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Seroprevalence of TBEV in bank voles from Poland—a long-term approach

Seroprevalence of TBEV in bank voles from Poland—a long-term approach Rodents are known to play a significant role as reservoir hosts for TBEV. During three sequential expeditions at 4-year intervals to three ecologically similar study sites in NE Poland, we trapped bank voles (Myodes glareolus) and then tested their blood for the presence of specific antiviral antibodies to TBEV. The strongest effects on seroprevalence were the extrinsic factors, site of capture of voles and year of sampling. Seroprevalence increased markedly with increasing host age, and our analysis revealed significant interactions among these three factors. Seroprevalence did not differ between the sexes. Therefore, based on the seroprevalence results, the dynamics of TBEV infection differ significantly in time, between local sub-populations of bank voles and with increasing host age. To fully understand the circulation of the virus among these reservoir hosts and in the environment, long-term monitoring is required and should employ a multi-site approach, such as the one adopted in the current study. Introduction rodents are the most important hosts for the immature Rodents, members of the most abundant and diversified stages of I. ricinus . There are five known routes for the mammalian order Rodentia , can pose a significant threat transmission and maintenance of TBEV. Ticks become to the health of humans, livestock, and wildlife because infected when feeding on a viremic host and maintain the 8 9 they are hosts for a wide range of pathogens and in some virus via transstadial or/and transovarial transmission , cases constitute important reservoir hosts for life- or through co-feeding on a non-viremic host . Sexual threatening zoonoses . transmission from male to female ticks is also known to The tick-borne encephalitis virus (TBEV), the causative occur . Consequently, all hematophagous stages of ticks agent of tick-borne encephalitis (TBE), is a zoonotic fla- can transmit the virus to mammalian hosts . Rodents vivirus in the family Flaviviridae that is endemic have been considered to play an essential role in main- throughout the northern Palearctic, spanning an area taining TBEV in nature by carrying persistent latent 13,14 from central and northern Europe and across Siberia to infections . Japan in the far east . TBEV is maintained in nature in a TBEV is the most important causative agent of arboviral cycle that includes tick vectors of the Ixodes persulcatus infections in Europe and is responsible for distressing complex and their vertebrate hosts. The most important neurologic symptoms in patients . Incidence of the dis- 4,5 vector in Central Europe is Ixodes ricinus , and small ease has greatly increased over the past decades, growing into a serious human threat, and changes in the spatial distribution of TBE cases have been concurrently 15,16 observed . Therefore, it is essential to identify the Correspondence: Maciej Grzybek (maciej.grzybek@gumed.edu.pl) Department of Tropical Parasitology, Medical University of Gdańsk, Gdańsk, endemic areas and to monitor the temporal changes of Poland this virus in order to ensure that suitable preventive Department of Parasitology, University of Warsaw, Warsaw, Poland measures are implemented successfully by human Full list of author information is available at the end of the article. These authors contributed equally: Jerzy M. Behnke, Anna Bajer © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Grzybek et al. Emerging Microbes & Infections (2018) 7:145 Page 2 of 8 Table 1 Seroprevalence of TBEV by year, site and host age Year Host age Site N 12 3 2002 Urwitałt 64 0.0 (0.0–23.8) 0.0 (0.0–13.4) 0.0 (0.0–13.4) Tałty 70 4.5 (0.2–22.2) 6.5 (1.9–17.4) 17.6 (5.0–41.7) Pilchy 69 0.0 (0.0–17.6) 11.5 (3.2–30.4) 8.3 (1.5–26.7) Overall by year 203 5.4 (3.7–7.8) 2006 Urwitałt 95 7.7 (1.4–24.6) 4.5 (0.7–17.7) 4.0 (0.2–19.6) Tałty 65 7.7 (1.4–24.6) 0.0 (0.0–22.2) 12.5 (3.5–31.0) Pilchy 67 24.1 (11.5–43.0) 38.5 (16.6–65.8) 76.0 (56.1–89.0) Overall by year 227 18.1 (14.7–21.9) 2010 Urwitałt 86 0.0 (0.0–20.8) 25.0 (12.9–41.9) 11.5 (3.2–30.4) Tałty 96 11.5 (3.2–30.4) 13.0 (3.7–32.4) 19.1 (8.8–36.6) Pilchy 56 11.8 (2.1–35.0) 50.0 (24.3–75.7) 32.1 (23.2–42.6) Overall by year 238 19.7 (16.2–23.8) Overall by age 8.7 (4.4–16.0) 13.7 (10.8–17.3) 20.8 (17.2–24.9) Overall 668 14.8 (12.5–17.5) Seroprevalence is presented as percentage and reported with + /−95% CL N number of bank voles tested, 1 immature juvenile voles, 2 mostly young adult voles, 3 breeding older animals communities living in or close to current endemic sites. In affect TBEV seroprevalence in this rodent species. Here, recent years, since the sudden and as of yet unexplained we report the results of our study, which was conducted increase in the incidence of TBEV infection in Poland in during three sequential expeditions at 4-year intervals to 1993, an average of 250 TBE cases each year have been study sites in the hyperendemic region of the country. recorded in the country with a mean incidence of 0.75 Our results are the first to report on the seroprevalence of cases/100,000 people . Incidence of TBEV infection is TBEV in wild rodents from Poland and make an impor- highest in northeastern regions of Poland, and these areas tant contribution to European datasets. Our study permits are considered to be a TBE-hyperendemic region of the future regional comparative analyses of the extent of this country (11.53 cases/100,000 inhabitants) . However, in viral agent in M. glareolus and the role of this particular contrast to other parts of Europe, there is still a gap in our host species in maintaining, perpetuating, and dis- knowledge about the extent of TBEV prevalence in bank seminating TBEV infections throughout the continent. voles (Myodes glareolus) in Poland, and in the exact role that they play as reservoirs of this virus in the region. Bank Results voles are one of the most common and widespread rodent The overall seroprevalence rate of TBEV was 14.8% species in European forests and are recognized as (12.5–17.5) (Table 1), but this rate varied significantly among the most important mammalian reservoir hosts of between surveys (YEAR × PRESENCE/ABSENCE of TBEV 14,20 2 TBEV . antibodies; χ = 24.07; P < 0.001) Bank voles sampled in We hypothesized that both extrinsic (temporal and 2006 and 2010 exhibited 2 to 2.5-fold higher ser- spatial) and intrinsic (age) factors play a major role in oprevalence rates than those sampled from 2002 (Table 1). affecting the seroprevalence of TBEV in bank voles and The site or location of sampling also had a significant consider it important to understand the role and relative effect (SITE × PRESENCE/ABSENCE of TBEV antibodies; importance of each of these factors in order to gain a χ = 36.2; P < 0.001), with the overall highest ser- greater insight into the local epidemiology of TBEV oprevalence rate recorded among bank voles from Pilchy infection. In this study, we aimed: (1) to assess the ser- (28.1% [20.2–37.5]). Bank voles collected from the other oprevalence of TBEV infection in bank voles in three two sites exhibited lower seroprevalence rates (Urwitałt = geographically separated but ecologically similar study 7.8% [5.5–10.8] and Tałty= 11.3% [8.6–14.6]). sites in the region and (2) to identify the intrinsic (host The TBEV seroprevalence rate was essentially identical age, sex) and extrinsic (year, study site) factors that most in both sexes (males = 14.9% [11.30–19.38] and females Grzybek et al. Emerging Microbes & Infections (2018) 7:145 Page 3 of 8 Fig. 1 Age related changes in TBEV seroprevalence by year of survey Fig. 3 Spatiotemporal dynamics of TBEV seroprevalence within study sites more complex interaction that included two extrinsic and one intrinsic factor (YEAR × SITE × SEX × PRESENCE/ ABSENCE of TBEV antibodies; χ = 14.07; P = 0.007). However, since seroprevalence rates did not differ sig- nificantly between the sexes overall, we did not explore this further. The spatiotemporal dynamics of seroprevalence rates are illustrated in Fig. 3 (YEAR × SITE × PRESENCE/ ABSENCE of TBEV antibodies; χ = 22.6; P < 0.001). Interestingly, seroprevalence rates were very similar and stable in bank voles from Tałty throughout the study Fig. 2 Age related changes in TBEV seroprevalence by the study site period, but displayed different dynamics at the other two sites. At Urwitałt, the seroprevalence rate increased slowly = 14.7% [11.21–19.04]) (NS), but differed significantly but constantly from 0% in 2002 to 16.3% in 