Fish possess numerous enzymatic antioxidant systems as part of their innate immunity. These systems have been poorly studied in
(rohu). The present study characterized and investigated the role of ...antioxidant genes in the defence mechanisms against two types of stressors, including infection and ammonia stress. Four key genes associated with antioxidant activity-catalase, glutathione peroxidase, glutathione S-transferase, and CuZn superoxide dismutase were successfully cloned and sequenced. These genes were found to be expressed in different tissues and developmental stages of rohu. The expression levels of these antioxidant genes in the liver and anterior kidney tissues of rohu juveniles were modulated in response to bacterial infection (
), parasite infection (
), poly I:C stimulation and ammonia stress. Additionally, the recombinant proteins derived from these genes exhibited significant antioxidant and antibacterial activities. These proteins also demonstrated a protective effect against
infection in rohu and had an immunomodulatory role. Furthermore, indirect ELISA assay systems were developed to measure these protein levels in healthy as well as
and ammonia-induced rohu serum. Overall, this study characterized and emphasised the importance of the antioxidant mechanism in rohu's defence against oxidative damage and microbial diseases.
Argulosis is one of the most unrestrained economically significant freshwater fish ectoparasitic diseases. Proper selection or normalization of the best reference gene governs the accuracy of results ...of gene expression studies using real-time PCR. Earlier studies in rohu carp (Labeo rohita) have used reference genes without proper validation. Here, seven candidate reference genes viz., acidic ribosomal protein (ARP0), glyceraldehyde 3-phosphate dehydrogenase, RNA polymerase II (RPo), elongation factor1α (EF1α), α- tubulin (AT), ribosomal protein L 10, and β-actin were evaluated using four algorithms (geNorm, BestKeeper, NormFinder and ∆Ct) followed by a comprehensive gene expression analysis using skin tissue of rohu at varied time points of experimental Argulus siamensis infection. ARP0 and EF1α were found to be the most stable whereas RPo and AT were considered as least stable genes based on basal expression level and variation in expression levels. Validation of candidate reference genes was undertaken by looking into the expression of six immune-related genes using the two most stable and two least stable genes as housekeeping genes in Argulus-infected rohu skin at different time points of infection. An increased expression of immune genes indicated the role of inflammation and the immune modulation process at the site of attachment of parasites in governing infection.
The outer-membrane proteins (OMPs) of Aeromonas hydrophila, an imperative fish pathogen accountable for massive economic losses to aquaculture industry, are found to be immunogenic and considered as ...potential vaccine candidates. In spite of development in the formulation of vaccine candidates against Aeromonas infection, no commercial preparation has been done so far; in addition, the molecular mechanisms of immunoprotection induced by various vaccine formulations in Indian major carp, Labeo rohita, are little known. The present study was undertaken to evaluate the modulation of immunity and expression of immune-related genes post-rOmpF (recombinant outer-membrane protein of A. hydrophila, a novel vaccine candidate) immunization and protective efficacy after A. hydrophila challenge. The rOmpF-immunized fish showed a variable expression of the immune-related genes, viz. toll-like receptor 22 (TLR), complement component 3 (C3), chemokine (CXCa), tumor necrosis factor-α (TNFα), interleukin 1β (IL-1β), manganese superoxide dismutase (MnSOD) and natural killer enhancing factor (NKEF) in the head kidney tissues, when compared to the control group at different time intervals post-vaccination. A significant increase in serum hemolysin titer, ceruloplasmin level and myeloperoxidase activity was observed on day 140 post immunization. Also, bacterial agglutination titer and antiprotease activity were significantly increased on day 42 post immunization. No significant change was observed in lysozyme activity. Challenge studies with live A. hydrophila on day 140 post-immunization of L. rohita significantly increased the relative percentage survival (∼44%) in the vaccinated group. The results suggest that the rOmpF could be used as a potential vaccine candidate to combat A. hydrophila infection in fish.
•The recombinant OmpF of A. hydrophila was assessed for its vaccine potential in Labeo rohita.•L. rohita immunized with the recombinant OmpF (rOmpF) generated specific immune response.•The rOmpF immunization stimulated the expression of IL1- β, C3, TLR22, TNF- α and CXCa chemokines.•The rOmpF-immunization improved the survival rate of L. rohita against A. hydrophila challenge.•The anti-rOmpF antisera specifically cross-reacted with all Aeromonas strains/isolates tested.
