A cross-sectional study was conducted from all outpatients who visited the Mongolian Medical Hospital in Tongliao city from August to September 2024. This hospital mainly treats patients with brucellosis. The brucellosis is diagnosed by clinical physicians according to the human brucellosis diagnosis standard (WS 269-2019). Th fresh blood samples were collected from all outpatients. The individual case investigation included patient name, gender, age, date of onset, contact history, medical history, medicant, etc. The clinical symptoms and signs included fever and limb soreness, etc. Laboratory tests include serum agglutination test (SAT). Physicians determined who needed to undergo serum tube agglutination test based on their condition.

3 mL venous blood from each outpatient was collected. Of 3 mL venous blood, 1mL was placed into a 3 mL sterile tube containing the anticoagulant (ethylene diamine tetraacetie acid, EDTA). Genomic DNA was extracted from 200 μL anticoagulated blood by using Blood/Cell/Tissue genomic DNA extraction kit (DNeasy Blood&Issue Kit QIAGEN, Germany). Remainder 2 mL blood was placed into a sterile tube without anticoagulants. And allowed it to stand at room temperature for 24 hours. The serum was separated and subjected to a tube agglutination test using Brucella antigen.

The DNA extraction from both positive and negative Brucella specimens was performed simultaneously during the process of extracting blood samples.

Based on the previously established method (Tian, 2021) [10], a nested-PCR brief description as follows: The first round PCR primers were F1:5'-GCGCATGCGAGATGGACGAA-3' and R1:5'-AGCAGACGAAGCCTTACAGATGAG-3'. The second round PCR primers: F2:5'-GAATGCGGTCAATGTTTTCTCGC-3' and R2:5'-ATATCTTCCGGGGCGAGTTGGTA-3'; the reaction system for the first round PCR included: 2 × Ex Taq Mastermix 12.5 μL, primer F1 and primer R1 each 0.4 μL (10 μ mol/L), DNA template 4 μL from blood, and double distilled water supplemented to 25 μL. PCR amplification reaction conditions: pre-denaturation at 95˚C for 4 min; denaturation at 95˚C for 30 sec, annealing at 55˚C for 30 sec, extension at 72˚C for 30 sec, 30 cycles; extend at 72˚C for 5 min. The reaction system and conditions for the second round were basically the same as those of the first round PCR. Except the primers F2 and R2, and the DNA template was 2 μL from the first round PCR products. The PCR products from the second round PCR will be sequenced using primers F2 and R2. The PCR amplification for positive and negative Brucella control samples was performed simultaneously during the process of PCR amplification for the DNA from blood samples.

Excel 2017 software was used for data organization and analysis. MEGA 5.1 software was used to construct an adjacent evolutionary tree to analyze the phylogenetic relationships by the unweighted pair group method arithmetic averages (UPGMA).

A total of 230 outpatients were enrolled in this study, including 156 males and 74 females, with a male-to-female ratio of 2.1:1. The median age was 47 years (range: 8 - 80 years), and the average age was 45.5 years. Among the participants, 97 (42.2%) were initial cases, 123 (53.5%) were follow-up cases, and 10 (4.3%) were health examiners.

Clinical presentations included fever only (24 cases, 10.4%), fever with limb soreness (42 cases, 18.3%), limb soreness only (101 cases, 43.9%), and asymptomatic infection (62 cases, 26.9%). One case presented with testicular pain. Regarding serum agglutination test (SAT) results, 53 patients (23.0%) had antibody titers ≥ 1:100, 51 (22.2%) had titers between 1:25 and 1:50, 78 (33.9%) were seronegative, and 48 (20.9%) were not tested. In terms of medication history before consultation, 116 patients (50.4%) had not taken any medication, while 114 (49.6%) reported prior use of medication. The onset duration was less than 1 month in 108 patients (46.9%), 1 - 6 months in 59 (25.7%), 6 months to 1 year in 24 (10.4%), and over 1 year in 29 (12.6%) (Table 1).

Table 1. Detection results on time of onset, clinical symptoms, drug in 230 outpatients.

