Gaurav Rathore

Gene cloning, expression and homology modeling of hemolysin gene from Aeromonas hydrophila.

Hemolysin is a significant toxin secreted by Aeromonas hydrophila, which contributes pathogenicity of fish to humans. The complete ORF of hemolysin gene (1886 bp) was amplified using PCR. It was cloned in TA and sub-cloned in pET28a vector then transformed into Escherichia coli BL21(DE3) codon plus RP cells expressed by the induction with 1.0 mM of IPTG. The expected size of expressed protein was 68.0 kDa estimated by migration in 12% SDS-PAGE. Anti-His monoclonal antibodies were used to substantiate the recombinant protein by Western blotting. The percent similarity between hemolysin of A. hydrophila with other hemolytic toxins revealed that the hemolysin/aerolysin/cytotoxin sequence varied from 99.35 to 50.40%. Homology modeling was used to construct 3-D structure of hemolysin of A. hydrophila with the known crystal 3-D structure (PDB: 1XEZ). This protein can be used for immunoassays and it is suitable for vaccine candidate against A. hydrophila infection.

Detection of aerolysin gene in Aeromonas hydrophila isolated from fish and pond water

Aerolysin is a hemolytic toxin encoded by aerolysin gene (1482 bp) that plays a key role in the pathogenesis of Aeromonas hydrophila infection in fish. New speciesspecific primers were designed to amplify 326 bp conserved region of aerolysin gene for A. hydrophila. Twenty-five isolates of A. hydrophila recovered from fish and pond water were studied for detection of aerolysin gene. Aerolysin gene was detected in 85% of the isolates during the study. The designed primers were highly specific and showed no cross reactivity with Escherichia coli, Aeromonas veronii, Vibrio cholerae, Flavobacterium spp., Chyseobacterium spp. and Staphylococcus aureus. The sensitivity limit of primers for detection of aerolysin gene in the genomic DNA of A. hydrophila was 5 pg.

Development of monoclonal antibodies to rohu [Labeo rohita] immunoglobulins for use in immunoassays.

Serum immunoglobulins [Ig] of rohu [Labeo rohita] were purified by affinity chromatography using bovine serum albumin as capture ligand. The purified rohu Ig [r-Ig] had a molecular weight [MW] of 880 kDa as determined with gel filtration chromatography. The heavy chain of r-Ig had an MW of 77.8 kDa and that of light chain was 26.4 kDa in SDS-PAGE. Purified r-Ig was used for the production of two anti-rohu Ig monoclonal antibodies [D7 and H4] that belonged to subclass IgG2b and IgG1, respectively. Both the MAbs were specific to heavy chain of r-Ig as seen in Western blotting. Anti-rohu Ig MAb was used as a diagnostic reagent in ELISA and immunocytochemical assays to demonstrate its application for sero-surveillance and for immunological studies in rohu. A competitive ELISA was used to demonstrate the antigenic relatedness of r-Ig with whole serum Ig of other fish species. Cross reactivity of anti-rohu Ig MAb was observed with serum Ig of Catla catla and Cirrihinus mrigala. No reactivity to serum Ig of Ophiocephalus striatus and Clarias gariepinus was seen. Anti-rohu Ig MAb was found to be suitable for the detection of pathogen specific [Edwardsiella tarda] antibodies in serum of immunized rohu by an indirect ELISA. In flow cytometry using D7 MAb, the mean percentage [+/-SE] of Ig positive cells in spleen and blood of rohu were found to be 64.85% [+/-2.34] and 51.84% [+/-2.55] of gated lymphocytes, respectively. Similarly, D7 MAb also stained 52.84% [+/-1.30] and 10.5% of gated lymphocytes in kidney and thymus, respectively. The anti-rohu Ig MAbs also showed specific staining of Ig bearing cells in spleen sections by the indirect immunoperoxidase test.

Development and characterization of three new diploid cell lines from Labeo rohita (Ham.).

