Pravata Kumar Pradhan

Detection of goldfish haematopoietic necrosis herpes virus (Cyprinid herpesvirus-2) with multi-drug resistant Aeromonas hydrophila infection in goldfish: First evidence of any viral disease outbreak in ornamental freshwater aquaculture farms in India

This outbreak report details of a mortality event where Cyprinid herpes virus-2 (CyHV-2) was detected in association with multidrug-resistant Aeromonas hydrophila infection in goldfish, Carassius auratus, from commercial farms. The goldfish exhibited large scale haemorrhages on the body, fins and gills, lepidorthosis, necrosed gills, protruded anus and shrunken eyes. White nodular necrotic foci in spleen and kidneys were noticed, along with necrosis and fusion of gill lamellae. Transmission electron microscopy of affected tissues revealed the presence of mature virus particles. Involvement of CyHV-2 was confirmed by PCR, sequencing and observed cytopathic effect in koi carp fin cell line along with experimental infection study. A bacterium isolated from the internal organs of affected fish was found to be pathogenic Aeromonas hydrophila having resistance to more than 10 classes of antibiotics. We postulate that CyHV-2 was the primary etiological agent responsible for this outbreak with secondary infection by A. hydrophila. The experimental infection trials in Labeo rohita and koi carp by intraperitoneal challenge with CyHV-2 tissue homogenates failed to reproduce the disease in those co-cultured fish species. This is the first report of a viral disease outbreak in organised earthen ornamental fish farms in India and bears further investigation.

Emergence of carp edema virus in cultured ornamental koi carp, Cyprinus carpio koi, in India

A disease outbreak was reported in adult koi, Cyprinus carpio koi, from a fish farm in Kerala, India, during June 2015. The clinical signs were observed only in recently introduced adult koi, and an existing population of fish did not show any clinical signs or mortality. Microscopic examination of wet mounts from the gills of affected koi revealed minor infestation of Dactylogyrus sp. in a few koi. In bacteriological studies, only opportunistic bacteria were isolated from the gills of affected fish. The histopathological examination of the affected fish revealed necrotic changes in gills and, importantly, virus particles were demonstrated in cytoplasm of gill epithelial cells in transmission electron microscopy. The tissue samples from affected koi were negative for common viruses reported from koi viz. cyprinid herpesvirus 3, spring viraemia of carp virus, koi ranavirus and red sea bream iridovirus in PCR screening. However, gill tissue from affected koi carp was positive for carp edema virus (CEV) in the first step of nested PCR, and sequencing of PCR amplicons confirmed infection with CEV. No cytopathic effect was observed in six fish cell lines following inoculation of filtered tissue homogenate prepared from gills of affected fish. In bioassay, the symptoms could be reproduced by inoculation of naive koi with filtrate from gill tissue homogenate of CEV-positive fish. Subsequently, screening of koi showing clinical signs similar to koi sleepy disease from different locations revealed that CEV infection was widespread. To our knowledge, this is the first report of infection with CEV in koi from India.

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.

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.

Establishment and characterization of a continuous cell line from thymus of striped snakehead, Channa striatus (Bloch 1793)

The establishment and characterization of a continuous cell line from the thymus of air-breathing fish Channa striatus are described. The cell line, designated C. striatus thymus (CST), has been subcultured over 71 times and shows optimal growth at 28°C in Leibovitz’s-15 (L-15) medium supplemented with 20% fetal bovine serum. The CST cells exhibited low plating efficiency which improved with increase in seeding density. The karyotype analysis revealed that CST cells have a normal diploid karyotype with 2n = 40. Partial amplification and sequencing of two mitochondrial genes, viz. 16S ribosomal RNA (rRNA) and cytochrome oxidase I, confirmed that the cell line originated from C. striatus. CST cells were successfully transfected indicating their potential application for expression of recombinant proteins. In immunocytochemical staining, CST cells showed characteristics of epithelial cells. These cells were sensitive to extracellular products of Vibrio cholerae MTCC 3904 as well as to heavy metal mercuric chloride. The CST cell line would be a useful tool in functional genomic studies such as RNA interference and gene knockout as well as for cytotoxicity studies.

