Not Available

Not AvailableViral haemorrhagic septicaemia virus (VHSV) is known to cause high mortality in olive flounder (Paralichthys olivaceus) at 15 °C, but no mortality at 20 °C. In order to understand the immune response at 15 °C and 20 °C against VHSV, differential expression kinetics of various innate immune-related genes in the kidney were analyzed in our previous study, and the current study is focused on immune responses in the liver. Fishes were intraperitoneally injected with VHSV (107.8 TCID50/fish) and reared at 15 °C and 20 °C. Viral copy number in the 15 °C group peaked at 1 dpi and remained high until 2 dpi; whereas in the 20 °C group, viral copies were much lower than in the 15 °C group. The expression kinetics results revealed significantly high levels of hepcidin expression in the 20 °C group in comparison to the 15 °C group at all time points. Cathepsin-L gene showed early upregulation at 12 hpi followed by a sharp decline at 1 dpi in both the groups; whereas, at 2–4 dpi, only the 20 °C group demonstrated subsequent upregulation of cathepsin-L transcript. The genes of complement pathways, C3 and factor-D showed prolonged downregulation trend in the 15 °C group against the normal expression level in the 20 °C group. Significant upregulation of ISG15, Mx, IL-1β and IL-8 genes were observed in the 20 °C group during 1–2 dpi in comparison with slower and lower responses in the 15 °C group. The MHC-II, MHC-I and CD8 gene transcripts showed significant upregulation only in the 20 °C group in contrast with downregulation in the 15 °C group, whereas, CD4 expression was very high at 12 hpi to 2 dpi in both the groups. The genes related to the apoptotic pathway, p53, HSP70, and caspase-3, also displayed consistent downregulation in the 15 °C group during the entire experimental period in comparison with the other group. In summary, it can be observed that most of the innate immune genes were under-expressed or have little to no response at 15 °C, whereas, the same genes exhibited high level of expression at 20 °C. Thus, it can be inferred that temperature plays a key role in facilitating the host liver to orchestrate a coordinative anti-VHSV immune response ultimately leading to host survival in the 20 °C group. Oppositely, the under-expression of important genes at 15 °C especially the genes related to the apoptotic pathway, cytotoxic T-cell pathway, and the complement system, resulted in failure of the host immune system to combat the viral proliferation.Not Availabl

Fish and Shellfish Immunology

Not AvailableRohu (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? 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? 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? 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?, 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

Aquaculture international

Not AvailableInterferon gamma (IFN-?) or type II interferon is a cytokine that is critical for innate and adaptive immunity against viral and some bacterial and protozoal infections. The importance of IFN-? in the immune system lies in its ability to inhibit viral replication directly and most importantly from its immunomodulatory effects. Previously, we successfully co-administered IFN-? along with GAPDH gene of Edwardsiella tarda as bicistronic DNA vaccine in Labeo rohita. In order to ascertain the individual role of IFN-?, the present study involves cloning and expression of 552-bp IFN-? open-reading frame (ORF) of L. rohita in striped snakehead (SSN-1) cell line using eukaryotic expression vector system (pQE-TriSystem) followed by transfection in peripheral blood lymphocytes (PBMCs) to evaluate its immunomodulatory ability in comparison to polyinosinic-polycytidylic acid (Poly I:C)-treated PBMCs. The 18.7-kDa protein, expressed in the pQE-IFN?-transfected SSN-1 cells, reacted with anti-His antibody in Western blot confirming it to be recombinant IFN-?, whereas the relative expression of IFN-?, iNOS, Mx, and IL-1? genes in PBMCs was quantified at 24 h and 48 h post treatment by qPCR. The comparative kinetics of all four genes showed significantly (p?<?0.05) high upregulation pattern in both pQE-IFN?-transfected cell group and Poly I:C-treated cell group demonstrating recombinant IFN-? as an equally efficient inducer like Poly I:C. Thus, our in vitro experiment results highlight the immunomodulatory potential of recombinant IFN-? as an analogue to synthetic Poly I:C which warranted future studies to further explore the potential of recombinant IFN-? as an effective vaccine adjuvant against different microbial invasion

