Candidatus Neoehrlichia mikurensis in Ticks from Migrating Birds in Sweden

Abstract

Candidatus Neoehrlichia mikurensis (CNM; family Anaplasmataceae) was recently recognized as a potential tick-borne human pathogen. The presence of CNM in mammals, in host-seeking Ixodes ticks and in ticks attached to mammals and birds has been reported recently. We investigated the presence of CNM in ornithophagous ticks from migrating birds. A total of 1,150 ticks (582 nymphs, 548 larvae, 18 undetermined ticks and two adult females) collected from 5,365 birds captured in south-eastern Sweden was screened for CNM by molecular methods. The birds represented 65 different species, of which 35 species were infested with one or more ticks. Based on a combination of morphological and molecular species identification, the majority of the ticks were identified as Ixodes ricinus. Samples were initially screened by real-time PCR targeting the CNM 16S rRNA gene, and confirmed by a second real-time PCR targeting the groEL gene. For positive samples, a 1260 base pair fragment of the 16S rRNA gene was sequenced. Based upon bacterial gene sequence identification, 2.1% (24/1150) of the analysed samples were CNM-positive. Twenty-two out of 24 CNM-positive ticks were molecularly identified as I. ricinus nymphs, and the remaining two were identified as I. ricinus based on morphology. The overall CNM prevalence in I. ricinus nymphs was 4.2%. None of the 548 tested larvae was positive. CNM-positive ticks were collected from 10 different bird species. The highest CNM-prevalences were recorded in nymphs collected from common redpoll (Carduelis flammea, 3/7), thrush nightingale (Luscinia luscinia, 2/29) and dunnock (Prunella modularis, 1/17). The 16S rRNA sequences obtained in this study were all identical to each other and to three previously reported European strains, two of which were obtained from humans. It is concluded that ornithophagous ticks may be infected with CNM and that birds most likely can disperse CNM-infected ticks over large geographical areas.

Fig 1. Phylogenetic relatedness of Candidatus Neoehrlichia mikurensis sequences.

The tree is based on the partial nucleotide sequence of the 16S rRNA gene of CNM and Anaplasma phagocytophilum. Bacterial sequences generated in this study (boldface) and previously reported sequences are denoted with GenBank accession number followed by information regarding sequence source. The tree was inferred using the neighbour joining method implemented in the SeaView software version 4 [27], utilizing 1000 bootstrap replications to determine support for inferred nodes. The tree was visualized using the MEGA software version 5.2 [28], and statistical supports of > 95% for inferred nodes are displayed. In the mid-point rooted tree, the branch between the CNM and the Anaplasma clade has been truncated in order to limit the tree size. The scale bar represents the number of substitutions per site. The CNM sequences were all 100% identical to each other as well as to three other European strains, including those detected in humans, and therefore appear in a single clade.