Pathogenicity of Elizabethkingia miricola causing mass mortalities of American bullfrog (Aquarana catesbeiana) and its induced host immune response.

In May 2024, outbreaks of massive mortality happened in the American bullfrog (Aquarana catesbeiana) farm in Honghu, Hubei Province China. A pathogenic strain of Elizabethkingia miricola, named as 2024EM05, was isolated and identified from diseased bullfrogs based on biochemical tests and molecular identification. Subsequently, the growth characteristic, pathogenicity and antibiotic sensitivity of the E. miricola strain, as well as the immune response of A. catesbeiana following E. miricola infection were both investigated. Virulence genes encoding polar flagella, capsule, urease accessory protein, isocitrate lyase, acyl carrier protein, T4SS effectors, outer membrane protein, O-antigen biosynthesis enzyme and porin protein were detected in E. miricola. The E. miricola 2024EM05 showed extensive antibiotic resistance to all tested aminoglycosides, cephalosporins, penicillin, carbapenems and sulfonamides. Challenge experiments confirmed the pathogenicity of isolated E. miricola, with a median lethal dosage (LD50) of 1.69 × 106 CFU/g for A. catesbeiana at 14 d post-infection. Severe histopathological changes, including extensive cell necrosis, hyaline degeneration and inflammatory cell infiltration, were observed in the diseased frog. Moreover, the activities of superoxide dismutase (SOD) and catalase (CAT) were significantly downregulated (P < 0.05), while the value of malondialdehyde (MDA) was significantly increased (P < 0.05) in serum of frog at 14th day post infection with E. miricola. The transcriptional profiles of immune associated genes (IL-8, IL-1β, IL-10, IFN-γ, TLR3, TLR4, CD4, CD8, CCL20, LZM, C3 and C4) in spleen and kidney of A. catesbeiana following E. miricola infection were determined by qRT- PCR assay. The results showed that all test genes were increased firstly and then decreased to the normal level, implying their regulatory role during the response of the host against bacterial invasion. The current study highlights that the presence of pathogenic E. miricola results in the mass mortalities of A. catesbeiana and provides valuable insights into the immune response of the American bullfrog after E. miricola infection.