Ribotype Groups Research Articles

Distribution of Borrelia species associated with Lyme disease in the subalpine forests of Nagano prefecture, Japan.

We surveyed the natural distribution of Borrelia species associated with Lyme disease in the subalpine forests of Nagano prefecture, Japan, during 1993-94. Tick-derived isolates (n = 112) from Ixodes persulcatus and rodent-derived isolates (n = 55) from Apodemus argenteus, Apodemus speciosus, Eothenomys andersoni, Eothenomys smithii, and Microtus montebelli were classified by rRNA gene restriction fragment length polymorphism analysis (RFLP ribotyping). Ribotype group IV (an intraspecific variant of Borrelia garinii) was predominant among the tick isolates. It was also isolated repeatedly from the rodents. Ribotype group III (Borrelia afzelii) was detected in low frequencies among the tick and rodent isolates. The data suggest that humans are likely to be exposed to the group IV when they are bitten by I. persulcatus ticks.

A phylogenetic analysis of Borrelia burgdorferi sensu lato isolates associated with Lyme disease in Japan by flagellin gene sequence determination.

We determined nearly complete flagellin gene sequences for Borrelia burdgdorferi sensu lato isolates (11 isolates obtained from Ixodes persulcatus ticks and patients in Hokkaido, Japan, and 1 European isolate) representing six different restriction fragment length polymorphism (RFLP) ribotype groups following cloning of the PCR-amplified genes. These sequences were aligned with those of representatives of the three Borrelia species associated with Lyme disease, and a phylogenetic tree was constructed by the Clustal method. On the Lyme disease borrelia portion of the tree, the species were clearly delineated into three different phylogenetic groups, in complete agreement with the division of B. burgdorferi sensu lato into three species. A phylogenetic analysis revealed that the representatives of RFLP ribotype groups IV, V, and VI clustered tightly with each other and belonged on the same branch as Borrelia garinii. We used the criteria that are currently used to delineate bacterial species and determined the levels of DNA relatedness for these Borrelia isolates. For the RFLP ribotype group IV, V, and VI isolates, the levels of DNA relatedness ranged from 79 to 88%, and the levels of relatedness to the reference strain of B. garinii ranged from 70 to 80%. The levels of DNA relatedness of the RFLP ribotype group IV, V, and VI isolates to the representatives of other species associated with Lyme disease ranged from 53 to 66%. All of these findings indicate that the RFLP ribotype group IV, V, and VI isolates should be included in the species B. garinii.

Outer surface protein C gene sequence analysis of Borrelia burgdorferi sensu lato isolates from Japan.

The nucleotide sequences of the outer surface protein C gene (ospC) from Borrelia burgdorferi sensu lato isolates representing six different restriction fragment length polymorphism (RFLP) ribotype groups were determined, and the deduced amino acid sequences were aligned in comparison with the previously published OspC protein sequences. The sequence similarity analysis revealed the high sequence variability of OspC protein, and the degree of amino acid similarity ranged from 53.8 to 100% among 25 isolates. It has been reported that the representatives belonging to the three species of B. burgdorferi sensu lato showed a species-specific amino acid sequence motif at positions 23 to 35 (B. Wilske, S. Jauris-Heipke, R. Lobentanzer, I. Pradel, V. Preac-Mursic, D. Rössler, E. Soutschek, and R.C. Johnson, J. Clin. Microbiol. 33:103-109, 1995). Alignment with the OspC sequences of RFLP ribotype group IV, V, and VI isolates revealed that a sequence motif of all the isolates was quite similar to that of Borrelia garinii. A phylogenetic analysis based on OspC protein sequences also showed that most of the Japanese isolates were closely related to the species B. garinii. THe RFLP ribotype group IV species is predominant among clinical isolates of Lyme disease patients, reservoir rodents, and adult ticks in Japan. Although the isolates differed from type strains of the three delineated genospecies in genetic and immunological characteristics, it is likely that the spirochetes diverged within the species level. Therefore, the representatives of ribotype groups IV, V, and VI appear to have evolved within B. garinii and to have adapted to an Asiatic habitat, and there appeared to be a sufficient ecological pressure to allow bacterial species level development.

Mixed infection of different Borrelia species among Apodemus speciosus mice in Hokkaido, Japan.

The wood mouse (Apodemus speciosus) serves as a wildlife reservoir for Lyme disease spirochetes in Hokkaido, Japan. To isolate Borrelia species, we captured 34 wood mice in an area where Borrelia species are endemic during October 1993. The earlobes (right and left), heart, spleen, and urinary bladder from each mouse were used as culture sources. As a result of culture 73 isolates from 21 mice were classified by rRNA gene restriction fragment length polymorphism analysis. Ribotype groups III (Borrelia afzelii) and IV (unknown species) were detected among those isolates. Thirty-one (77.5%) of 40 earlobe isolates were classified as group IV. In contrast, 6 (40.0%) of 15 heart isolates, 5 (50.0%) of 10 spleen isolates, and 7 (87.5%) of 8 urinary bladder isolates were B. afzelii. Seven mice showed mixed infection with B. afzelii and group IV. The data indicate that different Borrelia species can coexist in a reservoir host that is suitable for them.

