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Immunoglobulin (IgM) is the primary immunoglobulin essential for defense mechanisms in fish. It is difficult to reliably quantify IgM because a lack of standardization in methodology and limited availability of commercially reagents. In the present study, a polyclonal antibody was developed for the specific detection and quantification of IgM in Labeo rohita. Recombinant bicistronic NanoDNA plasmid (RBND Vac) encoding the glyceraldehyde-3-phosphate dehydrogenase gene of Edwarsiella tarda conjugated with poly (lactic-co-glycolic acid) - Chitosan (PLGA-Chit) was developed and its potential as a DNA vaccine, to prevent the infection of E. tarda in L. rohita was investigated. Two treatment groups [T1 - (PLGA-Chit-NPs-pDNA), T2 - (PLGA-NPs-pDNA) and one control group (T0 - 1 × PBS)] were utilized. Polyclonal antibody was developed to estimate IgM titers in the serum and mucosal associated tissues (MAT) using Enzyme-linked Immunosorbent Assay (ELISA) technique. Additionally, immune gene expression was studied using qRT-PCR. Vaccinated groups also exhibited a significant increase in the total serum protein, globulin concentration and relatively less mortality was observed in T1 group. IgM level in serum and mucosal tissues (skin, gill and gut) increased significantly days post vaccination compared to control group, also non-specific immune parameters (myeloperoxidase and lysozyme levels) showed significant improvement in vaccinated fish. Furthermore, histopathological examination confirmed minor damage in physiological structure of kidney and liver tissues in vaccinated fish. Knowledge of the immunoglobulin in L. rohita primed with RBND Vac complex provides the better protection against E. tarda. The normal physiology findings of this study will aid in monitoring changes in the health status of fish, when the animals undergo vaccination by immersion method.

Nile tilapia (Oreochromis niloticus) is one of the most important aquaculture species farmed worldwide. However, the recent emergence of tilapia lake virus (TiLV) disease, also known as syncytial hepatitis of tilapia, has threatened the global tilapia industry. To gain more insight regarding the host response against the disease, the transcriptional profiles of liver in experimentally-infected and control tilapia were compared. Analysis of RNA-Seq data identified 4640 differentially expressed genes (DEGs), which were involved among others in antigen processing and presentation, MAPK, apoptosis, necroptosis, chemokine signaling, interferon, NF-kB, acute phase response and JAK-STAT pathways. Enhanced expression of most of the DEGs in the above pathways suggests an attempt by tilapia to resist TiLV infection. However, upregulation of some of the key genes such as BCL2L1 in apoptosis pathway; NFKBIA in NF-kB pathway; TRFC in acute phase response; and SOCS, EPOR, PI3K and AKT in JAK-STAT pathway and downregulation of the genes, namely MAP3K7 in MAPK pathway; IFIT1 in interferon; and TRIM25 in NF-kB pathway suggested that TiLV was able to subvert the host immune response to successfully establish the infection. The study offers novel insights into the cellular functions that are affected following TiLV infection and will serve as a valuable genomic resource towards our understanding of susceptibility of tilapia to TiLV infection.

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 (LD50) 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.

Aim: The present experiment was undertaken to compare digestive, metabolic, antioxidant enzyme activities and immuno-biochemical responses in Labeo rohita fingerlings to some commonly used phytogenic feed additives. Methodology: Eleven experimental diets were prepared by supplementing fennel, coriander, cumin, fenugreek seed, turmeric, black pepper, peeled ginger, bay leaf, peeled onion bulb or peeled garlic clove meal at 1% inclusion level along with a control diet. Four hundred and ninety-five fingerlings (average weight 6.45±0.01 g) were distributed randomly in eleven experimental groups in triplicates with a stocking density of 15 fish per tank (400 l of water). Results: Turmeric, garlic or ginger meals appeared to be more effective than onion, fenugreek, cumin, coriander, fennel, black pepper and bay leaf meals for enhancing digestive, metabolic, antioxidant enzyme activities and innate immune functions. The physio-metabolic effects of phytogenic feed additives tested in Labeo rohita fingerlings were in the order of turmeric > garlic > ginger > onion > fenugreek > cumin > coriander > fennel > black pepper > bay leaf meal. Interpretation: The enhanced digestive and metabolic enzyme activities, antioxidant function, glucose homeostasis and improved innate immune function through modulation of the haemato-biochemical profile of Labeo rohita due to feeding of specific functional compounds present in turmeric, ginger and garlic meals compare to other phytogenic additives.

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.

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.

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.

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.

White spot syndrome virus (WSSV) is a pathogen causing significant economic losses to shrimp aquaculture worldwide. Previously, five genome sequences of the virus from farmed shrimp ( Penaeus vannamei and Penaeus monodon ) in India were reported, all originating from farms located on the east coast of the country. Here, we report three new and distinct WSSV genome sequences, two from shrimp ( P. vannamei ) farmed on the west coast of India and the third from the east coast.

