A novel grass carp pathogen Citrobacter amalonaticus induces severe intestinal inflammation

Abstract

Citrobacter amalonaticus is widely recognized as a conditionally pathogenic bacterium, primarily associated with meningitis, brain abscesses and urinary tract infections in humans. To date, no documented cases of C. amalonaticus causing disease in fish have been reported. However, our recent study identified this bacterium as a novel pathogen responsible for over 50 % morbidity in grass carp (Ctenopharyngodon idella). Through comprehensive morphological analysis, biochemical phenotype identification and 16S rRNA gene sequencing, this pathogen was confirmed as C. amalonaticus (ca). The lethal dose, 50 % (LD₅₀) of ca in grass carp was determined to be 2.04 × 10⁵ CFU/mL. Infected fish exhibited symptoms consistent with naturally diseased individuals, including ulcerated and hemorrhagic skin, abdominal distension, hemorrhagic and swollen cloaca, and severe ascites. Histopathological examination revealed extensive damage to the intestine, liver, spleen, and kidney, with the intestines exhibiting particularly severe lesions. The health status of the fish was assessed through various parameters, including hematological indices, intestinal enzyme activity 72 h post-infection and expression levels of inflammation-related genes at multiple time points (0, 24, 48, 72, 96 hpi). Results indicated significant changes, including increased white blood cell count, reduced digestive enzyme activity (e.g., trypsin, TRY), elevated oxidative stress enzyme activity (e.g., catalase, CAT), and upregulation of inflammatory genes (e.g., IL-8). Drug sensitivity testing demonstrated that ca was highly susceptible to ciprofloxacin, ofloxacin and vancomycin. This study confirms the pathogenicity of ca in fish and represents the first identification of C. amalonaticus infection in grass carp, causing severe intestinal inflammation. These findings provide valuable data for the prevention and treatment of this disease.