2010. At between host age classes (AGE × PRESENCE/ABSENCE Pilchy, there was a 6.4-fold increase in the TBEV ser- of TBEV antibodies; χ = 13.05; P < 0.001). Seropositivity oprevalence rate, which was as high as 46.3% in 2006 and was 2.4-fold higher in the oldest individuals compared to as low as 32.1% in 2010. the youngest (Table 1) and was at an intermediate level among age class 2 bank voles. Discussion Although the seroprevalence rate differed significantly In this paper, we have presented original data on the between surveys, it was confounded by an interaction with first study, and to-date the longest recorded period, of host age (YEAR × AGE × PRESENCE/ABSENCE of TBEV serological monitoring of TBEV infections in bank voles antibodies; χ = 11.43; P = 0.022) (Fig. 1). In 2002, the in Poland. The Mazury Lake District is a highly endemic seroprevalence rate of TBEV was lowest in age class 1 region for TBE in Poland (in 2010–2016: the mean inci- bank voles, but much higher or similar in age classes 2 dence was 3.17 cases/100,000 people) , and the average and 3. A similar pattern was recorded in 2010, but in 2006 percentage of seropositive patients with neurological the pattern was slightly different, with the seroprevalence symptoms was found to be 15.5% (12.9–18.9) in this rate being low and similar in age classes 1 and 2, but much region . Our results show high overall seroprevalence higher in age class 3 (Fig. 1). rate of TBEV antibodies (14.8%) in M. glareolus, one of The differences in the seroprevalence rates between the most common rodent species in the locality, sug- sites were also confounded by a significant interaction gesting an important role for this species as a reservoir with host age (SITE × AGE × PRESENCE/ABSENCE of host of TBEV in this region. These findings are not only of TBEV antibodies; χ = 13.05; P = 0.011) (Fig. 2). There considerable relevance to public health in the region but was a progressive increase in TBEV seroprevalence rates could also be important for other European regions with the increase in host age among bank voles from populated by M. glareolus. They complement earlier Pilchy. The highest seroprevalence rate among bank voles reports from Poland and other European countries sug- from Talty was also recorded in the oldest voles; but, in gesting frequent infection of Myodes (Clethrionomys) spp. 13,14,23–25 Urwitałt, no clear trend was apparent. We also observed a with TBEV . Grzybek et al. Emerging Microbes & Infections (2018) 7:145 Page 4 of 8 Once infected after feeding on viremic rodents, ticks We originally hypothesized that the dependence of TBEV transmit TBEV to new susceptible hosts during sub- on tick vectors and that the widespread distribution of sequent feeds, and continue to harbor the virus until they ticks in Polish forests where their final deer hosts are also 26 42,43 die . The prevalence of the virus in questing ticks is present , would essentially tend to negate any differ- generally low when compared to the reported ser- ences in these parameters between sites. However, as our oprevalence rate in rodents, e.g., 0.28% in Scandinavia , data revealed, this turned out not to be the case. Spatial 28,29 0.24% in Lithuania and 0.11–0.96% in Poland . How- differences in the seroprevalence of TBEV, combined with ever, TBEV infection in ticks enhances their questing temporal changes as discussed above, added another level activity . Our previous studies, carried out at the same of complexity to the epidemiology of TBEV infection in sites as the current study, showed a very high prevalence rodents. Thus, temporal changes in the prevalence were rate (80–100%) of tick infestations in woodland and fallow not consistent across sites, and for example, a sharp land rodents . Paziewska et al. (2010) also reported a increase in the prevalence of seropositivity was recorded high prevalence rate (81%) of juvenile stages of I. ricinus among bank voles from Pilchy between 2002 and 2006, on bank voles and heavy infestations with a high ratio of I. whereas the seroprevalence rates remained low among ricinus larvae to nymphs in forest rodents in contiguous bank voles from the other two sites. Therefore, short-term sites. Although M. glareolus may develop resistance to monitoring may be insufficient to fully understand the feeding ticks after