Megha Kadam Bedekar

Molecular cloning, characterisation and expression analysis of melanoma differentiation associated gene 5 (MDA5) of green chromide, Etroplus suratensis.

Innate immune system recognises pathogen-associated molecular patterns (PAMPs) by limited number of germline encoded and non-clonally developed pathogen recognition receptors (PRRs). Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are important cytosolic PRRs for sensing viral RNAs. The receptor encoded by melanoma differentiation associated gene 5 (MDA5), an RLR, recognises viral RNA and enhances antiviral response in host cells. The full-length MDA5 cDNA in Etroplus suratensis was cloned and found to have 3673 nucleotides encoding a polypeptide of 978 amino acids. The deduced amino acid sequence contains four main structural domains: two CARD domains in the N-terminal region, a DExDc (DEAH/DEAD box helicase domain), HELICc (C-terminal helicase) domain and a C-terminal regulatory domain (RD). Phylogenetic analysis revealed a close relationship of E. suratensis MDA5 (EsMDA5) with MDA5 of Neolamprologus brichardi and Oreochromis niloticus, both belonging to Cichlidae family. EsMDA5 transcripts were ubiquitously expressed in all the 12 tissues tested in healthy fish. Although, transcript level was found to be the highest in muscle, high expression was also detected in the spleen, head kidney and hindgut. In poly I:C-injected fish, EsMDA5 transcripts showed peak expression in the spleen, intestine and heart at 12h post-injection (hpi). However, in gill and kidney tissues, maximum up-regulation of EsMDA5 was observed at 6 and 48 hpi, respectively. Further, liver tissue showed an increasing trend in expression profile from 6 to 48 hpi. Interferon promoter stimulator-1 (IPS-1) gene, an adaptor triggering RIG-I- and MDA5-mediated type I interferon induction, also showed up-regulated expression at initial time-points in poly I:C-injected E. suratensis. The constitutive expression and up-regulation of EsMDA5 and the IPS-1 genes in different tissues indicate that EsMDA5 may play an important role in sensing viral PAMPs in conjunction with IPS-1.

Tissue specific expression profile of some immune related genes in Labeo rohita to Edwardsiella tarda infection

Abstract Rohu (Labeo rohita), an Indian Major Carp (IMC) is an economically important aquaculture species in India. Inspite of the technological advances, infectious diseases caused by viruses, bacteria and parasites have been a major limiting factor in the development and profitability of fish farms. At present, information regarding the immune status of the Indian major carps is limited. This lack of knowledge is a major impediment for establishment of effective preventive measures against broad spectrum of infectious agents. The present study was undertaken to examine the modulation of few immune‐regulatory genes: IgHC, NOD 1, TLR 22, iNOS and IL‐1&bgr; during experimental infection of E. tarda in L. rohita to understand their role in pathogenesis. Rohu fingerlings were intra‐peritoneally injected with Edwardsiella tarda (LD50 dose of 8.7 × 104 CFU/fish) and sampled for three immunologically important organs (kidney, liver and spleen) at different time intervals (zero hour or pre‐challenge and 6 h, 12 h, 24 h, 48 h and 96 h post challenge). For absolute quantification of genes by real time RT‐PCR, all the genes transcript were amplified from Poly I:C induced rohu lymphocytes and cloned in pTZ57R/T plasmid. Standard curves for each gene was generated from serially diluted plasmid bearing respective genes. Evaluation of copy number of different genes present in the tissue showed that the expression of IgHC, iNOS and IL‐1&bgr; was highest in kidney followed by spleen and least in liver. While for NOD 1 and TLR 22 gene, liver showed higher expression than kidney and spleen. Further, the expression of IgHC, INOS, TLR 22, NOD 1 and IL‐1&bgr; genes significantly differed (P < 0.05) in the E. tarda challenged fish when compared with pre‐challenged control fish. Among the five genes we studied, the basal expression of TLR 22 gene was highest. The result also depicts that iNOS and NOD 1 are immediate responsive genes as their expression reached maximum level at 6–24 h post infection (hpi) after which the expression declined. In contrast, TLR 22 and IgHC gene transcript showed enhanced expression during the late phase of with maximum expression observed after 48 hpi and 96 hpi respectively. IL‐1&bgr;, being the exception, showed high expression both at 24 hpi and 96 hpi. From this study, we conclude that these five immune genes have a definite role to play in the defense mechanism of host (L. rohita) against E. tarda. HighlightsPathogenesis related to Edwardsiella tarda on Labeo rohita was studied.E. tarda exposure modulates TLR22, NOD1, IgHC, iNOS, IL‐1&bgr; expression in L. rohita.Involvement of various immune pathways in disease processes was determined.Expression patterns demonstrated orchestration of various organs in host defences.Absolute quantization method used can be a referendum for various gene transcripts.

