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The available fully sequenced genome and genetic similarities compared to humans make zebrafish a prominent in vitro vertebrate model for drug discoveryscreening, toxicology, and radiation biology. Zebrafish also possess well developed immune systems which is ideal for studying infectious diseases. Fish skin confers immunity by serving as a physical barrier against the invading pathogens in the aquatic habitat. Therefore in vitro models from the skin tissue of zebrafish help to study the physiology, functional genes in vitro, wound healing, and pathogenicity of microbes. Hence the study aimed to develop and characterize a skin cell line from the wild-type zebrafish Danio rerio.A novel cell line designated as DRS (D. rerio skin) was established and characterized from the skin tissue of wild-type zebrafish, D. rerio, by the explant technique. The cells thrived well in the Leibovitz's -15 medium supplemented with 15% FBS and routinely passaged at regular intervals. The DRS cells mainly feature fibroblast-like morphology. The culture conditions of the cells were determined by incubating the cells at varying concentrations of FBS and temperature; the optimum was 15% FBS and 28 °C, respectively. Cells were cryopreserved and revived with 70-75% viability at different passage levels. Two extracellular products from bacterial species Aeromonas hydrophila and Edwardsiella tarda were tested and found toxic to the DRS cells. Mitochondrial genes, namely COI and 16S rRNA PCR amplification and partial sequencing authenticated the species of origin of cells. The modal diploid (2n) chromosome number of the cells was 50. The cell line DRS was found to be free from mycoplasma. The cells were transfected with pMaxGFP plasmid and tested positive for green fluorescence at 24-48 h post-transfection.The findings from this study thus confirm the usefulness of the developed cell line in bacterial susceptibility and transgene expression studies.

Nanoparticles (NPs) are small particles made of single or multiple materials having at least one dimension in nanoscale ranging from 1 to 100 nm. The outstanding features of NPs, such as larger surface area and high particle number per mass unit as compared to their bulk material, superior mechanical performance, possibility of surface functionalization makes them perfect candidate for wide applications. The main factors that make the NPs significantly different from the larger size material are the surface effects and quantum effects. These effects provide them many enhanced features in terms of mechanical, thermal, magnetic, electronic, optical, and catalytic properties. NPs can be of different shapes such as spherical, conical, cylindrical, tubular, and spiral. They are generally classified into three groups, organic, carbon-based, and inorganic NPs based on their composition. Organic NPs are made of proteins, carbohydrates, lipids, polymers, or any other organic compounds. Carbon-based NPs are made exclusively from carbon atoms while the inorganic NPs (for example metal NPs) are prepared differently from the other two classes.

ICAR-Directorate of Coldwater Fisheries Research, Bhimtal, Uttarakhand-263136

Indian leopards kept in zoos are fed solely on carabeef on bone (CBB) diets. Carabeef contains lesser or no carotenoids. Hence, the captive Indian leopard diets are suspected to be deficient in carotenoids while their wild counterparts acquire these pigments from their natural prey. Lutein is a vital carotenoid that plays its role as an antioxidant and immunomodulator. This experiment investigates the effect of lutein supplementation on antioxidant status, immunity, and stress in captive Panthera fusca fed CBB diets. Nine leopards were used based on 3 × 3 replicated Latin square designs in the experiment. Groups CON, LUT20, and LUT40 were supplemented with 0, 20, and 40 ppm of lutein, respectively. Each experiment comprised of 10 days of wash-out period, 11 days of adaptation, and 4 days of collection. Digestibility of crude protein (CP) was higher (p .01) in groups LUT20 and LUT40. Serum concentration of protein, globulin, urea (p .05), total carotenoids, total antioxidant capacity (TAC), catalase (CAT) activity, and lymphocyte transformation test (LTT) index were higher (p .001) in groups LUT20 and LUT40. Activity of superoxide dismutase (SOD) and serum concentration of immunoglobulin were higher (p .001) in group LUT20. Serum concentration of malonaldehyde (MDA) and fecal concentration of cortisol decreased (p .001) in groups LUT20 and LUT40. Serum concentration of total immunoglobulin (µg/ml) and LTT were higher in group LUT20. Fecal concentration of cortisol (ng/g) was lower in LUT20 and LUT40. The study concludes that supplementation of lutein at 20 ppm would improve antioxidant status and immunity and alleviate stress in captive Indian leopards.