Lectins are proteins that bind to the carbohydrate moieties on surface of bacteria, erythrocytes and other cells of invertebrates causing agglutination and mediate in recognition of foreign ...substances. In the present study, we isolated and characterized a lectin molecule present in the hemolymph of
Macrobrachium rosenbergii
, an important cultured freshwater prawn. Lectin in serum samples of adult prawns was assessed through hemagglutination (HA) test using rabbit RBC that showed a titre ranging from 16 to 64. This serum hemagglutinin was confirmed as a C-type lectin based on its dependency on calcium ions towards binding to rabbit RBCs. The hemagglutinin was also found to be stable at the pH range of 5.0–10.0 and temperature range of 10–40 °C. Of various sugars and glycoproteins tested in hemagglutination inhibition assay, the serum lectin was found specific only to
N
-acetylneuraminic acid and fetuin with respective minimum inhibitory concentrations at 50 mM and 0.31 mg/ml. Further, the lectin was purified by affinity chromatography on rabbit erythrocyte stroma, which showed hemagglutination with rabbit RBC. In electrophoretic analyses, the purified lectin showed one band with molecular weight of ~ 427 kDa in native gradient PAGE, and its two constituent polypeptide chains of ~ 81 and ~ 73 kDa in SDS-PAGE. These polypeptides were analysed in MALDI-TOF/TOF mass spectrometry and identified as hemocyanins. It was hence, concluded that hemocyanin in
M. rosenbergii
possesses lectin-like activity.
Linker histone H1 (LHH1) is an abundant nuclear protein that condenses chromatin to form higher-order structure. The present study reported cloning and sequencing of 942 bp of LHH1 from liver tissue ...of rohu, Labeo rohita, with a complete coding sequence of 792 bp of having 263 amino acids. The phylogenetic tree of L. rohita LHH1 (LrLHH1) shared maximum similarity with that of Carassius auratus. The three dimensional model and domain architecture of LrLHH1 protein was also predicted using Swiss-Prot and SMART domain software. The expression of LHH1 during ontogeny showed significantly higher transcript level in milt, unfertilized eggs and up to 3 h post-fertilization followed by a dramatic decrease thereafter. The tissue-specific expression showed constitutive expression of LrLHH1 in all examined tissues. The expression of LHH1 during different infection models, namely, bacteria (Aeromonas hydrophila); ectoparasite (Argulus siamensis) and poly I:C induction revealed modulation in the level of expression at varied time points post-exposure in the liver and anterior kidney tissues of rohu. However, a synthetic peptide derived from LHH1 sequence of rohu did not have any detectable antibacterial activity. The present study provided necessary information on the role of this protein during ontogeny and innate immunity in Indian major carp species.
Celotno besedilo
Dostopno za:
BFBNIB, DOBA, GIS, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
•First comparative analysis of the susceptibility of major cultured Indian fish species to A. siamensis infection.•L. rohita the most susceptible and C. idella the resistant carp ...species.•Differential early inflammatory response may govern susceptibility patterns.
Branchiuran ectoparasites of the genus Argulus can have extensive damaging effects on cultured fish. There exist no systematic studies that evaluate susceptibility or resistance of various carp species to Argulus sp. and the underlying mechanisms. The present study aimed at identifying the most susceptible and resistant cultured species, studying settlement and survival of parasite on these species, and finally unravelling the variations of immune response in both resistant and susceptible species. Fish from eight species (Labeo rohita, Cirrhinus mrigala, Catla catla, Hypophthalmichthys molitrix, Cyprinus carpio, Ctenopharyngodon idella, Carassius auratus, Labeo fimbriatus) were individually challenged with metanauplii of A. siamensis (100 metanauplii/fish) before rearing them in single tank in triplicate for 45 days. Based on the observed parasite load on each species, L. rohita was found to be the most susceptible and C. idella the resistant species. The settlement and survival of the parasite on L. rohita and C. idella was compared at 24, 48, 72 and 96h post experimental infection. Survival was significantly low at 72h onwards in C. idella indicating it is an unsuitable/poorly preferred host for A. siamensis. The inflammatory responses which are known to be related to susceptibility were analysed. Individuals of both the species were exposed to A. siamensis (100 parasites/fish), and after 24h and 3 d, skin samples directly from the attachment site and non-attachment sites were assessed for transcriptomic profiles of selected innate defence genes. Artificial skin abrasion permitted comparisons between abrasion associated injury and louse-associated injury. The inflammatory responses varied significantly between both species indicating their role in determining susceptibility of a host to A. siamensis. The expression of major histocompatibility class II and matrix metalloproteinase 2 was significantly higher in C. idella compared to L. rohita and therefore appeared to be involved in the early protective response against A. siamensis. It is essential to study the expression pattern of more participatory genes of the inflammation related pathways to understand species specific susceptible patterns.
Dactylogyrosis is caused by more than 900 reported species of
Dactylogyrus
(gill fluke), mostly in Cyprinoidei fish leading to low to severe mortality in different culture systems and also in ...rivers/lakes.
Dactylogyrus
has been reported worldwide where Cyprinoidei is endemic. In recent years, incidents of infection and infectivity of different species of
Dactylogyrus
have increased many-fold due to several predisposing factors like intensification of culture, environmental quality, climatic change, poor quality fish seed, etc. These parasites are host choosy, either generalist or specialist.