Establishment of a macrophage cell line from adherent peripheral blood mononuclear cells of Catla catla

Catla catla, commonly known as catla, is endemic to the riverine system in northern India, Indus plain and adjoining hills of Pakistan, Bangladesh, Nepal and Myanmar. This fish grows best at water temperatures between 25°C and 32°C. Catla, an important culture species, has the highest growth rate amongst Indian major carps. A number of cell lines have been reported from catla (Ishaq Ahmad et al. 2008, 2009a, b). However, cell lines of leucocytic origin have not been reported from this species to the best of our knowledge. Such a cell line would provide useful information regarding functions of leucocytes in teleost species. Macrophages are the multipotent cells of the immune system that play a central role in innate and adaptive immune response in teleosts. In the innate immune system, macrophages are believed to be the principal phagocytic cells in fish (Blazer 1991) and are considered to be important cells in disease resistance. Fish macrophages have a scavenging function (Ganassin and Bols 1998), bactericidal activity (Honda et al. 1986), larvicidal activity (Whyte et al. 1989) and tumouricidal activity (Mulero et al. 1994). These cells also act as professional antigen presenting cells and therefore, are required for eliciting specific immune response (Guidotti and Chisari 2001). In addition, macrophages are capable of producing a large array of proinflammatory, procoagulatory and immune-regulatory products (Ellsaesser and Clem 1994). These cells are distributed in most of the organs of the body, some of which are specialised, fully differentiated cells specific for their resident organ. The macrophage cell lines provide a useful tool for studying functions of these cells. Moreover, such a cell line may contribute to the development and characterisation of macrophage cell markers and may be used as a source of macrophage signal peptides such as cytokines and other factors influencing the growth and maturation of leucocytes. These cell lines can be used for preliminary screening of immunomodulatory substances and therefore can provide an alternative to animal usage for such experiments. A number of fish macrophage cell lines have been developed namely from peripheral blood (Faisal and Ahne 1990; Vallejo et al. 1991; Weyts et al. 1997), spleen (Ganassin and Bols 1998) and kidney (Wang et al. 1995; Dannevig et al. 1997) as well as peritoneal washings (Watanabe et al. 1997). To date, no macrophage cell line is available from any of the commercially important fishes of the country. In the present study, we report the establishment and characterization of a macrophage cell line designated as C. catla macrophage (CCM) cell line from adherent blood mononuclear cells of C. catla. The heparinised blood was collected from caudal vein of a healthy catla weighing 550 g, layered over Histopaque-1077 (Sigma-Aldrich, St. Louis, MO) and centrifuged at 400×g for 30 min. Mononuclear cells (MNCs) were collected, diluted with phosphate-buffered saline (PBS) and centrifuged at 250×g for 10 min. The pellet was suspended in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20 % fetal bovine serum (FBS). The cells were then seeded in 25 cm flasks (Nunc, Roskilde, Denmark) at a concentration of 1×10 cells ml and the flasks were incubated at 28°C. After 24 h, the non-adherent cells were removed and fresh medium was added. The adherent cells showed aggregation and multiplication at several places in the flask (Fig. 1A, B). A D. K. Chaudhary :N. Sood (*) : P. K. Pradhan :A. Singh : P. Punia :G. Rathore National Bureau of Fish Genetic Resources, Canal Ring Road, P.O. Dilkusha, Lucknow 226 002 Uttar Pradesh, India e-mail: sood_neeraj@rediffmail.com

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.

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

ABSTRACT 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±12g) 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 54KDa. 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. HIGHLIGHTSInfection with Aphanomyces invadans is one of the most destructive diseases of freshwater fishes.Inactivated germinated zoospores of A. invadans were evaluated as antigen for immunization.The antigen along with adjuvant Montanide rendered significant protection against infection with A. invadans.The protection was positively correlated with specific antibody level and total antiprotease activity.

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° C, with an optimum temperature of 28° 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.

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.