Amphiprion ocellaris (ocellaris clownfish) is one of the most commercially important marine ornamental fish. A cell line designated as OCF was developed for the first time from the caudal fin of this fish species. The cell line was maintained in Leibovitz’s—15 medium supplemented with 15% FBS (Fetal Bovine Serum) and was successfully subcultured up to 34 passages. The cell line was authenticated by sequencing mitochondrial cytochrome C oxidase subunit I (COI) and 16S rRNA genes. The growth rate of the OCF cell line was maximum in medium containing 20% FBS and 1% of 0.2 M NaCl at 28 °C. Chromosome analysis revealed 48 diploid chromosomes. The OCF cell line was transfected with the pMaxGFP plasmid vector with 7% efficiency and GFP expression was observed. The OCF cell line was used for testing nervous necrosis virus (NNV) susceptibility. Cytopathic effect (CPE) was observed in terms of plaque formation after virus inoculation. Nested PCR confirmed the susceptibility of the OCF cell line to NNV. The cell line was successfully cryopreserved by a slow freezing procedure at − 80 °C with a revival efficiency of 70–75%. The study revealed that the OCF cell line would be useful for virological studies. In addition, the cell line would play an important role as an in vitro tool for carrying out toxicological and biotechnological studies.

Antimicrobial peptides (AMPs) are considered alternatives to classical antibiotics and may become an excellent candidate for tackling antimicrobial resistance in aquaculture. Designing novel antimicrobial peptides for curbing antimicrobial resistance in aquaculture is paramount in one health approach. In this study, a short and compositionally simple peptide, KK16, was designed. KK16 is amphipathic with a net charge of + 6. Molecular docking results revealed that KK16 has a strong affinity towards two virulence proteins of Aeromonas sobria; aerolysin and outer membrane protein (omp). The peptide was synthesised using Fmoc-chemistry, and its antimicrobial efficacy was evaluated in vitro against A.sobria, A. salmonicida, Edwardsiella tarda, A. hydrophila, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis and methicillin-resistant S. aureus. The KK16 AMP showed potent activity against the tested bacterial pathogens as revealed by the MIC and MBC, ranging from 7.81 to 500 µM, and 15-900 µM, respectively. Moreover, the peptide was stable at higher temperatures and retained its activity in presence of serum and salt. The peptide displayed less haemolytic and cytotoxic activity even at higher concentrations. In peptide-DNA binding assay, KK16 showed its binding potential with bacterial genomic DNA and thus, may interfere with replication. Fluorescent microscopy revealed the uptake of propidium iodide by peptide treated bacterial cells, indicating its membrane disruption activity. In in vivo experiment, KK16 peptide completely inhibited the growth of Saprolegnia parasitica fungus at ≥ 30 µM peptide concentrations in embryonated fish eggs. The results indicate that KK16 peptide is stable, possess potent antibacterial and antifungal activity, less cytotoxic to host cells, and hence may prove to be a promising anti-infective agent for combating common bacterial and fungal infections.

