3D modeling and molecular dynamics simulation of an immune-regulatory cytokine,interleukin-10, from the Indian major carp, <Emphasis Type="Italic">Catla catla</Emphasis>

Interleukin-10 (IL-10) is a pleiotropic immune-regulatory cytokine that is expressed in various species of fish and higher vertebrates, and is activated during infection. In spite of its important role, IL-10 has not been well characterized either functionally or structurally in fish. To analyze its properties and function, we constructed a 3D model of IL-10 in the Indian major carp, the catla (Catla catla), which is a highly preferred fish species and the most commercially important one in the Indian subcontinent. The catla IL-10 model was constructed by comparative modeling using human IL-10 (2ILK) as the template, and a 5 ns molecular dynamics (MD) simulation was carried out to characterize its structural and dynamical features, which was validated by the SAVES, WHAT IF and MolProbity servers. Analysis using the VAST server revealed a comparatively low level of homology between catla and human IL-10 amino acids at the N-terminal (22.7%) compared to the C-terminal (38.29%). Six conserved domains (A–F) were predicted in catla that threaded well with human IL-10, but their putative interaction sites varied significantly. The amino acid residues in helices A and F differed in length between catla and human IL-10, which may lead to the differences in the IL-10/IL-10R complexes of these two species. The existence of two highly conserved amino acid residues (Cys5 and Cys10) in fish IL-10 but not in higher vertebrate (including human) IL-10 was analyzed in this 3D model. CastP, cons-PPISP and InterProSurf server identified several binding pockets with various probe radii, but Cys5 and Cys10 did not form any significant bonds relating to structural stabilization or protein–protein interactions.     

Expression analysis of immune response genes in fish epithelial cells following ranavirus infection

Ranaviruses (family Iridoviridae) are a growing threat to fish and amphibian populations worldwide. The immune response to ranavirus infection has been studied in amphibians, but little is known about the responses elicited in piscine hosts. In this study, the immune response and apoptosis induced by ranaviruses were investigated in fish epithelial cells. Epithelioma papulosum cyprini (EPC) cells were infected with four different viral isolates: epizootic haematopoietic necrosis virus (EHNV), frog virus 3 (FV3), European catfish virus (ECV) and doctor fish virus (DFV). Quantitative real-time PCR (qPCR) assays were developed to measure the mRNA expression of immune response genes during ranavirus infection. The target genes included tumour necrosis factor α (TNF-α), interleukin-1β (IL-1β), β2-microglobulin (β2M), interleukin-10 (IL-10) and transforming growth factor β (TGF-β). All ranaviruses elicited changes in immune gene expression. EHNV and FV3 caused a strong pro-inflammatory response with an increase in the expression of both IL-1β and TNF-α, whereas ECV and DFV evoked transient up-regulation of regulatory cytokine TGF-β. Additionally, all viral isolates induced increased β2M expression as well as apoptosis in the EPC cells. Our results indicate that epithelial cells can serve as an in vitro model for studying the mechanisms of immune response in the piscine host in the first stages of ranavirus infection.     

Comparative and evolutionary analysis of mitochondrial genes in Indian major carps

Direct sequencing of mitochondrial DNA regions such as cytochrome b, ATPase 6/8 and control region was performed to study comparative and evolutionary status of the three mitochondrial genes in Labeo rohita, Catla catla and Cirrhinus mrigala. DNA sequence alignment among species using specific software revealed comparative rates of divergence with considerably faster and more heterogeneous substitution rate for control region as compared to cytochrome b and ATPase 6/8. Despite the relatively high variability of control region, the overall levels of sequence divergence were low in coding regions. Two protein coding genes and the control region with varying degree of sequence divergence established two distinct groups which are genetically distant from each other exhibiting identical phylogenetic structure in IMCs. Closest relationship was between Labeo rohita and Catla catla indicating that they might have diverged from a common ancestral stock in genealogical lineage whereas Cirrhinus mrigala showed greater divergence with all the three DNA regions studied. Findings of this study will help to understand evolution of mitochondrial DNA genes in carps and facilitate future investigations on phylogeographic structure of Indian carps.     

