The organization of the ribosomal genes is unique in Borrelia burgdorferi in that the rrl (23S) and rrf (SS) genes are duplicated in tandem. Twelve Korean Borrelia isolates were classified by the method of rRNA gene restriction fragment length polymorphism (RFLP) and Southern hybridization. One of the isolates, KW-3, as well as the representative of ribotype group IV Ip89 appeared to have evolved within B. garinii. The size of the restriction bands of the 11 isolates showed different patterns from the USA and European isolates. The result by RFLP of rrf-rrl intergenic spacer region amplified by PCR showed a good correlation with that of Southern hybridization of rRNA gene. Therefore, the eleven Korean isolates could be classified as members of a new Borrelia group.
Borrelia burgdorferi Group* ; Borrelia burgdorferi* ; Borrelia* ; Classification* ; Genes, rRNA ; Korea* ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length* ; Ribotyping

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

Eleven Borrelia afzelii strains, isolated from Ixodes nipponensis and Apodemus agrarius in Korea, were characterized by groEL gene analysis. Results from previous studies suggested that the groEL gene, which encodes the 60-kDa heat shock protein GroEL, was useful for the differentiation of B. burgdorferi sensu lato. The B. afzelii isolates could be divided into two groups by the phylogenetic tree constructed by UPGMA method and Tsp509 I PCR-RFLP analysis. The result suggested that the groEL gene is useful for identification and characterization of B. burgdorferi sensu lato though a short DNA fragment (310 bp) of the gene was sequenced and compared each other, and that Korean B. afzelii strains are heterogeneous genotypically.
Animals ; Borrelia burgdorferi Group* ; Borrelia* ; DNA ; Heat-Shock Proteins ; Ixodes ; Korea* ; Murinae ; Population Characteristics

Eleven Borrelia afzelii strains, isolated from Ixodes nipponensis and Apodemus agrarius in Korea, were characterized by groEL gene analysis. Results from previous studies suggested that the groEL gene, which encodes the 60-kDa heat shock protein GroEL, was useful for the differentiation of B. burgdorferi sensu lato. The B. afzelii isolates could be divided into two groups by the phylogenetic tree constructed by UPGMA method and Tsp509 I PCR-RFLP analysis. The result suggested that the groEL gene is useful for identification and characterization of B. burgdorferi sensu lato though a short DNA fragment (310 bp) of the gene was sequenced and compared each other, and that Korean B. afzelii strains are heterogeneous genotypically.
Animals ; Borrelia burgdorferi Group* ; Borrelia* ; DNA ; Heat-Shock Proteins ; Ixodes ; Korea* ; Murinae ; Population Characteristics

Eight Borrelia burgdorferi strains, which had been isolated from Ixodes nipponensis and Apodemus agrarius captured in the Chungju area of Korea, were characterized by ospC gene sequence analysis. Nucleotide sequence similarity among the Chungju strains ranged from 83.6 to 100%. Deduced amino acid sequence similarity of the Chungju strains ranged from 75.4 to 100%. In a molecular phylogenetic analysis, the Chungju strains were separated from B. burgdorferi and B. garinii reference strains, and formed a cluster with B. afzelii reference strains. Three (KK2, KM4, and KK5), two (CJ2 and CJ21), and one (CJ3) Chungju strains formed clusters with OspC serotype 5, OspC serotype 8, and OspC serotype 7 reference strains, respectively. However, two Chungju strains (KK1 and KM10) formed a distinctive cluster that was separated from other strains of B. afzelii reference strains. These results suggest that Chungju strains are very heterogeneous in clonality.
Amino Acid Sequence ; Animals ; Base Sequence ; Borrelia burgdorferi Group* ; Borrelia burgdorferi* ; Borrelia* ; Chungcheongbuk-do* ; Genetic Heterogeneity* ; Ixodes ; Korea* ; Murinae ; Sequence Analysis*

