•Nanoconjugation of DNA vaccine against E. tarda using chitosan nanoparticles.•Rohu fingerlings were immunized through oral, immersion and injection routes.•CNPs-pGPD + IFN vaccinated fish exhibited ...high RPS post E. tarda challenged.•CNPs-pGPD + IFN immunized group showed higher specific and innate immune response.•Innate immune genes were significantly upregulated in immunized fish.
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.
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-1β 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-1β 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-1β 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-1β, 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.
•Pathogenesis related to Edwardsiella tarda on Labeo rohita was studied.•E. tarda exposure modulates TLR22, NOD1, IgHC, iNOS, IL-1β 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.
Tilapia lake virus (TiLV) is a serious pathogen of farmed Nile tilapia (
Oreochromis niloticus
) responsible for significant mortalities. In this study, we investigated a disease outbreak in ...cage-farmed Nile tilapia in India. The infected fish exhibited clinical signs such as severe scale loss, haemorrhage, exophthalmia, and fin and tail rot. The samples were screened for Tilapia lake virus (TiLV) by reverse-transcriptase PCR and also subjected to detailed bacteriological investigation to understand the association between TiLV and co-infecting bacterial pathogens. Bacteria were isolated from TiLV-infected and apparently healthy fish, and identified by conventional microbiological methods, followed by 16SrRNA gene sequencing. TiLV was detected by PCR in all the samples exhibiting clinical signs, while apparently healthy fish were negative for the virus. A total of 34 bacterial isolates belonging to the genera
Aeromonas
,
Staphylococcus
,
Enterococcus
,
Plesiomonas
,
Enterobacter
,
Bacillus
,
Lysinibacillus
,
Solibacillus
and
Exiguobacterium
were isolated from the virus-infected tilapia. However,
Aeromonas veronii
was found to be the most dominant bacterium isolated from the surface lesions and the internal organs of all infected fish. Antibiotic susceptibility testing revealed that
A. veronii
, by far, was susceptible to cephalosporins (ceftriaxone, cefotaxime, cefpodoxime), chloramphenicol, aminoglycosides (amikacin, gentamicin), ciprofloxacin and chloramphenicol. Experimental infection using intraperitoneally injected
A. veronii
reproduced the clinical signs of naturally infected Nile tilapia, and a lethal dose 50 (LD
50
) mortality was observed by day 7 post-challenge. Furthermore,
A. veronii
could be re-isolated from the experimentally infected fish. Based on this evidence, we propose that virulent
A. veronii
as a co-infecting bacterium can have an important role in the severity and outcome of the disease in Nile tilapia infected by TiLV.
Interferon gamma (IFN-γ) 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-γ 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-γ along with GAPDH gene of
Edwardsiella tarda
as bicistronic DNA vaccine in
Labeo rohita
. In order to ascertain the individual role of IFN-γ, the present study involves cloning and expression of 552-bp IFN-γ 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-IFNγ-transfected SSN-1 cells, reacted with anti-His antibody in Western blot confirming it to be recombinant IFN-γ, whereas the relative expression of IFN-γ, iNOS, Mx, and IL-1β 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-IFNγ-transfected cell group and Poly I:C-treated cell group demonstrating recombinant IFN-γ as an equally efficient inducer like Poly I:C. Thus, our in vitro experiment results highlight the immunomodulatory potential of recombinant IFN-γ as an analogue to synthetic Poly I:C which warranted future studies to further explore the potential of recombinant IFN-γ as an effective vaccine adjuvant against different microbial invasion.
Biotechnological advancements have paved newer avenues for developing and designing novel and effective vaccines for rendering protection from various types of infectious diseases. Use of immunogenic ...genes via plasmid DNA constitutes an important next-generation biotechnological approach to fish immunization. In addition, the use of nanotechnology has significantly addressed the issue of mucosal mode of DNA vaccine delivery in aquaculture. Taking together both these advance technologies, this chapter entails a detailed protocol for the development of a nano-conjugated bicistronic DNA vaccine using chitosan nanoparticles as delivery vehicle, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of Edwardsiella tarda as antigenic gene and interferon gamma (IFN-γ) gene of Labeo rohita as molecular adjuvant.