Aquaculture

Not AvailableDNA-based vaccination or genetic immunization is one of the most promising, effective and prophylactic measures to control aquatic animal diseases. This immunization strategy involves administration of eukaryotic antigen expression vectors (DNA vaccine) into the host that encode for an antigen under the control of a eukaryotic promoter which resulted into elicitation of strong cellular and humoral immune responses. In the present study, a bicistronic DNA vaccine (designated as pGPD+IFN) was constructed which contains an additional immune adjuvant gene (Interferon gamma gene of Labeo rohita) along with a regular antigenic gene (glyceraldehyde-3-phosphate dehydrogenase gene of Edwardsiella tarda) with the purpose to maximize the protective efficacy of the vaccine against E. tarda infection. After construction of the vaccine a pilot study was orchestrated in vitro to ascertain the positive co-expression of the dual genes in vaccine-transfected SSN-1 cell line. Successful co-expression of GAPDH and IFN-? genes in the transfected cell were confirmed by Western blot and RT-PCR respectively. Further, an in vivo vaccine trial was conducted in which rohu (L. rohita) fingerlings were intramuscularly (I/M) injected (initial and booster immunised) with two DNA constructs one group with pGPD+IFN and the other with pGPD (containing GAPDH gene only) and challenged with E. tarda (1 ? 105 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 and expression kinetics of immune-related iNOS gene. Evaluation of RPS analysis revealed that pGPD+IFN group recorded highest RPS of 63.16% while the pGPD vaccinated group showed 47.37% when compared with 63.33% cumulative mortality of control group. The results regarding respiratory burst activity, myeloperoxidase activity as well as antibody titre also showed pGPD+IFN group with highest activities at all the time points. Furthermore, the current study displayed pGPD+IFN group having significant (p < 0.05) upregulation of iNOS gene transcript at 24?48 h post-immunization (both initial and booster dose) as well as after challenge. Thus, from this study, we can conclude that the bicistronic vaccine can be an effective immunization strategy in orchestrating a coordinative immune response against E. tarda in L. rohita

Not Available

Not AvailableDNA-based vaccination or genetic immunization is one of the most promising, effective and prophylactic measures to control aquatic animal diseases. This immunization strategy involves administration of eukaryotic antigen expression vectors (DNA vaccine) into the host that encode for an antigen under the control of a eukaryotic promoter which resulted into elicitation of strong cellular and humoral immune responses. In the present study, a bicistronic DNA vaccine (designated as pGPD+IFN) was constructed which contains an additional immune adjuvant gene (Interferon gamma gene of Labeo rohita) along with a regular antigenic gene (glyceraldehyde-3-phosphate dehydrogenase gene of Edwardsiella tarda) with the purpose to maximize the protective efficacy of the vaccine against E. tarda infection. After construction of the vaccine a pilot study was orchestrated in vitro to ascertain the positive co-expression of the dual genes in vaccine-transfected SSN-1 cell line. Successful co-expression ofGAPDH andIFN-γ genesin the transfected cellwere confirmed byWestern blot and RT-PCR respectively. Further, an in vivo vaccine trial was conducted in which rohu (L. rohita)fingerlings were intramuscularly (I/M) injected (initial and booster immunised) with two DNA constructs one group with pGPD+IFN and the other with pGPD (containing GAPDH gene only) and challenged with E. tarda (1×105CFU/fish) at 35day post-initial vaccination. The protective immune responses were determined in terms of relative percentage survival (RPS), specific antibody production, non-specific immune response and expression kinetics of immune-related iNOS gene. Evaluation of RPS analysis revealed that pGPD+IFN group recordedhighestRPSof63.16%whilethepGPDvaccinatedgroupshowed47.37%whencomparedwith63.33% cumulativemortalityofcontrolgroup.Theresultsregardingrespiratoryburstactivity, myeloperoxidaseactivity as well as antibody titre also showed pGPD+IFN group with highest activities at all the time points. Furthermore,thecurrentstudydisplayedpGPD+IFNgrouphavingsignificant(p < 0.05)upregulationofiNOS gene transcript at 24–48h post-immunization (both initial and booster dose) as well as after challenge. Thus, from this study, we can conclude that the bicistronic vaccine can be an effective immunization strategy in orchestrating a coordinative immune response against E. tarda in L. rohita.Not Availabl

Vaccine

Not AvailableDNA-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 14days) 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 (8.7104CFU/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-1) 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

Not Available

Not AvailableDNA-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-1b) 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.Not Availabl