Objective To establish a brucellosis monitoring and testing technique applicable for the rapid field screening of natural epidemic diseases.Methods A rapid testing technique for Brucella was developed based on a double-antibody sandwich testing model using gradient-variant quantum dots as fluorescent tracers.The sensitivity,linearity,precision,and specificity of the technique were evaluated using suspensions of standard Brucella strains.Methodological comparisons across different sample types were conducted to assess the consistency of the test results.Results The gradient-variant quantum dots detection method was evaluated with standard Brucella strains,exhibiting a sensitivity of 1×103 CFU/mL and a linear correlation coefficient(r)of 0.994(95%CI,0.933-1.055).The maximum coefficient of variation was 12.94%in repeated tests,showing good specificity.A comparative assessment of 305 clinical samples was conducted using the Brucella gradient-variant quantum dots detection method,the Rose Bengal plate agglutination test(RBT),and the serum agglutination test(SAT),yielding a Kappa value of 0.95,indicating almost perfect agreement.Additionally,a comparative assessment of 110 environmental samples collected on-site was conducted using the Brucella gradient-variant quantum dots detection method and quantitative real-time PCR(qPCR).The Kappa values for aerosol collection fluid,surface wipes,and wool samples were all above 0.83,demonstrating near-perfect agreement.For fecal and soil samples,the Kappa values were above 0.62,indicating substantial agreement.Conclusion The Brucella detection method based on gradient-variant quantum dots technology is simple and can be conducted rapidly.The detection method demonstrates high sensitivity,linearity,precision,and specificity.It shows consistent performance in clinical sample testing.It is well-suited for field rapid screening of natural epidemic diseases in field settings and shows good application prospects in the monitoring,prevention,and rapid detection of zoonotic diseases.

Objective:To study the comparative genomic characteristics of Marmota himalayana-derived (referred to as marmota-derived) Brucella abortus (B.ab). Methods:The species and types of one strain of marmota-derived Brucella and one strain of human-derived Brucella isolated from the brucellosis epidemic area in Qinghai Province in the same year were identified. Meanwhile, DNA was extracted for whole genome sequencing and comparative genomics analysis (including phylogenetic tree construction, gene family clustering analysis, common/specific gene analysis, and genomic structural variation analysis, etc.). Results:Two Brucella strains from different hosts were identified as B.ab. By constructing a phylogenetic tree, the marmota-derived B.ab strain was grouped with strains from Heilongjiang Province and showed genetic correlation with strains from Russia. Human-derived B.ab strain was classified as a strain in Inner Mongolia Autonomous Region, Hebei Province, Beijing City, and Gansu Province. The multilocus sequence typing (MLST) of the two strains belonged to the ST2 type. Multiple locus variable-number tandem repeat analysis (MLVA) belonged to two new MLVA-8 and MLVA-11 genotypes, which were clustered in two subclusters of the same cluster and clustered with the strains from Inner Mongolia Autonomous Region, Xinjiang Uygur Autonomous Region, and Hebei Province. The pan-genome numbers of the marmota-derived B.ab and human-derived B.ab were 283 and 8, respectively; the number of core genes (common genes) was 68 and 2, respectively; and the number of unique genes was 3 and 4, respectively. The unique gene encoded proteins were inconsistent. In marmota-derived B.ab, the main ones were the ABC transporter ATP-binding protein, N-terminal acetyltransferase, and glucose/galactose transporter. The number of homologous genes of the marmota-derived B.ab and human-derived B.ab was 16 and 20, respectively; the number of translocation and inversion genes was 13 and 8, respectively; the number of deletion mutation genes was 11 and 14, respectively. Pathogenicity analysis showed that both strains had the mprF resistance gene, and the marmota-derived B.ab strain also carried bacitracin and macrolide resistance genes. Conclusions:Brucella exhibits cross-species genetic diversity. The proteins encoded by the unique genes of the marmota-derived B.ab mainly play a role in metabolic and epigenetic regulation. The strains cluster with B.ab strains from northern China, providing a reference for molecular epidemiology and pathogen tracing of B.ab infection.

