Monoclonal antibody generated against Nile tilapia (Oreochromis niloticus) IgT heavy chain using a peptide-based strategy.

Teleost IgT/Z plays a principal role in the defense mechanisms against infectious agents in the mucosal compartments and in systemic immunity. Previously, Nile tilapia (Oreochromis niloticus) IgT was discovered and characterized at transcription level. In this work, we generated a monoclonal antibody (mAb) that specifically recognized the Nile tilapia IgT. BALB/c mice were immunized with three synthetic peptides conjugated to KLH. The sequences of these peptides derived from the constant region of the Nile tilapia IgT heavy chain. ELISA and Western blotting confirmed the specificity of the polyclonal sera and the culture supernatant from a positive hybridoma clone. We observed immunoreactivity against a recombinant IgT fragment and native IgT in skin mucus. The anti-IgT mAb did not cross-react with purified tilapia IgM. Direct ELISA analysis allowed the quantification of skin mucus IgM and IgT concentrations. Flow cytometry analysis revealed differences in the percentage of IgT+ B cell populations between juveniles and adults in peripheral blood, head kidney and spleen lymphocytes and among the tissues analyzed. For further validation of the anti-IgT mAb utility, a recombinant vaccine candidate against sea lice (TT-P0 Ls) was injected into juvenile tilapia. Direct ELISA results revealed a differential secretion of skin mucus IgT and IgM after immunostimulation. In addition, the percentages of IgT+ B cells were determined at 7 days after booster and ex-vivo stimulation by flow cytometry. This mAb constitutes an important immunological tool to study the biological function and structural characteristics of tilapia IgT.

PACAP modulates the transcription of TLR-1/TLR-5/MyD88 pathway genes and boosts antimicrobial defenses in Clarias gariepinus.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multifunctional neuropeptide that belongs to the secretin/glucagon/GHRH/VIP superfamily. Some of these molecules have antimicrobial activity and they are capable of stimulating the immune system. The present work studied the antibacterial and immunostimulatory activity of PACAP-38 from African catfish Clarias gariepinus against the Gram-negative bacterium Pseudomonas aeruginosa in an in vivo test. PACAP-38 improved antimicrobial activity of skin mucus molecules against P. aeruginosa. The peptide modulates the gene expression profile of TLR-1, TLR-5, MyD88, IL-1ß, TNF-ɑ, IL-8, pardaxin, hepcidin and G/C-type lysozymes in skin, spleen and head kidney. The influenced exerted depended on the time after infection and tissue analyzed. This study provides the first evidence of a link between PACAP and antimicrobial peptides hepcidin and pardaxin. Our results suggest further use of PACAP as antimicrobial agent that could potentially be used to control disease in aquaculture.

Monoclonal antibody against Nile tilapia (Oreochromis niloticus) IgM heavy chain: A valuable tool for detection and quantification of IgM and IgM+ cells.

Nile tilapia (Oreochromis niloticus) is a freshwater fish, which is extensively cultivated worldwide and constitutes one of the model species for the study of fish immunology. Monoclonal antibodies are very advantageous molecular tools for studying teleost immune system. Specifically, monoclonal antibodies that react with immunoglobulins are used successfully in the study of the humoral immune response of several fish species. In the present study, we produced and characterized a monoclonal antibody against tilapia IgM heavy chain using a peptide-based strategy. The peptide sequence was selected from the surface-exposed region between CH3-CH4 domains. The specificity of the polyclonal serum and the hybridoma culture supernatant obtained by immunization with the peptide conjugated to keyhole limpet hemocyanin were evaluated by western blotting, both showing reactivity against tilapia serum IgM. The purified mAb was able to recognize secreted IgM by western blotting and ELISA and membrane IgM by flow cytometry. We also demonstrated that the antibody doesn't cross-react with a recombinant IgT fragment. This tool allowed us to study for the first time the stimulation of mucosal immunity after Pituitary Adenylate Cyclase Activating Polypeptide administration. Overall, the results demonstrated the utility of this mAb to characterize humoral immune response in O. niloticus.

Discovery of immunoglobulin T in Nile tilapia (Oreochromis niloticus): A potential molecular marker to understand mucosal immunity in this species.

