Objective To analyze the epidemiological surveillance results of human brucellosis in Shanxi Province from 2012 to 2017,to know the epidemic status of brucellosis,and to provide evidence for prevention and control of brucellosis.Methods Incidence date and surveillance date of disease outbreaks in Shanxi Province from 2012 to 2017 were collected,the retrospective analysis method was used to analysis the "three distribution" of brucellosis,outbreak situation and the results of serological and pathogenic surveillance in the 4 surveillance stations.Results A total of 36 220 brucellosis cases were reported from 2012 to 2017,the average incidence was 16.62/100 000;8 540 brucellosis cases were reported in 2014,with incidence 23.53/100 000;a total of 23 197 cases of brucellosis were reported mainly in Datong,Shuozhou,Jinzhong and Xinzhou,accounting for 64.04％ of the province total.The onset was seasonal,and the peak of the epidemic was from March to August,accounting for 67.23％ (24 350/36 220).The brucellosis cases were mainly youth (23 084),male (28 317),farmers and herdsman (32 616).In the 4 surveillance stations of the brucellosis,39 140 cases were investigated,of which 10 536 cases did serological test,in which 585 were positive for Brucella (5.55％).The highest positive rate of serological test was 9.50％ (226/2 738) which was found in Tianzhen.A total of 626 samples carried out pathogen culture,in which 107 strains of brucellosis were detected,the detection rate was 17.09％,and 106 strains Brucella were melitensis biovar 3 of the total strains except 1 mutant.Conclusions The reported incidence in Shanxi Province is in a decline tendency,but the situation of brucellosis epidemic is still relatively serious.It is suggested that the surveillance work should be strengthened;the epidemic situation of brucellosis should be mastered in time and effectively controlled.

Objective To screen the most suitable medium for Brucella drug susceptibility test, and observe the resistance of human derived Brucella to different antibiotics. Methods Totally 180 strains of Brucella isolated from 25 provinces (municipalities, autonomous regions) in recent years were taken as observation objects. Mueller-Hinton ( MH ) agar , MH blood agar and Brinell agar were used to carried out the drug susceptibility test in vitro, and to compare the results of drug susceptibility test of different medium; the most suitable Brucella drug susceptibility test medium was used to detect the resistance of human derived Brucella to Doxycycline, Rifampicin, Streptomycin, Levofloxacin, Moxifloxacin, Ceftriaxone sodium, Co-trimoxazole and Amoxicillin/Clavulanic acid by K-B drug sensitive paper, and to observe the formation of antibacterial ring around the drug sensitive paper. Results The growth of Brucella on the MH agar and MH blood agar were slower than that on the Brinell agar, and the antibacterial rings were not obvious. All the 180 strains of Brucella were sensitive to seven antibiotics such as Doxycycline, Rifampicin, Streptomycin, Levofloxacin, Moxifloxacin, Ceftriaxone sodium, and Amoxicillin/Clavulanic acid; and 70 strains of Brucella were resistant to Co-trimoxazole, accounting for 39％ (70/180); Brucella strains resistant to Co-trimoxazole were found in 21 provinces. Conclusions Brinell agar is the most suitable medium for Brucella susceptibility test. The human derived Brucella is resistant to Co-trimoxazole; the resistant strains are distributed in 21 provinces ( municipalities , autonomous regions ) . It is recommended that relevant departm ents of the province ( municipalities , autonomous regions ) carry out epidemiological investigations on the resistance of Brucella, and strengthen the monitoring of drug resistance in clinical drugs of brucellosis patients.

Objective: To understand the epidemiological characteristics of brucellosis in Hainan province. Methods: Automatic microbial identification system of Vitek 2 compact was used for the preliminary identification of 16 brucellosis cases in Hainan province from 2012 to 2017, and further confirmation was performed with traditional biological typing methods. The epidemiological and clinical characteristics of the patients were analyzed in combination with the results of serological and etiological tests for raised livestock. Results: Vitek 2 compact detection results showed that 12 strains were Brucella (B.) melitensis and 4 strains were Ochrobactrum anthropi. Traditional biological typing methods showed that 11 strains were B. melitensis biovar 3 and 5 strains were B. suis biovar 3. Sixteen cases were found in Dongfang, Lingao, Haikou, Wanning, Ledong and Ding’an with 1 case in 2012, 2 cases in 2013, 4 cases in 2014, 1 case in 2015, 2 cases in 2016 and 6 cases in 2017 respectively. At the same time, 745 sheep serum samples from the epidemic area (Dongfang) were collected for Brucella serum antibody detection, in which 47 were positive (6.3%). And B. melitensis biovar 3 was isolated from samples collected from sick sheep in Dongfang. Conclusions Vitek 2 compact is an simple and convenient method for Brucella identification, but it cannot replace traditional biological typing methods yet. The major epidemic strains of Brucella in Hainan were B. melitensis biovar 3 and B. suis biovar 3. The epidemic of brucellosis in Dongfang in 2017 indicated that brucellosis had spread from animal to human in Hainan, and it is very important to strengthen the prevention and control of brucellosis in Hainan.