repeated infestations, these rodent circulation of the virus within rodent populations. Based hosts still play a significant role as TBEV reservoirs, on our results, a reliable picture of how a given pathogen alongside Apodemus spp ., because the virus can persist is distributed spatially and how it fluctuates temporally in 13,14 in bank voles as a latent infection . The high pre- its host population can only be derived from studies uti- valence rate of antibodies against TBEV that we detected lizing a multi-site approach for monitoring microparasites in bank voles reinforces the idea that they play a role as and macroparasites in a chosen geographical region, reservoir hosts for TBEV, and thereby are a source of applied over many years. Such a long-term approach, with infection for human communities in the region, and regular sampling of wild rodent populations over a should not be underestimated. lengthy period of years, is more likely to capture crucial Year-to-year fluctuations in the prevalence and abun- unidirectional as well cyclical changes in prevalence and dance of other pathogens have been well documented in that will improve our understanding of the epidemiology 33–37 bank voles sampled from our study sites in the past . of TBEV in its rodent reservoirs. Our present data show that host age also plays an While some pathogen species have fluctuated markedly (e.g., some helminths and hemoparasites) or have even important role, significantly affecting the seroprevalence become locally extinct in our study sites, others have shown rate of TBEV. The seroprevalence rate of the virus was relative stability from year to year. The temporal dynamics higher among mature bank voles compared with younger of TBEV infection clearly placethisinfectiousagent among individuals. In the case of pathogens that cause chronic the former group since we found marked temporal varia- infections, the likelihood of being infected and the tions in seroprevalence rates of TBEV in bank voles. abundance of parasite burdens increase with the age of Populations of many rodent species, including bank voles, the host . The current work was based on the presence/ are also known to fluctuate markedly, exhibiting regular and absence of specific antibodies against TBEV, and hence predictable cycles over several years but can often fluc- positivity in our assay reflected the history of previous tuate without a predictable period between peak den- infections and not necessarily current infections. There- 33,39,40 sities . Similar and concurrent fluctuations have been fore, it was not unexpected to find that older animals were 40,41 observed in their ectoparasite populations . more likely to have experienced infection than juveniles. We also found significant differences in the ser- Bank voles are born in nests and spend most of their time 44,45 oprevalence TBEV between voles from different sites in the nests until they are capable of foraging outside , despite the documented similarity in the ecological so questing ticks are unlikely to be encountered until they structure and relative proximity of our three study sites become more mobile and spend more time in the vege- (Fig. 4). Our current results therefore, complement those tation surrounding nests. that we have previously reported on other pathogens Finally, the results presented in this paper provide a sig- (helminths and hemoparasites), and clearly establish that nificant and novel contribution to our understanding of the the site from which host populations are sampled is the seroprevalence rate of TBEV within bank vole populations. most important factor influencing prevalence and abun- Our data show that the dynamics of TBEV transmission dance of infection. Both parameters can vary markedly change markedly with time but not always to the same when derived from host populations living in different degree in sites in close proximity to one another. Future sites within the same geographical region, even when studies should establish exactly how peaks of infection can those sites are considered to be ecologically very similar. be accurately predicted locally. The patchy distribution of Grzybek et al. Emerging Microbes & Infections (2018) 7:145 Page 5 of 8 Fig. 4 Localizations of the study sites in the Mazury Lake District in N.E. Poland (Google Maps, 