Molecular characterization of Pasteurella testudinis isolated from desert tortoises (Gopherus agassizii) with and without upper respiratory tract disease.

Isolates of Pasteurella testudinis recovered from clinically healthy desert tortoises (Gopherus agassizii) and tortoises with upper respiratory tract disease (URTD) were characterized in an attempt to identify strains associated with disease. Eighty-nine isolates, 52 from ill and 37 from healthy tortoises collected from Nevada (USA), June 1990 to September 1991, were genomically fingerprinted and grouped based on ribotype similarity. Twelve isolates (six from ill and six from healthy tortoises) were further characterized with regard to whole-cell protein (WCP) and outer membrane protein (OMP) composition and their ability to survive in normal tortoise plasma. The 89 isolates were initially distributed into 33 distinct ribotype groups using the restriction enzyme EcoRI; five ribotypes contained over 50% of the isolates. Only one EcoRI ribotype was comprised of multiple isolates (n = 4) exclusively recovered from tortoises with URTD. When the ten EcoRI ribotypes that contained more than one isolate per ribotype were further studied using a second restriction enzyme, EcoRV, one EcoRI/EcoRV ribotype contained five isolates recovered from URTD tortoises and none from healthy animals. The EcoRI ribotype comprised of four isolates, all from tortoises with URTD, was further separated into three distinct groups with EcoRV. All 12 isolates studied grew equally well in normal tortoise plasma, and when broth-grown WCP and OMP profiles were evaluated, no proteins were unique to isolates from URTD tortoises. Iron-regulated OMP's were produced in three isolates examined, but these OMP's apparently were not virulence-related.

Lyme disease spirochetes in Japan: enzootic transmission cycles in birds, rodents, and Ixodes persulcatus ticks.

The ixodid tick, Ixodes persulcatus, serves as a vector of Borrelia species associated with Lyme disease in Hokkaido, Japan. The migratory birds of genera Emberiza and Turdus and the woodland rodents of genera Apodemus and Clethrionomys are the wildlife reservoirs. To systemize the enzootic transmission cycles, borreliae isolates were classified by ribosomal RNA gene restriction fragment length polymorphism (RFLP) analysis. Most (> 60%) isolates from the bird-feeding I. persulcatus larvae belonged to Borrelia garinii; the rest were classified as RFLP ribotype group IV (unknown species). In contrast, no B. garinii were found among isolates from rodent-feeding I. persulcatus larvae. These isolates were classified as Borrelia afzelii, group IV, and group V (unknown species). These observations suggest that two enzootic cycles in nature (bird-tick and rodent-tick) maintain borreliae specifically. The group IV species, which was predominant among clinical isolates from Lyme disease patients in Hokkaido, appears to be the most important pathogen for humans.

Comparison of methods for discrimination between strains of Listeria monocytogenes from epidemiological surveys.

Total cellular DNA from 28 strains of Listeria monocytogenes isolated from food implicated in food-borne illness and from patients with listeriosis was digested with the restriction endonucleases HindIII, HaeIII, and EcoRI. Following agarose gel electrophoresis, the fragments were subjected to Southern blot hybridization with a digoxigenin-labeled cDNA probe transcribed from Escherichia coli 16S and 23S rRNA. The patterns of bands from genomic (DNA fingerprints) and rDNA fingerprints (ribotypes) were used for classifying L. monocytogenes strains, and the resulting subtypes were compared with serotyping and multilocus enzyme electrophoresis classification schemes. A total of 15 distinct and identical groups were obtained when genomic DNA was digested with either HindIII or HaeIII. The most discriminating enzyme for ribotyping of strains was EcoRI, which divided the 28 strains of L. monocytogenes into 6 ribotype groups. DNA fingerprinting and ribotyping differentiated L. monocytogenes from other Listeria spp., including L. ivanovii, L. welshimeri, and L. innocua as well as the lactic acid bacteria Lactococcus lactis subsp. lactis and subsp. cremoris. L. monocytogenes strains isolated from four independent food-borne illness incidents were analyzed by all typing methods. Patient and product isolates were not distinguishable by serotyping, ribotyping, or multilocus enzyme electrophoresis. DNA fingerprinting was the only method capable of differentiating these strains, or conversely, of proving relatedness of patient-product pairs of isolates. This method was a relatively simple, sensitive, reproducible, and highly discriminating method for epidemiological tracking of L. monocytogenes implicated in food-borne illness.