Aeromonas salmonicida is the obligate pathogen of fishes having zoonotic potential. It is reported to cause considerable losses in world aquaculture. The current study has successfully demonstrated the induction of histopathological lesions in experimentally infected common carp. In the current study, the lethal concentration (LD50-96 h) of typical A. Salmonicida for common carp was found to be 1.5 × 107CFU mL−1. About 40% and 60% fish mortalities occurred after 72 h in the groups inoculated with 107 and 108 CFU mL−1 bacterial suspension, respectively. The fish challenged with A. salmonicida showed symptoms like abnormal swimming behaviour, lethargy, intra-abdominal fluid, haemorrhages on the ventral side of the body, vent and fins. The signs proceeded with the death of fish. In the histological sections, severe pathological alterations were reported in the tissue sections of internal organs. The microscopic observation showed sinusoidal and large blood vessel congestion in the liver, profuse haemorrhage, necrosis and infiltration of blood cells in the internal organs. The tubular architecture was lost with the infiltration of leucocytes in the kidney. In gills, more intense and prominent lamellar fusion was observed with leucocytic infiltration, telangiectasia and hyperplasia of lamellar epithelial cells. In summary, we have experimentally induced the typical A. salmonicida infection in common carp. The study will provide a research foundation for further studies on the host-pathogen interaction, therapeutics and epidemiology of A. salmonicida.

This study aimed to investigate the effectiveness of five natural plant extract compounds Curcumin (CUR); Eugenol (EUG), Cinnamaldehyde (CIN), Stigmasterol (ST) and Morin (MOR), on two species of Saprolegnia; Saprolegnia parasitica and S. australis. Selective compounds were screened for the minimum inhibitory concentration, first for anti-oomycetes activity and then mycelium growth inhibition, spore germination inhibition and colonisation test. Nitric oxide production and myeloperoxidase activity of the compounds were tested in head kidney leukocytes of rainbow trout, Oncorhynchus mykiss to assess the immunostimulatory potential. Molecular docking of effective compounds was carried out with effector proteins of S. parasitica to investigate the target binding sites. Among all, CUR could completely inhibit zoospore production and significantly (p ≤ .05) inhibit hyphal growth at 16 mg l−1 against S. parasitica and S. australis. CIN at the concentration of 50 mg l−1 completely inhibited hyphal growth of both Saprolegnia spp., although the zoospore production of S. parasitica and S. australis was reduced at 25 mg l−1 and 10 mg l−1. In the case of EUG, significant inhibition of the hyphal growth and germination of S. parasitica zoospores was observed at 50 mg l−1. ST and MOR did not show antioomycetes activity. The molecular docking results were consistent with in vitro studies, possibly due to the binding with the vital proteins (Plasma membrane ATPase, V-type proton ATPase, TKL protein kinase, Host targeting protein 1) of S. parasitica and ultimately inhibiting their activity. CUR and CIN showed increased nitric oxide production at the highest concentration of 250 and 256 mg l−1 but the value was not significant (p ≤ .05) with control. CUR showed significantly higher peroxidase activity (p ≤ .05) at a concentration of 256 mg l−1 though values were significantly similar with concentration from 16 to 128 mg l−1. The nitric oxide and total peroxidase activity of rainbow trout leukocytes in the case of CIN showed a significant difference only at 250 mg l−1 against the control. The results conclude that CUR, CIN showed the better anti-Saprolegnia activity and could be used as phyto-additives in aquaculture. Among all, the inclusion of CUR as phyto-additives will provide additional immunostimulatory activity.

In the present study, we have designed and synthesized a short compositionally simple peptide RY12WY having potent antimicrobial activity. The molecular docking study results showed that peptide has a strong affinity towards two protein targets of A. sobria; aerolysin and outer membrane protein (OMP). The MIC values ranged from 0.98 to 500 μM and MBC values ranged from 4 to 650 μM against the selected bacterial and fungal pathogens. The intense antimicrobial activity of RY12WY is reported against A. sobria, A. hydrophila, E. tarda, S. aureus, V. parahaemolyticus, P. aeruginosa and E.coli at low concentration.The peptide also showed good activity against A. salmonicida and S. parasitica zoospores. The peptide retained its antimicrobial activity at higher temperatures. Besides, it was active in the presence of physiological salts and serum.The peptide showed negligible haemolytic activity at 125 μM and HC50 was found to be 1437.10 μM. The DNA binding assay indicated that peptide can bind with the genetic material of the bacteria and may inhibit its replication. The bacterial viability assay reported that the peptide interferes with bacterial membrane integrity. To conclude, the results suggest that RY12WY could be a promising therapeutic agent in aquaculture and has possible application in food processing industry which warrants higher temperatures.