2018). Sites are approximately 10 km from one another in a NW-SE transect seropositivity among bank voles from local subpopulations, Materials and methods as reflected in the between-site differences in the ser- Ethical approval oprevalence rates that we found, is of fundamental interest, This study was carried out in accordance with the and sample coverage over a wider geographical range would recommendations found in the Guidelines for the Care and be more informative. Likewise, the relatively low prevalence Use of Laboratory Animals of the Polish National Ethics of the virus in the I. ricinus population compared to some Committee for Animal Experimentation. Formal permits other tick-transmitted pathogens (i.e., Anaplasma phago- were obtained, allowing for trapping of animals in the field cytophilum and Borrelia burgdorferi) (Stanczak et al. 2004) and for subsequent laboratory analysis of sampled materials. is puzzling, given the prevalence of TBEV in mammalian Our project was approved by the First Warsaw Local Ethics hosts. Future research should focus on resolving the enigma Committee for Animal Experimentation. of how the TBEV is maintained for long periods of time despite such a low prevalence in I. ricinus. Our study sites Study sites are located in a region of Poland that is extremely popular Our three study sites are located in the Mazury Lake with the tourists and thousands of summer holidaymakers District region in the northeastern corner of Poland who visit the Mazury Lake District each year. Therefore, the (Fig. 4). They are separated by natural barriers, i.e., lakes, high seroprevalence rate of TBEV in bank voles presents a and therefore are isolated from one another in ecological significant threat to public health, and a capacity to predict time. The host species is panmictic across the region, and peak-years and high risk sites may help to prevent human genetic studies have revealed that some gene flow exists cases of TBE and thereby contribute significantly to the between the three populations . The sites have been public health of local populations and visitors to the region. described comprehensively in our earlier paper . Grzybek et al. Emerging Microbes & Infections (2018) 7:145 Page 6 of 8 Collection of bank voles likelihood techniques based on log-linear analysis of Bank voles were sampled from mid-August to mid- contingency tables in the software package IBM SPSS September in 2002, 2006 and 2010. Trapping was carried Statistics Version 21 (IBM Corporation). This approach is out for 3–4 consecutive days at a time at each site. The based on categorical values of the factors of interest, methods used for trapping rodents and for sampling and which are used to fit hierarchical log-linear models to processing trapped animals have been thoroughly multidimensional cross-tabulations using an iterative 36,37,47 proportional-fitting algorithm and detects associations described by Behnke et al. . Three age classes were established according to the methods of Behnke et al. between the factors, one of which may be presence/ and Grzybek et al. using principal components analysis absence of anti-TBE-IgG antibodies against the TBE virus. of a range of morphological measures including body Initially, full factorial models were fitted, incorporating as weight and dried eye lens weight as follows: class 1— factors sex (2 levels: males and females), age (3 levels), immature juvenile bank voles; class 2—mostly young year (3 levels: 2002, 2006, and 2010), and site (3 levels: adult bank voles; and class 3—breeding older animals. Urwitałt, Tałty, and Pilchy). The presence or absence of Blood samples were collected directly from the heart by anti-TBE-IgG antibodies against the TBE virus (ser- cardiac puncture using a sterile 1.5 mL syringe immedi- oprevalence rate) was considered as a binary factor. All ately after death from over-exposure to an anesthetic. these five factors were fitted initially to all models that Blood was allowed to clot at room temperature. After were evaluated. For each level of analysis, beginning with separation of the blood clot, samples were centrifuged at the most complex model involving all possible main 5000 rpm for 10 min using an MPW High-Speed Brush- effects and interactions, those combinations that did not less Centrifuge. Serum was