Cloning and functional characterisation of natural killer enhancing factor-B (NKEF-B) gene of Labeo rohita: Anti-oxidant and antimicrobial activities of its recombinant protein.

Natural killer enhancing factor (NKEF) of peroxiredoxin family is an important innate immune molecule with having anti-oxidant activity. Although this gene has already been studied in a few fish species, it is yet to be identified and functionally characterised in Indian major carps. In the present study, the complete NKEF-B cDNA of rohu, Labeo rohita was cloned that encoded a putative protein of 197 amino acids. The phylogenetic study showed that L. rohita NKEF-B (LrNKEF-B) is closely related to NKEF-B of Cyprinus carpio and Danio rerio species. Tissue-specific expression of LrNKEF-B gene revealed the highest transcript level in the liver tissue. In the ontogeny study, the highest level of the expression was observed in milt and at 18 h post-development. The expression pattern of this gene was also studied in various pathogen models viz., Gram-negative bacteria (Aeromonas hydrophila), ectoparasite (Argulus siamensis) and a dsRNA viral analogue (poly I:C) in the liver and anterior kidney tissues of L. rohita juveniles. During A. hydrophila infection, the increase in expression of transcripts was observed at 3 h post-infection in both liver (15-fold) and anterior kidney (8-fold). In A. siamensis infection, the expression gradually increased up to 3 d post-infection in the anterior kidney, whereas in liver 3-fold up-regulation was noticed at 12 h post-infection. Similarly, during poly I:C stimulation, up-regulation of NKEF-B transcript was observed in anterior kidney from 1 h to 24 h post-stimulation and down-regulated afterwards whereas, the transcript level increased gradually from 6 h to 15 d post-stimulation in liver tissue. In vitro exposure to concanavalin, A and formalin-killed A. hydrophila upregulated NKEF-B gene expression in anterior kidney and peripheral blood leukocytes of L. rohita, however, down-regulated the same in the splenic leukocytes. A recombinant protein of LrNKEF-B (rLrNKEF-B) of 22 kDa was produced and it showed anti-oxidant activity by protecting supercoiled DNA and reducing insulin disulfide bonds. The minimum bactericidal concentration of this recombinant protein was found to be 4.54 µM against A. hydrophila and Staphylococcus aureus. Interestingly, rLrNKEF-B showed relative percent survival of 72.6 % in A. hydrophila challenged L. rohita, and the survival was found to be associated with a high level of expression of different cytokines, anti-oxidant genes and perforin in the rLrNKEF-B treated L. rohita. An indirect ELISA assay for estimation of NKEF was developed in L. rohita, and the concentrations of NKEF-B increased with time periods post A. hydrophila challenge viz., 0 h (42.56 ng/mL), 12 h (174 ng/mL) and 48 h (370 ng/mL) in rohu serum. Our results suggest a crucial role of LrNKEF-B in innate immunity against biotic stress and oxidative damage and also having antibacterial activity.

Development of a western blot method for detection of fish ectoparasite Argulus siamensis antigens.

A study was undertaken to develop a western blot method for detection of immunogenic proteins of fish ectoparasite, Argulus siamensis for its further use as potential vaccine candidates. Argulus antigens were prepared by homogenization and injected to rohu (Labeo rohita) juveniles for development of immune serum. The serum was used to immunostain the antigens in western blot. The other reagents added in sequence were rabbit anti-rohu serum, goat anti-rabbit ALP conjugate and substrate (BCIP-NBT). However, similar banding patterns were observed with both control and immunized rohu serum. Hence, a possible cross-reaction was suspected and verified in number of western blot experiments. A typical cross-reaction observed was of rabbit serum reacting directly with Argulus antigens. Hence, the rabbit anti-rohu serum was replaced with guinea pig anti-rohu serum. Another cross-reaction of goat anti-guinea pig ALP conjugate with rohu serum was eliminated by using goat anti-rabbit ALP conjugate with guinea pig serum. Thus, the final western blot method consisting of Argulus antigens → rohu serum → guinea pig anti-rohu serum → goat anti-rabbit ALP conjugate → substrate, yielded distinguishing results between control and Argulus-immunized rohu serum samples. The developed test has tremendous downstream applications, particularly in immunoproteomic studies of Argulus antigens.

Transcriptional analysis of immune-relevant genes in the mucus of Labeo rohita, experimentally infected with Argulus siamensis.

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 1ß) 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, ß2 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.

Transcriptional analysis of the common carp (Cyprinus carpio L.) immune response to the fish louse Argulus japonicus Thiele (Crustacea: Branchiura).

In the present study we investigated changes in transcription levels of a panel of selected immune relevant genes in peripheral blood leucocytes (PBL) and skin samples collected from carp exposed to larval Argulus japonicus. We show that in skin up-regulation of gene transcription of the chemokine CXCa, and to a lesser extent the chemokine receptor CXCR1 and the cytokine TNFalpha, are good indicators of parasite-induced skin damage at 2 days post-parasite exposure. Up-regulation of gene transcription corresponded well with an increase in leucocytes, probably neutrophilic granulocyte numbers in skin samples collected at the sites of infection. We show that time-point controls are essential when studying gene expression, especially in peripheral blood leucocytes (PBL). In addition, we demonstrate that non-infected control samples isolated from the skin of infected fish are suitable autologous controls, at least until after larval A. japonicus have undergone their first moult and begun to demonstrate increased mobility over their host's integument. The observed results are indicative of A. japonicus affecting the skin as a whole organ, particularly after the parasites' first moult, a phenomenon which has a great impact on correct skin sampling for RNA isolation.

Cortisol influences the host-parasite interaction between the rainbow trout (Oncorhynchus mykiss) and the crustacean ectoparasite Argulus japonicus.

The host-parasite interaction between the rainbow trout Oncorhynchus mykiss and the fish louse Argulus japonicus was investigated by administering low levels of dietary cortisol before infecting the fish with low numbers of the parasite. After 24 h, the dietary cortisol treatment elevated blood cortisol and glucose levels and stimulated the synthesis of secretory granules in the upper layer of skin cells. Infection with 6 lice per fish caused skin infiltration by lymphocytes, also in areas without parasites. The lymphocyte numbers in the blood at 48 h post-parasite infection were reduced. Other changes, typical for exposure to many stressors and mediated by cortisol, were also found in the epidermis of parasitized fish, although neither plasma cortisol nor glucose levels were noticeably affected. Glucocorticoid receptors were localized immunohistochemically and found in the upper epidermal layer of pavement and filament cells, and in the leucocytes migrating in these layers. Cortisol-fed fish had reduced numbers of parasites and the changes in the host skin are likely involved in this reduction. Thus a mild cortisol stress response might be adaptive in rejecting these parasites. Further, the data suggest that this effect of cortisol is mediated by the glucocorticoid receptor in the skin epidermis, as these are located directly at the site of parasite attachment and feeding in the upper skin cells that produce more secretory granules in response to cortisol feeding.