Development of cell lines from fish for identifying the pathogenesis of viral diseases and for vaccine production against viral and bacterial diseases is imperative where they are of commercial importance. Three new diploid fish cell lines (RF, RH, and RSB) were developed from fin, heart, and swim bladder of an Indian major carp, Labeo rohita, commonly called Rohu. All the cell lines were optimally maintained at 28°C in Leibovitz‐15 medium supplemented with 10% FBS. The propagation of RH and RSB cells was serum dependent, with a low plating efficiency (<16%), whereas RF cells showed 20% efficiency. The cytogenetic analysis revealed a diploid count of 50 chromosomes. The cells of RF and RSB were found to be epithelial, where as the cells of RH were mostly fibroblastic. The viability of the RF, RH, and RSB cell lines was 75, 70 and 72%, respectively after 6 months of storage in liquid nitrogen. The origin of the cell lines was confirmed by the amplification of 496 and 655 bp fragments of 16S rRNA and Cytochrome Oxidase Subunit I (COI) of mtDNA. The new cell lines would facilitate viral disease diagnosis and genomic studies.

Proteomic analysis of outer membrane proteins of Edwardsiella tarda

Aims:  The purpose of this study was to identify outer membrane proteins (OMPs) of Edwardsiella tarda.

Establishment of caudal fin cell lines from tropical ornamental fishes Puntius fasciatus and Pristolepis fasciata endemic to the Western Ghats of India.

Two new cell lines, PFF and CFF were established from the caudal fin of the Puntius fasciatus, and Pristolepis fasciata respectively. Since their initiation, these cell lines (PFF and CFF) have been subcultured in L-15 medium with 10% fetal bovine serum for more than 35 passages at 28°C and both the cell lines were characterized. Karyotyping analysis of PFF and CFF cells at 25th passage indicated that the modal chromosome number was 2n=50 and 2n=48 respectively. The cell line was cryopreserved in liquid nitrogen at -196°C and could be recovered from storage after six months with good cell viability. Polymerase chain reaction amplification of the fragments of two mitochondrial genes, 16S rRNA and COI confirmed that the cell lines originated from the respective species. The bacterial extracellular products from Vibrio cholerae MTCC3904 and Aeromonas hydrophila were found to be toxic to PFF and CFF. Both the cells were resistant to the marine viral nervous necrosis virus (VNNV). No CPE could be found in both cell lines inoculated with the fish samples and cell culture supernatants were demonstrated free of SVC, iridovirus and KHV by molecular methods. These results indicated the absence of SVC, iridovirus and KHV in the ornamental fishes collected from the Western Ghats of India.

Development and characterization of two new cell lines from common carp, Cyprinus carpio (Linn)

Two new cell lines (CCF and CCH) were established from fin and heart tissues of common carp, Cyprinus carpio. The cells were optimally maintained in Leibovitz-15 medium supplemented with 10% fetal bovine serum (FBS) and 10 ng/ml of basic fibroblastic growth factor (bFGF). The effects of temperature, concentration of FBS and bFGF on the growth of CCF and CCH cells were examined. The temperature ranged from 24 to 32°C for good growth of the cells. The growth rate of cells was higher in medium containing 10% FBS and the addition of bFGF to the medium significantly increased the growth rate. The CCF cells were found to be epithelial, while the CCH cells were fibroblastic in nature. The cytogenetic analysis of the cell lines revealed a diploid number of 100 chromosomes in C. carpio. The viability of CCF and CCH cell lines were 70 and 72%, respectively, after six months of storage in liquid nitrogen (-196° C). Molecular characterization of the cell lines using 16S rRNA and Cytochrome Oxidase Subunit I (COI) revealed the origin of the cell lines. These new cell lines will be useful for isolation of fish viruses and other in vitro biotechnological studies.