Bioactive compounds present in Parkia javanica seeds are reported to have anticancer effect. The present study was taken up to evaluate the apoptosis inducing ability of methanol extract of Parkia javanica seeds in cancer cells. The extract was found to cause 50 % cell death in HeLa and MCF-7 cells at 0.54 and 0.74 mg/ml, respectively, which is lower than that of normal healthy cells. In haematoxylin-eosin and fluorescent staining of the extract-treated cancer cells, chromatin condensation and nuclear fragmentation were observed. Fluorescent-activated cell sorting analysis using Annexin V-fluorescein isothiocyanate/propidium iodide also revealed the presence of apoptotic and secondary necrotic cell populations in the extract-treated cancer cells. In gel electrophoresis, DNA fragmentation, a biochemical hallmark of apoptosis was also observed. Caspase-3, an enzyme activated both in extrinsic and intrinsic pathways of apoptosis was detected in extract-treated HeLa cells by immunocytochemistry. The results of the present study suggested that Parkia javanica seed has the ability to induce apoptosis in cancer cells and could be considered in future endeavours for development of safer natural anticancer agent.

Kisspeptin 1 is a neuropeptide hormone of the RFamide family, which act as an upstream regulator of brain-pituitary-gonad (BPG) axis in most vertebrates including teleosts. In the present study, a 16 amino acid long putative mature bioactive peptide (kiss 1) from preprokisspeptin 1 of golden mahseer, Tor putitora (Hamilton, 1822), was synthesized and characterized using an integrated (experimental and in silico) approach. The far-UV circular dichroism (CD) spectrum of this peptide was evaluated both in aqueous and membrane mimicking solvents (TFE, HFIP and Dioxane). The results indicate that kiss 1 peptide adopted helical, turn and β conformations in membrane like environments. The near-UV CD spectroscopy was also carried out to examine the tertiary packing around aromatic residues of kiss 1 peptide and the peptide-membrane complex. The kiss 1 peptide exhibited little signal in water, but a prominent negative band was observed at around 275 nm when membrane mimetic solution was added. The observed ordered conformations of kiss 1 peptide in the different solvents indicated its potential biological activity which could enhance the secretion of gonadotropin-releasing hormone (GnRH) at BPG axis. The conformational information generated from the present study reinforces the application prospects of bioactive synthetic peptide analogs of kisspeptin 1 in improving the reproductive performances of important cultivable fish species.

The peptide dendrimer provides novel strategies for various biological applications. Assembling of peptide in macromolecular structure is expected to give rational models as drugs, their delivery and diagnostic reagents. Improved understanding of virus structure and their molecular interactions with ligands have paved the way for treatment and control of emerging and re-emerging viral diseases. This review presents a brief account of a synthetic peptide dendrimer used for diagnostic, therapeutic and prophylactic applications. The designs comprise of multiple antigenic peptides which are being used as alternate synthetic antigens for different viruses.