Characterization of IgM of Indian major carps and their cross-reactivity with anti-fish IgM antibodies

Indian major carps (IMC), rohu (Labeo rohita), catla (Catla catla) and mrigal (Cirrhinus mrigala) were immunized with bovine serum albumin and the serum immunoglobulin M (IgM) was purified by affinity chromatography. The heavy and light chain of IgM of all the three species of IMC were about 88 and 26 kDa, respectively. Anti-fish IgM antibody against all the three species were raised in mice and the reaction of anti-fish IgM antibodies with IgM of all the three species of IMC were studied by Western blot. The anti-fish IgM antibodies reacted strongly with the heavy chain of the same species against which it was raised while the reactions with the heavy chain of other species were milder indicating some degree of epitope sharing among the heavy chains of IgM of IMCs. However, there was no cross-reaction with the light chain of any of the IgM.     

Establishment and characterization of an epithelial cell line from thymus of Catla catla (Hamilton, 1822)

A cell line, CTE, derived from catla (Catla catla) thymus has been established by explant method and subcultured for more than 70 passages over a period of 400 days. The cell line has been maintained in L-15 (Leibovitz) medium supplemented with 10% fetal bovine serum. CTE cell line consists of homogeneous population of epithelial-like cells and grows optimally at 28 °C. Karyotype analysis revealed that the modal chromosome number of CTE cells was 50. Partial amplification, sequencing and alignment of fragments of two mitochondrial genes 16S rRNA and COI confirmed that CTE cell line originated from catla. Significant green fluorescent signals were observed when the cell line was transfected with phrGFP II-N mammalian expression vector, indicating its potential utility for transgenic and genetic manipulation studies. The CTE cells showed strong positivity for cytokeratin, indicating that cell line was epithelial in nature. The flow cytometric analysis of cell line revealed a higher number of cells in S-phase at 48 h, suggesting a high growth rate. The extracellular products of Vibrio cholerae MTCC 3904 were toxic to the CTE cells. This cell line was not susceptible to fish betanodavirus, the causative agent of viral nervous necrosis in a large variety of marine fish.     

Effect of acclimated temperature on thermal tolerance,immune response and expression of HSP genes in Labeo rohita,Catla catla and their intergeneric hybrids

The ability of a species and population to respond to a decrease or an increase in temperature depends on their adaptive potential. Here, the critical thermal tolerance (CTmax and CTmin) of four populations: Labeo rohita, Catla catla, and their reciprocal hybrids L. rohita♀× C. catla♂ (RC) and C. catla♀ × L. rohita♂ (CR) being acclimatized at four acclimation temperatures (22, 26, 30 and 34 °C) were determined. All populations indicated substantial variations (P < 0.05) in CTmax and CTmin values. L. rohita displayed, comparatively the highest CTmax with largest total and intrinsic polygon zones as well as the upper and lower acquired thermal tolerance zones followed by RC and CR hybrids, while C. catla showed significantly the highest CTmin value and the smallest intrinsic and acquired thermal tolerance zones. Both hybrids illustrated low parent heterosis (≤11%). Additionally, the highest expression of Hsp70 and Hsp90 (heat shock proteins) genes, serum lysozyme level, respiratory burst activity and lowest lipid peroxidation level under lower and higher temperature shock further illustrated strong physiological mechanism of L. rohita in contrast to C. catla, to deal with acute temperature, while hybrids, especially F1 RC hybrid appeared as a good option to replace C. catla in relatively higher and lower temperature areas.     

Chlamydia psittaci plasmid-encoded CPSIT_P7 induces macrophage polarization to enhance the antibacterial response through TLR4-mediated MAPK and NF-κB pathways

Although the protective effects of Chlamydia psittaci plasmid-encoded protein CPSIT_P7 as vaccine antigens to against chlamydial infection have been confirmed in our previous study, the function and mechanism of CPSIT_P7 inducing innate immunity in the antibacterial response remain unknown. Here, we found that plasmid protein CPSIT_P7 could induce M1 macrophage polarization upregulating the genes of the surface molecule CD86, proinflammatory cytokines (TNF-α, IL-6, and IL-1β), and antibacterial effector NO synthase 2 (iNOS). During M1 macrophage polarization, macrophages acquire phagocytic and microbicidal competence, which promotes the host antibacterial response. As we observed that CPSIT_P7-induced M1 macrophages could partially reduce the infected mice pulmonary Chlamydia psittaci load. Furthermore, CPSIT_P7 induced M1 macrophage polarization through the TLR4-mediated MAPK and NF-κB pathways. Collectively, our results highlight the effect of CPSIT_P7 on macrophage polarization and provide new insights into new prevention and treatment strategies for chlamydial infection.     

