OBJECTIVEHuman Lyme Borreliosis (LB), which is caused by Borrelia burgdorferi sensu lato (B. burgdorferi), has been identified as a major arthropod-borne infectious disease in China. We aimed to develop a multiple locus variable-number tandem repeat (VNTR) analysis (MLVA) assay for the genotyping of Borrelia burgdorferi strains detected in China.

METHODSB. garinii PBi complete 904.246 kb chromosome and two plasmids (cp26 and lp54) were screened by using Tandem Repeats Finder program for getting potential VNTR loci, the potential VNTR loci were analyzed and identified with PCR and the VNTR loci data were analyzed and MLVA clustering tree were constrcted by using the categorical coefficient and the unweighted pair-group method with arithmetic means (UPGMA).

RESULTSWe identified 5 new VNTR loci through analyzing 47 potential VNTR loci. We used the MLVA protocol to analyse 101 B. burgdorferi strains detected in China and finally identified 51 unique genotypes in 4 major clusters including B. burgdorferi sensu stricto (B.b.s.s), B. garinii, B. afzelii, and B. valaisiana, consistent with the current MLSA phylogeny studies. The allele numbers of VNTR-1, VNTR-2, VNTR-3, VNTR-4, and VNTR-5 were 7, 3, 9, 7, and 6. The Hunter-Gaston index (HGI) of five VNTR loci were 0.79, 0.22, 0.77, 0.71, and 0.67, respectively. The combined HGI of five VNTR loci was 0.96. Clustering of the strains of Xinjiang, Inner Mongolia and Heilongjiang was confirmed, and this situation was consistent with the close geographical distribution of those provinces.

CONCLUSIONThe MLVA protocol esytablished in this study is easy and can show strains' phylogenetic relationships to distinguish the strains of Borrelia species. It is useful for further phylogenetic and epidemiological analyses of Borrelia strains.

Borrelia burgdorferi Group ; genetics ; China ; Genotyping Techniques ; Minisatellite Repeats

Animals ; Borrelia burgdorferi ; genetics ; China ; DNA, Bacterial ; genetics ; isolation & purification ; Rodentia ; microbiology ; Sequence Analysis, DNA

OBJECTIVETo learn the existence of natural focus of Lyme disease and its distribution.

METHODSA semi-nested polymerase chain reaction (PCR) method was developed for detection and genotyping of Borrelia burgdorferi on basis of outer surface protein A (OspA) gene. Ticks and mice collected from 6 forest areas in Beijing were detected with above methods. The positive PCR products were cloned and sequenced. The sequences were compared with published sequences for homology. IFA as used to detect IgG antibody on Borrelia burgdorferi. Lyme disease spirochete were isolated from H. longicornis were also attempted.

RESULTSB. Burgdorferi sensu lato were detected from 939 ticks and 250 mice specimens collected from above 6 study sites using primer pairs OA(1)/OA(4) and SL/OA(4). Only the specimens collected from Dongling mountain showed positive amplification. One in three adult Ixodes persulcatus with one of 57 nymph Ixodes persulcatus showed positive while 9 of 119 (7.56%) mice specimens showed positive, of which 8 were B. grinii and one B. afzelii. In this study, we attempted to isolate B. burgdorferi sensu lato strains from 160 H. longicornis ticks (20/group) but failed. Serological survey showed a 9.1% (5/55) infection rate with B. burgdorferi sensu lato in the mice of Dongling mountain forest areas.

CONCLUSIONSThe natural focus of Lyme disease including B. garinii and B. afzelii might have existed in Dongling mountain of Mentougou district, Beijing. Ixodes persulcatu and mice may serve as vectors and reservoirs, respectively.

Animals ; Antigens, Surface ; genetics ; Bacterial Outer Membrane Proteins ; genetics ; Bacterial Vaccines ; Borrelia burgdorferi ; genetics ; Borrelia burgdorferi Group ; classification ; genetics ; Humans ; Ixodes ; microbiology ; Lipoproteins ; Lyme Disease ; microbiology ; Mice ; Phylogeny ; Polymerase Chain Reaction ; methods

OBJECTIVETo explore the fact that the east border of Heilongjiang had been a lyme disease natural focus,we investigated the species and distribution of ticks and isolated bacteria from ticks and identified genomic species of Borrelia burdorferi sensu lato. This study provided evidence for prevention and control of lyme disease.

