Virulent and Multi-drug-Resistant Edwardsiella tarda Infection in Oscar Fish: Unveiling the Threat of Mass Mortality and AMR Dissemination.

The Oscar fish (Astronotus ocellatus) is among the most commonly domesticated and exported ornamental fish species from Kerala. The ornamental fish industry faces a significant challenge with the emergence of diseases caused by multi-drug-resistant bacteria. In the present study, six isolates were resolved from the diseased Oscar fish showing haemorrhages, necrosis, and loss of pigmentation. After phenotypic and genotypic characterization, the bacteria were identified as Edwardsiella tarda, Klebsiella pneumoniae, Enterococcus faecalis, Escherichia coli, Brevibacillus borstelensis, and Staphylococcus hominis. Experimental challenge studies in healthy Oscar fish showed that E. tarda caused 100% mortality within 240 h with 6.99 × 106 CFU/fish as LD50 and histopathology revealed the typical signs of infection. The pathogen was re-recovered from the moribund fish thereby confirming Koch's postulates. E. tarda was confirmed through the positive amplification of tarda-specific gene and virulence genes viz., etfD and escB were also detected using PCR. Antibiotic susceptibility tests using disc diffusion displayed that the pathogen is multi-drug-resistant towards antibiotics belonging to aminoglycosides, tetracyclines, and quinolones categories with a MAR index of 0.32, which implicated the antibiotic pressure in the farm. Plasmid curing studies showed a paradigm shift in the resistance pattern with MAR index of 0.04, highlighting the resistance genes are plasmid-borne except for the chromosome-borne tetracycline resistance gene (tetA). This study is the first of its kind in detecting mass mortality caused by E. tarda in Oscar fish. Vigilant surveillance and strategic actions are crucial for the precise detection of pathogens and AMR in aquaculture.

Investigation of antibiotic-resistant vibrios associated with shrimp (Penaeus vannamei) farms.

For the sustainable farming of disease-free and healthy shrimps, antimicrobial use is frequent nowadays in shrimp-cultured system. Considering the serious impact of global antimicrobial resistance (AMR), the present study was focused to investigate the prevalence of antimicrobial-resistant vibrios among infected shrimps (Penaeus vannamei) from two brackish water-cultured farms. Diverse species of vibrios viz. V. alginolyticus, V. parahaemolyticus, V. cholerae, V. mimicus, and V. fluvialis along with Aeromonas hydrophila, A. salmonicida and Shewanella algae were recovered from the shrimps on TCBS medium. Shannon-Wiener diversity index and H' (loge) were 1.506 and 1.69 for the isolates from farm 1 and farm 2, respectively. V. alginolyticus was found to be the most resistant isolate by showing multiple antibiotic resistance (MAR) index of 0.60 followed by V. mimicus (0.54) and V. parahaemolyticus (0.42). Among the 35 antibiotics of 15 different classes tested, tetracyclines, beta-lactams and cephalosporins were found as the most resistant antibiotic classes. All the isolates possessed a MAR index > 0.2 and the majority exhibited minimum inhibitory concentration (MIC) > 256 mcg/ml, thereby indicating the excess exposure of antibiotics in the systems. An enhanced altered resistance phenotype and a significant shift in the MAR index were noticed after plasmid curing. Public health is further concerning because plasmid-borne AMR is evident among the isolates and the studied shrimp samples are significant in the food industry. This baseline information will help the authorities to curb antimicrobial use and pave the way for establishing new alternative strategies by undertaking a multidimensional "One-Health" approach.

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.

Reassortment and evolutionary dynamics of tilapia lake virus genomic segments.

The tilapia lake virus (TiLV), a highly infectious negative-sense single-stranded segmented RNA virus, has caused several outbreaks worldwide since its first report from Israel in 2014, and continues to pose a major threat to the global tilapia industry. Despite its economic importance, little is known about the underlying mechanisms in the genomic evolution of this highly infectious viral pathogen. Using phylogenomic approaches to the genome sequences of TiLV isolates from various geographic regions, we report on the pervasive role of reassortment, selection, and mutation in TiLV evolution. Our findings provided the evidence of genome-wide reassortment in this newly discovered RNA virus. The rate of non-synonymous (dN) to synonymous (dS) substitutions was less than one (dN/dS = 0.076 to 0.692), indicating that each genomic segment has been subjected to purifying selection. Concurrently, the rate of nucleotide substitution for each genomic segment was in the order of 1-3 × 10-3 nucleotide substitutions per site per year, which is comparable to the rate of other RNA viruses. Collectively, in line with the results of the previous studies, our results demonstrated that reassortment is the dominant force in the evolution and emergence of this highly infectious segmented RNA virus.

