Discovery of novel Rickettsiella spp. in ixodid ticks from Western Canada

Abstract

The genomic DNA from four species of ixodid ticks in western Canada was tested for the presence of Rickettsiella by PCR analyses targeting the 16S rRNA gene. Eighty-eight percent of the Ixodes angustus (n = 270), 43% of the I. sculptus (n = 61), and 4% of the I. kingi (n = 93) individuals examined were PCR positive for Rickettsiella, whereas there was no evidence for the presence of Rickettsiella in Dermacentor andersoni (n = 45). Three different single-strand conformation polymorphism profiles of the 16S rRNA gene were detected among amplicons derived from Rickettsiella-positive ticks, each corresponding to a different sequence type. Furthermore, each sequence type was associated with a different tick species. Phylogenetic analyses of sequence data of the 16S rRNA gene and three other genes (rpsA, gidA, and sucB) revealed that all three sequence types were placed in a clade that contained species and pathotypes of the genus Rickettsiella. The bacterium in I. kingi represented the sister taxon to the Rickettsiella in I. sculptus, and both formed a clade with Rickettsiella grylli from crickets (Gryllus bimaculatus) and "R. ixodidis" from I. woodi. In contrast, the Rickettsiella in I. angustus was not a member of this clade but was placed external to the clade comprising the pathotypes of R. popilliae. The results indicate the existence of at least two new species of Rickettsiella: one in I. angustus and another in I. kingi and I. sculptus. However, the Rickettsiella strains in I. kingi and I. sculptus may also represent different species because each had unique sequences for all four genes.

FIG 1

SSCP profiles of representative amplicons of the 16S rRNA gene of Rickettsiella from the total gDNA of Ixodes angustus (lanes 1 to 7 and lanes 19 to 25), I. kingi (lanes 17 and 18), and I. sculptus (lanes 8 to 16).

FIG 2

Neighbor-joining tree depicting the relationships of the 16S rRNA gene sequences of “Rickettsiella angustus,” “R. kingi,” “R. sculptus,” and other species and pathotypes of the genus Rickettsiella. The scale bar represents the inferred substitutions per nucleotide site. The relative support for clades in the tree produced from the NJ and MP analyses are indicated above and below branches, respectively.

FIG 3

Phylogenetic trees depicting the relationships of “‘Rickettsiella angustus,” “R. kingi,” “R. sculptus,” and other species and pathotypes of the genus Rickettsiella based on NJ analyses of the nucleotide and amino acid sequences of the rpsA, gidA, and sucB genes, and the concatenation of the sequence data for these three genes. The scale bar represents the inferred substitutions per nucleotide site. The relative support for clades in the tree produced from the NJ and MP analyses are indicated above and below branches, respectively.