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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• Development of a shrimp cell culture system using various tissues from P.vannamei. Based on the amino acid composition of the hemolymph five different media were formulated. • HBSCM-5 with supplements showed a healthy morphology and cell proliferation was observed. • The hemocytes, heart, lymphoid tissue, hepatopancreas and gill cultures grew well and growth was sustain for a maximum period of 20, 25, 27, 10 &15 days. • Hemocyte culture was used for various applications such as WSSV replication, bacterial ECP toxicity. • The identity of the developed cell corroborated by PCR amplification of the mitochondrial COI. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Moribund koi carp, Cyprinus carpio koi, from a farm with 50% cumulative mortality were sampled with the aim of isolating and detecting the causative agent. Three bacterial species viz., Citrobacter freundii (NSCF-1), Klebsiella pneumoniae (NSKP-1) and Proteus hauseri [genomospecies 3 of Proteus vulgaris Bio group 3] (NSPH-1) were isolated, identified and characterized on the basis of biochemical tests and sequencing of the 16S rDNA gene using universal bacterial primers. Challenge experiments with these isolates using healthy koi carp showed that P. hauseri induced identical clinical and pathological states within 3 d of intramuscular injection. The results suggest P. hauseri (NSPH-1) was the causative agent . In phylogenetic analysis, strain NSPH-1 formed a distinct cluster with other P. hauseri reference strains with ≥99% sequence similarity. P. hauseri isolates were found sensitive to Ampicillin, Cefalexin, Ciprofloxacin and Cefixime and resistant to Gentamycin, Oxytetracycline, Chloramphenicol, and Kanamycin. The affected fish recovered from the infection after ciprofloxacin treatment. [ABSTRACT FROM AUTHOR]

Highlights: [•] PFF cell line was developed from the fin of the Puntius fasciatus. [•] CFF was developed from the fin of the Pristolepis fasciata. [•] SVC, iridovirus and KHV could not be detected from the fishes. [•] The ECPs of Vibrio cholerae and Aeromonas hydrophila were toxic to both the cells. [ABSTRACT FROM AUTHOR]

Aeromonas spp. cause significant mortality and high economic losses in Nile tilapia, Oreochromis niloticus culture worldwide. In the present study, we isolated and confirmed the involvement of four bacteria from diseased Nile tilapia showing signs of bilateral exophthalmia in Tamil Nadu, India. The bacterial isolates were identified as Citrobacter freundii, Pseudomonas aeruginosa , Acinetobacter juvenii and Aeromonas veronii based on biochemical and 16S rRNA gene sequence analysis. All the four isolates failed to produce positive CAMP reaction on 5% sheep blood agar and no cytopathic effect was observed in OnlL cell line after inoculating filtrate of the tissue homogenates prepared from the affected Nile tilapia. Experimental challenge infection assays revealed that out of the four, A. veronii isolate alone killed 100% of experimental fish within 120 h and the bacteria could be subsequently re-isolated from the dead fish. The LD50 values of A. veronii were found to be 105.35 CFU/fish. The bacterial isolate exhibited cytotoxic effects on onlL cells and haemolytic activity on 5% sheep blood agar and was found positive for virulence genes such as enterotoxin act and haemolytic toxin aerA. This study described a virulent A. veronii isolated from Nile tilapia with bilateral exophthalmia and eye lesions as that exhibited by the well known outbreak causing pathogen, Streptococcus agalactiae. In this study, A. veronii isolates were also found sensitive to cefixime, ciprofloxacin, chloramphenicol, kanamycin, cifixime/clavulanic acid, suggesting the possible application of these antibiotics in aquaculture. • Aeromonas veronii was isolated from Nile tilapia with bilateral exophthalmia. • Experimental challenge infection assays revealed that A. veronii isolate killed 100% of fish and could be reisolated. • The LD50 values of A. veronii were found to be 105.35 CFU/fish. • The affected fish recovered from the infection after ciprofloxacin treatment. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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 400days. 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 48h, 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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Sea cage farming of Asian seabass is a prospective aquaculture activity in India. Reports from India on betanodavirus-induced mortality in marine fish culture are not many. An outbreak of viral encephalopathy and retinopathy associated with red-spotted grouper nervous necrosis virus (RGNNV) genotype of betanodavirus in Asian seabass juveniles reared in indoor tanks and sea cages is described in the present paper. Mortality was less severe in cages when compared to tanks. Sequence analysis of the coat protein gene indicated that the virus belonged to RGNNV genotype but found to be distinct from other reported RGNNV isolates. Virus was able to induce cytopathic effects in fish cell lines. Light microscopic features of the disease are also described. [ABSTRACT FROM AUTHOR]