Among 230 outpatients, 100 (43.5%) tested positive for the BrucellaIS711 gene. Of these positive samples, 70 (70.0%) were identified asB.abortus, comprising 66 (94.3%) biovar 1 and 4 (5.7%) biovar 2. A total of 27 (27.0%) positive sequences were identified asB. melitensis, distributed as biovar 1 (n = 2, 7.4%), biovar 2 (n = 3, 11.1%), and biovar 3 (n = 22, 81.5%). The remaining 3 (3.0%) were B.suis, including one biovar 1 and two biovar 2 (Table 2).

Table 2. Distribution of 100 Brucella IS711-positive samples.

Among initial cases (n = 97), the positivity rate was 43.3% (42/97), while among follow-up cases (n = 123), it was 44.7% (55/123). Among asymptomatic health examiners (n = 10), the positivity rate was 30.0% (3/10). Regarding species distribution, initial and follow-up cases demonstrated high consistency: B.abortus accounted for 69.0% (29/42) and 70.9% (39/55), respectively; B. melitensis accounted for 26.2% (11/42) and 27.3% (15/55), respectively; and B.suis constituted a minor proportion (4.8% in initial cases and 1.8% in follow-up cases). The biovar composition remained similarly stable: B.abortus was predominantly biovar 1 (96.6% in initial cases and 92.3% in follow-up cases), while B. melitensis was predominantly biovar 3 (63.6% in initial cases and 93.3% in follow-up cases). Among the positive samples from health examiners, two of B.abortus biovar 1 and one strain of B. melitensis biovar 3 were identified. Overall, no significant differences in IS711 detection rates were observed across different case types, and the species/biovar distribution patterns remained consistent, suggesting that this molecular assay demonstrates consistent diagnostic performance across patients with varying clinical statuses and indicating the persistent circulation of dominant strains in this region.

Of 230 outpatients, 1) 10.43% (24/230) was fever only. 41.67% (10/24) was positive for IS711 gene including 60.00% (6/10) wasB.abortus, 30.00% (3/10) wasB. melitensis, and 10.00% (1/10) wasB.suis. 2) 18.26% (42/230) was fever and limb soreness. 42.86% (18/42) was positive for IS711 gene including 72.22% (13/18) wasB.abortus, 22.22% (4/18) wasB. melitensis, and 5.56% (1/18) was B.suis. 3) 43.91% (101/230) was limb soreness only. 40.59% (41/101) was positive for IS711 gene including 68.29% (28/41) was B.abortus and 31.71% (13/41) was B. melitensis. 4) 26.96% (62/230) was asymptomatic cases. 48.39% (30/62) was positive for IS711 gene including 63.33% (19/30) was B.abortus biovars 1, 10.00% (3/30) was B. abortus biovars2, 20.00% (6/30) was B. melitensisbiovars 3, 3.33% (1/30) was B. melitensisbiovars 1, 3.33% (1/30) was 1 B.suis biovars 2. 5) There was one case had testicular pain, which was B.abortus biovars 1 (Table 1).

Of 230 outpatients, 1) 23.04% (53/230) with serum antibody levels was above 1:100, and 43.40% (23/53) was positive for IS711 gene. Among them, 52.17% (12/23) was B.abortus biovars 1, 13.04% (3/23) wasB.abortus biovars 2, 4.35% (1/23) was B. melitensisbiovars 1, 26.09% (6/23) was B. melitensisbiovars 3, 4.35% (1/23) was B. suis biovars 1. 2) 22.17% (51/230) with serum antibodies was at 1:25-1:50. And 39.22% (20/51) was positive for IS711 gene including 65.00% (13/20) was B.abortus biovars 1, 5.00% (1/20) was B.abortus biovars 2, 5.00% (1/20) was B. melitensisbiovars 1, 20.00% (4/20) was B. melitensisbiovars 3, 5.00% (1/20) was B.suis biovars 2. 3) 33.91% (78/230) was negative serum antibodies, and 38.46% (30/78) was positive for IS711 gene. 70.00% (21/30) was B.abortus biovars 1, 3.33% (1/30) was B. melitensisbiovars 1, 6.66% (2/30) was B. melitensisbiovars2, 16.67% (5/30) was B. melitensisbiovars 3, 3.33% (1/30) was B.suis biovars 2. 4) 20.87% (48/230) was not tested for SAT, and 56.25% (27/48) was positive for IS711 gene. Among them, 74.07% (20/27) was B.abortus biovars 1, 25.93% (7/27) was B. melitensisbiovars 3 (Table 3).

