To prepare and identify the monoclonal antibody against Borrelia burgdorferi in order to provide basis for the detection of Lyme spirochetes and diagnosis of Lyme disease.Spleen cells were prepared from BALB/c mice which was immunized by the whole proteins of B.garinii strain PD91.And then they were fused with SP2/0 myeloma cells.The positive clones were picked out and identified by ELISA and WB.The positive clones were subcloned two or three times to obtain the monoclonal cells.Ten hybridoma cell strains which could produce the monoclonal antibody against B.burgdorferi were obtained,in which four is against OspA,three against OspB and three against OspC.Three kinds of monoclonal antibodies against B.burgdorferi have been successfully prepared.And they can be used in the diagnosis of Lyme disease and detection of Lyme spirochetes.
The objective of this study was to understand the status of rats,the reservoir hosts of Borrelia burgdorferi,in the six representative provinces in China including Jilin,Shanxi,Gansu,Qinghai,Guizhou and Hunan.The rats were catched in two different areas in each province and the pathogens were isolated and cultured.Nested-PCR was used to test Borrelia burgdorferi in spleen,kidney and bladder of rats.The sequence analysis was used to identify the genotype.Results indicated that two strains of Borrelia burgdorferi were obtained from apodemus in Guizhou;the specific fragments of Borrelia burgdorferi were obtained in spleen and kidney of rats from all representative provinces in China expect Guizhou;the PCR positive rate was higher in Huangnan(28.85%)and Shimen(19.6%);there were statistical differences in the areas.The sequence analysis shows that the genotype in Jilin,Qinghai,Gansu and Shanxi belongs to Borrelia garinii,and in Hunan belongs to Borrelia valaisiana.The results indicated that there were different infection rate in rats infected in the six provinces,thus further investigation should conduct for the primary reservoir hosts and for the pathogens carrying situation in different areas of China.
In order to investigate geographic distribution of Lyme disease thoroughly in nine districts of Jilin Province. infection rate of humans and animals, germ-carrying rate of intermediary ticks, action regulation of ticks, time of rise and fall and life conditions is registered and investigated according to the individual cases schedule table of epidemiology of Lyme disease,the disposition of natural foci and geographical landscapes of Lyme disease in Jilin Province is analyzed,the investigation data of mountain areas, half-mountain areas, plains is treated statistically.It is found that biting rate of ticks of 3561 crow is 91.00% and infection rate of 2292 crow is 6.20% in 53 small towns, 33 cities and towns, 9 territories of Jilin Province. In 2499 investigations of horses,oxes,sheep,mice,and dogs infection rate is 22.13%. The germ-carrying rate of 3570 catched ticks is 35.80%. It is concluded that results of investigation showed that there are natural foci of Lyme disease in nine districts of Jilin Province generally. Feature spots of tourism are important natural foci of Lyme disease. Lyme disease is not a disease of forestry type.Pathogen gene classification is confirmed by molecular biology method that at least there are two kinds in Jilin province.
The Mycobacterium chelonae/abscessus(M.chelonae/abscessus) complex belongs to the rapidly growing genus Mycobacterium(RGM).It is one of the most important pathogenic members of Mycobacterium leading to nosocomial infections and outbreaks.It includes members of M.chelonae,M.immunogenum,M.abscessus,M.massiliense,and M.bolletii.In order to investigate the epidemiological characteristics of the M.chelonae/abscessus complex in China and to conduct the molecular methods for species identification of M.chelonae/abscessus,we collected clinical M.chelonae/abscessus complex strains identified by phenotypic tests.Members were verified by sequencing of 16S rRNA.Species and subspecies were identified by hsp65 and rpoB PCR-RFLP methods.In total,27 clinical specimens were identified as Mycobacterium chelonae/abscessus complex by phenotypic tests.16s rRNA gene sequence analysis of all 27 clinical samples shared over 99.7% similarity with M.chelonae and M.abscessus.Species identification with hsp65 PCR-RFLP and rpoB PCR-RFLP revealed that 18 specimens were M.abscessus and 4 were M.absecces.The remaining 5 samples displayed a pattern that failed to match any previously reported pattern.Thus,this might represent a novel species that is part of the Mycobacterium chelonae/abscessus complex.We identified that a majority of the chronic lung infection in China is caused by the M.chelonae/abscessus complex.Specifically,the M.abscessus species might be the most infectious,while other species in the complex can still cause infection.Interestingly,there may be a novel or previously unidentified species that is a part of the complex.Finally,we show that species identification can be carried out more accurately by combined use of hsp65 and rpoB PCR-RFLP.
To analyze the genotypes of Mycobacterium tuberculosis clinical isolates in Zhejiang province with spacer oligonucleotide typing(spoligotyping) and multiple loci variable number tandem repeat(VNTR) analysis(MLVA),70 clinical strains of Mycobacterium tuberculosis isolated from Zhejiang province were selected randomly.After conventional cultivation,the bacterial DNA was extracted from these isolated strains and the PCR technology was used to amplify the total DR region with spoligotyping and to amplify simultaneously 15 loci of VNTR with MLVA.The clustering of genotypes was analyzed with BioNumberies(Version 5.0).Through spoligotyping,these clinical strains of T.tuberculosis could be categorized into two families:the Beijing family(70%) and non-Beijing family(30%).In the Beijing family,89.8%(44/49) belonged to the typical strains and these strains could be categorized into 4 genogroups with MLVA,i.e.I,II,III,IV genogroups,in which I genogroup(8.5$) consisted of 5 genotypes;II genogroup(18.3%) consisted of 13 genotypes' III genogroup(70.4%) consisted of 38 genotypes and IV genogroup(2.8%) consisted of 2 genotypes.The results using these two methods of genotyping matched quite well,from which all the Beijing family strains were MLVA-III and the non-Beijing family strains were distributed among MLVA-I,II,IV.All the 70 strains of M.tuberculosis could be divided into 58 genotypes including 53 unique genotypes by means of MLVA,while they could be divided into 18 genotypes including 12 unique genotypes by means of spoligotyping.Results of the susceptibility tests against the first-line drugs indicated that 71.4%(35/49) of the Beijing family strains were sensitive,while 28.6%(14/49) were resistant,but in the non-Beijing family strains,66.7% were sensitive and 33.3% were resistant.However,no significant difference existed between them as demonstrated by chi-square test(χ2=0.158,P0.05).From these observation,it is evident that significant genetic polymorphism exists among the clinical strains of M.tuberculosis isolated from Zhejiang province with the predominantly prevalent strains of Beijing family and without obvious association on the drug-resistance.It is also demonstrated that MLVA should be superior to spoligotyping to analyze the genotypes of M.tuberculosis,especially the Beijing family strains.
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