Pathogenic New World Relapsing Fever Borrelia in a Myotis Bat, Eastern China, 2015.

We identified Candidatus Borrelia fainii, a human pathogenic bacterium causing New World relapsing fever in a Myotis bat in eastern China. This finding expands knowledge about the geographic distribution of Borrelia spp. and the potential for infection with New World relapsing fever in China.

Borrelia is a genus comprising 3 groups of spirochetes: the Lyme disease group, the relapsing fever group, and a nonconformist third group. Typically, Lyme disease borreliae are transmitted by hard ticks and have a worldwide distribution, but most relapsing fever Borrelia are transmitted by soft ticks, except for louse-borne B. recurrentis. Relapsing fever borreliae are further classified into 2 subgroups, New World relapsing fever (NWRF) Borrelia and Old World relapsing fever, on the basis of epidemic regions and the genetic lineage of the causative agent. B. lonestari and B. miyamotoi are transmitted by hard ticks, but are more closely related to relapsing fever borreliae than to Lyme disease borreliae and are distributed both in the New World (North and South America and Oceania) and the Old World (Europe, Asia, and Africa). The nonconformist third group includes an orphan Borrelia species named B. turicata (). We identified Candidatus Borrelia fainii in a Myotis bat from eastern China.

During March–October 2015, with the help of local farmers, we caught a total of 145 bats from various niches in Mengyin County, Shandong Province, China, using mist nets or butterfly nets and took tissue samples (liver, spleen, lung, or kidney). The captured bats included 4 Rhinolophus ferrumequinum and 14 Rh. pusillus from a karst cave, 26 Eptesicus serotinus from 2 farm houses, 34 Myotis fimbriatus and 10 M. ricketti from a city sewer, and 57 M. pequinius from a cave. We extracted DNA from the bat tissue, then screened for Borrelia by amplifying the rrs, flaB, and glpQ genes using methods described previously (–); after gel extraction, we cloned the PCR products into pMD19 T-vectors (TaKaRa, https://www.takarabio.com) for sequencing.

We found 1 M. ricketti bat positive for Borrelia (GenBank accession nos. MG832412 for rrs, MG832413 for flaB, and MG921625 for glpQ). BLAST searches showed that rrs exhibited 99.7% (1,491/1,495 bp) identity with Candidatus Borrelia fainii (accession no. LC382043), flaB exhibited 97.9% (756/772 bp) identity with B. turicatae (accession no. CP015629), and glpQ exhibited 97.6% (859/880 bp) identity with B. parkeri (accession no. AY934633).

We performed multilocus sequence typing (MLST) by amplifying 8 housekeeping genes (clpA, clpX, nifS, pepX, pyrG, recG, rplB, and uvrA) with degenerate primers from the Borrelia MLST database (https://pubmlst.org/borrelia). Sequence query showed that all 8 housekeeping genes were novel alleles, which were assigned the following novel allele numbers: clpA (298), clpX (261), nifS (235), pepX (264), pyrG (277), recG (292), rplB (254) and uvrA (268); the Borrelia species of this study was designated sequence type (ST) 927.

Sequences of the 8 housekeeping genes were concatenated in the order clpA, clpX, nifS, pepX, pyrG, recG, rplB, and uvrA and imported into MEGA7 (MEGA, https://www.megasoftware.net) for phylogenetic analysis. We constructed a phylogenetic tree using the maximum-likelihood method with the Kimura 2-parameter model. The ST927 Borrelia species was phylogenetically closely related to multiple NWRF Borrelia species, including B. turicatae, B. parkerii, and Candidatus Borrelia johnsonii, which are endemic in the United States, as well as Candidatus Borrelia fainii, which was recently identified in Zambia (Figure).

Figure

Phylogenetic analysis of Borrelia species based on 8 concatenated housekeeping genes (clpA-clpX-nifS-pepX-pyrG-recG-rplB-uvrA). Bold indicates Borrelia species identified in study of pathogenic New World relapsing fever Borrelia in a Myotis bat, eastern China, 2015. The tree was constructed by using the maximum-likelihood method in MEGA7 (https://www.megasoftware.net). Bootstrap values were calculated with 1,000 replicates. There were a total of 4,776 positions in the final dataset. Reference sequences of Borrelia species were downloaded from the Borrelia MLST database; the corresponding sequence type (ST) number of each Borrelia species is shown in parentheses. For Candidatus Borrelia fainii, the GenBank accession number is shown instead of an ST number because the 8 housekeeping gene sequences of Candidatus Borrelia fainii were only submitted to GenBank and no ST number was assigned. Scale bar indicates 5% divergence.

We calculated pairwise genetic distances using the Kimura 2-parameter model and identified relapsing fever Borrelia spp. using the threshold of 98.3% similarity and genetic distance 0.017 (). Genetic distance analysis of the 8 concatenated housekeeping genes (4,776 bp) revealed a value of 0.015 compared with Candidatus Borrelia fainii, strain Qtaro. Thus, the Borrelia sp. in our study was identified as Candidatus Borrelia fainii.

There have been several reports of NWRF Borrelia spp. in the Old World, although exclusively in Africa. A new human pathogenic Borrelia spp. was identified in Ornithodoros ticks from Tanzania that grouped together with NWRF borreliae rather than the relapsing fever–inducing spirochetes previously known to be endemic in East Africa (). Another study described the discovery of a NWRF Borrelia, Candidatus Borrelia kalaharica, in a traveler returning from the Kalahari Desert (). Finally, a 2019 study reported on a NWRF Borrelia–like spirochete, Candidatus Borrelia fainii, recently isolated from a febrile patient as well as from bats and bat ticks in Zambia ().

Whether bats are reservoirs for Borrelia remains inconclusive. A new Old World relapsing fever Borrelia species, CPB1, was found responsible for the death of a Pipistrelle bat in the United Kingdom () and was also detected in bat soft ticks in France (). A recent study found that bats and bat soft ticks collected from a cave in Zambia showed a high infection rate for Candidatus Borrelia fainii, and the authors proposed that bats contribute to the environmental cycle of Candidatus Borrelia fainii as hosts and bat soft ticks as vectors (). For this study, we found only 1 bat infected with Candidatus Borrelia fainii, and it remains unclear whether bats serve as reservoirs of this Borrelia species. However, with the discovery of Candidatus Borrelia fainii in China, both health officials and physicians should pay attention to its potential emergence.