Patterns of Tick-Borne Encephalitis Virus Infection in Rodents in Slovenia

Abstract

Tick-borne encephalitis virus (TBEV) is the most important causative agent of arboviral infection in Europe, causing neurologic symptoms. The incidence of the disease has greatly increased over the past decades, and in the meantime, some changes in spatial distribution of TBE cases have been observed. Therefore, it is important to recognize the distribution of endemic areas, to use preventive measures successfully. In this study, rodents from all over Slovenia were evaluated as suitable sentinels for TBEV distribution. Rodents from four species (Myodes glareolus, Apodemus flavicollis, Apodemus sylvaticus, and Apodemus agrarius) were screened for the presence of TBEV antibodies with immunofluorescence assay; the antibodies were detected in 5.9% of sera. The prevalence of infection varied according to the rodent species and according to the region of trapping. Select rodents were also screened for the presence of TBEV RNA in several organs. Both analyses showed higher rate of infection in bank voles, which also produced higher titers of anti-TBEV antibodies and a higher TBEV RNA viral load compared with mice. The regional prevalence of infection in rodents can be correlated with the incidence of disease. Molecular results indicate that the virus can be detected in the organs of the rodents for longer periods, indicating prolonged infections of the rodent hosts by the virus. Rodents can therefore be used as a useful indicator of the circulation of TBEV in an area.

Introduction

T ick-borne encephalitis virus (TBEV) is the causative agent of the most prevalent arboviral infection in Europe. Three subtypes have been identified: European (TBEV-Eu), Far Eastern (TBEV-FE), and Siberian (TBEV-Sib) (Ecker et al. 1999). The vector for transmission of TBEV-Eu is the tick Ixodes ricinus, whereas Ixodes persulcatus is the vector for transmission of the other two subtypes (Gritsun et al. 2003, Suss 2003).

The distribution of TBE is focal and the endemic risk areas according to Pavlovsky (1964) arise as a consequence of favorable complex of flora, fauna, and abiotic environment. The hosts in the concept of natural foci are carriers of the pathogen, without necessarily contributing to the transmission of the pathogen. Many different vertebrates have been implicated in both the maintenance and circulation of TBEV. Large mammal hosts, such as deer, are accidental hosts of the virus and they do not promote transmission of the virus but they do have an important role in the maintenance of the vector and, consequently, TBEV in the endemic areas (Carpi et al. 2008, Pugliese and Rosa 2008). The major role in TBEV circulation in Central Europe seems to belong to Apodemus flavicollis and Myodes glareolus, not only because they are abundant in the regions where TBE incidence is high, but also because they are excellent hosts for both nymphal and larval stages of the tick. They, as other rodent species, are vital, because they promote TBEV transmission by viremic or nonviremic transmission (Labuda et al. 1996, 1997, Randolph et al. 2002, Rosa et al. 2007).

Slovenia is a very heterogeneous area due to different abiotic (climatic, tectonic, edaphic, orographic, and lithologic) conditions, which consequently lead to various ecological conditions across the country. Tick-borne encephalitis is a notifiable disease in Slovenia and it has one of the highest average annual incidences in Europe (14 cases per 100,000 inhabitants per year) (Arnež and Avšič-Županc 2009). The incidence varies in different regions in Slovenia, ranging from 0 in the coastal region to 42 per 100,000 inhabitants in the northern and central part of the country (www.ivz.si). The predominant tick species in Slovenia is I. ricinus (Knap et al. 2009). The TBEV infection rate among ticks was previously shown to vary from 0 to 1.9%, depending on the region of sampling and the year of investigation (Durmisi et al. 2011).

The aim of the present study was to establish a possible correlation between TBE incidence and TBEV prevalence in rodents in Slovenia, thereby also providing a tool that can be used as sentinels for TBEV monitoring. Further, the study was focussed to ascertain any likely variation in viral load between rodent species and different tissues examined.

Materials and Methods

Trapping of rodents

Rodents were captured between 1990 and 2009 all over Slovenia, but not on all sites every year. We used live and Elliot special and Sherman traps. We set the traps overnight in lines of 30 traps at the distance of 5–7 m, if possible, over a rodent hole or near a tree trunk. Traps were baited with mixed sardines and oatmeal. Cotton fiberfill was placed in the traps to provide nesting material and to reduce trap-associated deaths. Each night, we set 150 traps. The trapped animals were brought to the laboratory, where species was determined and weight, size, and sex were recorded. The animals were sacrificed, and blood and sera samples were collected along with the internal organs (heart, lung, liver, kidney, spleen, bladder, and brain) and stored at −70°C until analysis. In addition to A. flavicollis and M. glareolus, 18 other rodent and insectivore species were captured.

