OBJECTIVE To isolate and identify the pathogen of enteritis in Whitmania pigra to provide basis for its scientific prevention and treatment. METHODS Illumina high-throughput sequencing technique was used to analyze and compare the diversity and difference of intestinal microbial community structure between enteritis and healthy leeches, and to identify the potential pathogens of enteritis in Whitmania pigra. The pathogenic strains were isolated and purified from the intestinal tract of diseased leeches by pure microbial culture, and the sequence of 16S rDNA gene was analyzed to determine its taxonomic status. Koch's back staining method and intestinal pathological section were used to verify the pathogenicity of the Isolated strains, and the drug sensitivity was evaluated by disk diffusion method (K-B method). RESULTS The results of the Illumina high-throughput sequencing showed that compared with healthy leeches, the intestinal microbial community diversity of diseased leeches decreased significantly, and the relative abundance of Aminobacter, Lactobacillus and Prevotella decreased significantly(P<0.05). The relative abundance of Citrobacter, a common aquatic pathogen, increased significantly, reaching 60.92%, while that in the healthy leeches was 0.01%, suggesting that Citrobacter may be a potential pathogen. A strain of Citrobacter freundii SZ01 was isolated and identified from the intestinal tract of diseased leeches under the culture conditions of Citrobacter. Regression infection showed that the infection of leechs by SZ01 strain was consistent with that of natural infection. Antimicrobial sensitivity test showed that SZ01 strain was resistant to 10 antibiotics including cefalexin and ampicillin, moderately sensitive to five antibiotics including tetracycline and minocycline, and sensitive to 12 antibiotics including gentamicin, cefoperazone, and kanamycin. CONCLUSION High-throughput sequencing technique can effectively identify the pathogen of enteritis in Whitmania pigra. This study provides a basis for the prevention and treatment of Whitmania pigra enteritis and has reference significance for the application of high-throughput sequencing technique in the rapid isolation and identification of intestinal pathogens in aquatic animals.