Evaluation of in vitro and in vivo potential of Bacillus subtilis MBTDCMFRI Ba37 as a candidate probiont in fish health management.

Bacillus subtilis MBTDCMFRI Ba37 (B. subtilis Ba37), an antibacterial strain isolated from the tropical estuarine habitats of Cochin, was evaluated for in vitro and in vivo potential, and its application as a candidate probiont in fish health management. B. subtilis Ba37 was characterized using their morphological and biochemical properties. It exhibited exoenzymatic activities, tolerance to various physiological conditions and a wide spectrum of antibacterial activity against aquaculture pathogens such as Vibrio and Aeromonas. In co-culture assay, B. subtilis Ba37 inhibited Vibrio anguillarum O1 (V. anguillarum O1) even with the initial cell count of 104 CFUmL-1. Cytotoxicity assay performed using the cell free supernatant (CFS) of B. subtilis Ba37 revealed its non toxic nature. A twenty one days of feeding trial was conducted in juveniles of Etroplus suratensis (E.suratensis) by administrating B. subtilis Ba37 to evaluate its efficacy on growth, immune parameters and antioxidant enzyme activities. Overall the supplementation of B. subtilis Ba37 enhanced significantly (P < 0.05) the survival rate, weight gain, specific growth (SGR), feed conversion ratio (FCR), protein efficiency ratio (PER), and feed efficiency (FE) of the fed animals as compared with the control. The immune parameters and antioxidant activities such as total protein, alkaline phosphatase (ALP), superoxide dismutase (SOD) and catalase were also improved significantly (P < 0.05) while serum alanine aminotransferase (SGOT) and serum aspartate aminotransferase (SGPT) activities were decreased slightly than the control. After fifteen days of challenge test, the fish fed with B. subtilis Ba37 showed higher relative percentage survival (RPS) than the control. Thus the study indicated the advantages of B. subtilis Ba37 to be used as a candidate probiont, which could be effectively utilized in managing diseases in aquaculture systems and to improve the health of the host.

Antibiotic-resistant Enterobacteriaceae from diseased freshwater goldfish.

Goldfish farming gained more attention among the ornamental fishes in aquaculture industry. The occurrence of bacterial infections and further antimicrobial treatment lead to the major crisis of antibiotic resistance in aquaculture. We have isolated diverse enterobacteriaceae groups which affect the goldfish and identified their response towards 46 antimicrobials of 15 different classes. Thirteen significant bacterial isolates such as Edwardsiella tarda, Serratia marcescens, Klebsiella aerogenes, Proteus penneri, P. hauseri, Enterobacter cloacae, E. cancerogenus, E. ludwigii, Citrobacter freundii, E. coli, Kluyvera cryocrescens, Plesiomonas shigelloides and Providencia vermicola were recovered from the infected fish with the Shannon-wiener diversity index of 2.556. Multiple antibiotic resistance (MAR) index was found to be maximum for P. penneri (0.87) and minimum for C. freundii and E. cloacae (0.22), highlighting the hyper antibiotic selection pressure in the farm. The minimum concentration of antibiotics required to inhibit most of the resistant isolates was found to be > 256 mcg/ml. All the isolates were susceptible towards ciprofloxacin. Plasmid curing and further AMR tests could reveal the location of antibiotic resistance genes mainly as plasmids which determine the large extent of AMR spread through horizontal gene transfer. This study is the first of its kind to investigate the antimicrobial resistance profile of enterobacteriaceae recovered from goldfish, before and after plasmid curing.

Establishment and characterization of fantail goldfish fin (FtGF) cell line from goldfish, Carassius auratus for in vitro propagation of Cyprinid herpes virus-2 (CyHV-2).