A rapid label-free visual assay for the detection of viral RNA using peptide nucleic acid (PNA) probes and gold nanoparticles (AuNPs) is presented in this study. Diagnosis is a crucial step for the molecular surveillance of diseases, and a rapid visual test with high specificity could play a vital role in the management of viral diseases. In this assay, the specific agglomerative behavior of PNA with gold nanoparticles was manipulated by its complementation with viral RNA. The assay was able to detect 5-10 ng of viral RNA from various biological samples, such as allantoic fluids, cell culture fluids and vaccines, in 100 μl of test solution. The developed assay was more sensitive than a hemagglutination (HA) test, a routine platform test for the detection of Newcastle disease virus (NDV), and the developed assay was able to visually detect NDV with as little as 0.25 HA units of virus. In terms of the specificity, the test could discriminate single nucleotide differences in the target RNA and hence could provide visual viral genotyping/pathotyping. This observation was confirmed by pathotyping different known isolates of NDV. Further, the PNA-induced colorimetric changes in the presence of the target RNA at different RNA to PNA ratios yielded a standard curve with a linear coefficient of R(2)=0.990, which was comparable to the value of R(2)=0.995 from real-time PCR experiments with the same viral RNA. Therefore, the viral RNA in a given samples could be quantified using a simple visual spectrophotometer available in any clinical laboratory. This assay may find application in diagnostic assays for other RNA viruses, which are well known to undergo mutations, thus presenting challenges for their molecular surveillance, genotyping and quantification.

The innate immune system recognizes pathogens through pattern recognition receptors (PRRs), and toll-like receptors (TLRs) are one of the most important PRRs. TLR3 is a unique member of TLR family that recognizes double-stranded RNA (dsRNA), a viral replication intermediate. There is a variation in its response among diverse fish species toward the same stimulants. We identified and cloned TLR3 from Indian snow trout, Schizothorax richardsonii and carried out its expression analysis in un-induced and poly (I:C) challenged fish. It has an open reading frame (ORF) of 2712 bases that encodes a polypeptide of 904 amino acids. The molecular weight of the polypeptide was predicted to be 102.4482 kDa with an isoelectric point of 7.40. Quantitative real time PCR (qRT-PCR) was carried out after 24 hours of poly (I:C) treatment and expression of TLR3 was analyzed in different tissues. As compared with untreated fish the poly (I:C) challenged fish revealed significantly high expression of TLR3 in kidney followed by liver and gills.

Complete cDNA sequences of kiss1 ( gmkiss1 ) and its receptor kiss1r ( gmkiss1r ) were cloned and characterized from brain tissue of adult golden mahseer ( Tor putitora ). Thereafter, quantification of gmkiss1 and gmkiss1r mRNA expression in brain-pituitary-gonad (BPG) axis of male and female golden mahseer was carried out using quantitative real-time (qRT)-PCR assay during an annual reproductive cycle, at different gonadal development stages. The gmkiss1 cDNA was 508 bp, with 330 bp open reading frame (ORF), encoding a precursor protein of 109 amino acids, whereas gmkiss1r cDNA was 1383 bp with an ORF of 1004 bp, which encodes a 334 amino acid protein residue. The qRT-PCR study shows that gmkiss1 and gmkiss1r are expressed in brain, pituitary and gonads of both the sexes of golden mahseer. An apparent sexual dimorphism in transcript level of gmkiss1 and gmkiss1r in brain and gonads was evident during the reproductive cycle. Overall, in brain, testis and ovary, the gmkiss1 and gmkiss1r mRNA expression level was comparatively higher during the initial stages of gonadal development, than that of spermiation or ovulation stage. In pituitary of both the sexes, throughout the gonadal development, consistently low transcript level of gmkiss1 and gmkiss1r was observed. The gmkiss1 mRNA expression level in brain and ovary of female golden mahseer was several folds higher than the brain and testis of male fish. In conclusion, we confirm the presence of kiss1 and its receptor in golden mahseer, and results of our study strongly suggested the involvement of kisspeptin1 system in gonadal development and annual reproductive cycle of this species.