Objective: To analyze the epidemiological characteristics of human brucellosis and trace back source of infection of human brucellosis in Hunan province during 2010-2018, and provide evidence for the prevention and control of human brucellosis. Methods: The surveillance data of human brucellosis in Hunan during 2010-2018 were analyzed with software Excel 2016 and ArcGIS 10.5, the epidemic characteristics were described using cases number, constituent ratio and rate. The conventional biotype methods were used for the identification of Brucella species, UTS-PCR was applied to further confirm the results from conventional biotype detections, then six virulence genes of two clinical Brucella strains were detected by PCR assay. Cluster analysis of two Brucella strains were performed with Multiple locus variable-number tandem repeat analysis (MLVA) for the investigation of the infection source of human brucellosis. Results: From 2010 to 2018, a total of 728 human brucellosis cases were reported in Hunan with the annual incidence rate of 0.12/100 000. The incidence rate was 2.50/100 000 in Chenzhou and 1.90/100 000 in Yongzhou, higher than those in other areas. The number of counties reporting cases increased from 5 in 2010 to 69 in 2018. Most cases were reported in age group 45-54 years, accounting for 38.32% (279/728). The cases in farmers accounted for 59.07% (430/728) of the total. The male to female ratio of the cases was 2.75 ∶ 1. The reported case number was highest during May-July, accounting for 45.33% (330/728). The incidence was high in summer and autumn, and the peak was in May. The conventional identification showed that two strains were all Brucella melitensis biovar 1, consistent with UTS-PCR amplification results. Six virulence genes were found in two isolated strains, suggesting that the Brucella melitensis strains in this study had strong virulence. MLVA results confirmed that two strains detected in Hunan had complete identical MLVA-16 genotype with strains isolated from goat and camel in Inner Mongolia Autonomous Region, indicating that there was molecular epidemiology relationship between these strains and the source of infection were originated from Inner Mongolia. Conclusions The epidemic of human brucellosis in Hunan is becoming serious, and disease has spread to general population and non-epidemic areas. Two Brucella melitensis strains detected in Hunan were originated from Inner Mongolia. The quarantine and inspection in animal transportation should be strengthened to prevent human outbreaks of brucellosis.

Objective To understand the infected strains and prevalence of brucellosis in occupational population in Tacheng and Kashgar regions,Xinjiang Uygur Autonomous Region.Methods In September 2015,blood samples from occupational population (including herders,semi-agricultural and semi-pastoral,veterinarians) and non-occupational population (including students and cadres) were collected to detect Brucellaspecific antibody and bacterial nucleic acids by rose bengal plate test (RBPT),serological standard tube agglutination test (SAT) and PCR methods,respectively.The positive products of PCR were sent to Shanghai Sangon Biotechnology Co.,LTD.Then the sequence results were retrieved online using the basic alignment search tool (BLAST) in GenBank web page and uploaded to NCBI (National Center for Biotechnology Information).Results A total of 546 blood samples were tested,including 300 males,aged (55-± 15) years old,and 246 females,aged (54 ± 12) years old.The positive rates were 17.58％ (96/546) and 6.78％ (37/546) in 546 blood samples by serological method and genetic markers targeting omp22 and omp2,respectively.The positive rates were statistically significant (x2 =29.8,P ＜ 0.05).Additionally,based on BLAST analysis of outer membrane protein omp22 and omp2 genes,the positive products were identified as Brucella abortus,and the sequence similarity was 100.00％ (253/253,863/863 bp) to Brucella abortus strain Wisconsin genome assembly,chromosome (LT651712).Conclusions Brucellosis has a high infection rate in the occupational population of some animal husbandry-based groups in Xinjiang.The infection strain is abortive species Brucella,and health education for the occupational population and prevention of brucellosis should be strengthened to reduce the infection rate.

