Isolation，identification and pathogenicity analysis of Aeromonas hydrophila from farmed bullfrog（Rana catesbeiana）in Guangxi

【Objective】This study aimed to identify the pathogenic bacteria causing the morbidity and death of bullfrog（Rana catesbeiana），and to understand their pathogenicity，histopathological characteristics，drug resistance and the carrying of drug resistance genes，it would provide reference for the clinical pathological diagnosis and scientific prevention and treatment of bullfrog.【Method】Conventional bacterial isolation and purification methods were used to isolate dominant strains from the lesion tissues of diseased bullfrog. The pathogenic bacteria were identified by observing the morphological characteristics，physiological and biochemical characteristics and 16S rRNA gene sequence of the isolated strains. The drug resistance was determined by disk diffusion method，the carrying status of drug resistance genes and virulence genes was analyzed by PCR amplification，and the pathogenicity of the pathogenic bacteria was determined by artificial regression infection test. Histopathology was used to study the tissue characteristics of infected bullfrog.【Result】A dominant strain（NFAH-5）was isolated from the leg of the infected bullfrog. Based on the morphological，physiological and biochemical characteristics，hemolysis characteristics and molecular identification results，the isolated strain was identified as Aeromonas hydrophila. The strain NFAH-5 was sensitive to six antimicrobial drugs including doxycycline，polymyxin B and gentamicin，and was resistant to twelve antibiotics such as cporadine，enrofloxacin and amoxicillin. According to PCR test of the resistance genes，the isolate carried nine resistance genes such as Ant（3″）-I，Intl1 and parC，which was basically consistent with the drug resistance phenotype.Virulence gene PCR test revealed that isolate NFAH-5 carried seven virulence genes，including Ser，aer，Lip，exu，LuxS，act，and aha. Strain NFAH-5 infected bullfrog with a median lethal dose（LD₅₀）of 1.55×10⁶ CFU/mL. Infection-affected bullfrog had congestion and redness on the underside of the limbs，reddish-brown or yellow liver enlargement，intestinal emptiness or bleeding，and renal congestion. Symptoms were similar to those of naturally diseased bullfrog. The liver cells of the diseased bullfrog were degenerated；the red and white medulla of the spleen were difficult to distinguish，and a large amount of blood-derived pigmentation，glomerular atrophy，and hyaline degeneration of the renal tubules were observed.【Conclusion】A. hydrophila is the pathogenic bacterium that causes red leg disease in breeding bullfrog in Nanning，Guangxi. Combining the list of drugs approved for use in the document Paper on Aquaculture Drugs No. 2 in 2022 by Ministry of Agriculture and Rural Affairs，the sensitive drug doxycycline can be used to control it.