Wild Rodents and Novel Human Pathogen Candidatus Neoehrlichia mikurensis, Southern Sweden

We examined small mammals as hosts for Anaplasmataceae in southern Sweden. Of 771 rodents, 68 (8.8%) were infected by Candidatus Neoehrlichia mikurensis, but no other Anaplasmataceae were found. Candidatus N. mikurensis has recently been found in human patients in Germany, Switzerland, and Sweden, which suggests that this could be an emerging pathogen in Europe.

T he rickettsial family Anaplasmataceae contains several tick-transmitted bacteria of considerable medical and veterinary importance, including known human pathogens such as Anaplasma phagocytophilum and Ehrlichia chaffeensis. Recently, a case of human disease in a Swedish patient with recurrent fever episodes caused by another member of Anaplasmataceae was reported. The infectious agent was shown by PCR amplifi cation of the 16S rRNA gene to be identical to Candidatus Neoehrlichia mikurensis (1). This published report was followed by the descriptions of a patient from Switzerland (2) and 2 German patients with severe febrile illness caused by Candidatus N. mikurensis (3). This member of the family Anaplasmataceae was fi rst discovered in ticks in the Netherlands in 1999; it was originally designated as an Ehrlichia spp.-like species (4). Similar organisms were later detected in Russia (5) as well as in other parts of Europe (6). In 2004, a closely related organism was detected in Japan in wild rodents and was named Candidatus N. mikurensis (7). In the United States, a similar organism (Candidatus N. lotori) has been detected in wild raccoons (8). The distribution and reservoir hosts of Candidatus N. mikurensis in Europe are largely unknown. It has, however, been detected in a blood sample from a single bank vole in Italy (9), indicating that rodents are possible reservoir hosts.
In this study, we collected blood samples from 7 small mammal species (rodents and shrews) at 5 localities in southern Sweden to investigate their role as hosts for Anaplasmataceae and to determine whether these small mammals serve as natural hosts for Candidatus N. mikurensis.

The Study
From May through October 2008, we trapped small mammals at 5 sites in southern Sweden ( Figure 1). Animals were captured with live traps and released after sampling. Our primary objective was to obtain samples from bank voles, Myodes glareolus, but other species were also sampled when captured. A total of 829 animals of 7 species were caught (Table). Approximately 20 μL of blood was taken from each animal. A nested PCR was performed with primers for Anaplasmataceae specifi c for the 16S rRNA gene (10).
All amplifi ed fragments were sequenced, and a BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) search showed that 68 animals were infected by Candidatus N. mikurensis. The primers were chosen to be specifi c for bacteria belonging to the families Rickettsiaceae and Anaplasmataceae (10) The groEL sequence was identical to the isolate from Germany (1,233/1,233 bp) but differed slightly from the isolate from Switzerland (1,072/1,084 bp, 98.9% pairwise identity). A phylogenetic network containing unique 16s rRNA sequences from Candidatus N. mikurensis available at the National Center for Biotechnology Information was made with the program Network 4.5.1.6 (www.fl uxusengineering.com) by using the median-joining algorithm (11) (Figure 2). The prevalence of Candidatus N. mikurensis at the 5 sites ranged from 0% to 12.5%, and Bartonella spp. occurred in 33.7% to 45.0% of the animals (Figure 1). The prevalence of Candidatus N. mikurensis and Bartonella spp. in each host species is given in the Table. Candidatus N. mikurensis occurred in all 4 rodent species, but not in the shrews; the difference in prevalence between rodents and shrews was statistically signifi cant (p = 0.011, by Fisher exact test). Bartonella spp. were found in all rodent species and the common shrew.

Conclusions
In the present fi eld survey of Anaplasmataceae in Swedish rodents, we have amplifi ed 16s rRNA and groEL sequences identifi ed as Candidatus N. mikurensis identical to sequences obtained from humans. This organism has been amplifi ed from humans with febrile illness on ≥4 occasions (1-3), demonstrating that Candidatus N. mikurensis can cause human infections, at least occasionally.
Apart from the fi rst human case, to our knowledge, Candidatus N. mikurensis has not been detected in Sweden. However, we identifi ed Candidatus N. mikurensis at 4 of 5 sites, indicating that this organism is widespread in southern Sweden. Identical or similar sequences have been detected in Ixodes ricinus ticks from several European countries (3)(4)(5)(6)13), showing that it is distributed over a large area of Europe. In contrast, we found no evidence of infection by any other Anaplasmataceae. I. ricinus is the dominating tick species in Sweden, and animals at all our study sites were heavily infested with larvae of this species and occasionally with nymphs (M. Andersson and L. Råberg, unpub. data). Reservoir hosts are essential for several tick-borne pathogens that lack the capacity for transovarial transmission from female ticks to larvae, such  (3) and Switzerland (2). The Japanese reference strain TK4456 (7) showed 99.2% similarity with our sequence, and the North American Candidatus N. lotori strain (12) showed 98.3% similarity.
as A. phagocytophilum (14). Approximately 70% of the tick larvae in Sweden engorge on small mammals, such as voles, mice, and shrews (15). The prevalence of Candidatus N. mikurensis was similar in all investigated rodent species in our study with a mean value of 8.8%. These results are consistent with several studies in Japan, which also found Candidatus N. mikurensis in several different rodent species (7,10). This fi nding indicates that rodents play a role in the natural cycle of Candidatus N. mikurensis in Europe and that rodents are likely to be competent reservoir hosts. In contrast, the closely related Candidatus N. lotori, found in the United States, seems to use raccoons rather than rodents as hosts (12). Whether the European variant of Candidatus Neoehrlichia is capable of infecting animals other than rodents and humans remains to be investigated. We found the organism was completely absent in shrews, which suggests that they might not be competent hosts for these bacteria.
We conclude that Candidatus N. mikurensis is geographically widespread in southern Sweden and that several rodent species, the main source of blood meals for larvae of I. ricinus in Sweden (15), can be infected. The relatively high prevalence in common rodent species suggests that the risk for humans and domestic animals to encounter infected I. ricinus nymphs and adults is substantial. Thus, Candidatus N. mikurensis should be considered when diagnosing bacterial infections associated with tick bites.