METHODSTicks were caught by flagging method and Direct immunofluorescence method was used to detect the rate of bacteria borne by the tick. BSK UI culture medium was used to isolate the agent and Specific McAbs were used to identify the bacteria. SDS-PAGE protein profile and PCR-RFLP method were also used to identify the species of Spirochetes.

RESULTSTicks, collected from China-Russia border of east Heilongiiang province were classified including Ixodes persulcatus Schulze, Dermacentor sivarum Olener, Haemaphysalis concinna Kock,and Haemaphysalis japonica Kock. We found that the distributon of ticks was different under different circumstances and the predominant species were also different in different ports. The rate of bacteria borne by Iodes persulaatus Schulze was 31.4% ,by Dermacentor sivarum Olener and Haemaphysalis concinna Kock were 2.2% and 3.8%, respectively. However,it was negative for Haenaphysalis japonica Kock. Spirochetes isolated from Ixodes persulcatus Schulze were collected from Dongning and Tongjiang while Genomic species of Spirochetes, isolated from ticks of the border belonged to B. garinii.

CONCLUSIONAll the results showed that the east border of Heilongjiang province was the natural focus of lyme disease.

Animals ; Arachnid Vectors ; classification ; microbiology ; Borrelia burgdorferi ; classification ; genetics ; isolation & purification ; China ; Humans ; Lyme Disease ; microbiology ; Russia ; Ticks ; classification ; microbiology

Animals ; Borrelia burgdorferi Group ; classification ; genetics ; isolation & purification ; China ; Genotype ; Lyme Disease ; microbiology ; transmission ; Rats ; Ticks ; microbiology

Objective: To study the polymorphism in P66 and its human B-cell epitopes of Methods: Polymerase chain reaction (PCR) and sequencing were used to obtain the P66 sequences of 59 Chinese Results: Results showed that genetic and amino acid diversity presented in the 66 kD protein of all 59 Chinese strains, especially in Conclusion In P66 of 59 Chinese strains, polymorphisms were widely distributed. More importantly, the P66 amino acid sequences of
Bacterial Proteins/genetics* ; Borrelia burgdorferi/genetics* ; China ; Cluster Analysis ; Epitopes, B-Lymphocyte/genetics* ; Genetic Markers ; Genotype ; Humans ; Mutation ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Porins/genetics*

OBJECTIVETo recombine OspC gene from Borrelia burgdorferi PD91 of China and expressed it in E. coli for early diagnosis of Lyme disease.

METHODSThe OspC gene was amplified from the genome of Borrelia burgdorferi PD91 strain by polymerase chain reaction and recombined with plasmid PET-11D. The recombinant plasmid PET-11D-OspC was identified with PCR, restriction endonuclease analysis and sequencing. The antigenicity was verified with Western Blot.

RESULTSOspC gene was cloned correctly into vector PET-11D. The resultant sequence was definitely different from the published sequence. The recombinant OspC seemed to have had strong antigenicity.

CONCLUSIONThe findings laid basis for the studies on early diagnosis of Lyme disease.

Antigens, Bacterial ; Bacterial Outer Membrane Proteins ; genetics ; immunology ; Blotting, Western ; Borrelia burgdorferi Group ; immunology ; Escherichia coli ; genetics ; Humans ; Lyme Disease ; diagnosis ; Polymerase Chain Reaction ; Recombinant Proteins ; analysis ; immunology

OBJECTIVETo understand the coinfection status of Borrelia burgdorferi sensu lato (B.b.s.l) and spotted fever group Rickettsia (SFGR) in Hunchun of Jilin province, China.

METHODSPolymerase chain reaction (PCR) was used to detect the 5S-23S rRNA intergenic spacer of B. b. s. l and ompA of SFGR in ticks was collected in Hunchun,Jilin province. The amplification products of positive ticks were sequenced, and phylogenetic analysis was conducted by PHYLIP software package.