Fundamentals of Fish Vaccination Bedekar, Megha Kadam; Kole, Sajal
Methods in molecular biology (Clifton, N.J.),
2022, Volume:
2411
Journal Article
Fish health management has become a critical component of disease control and is invaluable for improved harvests and sustainable aquaculture. Vaccination is generally accepted as the most effective ...prophylactic measure for fish disease prevention, on environmental, social, and economic grounds. Although the historical approach for developing fish vaccines was based on the principle of Louis Pasteur's "isolate, inactivate and inject," but their weak immunogenicity and low efficacies in many cases, have shifted the focus of fish vaccine development from traditional to next-generation technologies. However, before any fish vaccine can be successfully commercialized, several hurdles need to be overcome regarding the production cost, immunogenicity, effectiveness, mode of administration, environmental safety, and associated regulatory concerns. In this context, the chapter summarises the basic aspects of fish vaccination such as type of vaccine, modalities of vaccine delivery, the immunological basis of fish immunization as well as different challenges associated with the development process and future opportunities.
A serological test for screening of TiLV in Oreochromis niloticus would be useful for the epidemiological investigations. Using polyclonal antisera against TiLV (TiLV-Ab), an indirect enzyme-linked ...immune sorbent assay (iELISA) was developed for the detection of TiLV antigen in fish tissue and mucus. After a cutoff value was established and antigen and antibody concentrations were optimized, the iELISA's sensitivity and specificity were assessed. We found the ideal dilutions of TiLV-Ab as 1: 4000 and secondary antibody as 1:65,000. High analytical sensitivity and moderate specificity were displayed by the developed iELISA. The Positive and Negative Likelihood Ratio (LR+, LR-) were 1.75 and 0.29, respectively. The estimated Positive and Negative Predictive Values (PPV and NPV) of the test were 76.19% and 65.62%, respectively. The accuracy of the developed iELISA was estimated as 73.28%. An immunological survey was performed using the developed iELISA with samples from the field and 155/195 fishes tested positive, indicating a 79.48% TiLV antigen positives. Among the pooled organs and mucus tested, the highest positive rate of 92.3% (36/39) is observed in mucus compared to other tissues, and least positive rate is found in liver of 46% (18/39). The newly designed iELISA proved sensitive and may be helpful for extensive examinations of TiLV infections and monitoring disease status even from apparently healthy samples using a non-invasive technology by collecting mucus as sample for iELISA.
•Using TiLV-Ab, a sensitive iELISA for the direct detection of TiLV antigen in fish tissue and mucus samples was developed.•The estimated PPV and NPV of the test were 76.19% and 65.62%, respectively, and the accuracy was estimated as 73.28%..•The highest TiLV positive rate is observed in the mucus samples compared to tissues (kidney, liver, and brain)..•Extensive examination of infection can be done even from apparently healthy fishes using non-invasive collection of mucus.
Polymeric immunoglobulin receptor (pIgR) is a protein that transports Immunoglobulins (Igs) from epithelial cells into the external secretion system of the animal. In the present study, we ...characterized the partial pIgR gene from Labeo rohita and analyzed its expression in response to the pDNA (pGPD-IFN) vaccine, and studied its correlation with the expression of IgM, in the mucosal-associated lymphoid tissues (MALT). The significant (p < 0.05) up-regulation of pIgR mRNA was observed in the mucosal tissues of vaccinated fish by qRT-PCR. The highest expression of the gene was detected in gill tissue at 15 days post-vaccination (dpv) followed by skin and gut at 30 dpv. The expression of IgM also showed a similar pattern and indicated a direct correlation of pIgR expression in all the tested tissues. The protective immune response of the DNA vaccine was measured as the relative percentage of survival (RPS) by challenging vaccinated fish with live Edwardsiella tarda (1 × 105 CFU/Fish). The result showed a significantly high relative percentage survival (RPS) in the vaccinated group (47.05%) compared to the control group. Many factors contributing to the immune response of the vaccine. One of the most critical aspects is the rise in IgM level in local tissues. In other higher vertebrates, pIgR is reported to act as the transporter of IgM. The positive correlation in the expression of pIgR and IgM observed in the present study demonstrated the possible role of pIgR as the transporter of IgM in L. rohita. The study suggests the possibility of the secretory nature of IgM in fish.