To detect Rickettsia, we have developed a nested PCR method amplifying the groEL gene. Rickettsia strains were successfully amplified by this PCR method but the microorganisms causing other febrile diseases, such as Orientia tsutsugamushi, Coxiella burnetii, Ehrlichia sennetsu, Borrelia burgdorferi sensu lato, Borrelia hermsii, and Leptospira interrogans were not amplified. This PCR assay was applied to detect Rickettsia DNA from 100 ticks. Sixteen Haemaphysalis longicornis ticks were positive by this PCR assay. These results suggest that the new nested PCR method might be sensitive and useful for discrimination between Rickettsia and other febrile disease-causing microorganisms.
Borrelia ; Borrelia burgdorferi Group ; Coxiella burnetii ; Discrimination (Psychology) ; DNA ; Leptospira interrogans ; Neorickettsia sennetsu ; Orientia tsutsugamushi ; Polymerase Chain Reaction* ; Rickettsia* ; Ticks

Spirochetes were isolated from the midgut of Ixodes persulcatus ticks captured at Chungju, Korea and identified as Borrelia afzelii strains by polymerase chain reaction. To determine the pathogenicity of the B. afzelii strains isolated in Korea, the microbiological and pathological features of Lyme disease were observed in C3H/He mice after intraperitoneal inoculation of the fresh isolate of B. afzelii strain. The results are summarized as follows 1) The Borrelia were detected in the tissues of heart, spleen, kidney, urinary bladder and knee joint within 7 days after inoculation of infection by dark field microscopic examination. The isolation rate from heart, urinary bladder and joint was significantly higher than the rate from spleen, kidney, and blood samples. 2) The Borrelia was detected in heart muscle by indirect immunofluorescent antibody test. 3) Antibody to the Borrelia was detected as early as one week after inoculation. 4) The marked tropism of the Borrelia was observed in myocardial, urinary tract and joint tissue. The main pathological features are inflammation in tissues of heart, kidney, joint and urinary bladder. From these results, the Borrelia afzelii strain isolated in Korea were determined as pathogenic strain.
Animals ; Borrelia burgdorferi Group* ; Borrelia burgdorferi* ; Borrelia* ; Chungcheongbuk-do ; Heart ; Inflammation ; Ixodes ; Joints ; Kidney ; Knee Joint ; Korea* ; Lyme Disease ; Mice ; Myocardium ; Pathology ; Polymerase Chain Reaction ; Spirochaetales ; Spleen ; Ticks ; Tropism ; Urinary Bladder ; Urinary Tract ; Virulence*

OBJECTIVETo study the technique of Western blot for the diagnosis of Lyme disease caused by Borrelia afzelii in China and to establish the standard criteria by operational procedure.

METHODSFP1, which is the representative strain of B. afzelii in China, was analyzed by SDS-PAGE, electro transfer and immunoblotting assays. The molecular weights of the protein bands of FP1 were analyzed by Gel-Pro analysis software. In a study using 451 serum samples (159 patients with Lyme disease and 292 controls), all observed bands were recorded. The accuracy of the WB as a diagnostic test was established by using the ROC curve and Youden index.

RESULTSCriteria for a positive diagnosis of Lyme disease were established as at least one band of P83/100, P58, P39, OspB, OspA, P30, P28, OspC, P17, and P14 in the IgG test and at least one band of P83/100, P58, P39, OspA, P30, P28, OspC, P17, and P41 in the IgM test. For IgG criteria, the sensitivity, specificity and Youden index were 69.8%, 98.3%, and 0.681, respectively; for IgM criteria, the sensitivity, specificity and Youden index were 47%, 94.2%, and 0.412, respectively.

CONCLUSIONEstablishment of WB criteria for B. afzelii is important in validating the diagnostic assays for Lyme disease in China.

Blotting, Western ; methods ; Borrelia burgdorferi Group ; pathogenicity ; China ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Humans ; Lyme Disease ; diagnosis ; microbiology

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

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