BACKGROUND: Herpesviral hematopoietic necrosis disease, caused by cyprinid herpesvirus-2 (CyHV-2), is responsible for massive mortalities in the aquaculture of goldfish, Carassius auratus. Permissive cell lines for the isolation and propagation of CyHV-2 have been established from various goldfish tissues by sacrificing the fish. Here, we report the development of a cell line, FtGF (Fantail Goldfish Fin), from caudal fin of goldfish using non-lethal sampling. We also describe a simple protocol for successful establishment and characterization of a permissive cell line through explant method and continuous propagation of CyHV-2 with high viral titer using this cell line. METHODS: Caudal fin tissue samples were collected from goldfish without killing the fish. Cell culture of goldfish caudal fin cells was carried out using Leibovitz's L-15 (L-15) medium containing 20% FBS and 1X concentration of antibiotic antimycotic solution, incubated at 28 °C. Cells were characterized and origin of the cells was confirmed by sequencing fragments of the 16S rRNA and COI genes. CyHV-2 was grown in the FtGF cells and passaged continuously 20 times. The infectivity of the CyHV-2 isolated using FtGF cells was confirmed by experimental infection of naïve goldfish. RESULTS: The cell line has been passaged up to 56 times in L-15 with 10% FBS. Karyotyping of FtGF cells at 30th, 40th and 56th passage indicated that modal chromosome number was 2n = 104. Species authentication of FtGF was performed by sequencing of the 16S rRNA and COI genes. The cell line was used for continuous propagation of CyHV-2 over 20 passages with high viral titer of 107.8±0.26 TCID50/mL. Following inoculation of CyHV-2 positive tissue homogenate, FtGF cells showed cytopathic effect by 2nd day post-inoculation (dpi) and complete destruction of cells was observed by the 10th dpi. An experimental infection of naïve goldfish using supernatant from infected FtGF cells caused 100% mortality and CyHV-2 infection in the challenged fish was confirmed by the amplification of DNA polymerase gene, histopathology and transmission electron microscopy. These findings provide confirmation that the FtGF cell line is highly permissive to the propagation of CyHV-2.

Antimicrobial Resistance analysis of Pathogenic Bacteria Isolated from Freshwater Nile Tilapia (Oreochromis niloticus) Cultured in Kerala, India.

Aquaculture of popular freshwater species, Nile tilapia (Oreochromis niloticus), accounts for around 71% of the total global tilapia production. Frequent use of antibiotics for treating bacterial infections in tilapia leads to the emergence of antimicrobial resistance. To mitigate the issue, proper evaluation methods and control strategies have to be implemented. This study was aimed to analyze the antimicrobial resistance of bacterial isolates from the infected Nile tilapia cultured in freshwater. The recovered isolates were identified as Pseudomonas entomophila, Edwardsiella tarda, Comamonas sp, Delftia tsuruhatensis, Aeromonas dhakensis, A. sobria, A. hydrophila, A. lacus, Plesiomonas shigelloides and Vogesella perlucida through phenotypic and genotypic analyses. Using Primer-E software, Shannon Wiener diversity index of the isolates was determined as H' (loge) = 2.58. Antibiotic susceptibility test of the recovered strains through disk diffusion using 47 antibiotics, showed an elevated resistance pattern for Aeromonas hydrophila, Pseudomonas entomophila and Comamonas with higher multiple antibiotic resistance indexes (MAR index > 0.3). The minimum inhibitory concentration of antibiotics was > 256 mcg/ml for most of the resistant isolates. Meanwhile, all the recovered isolates were susceptible to amikacin, aztreonam, kanamycin, cefalexin, cefotaxime, levofloxacin, norfloxacin, piperacillin, and polymyxin-B.

Epizootics of epizootic ulcerative syndrome among estuarine fishes of Kerala, India, under post-flood conditions.

