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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.
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Objective To investigate the HOOF genotyping characteristics of 83 Brucella (B.)melitensis strains isolated in Ulanqab of Inner Mongolia Autonomous Region from 2012 to 2015.Methods A total of 83 B.melitensis strains were detected by convention identification and AMOS-PCR,then HOOF protocol with eight VNTR locus were used for the genotyping of the strains,and the allelic diversity of each VNTR locus and the discriminatory power of VNTR typing of HOOF were assessed by Hunter-Gaston Discriminatory index.BioNumerics 5.0 was used for phylogenetic analysis and constructing dendrogram.Results All of the isolates were identified as B.melitensis strains by two identification methods.The complete eight VNTR locus had higher polymophism and diversity index was 0.998;and diversity index of six locus (1,2 and 4-7) were ≥0.678,discriminatory power of HOOF was mainly from this six higher diversity index locus.The 83 B.melitensis strains were classified into eight clusters and 76 genotypes,6 shared genotypes included 13 isolates,indicating that these brucellosis cases had epidemiological link,the other 70 strains had distinct genotypes,indicating that these cases had no epidemiological link.Conclusions The epidemic of human brucellosis in Ulanqab was characterized by local and sporadic outbreaks.Cross infection was related with the transfer of the sources of infection.
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Objective To explore the change and value of interleukin (IL)-2,IL-6 and tumor necrosis factor (TNF)-α in the serum samples of patients with brucellosis.Methods The levels of serum IL-2,IL-6 and TNF-α were measured using the enzyme linked immunosorbent assay (ELISA) method in 155 patients with brucellosis in different clinical stages (including 69 cases in acute,34 cases in subacute and 52 cases in chronic periods) and 50 healthy controls.IL-2 was used to represent the helper T cell (Th)-type 1 cytokines,IL-6 represent Th2 type cytokines,and the Th1/Th2 ratio in different clinical stages was compared.Results The expression level of serum IL-2 in patients with brucellosis was different (ng/L:acute:10.15 ± 2.01;subacute:9.53 ± 1.68;chronic:6.76± 1.31;control:47.25 ± 5.68),the differences were statistically significant (F =74.921,P ＜ 0.05).The expression levels of serum IL-2 in acute,subacute and chronic periods of brucellosis patients were significantly lower than that of healthy controls (all P ＜ 0.05).The expression levels of serum IL-2 in acute and subacute periods of brucellosis patients were higher than that of chronic period patients (all P ＜ 0.05).The expression of IL-6 and TNF-α in serum of patients in different clinical stages were different (ng/L:acute:615.22± 341.07,802.55 ± 479.53;subacute:478.45 ± 105.33,680.21 ± 366.95;chronic:306.37 ± 96.12,455.36 ± 176.27;control:121.45 ± 30.16,87.51 ± 24.03),the differences were statistically significant (F =57.692,63.210,all P ＜ 0.05).The levels of serum IL-6 and TNF-o were significantly higher in patients with brucellosis in acute,subacute and chronic clinical stages than that of healthy controls (all P ＜ 0.05).Serum IL-6 and TNF-α levels in acute period of brucellosis patients were higher than those of subacute and chronic periods patients (all P ＜ 0.05);the levels of serum IL-6 and TNF-α in subacute period of brucellosis patients were also significantly higher than that of chronic period patients (all P ＜ 0.05).The Th1/Th2 ratio was different (acute:0.02 ± 0.00;subacute:0.02 ± 0.00;chronic:0.23 ± 0.02;control:0.41 ± 0.06) in clinical patients with brucellosis,the differences were statistically significant (F =17.843,P ＜ 0.05),the value of Th1/Th2 in acute and subacute period patients were lower than that of chronic period (all P ＜ 0.05).Conclusion IL-2,IL-6 and TNF-α may play an important role in the pathogenesis of brucellosis,and their dynamic changes can reflect the progression and outcome of brucellosis.
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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.