Monoclonal antibodies to snakehead, Channa striata immunoglobulins: Detection and quantification of immunoglobulin-positive cells in blood and lymphoid organs

Snakehead Channa striata is an important freshwater food fish in many Southeast Asian countries. Three monoclonal antibodies (C9, C10 and D10) were developed against purified serum immunoglobulins of Channa striata (Cs-Ig) and characterized. C9 and D10 MAbs were specific to heavy chain, while C10 MAb detected only unreduced Cs-Ig in western blotting. In competitive ELISA, C9 and C10 MAbs were specific to C. striata Ig and showed no cross reactivity with serum Ig of other fish species i.e. Channa punctatus, Channa marulius, Clarias batrachus and Labeo rohita. D10 MAb showed reactivity to serum Ig of C. striata and C. marulius. In FACS analysis of gated lymphocytes, the percentage of Ig+ cells detected by C9 MAb was 18.2%, 27.7% and 10.3% in blood, spleen and kidney, respectively (n=3, body weight 500-600 g). However, only a few cells (0.5%) were found to be Ig+ in thymus (n=5). C9 MAb was also successfully employed to demonstrate Ig+ cells in blood smears and formalin fixed sections of spleen and kidney. These findings suggest that the spleen plays an important role in humoral immunity as compared to head kidney. Further, these MAbs can be useful immunological tool in monitoring health status of cultured C. striata.

Production of monoclonal antibodies specific to major outer membrane protein of Edwardsiella tarda

Edwardsiella tarda is an important cause for hemorrhagic septicemia in fish and gastro and extra-intestinal infections in humans. Monoclonal antibodies (MAbs) were produced against outer membrane proteins (OMPs) of E. tarda ET-7, isolated from diseased snakehead (Ophiocephalus punctatus). Two stable hybridoma clones, designated as 3F10 and 2C3 MAbs were found to be potentially specific for E. tarda by indirect enzyme linked immunosorbent assay (ELISA). These MAbs recognized major immunogenic OMP band at 44kDa in Western blotting. Both MAbs belonged to the IgG1 isotype and recognized different epitopes of OMP as seen by competitive ELISA. These MAbs strongly reacted with all 17 isolates of E. tarda used in our study by indirect ELISA and Western blotting. Interestingly, no reaction was observed with the reference strain of E. tarda (MTCC 2400). The sensitivity of 3F10 MAb to detect whole cells of E. tarda was up to a level of 1x10(4)CFU/ml in indirect ELISA. No cross-reactivity of MAbs were seen with Escherichia coli, Salmonella arizonae, Pseudomonas fluorescens, Aeromonas hydrophila, Vibrio cholerae, Flavobacterium ferrugineum and Mycobacterium tuberculosis. These MAbs could be used for specific detection of E. tarda infection in fish by immunoassays.

Establishment and characterization of an epithelial cell line from thymus of Catla catla (Hamilton, 1822).

A cell line, CTE, derived from catla (Catla catla) thymus has been established by explant method and subcultured for more than 70 passages over a period of 400 days. The cell line has been maintained in L-15 (Leibovitz) medium supplemented with 10% fetal bovine serum. CTE cell line consists of homogeneous population of epithelial-like cells and grows optimally at 28°C. Karyotype analysis revealed that the modal chromosome number of CTE cells was 50. Partial amplification, sequencing and alignment of fragments of two mitochondrial genes 16S rRNA and COI confirmed that CTE cell line originated from catla. Significant green fluorescent signals were observed when the cell line was transfected with phrGFP II-N mammalian expression vector, indicating its potential utility for transgenic and genetic manipulation studies. The CTE cells showed strong positivity for cytokeratin, indicating that cell line was epithelial in nature. The flow cytometric analysis of cell line revealed a higher number of cells in S-phase at 48 h, suggesting a high growth rate. The extracellular products of Vibrio cholerae MTCC 3904 were toxic to the CTE cells. This cell line was not susceptible to fish betanodavirus, the causative agent of viral nervous necrosis in a large variety of marine fish.