•Characterization of polymeric immunoglobulin receptor gene (pIgR) in L. rohita•mRNA expression of pIgR and IgM gene in mucosal associated lymphoid tissues in response to DNA vaccine and E. tarda infection•Vaccine efficacy study against E. tarda infection in terms of relative percentage survival (RPS).
DNA vaccine is an important tool to elicit both humoral and cellular immunity. Present study investigates mucosal immune response of Labeo rohita (15 ± 04 g) to plasmid DNA (pDNA) vaccine ...macromolecule complexed with nanoparticles (NPs). Poly lactic-co-glycolic acid (PLGA), Chitosan (Chit) and PLGA-Chit-NPs were synthesized by double emulsion solvent evaporation method. Synthesized NPs were complexed with pDNA (pGPD + IFN) vaccine construct. Size and zeta potential of PLGA-NPs, Chit-NPs and PLGA-Chit-NPs-pDNA complex were recorded to be 120 nm and +0.5 mV, 117 nm and +32 mV, 189 nm and +11 mV, respectively. Immunization by immersion was carried out in two groups receiving PLGA-Chit-NPs-pDNA (T1) and PLGA-NPs-pDNA (T2) respectively. After immersion, samples were collected on 0, 2, 4, 7, 15 and 30 days from mucosa-associated lymphoid tissues (MALT) for mRNA expression studies of IgM, IgD and IgZ using qRT-PCR. Significant up-regulation of the mRNA expression of IgM, IgD, and IgT were observed in MALT in immunized fish compared to control. After 30 days post-immunization fish were infected with a virulent strain of Edwardsiella tarda. The highest relative percentage survival was observed in T1 (64.7%) compared to T2. The study showed better efficiency of pDNA-PLGA-Chit-NPs compared to pDNA-PLGA-NPs for inducing adaptive mucosal immunity in fish.
•Growth enhancing potential of turmeric with combination of ginger or garlic.•Dietary combination of turmeric with ginger or garlic enhances the digestive, metabolic and anti-oxidative enzyme ...activities.•These feed additives can be used in aquafeed as the potential natural remedies to modulate innate immune function.
A 45-day feeding trial was conducted to screen the effective combinations of some commonly used phytogenic feed additives (turmeric, ginger, and garlic) on growth, body composition, digestive, metabolic, antioxidant enzyme activity and innate immune functions of Labeo rohita fingerlings. Five experimental diets were formulated viz., Control (no phytogenic additive), T1 (turmeric + ginger), T2 (turmeric + garlic), T3 (ginger + garlic) and T4 (turmeric + ginger + garlic) at 1% inclusion level keeping an equal proportion of each additive used in the combination. Two hundred and twenty-five fish (average weight 2.75 ± 0.01 g) were distributed randomly into five experimental groups in triplicates with 15 fish in each tank containing 400 L water. Significantly higher weight gain, specific growth rate, protein efficiency ratio and low feed conversion ratio was found in the T1, T2 and T3 groups while the T4 group showed significantly lower growth performance (P < 0.05) in comparison to control and other treatments. Significantly higher digestive enzyme activities were found in all phytogenic additive fed groups while higher (P < 0.05) metabolic enzyme activities of liver and muscle were found only in the T1 group. Antioxidant enzyme activities were higher (P < 0.05) in T1 and T2 groups. Serum glucose, triglyceride, and whole-body lipid content significantly decreased due to feeding of phytogenic additives. From the present study, it can be concluded that combination of turmeric with ginger or garlic with equal proportion at 1% inclusion level can be useful to improve growth, digestive, metabolic, antioxidant enzyme activities and health status of Labeo rohita fingerlings.