The chitosan nanoparticles (CNPs) conjugated bicistronic DNA vaccine (pGPD+IFN) containing GAPDH gene of Edwardsiella tarda and IFN-γ gene of Labeo rohita was found to induce high protective immunity in L. rohita against virulent E. tarda challenge in our previous study. However, for conducting vaccination trials in aquaculture farms, it is essential to circumspect various biosafety issues concerning the use of DNA vaccines in food fishes as per international guidelines. In this context, the present study was undertaken to evaluate the persistence, bio-distribution and environmental transmission of the DNA vaccine (pGPD+IFN) to provide the basis for commercial application. The experimental study involves complexation of the DNA construct with chitosan coated PLGA (poly lactide-co-glycolide) nanoparticles (Chi/PLGA NPs) in order to enhance the vaccine uptake by the host through immersion route. The mean particle size (267.4 nm) and zeta potential (+27.mV) of the Chi/PLGA- pGPD+IFN vaccines assists in giving higher stability to the pDNA as well as facilitate in easy uptake and wide tissue distribution. The bio-distribution and persistence results showed that the DNA vaccine was localised at the immunization administration surfaces of the host (gill, gut and skin+muscle and not in kidney and liver) and remained persistent for 14–30 days post immersion treatment. The low copy numbers (

We investigated the subchronic immunotoxicity of the phosphoramidothioate organophosphorous insecticide, acephate in white leghorn cockerels (WLH). The cockerels were divided into five groups; C1 (plain control), C2 (vehicle control), T1, T2, and T3 which received acephate suspended groundnut oil for 60 days at doses of 21.3, 28.4 and 42.6 mgkg− 1respectively. The live body weight gain, absolute and relative weights of the spleen, thymus, and bursa of Fabricius, hemoglobin (Hb), total erythrocyte counts (TEC), packed cell volume (PCV) and lymphocytes were significantly decreased. However, monocytes, eosinophils, heterophils, and basophils were significantly increased. Total protein, albumin and albumin to globin ratio, the antibody response to RD-F and delayed-type hypersensitivity response to DNCB dye or PHA-P, erythrocyte and brain Acetylcholinesterase activity was also significantly reduced in T2 and T3. At 40 and 60 days of acephate exposure, nitrate and nitric oxide production by RD-F and mitogen Con A stimulated peripheral blood and splenic lymphocytes, as well as lymphocyte proliferation in response to antigen RD-F and mitogen Con A stimulation, were significantly decreased in groups T2 and T3. Furthermore, dose-dependent increases in the frequency of micronuclei formation, varying intensity serum protein bands with different protein fractions (14.85KDa), and splenic DNA laddering (180 bp) were observed in groups T2 and T3. Histopathologically, the spleen and bursa showed morphological changes and mild lymphocyte depletion. In conclusion, low-level acephate exposure may affect acetylcholinesterase, lymphocytes, and immune responses in cockerels. As a result, it should be considered when assessing immunotoxicity and the risk to human and animal health.

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

In the present study, ontogenetic expression of different innate immune genes in the Toll pathway of tiger shrimp, Penaeus monodon, such as TLR, myeloid differentiation factor 88 (MyD88) and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), was investigated in different developmental stages. Ontogenetic expression by real-time PCR revealed constitutive expression of these genes in all the developmental stages selected. TLR expression was found to be the highest in PL4, whereas MyD88 and TRAF6 showed the highest expression in eggs. The ubiquitous expression of TLR, MyD88 and TRAF6 in different developmental stages of P. monodon indicates the role of these genes in protecting the animals during early development. Immersion challenge of PL 18 with V. harveyi resulted in significant upregulation of TRAF6 at all time-points and significant upregulation of TLR at most of the time-points selected; however, MyD88 showed differential modulation pattern. In contrast to the bacterial challenge, WSSV infection in PL18 did not show any significant change in the expression of TRAF6, except for a downregulation observed at 12 to 48 hpi. However, TLR and MyD88 showed moderate increase in their expression, especially at late time-points. The responses of these genes to V. harveyi and WSSV immersion challenges in the juveniles (average body weight 3 g) of P. monodon were investigated in selected tissues including gill, hepatopancreas, and different parts of gastrointestinal tract such as foregut (stomach), midgut and hindgut. Temporal expression analysis revealed complete downregulation of PmMyD88 at most of the time-points in the gill following V. harveyi challenge and significant induction of PmTRAF6 at all time-points following WSSV infection. Immersion challenge with V. harveyi resulted in enhanced expression of TRAF6 in stomach and MyD88 in hepatopancreas showed similar pattern of expression post-WSSV challenge. Other tissues showed varying levels of induction of these genes at different time-points following pathogen challenge. The results of the present study suggest that both bacterial and viral challenges through immersion modulates the genes involved in the Toll pathway, and this might play an important role in the immune defense of post-larvae and juveniles of P. monodon.

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.