Screening of the rodent sera

The sera were diluted to a ratio of 1:10 and tested using indirect immunofluorescence assay on spot slides containing Vero E6 cells infected with TBEV (strain: Ljubljana I, U27494). In brief, the diluted serum was allocated on the slides. The slides were incubated for 30 min at room temperature and washed in phosphate-buffered saline (PBS), and afterward, anti-mouse IgG conjugate (A7506; Sigma) dilution at the ratio of 1:128 was added. Another incubation at room temperature followed. Prior to examination under a fluorescent microscope (Eclipse 80i; Nikon), the slides were again washed in PBS and dried. The sera were considered positive when the characteristic TBEV cytoplasmic fluorescence was observed. The rodent sera samples that were previously established as positive and PBS were used as positive and negative controls. The sera from seropositive rodents were further diluted to establish the end-point titer.

RNA isolation and PCR screening

Rodents (698 animals) collected from 2000 to 2008 were screened for the presence of TBEV RNA. The presence of the viral RNA was established in either the spleen or kidney samples. Total RNA was extracted from either rodent sample with Trizol^® Reagent (Invitrogen Life Technologies™) according to the manufacturer's instructions. Extracted RNAs were pooled in groups of 10. Viral RNA was detected in pools, using a previously described probe-specific real-time PCR protocol, which amplifies a 67-bp-long segment of the 3′ noncoding region of the TBEV genome (Schwaiger and Cassinotti 2003). Synthetic wild-type RNA transcripts were produced to be used as quantitative calibrators. Internal control and standards were prepared as described by Drosten et al. (2006). Pools in which TBEV RNA was detected were further analyzed and the infected animal was determined by screening RNA isolates from the individual animals. Afterward, RNA was isolated from the remaining organs and tissues of the selected infected animals: lung, liver, heart, brain, and blood clot (Table 2). Viral load was determined in all of the isolated samples using the method described earlier.

Statistical analysis

Statistical analysis was performed using PASW statistics (version 18.0.0; SPSS, Inc.). The relationship between the incidence of TBEV and the status of infection in rodents was evaluated using the χ ² test. Pearson's coefficient was calculated to establish correlation. Mann–Whitney U test was used for intergroup comparison. p-Values of <0.05 were considered to be statistically significant.

Results

Although other species of rodents and insectivores were captured during our study, only yellow-necked mice (A. flavicollis), wood mice (Apodemus sylvaticus), striped field mice (Apodemus agrarius), and bank voles (M. glareolus) were caught in sufficient numbers to merit any statistical analysis. The presence of antibodies against TBEV was surveyed in all of the mentioned rodent species with available serum samples. Specific antibodies were detected in 83 of 1401 (5.9%) rodents. The number of animals with detected antibodies against TBEV varied according to rodent species (p<0.05). The highest overall prevalence of infection was established in bank voles (12.5%), followed by the wood mice, yellow-necked mice, and striped field mice (9.6%, 2.4%, and 3.9%, respectively) (Table 1).

Table 1.

Prevalence of Tick-Borne Encephalitis Virus Infection in Rodents Captured in Slovenia, Established by Specific Anti-Tick-Borne Encephalitis Virus Antibody Detection Using IFA

Species	Prevalence of infection ^a
Apodemus flavicollis	33/820 (3, 9%)
Apodemus sylvaticus	7/66 (9, 6%)
Apodemus agrarius	4/160 (2, 4%)
Myodes glareolus	39/272 (12, 5%)
Total	83/1401 (5, 9%)

The numbers of infected and tested individuals by species are provided; prevalence of infection is given within parentheses.

IFA, immunofluorescent assay.

The presence of antibodies in rodents was also analyzed according to regions and compared with the incidence of disease (Fig. 1). The statistical analysis showed a significant correlation between the two variables (r=0.67, p<0.05), confirming that the regions with higher prevalence of TBEV in rodents had higher incidence of TBE (Fig. 2). We also proved that the incidence of disease in smaller units—municipalities—differed according to the information whether or not the antibodies were detected in the rodents in the region. A statistically significant lower incidence was found in areas where no TBEV antibodies were detected in rodents (χ ² test, p<0.05).

FIG. 1.

Map of municipalities in Slovenia, showing sites where tick-borne encephalitis virus (TBEV) was detected in rodents (dots) and municipalities (gray) where rodents were captured. Smaller map is the TBE incidence map of Slovenia, where darker colors represent areas with higher TBE incidence.

FIG. 2.

Correlation between the prevalence of infection in rodents (specific TBEV antibody prevalence) and the incidence of disease in Slovenian health protection agency regions.

Comparing the antibody titers in the three rodent species, we found that the bank voles had significantly higher concentrations of antibodies compared with wood mice and yellow-necked mice (χ ² test, p<0.05; Table 2).

Table 2.

