Abstract
By using multilocus sequence analysis, five Borrelia valaisiana-related strains isolated from rodents and ticks in southwestern China were eventually classified as a new genospecies of B. burgdorferi sensu lato rather than B. valaisiana. The finding explained the differences in transmission cycle and phenotype between B. valaisiana strains from Europe and B. valaisiana-related strains from eastern Asia.
Strains of Borrelia burgdorferi sensu lato, the causative agent of Lyme borreliosis, have been divided into at least 13 genospecies (7, 8, 10, 15). The bacterium is maintained mainly in natural foci through the transmission cycles of Ixodes ticks and a wide variety of vertebrate hosts (15). Different B. burgdorferi sensu lato genospecies are distributed unevenly throughout the world and are associated with distinct ecologic features (15).
To date, five established genospecies and a group of B. valaisiana-related strains have been isolated in mainland China (7, 14, 16). B. garinii and B. afzelii are major genospecies in natural foci of northern China and are maintained mainly in a tick-rodent cycle (16). Some B. valaisiana-related strains and B. sinica strains were recently isolated in some regions of the Yangtze River valley (7, 14). B. valaisiana-related strains were once tentatively classified as B. afzelii based on the phylogenetic analysis of the rrs gene (2) and then considered to be B. valaisiana based on the phylogenetic analysis of the rrf-rrl intergenic spacer, the rrs gene, and the flagellin gene (4). Recently, Masuzawa et al. suggested that these isolates should be classified as a new genospecies based on a similar phylogenetic analysis (5). In order to clarify the exact taxonomy of B. valaisiana-related strains and explain the differences in transmission cycle and phenotype between B. valaisiana-related strains and B. valaisiana strains, we examined five B. valaisiana-related strains isolated from Guizhou Province in southwestern China by multilocus sequence analysis (MLSA), which was confirmed to surpass the discrimination power of whole-genome DNA-DNA hybridization (the “gold standard” in taxonomy) for B. burgdorferi sensu lato genospecies definition (8, 10). Moreover, new species B. spielmanii and B. californiensis were also confirmed and validated by MLSA (8, 10).
Borrelia strains and culture conditions.
The five strains used in this study were isolated either from ticks or from the urinary bladders of rodents in Guizhou Province in southwestern China in May 2006, as described previously (1). Strain QTMP2 was from Ixodes granulatus fed from Niviventer fulvescens, strains QSYSP3 and QSYSP4 were from Haemaphysalis longicornis fed from Apodemus agrarius, strain QSDS4 was from A. agrarius, and strain QLZSP1 was from I. granulatus fed from A. agrarius. These strains belong to the B. valaisiana-related group rather than the B. sinica and other B. burgdorferi sensu lato genospecies according to the results of restriction fragment length polymorphism analysis and the analysis of rrf-rrl intergenic spacer sequences (Table 1 and Fig. 1).
TABLE 1.
Restriction fragment length polymorphism analysis of the rrf-rrl intergenic spacer regions of B. valaisiana-related strains from Guizhou and B. sinica strains
| Strain | Geographic origin | Amplicon size (bp) | Restriction fragment lengths (bp) with:
|
|
|---|---|---|---|---|
| DraI | MseI | |||
| QTMP2 | Guizhou | 255 | 145, 52, 51, 7 | 144, 53, 28, 23, 7 |
| QSYSP3 | Guizhou | 254 | 144, 52, 51, 7 | 143, 53, 28, 23, 7 |
| QSYSP4 | Guizhou | 254 | 173, 81 | 148, 60, 24, 22 |
| QSDS4 | Guizhou | 254 | 173, 81 | 148, 60, 24, 22 |
| QLZSP1 | Guizhou | 246 | 144, 102 | 143, 61, 28, 14 |
| B. sinica CMN3 | Sichuan | 235 | 157, 49, 29 | 105, 50, 38, 29, 13 |
| B. sinica CMN1a | Sichuan | 236 | 144, 50, 42 | 105, 51, 38, 29, 13 |
| B. sinica CWO1 | Anhui | 236 | 144, 50, 42 | 105, 51, 38, 29, 13 |
FIG. 1.
Unrooted NJ phylogenetic tree based on sequences of the rrf-rrl intergenic spacer determined by using the PHYLIP (version 3.65) software package. Numbers at the branch nodes represent bootstrap values as the proportions of 1,000 replications. Isolates used in this study and other strains from China are indicated in bold type. The geographic sources of reference strains are marked after the names of the strains, as follows: Ja for Japan, Eu for Europe, USA for the United States, and Ko for Korea. s.s., sensu stricto.