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Objective To establish genotyping methods for rapid identification of Brucella melitensis (B.melitensis) biovar 1,2 and 3 and to verify these method.Methods Single nucleotide polymorphism of RpoB gene and tandem repeat sequence (TRS) Bru42 of standard reference strain 16M were used to design primers,then the RpoB-PCR and TRS-PCR method were established for identification of B.melitensis standard reference strains,these two methods were used to identify clinical isolates of B.melitensis and compared with the conventional methods.Results The results of B.melitensis standard reference strains (biotype 1,2,3) identified by RpoB-PCR and TRS-PCR were consistent with those of the conventional identification methods.Totally 50 clinical isolates [including B.melitensis biovar 1 (17),2 (3) and 3 (30)] were identified as RpoB-2 genotype,only one B.melitensis biovar 1 strain was identified as RpoB-3 genotype.Genotype identification results of standard reference strains and clinical isolates with the same biotype were not exactly the same.Fothermore,TRS-PCR experiment displayed that 51 clinical isolates were all genotype 2 of B.melitensis (genotype TRS-2).Conclusions There is no clear relationship between biovars and genotypes within B.melitensis,and significant difference exists between B.melitensis standard reference strains and clinical isolates within RpoB gene.Bru42 can not be used for genotyping clinical isolates of B.melitensis.
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Objective To establish genotyping methods for rapid identification of Brucella melitensis (B.melitensis) biovar 1,2 and 3 and to verify these method.Methods Single nucleotide polymorphism of RpoB gene and tandem repeat sequence (TRS) Bru42 of standard reference strain 16M were used to design primers,then the RpoB-PCR and TRS-PCR method were established for identification of B.melitensis standard reference strains,these two methods were used to identify clinical isolates of B.melitensis and compared with the conventional methods.Results The results of B.melitensis standard reference strains (biotype 1,2,3) identified by RpoB-PCR and TRS-PCR were consistent with those of the conventional identification methods.Totally 50 clinical isolates [including B.melitensis biovar 1 (17),2 (3) and 3 (30)] were identified as RpoB-2 genotype,only one B.melitensis biovar 1 strain was identified as RpoB-3 genotype.Genotype identification results of standard reference strains and clinical isolates with the same biotype were not exactly the same.Fothermore,TRS-PCR experiment displayed that 51 clinical isolates were all genotype 2 of B.melitensis (genotype TRS-2).Conclusions There is no clear relationship between biovars and genotypes within B.melitensis,and significant difference exists between B.melitensis standard reference strains and clinical isolates within RpoB gene.Bru42 can not be used for genotyping clinical isolates of B.melitensis.
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Objective To investigate the HOOF genotyping characteristics of 83 Brucella (B.)melitensis strains isolated in Ulanqab of Inner Mongolia Autonomous Region from 2012 to 2015.Methods A total of 83 B.melitensis strains were detected by convention identification and AMOS-PCR,then HOOF protocol with eight VNTR locus were used for the genotyping of the strains,and the allelic diversity of each VNTR locus and the discriminatory power of VNTR typing of HOOF were assessed by Hunter-Gaston Discriminatory index.BioNumerics 5.0 was used for phylogenetic analysis and constructing dendrogram.Results All of the isolates were identified as B.melitensis strains by two identification methods.The complete eight VNTR locus had higher polymophism and diversity index was 0.998;and diversity index of six locus (1,2 and 4-7) were ≥0.678,discriminatory power of HOOF was mainly from this six higher diversity index locus.The 83 B.melitensis strains were classified into eight clusters and 76 genotypes,6 shared genotypes included 13 isolates,indicating that these brucellosis cases had epidemiological link,the other 70 strains had distinct genotypes,indicating that these cases had no epidemiological link.Conclusions The epidemic of human brucellosis in Ulanqab was characterized by local and sporadic outbreaks.Cross infection was related with the transfer of the sources of infection.