Objective:To study the comparative genomic characteristics of Marmota himalayana-derived (referred to as marmota-derived) Brucella abortus (B.ab). Methods:The species and types of one strain of marmota-derived Brucella and one strain of human-derived Brucella isolated from the brucellosis epidemic area in Qinghai Province in the same year were identified. Meanwhile, DNA was extracted for whole genome sequencing and comparative genomics analysis (including phylogenetic tree construction, gene family clustering analysis, common/specific gene analysis, and genomic structural variation analysis, etc.). Results:Two Brucella strains from different hosts were identified as B.ab. By constructing a phylogenetic tree, the marmota-derived B.ab strain was grouped with strains from Heilongjiang Province and showed genetic correlation with strains from Russia. Human-derived B.ab strain was classified as a strain in Inner Mongolia Autonomous Region, Hebei Province, Beijing City, and Gansu Province. The multilocus sequence typing (MLST) of the two strains belonged to the ST2 type. Multiple locus variable-number tandem repeat analysis (MLVA) belonged to two new MLVA-8 and MLVA-11 genotypes, which were clustered in two subclusters of the same cluster and clustered with the strains from Inner Mongolia Autonomous Region, Xinjiang Uygur Autonomous Region, and Hebei Province. The pan-genome numbers of the marmota-derived B.ab and human-derived B.ab were 283 and 8, respectively; the number of core genes (common genes) was 68 and 2, respectively; and the number of unique genes was 3 and 4, respectively. The unique gene encoded proteins were inconsistent. In marmota-derived B.ab, the main ones were the ABC transporter ATP-binding protein, N-terminal acetyltransferase, and glucose/galactose transporter. The number of homologous genes of the marmota-derived B.ab and human-derived B.ab was 16 and 20, respectively; the number of translocation and inversion genes was 13 and 8, respectively; the number of deletion mutation genes was 11 and 14, respectively. Pathogenicity analysis showed that both strains had the mprF resistance gene, and the marmota-derived B.ab strain also carried bacitracin and macrolide resistance genes. Conclusions:Brucella exhibits cross-species genetic diversity. The proteins encoded by the unique genes of the marmota-derived B.ab mainly play a role in metabolic and epigenetic regulation. The strains cluster with B.ab strains from northern China, providing a reference for molecular epidemiology and pathogen tracing of B.ab infection.

Objective:To analyze the surveillance results of human brucellosis in Qinghai Province, to study the epidemic characteristics of brucellosis, and to provide scientific basis for formulating brucellosis prevention and control strategies and measures.Methods:By using descriptive epidemiological method, surveillance data from 4 national and 26 provincial brucellosis surveillance sites in Qinghai Province in 2022 and brucellosis related information from the "Disease Surveillance Information Reporting and Management System" in 2022 were collected and summarized. The population, time, regional distribution and epidemic characteristics of brucellosis in Qinghai Province were analyzed.Results:In 2022, a total of 12 483 people were monitored at 4 national and 26 provincial brucellosis surveillance sites. In Rose Bengal plate agglutination test (RBPT), 714 patients were positive, and the positive rate was 5.72% (714/12 483). In tube agglutination test (SAT), 508 individuals tested positive, and the positive rate was 4.07% (508/12 483). There were 1 156 reported cases in 2022, including 910 males and 246 females, with a gender ratio of 3.70 ∶ 1.00. The age range was mainly between 30 and 59 years old, accounting for 77.68% (898/1 156). Most of them were farmers engaged in breeding and transportation, accounting for 81.40% (941/1 156). The onset time was concentrated from June to August, accounting for 50.26% (581/1 156). The distribution area was mainly in the northeast of Qinghai Province (Haibei Tibetan Autonomous Prefecture, Xining City, Haidong City), accounting for 86.33% (998/1 156).Conclusions:The epidemic of human brucellosis in Qinghai Province is still relatively serious, especially in the northeastern agricultural area. Comprehensive measures should be taken to further curb the outbreak of human brucellosis.