Strains were cultivated at 32°C in BSK-H medium (Sigma, St. Louis, MO) supplemented with 6% rabbit serum and 1% antibiotic mixture for Borrelia (Sigma, St. Louis, MO).
DNA extraction.
DNA extraction was performed as described previously (8). Briefly, Borrelia cultures were harvested by centrifugation (10,000 × g for 20 min). The bacterial pellet was washed in phosphate-buffered saline and resuspended. The DNA was extracted from the centrifugation pellet of cultivated isolates by boiling the pellet in water at 100°C for 10 min, and the DNA samples were stored at −20°C until use.
MLSA.
Seven loci, rrs, hbb, groEL, recA, fla, ospA, and the rrf-rrl intergenic spacer, were used for MLSA and amplified under the conditions described previously (10). Amplification products were gel purified using a QIAquick gel extraction kit (Qiagen, Hilden, Germany) and sequenced with an automated DNA sequencer (ABI Prism 377; Perkin-Elmer, Foster City, CA). Corresponding sequences of 14 other control strains from Europe, North America, and eastern Asia, representing B. burgdorferi sensu stricto, B. garinii, B. afzelii, B. valaisiana, and B. lusitaniae species, were derived from GenBank and used for comparison in this study. Subsequently, the sequences of the seven loci of each strain were concatenated, resulting in sequences of 2,100 bp for MLSA.
The Clustal X software (version 1.8) (13) and the PHYLIP software package (version 3.63) (9) were used for sequence alignment and phylogenetic analyses, respectively. The phylogenetic trees were constructed using the neighbor-joining (NJ) method (11) and DNA maximum-likelihood (DNAML) software (12). The stability of each tree was evaluated by bootstrap analysis with 1,000 replications. The distance matrix for the aligned sequences was generated by using the DNAStar software package.
In the phylogenetic trees for the concatenated sequences of the seven loci, the Guizhou strains clustered closely into a branch and clearly were separated from all other species by whatever method used, supported by bootstrap analysis with 1,000 replications (Fig. 2). The concatenated sequences of the Guizhou strains were 98.4 to 99.8% identical to one another, whereas they were 96.4 to 96.9% related to those of B. valaisiana strains and only 92.5 to 94.9% related to those of other B. burgdorferi sensu lato genospecies on the basis of the sequence identity matrix (Table 2). According to the MLSA sequence identity cutoff of 97.9% to differentiate B. burgdorferi sensu lato genospecies (8, 10), the Guizhou strains were classified as a novel genospecies of B. burgdorferi sensu lato rather than B. valaisiana. Since B. valaisiana-related strains were isolated in Guizhou Province in the Yangtze River valley, these B. valaisiana-related strains were designated B. yangtze sp. nov.
FIG. 2.
Phylogenetic trees based on concatenated sequences of seven loci, rrs, hbb, groEL, recA, fla, ospA, and the rrf-rrl intergenic spacer, as determined by the DNAML (a) and NJ (b) methods using the PHYLIP (version 3.65) software package. Numbers at the branch nodes represent bootstrap values as the proportions of 1,000 replications. Isolates in this study are indicated in bold type. The geographic sources of reference strains are marked after the names of the strains, as follows: Eu for Europe, USA for the United States, and Ja for Japan. s.s., sensu stricto.
TABLE 2.