Epizootic ulcerative syndrome (EUS), primarily caused by the water mold Aphanomyces invadans, is an OIE-notifiable disease, having potential impacts on fisheries. We report EUS epizootics among estuarine fishes of Kerala, India, during 2018, under post-flood conditions 3 decades after its primary outbreak. Six fish species (Mugil cephalus, Platycephalus sp., Scatophagus argus, Arius sp., Planiliza macrolepis and Epinephelus malabaricus) were infected, including the first confirmed natural case in E. malabaricus and P. macrolepis. Salinity, surface temperature, dissolved oxygen and pH of resident water during the epizootic were <2 ppt, 25°C, 4.1 ppm and 7.0. The presence of zoonotic bacterial pathogens (Aeromonas veronii, Shewanella putrefaciens, Vibrio vulnificus and V. parahaemolyticus) in tissues of affected fish indicates that EUS-infected fish may pose a public health hazard if not handled properly. Lack of clinical evidence in the region during the last 3 decades, a high number of affected fishes, including 2 new fish species, the severity of skin lesions and very low water salinity (<2 ppt) during the outbreak in contrast to historical water salinity records suggest relatively recent invasion by A. invadans. Phylogenetic analysis based on the internal transcribed spacer region of the rRNA gene showed that the same clone of pathogen has spread across different continents regardless of fish species and ecotypes (fresh/estuarine environments). Altogether, the present study provides baseline data which can be applied in EUS management strategies within brackish-water ecosystems. We recommend strict surveillance and development of sound biosecurity measures against the disease.

Unravelling the menace: detection of antimicrobial resistance in aquaculture.

One of the major problems to be addressed in aquaculture is the prominence of antimicrobial resistance (AMR). The occurrence of bacterial infections in cultured fishes promotes the continuous use of antibiotics in aquaculture, which results in the selection of proliferated antibiotic-resistant bacteria and increases the possibility of transfer to the whole environment through horizontal gene transfer. Hence, the accurate cultivation-dependent and cultivation-independent detection methods are very much crucial for the immediate and proper management of this menace. Antimicrobial resistance determinants carrying mobile genetic transfer elements such as transposons, plasmids, integrons and gene cassettes need to be specifically analysed through molecular detection techniques. The susceptibility of microbes to antibiotics should be tested at regular intervals along with various biochemical assays and conjugation studies so as to determine the extent of spread of AMR. Advanced omic-based and bioinformatic tools can also be incorporated for understanding of genetic diversity. The present review focuses on different detection methods to unearth the complexity of AMR in aquaculture. This monitoring helps the authorities to curb the use of antibiotics, commencement of appropriate management measures and adequate substitute strategies in aquaculture. The long battle of AMR could be overcome by the sincere implementation of One Health approach. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of antibiotics and increased antimicrobial resistance (AMR) are of major concerns in aquaculture industry. This could result in global health risks through direct consumption of cultured fishes and dissemination of AMR to natural environment through horizontal gene transfer. Hence, timely detection of the antimicrobial-resistant pathogens and continuous monitoring programmes are inevitable. Advanced microbiological, molecular biological and omic-based tools can unravel the menace to a great extent. This will help the authorities to curb the use of antibiotics and implement appropriate management measures to overcome the threat.

Establishment and cryopreservation of a cell line derived from caudal fin of endangered catfish Clarias dussumieri Valenciennes, 1840.

We describe a new cell line, Clarias dussumieri fin (ClDuF), from the caudal fin of C. dussumieri using the explant technique followed by cryopreservation. The cryopreserved CiDuF cells were validated for quality and other characteristics. They showed typical epithelial morphology in vitro and epithelial cells outgrew their fibroblast cells after the fifth passage. ClDuF cells had a characteristic sigmoid curve with population doubling in 24 h. Immunotyping of the ClDuF cells against cytokeratin suggested the epithelial lineage. Chromosome analysis showed normal diploid (2n = 50) numbers and the cells did not contain any contamination, including Mycoplasma and other microbes. Partial sequencing of fragments of mitochondrial 16s rRNA and COI genes of ClDuF confirmed that the cell line was initiated from C. dussumieri. Cells at the 10th and 25th passages had more than 80% and 70% viability in the culture, respectively, after 6 months of storage at LN2 . These ClDuF cells were morphologically identical to the cells before freezing and the genetic resource of C. dussumieri was preserved. The species-specific cells can serve as a valuable source for virus isolation, conservation and cloning of somatic cells.

Diversity of antimicrobial-resistant pathogens from a freshwater ornamental fish farm.

