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Figure. Locations of primary health care centers in study of Spiroplasma ixodetisin ticks removed from humans, Sweden and in the Åland Islands, Finland. Maps indicate all primary health care...

This study is a part of the Tick-Borne Diseases (TBD) STING study, a prospective multicenter study in Sweden and in the Åland Islands, Finland. The study enrolled 2,327 healthy tick-bitten participants (>18 years of age) at primary healthcare centers (PHCs) in 4 geographic regions (9) (Figure) through public advertisements during 2008–2010. The Regional Ethics Review Board in Linköping, Sweden, and the Åland Health Care Ethics Committee approved the study.

We homogenized tick specimens, extracted total nucleic acids, and reverse-transcribed them to cDNA (9) (Appendix). We also extracted DNA from blood plasma collected at inclusion (at the time of the tick bite) and at follow-up 3 months later from participants bitten by a S. ixodetis–positive tick. We detected S. ixodetis in ticks and plasma using a species-specific TaqMan real-time PCR targeting a 170-bp fragment of the RNA polymerase β subunit (10), then performed nucleotide sequencing and verification with BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). We analyzed all S. ixodetis–positive ticks for the presence of nucleic acid from Borrelia burgdorferi sensu lato, B. miyamotoi, tick-borne encephalitis virus, Anaplasma phagocytophilum, Neoehrlichia mikurensis, Babesia spp., and Rickettsia spp. by real-time PCR (11) (Appendix Table 1).

The study consisted of 2,735 I. ricinus ticks: 1,823 nymphs, 689 adults, 118 larvae, and 105 ticks for which we could not determine developmental stage (Table 1). The blood feeding time range was <24 to >72 hours (Appendix Table 2). Most (n = 1,156) ticks were collected in southcentral Sweden, followed by the Åland Islands (n = 950), southernmost Sweden (n = 605), and northern Sweden (n = 24) (Table 2). In total, 72 ticks (2.6%) were positive for S. ixodetis; of those, 60 showed >99.42% sequence identity with S. ixodetis strain sHm (GenBank accession no. AP026933.1). All plasma samples from study participants bitten by a S. ixodetis–containing tick yielded negative PCR results.

We found no statistical differences in the prevalence of S. ixodetis among the different developmental stages of the ticks (Table 1); however, we found a statistically significant difference in geographic distribution of S. ixodetis–positive ticks between southcentral Sweden and southernmost Sweden (p = 0.0004), southcentral Sweden and the Åland Islands (p = 0.00005), and southernmost Sweden and the Åland Islands (p<0.00001). No ticks collected in northern Sweden were positive for S. ixodetis.

In total, 26 (36%) of the S. ixodetis–positive ticks contained >1 additional pathogens, mainly species in the B. burgdorferi s.l. complex (n = 14), of which most were B. afzelii. Coexistence of S. ixodetis and other tickborne pathogens was less common. Fifteen of the ticks with coexisting pathogens were nymphs, and 11 were adult females. Three ticks carried 3 pathogens: S. ixodetis, B. burgdorferi s.l., and N. mikurensis (Appendix Table 2). We found no statistically significant difference between observed frequency (17%) and expected frequency (19%) for coexistence between S. ixodetis and B. burgdorferi s.l. bacteria, suggesting that coexistence does not appear more often than expected by chance based on the prevalences of the individual pathogens (p = 0.738). We found statistically significant differences in geographic distribution of ticks with coexistence, regardless of pathogens, between southcentral Sweden and southernmost Sweden (p = 0.0004), southcentral Sweden and the Åland Islands (p = 0.04), and southernmost Sweden and the Åland Islands (p<0.00001).

The overall prevalence of S. ixodetis in I. ricinus ticks removed from humans was 2.6%, with statistically significant differences in distribution between geographic areas. S. ixodetis–positive ticks were found in all developmental stages, including larvae, suggesting transovarial transmission of the bacterium. The prevalence in our study is consistent to previous studies showing a prevalence of 0.4%–3% in questing ticks (1,2). The number of ticks analyzed from northern Sweden was low, which can explain the negative results in this area. However, because of climate changes and raised temperatures, more suitable habitats for ticks and hosts might result in the spread of S. ixodetis–infected ticks into new areas (12). Few S. ixodetis–positive ticks were detected in the Åland Islands, an area with high tick density, highly endemic for B. burgdorferi s.l. and tick-borne encephalitis virus (9,13). That finding indicates that endemic areas for one pathogen may not be endemic for others.

The negative PCR results in plasma from participants who did not necessarily show symptoms related to tickborne diseases were consistent with a previous study (3). In that study, patients with influenza-like symptoms and erythema migrans also showed negative results in blood after being bitten by a tick carrying S. ixodetis. Even though S. ixodetis bacteria have been detected in blood (4), knowledge of the optimal time of sampling or the frequency of the pathogen in blood is limited. The inclusion sample was collected only days after the tick bite, perhaps before a potential bacteremia, and the follow-up sample was collected 3 months after a potential acute infection, which may be the reason for the negative outcome. No time lag for transmission has been established as of November 2025; because S. ixodetis is located in the midgut of the tick, we hypothesize that the time lag could be similar to that of Borrelia spp. transmission, 24–48 hours (9).

Most of the ticks carrying >1 pathogen contained both S. ixodetis and B. burgdorferi s.l. bacteria. That finding was not surprising because Borrelia spp. bacteria, mainly B. afzelii, are the most common pathogens found in questing ticks in Europe (14).

Our study used samples collected >15 years ago; although our findings might not reflect the current situation, they provide relevant epidemiologic insights into the prevalence and geographic distribution of S. ixodetis. Although we were unable to sequence all S. ixodetis–positive ticks, mainly because of high cycle threshold values (>35), we believe the real-time PCR findings are trustworthy because primers and probe are designed for species-specific detection.

Further studies are needed to expand our understanding of prevalence, geographic distribution, and the possibility of co-infection of tickborne S. ixodetis. Our results indicate low risk of being infected by S. ixodetis after a tick bite; however, spiroplasmosis and co-infections should be considered as differential diagnoses in cases of fever after a tick bite (4,8).