Objective:To analyze the surveillance results of human brucellosis in Qinghai Province, to study the epidemic characteristics of brucellosis, and to provide scientific basis for formulating brucellosis prevention and control strategies and measures.Methods:By using descriptive epidemiological method, surveillance data from 4 national and 26 provincial brucellosis surveillance sites in Qinghai Province in 2022 and brucellosis related information from the "Disease Surveillance Information Reporting and Management System" in 2022 were collected and summarized. The population, time, regional distribution and epidemic characteristics of brucellosis in Qinghai Province were analyzed.Results:In 2022, a total of 12 483 people were monitored at 4 national and 26 provincial brucellosis surveillance sites. In Rose Bengal plate agglutination test (RBPT), 714 patients were positive, and the positive rate was 5.72% (714/12 483). In tube agglutination test (SAT), 508 individuals tested positive, and the positive rate was 4.07% (508/12 483). There were 1 156 reported cases in 2022, including 910 males and 246 females, with a gender ratio of 3.70 ∶ 1.00. The age range was mainly between 30 and 59 years old, accounting for 77.68% (898/1 156). Most of them were farmers engaged in breeding and transportation, accounting for 81.40% (941/1 156). The onset time was concentrated from June to August, accounting for 50.26% (581/1 156). The distribution area was mainly in the northeast of Qinghai Province (Haibei Tibetan Autonomous Prefecture, Xining City, Haidong City), accounting for 86.33% (998/1 156).Conclusions:The epidemic of human brucellosis in Qinghai Province is still relatively serious, especially in the northeastern agricultural area. Comprehensive measures should be taken to further curb the outbreak of human brucellosis.

Objective:To investigate the diagnostic value of 99mTc-methylenediphosphonate(MDP) whole body bone scintigraphy in early brucellosis patients with bone and joint injuries. Methods:According to the Diagnosis for Brucellosis (WS 269-2019), combined with epidemiological history, clinical manifestations and serological tests, from November 2020 to April 2021, 15 early brucellosis patients (the course of disease was within 6 months) who had not received any drug treatment diagnosed at the Department for Brucellosis Prevention and Control, Qinghai Institute for Endemic Disease Prevention and Control were selected as the research subjects, and 99mTc-MDP whole body bone scintigraphy was performed on the patients to evaluate the images and analyze the pathological changes. Results:The 99mTc-MDP whole body bone scintigraphy of 15 patients with early brucellosis showed abnormalities, and the abnormal concentration of radionuclides mainly occurred in the 8th to 12th thoracic vertebrae (T8-12), the 1st to 2nd lumbar vertebrae (L1-2) and L4-5. Among them, the thoracic vertebrae abnormalities were T8, T9, T10, T11 and T12 in 1 case each; lumbar vertebrae abnormalities were 1 case of L1, 1 case of L2, 4 cases of L4, and 5 cases of L5. Conclusions:The 99mTc-MDP whole body bone scintigraphy is abnormal in patients with early brucellosis. Bone scintigraphy has certain value in the diagnosis of bone and joint injuries in patients with early brucellosis.

Objective:To observe multiple locus variable-number tandem repeat analysis (MLVA) typing of Brucella isolated from Himalayan marmot in Qinghai-Tibet Plateau of Qinghai Province, and to explore the relationship between the strains and strains previous isolated from Qinghai Province. Methods:Blood samples of Himalayan marmot were collected in Qinghai-Tibet Plateau of Qinghai Province from March 2019 to October 2020. Pathogens were isolated and cultured from Brucella antibody positive samples identified by using the rose bengal test (RBT). Conventional biological methods and molecular biological methods (BCSP31-PCR and AMOS-PCR) were used for strain identification. At the same time, MLVA method was used to genotype the isolated strains, and cluster analysis was used to analyze the genetic relationships between the strains based on the genotype of 70 Brucella isolated from different hosts in Qinghai Province. Results:A total of 1 466 blood samples of Himalayan marmot were collected from Qinghai-Tibet Plateau. Two strains of Brucella were isolated and cultured from 64 RBT-positive samples, named QH2013054 and QH2013062, respectively. They were identified as Brucella ovis biotype Ⅲ by conventional and molecular biological methods. The MLVA genotyping results showed that QH2013054 and QH2013062 were different at the Bru16 locus, indicating different MLVA genotypes. Cluster analysis showed that strain QH2013054 had the same MLVA genotype as 7 strains, among which 6 strains were from 3 farmers and 3 sheep from the same family in Gonghe County, and 1 strain was from a farmer in Menyuan Hui Autonomous County. The strain QH2013062 had the same MLVA genotype as 4 strains, including 3 strains from 3 farmers in Menyuan Hui Autonomous County and 1 strain from a farmer in Tu Autonomous County of Huzhu. Conclusions:The strains of Brucella isolated from Himalayan marmot in Qinghai-Tibet Plateau of Qinghai Province have the same MLVA genotype as some strains of Brucella isolated from humans and sheep in Qinghai Province. It is speculated that the host humans, sheep and Himalayan marmot in Qinghai-Tibet Plateau may have a common source of infection.

