Brucellosis ; China ; Epidemics ; Humans

Objective To sequence an atypical Brucella strain 16S rDNA,and to evaluate the feasibility of 16S rDNA sequencing method for identification of Brucella.Methods Preliminary identification of atypical strains was carried out with conventional method.Strain DNA was extracted,and 16S rDNA complete sequence was bidirectional sequenced,and Blast in NCBI and DNAMAN software were used for comparison of the sequence identities of the 16S rDNA.Moreover,16S rDNA complete sequence of the stains those were known to cross-react serologically with Brucella was downloaded from GenBank,MEGA 6.0 was used to construct the phylogenetic tree.Results The conventional identification results revealed that it was an atypical Brucella,the gene similarity between the sequences of the test strain 16S rDNA and Brucella was 99％,between 16S rDNA sequence of Brucella abortus 544A and Brucella melitensis 16M was 96.99％.Phylogenetic tree revealed that the test stain was a Brucella,and closely related to Brucella abortus 544A and Brucella melitensis 16M.Conclusion The test strain is an atypical Brucella,and 16S rDNA sequencing analysis is a simple,rapid,and accurate identification method for atypical Brucella.

To investigate the the possibility to utilize the cellular fatty acid (CFA) information as a method in Brucella typing, 90 Brucella strains were subjected to the study on CFAs, and all the experimental strains were inoculated on Brucella Agar plates for 48 hours. After that, cells were harvested, saponificated, methylated and extracted to provide fatty acids methylesters for gas chromatography analysis. Based on the CFAs data matrix, dendrogram of 90 experimental strains was generated by SPSS11.5 software package. As shown in the dendrogram, 90 Brucella strains could be divided into 5 clusters. The first cluster included some species of Brucella abortus,Brucella melitensis,Brucella suis, Brucella ovis; and some of the variant strains of Brucella abortus and Brucella melitensis and the typical strain of Brucella neotomae. The second cluster included typical strains of Brucella suis (1,2,3 and 5 types); vaccine strains of Brucella suis S2; vaccine strains of Brucella melitensis M28、Rev.1 and typical strain of Brucella ovis. The third cluster included some of Brucella melitensis; some of the variant strains of Brucella melitensis; some of Brucella abortus(3,6 types); Brucella canis and Brucella ovis. The fourth cluster was the typical strain of Brucella canis.and the fifth cluster included some of Brucella melitensis(1 type); some of Brucella abortus (1 type); some of the variant strains of Brucella melitensis and Brucella suis(1,3 type). It is apparent that CFAs information can be used in brucella typing. and Brucella suis and Brucella canis can be distinguished by the difference in the CFA contents of 3 fatty acids 19:0CYCLOω8c, 18:1ω7c and 16:0. The results of CFAs typing in Brucella species show that Brucella canis includes 2 biovars at least and the high homologization of Brucella abortus (3 type) and Brucella abortus(6 type) can be found.

The aim of this study is to develop a rapid and accurately species typing method for Brucella isolates by using High Resolution Melting (HRM ) analysis .Six pairs of primers were used according to the reference for the sequence of pur‐pose gene .Nineteen biotypes of six species Brucella standard strains were identified by PCR‐HRM analysis and this analysis was used to detect the 35 clinical isolates .Results showed Brucella amplified specific melting curves were different from con‐trasted strains with primer Bspp .The six species Brucella standard strains have own characteristic curve shape from each oth‐ers by PCR‐HRM analysis with five pairs of primers .Thirty‐five clinical isolates of Brucella have entirely consistent with PCR‐HRM curve shape with Brucella melitensis standard strains .So ,PCR‐HRM analysis methods can accurately identify Brucella strains ,especially clinical isolated Brucella melitensis ,and may be used in clinical microbiology laboratories .

To explore the possibility to type the Brucella strains isolated in Guangdong province with analytical method to detect the fatty acid components and to collect the basic data of fatty acid components of Brucella strains, 29 strains of Brucella were selected for analysis on the bacterial fatty acid components and the cluster analysis on the collected data was performed with Sherlock analysis soft-ware (MIDI). It was demonstrated that the main fatty acid components of Brucella strains isolated in Guangdong province were 19∶0 cycloω8c acid, 16∶0 acid and 18∶0 acid. The content of 19∶0cycloω8c acid was highest in B.abortus, followed by B.melitensis and lowest in B.suis.-In addition, the content differences of 19∶0cycloω8c and 18∶0 acid between B. melitensis and Brucella suis were statistically significant; and that of 19∶0cycloω8c and 18∶0 acid between strains isolated in 1965 and those isolated in recent 3 years was statistically significant. It was also shown that the fatty acid components of Brucella strains were stable, but the contents of fatty acid components were different in different species.-It is evident that at certain euclidean distance, 3 species of Brucella can be differentiated in species level.

Brucellacapt is a new serological test based on immunocapture-agglutination technique that has been used in many countries ,but there was no related article reported in China .The value of the Brucellacapt method in diagnosing human brucellosis was evaluated in this study .Among 120 suspected patients ,75 patients and 45 negatives were diagnosed by SAT and RBPT method combination with their clinical symptoms .Sera from all 120 people were tested by the method of Brucella-capt and iELISA ,and the results were ,consequently ,analyzed and compared .It showed that sensitivity ,specificity ,consis-tency rate ,Kappa value ,and area under ROC curve were found to be 82 .7% ,88 .9% ,85 .0% ,0 .69 ,and 0 .86 ,respectively for Brucellacapt ,whereas they were found to be 90 .7% ,64 .4% ,80 .8% ,0 .57 ,and 0 .78 for iELISA .In conclusion ,specific-ity ,consistency rate ,Kappa value ,and area under ROC curve were higher in Brucellacapt method than that in iELISA .How-ever ,the sensitivity of iELISA is higher .

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 get knowledge of the molecular epidemiological characteristics of human derived Brucella isolated in Hohhot,and to provide experimental basis in guiding prevention and treatment of Brucella infection.Methods Twenty-seven Brucella isolates derived from patients in Affiliated Hospital of Inner Mongolian Medical University from 2013 to 2015 were identified by routine bacteriological methods and molecular methods.Multiple-locus variable number tandem repeats analysis (MLVA-16) was used to detect molecular typing and do cluster analysis.Sixteen virulent genes were detected and analyzed by polymerase chain reaction (PCR).Results Twenty-seven Brucella isolates were identified as Brucella melitensis (B.melitensis) by routine bacteriological methods and PCR.Out of them,six isolates were B.melitensis biovar 1,and twenty-one isolates were B.melitensis biovar 3.MLVA-16 analysis showed that seven genotypes were obtained from nine Brucella isolates,which showed significant difference in variable number of tandem repeats,which suggested that they originated from sporadic outbreak.Moreover,two isolates were clustered into the same clade,which suggested they were epidemiologically correlated and may be derived from the same origin.Sixteen virulent genes were detected in all of the twenty-seven isolates.Conclusions Brucella isolates from patients in Hohhot are mainly B.melitensis biovar 3 and B.melitensis biovar 1,and the distribution profile of multiple virulence genes is similar.Some isolates have showed epidemic correlation,and the epidemic mechanism should be further explored.

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 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.