Monoclonal antibodies against recombinant GAPDH of Edwardsiella tarda reveal the conserved nature of the protein

ABSTRACT The outer membrane protein, encoded by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, of Edwardsiella tarda is a highly conserved immunogenic protein. The GAPDH was cloned and expressed in Escherichia coli. The purified protein was used to produce mouse monoclonal antibodies (MAbs). Four stable hybridomas producing MAbs (3G12, 4E9, 5A11 and 9G1) against rGAPDH were obtained. The heavy chains of antibodies produced by the hybridomas were of the isotypes IgG1 and IgM. Cross reactivity of MAbs (3G12 and 9G1) was observed with GAPDH of Aeromonas hydrophila and Micrococcus luteus. MAbs 3G12 and 4E9 reacted with Vibrio cholerae, Salmonella enterica and Penaeus monodon tissues but not with vertebrate GAPDH. None of the MAbs reacted with Staphylococcus aureus. The results indicate that the level of conservation of GAPDH is high among evolutionarily close species. The MAbs developed will be a useful tool to study the evolutionarily conserved and functionally diverse GAPDH.

Inhibition of Infectious Bursal Disease Virus by Vector Delivered SiRNA in Cell Culture

Infectious Bursal Disease (IBD) is major threat to poultry industry. It causes severe immunosuppression and mortality in chicken generally at 3 to 6 weeks of age. RNA intereference (RNAi) emerges as a potent gene regulatory tool in last few years. The present study was conducted to evaluate the efficiency of RNAi to inhibit the IBD virus (IDBV) replication in-vitro. VP2 gene of virus encodes protein involved in capsid formation, cell entry and induction of protective immune responses against it. Thus, VP2 gene of IBDV is the candidate target for the molecular techniques applied for IBDV detection and inhibition assay. In this study, IBDV was isolated from field cases and confirmed by RT-PCR. The virus was then adapted on chicken embryo fibroblast cells (CEF) in which it showed severe cytopathic effects (CPE). The short hairpin RNA (shRNAs) constructs homologous to the VP2 gene were designed and one, having maximum score and fulfilling maximum Reynolds criteria, was selected for evaluation of effective inhibition. Selected shRNA construct (i.e., VP2-shRNA) was observed to be the most effective for inhibiting VP2 gene expression. Real time PCR analysis was performed to measure the relative expression of VP2 gene in different experimental groups. The VP2 gene was less expressed in virus infected cells co-transfected with VP2-shRNA as compared to mock transfected cells and IBDV+ cells (control) at dose 1.6 µg. The result showed ∼95% efficient down regulation of VP2 gene mRNA in VP2-shRNA treated cells. These findings suggested that designed shRNA construct achieved high level of inhibition of VP2 gene expression in-vitro.