Dactylogyrus
infection causes major changes in the anatomy and physiology of gill function. Necrosis, hyperplasia and hypertrophy of gill lamellae are the common pathologies observed during infection, which in turn invites other secondary pathogens leading to heavy mortalities. Currently, very few drugs or chemicals are available for controlling dactylogyrosis. Very few attempts have been made to develop vaccines against this important parasite. Here, we review the innate and adaptive immune responses modulated by
Dactylogyrus
infection and a hypothetical immunological model has been proposed to understand host-pathogen interaction based on our findings. The information collected and analysed in this review on the biology and host-pathogen interaction will help in the development of prophylactic and other sustainable management measures to reduce the loss associated with dactylogyrosis.
Ceruloplasmin is an ancient multicopper oxidase evolved to insure a safe handling of oxygen in some metabolic pathways of vertebrates. The current knowledge of its structure provides a glimpse of its ...plasticity, revealing a multitude of binding sites that point to an elaborate mechanism of multifunctional activity. Ceruloplasmin is highly conserved throughout the vertebrate evolution. Cupredoxin, a multi-cupper blue protein is believed to be the evolutionary precursor of ceruloplasmin with three trinuclear and three mononuclear copper binding sites. There are 20 copper-binding residues in ceruloplasmin gene out of which 16 residues are conserved in fish. This ceruloplasmin gene is being characterized in zebrafish (Danio rerio), rohu (Labeo rohita), Indian medaka (Oryzias melastigama), catfish (Ictalurus punctatus), icefish (Chionodraco rastrospinosus), goldfish (Carassius auratus) and yellow perch (Perca flaviscens). The complete coding sequence of fish ceruloplasmin gene is around 3.2 kb which codes for 1000 to 1100 amino acid residues. The size of ceruloplasmin gene sequence in fish ranges around 13 kb containing 20 exons and 19 introns. Liver is the major site of synthesis in fish. Increased expression of this gene during bacterial infection in channel catfish and rohu suggested its potential involvement in bacterial disease response in fish. It has been found to serve as an indirect marker for selection against Aeromonas hydrophila resistance in rohu carp. Ceruloplasmin expression is also evident during parasitic infection in few fish species. The role of this gene is well studied during inflammatory response to hormonal, drug and heavy metal mediated toxicity in fish. Overall, ceruloplasmin represents an example of a 'moonlighting' protein that overcomes the one gene-one structure-one function concept to follow the changes of the organism in its physiological and pathological conditions.
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•Ceruloplasmin (Cp) is a highly conserved and moonlighting protein with multifunctional activity.•Cp is a multicopper oxidase evolved to insure a safe handling of oxygen in metabolic pathways.•It serves as an indirect marker for selection against A. hydrophila resistance in rohu carp.•Its role has been identified during development, pathogen attack and inflammatory response.
spp. are important ectoparasites of fish, and the current approach of their control using chemical pesticides has numerous drawbacks. Vaccination is a promising alternative but identification of ...protective antigens is a limiting step. The ribosomal protein P0, essential for protein synthesis, has been studied as a vaccine candidate. We generated sequence information of the P0 protein of the ectoparasite
and the host
. The region of the parasite P0 protein with less sequence similarity with that of the host P0 protein and high predicted antigenicity was used for peptide synthesis. The peptide was conjugated with keyhole limpet hemocyanin (KLH) for immunization of rohu at a dose of 1.5 μg/g body weight. Dot blot assays confirmed production of antibodies against pP0-KLH in immunized fish. We evaluated the efficiency of pP0-KLH as a vaccine antigen by challenge of the immunized fish with
.
. Although there was no significant difference in parasite load between both groups, a reduced and delayed mortality of 59% (15 days post-infection) in immunized group was noticed as compared to 75% mortality (within 7–15 days post-infection) in control group. The partial protection observed indicated the need for further optimization of this molecule to develop it into a vaccine candidate.
The knowledge of mucosa-associated molecular events that occur during infections is scarce despite the well-established importance of mucus in fish immunity. Using qRT-PCR, we analyzed the immune ...gene expression patterns in mucus of Labeo rohita experimentally infected with an ectoparasite Argulus siamensis. Mucus samples were collected at 0 h, 12 h, 24 h, 3 d, 7 d, 15 d, and 30 d post challenge of L. rohita with metanauplii of A. siamensis. All interleukins studied herein (IL 6, IL 15, and IL 1β) showed significant upregulation of expression levels in mucus of A. siamensis-infected fish compared to control samples. Further, the expression levels of molecules involved in pathogen recognition, toll like receptor 22, and pathogen presentation, β2 microglobulin, were found to be significantly upregulated in experimental samples until 7 d post challenge compared to control samples. The upregulated expression of lysozyme G at all time points post infection indicated the early activation of acute phase responses in mucus of infected L. rohita. Moreover, the expression levels of natural killer cell enhancing factor B were found to be higher in infected fish than they were in the control fish. The early upregulation of the immune genes observed herein reinforces the role of mucus as the first line of defense against pathogenic assault; furthermore, it expands our understanding of mucosal-immune responses to A. siamensis infection, which can aid development of immunological interventions.
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