collected and stored at −80 °C contribute significantly to explaining variation in the data until the samples could be analyzed upon completion of were eliminated stepwise beginning with the highest level the fieldwork. interaction (backward selection procedure). A minimum sufficient model was then obtained, for which the like- Immunochemical analysis by ELISA lihood ratio of χ was not significant, indicating that the We carried out ELISAs for the quantitative determina- model was sufficient in explaining the data. The impor- tion of anti-TBE-IgG antibodies using the IMMUNO- tance of each term in interactions involving ser- ZYM® FSME (TBE) IgG All Species Kit (PROGEN oprevalence in the final model was assessed by the Biotechnik GmbH, Germany) and according to the probability that its exclusion would alter the model sig- manufacturer’s instructions. In total, we analyzed 668 nificantly and these values are given in the text. The bank vole sera. The optical density was measured at a remaining terms in the final model that did not include wavelength of 450 nm (0.1 s) using a PerkinElmer Victor 3 seroprevalence (for example, variation among sites in the Multilabel Plate Counter. Calculation of anti-TBE-IgG number of animals of each sex sampled [site × sex]) are concentration was performed quantitatively using the not given but can be made available from the authors on reference curve. The optical density at 450 nm was request. transformed into Vienna units (VIEU). Samples were Acknowledgements scored as negative for anti-TBE-IgG antibodies if VIEU/ We thank the University of Nottingham, Warsaw University, University of Life mL was <63, as borderline if VIEU/mL was in the range of 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 63–126, and as positive if VIEU/mL was >126. All bor- Grabowski Fund. A.B. was supported by the Polish State Committee for derline samples were tested twice, and if the second test Scientific Research and the British Council’s Young Scientist Programme. M.G. confirmed a borderline score for a sample, it was was supported by the Ministry of Science and Higher Education in Poland, Fellowship for Outstanding Scientists (428/STYP/11/2016). treated subsequently as negative. Otherwise, previously borderline samples were considered to be positive or Author contributions negative according to the value of the score derived on The study was conceived and designed by M.G., B.B., J.M.B., and A.B. retesting. Supervision of the long-term monitoring of bank vole populations in the region was conducted by J.M.B. and A.B. Samples were collected in the field by J.M.B., A.B., M.A., K.T., J.B.B., and M.G. The immunological analysis and laboratory Statistical analysis work was conducted by M.G., A.S., K.S., L.G., and J.P. Data analysis was carried Seroprevalence values (percentage of seropositive ani- out by MG, AB, and JMB. The manuscript was written by M.G., B.B., A.B., J.S., and J.M.B. in consultation with all co-authors. M.G., A.B., J.S., J.P., and J.M.B. revised mals) are given with 95% confidence limits in parenthesis the manuscript. All authors accepted the final manuscript version. 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Parasitology 123,401–414 (2001). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Emerging Microbes & Infections Springer Journals

Seroprevalence of TBEV in bank voles from Poland—a long-term approach

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Springer Journals
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Copyright © 2018 by The Author(s)
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Biomedicine; Biomedicine, general; Immunology; Medical Microbiology; Microbiology; Antibodies; Vaccine
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Abstract