Nanoconjugation of bicistronic DNA vaccine against Edwardsiella tarda using chitosan nanoparticles: Evaluation of its protective efficacy and immune modulatory effects in Labeo rohita vaccinated by different delivery routes

DNA-based immunization has proven to be an effective prophylactic measure to control aquatic animal diseases. In order to improve the efficiency of vaccine against fish pathogen, novel delivery mechanism needs to be adopted. In the present study we nanoconjugated the previously constructed DNA vaccine (pGPD + IFN) with chitosan nanoparticles (CNPs) by complex coacervation process. After construction of the vaccine, an in vivo vaccination trial was conducted in which 2 groups of rohu (L. rohita) fingerlings were vaccinated with CNPs-pGPD + IFN, one group by oral route (incorporated in feed for 14 days) and the other by immersion route (primary and booster immunised), whereas, a third group was intramuscularly (I/M) injected (initial and booster immunised) with naked pGPD + IFN and subsequently challenged with E. tarda (8.7 × 104 CFU/fish) at 35-day post initial vaccination. The protective immune responses were determined in terms of relative percentage survival (RPS), specific antibody production, non-specific immune response, expression kinetics of immune-related genes and pathological manifestation. Evaluation of RPS analysis revealed that CNPs-pGPD + IFN groups recorded highest RPS (81.82% and 72.73% in oral and immersion vaccinated fish group respectively) while the naked pGPD + IFN injected group showed 63.62% RPS when compared with 55% cumulative mortality of control group. In addition, NBT, myeloperoxidase activity, serum lysozyme activity and specific antibody titre in case of CNPs-pGPD + IFN groups showed higher activities during all the time points. Furthermore, CNPs-pGPD + IFN groups showed significant (p < 0.05) upregulation of different immune gene transcripts (IgHC, iNOS, TLR22, NOD1 and IL-1β) in three immunologically important tissues post immunization (both primary and booster dose) as well as after challenge. Thus, from this study, we can conclude that oral or immersion vaccination with CNPs-pGPD + IFN can orchestrate an effective immunisation strategy in organizing a coordinative immune response against E. tarda in L. rohita exhibiting minimum stress to the host with maximum efficacy.

Detection of very virulent infectious bursal disease virus from a field outbreak in Central India

In order to detect infectious bursal disease virus (IBDV), bursal tissue was collected from 10 IBD-suspected birds from a 30-day-old, IBDV-vaccinated commercial broiler chicken flock of 2000 birds exhibiting clinical signs suggestive of infectious bursal disease (IBD). The presence of IBDV was confirmed by partial amplification of the VP2 gene by reverse transcription and polymerase chain reaction. Isolates were identified as very virulent strains of IBDV (vvIBDV) by nucleotide sequence analysis. The comparison of the VP2 nucleotide sequences among the isolates revealed the presence of single-nucleotide polymorphisms in the VP2 gene of IBDV in the same flock. The comparative analysis indicated that these viruses were genetically close to the vvIBDVs previously detected in India. Our analysis provided information about the existence of vvIBDV in Central India.

Molecular cloning, characterization and expression profiling of galectin-9 gene from Labeo rohita (Hamilton, 1822)

&NA; Galectin‐9 is a b‐galactoside‐binding tandem repeat galectin that regulates many cellular functions, ranging from cell adhesion to pathogen recognition. In spite of extensive study of mammalian galectin importance in immune system, little is known about that of fish. To study the normal expression and immune response of Labeo rohita to pathogens, a tandem‐repeat galectin‐9 from Labeo rohita was identified and named LrGal‐9. Its full‐length cDNA was 1534 bp encoded 291 amino acids (35.12 KDa), shared the highest 81% identity with the galectin‐9 of Danio rerio. LrGal‐9 identified in this study lacked signal peptide and a transmembrane domain like galectin‐9 members reported in other fishes. Quantitative PCR showed that LrGal‐9 was lowly expressed in gill, muscle, heart, highly expressed in tested immune tissues (intestine, kidney, liver, spleen) in normal body. After Aeromonas hydrophila challenge, LrGal‐9 was remarkably increased in all tested immune tissues in a time‐dependent manner. These results suggest that LrGal‐9 plays a role in innate immunity in Labeo rohita. HighlightsExpression and immune response of L. rohita to pathogens, a tandem‐repeat gal9 was identified and named LrGal‐9.LrGal‐9 was lowly expressed in gill, muscle, heart and highly expressed in intestine, kidney, liver and spleen.LrGal‐9 plays an important role in innate immunity in Labeo rohita.