Establishment and characterization of a continuous cell line from caudal fin of Labeo calbasu (Hamilton, 1822).

Labeo calbasu is an important food fish and candidate species for diversification of carp aquaculture. In the present study, we have established a continuous cell line, designated as L. calbasu fin (LCF), from caudal fin of L. calbasu using explant method. The cell line has been subcultured for over 73 passages and the LCF cells show optimal growth in Leibovitz's L-15 medium supplemented with 20% fetal bovine serum at a temperature of 28°C. In karyotype analysis, the modal chromosome number of LCF cells at 35th passage was found to be 50. The amplification and sequencing of partial fragments of mitochondrial genes, namely 16S rRNA and COI from LCF cells confirmed the origin of cell line from L. calbasu. The LCF cells could be successfully transfected with GFP reporter gene, indicating suitability of these cells for expression of foreign genes. Further, following inoculation with supernatant from Tilapia lake virus (TiLV) infected cell line, no cytopathic effects were observed in the LCF cells and cell pellet was negative for TiLV in RT-PCR, indicating that LCF cells were not susceptible to TiLV. The developed cell line has been submitted to National Repository of Fish Cell Lines being maintained at ICAR-National Bureau of Fish Genetic Resources, Lucknow (accession no. NRFC063). The newly developed LCF cell line would be helpful in investigating diseases affecting this candidate species particularly the ones suspected to be of viral etiology, and for cytotoxicity and transgenic studies.

Ontogeny and tissue-specific expression of immune-relevant genes in Catla catla (Hamilton).

India is the second largest fish producing country in the world with production of 12.6 million tonnes (mt) in 2017-18, and Indian major carps (IMCs) contribute bulk of this fish production. Catla, Catla catla is the fastest growing species among IMCs. However, the survival rate of catla during larval rearing is normally lower than the other IMCs i.e rohu Labeo rohita, and mrigal Cirrhinus mrigala. Continuous efforts are devoted for the identification of nutritional and environmental requirements of fish larvae in order to reduce hatchery mortalities. However, very little information is available regarding physiology of the immune system, especially during the late larval and juvenile stages. Hence, understanding the ontogenetic development of immune-relevant genes in the larval stages of catla will serve as the markers for the development of immune competence and thereby, will be beneficial in developing effective immune intervention strategies. In the present study, expression profiles of some of the important innate (IL-1ß, IL-10, iNOS and C3) and adaptive immune (RAG-1, Ikaros, IgM and IgZ) genes during ontogenetic developmental stages and in different tissues of catla were investigated using quantitative real-time PCR. The results revealed that immune genes IL-1ß, C3, IgM and IgZ were detected in the unfertilized eggs indicating their maternal inheritance. Immune genes, IL-1ß, IL-10 and iNOS were expressed significantly during initial larval developmental stages whereas C3, RAG-1, Ikaros, IgM and IgZ showed significant expression during advanced stages of larval development in catla i.e. from 23 days post hatch (dph). Study of tissue distribution pattern of the genes indicated that innate immune genes were ubiquitously expressed in different tissues with varying degree of expression, whereas adaptive immune genes were predominantly expressed in lymphoid organs of catla. The information thus generated will improve knowledge on the maturation of the immune system in catla and will aid in deciding the appropriate age for vaccination in this teleost species.

Expression of immune genes in Indian major carp, Catla catla challenged with Flavobacterium columnare.

Columnaris disease, caused by Flavobacterium columnare, is one of the important bacterial diseases responsible for large-scale mortalities in numerous freshwater fishes globally. This disease can cause up to 100% mortality within 24 h of infection and is considered to be a cause of concern for aquaculture industry. Despite being a serious disease, scarce information is available regarding host-pathogen interaction, particularly the modulation of different immune genes in response to F. columnare infection. Therefore, in the present study, an attempt has been made to study expression of important immune regulatory genes, namely IL-1ß, iNOS, INF-γ, IL-10, TGF-ß, C3, MHC-I and MHC-II in gills and kidney of Catla catla following experimental infection with F. columnare. The expression analysis of immune genes revealed that transcript levels of IL-1ß, iNOS, IL-10, TGF-ß, C3 and MHC-I were significantly up-regulated (p < 0.05) in both the organs of the infected catla. IFN-γ and MHC-II were up-regulated in gills of infected catla whereas, both the genes showed down-regulation in kidney. The results indicate that important immune genes of C. catla are modulated following infection with F. columnare. The knowledge thus generated will strengthen the understanding of molecular pathogenesis of F. columnare in Indian major carp C. catla.

Innate immune response against an oomycete pathogen Aphanomyces invadans in common carp (Cyprinus carpio), a fish resistant to epizootic ulcerative syndrome.

Infection with Aphanomyces invadans, also known as epizootic ulcerative syndrome, is a destructive disease of freshwater and brackishwater fishes. Although more than 130 species of fish have been confirmed to be susceptible to this disease, some of the commercially important fish species like common carp, milk fish and tilapia are reported to be resistant. Species that are naturally resistant to a particular disease, provide a potential model to study the mechanisms of resistance against that disease. In the present study, following experimental infection with A. invadans in common carp Cyprinus carpio, sequential changes in various innate immune parameters and histopathological alterations were monitored. Some of the studied innate immunity parameters viz. respiratory burst, alternative complement and total antiproteases activities of the infected common carp were higher compared to control fish, particularly at early stages of infection. On the other hand, some parameters such as myeloperoxidase, lysozyme and alpha-2 macroglobulin activities were not altered. Histopathological examination of the muscle at the site of injection revealed well developed granulomas at 12 days post infection, with subsequent regeneration of muscle fibers. From the results, it could be inferred that innate defense mechanisms of common carp are able to neutralize the virulence factors secreted by A. invadans, thereby, preventing its invasive spread and containing the infection. The results obtained here will help to better understand the mechanisms underlying resistance against A. invadans infection.

Innate immune response of Indian major carp, Labeo rohita infected with oomycete pathogen Aphanomyces invadans.

The fish pathogenic oomycete Aphanomyces invadans is the causative agent of epizootic ulcerative syndrome (EUS), a fish disease of international significance and reportable to the World Organisation for Animal Health. In spite of the current and potential impact of A. invadans infection on fisheries and aquaculture sectors of the world, very little is known about the host-A. invadans interactions. In the present study, following experimental infection with A. invadans in one of the Indian major carps, Labeo rohita, sequential changes in various innate immune parameters were monitored. The results indicated that at early stages of infection, no significant changes in any of the studied innate immune parameters were observed. However, at the advanced stages of infection from 6 to 12 days post infection (dpi), the respiratory burst and alternate complement activity were significantly higher whereas lysozyme, antiproteases and α-2 macroglobulin values were significantly lower than the control group and also from the infected group at earlier stages of infection. Since, the possibility of vaccination of fish against A. invadans appears remote due to difficulties in eliciting a specific antibody response, the information generated in the present study could be useful for developing strategies for improving resistance to A. invadans infection by stimulating the innate immunity through immunomodulation.