Objective To analyze the etiological characteristics of human Brucella strains isolated, and to improve the precision in control and prevention of brucellosis. Methods In 2016, blood samples were collected from patients in Jingyuan County Gansu Province, and tested via the Rose-Bengal Plate Agglutination Test (RBPT) and the tube agglutination test methods,and serological positive blood samples were inoculated to bidirectional blood culture bottle for culturing, and further identified by traditional biological classification method and the Brucella abortus, Brucella melitensis, Brucella ovis, and Brucella suis species-specific PCR (AMOS-PCR). Multiple-locus variable number tandem repeat sequence analysis (MLVA) -16 was used to detect molecular typing and do cluster analysis. Results The isolated strain was identified by the traditional biological classification method, bacteria could grow in thionine and reddened dye, A and M factors agglutination tests were positive, Bk2phage treatment of bacterial strain cracking, but Tb, Wb phages were not cracked. AMOS-PCR amplification result showed a 731 bp band, which was a strain of Brucella melitensis. The results of MLVA-16 showed that there was a difference in the number of repeats on some Variaable Number of Tandem Repeat(VNTR)sites of the isolated strain. Clustering analysis showed that, the isolated strain was clusted into the same clade with the clustering of Brucella melitensis type 3 from GS-201605 in Gansu. And the clustering was similar compared with that of Zhejiang, Guangdong, Fujian and Yunnan. Conclusion Human brucellosis is a inputting transmission in Gansu Province, there is a genetic variation of genotype 3 sheep Brucella between Gansu Province and other domestic provinces.

Objective To observe the levels of peripheral blood cytokines interferon-γ (IFN-γ)and interleukin-4 (IL-4) in patients with acute and chronic brucellosis before and during treatment,and to understand the differences of two immunocytokines in acute and chronic stage of brucellosis,and the effect of antibacterial therapy on these two cytokines to provide immunological basis for clinical evaluation of the therapeutic effect of brucellosis.Methods Research subjects were 36 pre-treatment acute brucellosis and 36 pre-treatment chronic brucellosis and 36 dur-treatment acute brucellosis and 36 dur-treatment chronic brucellosis,which were selected from Ulanqab Center for Endemic Disease Prevention and Control of Jining City with 25 local healthy persons as healthy controls.The levels of IFN-γ and IL-4 in serum were measured by enzyme-linked immunosorbent assay (ELISA) in patients with acute and chronic brucellosis and control group before and during treatment.Parameters of IFN-γ and IL-4 were analyzed with One-Way ANOVA analysis in pre-treatment and dur-treatment acute brucellosis,chronic brucellosis and control groups.Results The means of IFN-γ [(462.79 ± 47.94),(431.92 ± 40.39),(280.50 ± 40.48) ng/L] and IL-4 [(606.11 ± 51.86),(550.66 ± 51.56),(383.24 ± 53.98) ng/L] were significantly different in the three groups before treatment (F =141.84,139.28,P ＜ 0.05);Compared to control group,the levels of IFN-γ and IL-4 in acute brucellosis and chronic brucellosis were significantly increased before treatment (P ＜ 0.05).The levels of IFN-γand IL-4 in the acute brucellosis were significantly increased compared to those of chronic brucellosis before treatment (P ＜ 0.05).After about ten days antibiotic therapy,the means of IFN-γ [(356.05 ± 43.75),(368.61 ± 35.69),(280.50 ± 40.48) ng/L] and IL-4 [(487.31 ± 51.59),(496.73 ± 48.70),(383.24 ± 53.98) ng/L] were significantly different in the three groups (F =39.57,41.99,P ＜ 0.05).Compared to control group,the levels of IFN-γ and IL-4 in acute brucellosis and chronic brucellosis were significantly increased during treatment (P ＜ 0.05).The levels of IFN-γand IL-4 in the acute brucellosis were not significantly different compared to those of chronic brucellosis during treatment (P ＞ 0.05).Conclusion Different immunological characteristics of cytokines in peripheral blood of patients with acute and chronic brucellosis before treatment have affected the therapeutic effect and clinical outcomes.