Immune responses and protection in catla (Catla catla) vaccinated against epizootic ulcerative syndrome

Three different antigenic preparations from the epizootic ulcerative syndrome (EUS) pathogen Aphanomyces invadans were evaluated as vaccine candidate in catla (Catla catla). Anti-catla enzyme immunoconjugate was prepared after isolating catla immunoglobulin and raising hyperimmune sera against it, in rabbit. Three antigens namely, fungal extract (FE), fungal extract mixed with Freund’s incomplete adjuvant (FIA) in a 1:1 (v/v) ratio (FE + A) and extra cellular product (ECP) were prepared and three groups of catla were vaccinated intramuscularly with all these antigens (200 μg/fish). Different cellular and humoral immune responses were measured for the entire vaccinated and control group on 0th, 5th, 15th and 25th day post vaccination. Thirty days after the vaccination, the fish were challenged with an A. invadans zoospore dose of 1 × 10⁵ ml⁻¹ and mortality and relative percent of survival (RPS) were recorded. Study of cellular immunological parameters including antigen-specific leukocyte proliferation, antigen-specific nitric oxide production and superoxide anion production showed significantly higher (p < 0.05) values, in general, on 5th and 15th day post vaccination than the 0th day. Among all the antigenic groups, FE + A showed most significant response compared to the other groups. Among the humoral immune responses, lysozyme activity showed almost similar trend like cellular parameters. Anti-Aphanomyces antibody production was measured by enzyme-linked immunosorbent assay (ELISA) and it was increased with increasing days of vaccination in all the vaccinated groups with the highest observed on 25th day. Among the antigens, FE + A showed the highest antibody production following vaccination. The result of the homologous pathogen challenge study showed reduction in mortality in all the vaccinated groups. However, this reduction was not statistically significant (p > 0.05). Increased immune responses and protection have important implications with regard to the control of EUS by vaccination.     

Comparative structural analysis of myosin light chains and gene duplication in fish

Origin and structure of myosin light chain (MLC) proteins have been studied by comparative analysis of fish mlc1, mlc2, and mlc3 genes encoding MLC1, MLC2, and MLC3, respectively. The exon-intron structure of these genes has been analyzed in zebrafish Danio rerio, loach Misgurnus fossilis, fugu Takifugu rubripes, and Nile puffer Tetraodon fahaka. We propose that mlc1 and mlc3 are homologues genes originated by fish-specific whole genome duplication (paralogs). This is supported by high sequence similarity between mlc1 and mlc3 as well as by the exon-intron structure of these genes and their localization on different chromosomes. Exons 2 to 5 of mlc1 and mlc3 are highly conserved and have similar splicing sites. A paralog gene of mlc2 resulting from a similar duplication event has been identified in zebrafish genome. Expression of mlc1 paralog is limited to the larval stages of Danio rerio and to regenerating tissues of the adult fish. There is a possibility that the paralog of mlc1 encodes larval myosin light chain protein (larval MLC) previously reported in a number of fish species.     

Culture-based fisheries in minor perennial reservoirs in Sri Lanka: variability in production, stocked species and yield implications