Selected Rodents and Their Assigned End-Point Tick-Borne Encephalitis Virus Antibody Titers, Average Detected Viral Load in Specific Rodents, and Tissues in Which Tick-Borne Encephalitis Virus was Detected

				Tissue
	Rodent species	Sex	IFA end-point titer	Spleen	Kidney	Lungs	Liver	Heart	Serum	Blood clot	Brain	Average viral load (copies/mL)
1	A. flavicollis	M	1:10	+	−	−	−	−	−	−	−	2.55E+03
2	A. flavicollis	M	1:40	−	−	−	−	−	−	+	−	6.38E+02
3	A. flavicollis	F	1:40	+	−	−	−	−	^a	−	+	3.70E+05
4	A. flavicollis	F	1:40	−	−	−	−	−	−	−	+	6.48E+00
5	A. flavicollis	M	1:10	+	−	−	−	−	−	−	^a	1.18E+03
6	A. flavicollis	M	0	+	−	+	−	−	^a	^a	−	5.11E+01
7	A. flavicollis	M	1:10	+	−	+	−	−	−	−	−	1.23E+05
8	A. sylvaticus	M	1:80	+	−	−	−	−	−	−	+	1.49E+03
9	A. sylvaticus	M	1:80	+	−	−	−	+	−	+	−	3.70E+03
10	M. glareolus	F	1:80	+	+	+	+	+	+	+	+	1.53E+08
11	M. glareolus	F	1:640	+	−	+	−	−	+	^a	+	3.88E+03
12	M. glareolus	M	1:1280	+	+	+	+	+	+	^a	+	1.06E+06
13	M. glareolus	F	1:640	+	+	+	+	+	+	^a	+	1.77E+06
14	M. glareolus	F	1:640	+	+	+	+	+	−	+	+	8.23E+04
15	M. glareolus	F	0	+	+	+	+	+	+	+	^a	3.22E+08

No sample available.

Using quantitative one-step real-time RT-PCR, the viral load was measured in the tissues of rodents in which TBEV RNA was detected during preliminary screening. The study revealed that the median viral load detected in M. glareolus was significantly higher than the viral load detected in A. flavicollis and A. sylvaticus (p<0.001; Fig. 3). The viral load ranged from 0 to 2.5×10⁹ copies/mL in M. glareolus and from 0 to 2.94×10⁶ in Apodemus rodents. A significant variation of viral load was observed between various examined tissue samples (p<0.05). In two of the three rodent species, the highest virus load was measured in spleen and brain samples (Fig. 4). The virus was usually detected in the majority of the surveyed tissues of M. glareolus, but this was not the case in Apodemus mice. In A. flavicollis and A. sylvaticus, TBEV was usually detected in the spleen and brain tissue (Table 1).

FIG. 3.

Comparison of the average TBEV RNA viral load in different rodent species.

FIG. 4.

TBEV viral load in three species of rodents measured in different tissues.

Discussion

TBE is the most important tick-borne viral zoonosis in Europe, where more than 8000 cases were reported annually in the past two decades (Suss 2008). The incidence of the disease increased by 400% from 1974 to 2003 and the disease also spread to the regions where this viral infection was previously unknown (Randolph and Šumilo 2007, Suss 2008). Slovenia is one of the regions in Europe with the highest TBE incidence. It is therefore important to recognize the high-risk areas to channel there the execution of preventive measures.

In the past years, many studies have been made on TBEV prevalence in ticks to recognize the risk areas (Suss et al. 1997, Suss et al. 2006, Golovljova et al. 2008, Carpi et al. 2009, Barandika et al. 2010, Durmisi et al. 2011, Gaumann et al. 2010). What all these studies have in common is that the number of screened ticks had to be enormous to find at least some TBEV. In the study performed in Slovenia during 2005 and 2006, almost 4800 ticks were tested for the presence of viral RNA. Although the ticks were pooled, around 700 RNA isolations were performed and followed by PCRs. The overall prevalence confirmed in this study was 0.47%. The study confirmed the circulation of the virus in the areas with high TBE incidence (Durmisi et al. 2011), but the amount of work and time devoted to establish the presence of the virus in the area was enormous compared with the work invested in determining the prevalence in rodents. One of the locations (Rakovnik) was sampled for ticks and rodents in the same year; 458 nymph and adult Ixodes ricinus ticks were sampled, pooled, and investigated for the presence of TBEV virus by PCR. The established infection rate was 0.9% (Durmisi et al. 2011). During the same year, 24 rodents (6 A. flavicollis, 3 A. sylvaticus, and 12 M. glareolus) were captured in the same location, and in 12.5%, specific TBEV antibodies were detected by the immunofluorscent assay. Both studies showed that the virus circulated in the area, but the time and effort invested in the second approach (the rodent survey) was considerably lower.

