Rodents play a role as reservoir hosts for tick-borne encephalitis virus (TBEV). We trapped 77 voles of the genus Microtus and tested their blood using an enzyme-linked immunosorbent assay for the presence of antibodies against TBEV. We detected TBEV antibodies in three adult individuals (4%). Voles might play a role in the circulation of TBEV.

Tick-borne encephalitis (TBE) is one of the most important flaviviral human diseases in Europe, and is of considerable public health importance, with more than 2,600 cases reported in Europe and 283 reported cases in Poland in 2016 (European Centre for Disease Prevention and Control 2018). In Europe, tick-borne encephalitis virus (TBEV) is transmitted by the bite of infected Ixodes ricinus ticks in central and western Europe or Ixodes persulcatus ticks in eastern Europe (Suss 2011).

Small sylvatic rodents (e.g., Myodes glareolus, Apodemus spp.) in Europe are also important hosts for the larval and nymph stages of I. ricinus (Mihalca and Sándor 2013) and are known to carry persistent latent TBE infections (Zöldi et al. 2015), likely helping to maintain TBE virus in the wild (Weidmann et al. 2006; Knap et al. 2012; Grzybek et al. 2018).

Identification of hosts that carry zoonotic pathogens is a necessary initial step in understanding the disease threat arising from wild animals and is crucial for mitigation of relevant human diseases that are zoonotic (Grzybek et al. 2019). Therefore, we assessed prevalence of TBEV antibodies in one of the most common rodent genera in Poland and throughout Europe, the voles of the genus Microtus.

We followed all relevant rules governing experimental conditions and care of laboratory and wild animals required by the European Union and the Polish Law on Animal Protection. Our project was approved by the First Warsaw Local Ethics Committee for Animal Experimentation (no. 148/2011 and 406/2013).

The study site was located in the Mazury Lake District region in the northeastern corner of Poland (Urwitałt, near Mikołajki; 53°48′50.3″N, 21°39′07.2″E). The abandoned, previously intensively cultivated fields in the study site had become vegetated with mixed scrub and long grass (Tołkacz et al. 2017, 2018).

We trapped voles in August 2013 during the late summer season. Voles were live-trapped using mixed bait comprising apple, carrot cucumber, and grain. Two traps were set every 10 m along the trap lines at dusk. The following morning traps were checked and closed to prevent animals from entering during daytime and to avoid losses from excessive heat from exposure of traps to direct sunlight. Traps were then rebaited and reset on the following afternoon. All traps were also closed during periods of intensive rainfall.

The necropsies were carried out under terminal isoflurane (Baxter, Deerfield, Illinois, USA) anaesthesia. Animals were weighed to the nearest gram, and total body length and tail length were measured in millimetres. Animals assigned to three age classes (juveniles, young adults, and adults), based on body weight and nose-to-anus length together with reproductive condition (scrotal, semiscrotal, or nonscrotal testicles for males; lactating, pregnant, or receptive for females; Pawelczyk et al. 2004; Tołkacz et al. 2017, 2018). Initially, vole species were distinguished based on their appearance (fur color: gray and yellowish hair with brighter belly and legs, M. arvalis; brown-reddish fur with dark belly and legs, M. agrestis; dark brown fur with dark belly and black legs, M. oeconomus) and on body weight and body measurements. Finally, we confirmed the species identity of each by examination of the lower molars M1 and M2 and the second upper molar M2, especially to distinguish between juvenile individuals of M. oeconomus and M. agrestis (Pucek 1981).

Blood samples were collected directly from the heart using a sterile 1.5 mL syringe immediately after death from over-exposure to anesthetic. Samples were centrifuged at 1.9 × G for 10 min using an MPW High-Speed Brushless Centrifuge (MPW, Warsaw, Poland). Serum was collected and stored at –80 C until the samples could be analyzed.

We used an enzyme-linked immunosorbent assay for quantitative determination of IgG antibodies against the TBE virus using IMMUNOZYM® FSME (TBE) IgG All Species (PROGEN Biotechnik GmbH, Heidelberg, Germany), according to the manufacturer's instructions. The optical density was measured at a wavelength of 450 nm (0.1 s) using a Victor 3 Multilabel Plate Counter (Perkin Elmer, Waltham, Massachusetts, USA). Calculation of anti-TBE-IgG concentration was performed using the reference curve. The optical density at 450 nm was transformed into Vienna units (VIEU). Samples were scored TBE-IgG antibody-negative if VIEU/mL was <63; borderline if it was 63–126; and positive if it was >126. Seroprevalence values (percentage of seropositive animals) are given with 95% confidence intervals in parenthesis calculated by bespoke software based on the tables of Sokal and Rohlf (Sokal and Rohlf 1995).

In total, we analysed 77 vole sera. The overall seroprevalence of TBEV was 3.9% (1.0–11.8), based on three positive samples. These were all adult animals, one male and one female M. arvalis, and one male M. oeconomus. No M. agrestis were found to be seropositive (Table 1). Our findings confirmed that both M. arvalis and M. oeconomus might participate in the circulation of TBEV in nature and the observation that all three positive animals were adults is consistent with well-documented observations that the chances of acquiring infection increase with age of the host for parasites and other infectious organisms that cause chronic infections (Grzybek et al. 2015). Detection of antibody indicates that the rodent has been exposed to the virus, but cannot identify whether the infection is current, especially as the rodents were adults and might have been exposed in the past. Future studies should explore whether TBE virus can be directly detected in these species of voles, to determine the role of Microtus spp. in TBEV circulation.

Table 1

Seroprevalence of tick-borne encephalitis virus in three Microtus spp. in Poland determined by use of an enzyme-linked immunosorbent assay for antibodies against tick-borne encephalitis virus. Prevalence is reported in percentages. CI = confidence interval.

Seroprevalence of tick-borne encephalitis virus in three Microtus spp. in Poland determined by use of an enzyme-linked immunosorbent assay for antibodies against tick-borne encephalitis virus. Prevalence is reported in percentages. CI = confidence interval.
Seroprevalence of tick-borne encephalitis virus in three Microtus spp. in Poland determined by use of an enzyme-linked immunosorbent assay for antibodies against tick-borne encephalitis virus. Prevalence is reported in percentages. CI = confidence interval.

Our results, albeit based on the detection of only three infected animals in the total sample of 77 screened voles, nevertheless provide preliminary data to further explore the full range of potential rodent hosts. Voles of the genus Microtus are common and abundant rodent species in Europe, and in peak seasons their numbers can exceed millions of individuals in suitable habitats (Forbes et al. 2014), so a prevalence of just 3–4% could be reflected in many thousands of infected animals at national levels in countries located in endemic regions. Therefore, future studies should search for the TBE virus within greater number of individuals to confirm their role as reservoirs of the virus.

We thank the University of Nottingham, Warsaw University, University of Life Sciences in Lublin, and the Medical University of Gdańsk for financial support. J.M.B. was supported by the Royal Society, the British Ecological Society, and the Grabowski Fund. A.B. was supported by the Polish State Committee for Scientific Research and British Council's Young Scientist Programme. M.G. was supported by the Ministry of Science and Higher Education in Poland, Fellowship for Outstanding Scientists (428/STYP/11/2016). M.G. thanks Alicja Rost and Ewa Zieliniewicz for their assistance in laboratory.

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Author notes

5These authors contributed equally to this work