Objective To identify Brucella species by means of bacteriological and polymerase chain reaction(PCR)methods,and to understand the drug susceptibility by in vitro susceptibility test of these strains to eight antimicrobial drugs.Methods The isolated Brucella strains were identified by standard method with conventional positive serum experiment,monophase specificity serum(A,M and R) agglutination experiment and brucella phage splitting experiment(Tb and BK2).Reference strains were set as control group.Molecular typing was performed by polymerase chain reaction(PCR)targeting Brucella surface protein 31(BCSP-31)and(abortus melitensis ovis suis,AMOS)-PCR assay which is able to distinguish among B.abortus,B.melitensis,B.ovis and B.porcine.Microdilution broth method was used to determine the minimum inhibitory concentrations(MIC)of 8 antibiotics to 27 Brucella strains isolated from blood culture,including azithromucin,ciprofloxacin,levofloxacin,doxycycline,rifampicin, gentamicin,acheomycin,and streptomycin.Results Twenty-seven strains were identified as B. melitensis,of which 21 were B.melitensis biovar 3 isolates,6 were B.melitensis biovar 1.The BCSP-31-PCR confirmed that all 27 isolates were Brucella.spp.AMOS-PCR assay confirmed that all isolates were B.melitensis.All isolates were susceptible to ciprofloxacin,levofloxacin,doxycycline,gentamicin, acheomycin,and streptomycin.Doxycycline was the most effective antibiotic(MIC900.064 mg/L),while rifampicin was moderately active to 3 isolates(MIC 2 mg/L).Conclusions Brucella isolates are susceptible in vitro to the antibiotics recommended by world health organization.Regular evaluation of antibiotic susceptibilities of Brucella strains is helpful for epidemiological investigation and antibiotic resistance monitor.

Objective We compared peripheral blood nucleotide-binding oligomerization domain-leucinerich repeats containing pyrin domain 3 (NLRP3),cysteinyl aspartate specific proteinase-1 (Caspase-1),interleukin-1β (IL-1β) and IL-18 between acute and chronic brucellosis patients before treatment and revealed their immune characteristics,to find targets for immune intervention of brucellosis.Methods From March to April 2016,42 acute and 42 chronic brucellosis patients were selected from Ulanqab Center for Endemic Disease Prevention and Control as the research subjects,and 20 local healthy persons were selected as healthy control.Brucellosis were diagnosed according to the "Diagnostic Criteria of Brucellosis".Enzyme-linked immunesorbent assay (ELISA) method was used to determine serum levels of NLRP3,Caspase-1,IL-1β and IL-18.Results The expression levels of NLRP3,Caspase-1,IL-1β and IL-18 [(1 264.40 ± 424.74),(1 350.67 ± 468.93),(192.96 ± 61.52),(162.74 ±54.23),(172.44 ± 60.56),(120.10 ± 61.52),(47.23 ± 13.79),(46.68 ± 14.72),(27.71-± 8.71),(202.23 ± 65.24),(169.19 ± 54.33),(108.62 ± 41.39) ng/L] were compared in acute,chronic and control groups,the differences were statistically significant (F =61.96,6.26,16.68,18.31,P ＜ 0.01).Compared to control group,the levels of NLRP3,Caspase-1,IL-1β and IL-18 in acute and chronic groups were significantly increased (P ＜ 0.05);compared to chronic group,the levels of IL-18 in acute group were significantly increased (P ＜ 0.05),and NLRP3,Caspase-1 and IL-1 β levels were not statistically different (P ＞ 0.05).Conclusions This study has showed that the expression of NLRP3 inflammasome in innate immunity is not significantly different between acute brucellosis and chronic brucellosis.The difference of IL-18 levels between acute and chronic brucellosis may affect their immune response.

Objective To identify the Brucella strains isolated from sheep blood in Jingyuan County Gansu Province,and to provide scientific basis for Brucellosis control and prevention.Methods In 2016,blood samples from suspected cases of sheep were collected in the home of Brucellosis patient of Jingyuan County,and cultured with vaccination double blood bottles via the enrichment method,and the Brucella strains isolated from sheep were identified by traditional bio-typing,genus specific Brucella surface protein 31 PCR (BSCP31-PCR) and Brucella abortus,Brucella melitensis,Brucella ovis,Brucella suis species-specific PCR (AMOS-PCR),respectively.Results The isolated strain was identified using the traditional biological classification method,bacteria could grow in thionine and fuchsin dyes,A and M factors serum agglutination tests were positive,Bk2 phage lysis test was positive,but Tb and Wb phage lysis tests were negative;and the results of BSCP31-PCR of tested strain was 223 bp band same to Brucella;the results of AMOS-PCR was 731 bp band same to Brucella melitensis.Conclusion Conventional bio-typing test assisted with molecular techniques has confirmed that the isolated Brucella strain from sheep blood is Brucella melitensis type 3 in Jingyuan County Gansu Province.