Joy Krushna Jena

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.

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.

DNA sequence information resolves taxonomic ambiguity of the common mud crab species (Genus Scylla) in Indian waters

Abstract For several years, mud crabs of genus Scylla have been misidentified owing to their high morphological plasticity and the absence of distinct morphological diagnostic characters. The taxonomic confusion of genus Scylla de Haan is considered to be a primary constraint to the development of aquaculture. Although genus Scylla was revised using morphological and genetic characteristics, taxonomy of Scylla species occurring in India is still not clear. In this study, partial sequences of two mitochondrial genes, 16S rRNA and CO1 (Cytochrome C oxidase subunit I) in populations of Scylla spp. obtained from eleven locations along the Indian coast were used to differentiate and resolve taxonomical ambiguity of the mud crab species in India. The sequences were compared with previously published sequences of Scylla spp. Both trees generated based on 16S rRNA and CO1 indicated that all S. tranquebarica morphotypes obtained during this study and S. tranquebarica sequences submitted previously from Indian waters reciprocally monophyletic with reference sequence of S. serrata. Both sequence data and morphological characters revealed that the species S. serrata (Forskal) is the most abundant followed by S. olivacea. Further, the 16S rRNA and COI haplotypes of Indian S. tranquebarica obtained in the study significantly differed with the known S. tranquebarica by 6.7% and 10.6% respectively whereas it differed with known S. serrata by 0.0–0.7% only, a difference that was not statistically significant. From these studies it is clear that “S. tranquebarica” commonly reported from India should be S. serrata (Forskal).

A new fish cell line derived from the caudal fin of freshwater angelfish Pterophyllum scalare: development and characterization

In this study, a new cell line derived from the caudal fin of the freshwater angelfish Pterophyllum scalare was developed and characterized. The cell line was designated angelfish fin (AFF) and subcultured 44 times since its development. These cells grew well in Leibovitzs -15 medium supplemented with 10% foetal bovine saline (FBS) at 28° C and the modal chromosome number (2n) was 48. The AFF cell-line is mainly comprised of epithelial cells as confirmed by immunocytological technique using anti-cytokeratin antibodies, an epithelial cell marker. This cell line was tested for growth in a temperatures range from 20 to 37° C and at various FBS concentrations of 5-20% at 28° C. The cell line was cryopreserved at different passage levels and revived successfully with 80% survival rate. Polymerase chain reaction amplification and sequencing of partial mitochondrial 16s rRNA and coI genes confirmed that the AFF cell-line originated from angelfish. Mycoplasma sp. contamination was not detected in AFF cells and checked by Hoechst 33258 fluorescence staining. At the 42nd passage the cells were transfected with 2 μg of pAcGFP1-N1 expression vector. The AFF cells exhibited cytotoxic effects when exposed to the bacterial extra cellular products from Serratia marcescens and Proteus hauseri. The AFF cells and cells from kidney and brain did not show cytopathic effect when exposed to cyprinid herpes virus2 and viral nervous necrosis virus. The newly developed AFF cell line will be useful for the isolation of viruses affecting angelfishes, such as iridoviruses, in the future.

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.

WGSSAT: A High-Throughput Computational Pipeline for Mining and Annotation of SSR Markers From Whole Genomes

Mining and characterization of Simple Sequence Repeat (SSR) markers from whole genomes provide valuable information about biological significance of SSR distribution and also facilitate development of markers for genetic analysis. Whole genome sequencing (WGS)-SSR Annotation Tool (WGSSAT) is a graphical user interface pipeline developed using Java Netbeans and Perl scripts which facilitates in simplifying the process of SSR mining and characterization. WGSSAT takes input in FASTA format and automates the prediction of genes, noncoding RNA (ncRNA), core genes, repeats and SSRs from whole genomes followed by mapping of the predicted SSRs onto a genome (classified according to genes, ncRNA, repeats, exonic, intronic, and core gene region) along with primer identification and mining of cross-species markers. The program also generates a detailed statistical report along with visualization of mapped SSRs, genes, core genes, and RNAs. The features of WGSSAT were demonstrated using Takifugu rubripes data. This yielded a total of 139 057 SSR, out of which 113 703 SSR primer pairs were uniquely amplified in silico onto a T. rubripes (fugu) genome. Out of 113 703 mined SSRs, 81 463 were from coding region (including 4286 exonic and 77 177 intronic), 7 from RNA, 267 from core genes of fugu, whereas 105 641 SSR and 601 SSR primer pairs were uniquely mapped onto the medaka genome. WGSSAT is tested under Ubuntu Linux. The source code, documentation, user manual, example dataset and scripts are available online at https://sourceforge.net/projects/wgssat-nbfgr.

Identification and characterization of SSRs in Clarias batrachus and their application in population study

Magur Clarias batrachus is an indigenous catfish, commonly found in India, Nepal, Bhutan and Bangladesh. Presently, the populations of magur have declined rapidly in their natural habitat mainly due to overexploitation and habitat degradation. Understanding the population genetic structure of the species has significance in improvement of stocks and in conservation of the species. In the present study, simple sequence repeat (SSR) markers were used to differentiate the populations of magur, collected from three geographic locations. For this, a total of 31,814 SSRs were mined from the de novo assembled pooled of whole genome sequence data of C. batrachus. A bioinformatics pipeline with stringent criteria was applied to analyze the data which resulted in selection of 30,142 microsatellite loci falling in the intergenic region. Out of the 25 loci selected for primer development, 16 loci were successfully amplified and 9 loci were found to be polymorphic in this species. The average observed as well as expected heterozygosity in the loci across different stocks varied from 0.652 to 0.688 and 0.864 to 0.873, respectively. These three populations were further segregated into two clusters based on the NJ genetic distance. The Lucknow population formed one cluster, while the Bhubaneswar and Kolkata populations constituted another cluster. A comparable finding was also deduced from the STRUCTURE analyses. The results revealed significant variation among the populations of C. batrachus under study.