Sequence identities of strains based on the concatenated sequences of seven loci selected for MLSA
| Strain | % Sequence identity to:
|
||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| BO23 | Pgau | VS461 | 212 | B31 | IP1 | 20047 | NT29 | PBi | PotiB1 | PotiB2 | UK | VS116 | Am501 | QLZSP1 | QTMP2 | QSYSP3 | QSYSP4 | QSDS4 | |
| B. afzelii BO23 | 100.0 | 99.5 | 99.8 | 93.8 | 93.8 | 94.1 | 94.4 | 94.0 | 94.1 | 93.0 | 92.9 | 94.4 | 94.4 | 94.2 | 93.8 | 93.4 | 93.6 | 93.3 | 93.6 |
| B. afzelii Pgau | 99.6 | 93.8 | 93.9 | 94.1 | 94.4 | 94.3 | 94.3 | 93.0 | 93.2 | 94.7 | 94.7 | 94.5 | 94.1 | 93.7 | 93.9 | 93.6 | 93.8 | ||
| B. afzelii VS461 | 93.8 | 93.8 | 94.0 | 94.2 | 94.0 | 94.1 | 92.9 | 92.8 | 94.4 | 94.4 | 94.2 | 93.8 | 93.4 | 93.6 | 93.4 | 93.6 | |||
| B. burgdorferi sensu stricto 212 | 99.6 | 99.7 | 93.5 | 93.8 | 93.6 | 93.0 | 93.0 | 94.0 | 93.9 | 93.8 | 93.2 | 93.1 | 93.3 | 93.2 | 93.4 | ||||
| B. burgdorferi sensu stricto B31 | 99.8 | 93.5 | 93.8 | 93.5 | 92.9 | 92.9 | 93.8 | 93.7 | 93.9 | 93.3 | 93.0 | 93.3 | 93.2 | 93.4 | |||||
| B. burgdorferi sensu stricto IP1 | 93.7 | 94.0 | 93.7 | 93.2 | 93.2 | 94.1 | 94.0 | 94.1 | 93.4 | 93.3 | 93.5 | 93.4 | 93.6 | ||||||
| B. garinii 20047 | 97.6 | 98.2 | 93.1 | 93.1 | 94.6 | 94.4 | 94.6 | 94.3 | 94.0 | 94.2 | 94.0 | 94.5 | |||||||
| B. garinii NT29 | 98.3 | 93.4 | 93.4 | 94.9 | 94.8 | 94.8 | 94.4 | 94.2 | 94.4 | 94.1 | 94.4 | ||||||||
| B. garinii PBi | 92.9 | 92.9 | 94.7 | 94.6 | 94.7 | 94.4 | 94.1 | 94.3 | 94.0 | 94.3 | |||||||||
| B. lusitaniae PotiB1 | 99.7 | 93.5 | 93.4 | 93.9 | 93.0 | 92.9 | 93.1 | 92.5 | 93.0 | ||||||||||
| B. lusitaniae PotiB2 | 93.7 | 93.7 | 94.0 | 93.0 | 93.0 | 93.2 | 92.7 | 93.1 | |||||||||||
| B. valaisiana UK | 99.7 | 98.8 | 96.9 | 96.6 | 96.8 | 96.8 | 96.9 | ||||||||||||
| B. valaisiana VS116 | 98.7 | 96.7 | 96.4 | 96.7 | 96.7 | 96.7 | |||||||||||||
| B. valaisiana Am501 | 96.6 | 96.5 | 96.7 | 96.7 | 96.7 | ||||||||||||||
| QLZSP1 | 98.8 | 98.9 | 98.4 | 98.9 | |||||||||||||||
| QTMP2 | 99.8 | 98.8 | 98.6 | ||||||||||||||||
| QSYSP3 | 99.0 | 98.8 | |||||||||||||||||
| QSYSP4 | 98.9 | ||||||||||||||||||
| QSDS4 | 100.0 | ||||||||||||||||||
The geographical distribution of B. valaisiana-related spirochetes remains to be investigated. The five B. valaisiana-related strains used in this study were isolated from I. granulatus and H. longicornis ticks and an A. agrarius rodent in southwestern China. In addition, strains of the same species have been isolated previously from I. granulatus ticks, I. nipponensis ticks, and a variety of rodents, including A. agraricus, N. confucianus, Rattus losea, Mus formosanus, R. norvegicus, M. calori, Crocidura watasei, and Suncus murinus, in some areas of eastern Asia, including Japan, Korea, Taiwan, and the Zhejiang Province of mainland China (2, 4-7). These findings suggested that these B. valaisiana-related spirochetes probably circulate between their rodent reservoir hosts and tick vectors in eastern Asia. However, in Europe B. valaisiana has been found previously in various avian reservoirs but never in rodent hosts (15). Moreover, phenotypic differences between B. valaisiana isolates found in Europe and the B. valaisiana-related strains isolated in eastern Asia may also exist, since the detection of Borrelia sensitivities to the sera of the reservoir hosts showed that rodent serum lyses B. valaisiana from Europe while bird serum does not (3). It remains to be determined whether B. valaisiana-related strains can cause a disease in humans.
Nucleotide sequence accession numbers.
Sequences of the Guizhou strains determined in this study have been deposited in GenBank with the following accession numbers: rrf-rrl intergenic spacer sequences, EU247839 to EU247843; rrs gene sequences, EU135593 to EU135597; fla gene sequences, EU135599 to EU135602; ospA gene sequences, EU325674 to EU325678; recA gene sequences, EU390784 to EU390788; groEL gene sequences, EU390794 to EU390798; and hbb gene sequences, EU390789 to EU390793.
Acknowledgments
This study was supported by the National Science Fund for Distinguished Young Scholars (grant no. 30725032).
Footnotes
Published ahead of print on 9 July 2008.
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