RESULTSThe infection rate of B. b. s. l was 36.0% in Ixodes persulcatus ticks and the SFGR was discovered in I. persulcatus ticks,with an infection rate of 2.0%. The coinfection rate of both agents was 2.0%. In 327 Dermacentor siltarum ticks, the positive rates of B. b. s. l and SFGR were 30.9% and 29.1% respectively. 55 ticks (16.8%) were coinfected with the two pathogens. The sequence analysis of B. b. s. l showed that the B. b. s. l in Jilin area, which were highly homologous, all belonged to B. garinii genotypes. The sequence analysis of SFGR positive products showed that the DNA secquence of the newly detected agent (JL-95) was close to the two previously described rickettsiae which were detected in I. ricinus from Slovakia (called IRS3 and IRS4). Phylogenetic relationships inferred from the comparison of these sequences with those of other genus Rickettsiae indicated that JL-95, IRS3 and IRS4 constituted a new rickettsial genotype and formed a separate cluster among the spotted fever group Rickettsiae.

CONCLUSIONCoinfection of B. b. s. l and SFGR existed in Hunchun, Jilin province. The sequencing of specific fragment confirmed a new SFGR which was different from other rickettsiae known in China.

Animals ; Borrelia burgdorferi Group ; genetics ; isolation & purification ; China ; DNA, Bacterial ; analysis ; Genotype ; Lyme Disease ; veterinary ; Phylogeny ; Polymerase Chain Reaction ; Rickettsia ; genetics ; isolation & purification ; Rickettsia Infections ; veterinary ; Ticks ; microbiology

A set of universal loop-mediated isothermal amplification (LAMP) primers targeting the fla gene was designed to detect Borrelia burgdorferi sensu lato (B. burgdorferi s.l.) in human samples. The sensitivity of LAMP was 20 copies/reaction, and the assay did not detect false positives among 11 other related bacteria. A positive LAMP result was obtained for 9 of the 24 confirmed cases and for 12 of 94 suspected cases. The positive rate of LAMP was the same as that of nested PCR. The LAMP is a useful diagnostic method that can be developed for rapid detection of B. burgdorferi s.l. in human sera. Combination of the LAMP and nested PCR was more sensitive for detecting B. burgdorferi s.l. in human serum samples.
Borrelia burgdorferi Group ; genetics ; isolation & purification ; China ; DNA, Bacterial ; genetics ; isolation & purification ; Humans ; Lyme Disease ; diagnosis ; Nucleic Acid Amplification Techniques ; methods ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

OBJECTIVETo study the cloning and expression of flagellin gene from Chinese Borrelia burgdorferi, PD91 strain and to evaluate the feasibility of using recombinant protein as diagnostic antigen when comparing the gene sequence with flagellin gene from North American Borrelia burgdorferi B31.

METHODSThe piece of genes coding flagellin from Chinese Borrelia burgdorferi PD91 by polymerase chain reaction (PCR) method was obtained, and constructed recombinant plasmid, before transformed into E. coli BL21 strain, and induced. The recombinant plasmid was identified with enzyme cutoff and gene sequence comparison. Efficient expression strain was selected and the expression product was analyzed with sodium amplified polymorphic-polyacrylamide gel electrophoresis (SDS-PAGE) and Western-blot method.

RESULTSThe recombinant protein (r-flagellin) expressed in host bacteria was successful. By means of western-blot assay, the immunological response showed the same antigenicity between r-flagellin and PD91 flagellin. The piece of genes coding flagellin of PD91 was 1011 bp, but when comparing with that of North American Borrelia burgdorferi it showed 94.70% homology. Homology between the sequence of amino acid of the r-flagellin and that of B31 flagellin was 95.85%.

CONCLUSIONFlagellin gene of Borrelia garinii of Chinese Lyme disease spirochete was successfully cloned and expressed for the first time. It was proved that the immunoreactivity of r-flagellin was the same as the natural flagellin.

Amino Acid Sequence ; Base Sequence ; Borrelia burgdorferi ; genetics ; isolation & purification ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Flagellin ; biosynthesis ; genetics ; Humans ; Lyme Disease ; microbiology ; Molecular Sequence Data ; Plasmids ; genetics ; Recombinant Proteins ; biosynthesis ; genetics