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Objective This study was designed to compare the mRNA expression of Toll-like receptors 2 (TLR2) and Toll-like receptors 4 (TLR4) in monocytes between brucellosis cases and healthy controls,to explore the role and value of TLR2 and TLR4 in the pathogenesis of brucellosis.Methods From March to June in 2015,a total of 155 brucellosis cases at different clinical stages were diagnosed in Ulanqab Center for Endemic Disease Prevention and Control and 50 healthy controls were selected as research subjects.Subjective clinical symptoms of all patients were observed.Serological detection of all cases was done using serum tube agglutination test (SAT).The mRNA expression of TLR2 and TLR4 in peripheral blood cell was detected by real-time PCR technique.Results Fever,hyperhidrosis,weak,bone ache and hepatosplenomegaly were found in patients of acute (69 people),subacute (34 people) and chronic (52 people) periods.The incidences of fever and hyperhidrosis in acute and subacute periods were higher than that in chronic period,and the differences were statistically significant (x2 =58.427,26.190,all P ＜ 0.01).The incidence of bone ache in chronic period was higher than that in acute period,and the difference was statistically significant (x2 =9.264,P ＜ 0.01).In the agglutination titer of 1:800 and higher,the positive rate of chronic period was lower than those in acute and subacute periods,and the differences were statistically significant (x2 =14.302,8.682,all P ＜ 0.01).The mRNA expression levels of TLR2 and TLR4 were different among different clinical periods of brucellosis patients,and the differences were statistically significant (F =17.502,24.931,all P ＜ 0.01).The expression levels of TLR2 mRNA (8.67 ± 2.39) and TLR4 mRNA (12.38 ± 3.87) in acute period of brucellosis patients were higher than those of subacute period (5.21 ± 1.76,7.62 ± 2.21),chronic period (1.25 ± 0.47,1.72 ± 0.55) and healthy control (1.17 ± 0.23,1.43 ± 0.62),and the differences were statistically significant (all P ＜ 0.01).The expression levels of TLR2 and TLR4 mRNA in subacute period of brucellosis patients were higher than those of chronic period and healthy controls,and the differences were statistically significant (all P ＜ 0.01).The expression levels of TLR2 and TLR4 mRNA were not significantly different (all P ＞ 0.05) between chronic period and healthy controls.Conclusion TLR2 and TLR4 may be the specific recognizing receptors participated in the immune response in brucellosis and associated with the occurrence and development of brucellosis.
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Objective To study the characteristics of liver function damage in brucellosis and provide theoretical basis for clinical treatment.Methods One hundred and thirty-six hospitalized patients with brucellosis from Endemic Disease Prevention and Control Center of Wulanchabu City from January 2011 to October 2012 were selected randomly as the research subjects.Parameters of Alanine transaminase (ALT),Aspartate transaminase (AST),Alkaline phosphatase (ALP),Gamma glutamyltransferase (γ-GT),Total protein (TP),Albumin (ALB),Albumin/globulin (A/G),total bilirubin (TBIL) and Direct bilirubin (DBIL) were determined.The means of liver function parameters with mid-value of all the reference ranges were compared by one sample t test;Parameters of liver function were analyzed in 70 acute and 66 chronic brucellosis by two independent-samples t test.Results The means of ALT,AST,ALP,γ-GT,TP,ALB,TBIL and DBIL ((x):36.18,28.20,95.87,29.29 U/L,65.08,40.84 g/ L and 19.76,5.86 μmol/L) in the 136 samples of brucellosis serum were significantly different from the medians of their reference ranges ((x):20.50,18.50,85.00,24.50 U/L,69.00,43.50 g/L and 11.80,3.40 μ mol/L,t =9.46,9.19,4.55,2.22,-2.71,-4.99,5.58,11.32,all P ＜ 0.05);A/G was not significantly different ((x):2.14,2.00;t =0.94,P ＞ 0.05);Parameters of liver function were not significantly different between acute and chronic brucellosis (all P ＞ 0.05).Conclusions This study confirmed that the liver function of the 136 patients with brucellosis is impaired.The level of the liver function impairment is usually mild and moderate and no difference in acute and chronic brucellosis is found.
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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.