Objective: Human brucellosis poses a substantial public health threat in Tongliao; however, the species diversity of Brucella strains infecting humans remains unclear. Methods: A cross-sectional study was conducted among consecutive outpatients at a brucellosis hospital (August-September 2024). Demographic, clinical, and symptom data were collected via questionnaire. Serum samples were tested with SAT, and Brucella species/biovars were identified by nested-PCR. Sequencing data were used for phylogenetic analysis (neighbor-joining tree) to assess strain clustering. Results: The cohort comprised 42.17% (97/230) initial cases, 53.48% (123/230) follow-up cases, and 4.35% (10/230) health examiners. Clinical presentations included fever only (10.43%), fever with limb soreness (18.26%), limb soreness only (43.91%), and asymptomatic infection (26.96%). Serologically, 23.04% (53/230) had serum agglutination test (SAT) titers ≥ 1:100, 22.17% (51/230) had titers of 1:25 - 1:50, and 33.91% (78/230) were negative. Nearly half (49.57%, 114/230) had taken medication prior to consultation. Brucella was detected in 43.48% (100/230) of cases by nested-PCR. Species distribution among positive samples was B. abortus (70.00%), B. melitensis (11.74%), and B. suis (3.00%), with multiple biovars identified for each species. Phylogenetic analysis of 100 sequences resolved them into 9 distinct clades (Groups 1 - 9), revealing clear biovar-clade associations: B. abortus biovar 1 clustered in Groups 1 and 8, biovar 2 in Group 2; B. melitensis biovars 1, 2, and 3 were respectively assigned to Groups 5, 4, and 3/9; B. suis biovars 1 and 2 were placed in Groups 6 and 7. Conclusion: This study first reveals the high species and genetic diversity of Brucella infecting humans in Tongliao, showing a dominance by B. abortus with co-circulation of B. melitensis and B. suis. The high diversity indicates complex epidemic origins and control challenges, calling for integrated strategies and enhanced molecular surveillance.
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