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

DNA-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-γ 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

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

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.

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.

Aquaculture has emerged as a fast-growing food-producing sector in the world in recent years; however, infectious diseases of bacterial, viral, mycotic, and parasitic origin are the most significant restrictive agents in the improvement of intensive aquaculture. In view of the constant threat of various diseases to the aquaculture industry, the need for prophylactic measures has been felt worldwide for more than three decades. Immunoprophylaxis or vaccination strategies are regarded as the most efficient and economical remedial measure in protecting the health of fish and aquaculture animals from various infectious agents. However, traditional vaccines proved to be inefficient in many cases to address the fish disease problem. Biotechnological advancements have paved newer avenues for developing and designing novel and effective vaccines, as well as improving existing vaccines for rendering protection from various types of infectious diseases. Current advances in fish vaccinology offer valuable opportunities to discover new vaccine candidates to combat fish pathogens for which vaccines are still lacking. This chapter focuses on the usage of biotechnology in the areas of fish vaccinology—current knowledge, recent advances, and future perspectives of various new generation vaccines for the aquaculture industry.

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

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

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 ( Es MDA5) with MDA5 of Neolamprologus brichardi and Oreochromis niloticus , both belonging to Cichlidae family. Es MDA5 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, Es MDA5 transcripts showed peak expression in the spleen, intestine and heart at 12 h post-injection (hpi). However, in gill and kidney tissues, maximum up-regulation of Es MDA5 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 Es MDA5 and the IPS-1 genes in different tissues indicate that Es MDA5 may play an important role in sensing viral PAMPs in conjunction with IPS-1.

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 µ g. 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.

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.

Skeletal muscle is the major component of lean tissue that is used for consumption, and myostatin is a negative regulator of skeletal muscle growth. Downregulation of this gene therefore offers a strategy for developing superior animals with enhanced muscle growth. Knockdown of myostatin was achieved by RNA interference technology. The anti-myostatin shRNA were designed and stably transfected in caprine fibroblast cells. The reduced expression of target gene was achieved and measured in clonal fibroblast cells by real-time PCR. Two single-cell clones induced significant decrease of myostatin gene expression by 73.96 and 72.66 %, respectively (P < 0.05). To ensure the appropriate growth of transfected cell, seven media were tested. The best suited media was used for transfected fibroblast cell proliferation. The findings suggest that shRNA provides a novel potential tool for gene knockdown and these stably transfected cells can be used as the donor cells for animal cloning.

Infectious bronchitis (IB) is an acute and contagious disease of poultry. The spike glycoprotein (S) of IB virus is a dimmer and is cleaved into two glycopolypeptides, S1 and S2 post-translationally. S1 gene defines the serotype and plays a major role in induction of protective immunity. Eukaryotic expression systems are frequently employed for the production of recombinant S1 proteins as it is highly glycosylated protein. In present study the S1 gene amplified from isolated field strain of IBV was cloned into eukaryotic expression vector and express in vero cell line. The pQE-TriSystem vector was used as eukaryotic expression vector to express the corresponding protein. The successful expression was confirmed at 24 and 48 hrs post transfection by Reverse Transcriptase-PCR. These promising observations emphasize the need of expression of S1 gene recombinant protein for the development of effective recombinant DNA vaccine against IB in near future.

Phytases are enzymes which hydrolyze phytate. Bacillus species are known to produce a thermostable phytase. The Bacillus subtilis strain NCIM-2712 was chosen for cloning of phy gene. Primers were designed for phy gene amplification using the phy gene sequence of B. subtilis (AF298179). A sequence of 1059 bp characteristic of phy gene was obtained on PCR amplification. This gene was cloned into InsT/A cloning vector and the positive clones were confirmed by colony PCR with gene specific primers and restriction digestion. Phytase is a promising candidate for feed applications. The cloned gene obtained in this study will have potential for producing recombinant enzyme, which would enhance the feed quality for poultry and piggery by supplementing it in their diets.

In India the most common vaccine strain against infectious bronchitis (IB) virus (IBV) is Mass strain (M41). Most of the organized and unorganized poultry farms use Mass strain for vaccination of parent stock. But even after taking all precautions the incidences of IB outbreak are common in poultry population. IBV, a major pathogen of poultry flocks, circulates in the form of several genotypes and serotypes. Only a few amino acid changes in the S1 subunit of wild type proteins may results in mutants unaffected by current vaccine. In the year 2008 one strains of IBV was isolated from vaccinated chicken flocks. The results from sequencing of S1 gene showed that this strain was distinct from classic IBV strains of H120, M41 etc. Compared to H120 and M41 vaccine strain, point mutation occurred at many positions in the S1 protein of this field strain. The homology of the nucleotide and amino acid sequences of the S1 gene of this isolate was 79.0%-99.6% and 74.5%-98.8%, respectively with relation to major vaccine strains used worldwide. The results from this study indicate that different IBV strains cocirculate in the chicken population in India.