Table 3. Detection results on IS711 gene in different serum antibody titers.

Of 230 outpatients, 50.43% (116/230) was not use medicant before seeking medical treatment, 38.79% (45/116) was positive for the IS711 gene including 68.89% (31/45) was B.abortus, 26.67% (12/45) was B. melitensis, and 4.44% (2/45) was B.suis. 49.57% (114/230) was drug users before seeking medical treatment, 48.25% (55/114) was positive for the IS711 gene including 70.91% (39/55) was B.abortus, 27.27% (15/55) was B. melitensis, and 1.82% (1/55) was B.suis.

Of 230 outpatients, 1) 46.96% (108/230) was less than 1 month for onset time. Among them, 38.89% (42/108) was positive for IS711 gene including 71.43% (30/42) was B.abortus, 23.81% (10/42) was B. melitensis, and 4.76% (2/42) was B.suis; 2) 49.15% (29/59) was positive for IS711 gene in 59 cases with 1 month to 6 months for onset time including 68.97% (20/29) was B.abortus, 27.59% (8/29) was B. melitensis, and 3.45% (1/29) was B.suis; 3) 10.43% (24/230) was 6 months to 1 year for onset time. Among them, 66.67% (16/24) was positive for IS711 gene including 56.25% (9/16) was B.abortus and 43.75% (7/16) was B. melitensis; 4) 12.61% (29/230) was over 1 year. Among them, 34.48% (10/29) was positive for IS711 gene including 90.00% (9/10) was B.abortus and 10.00% (1/10) was B. melitensis; 5) 4.35% (10/230) was health examiner. Among them, 30.00% (3/10) was positive for IS711 gene including 2 B.abortus and 1 B. melitensis.

In the Left Banner area of Tongliao city, 52.17% (12/23) of outpatients were IS711-positive, comprising 8 B.abortus biovar 1 and 4 B. melitensis biovar 3. In Zhalai Banner (Tongliao), 66.67% (4/6) were positive, with equal distribution of B.abortusand B. melitensis. Kailu region (Tongliao) showed 30.77% (4/13) positivity, all identified as B.abortus. Notably, all 4 cases from Shenyang (Liaoning province) were B. melitensis biovar 3, while 3 cases from Heilongjiang province were exclusively B.abortus biovar 1. These findings suggest regional variations in both detection rates and species distribution, with mixed B.abortus and B. melitensis circulation in some Tongliao areas versus single-species dominance in other regions, potentially reflecting distinct epidemiological patterns or exposure sources across geographical areas.

Figure 1. Cluster analysis of 100 sequences of theBrucella IS711 gene. Note: Cluster analysis revealed that these sequences formed nine distinct groups, with each group corresponding to specific species and biovars. This clustering pattern demonstrates a strong correlation between IS711 sequence variation and the classical species/biovar classification.

Cluster analysis of IS711 sequences from 100 Brucella sequences were divided into 9 groups (Group 1 to Group 9) (Figure 1). Group 1 consists of 42 B.abortusbiovars 1. Group 2 consists of 4 B.abortusbiovars 2. Group 3 consists of 7 B. melitensis biovars 3. Group 4 consists of 3 B. melitensis biovars 2. Group 5 consists of 2 B. melitensis biovars 1. Group 6 consists of 1 B.suisbiovars 1. Group 7 consists of 2 B.suis biovars 2. Group 8 consists of 24 B.abortusbiovars 1. Group 9 consists of 15 B. melitensis biovars 3. This clustering pattern demonstrates a strong correlation between IS711 sequence variation and the classical species/biovar classification, with each biovar forming distinct phylogenetic clusters, while the separation of B.abortus biovar 1 into two groups (Group 1 and 8) and B. melitensis biovar 3 into two groups (Group 3 and 9) suggests potential genetic diversity within these predominant biovars in the study region.