According to our study, the prevalence of TBEV antibodies in rodents is a good indicator for the presence of TBE foci in the area (Fig. 1). It can be also a predictive measure of the intensity of TBE foci, as the prevalence of antibodies in rodents correlates with the known incidence of TBE in the searched areas (Fig. 2). Although the amount of rodents has to be significant to find a correlation, even a relatively small number of rodents can indicate the presence or absence of the virus in an area, but a high variation in sample size and a relatively long period, spanning over several years, of capturing rodents should not be considered without reservations in coming to definite conclusions. Trends though can nonetheless be seen. Additionally, temporal analysis was not possible in our study because of the fact that the sampling was not conducted consistently in the same area over a longer period of time. Therefore, it was not possible to establish whether a certain year with the high prevalence of specific TBEV antibodies in rodents was indeed connected with the high incidence of TBEV in the particular year or whether the high prevalence of TBEV antibodies in rodents is just a consequence of sampling in a high-risk area.

Another important observation in our study was that M. glareolus rodents are more often infected with TBEV then the Apodemus sp. rodents. This is in concordance with the few previous studies conducted in Europe in the past, although it has been implied that the rate of infection in a specific species might depend on the year (Kozuch et al. 1995, Weidmann et al. 2006). No such analysis could be made in our study, because not the same sites were sampled throughout the duration of the study. The prevalence of infection seems to be higher in bank voles, whereas some previous studies indicated that the tick burden is higher on A. flavicollis compared with M. glareolus (Humair et al. 1993, Kiffner et al. 2011). Also, M. glareolus develops resistance to ticks (Dizij and Kurtenbach 1995). All of these factors should lead to higher prevalence of infection in mice, but both the epidemiological studies in naturally infected rodents as well as the studies on experimentally infected animals showed a different picture (Heigl and von Zeipel 1966, von Zeipel and Heigl 1966, Kozuch et al. 1990, Labuda et al. 1996, 1997).

In previous studies, when animals were infected in the laboratory, it was confirmed that M. glareolus rodents produce higher viremia and higher antibody titers in comparison to the rodents from the Apodemus species (Heigl and von Zeipel 1966, von Zeipel and Heigl 1966, Kozuch et al. 1990, Labuda et al. 1993). Our study established higher TBEV RNA values and anti-TBEV antibody titers in M. glareolus rodents compared with the two analyzed Apodemus species. All these data indicate that M. glareolus rodents might be better hosts for systemic transmission of TBEV. On the other side, the importance of M. glareolus rodents in TBEV circulation still needs to be considered with care, especially in view of the cofeeding experiments, which showed that the most important host for virus transmission is A. flavicollis (Labuda et al. 1997). As for the role of both species in the maintenance of TBEV in natural foci, further studies are needed.

In our study, we confirmed that a very high TBEV RNA viral load can be above all found in the spleen and often in the brain even when viremia is not detectable anymore. In one A. flavicollis, the viral RNA was detected in high concentrations (10⁴) in the brain only. Further, we established the presence of viral RNA in 8 of 14 tested animal blood clots and/or sera samples (Table 2). This is a high percentage of viremic animals, especially considering the previously established short duration of viremia (Heigl and von Zeipel 1966). This might indicate that the more advanced modern methods are able to detect viral RNA for longer periods of time. Therefore, it is likely that the duration of viremia is actually longer than previously believed and viral RNA can also be confirmed even after the detection of TBEV-specific antibodies. A study performed by Achazi et al. (2011) also confirmed the presence of the viable TBE virus in experimentally infected Microtus arvalis voles 100 dpi in some organs, indicating long-term presence of the virus in the mentioned rodent species (Achazi et al. 2011). Although we cannot estimate the time of infection from our study of naturally infected animals, it is likely that the viremia lasts longer than previously assumed and that TBEV can be detected in organs long after the infection; the same has also been established in other recent studies (Achazi et al. 2011, Tonteri et al. 2011). Some studies also suggest that small mammals might provide a means for virus overwintering (Kozuch 1963, Tonteri et al. 2011). This is indicated by long-term virus presence in the brain samples of these animals, but further studies are still needed to elucidate these findings. All of the recent findings from our and other studies imply that rodents play an even more important role in the circulation of the TBEV in natural foci as previously believed.

The importance of rodents in the transmission of the virus has been firmly established in the past decades, but their role in the maintenance of the virus in nature still raises a lot of questions. The results from our study revealed that rodents can be used for confirming the circulation of TBEV in an area. This approach produces comparable or even better results than investigations of ticks and, most probably, it is more cost effective and less time consuming and labor intensive.

Footnotes

Acknowledgments

The authors thank everybody who helped with the field work through the years and Darja Duh for establishing the molecular methods.

Disclosure statement

No competing financial interests exist.

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