Objective:To analyze the results of serum erythropoietin (EPO) in adults patients with Kaschin-Beck disease (KBD) in Qinghai Province.Methods:According to the "Diagnosis of Kaschin-Beck Disease" (WS/T 207-2010), by using clinical examination and X-ray, adults over 20 years old in KBD areas of Xinghai County and Guide County, Hainan Tibetan Autonomous Prefecture, Qinghai Province, were divided into KBD case group ( n = 109) and internal control group ( n = 95) in July 2019. At the same time, healthy people were selected as external control group ( n = 90) in Xunhua County. Then 2 ml fasting cubital venous blood was collected from the target population to separate serum. The serum EPO level was determined by enzyme-linked immunosorbent assay (ELISA). Results:There was no significant difference in age and sex ratio among the 3 groups ( F = 0.73, P = 0.484; χ 2 = 1.03, P = 0.611). There was significant difference in serum EPO levels among the 3 groups [KBD case, internal and external control groups: (30.74 ± 26.23), (19.73 ± 11.53) and (10.83 ± 4.48) U/L, F = 26.51, P < 0.001]. Multiple comparisons showed that there were statistically significant differences in serum EPO levels between KBD case group and the internal and external control groups ( P < 0.05), but there was no significant difference between the internal and external control groups ( P > 0.05). Conclusions:The serum EPO level in adult KBD patients in Qinghai Province is increased significantly.

Objective:To master the epidemic trend of human brucellosis in Qinghai Province, so as to provide basis for scientific prevention and control of the disease.Methods:In 2019 and 2020, at the national and provincial brucellosis monitoring sites in Qinghai Province, a total of 18 counties (cities and districts, hereinafter referred to as counties), no less than 400 serum samples were sampled every year for brucellosis Rose-Bengal plate agglutination test (RBPT) and serum tube agglutination test (SAT), which would be tested and judged according to the criteria of "Diagnosis for Brucellosis" (WS 269-2019).Results:In 2019, a total of 1 612 people were monitored in national brucellosis monitoring sites, 93 were RBPT positive, 54 were SAT positive, 54 were diagnosed, and the prevalence rate was 3.35% (54/1 612). In 2020, 1 677 people were monitored in national brucellosis monitoring sites, 151 were RBPT positive, 80 were SAT positive, 80 were diagnosed, and the prevalence rate was 4.77% (80/1 677). There were significant differences in RBPT positive rate, SAT positive rate and prevalence rate among national monitoring sites between the two years (χ 2 = 12.52, 4.24, 4.24, P < 0.05). In 2019, a total of 6 043 people were monitored in provincial brucellosis monitoring sites, 128 were RBPT positive, 91 were SAT positive, 87 were diagnosed, and the prevalence rate was 1.44% (87/6 043). In 2020, 5 664 people were monitored, 108 were RBPT positive, 59 were SAT positive, 52 were diagnosed, and the prevalence rate was 0.92% (52/5 664). There was no significant difference in RBPT positive rate among provincial monitoring sites between the two years (χ 2 = 0.66, P = 0.416), and the differences in SAT positive rate and prevalence rate were statistically significant among provincial monitoring sites between the two years (χ 2 = 4.98, 14.57, P < 0.05). Conclusion:In 2019 and 2020, there are human brucellosis in national and provincial brucellosis monitoring sites in Qinghai Province.

Objective:Combined with the actual work requirements of brucellosis in Qinghai Province, to design a management information system for brucellosis patients, in order to realize the information of diagnosis and treatment of brucellosis patients.Methods:Based on the theory of management information system, according to the functional requirements of the actual work and performance parameters, the general framework of management information system was designed, the establishment of the management information system was gradually completed, and the system was improved through the system testing.Results:The system was easy to use and easy to operate, and patient's detailed information, diagnosis and treatment results and treatment plan could be obtained quickly. At the same time, follow-up, trace investigation, and brucellosis prevention and treatment propaganda could be carried out.Conclusions:The established brucellosis management information system can realize the electronic management of patient information, master the disease development and diagnosis and treatment results of brucellosis patients. Through follow-up investigation, the system can increase active screening and early warning of suspected cases, and provide health education to realize early diagnosis and early treatment of the patients.