Rodents are known to play a significant role as reservoir hosts for TBEV. During three sequential expeditions at 4-year intervals to three ecologically similar study sites in NE Poland, we trapped bank voles (Myodes glareolus) and then tested their blood for the presence of specific antiviral antibodies to TBEV. The strongest effects on seroprevalence were the extrinsic factors, site of capture of voles and year of sampling. Seroprevalence increased markedly with increasing host age, and our analysis revealed significant interactions among these three factors. Seroprevalence did not differ between the sexes. Therefore, based on the seroprevalence results, the dynamics of TBEV infection differ significantly in time, between local sub-populations of bank voles and with increasing host age. To fully understand the circulation of the virus among these reservoir hosts and in the environment, long-term monitoring is required and should employ a multi-site approach, such as the one adopted in the current study. Introduction rodents are the most important hosts for the immature Rodents, members of the most abundant and diversified stages of I. ricinus . There are five known routes for the mammalian order Rodentia , can pose a significant threat transmission and maintenance of TBEV. Ticks become to the health of humans, livestock, and wildlife because infected when feeding on a viremic host and maintain the 8 9 they are hosts for a wide range of pathogens and in some virus via transstadial or/and transovarial transmission , cases constitute important reservoir hosts for life- or through co-feeding on a non-viremic host . Sexual threatening zoonoses . transmission from male to female ticks is also known to The tick-borne encephalitis virus (TBEV), the causative occur . Consequently, all hematophagous stages of ticks agent of tick-borne encephalitis (TBE), is a zoonotic fla- can transmit the virus to mammalian hosts . Rodents vivirus in the family Flaviviridae that is endemic have been considered to play an essential role in main- throughout the northern Palearctic, spanning an area taining TBEV in nature by carrying persistent latent 13,14 from central and northern Europe and across Siberia to infections . Japan in the far east . TBEV is maintained in nature in a TBEV is the most important causative agent of arboviral cycle that includes tick vectors of the Ixodes persulcatus infections in Europe and is responsible for distressing complex and their vertebrate hosts. The most important neurologic symptoms in patients . Incidence of the dis- 4,5 vector in Central Europe is Ixodes ricinus , and small ease has greatly increased over the past decades, growing into a serious human threat, and changes in the spatial distribution of TBE cases have been concurrently 15,16 observed . Therefore, it is essential to identify the Correspondence: Maciej Grzybek (maciej.grzybek@gumed.edu.pl) Department of Tropical Parasitology, Medical University of Gdańsk, Gdańsk, endemic areas and to monitor the temporal changes of Poland this virus in order to ensure that suitable preventive Department of Parasitology, University of Warsaw, Warsaw, Poland measures are implemented successfully by human Full list of author information is available at the end of the article. These authors contributed equally: Jerzy M. Behnke, Anna Bajer © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Grzybek et al. Emerging Microbes & Infections (2018) 7:145 Page 2 of 8 Table 1 Seroprevalence of TBEV by year, site and host age Year Host age Site N 12 3 2002 Urwitałt 64 0.0 (0.0–23.8) 0.0 (0.0–13.4) 0.0 (0.0–13.4) Tałty 70 4.5 (0.2–22.2) 6.5 (1.9–17.4) 17.6 (5.0–41.7) Pilchy 69 0.0 (0.0–17.6) 11.5 (3.2–30.4) 8.3 (1.5–26.7) Overall by year 203 5.4 (3.7–7.8) 2006 Urwitałt 95 7.7 (1.4–24.6) 4.5 (0.7–17.7) 4.0 (0.2–19.6) Tałty 65 7.7 (1.4–24.6) 0.0 (0.0–22.2) 12.5 (3.5–31.0) Pilchy 67 24.1 (11.5–43.0) 38.5 (16.6–65.8) 76.0 (56.1–89.0) Overall by year 227 18.1 (14.7–21.9) 2010 Urwitałt 86 0.0 (0.0–20.8) 25.0 (12.9–41.9) 11.5 (3.2–30.4) Tałty 96 11.5 (3.2–30.4) 13.0 (3.7–32.4) 19.1 (8.8–36.6) Pilchy 56 11.8 (2.1–35.0) 50.0 (24.3–75.7) 32.1 (23.2–42.6) Overall by year 238 19.7 (16.2–23.8) Overall by age 8.7 (4.4–16.0) 13.7 (10.8–17.3) 20.8 (17.2–24.9) Overall 668 14.8 (12.5–17.5) Seroprevalence is presented as percentage and reported with + /−95% CL N number of bank voles tested, 1 immature juvenile voles, 2 mostly young adult voles, 3 breeding older animals communities living in or close to current endemic sites. In affect TBEV seroprevalence in this rodent species. Here, recent years, since the sudden and as of yet unexplained we report the results of our study, which was conducted increase in the incidence of TBEV infection in Poland in during three sequential expeditions at 4-year intervals to 1993, an average of 250 TBE cases each year have been study sites in the hyperendemic