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.

Effect of immunization of rohu Labeo rohita with inactivated germinated zoospores in providing protection against Aphanomyces invadans.

Infection with Aphanomyces invadans is one of the most destructive diseases of freshwater fishes. Indian major carps, the dominant cultured species in the Indian sub-continent are highly susceptible to this disease. Till date, there is no effective treatment for control of this disease and immunization can be one of the strategies to reduce disease-related losses. In the present study, inactivated germinated zoospores of A. invadans were evaluated as antigen in conjunction with and without adjuvant Montanide™ ISA 763 A VG, for assessing their efficacy in rendering protection against A. invadans infection. For the experiment, rohu Labeo rohita, (n = 160, 74 ±â€¯12 g) were divided into 4 groups (C, A, G and GA) with 40 fish in each group. The fish in groups i.e., C, A, G and GA were injected intraperitoneally with PBS, adjuvant emulsified with PBS, inactivated germinated zoospores, and inactivated germinated zoospores emulsified with adjuvant, respectively. After 21 days of immunization, the fish were given a booster dose as above. After 7 days of the booster dose, the fish were challenged with zoospores of A. invadans to determine the relative percent survival (RPS). The results revealed that all the fish in C, A and G group succumbed to infection (0% RPS), although there was delayed mortality in fish from A and G groups in comparison to the C group. However, the fish in GA group showed significantly higher (P < 0.05) protection (66.7% RPS). In addition, following booster immunization (before challenge), the antibody level in the GA group was significantly higher (P < 0.05) than the control group. In western blotting, sera from G and GA groups showed reactivity with peptides of about 54 KDa. Following challenge (on 14th day), the antibody level as well as total antiprotease activity in fish of all the groups was significantly decreased in comparison to pre-challenge, except fish in GA group not exhibiting any gross lesions. In addition, sera of surviving fish of GA group showed significant inhibition of germination of zoospores and germlings growth in comparison to other groups (P < 0.05). Further, histopathological examination of the muscle tissue revealed that, in fish of GA group without any gross lesions, there were well developed granulomas and extensive mononuclear cell infiltration restricted to the site of injection, whereas in other groups, there was extensive myonecrosis with proliferating hyphae. These preliminary findings indicate that inactivated germinated zoospores of A. invadans in combination with adjuvant could stimulate good immune response and confer remarkable protection in rohu.

First report of cutaneous infiltrative lipoma in goldfish Carassius auratus.

Goldfish Carassius auratus is the most popular ornamental species, widely present in private and public aquaria. In the present case, 2 goldfish exhibited bilateral, multiple, variably sized, round, pale-white, soft, protruding masses on the body. The microscopic examination of the masses revealed well-differentiated adipocytes infiltrating the subcutaneous skeletal muscle bundles. The histological lesions were consistent with infiltrative lipoma. To our knowledge, this is the first report of cutaneous infiltrative lipoma in goldfish.

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.

Development, characterization and application of a new epithelial cell line from caudal fin of Pangasianodon hypophthalmus (Sauvage 1878).

A cell line, designated as PHF, has been established from caudal fin of Pangasianodon hypophthalmus. The cell line was developed using explant method and PHF cells have been subcultured for more than 72 passages over a period of 14 months. The cells were able to grow at temperatures between 24 and 32°â€¯C, with an optimum temperature of 28°â€¯C. The growth rate of PHF cells was directly proportional to FBS concentration, with optimum growth observed at 20% FBS concentration. On the basis of immunophenotyping assay, PHF cells were confirmed to be of epithelial type. Karyotyping of PHF cells revealed diploid number of chromosomes (2n = 60) at 39th and 65th passage, which indicated that the developed cell line is chromosomally stable. The origin of the cell line was confirmed by amplification and sequencing of cytochrome oxidase c subunit I and 16S rRNA genes. The cell line was tested for Mycoplasma contamination and found to be negative. The cells were successfully transfected with GFP reporter gene suggesting that the developed cell line could be utilized for gene expression studies in future. The cell line could be successfully employed for evaluating the cytotoxicity of heavy metals, namely mercuric chloride and sodium arsenite suggesting that PHF cell line can be potential surrogate for whole fish for studying the cytotoxicity of water soluble compounds. The result of virus susceptibility to tilapia lake virus (TiLV) revealed that PHF cells were refractory to TiLV virus. The newly established cell line would be a useful tool for investigating disease outbreaks particularly of viral etiology, transgenic as well as cytotoxicity studies.

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.