Sri Lanka is blessed with a large number of irrigation reservoirs. Depending on their hydrological regimes they are broadly categorized into perennial and seasonal reservoirs, and are secondarily used for inland fisheries. The Ministry of Fisheries and Aquatic Resources in Sri Lanka initiated a programme to introduce culture‐based fisheries in minor perennial reservoirs (< 250 ha) whereby 15 minor perennial reservoirs situated in eight administrative districts were selected for introduction of culture‐based fisheries in its initial year of implementation. These reservoirs were stocked with fish fingerlings of catla Catla catla, rohu Labeo rohita, common carp Cyprinus carpio, Nile tilapia Oreochromis niloticus and post‐larvae of freshwater prawn Macrobrachium rosenbergii. Stocking commenced in 2004; stocking data from 2004, 2005 and 2006 are considered in this study. Stocking densities varied from 217 to 870 ha^?1 year^?1, with an average density of 425 ha^?1 year^?1. Fish catch statistics for 2005, 2006 and 2007 were considered; high variability in fish yield among reservoirs as well as in some reservoirs between the years were observed. Mean annual fish yield from all 15 reservoirs increased from 57.3 to 208.1 kg ha^?1 year^?1 after the introduction of culture‐based fisheries. Overall fish production in all 15 reservoirs increased by 263%, ranging from 42.8 to 1344%. Overall contributions of Nile tilapia, catla, rohu, common carp and freshwater prawn to fish harvest were 47.4, 27.2, 16.9, 4.3 and 0.7%, respectively. Biomass gain per fingerling stocked with respect to catla, rohu, common carp and freshwater prawn are 0.51, 0.42, 0.26 and 0.04 kg, respectively, indicating attractive monetary gains through stocking of these species. Results indicate the viability of culture‐based fisheries in minor perennial reservoirs. Possible reasons for high variability in fish yield and areas for follow‐up studies are discussed.     

Dietary omega-3 fatty acids enhance the B1 but not the B2 cell immune response in mice with antigen-induced peritonitis

The effects of omega-3 fatty acids on the adaptive immune response have mainly been analysed in vitro with varying results. How omega-3 fatty acids affect the adaptive immune response in vivo is largely unknown. This study examined the effects of dietary fish oil on the adaptive immune response in antigen-induced inflammation in mice, focusing on its effects on B cells and B cell subsets. Mice were fed a control diet with or without 2.8% fish oil, immunized twice with methylated BSA (mBSA) and peritonitis induced by intraperitoneal injection of mBSA. Serum, spleen and peritoneal exudate were collected prior to and at different time points after induction of peritonitis. Serum levels of mBSA-specific antibodies were determined by ELISA and the number of peritoneal and splenic lymphocytes by flow cytometry. The levels of germinal center B cells and IgM⁺, IgG⁺ and CD138⁺ cells in spleen were evaluated by immunoenzyme staining. Mice fed the fish oil diet had more peritoneal B1 cells, more IgM⁺ cells in spleen and higher levels of serum mBSA-specific IgM antibodies compared with that in mice fed the control diet. However, dietary fish oil did not affect the number of peritoneal B2 cells, splenic IgG⁺ or CD138⁺ cells or serum levels of mBSA-specific IgG antibodies in mice with mBSA-induced peritonitis. These results indicate that dietary fish oil can enhance the adaptive immune response, specifically the B1 cell response, which may lead to better protection against secondary infection as well as improvement in reaching homeostasis following antigenic challenge.     

Inducible nitric oxide synthase-deficient mice show exacerbated inflammatory process and high production of both Th1 and Th2 cytokines during paracoccidioidomycosis

Paracoccidioidomycosis, the major systemic mycosis in Latin America, is caused by fungus Paracoccidioides brasiliensis. To analyze the influence of inducible nitric oxide synthase (iNOS) in this disease, iNOS-deficient (iNOS^−/−) and wild-type (WT) mice were infected intravenously with P. brasiliensis 18 isolate. We found that, unlike WT mice, iNOS^−/− mice did not control fungal proliferation, and began to succumb to infection by day 50 after inoculation of yeast cells. Typical inflammatory granulomas were found in WT mice, while, iNOS^−/− mice presented incipient granulomas with intense inflammatory process and necrosis. Additionally, splenocytes from iNOS^−/− mice did not produce nitric oxide, however, their proliferative response to Con-A was impaired, just like infected WT mice. Moreover, infected iNOS^−/− mice presented a mixed pattern of immune response, releasing high levels of both Th1 (IL-12, IFN-γ and TNF-α) and Th2 (IL-4 and IL-10) cytokines. These data suggest that the enzyme iNOS is a resistance factor during paracoccidioidomycosis by controlling fungal proliferation, by influencing cytokines production, and by appeasing the development of a high inflammatory response and consequently formation of necrosis. However, iNOS-derived nitric oxide seems not being the unique factor responsible for immunosuppression observed in infections caused by P. brasiliensis.