a) Introduction: Mahseers, belong to the family Cyprinidae, are well renowned for the excellent game as well as food i¬shes. Various aspects simultaneously considered towards the domestication of this fish species also include the study of fish health issues, preventive and remedial programmes for sustainable aquaculture. Experimental studies can be conducted in the control condition to study the pathological symptoms during bacterial infection. Considering the bacterial pathogens as a major constraint for aquaculture, the present study was carried out to standardize the bacterial concentration of A. hydrophila for the experimental challenge of Golden Mahseer for the first time, and various clinical changes were also monitored during the infection. Materials and Methods: A total of 140 live fishes were acclimatized to the laboratory conditions for experimental study. A constant water quality was monitored during the acclimation period and throughout the experiment. The test group was injected with A. hydrophila and various physiological, biochemical and tissue level changes were observed during the experiment in comparison to phosphate buffer saline (PBS) injected control group. b) Results: LD50 value of A. hydrophila for Golden Mahseer was successfully standardized. Clinical signs including weakness, slower movement, swimming closer to the surface, fin haemorrhages and red patches at the gut region were observed. Enlargement of spleen followed by tissue necrosis along with signs of haemorrhagic septicaemia was also seen in infected fishes. The necrosis of hepatocytes was observed in the histological section of liver tissue. The bacterial infection increases the superoxide dismutase activity and cortisol level in Golden Mahseer. c) Conclusion: The LD50 value of A. hydrophila for experimental challenge studies on Golden Mahseer is 1.74 × 105 cfu per 100 g of body weight. The symptoms of severe bacterial haemorrhage were observed. Necrosis of hepatocytes was observed in infected fishes. The superoxide dismutase activity and cortisol level also increased in infected fishes. A. hydrophila was confirmed to be the etiological agent which was re-isolated using spread plate method and confirmed by 16s rRNA sequencing.

Mammalian beta-defensins are innate host defense peptides with potential as a therapeutic agent. Present study reports expression of beta-defensin mRNA transcript in teat mucosal layer of Bubalus bubalis, which is homologous to lingual antimicrobial peptide (LAP) of cattle. The PCR-amplified and cloned full-length cDNA encodes for a putative peptide of 64 residues ( approximately 7 kDa) with pI of 11.324 and possess characteristic features of beta-defensin family including presence of six evolutionary conserved cysteine residues. The first 21 amino acids constitute a putative signal peptide, suggesting that it is a secretory protein. The peptide shares 85%-100% sequence identity with beta-defensins of cattle (Bos taurus). Nonetheless, the maximum identity at nucleotide (100%) and amino acid (98.5%) level was with cattle LAP, which is in consistent with phylogeny analysis report. Hence, the cloned beta-defensin has been designated as buffalo LAP. This study envisages role of beta-defensin peptides in innate immunity of mammary gland.

Development and evaluation of various experimental animal models for study of various aspects of human and animal cancers is a continuous requirement in cancer research. In the present study, pathology of LA7 cell induced tumours were studied in 50 female Sprague Dawley rats of about 30 days of age which were divided into two groups. Group A (experimental) included 36 rats which were administered with LA7 cells at the dose rate of 6x106 cells in 200μl subcutaneously. Group B (n = 14) served as control. After development of palpable tumours, 3 animals each were sacrificed at three days interval till 30 days and three animals were sacrificed on 45th day. Two animals each were sacrificed at weekly interval from the control group. During 45 days of experimental period, total 33 (91.66%) mammary tumours were induced in Group A. The average number of tumours per animal (Tumour frequency) was found to be 1.0 tumour. The appearance of first tumour was observed at 3rd to 7th day post injection (DPI) of cells. The average latency period was found to be 4.82 ± 0.29 day. Out of 33 induced tumours, 12 tumours were growing and 11 regressing and 10 were of regressed type. Within the experiment period, 10 tumours completely regressed after attaining growth stage and were found to regress to original detectable size or smaller, as early as on 9th DPI and as late as on 27th DPI. Histologically, the tumours were mostly undifferentiated or poorly differentiated carcinomas. Only five tumours showed either distinct acinar pattern or cystic appearance. The tumour induction with LA7 cells was found to be very fast with very high rate of induction. However, due to autoregressive nature of tumour, use of this model for different experiments need to be done cautiously.