Detection of Candidatus Neoehrlichia mikurensis in Norway up to the northern limit of Ixodes ricinus distribution using a novel real time PCR test targeting the groEL gene

Abstract

Background: Candidatus Neoehrlichia mikurensis is an emerging tick-borne pathogen. It is widely distributed in Ixodes ricinus ticks in Europe, but knowledge of its distribution in Norway, where I. ricinus reaches its northern limit, is limited. In this study we have developed a real time PCR test for Ca. N. mikurensis and used it to investigate the distribution of Ca. N. mikurensis in Norway.

Results: Real time PCR targeting the groEL gene was developed and shown to be highly sensitive. It was used to detect Ca. N. mikurensis in 1651 I. ricinus nymphs and adults collected from twelve locations in Norway, from the eastern Oslo Fjord in the south to near the Arctic Circle in the north. The overall prevalence was 6.5% and varied locally between 0 and 16%. Prevalence in adults and nymphs was similar, suggesting that ticks acquire Ca. N. mikurensis predominantly during their first blood meal. In addition, 123 larvae were investigated; Ca. N. mikurensis was not found in larvae, suggesting that transovarial transmission is rare or absent. Sequence analysis suggests that a single variant dominates in Norway.

Conclusions: Ca. N. mikurensis is widespread and common in ticks in Norway and reaches up to their northern limit near the Arctic Circle. Ticks appear to acquire Ca. N. mikurensis during their first blood meal. No evidence for transovarial transmission was found.

Keywords: Ixodes ricinus; Neoehrlichia mikurensis; Norway; Scandinavia; Tick-borne diseases; Ticks.

Fig. 1

Multiple sequence alignment of the PCR target regions of groEL in Ca. N. mikurensis (CNM), Ca. N. lotori and selected Ehrlichia species. Dots indicate identity to the reference sequence, AB084583; letters indicate differences; hyphens indicate gaps or no sequence. The target regions for the primers and probe are highlighted in yellow and green respectively. Mismatches within the primer/probe target regions that give stable G:T basepairs are highlighted in blue. Destabilising mismatches (variants resulting in A:C, purine:purine or pyrimidine:pyrimidine) are highlighted in red. For reasons of space, sequence accession numbers for Ca. N. lotoris, Ehrlichia ewingii, Candidatus E. shimanensis, two sequence variants of E. ruminantium and E. chafeensis have been omitted from the figure; these are: EF633745, AF195273, AB074462, AB625796, DQ647005 and JQ085941 respectively

Fig. 2

Comparison of TaqMan MGB Probe and SYBR green PCR. Amplification of a dilution series of an I. ricinus sample positive for Ca. N. mikurensis. Dilutions are 1:4, 1:20, 1:100, 1:500 and 1:2500 respectively. Green curves are for SYBR-green, red curves are for TaqMan MGB probe. The signals to the lower right of the amplification curves below the yellow-green threshold line are background noise from the 1:2500 dilutions

Fig. 3

a Amplification curves for a 10x dilution series of pNeo containing from 1.6 × 10⁹ copies (leftmost curves) to 1.6 × 10⁰ copies (rightmost curves). b Standard curve of Cq values (CT) derived from (a) plotted against number of copies of the groEL gene (quantity; logarithmic scale)

Fig. 4

Map of Norway showing collection locations and the proportion of adult and nymphal ticks positive for Ca. N. mikurensis at each location. Location numbers correspond to location numbers in Table 5. The areas of the pie charts are proportional to the number of ticks. Collection 4 is not included as it includes only larvae. The locality is the same as collection 5