The indiscriminate use of antimicrobials in aquaculture results in antibiotic selection pressure and proliferation of antimicrobial resistant (AMR) bacteria. Frequent assessment of antimicrobial resistance in aquaculture environment is inevitable so as to reduce the passage of clinically important AMR from aquatic to other environment. The present study analysed the antimicrobial resistance of pathogens associated with diseased koi carp and goldfish from an ornamental fish farm. Phenotypic and genotypic characterization of the recovered isolates from both fishes revealed significant pathogens in aquaculture such as Aeromonas, Edwardsiella tarda, Acinetobacter, Lactococcus, Citrobacter, Enterobacter and Comamonas. Shannon-Wiener diversity of koi isolates (2·359) was found to be higher than that of goldfish (1·864). Antibiotic susceptibility testing using disc diffusion with 47 antibiotics revealed significant resistance pattern of Acinetobacter, Comamonas, Klebsiella and Enterobacter from goldfish and Edwardsiella, Aeromonas, Lactococcus, Enterobacter and Acinetobacter from koi with higher multiple antibiotic resistance indexes (>0·3). The minimum inhibitory concentration of antibiotics for the major resistant isolates was found to be very high with >256 µg. All the isolates were susceptible to amoxicillin, kanamycin, cefepime, cefexime, cefotaxime, ceftazidime, doripenem, ciprofloxacin and norfloxacin, recommending their successful application in the farm. SIGNIFICANCE AND IMPACT OF THE STUDY: Antimicrobial resistance is a major threat faced in aquaculture industry. The current study provides baseline information regarding the antibiotic resistance patterns of diverse pathogens recovered from ornamental koi carp and goldfish. The higher MAR index of pathogens and greater MIC of antibiotics for the resistant isolates highlighted the intense use of antibiotics in aquaculture farm. The potential of the pathogens to exhibit resistance even towards the new generation antibiotics remind the need of prudent use of antibiotics and continuous monitoring and surveillance programmes.

The Development and Characterization of a Cell Culture System from Indian Mud Crabs Scylla serrata.

Commercially available culture media and supplements were tested for their potential to produce primary cell cultures from tissues of Indian mud crabs Scylla serrata. Eight commercially available culture media from Sigma-Aldrich (Leibovitz's L-15, Medium 199, Grace's Insect Medium, Minimal Essential Medium, Dulbecco's Modified Eagle Medium, TC-100 Insect Medium, IPL-41 Insect Medium, and Roswell Park Memorial Institute) were examined. Three different supplements (amino acid and sugar [AS], crab muscle extract [CME], and natural seawater [NSW]) were also examined. The hemocyte culture appeared to grow well for a maximum period of 21 d in 2 × L-15 medium supplemented with AS and 15% fetal bovine serum (FBS). Partial amplification and sequencing of the cytochrome oxidase subunit I (COI) gene confirmed that the primary hemocytes originated from Indian mud crabs. The effects of four metals on hemocyte viability were evaluated using the MTT assay. Of the four metals examined (arsenic, lead, cobalt, and nickel), cobalt and nickel were more toxic to the crab cells than the other metals. Both acridine orange/ethidium bromide and Hoechst staining showed the presence of apoptosis and necrosis in metal-treated groups, which suggests that metals in an aquatic environment induce death of the Indian mud crab's hemocytes. The hemocyte primary cell culture was also used to study the cytotoxicity effect of bacterial extracellular products from Vibrio harveyi and white spot syndrome virus. This study demonstrates that hemocyte primary cell culture can be used as a tool to study viral and bacterial pathogenesis and to assess the cytotoxicity of pollutants present in aquatic environments.

Medium optimization and characterization of cell culture system from Penaeus vannamei for adaptation of white spot syndrome virus (WSSV).

The lack of shrimp cell lines and difficulty in establishing shrimp cell culture systems, with an appropriate medium is a major concern in the aquaculture sector. The present study attempts to address this issue by developing an in vitro cell culture system from various tissues (hemocytes, heart, lymphoid tissue, hepatopancreas, gill, eye stalk, and muscle) of Penaeus vannamei (P.vannamei) using commercially available L-15 medium. The cell culture medium was formulated using five different media such as HBSCM-1, HBSCM-2, HBSCM-3, HBSCM-4, and HBSCM-5 containing L-proline and glucose with fetal bovine serum (FBS) supplements. Among the different media used, the HBSCM-5 medium with supplements showed good attachment and proliferation of cells with fibroblast-like, epithelioid, round, and adherent cell morphology in hemocyte culture. The same medium was further screened using different tissues to enhance the cell growth. The hemocytes, heart, and lymphoid tissue cells were passaged five times and maintained up to 20 days. Hepatopancreas and gill cells initially showed good morphological features and survived for more than ten days following subculture cells. Eye stalks and muscle cells perished within five days and did not show any unique morphology. The primary hemocyte cells were subjected to species identification, using cytochrome oxidase subunit I (COI) gene. To assess the primary hemocyte cell culture, cells were used for in vitro propagation of white spot syndrome virus (WSSV) and confirmed by the conventional polymerase chain reaction (PCR). Similarly, the primary cells were treated with bacterial extracellular products (ECPs) from Vibrio parahaemolyticus and Vibrio harveyi, to evaluate the cytotoxicity.