Immunoglobulin molecules play an important role in the immune defense system in all jawed vertebrates, by protecting the organism from a wide variety of pathogens. Nile tilapia (Oreochromis niloticus) is extensively cultivated worldwide, with a strong established market demand. It constitutes one of the model species for the study of fish immunology and its genome is currently fully sequenced. The presence of the immunoglobulin M gene in this species is well documented, as well as its major role in systemic immunity. To date, the IgT gene from O. niloticus has not been identified and, therefore, no information is available on the role of this immunoglobulin isotype in the immune response in tilapia. In the present work, novel secreted and membrane immunoglobulin T isotypes and a fragment of IgM were isolated from tilapia head kidney lymphocytes. Their transcriptional profiles were analyzed by quantitative PCR in larval development and in different tissues of healthy or lipopolysaccharide/Edwardsiella tarda-challenged tilapia adults. The presence of IgT and IgM were detected in early stages of larval development. Additionally, these genes exhibited differential expression profiles in basal conditions and after E. tarda infection in adult tilapia, in accord with the proposed effector functions of these immunoglobulins in the systemic and mucosal compartments. Our results suggest the potential involvement of this new Ig in mucosal immunity in tilapia.

Novel IFNγ homologue identified in Nile tilapia (Oreochromis niloticus) links with immune response in gills under different stimuli.

Interferon gamma (IFN-γ) has important roles in both innate and adaptive immune responses. This cytokine plays a very important role in defining Th1 immune response in all vertebrates. In the present study, we identified and isolated for the first time the gene coding for Nile tilapia (Oreochromis niloticus) IFNγ from spleen lymphocytes. The isolated tilapia IFNγ has between 24 and 62% of amino acid identity as compared to reported sequences for other teleost fishes. It has close phylogenetic relationships with IFNγ molecules belonging to the group of Perciforms and presents the typical structural characteristics of gamma interferon molecules. The tissue expression analysis showed that IFNγ is expressed constitutively in head kidney, skin, intestine, muscle and brain. Its expression was not detected in gills by conventional RT-PCR. However, under conditions of stimulation with Poly I:C and LPS, IFNγ expression was up-regulated in gills after 24 h post-stimulation. IFNγ expression was also induced in gills 24 h after Edwardsiella tarda infection suggesting its important role in immunity against intracellular bacteria. The recombinant protein produced in Escherichia coli induced Mx gene transcription in head kidney primary culture cells. These results are the first steps to characterize the role of tilapia IFNγ in the defense against pathogens in tilapia. Furthermore, the isolation of this molecule provides a new tool to characterize the cellular immune response to various stimuli in this organism.

Recombinant novel pituitary adenylate cyclase-activating polypeptide from African catfish (Clarias gariepinus) authenticates its biological function as a growth-promoting factor in low vertebrates.

Nowadays, the studies of pituitary adenylate cyclase-activating polypeptide (PACAP)-related peptide (PRP) and PACAP in non-mammalian vertebrates, especially in fish, have paid attention mainly to the localization, cloning, and structural evolution of the peptides, but very little is known about its biological functions as growth-promoting factors in low vertebrates. In this work, we have cloned and characterized the PRP/PACAP cDNA from the commercially important North African catfish Clarias gariepinus. The sequence obtained agrees with the higher conservation of PACAP than of PRP peptide sequences. We have reported for the first time the recombinant expression of fish PRP and PACAP in mammalian cells and bacteria and also demonstrated that the growth rate of fish is enhanced by both PRP and PACAP recombinant peptides. The results obtained in vivo in three different fish species, catfish (C. gariepinus), tilapia (Oreochromis niloticus), and carp (Cyprinus carpio) support the finding that PACAP rather than PRP plays a primordial role in growth control in teleost fish. This finding could help to elucidate the neuroendocrine axis proposed to explain the hypothalamic regulation of growth in non-mammalian vertebrates.

Myostatin gene silenced by RNAi show a zebrafish giant phenotype.

Myostatin is a member of the transforming growth factor-beta (TGF-beta) family that functions as a negative regulator of skeletal muscle development and growth. Recently, it has been reported that the transgenic zebrafish expressing myostatin prodomain exhibited an increased number of fiber in skeletal muscle. Other novel results suggest that myostatin plays a mayor role during myogenesis, apart from inhibition of proliferation as well as differentiation. We have investigated the ability of double-stranded RNA (dsRNA) to inhibit myostatin function in the zebrafish. By microinjection dsRNA, corresponding to biologically active C-terminal domain from aminoacid 268 to end codon of tilapia myostatin protein, we produced an increased body mass in treated fish. The dsRNA injection in early development stage in zebrafish produced hyperplasia or hypertrophy. In addition, the interference of gene function showed a strong dependence on the amount of dsRNA.