j) exhibit good anti-microbial activity. In addition, in silico study was carried out to determine the specific binding affinity of the diamine halogen substituted benzimidazole derivatives to the specific target proteins. In silico analysis revealed a high correlation between docking results and experimental results. Finally, benzimidazole demonstrated significant antibacterial and antifungal activity. Zebrafish embryos were subjected to In vivo toxicological test found that all of the benzimidazole compounds (3a-l) were non-toxic and had low embryotoxicity after 96 h, with an LC50 of 36.425 µg, which could facilitate the design of novel antimicrobial agents using a cost-effective method.

Sustainable development through the bio-fabrication of ecofriendly ZnO nanoparticles and its approaches to toxicology and environmental protection.

Mosquito control is becoming more difficult as a result of the rise in resistance to toxic chemical insecticides. The insecticides of bio-fabrication sources may serve as a convenient alternative to environmentally acceptable methods in the future. The larvicidal and pupicidal activities of bio-fabricated zinc oxide nanoparticles (ZnO NPs) on the different instar larvae and pupae of Anopheles subpictus Grassi (Malaria vector) and Culex quinquefasciatus Say (lymphatic filariasis) were investigated in this study. The results recorded from XRD, FTIR, SEM-EDX, and TEM analyses confirmed the bio-fabrication of ZnO NPs. Such nanoparticles were nearly spherical and agglomerated with a size of 34.21 nm. GC-MS analysis of methanol extract revealed the compound, stigmasterol (C29H48O) as major one. Mosquito larvae and pupae of targeted mosquito were tested against varied concentrations of the bio-fabricated ZnO NPs and methanol extract of Vitex negundo for 24 h. The maximum activity was recorded from ZnO NPs against the larvae and pupae of A. subpictus LC50 which were 1.70 (I), 1.66 (II), 1.93 (III), 2.48 (IV), and 3.63 mg/L (pupa) and C. quinquefasciatus LC50 were 1.95 (I), 2.63 (II), 2.90 (III), 4.32 (IV), and 4.61 mg/L (pupa) respectively. ZnO NPs exhibited strong DPPH radical and FRAP scavengers compared to the aqueous extract of V. negundo. Also, V. negundo leaf methanol extract (VNLME) and ZnO NPs were evaluated for their cytotoxicity on HeLa cells, which exhibited the IC50 values of 72.35 and 43.70µg/mL, respectively. The methylene blue (MB) dye, which is harmful to both aquatic and terrestrial life, was degraded using the biosynthesized ZnO nanoparticles. At 664 nm, 81.2% of the MB dye had degraded after 120 min of exposure to sunlight. Overall, our results revealed that ZnO NPs are the perfect biological agent and economical for the control of malaria, filariasis vectors, antioxidant, HeLa cells, and MB blue dye degradation under sunlight irradiation.

Development and characterization of five novel cell lines from snubnose pompano, Trachinotus blochii (Lacepede, 1801), and their application in gene expression and virological studies.

Five novel permanent cell lines have been established from gill, heart, kidney, eye and fin of snubnose pompano, Trachinotus blochii. They were designated as snubnose pompano gill (SPG), snubnose pompano heart (SPH), snubnose pompano kidney (SPK), snubnose pompano eye (SPE) and snubnose pompano fin (SPF), respectively. All these cell lines were characterized and cryopreserved successfully at different passage levels. Cell lines were passaged every alternate day; SPG, SPH, SPK, SPE and SPF cell lines attained passage levels of 68, 74, 82, 79 and 106, respectively, since the initiation of their development in 2019. The cell lines grew well in Leibovitz's 15 medium containing 15% foetal bovine serum at 28°C. Immunophenotyping of the cell lines revealed the presence of fibronectin and pancytokeratin. No mycoplasma contamination was found. The transfection study revealed the gene expression efficiency of these cell lines by expressing the green fluorescent protein (GFP). The authentication on origin of cell lines from T. blochii was confirmed by amplification of species-specific mitochondrial cytochrome oxidase I gene. The results showed the susceptibility of these cell lines to fish nodavirus (FNV) and tilapia lake virus (TiLV) and resistance to cyprinid herpesvirus 2 (CyHV-2). The FNV infection in the cell lines was confirmed by RT-PCR, Western blot, ELISA and immunocytochemistry, while TiLV infection was confirmed by RT-PCR assay. These results revealed that these cell lines are suitable for virological and foreign gene expression studies.

Field-Usable Lateral Flow Immunoassay for the Rapid Detection of White Spot Syndrome Virus (WSSV).

BACKGROUND: White spot disease (WSD), a major threat to sustainable aquaculture worldwide, is caused by White spot syndrome virus (WSSV). The diagnosis of WSD relies heavily on molecular detection of the virus by one-step PCR. These procedures are neither field-usable nor rapid enough considering the speed at which the virus spreads. Thus, development of a rapid, reliable and field-usable diagnostic method for the detection of WSSV infection is imperative to prevent huge economic losses. METHODS/PRINCIPAL FINDINGS: Here, we report on the development of a lateral flow immunoassay (LFIA) employing gold nanoparticles conjugated to a polyclonal antibody against VP28 (envelope protein of WSSV). The LFIA detected WSSV in ~20 min and showed no cross-reactivity with other shrimp viruses, viz. Monodon Baculovirus (MBV), Hepatopancreatic parvovirus (HPV) and Infectious Hypodermal and Hematopoietic Necrosis virus (IHHNV). The limit of detection (LOD) of the assay, as determined by real-time PCR, was 103 copies of WSSV. In a time course infectivity experiment, ~104 WSSV particles were injected in Litopenaeus vannamei. The LFIA could rapidly (~ 20 min) detect the virus in different tissues after 3 h (hemolymph), 6 h (gill tissue) and 12 h (head soft tissue, eye stalk, and pleopod) of infection. Based on these findings, a validation study was performed using 75 field samples collected from different geographical locations in India. The LFIA results obtained were compared with the conventional "gold standard test", viz. one-step PCR. The analysis of results in 2x2 matrix indicated very high sensitivity (100%) and specificity (96.77%) of LFIA. Similarly, Cohen's kappa coefficient of 0.983 suggested "very good agreement" between the developed LFIA and the conventional one-step PCR. CONCLUSION: The LFIA developed for the rapid detection of WSSV has an excellent potential for use in the field and could prove to be a boon to the aquaculture industry.