Diversity unearthed by the estimated molecular phylogeny and ecologically quantitative characteristics of uncultured Ehrlichia bacteria in Haemaphysalis ticks, Japan

Abstract

Ehrlichia species are obligatory intracellular bacteria transmitted by arthropods, and some of these species cause febrile diseases in humans and livestock. Genome sequencing has only been performed with cultured Ehrlichia species, and the taxonomic status of such ehrlichiae has been estimated by core genome-based phylogenetic analysis. However, many uncultured ehrlichiae exist in nature throughout the world, including Japan. This study aimed to conduct a molecular-based taxonomic and ecological characterization of uncultured Ehrlichia species or genotypes from ticks in Japan. We first surveyed 616 Haemaphysalis ticks by p28-PCR screening and analyzed five additional housekeeping genes (16S rRNA, groEL, gltA, ftsZ, and rpoB) from 11 p28-PCR-positive ticks. Phylogenetic analyses of the respective genes showed similar trees but with some differences. Furthermore, we found that V1 in the V1-V9 regions of Ehrlichia 16S rRNA exhibited the greatest variability. From an ecological viewpoint, the amounts of ehrlichiae in a single tick were found to equal approx. 6.3E+3 to 2.0E+6. Subsequently, core-partial-RGGFR-based phylogenetic analysis based on the concatenated sequences of the five housekeeping loci revealed six Ehrlichia genotypes, which included potentially new Ehrlichia species. Thus, our approach contributes to the taxonomic profiling and ecological quantitative analysis of uncultured or unidentified Ehrlichia species or genotypes worldwide.

Figure 1

Phylogenetic tree of p28 clones based on 88–91 amino acid sequences from uncultured Ehrlichia bacteria and their close relatives. The tree was constructed using the maximum likelihood method with the Jones–Taylor–Thornton model and “all sites” for gaps/missing data treatment. The numbers on the branch node of the tree indicate bootstrap values from 1000 replications. The scale bar indicates the evolutionary distance. The p28 clones of the uncultured Ehrlichia from a tick individual are indicated by characters with the same color. The accession numbers are in parentheses.

Figure 2

Phylogenetic analyses of five housekeeping genes from uncultured Ehrlichia bacteria based on nucleotide sequences. (A) 16S rRNA (1396–1397 bp), (B) groEL (427 bp), (C) gltA (430 bp), (D) ftsZ (321 bp), and (E) rpoB (271 bp). These trees were constructed using the maximum likelihood method with the Kimura two-parameter model and “complete deletion” for gaps/missing data treatment. The bootstrap values were obtained from 1000 replications. Bootstrap values higher than 40 are shown in the tree branches. The scale bar indicates the evolutionary distance, and the accession number of each sequence is shown in parentheses. The uncultured Ehrlichia bacteria from tick individuals in this study are shown by characters with the same colors on the respective trees of five genes.

Figure 3

Alignment of the V1 region of 16S rRNA sequences from 33 Ehrlichia bacteria, including 11 uncultured ehrlichiae, in this study. The V1 region of the Ehrlichia 16S rRNA alignment is boxed with a red line.

Figure 4

Core-partial-RGGFR-based phylogenetic analysis of uncultured Ehrlichia bacteria and cultured Ehrlichia species with genome sequence data. Tree based on five housekeeping genes (16S rRNA-groEL-gltA-ftsZ-rpoB) of uncultured Ehrlichia bacteria in this study and cultured Ehrlichia species for which complete genome sequence data have previously been obtained were constructed using the maximum likelihood method with the Kimura two-parameter model and “complete deletion” for gap/missing data treatment. The bootstrap values from 1000 replications are shown on the branch nodes. The scale bar indicates the evolutionary distance. The uncultured Ehrlichia bacteria in this study are shown by the colored characters, and their “genotypes” that were estimated based on the core-partial-RGGFR alignment and summarized in Table 4 are shown on the right side. The accession numbers and the location of isolated or identified Ehrlichia species or genotypes are shown in parentheses.