In this study, we investigated small mammals, their ticks and questing I. ricinus for the occurrence of DNA of CNM in areas with sympatric I. ricinus and D. reticulatus populations. Rodents have been found to harbour CNM and this group of animals has thus been suggested as reservoir hosts [12–14, 28–31]. The high infection rates of yellow-necked mice and bank voles in the present study corroborate this hypothesis. DNA of CNM was also detected in a striped field mouse, but the number of animals (n=3) was too low to draw a conclusion from this finding. We found the highest percentage of infected animals in August, when the developmental stages of both I. ricinus and D. reticulatus are most active [4, 32]. Rodents were still infected in October and November, but DNA was not detected in the animals in spring. Hence, it may be that the rodents cannot carry the infection over the winter. All the same, the number of animals caught in spring was low, and to prove this hypothesis, more systematic studies with a larger number of animals are needed.
We compared different organs as material for detection of DNA of CNM in epidemiological studies. During experimental infection of rats, infection was not observed after 15 days in blood, spleen and liver, weak infection in the spleen after 30 days, and infection in all three sample types after 60 days. Histologically, the agent was recognized in the spleen sinus endothelial cells forming intracellular inclusions in the cytoplasm. Consequently, blood does not seem to be an ideal target material, because infection may be detectable only after more than 30 days, assuming the course of infection in mice is similar to the one observed in rats. Experimentally infected mice did not have detectable CNM-DNA in their spleen after 10 days . Our finding that blood samples were negative when other organs were positive may therefore reflect different time points of infection , but further experimental infections of mice are lacking thus far. According to our own results and a comparison with experimental infection in the literature, we conclude that spleen and kidney are the best organ material for detection of DNA of CNM in epidemiological studies involving rodents. Skin seems unsuitable for the detection of DNA of CNM. At any rate, all results taken together strongly argue for a systemic course of infection [12, 13], which is supported by observations in human patients. Fehr et al. and von Loewenich et al. detected DNA of CNM in peripheral blood samples of human patients. Peková et al. were able to show the agent in patients’ granulocytes in transmission electron microscopy. In spite of this, the agent has as yet not been observed in blood smears.
In comparison to previous studies from other areas in Europe, the prevalence rates in the small rodents in the present study were high and similar to the initial investigations in Japan where 7 out of 15 wild caught Norway rats were infected . In Sweden, between 4 and 10% of rodents (bank voles, field voles (Microtus agrestis), wood mice (A. sylvaticus) and yellow-necked mice), but no shrews were infected. In different sites in Sweden the prevalence ranged from 0% to 12.5% . The fact that blood was used in that study may account for the lower prevalence compared to our study. In the Netherlands, spleens were investigated and prevalence was higher: 21.7% of wood mice, 25% of common voles, and 11.4% of bank voles, but none of the two yellow-necked mice were infected. Shrews were not infected either . Our findings strongly support the hypothesis that rodents may be competent reservoir hosts and that they may play an important role in the endemic cycle of CNM . In line with the findings in Sweden and the Netherlands, we did not detect CNM in any insectivore species [13, 29]. With a prevalence about twice as high as in questing ticks a reservoir function of the rodents seems highly likely at least for the season of the year when ticks are active.
Altogether 6.5% of host-attached I. ricinus ticks (larvae and nymphs) were positive, which is lower in comparison to questing ticks. When interpreting this result, it has to be taken into account that the questing ticks and the rodents and their ticks were from different years (2009: questing ticks; 2010/11: rodents and their ticks). Tick-host-pathogen cycles are influenced by a lot of factors, many of which are still largely unknown and may account for differences in prevalence rates at different time points. Variations have, for example, been shown in the prevalence over several years with A. phagocytophilum in ticks .
Previously investigated questing D. reticulatus collected from vegetation had no detectable DNA of CNM in them [13, 34]. All positive developmental stages of D. reticulatus from the present study were from bank voles that were positive for CNM in the blood; therefore it may be assumed that the detection of CNM-DNA represents the blood meal. The same can be said for the I. ricinus larvae from the small mammals, as they were all engorged. It is not known yet whether transovarial transmission occurs, but 55 larval pools from questing larvae were negative . Unfortunately, we did not have questing larvae available for the present study. It seems likely that CNM cannot be transmitted transovarially and experimental evidence for transstadial transmission is also lacking, thus, an experimental setting to uncover the epidemiological transmission cycle is needed.
Average prevalences of CNM in questing I. ricinus or I. persulcatus ticks in several Eurasian countries range from 0% to 16.7% [9, 13, 14, 22, 23, 35–42]. Thereby, the prevalence rates in I. ricinus seem to average around 6%, whereas the prevalence in I. persulcatus seems to be lower, reaching up to 3.8%, but staying in general around or below 1% [23, 30, 43]. Most previous studies used conventional PCR; sometimes combined with sequencing and/or hybridization with oligonucleotide probes for species identification. The increased sensitivity of real-time PCR used in the present and another study may also account for the increased detection rate of DNA of CNM in these studies [13, 23]. In our investigated area, the rodents and ticks seem to provide a very efficient system for CNM to thrive on and to develop such high prevalence. Whether this persists over time needs further systematic and longitudinal investigations in both ticks and host species.
Co-infections of CNM in ticks in our study with R. helvetica, A. phagocytophilum and B. microti may be explained by using the same (suggested) reservoir host animals [3, 44, 45]. No difference was found in prevalence between males and females in infection rates, which is in line with our findings . Data from northern Italy found females significantly more often co-infected with more than one pathogen , whereas in our study, there was a tendency in the opposite direction comparing female ticks with male ticks.