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.

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.

Candidatus Actinochlamydia pangasiae sp. nov. (Chlamydiales, Actinochlamydiaceae), a bacterium associated with epitheliocystis in Pangasianodon hypophthalmus.

Chlamydial infections are recognised as causative agent of epitheliocystis, reported from over 90 fish species. In the present study, the farmed striped catfish Pangasianodon hypophthalmus (14-15 cm, 70-90 g) with a history of cumulative mortality of about 23% during June and July 2015, were brought to the laboratory. The histopathological examination of gills from the affected fish revealed presence of granular basophilic intracellular inclusions, mostly at the base of the interlamellar region and in gill filaments. A concurrent infection with Trichodina spp., Ichthyobodo spp. and Dactylogyrus spp. was observed in the gills. The presence of chlamydial DNA in the gills of affected fish was confirmed by amplification and sequencing of 16S rRNA gene. BLAST-n analysis of these amplicons revealed maximum similarity (96%) with Candidatus Actinochlamydia clariae. On the basis of phylogenetic analysis, it was inferred that the epitheliocystis agents from striped catfish were novel and belonged to the taxon Ca. Actinochlamydia. It is proposed that epitheliocystis agents from striped catfish will be named as Ca. Actinochlamydia pangasiae. The 16S rRNA gene amplicons from novel chlamydiae were labelled and linked to inclusions by in situ hybridisation. This is the first report of epitheliocystis from India in a new fish host P. hypophthalmus.

Effects of concentration and gas flow rate on the removal of gas-phase toluene and xylene mixture in a compost biofilter.

The aim of this work was to study the performance of a compost/ceramic bead biofilter (6:4 v/v) for the removal of gas-phase toluene and xylene at different inlet loading rates (ILR). The inlet toluene (or) xylene concentrations were varied from 0.1 to 1.5gm-3, at gas flow rates of 0.024, 0.048 and 0.072m3h-1, respectively, corresponding to total ILR varying between 7 and 213gm-3h-1. Although there was mutual inhibition, xylene removal was severely inhibited by the presence of toluene than toluene removal by the presence of xylene. The biofilter was also exposed to transient variations such as prolonged periods of shutdown (30days) and shock loads to envisage the response and recuperating ability of the biofilter. The maximum elimination capacity (EC) for toluene and xylene were 29.2 and 16.4gm-3h-1, respectively, at inlet loads of 53.8 and 43.7gm-3h-1.

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.

First description of the female Elimaea melanocantha (Walker, 1869) (Orthoptera: Tettigoniidae: Phaneropterinae) from India.

The genus Elimaea Stal, 1874 was established for the type species Phaneroptera subcarinata Stal 1861 (Hongkong), the type genus of the Phaneropterinae. These insects are poorly known from India. Relevant important work on the taxonomy and distribution of Tettigoniidae of northeastern India include those of Barman & Srivastava (1976), Shishodia (2000), Shishodia & Tandon (2000), Barman (2003), Shishodia et al. (2003 & 2010) and, recently, Nagar et al. (2014, 2015a-b) have reported new and additional species of Phaneropterinae from India. The tribe Elimaeini Yakobson, 1905 (group Elimaeae Brunner, 1878) is known to comprise three Oriental genera, Elimaea Stal, 1874; Hemielimaea Brunner, 1878; and Ectadia Brunner, 1878. Karny (1926) divided the genus into three subgenera: Orthelimaea, Rhaebelimaea and Elimaea s. str., based on the shape of the anterior femora and the branching in the radius sector of the tegmen. Of these, Elimaea is most speciose and quite widespread in the Indian sub-continent. Gorochov (2013) reported two species, Elimaea melanocantha (Walker, 1869) and Elimaea nigrosignata Bolivar, 1900 in the group Neoelimaea. The terminology used is that of Ingrisch (1998) and Rentz and Colless (1990).