An outbreak of disease affecting intensively cultured Singhi (Stinging Catfish, Heteropneustes fossilis) occurred in Tripura, India. Affected fish exhibited external lesions and elevated mortality. The study aimed to identify and describe the organism associated with this outbreak, confirm its pathogenicity in Singhi through experimental infection, quantify the median lethal dose, evaluate host responses, and determine the isolate's antimicrobial resistance profile.
A bacterial isolate, designated COF_AHE25, was recovered from clinically affected fish showing white patches, skin ulcers, hemorrhages and fin rot. Identification combined phenotypic and biochemical testing with molecular confirmation by sequencing of the 16S ribosomal RNA gene and phylogenetic analysis. Experimental pathogenicity trials were performed by challenging Singhi and monitoring cumulative mortality daily. The median lethal dose (LD50) was estimated from dose–response mortality data. Host responses were assessed using hematological, biochemical and immunological assays. Antimicrobial susceptibility testing used the Kirby–Bauer disk diffusion method following Clinical and Laboratory Standards Institute (CLSI) guidelines.
The recovered isolate, COF_AHE25, underwent standard phenotypic and biochemical evaluation and was molecularly confirmed by 16S rRNA gene sequencing. Sequence data were deposited (GenBank accession no. ON798627). Phylogenetic analysis supported identification of the isolate as Citrobacter freundii. This identification represents the first reported association of C. freundii with clinical disease in cultured Singhi.
Experimental infection with strain COF_AHE25 reproduced clinical signs similar to those observed during the natural outbreak. In experimentally infected Singhi, clinical manifestations included external lesions consistent with those from field cases. Mortality began at 4 days postinfection and progressed in a dose-dependent manner. The median lethal dose (LD50) for the isolate was estimated at 2.5 × 10^7 colony forming units (CFU) per fish, providing a quantitative measure of virulence in this host species.
Infected fish exhibited significant alterations in hematological, biochemical and immunological parameters. The pattern of changes was consistent with systemic bacterial septicemia, indicating that infection with this Citrobacter freundii strain produced systemic pathophysiology rather than limited superficial disease. Specific numerical values for the measured parameters were reported in the source article; the overall finding was statistically significant perturbation of host responses associated with morbidity and mortality.
Antimicrobial susceptibility testing used the Kirby–Bauer disk diffusion method interpreted according to CLSI guidelines. The isolate COF_AHE25 exhibited multidrug resistance. Reported resistances included tetracycline and erythromycin, resistance to several β-lactam antibiotics (penicillin, oxacillin and cefoxitin), and resistance to vancomycin. These findings indicate limited susceptibility to several antimicrobials commonly used in freshwater aquaculture and underscore the challenge of treating such infections in intensive production systems.
The study establishes Citrobacter freundii strain COF_AHE25 as a pathogenic bacterium capable of causing clinical disease and significant mortality in intensively farmed Singhi (Heteropneustes fossilis). The experimentally determined LD50 (2.5 × 10^7 CFU/fish) and reproduction of outbreak-like clinical signs confirm virulence in this host. Observed systemic host responses are consistent with septicemia.
The detection of multidrug resistance, including resistance to agents frequently used in aquaculture, highlights the need for strengthened disease surveillance, responsible antimicrobial stewardship, and improved health management in intensive fish farming. Specific control measures, choice of therapeutic agents, and on-farm interventions were not detailed here beyond the antimicrobial susceptibility findings; implementation should follow local regulations and veterinary guidance.
Funding sources reported included the National Fisheries Development Board (NFDB), Hyderabad, India, and the Department of Fisheries, Ministry of Fisheries, Animal Husbandry and Dairying, Government of India, under the Pradhan Mantri Matsya Sampada Yojana (PMMSY). The authors declared no conflicts of interest related to this research.
Notes: This summary and rewrite use only information contained in the source article abstract and associated PubMed entry. Where the source reported results without exhaustive numerical detail (for example, full parameter values for hematological or biochemical assays), those detailed values were not reproduced here because they were not provided in the source abstract.