Large-Scale Survey for Tickborne Bacteria, Khammouan Province, Laos

We screened 768 tick pools containing 6,962 ticks from Khammouan Province, Laos, by using quantitative real-time PCR and identified Rickettsia spp., Ehrlichia spp., and Borrelia spp. Sequencing of Rickettsia spp.–positive and Borrelia spp.–positive pools provided evidence for distinct genotypes. Our results identified bacteria with human disease potential in ticks in Laos.

We extracted DNA by using the NucleoSpin 8 Virus Extraction Kit (Macherey-Nagel, Düren, Germany). Pools were screened by using single quantitative real-time PCRs specific for Rickettsia spp.  Table 1). Five microliters of diluted (1:10) template containing 1× Platinum Supermix-UDG (Invitrogen, Carlsbad, CA, USA) and bovine serum albumin (40 mg/mL) were used for each assay. Positive and nontemplate controls were included in each run. Screening by PCR was performed once per sample. In concordance with published guidelines, results were considered positive if they had a cycle quantitation (Cq) value <40 and likely positive if they had a Cq value 40-45 (9).
Sequencing was attempted for pools with C q values <40 (online Technical Appendix Table 2) and performed by Macrogen (Seoul, South Korea). Consensus sequences were analyzed by using CLC Main Workbench 7 (http://www.clcbio.com/products/clc-main-workbench/) and BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and submitted to GenBank. Phylogenetic trees were constructed by using the Kimura 2-parameter model and the neighbor-joining method. Bootstrap values were determined by using 1,000 replications.
Three novel genotypes ( Table 2) were identified that might be new species. Candidatus Rickettsia laoensis (pool 447) was identified in 1 Haemaphysalis sp. pool. Phylogenetic analysis of 2845-2920-bp concatenated sequences of gltA, sca4, and ompB genes suggested that this bacteria belonged to the R. massiliae group of rickettsiae (online Technical Appendix Figure 3). Candidatus Rickettsia mahosotii (pools 81 and 372) was identified in Haemaphysalis spp. and A. testudinarium pools. Phylogenetic analysis of gltA, sca4, and ompB genes suggested that this bacteria belonged to the R. rickettsii group (online Technical Appendix Figure 3). Candidatus Rickettsia khammouanensis was identified in 1 Haemaphysalis sp. nymph pool (pool 120). Phylogenetic analysis of gltA, 17-kDa, and ompB genes suggested a relationship with the R. helvetica group (online Technical Appendix Figure 4).
In addition, 15 A. testudinarium pools showed dual peaks for 17-kDa gene sequences, which suggested the presence of R. tamurae and Rickettsia sp. ATT. Sequencing of sca4, ompA, and ompB genes from 1 of these pools (pool 239) identified unique sequences ( Table 2; online Technical Appendix Figure 4).
No pools were positive for Anaplasma spp., but 2 were likely positive (Table 1). Although not all pools were tested for Coxiella spp. (n = 511), 1 pool (0.2%) was positive, and 4 pools were likely positive for C. burnetti. No confirmatory sequences were obtained from these pools. The 1 tick that contained a blood meal (A. testudinarium nymph) showed negative results by screening PCRs.

Conclusions
This study provides evidence that Rickettsia spp., Borrelia spp., and Ehrlichia spp. are present in ticks in Laos. Several Rickettsia spp. identified in this study are human pathogens. Infections with R. tamurae (2) and R. japonica are well described in Southeast Asia (10). However, the pathogenicity of Rickettsia sp. TwkM01 (11), Rickettsia sp. ATT (12), Rickettsia sp. kagoshima6 genotypes (13) and potential novel Candidatus Rickettsia laoensis, Candidatus Rickettsia mahosotii, and Candidatus Rickettsia khammouanensis is unknown. Candidatus Rickettsia khammouanensis is phylogenetically related to R. helvetica, for which there is serologic evidence for its role as a human pathogen in Laos (2). Unique ompA, ompB, and sca4 sequences identified in this study (  Rickettsia sp. ATT (12), which was previously believed to be identical to R. tamurae (14), and suggests that it might be a distinct species. Further studies, including whole-genome sequencing, are required to identify and confirm these novel genotypes and understand their role in human disease. Borrelia spp. sequences identified in Haemaphysalis spp. pools were shown to have high concordance with the Shiretoko Haemaphysalis Borrelia isolated from Haemaphysalis spp. ticks and deer in Japan (15). The species belongs to the relapsing fever group of Borrelia and is related to B. lonestari.
Sequence data for Ehrlichia spp. indicated the presence of these bacteria but were not sufficient to identify them to the species level. The C q values were high (40-45) for Anaplasma spp., but no sequence data were obtained. Coxiella spp. were screened by using primers for IS1111, which are not specific for C. burnetii, and no confirmatory sequence data were obtained. Because of limited reagents, screening of all 768 pools for Coxiella spp. was not completed. Further work is required to investigate the presence of these bacteria in Laos.
Our study had several limitations. First, pooling of ticks precludes an accurate assessment of prevalence of bacterial pathogens. Second, sequences obtained from some A. testudinarium pools had dual peaks, suggestive of multiple infections, and could therefore not be interpreted. Third, ticks were collected only from 1 area in Laos

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