region of the country. recorded in the country with a mean incidence of 0.75 Our results are the first to report on the seroprevalence of cases/100,000 people . Incidence of TBEV infection is TBEV in wild rodents from Poland and make an impor- highest in northeastern regions of Poland, and these areas tant contribution to European datasets. Our study permits are considered to be a TBE-hyperendemic region of the future regional comparative analyses of the extent of this country (11.53 cases/100,000 inhabitants) . However, in viral agent in M. glareolus and the role of this particular contrast to other parts of Europe, there is still a gap in our host species in maintaining, perpetuating, and dis- knowledge about the extent of TBEV prevalence in bank seminating TBEV infections throughout the continent. voles (Myodes glareolus) in Poland, and in the exact role that they play as reservoirs of this virus in the region. Bank Results voles are one of the most common and widespread rodent The overall seroprevalence rate of TBEV was 14.8% species in European forests and are recognized as (12.5–17.5) (Table 1), but this rate varied significantly among the most important mammalian reservoir hosts of between surveys (YEAR × PRESENCE/ABSENCE of TBEV 14,20 2 TBEV . antibodies; χ = 24.07; P < 0.001) Bank voles sampled in We hypothesized that both extrinsic (temporal and 2006 and 2010 exhibited 2 to 2.5-fold higher ser- spatial) and intrinsic (age) factors play a major role in oprevalence rates than those sampled from 2002 (Table 1). affecting the seroprevalence of TBEV in bank voles and The site or location of sampling also had a significant consider it important to understand the role and relative effect (SITE × PRESENCE/ABSENCE of TBEV antibodies; importance of each of these factors in order to gain a χ = 36.2; P < 0.001), with the overall highest ser- greater insight into the local epidemiology of TBEV oprevalence rate recorded among bank voles from Pilchy infection. In this study, we aimed: (1) to assess the ser- (28.1% [20.2–37.5]). Bank voles collected from the other oprevalence of TBEV infection in bank voles in three two sites exhibited lower seroprevalence rates (Urwitałt = geographically separated but ecologically similar study 7.8% [5.5–10.8] and Tałty= 11.3% [8.6–14.6]). sites in the region and (2) to identify the intrinsic (host The TBEV seroprevalence rate was essentially identical age, sex) and extrinsic (year, study site) factors that most in both sexes (males = 14.9% [11.30–19.38] and females Grzybek et al. Emerging Microbes & Infections (2018) 7:145 Page 3 of 8 Fig. 1 Age related changes in TBEV seroprevalence by year of survey Fig. 3 Spatiotemporal dynamics of TBEV seroprevalence within study sites more complex interaction that included two extrinsic and one intrinsic factor (YEAR × SITE × SEX × PRESENCE/ ABSENCE of TBEV antibodies; χ = 14.07; P = 0.007). However, since seroprevalence rates did not differ sig- nificantly between the sexes overall, we did not explore this further. The spatiotemporal dynamics of seroprevalence rates are illustrated in Fig. 3 (YEAR × SITE × PRESENCE/ ABSENCE of TBEV antibodies; χ = 22.6; P < 0.001). Interestingly, seroprevalence rates were very similar and stable in bank voles from Tałty throughout the study Fig. 2 Age related changes in TBEV seroprevalence by the study site period, but displayed different dynamics at the other two sites. At Urwitałt, the seroprevalence rate increased slowly = 14.7% [11.21–19.04]) (NS), but differed significantly but constantly from 0% in 2002 to 16.3% in 2010. At between host age classes (AGE × PRESENCE/ABSENCE Pilchy, there was a 6.4-fold increase in the TBEV ser- of TBEV antibodies; χ = 13.05; P < 0.001). Seropositivity oprevalence rate, which was as high as 46.3% in 2006 and was 2.4-fold higher in the oldest individuals compared to as low as 32.1% in 2010. the youngest (Table 1) and was at an intermediate level among age class 2 bank voles. Discussion Although the seroprevalence rate differed significantly In this paper, we have presented original data on the between surveys, it was confounded by an interaction with first study, and to-date the longest recorded period, of host age (YEAR × AGE × PRESENCE/ABSENCE of TBEV serological monitoring of TBEV infections in bank voles antibodies; χ = 11.43; P = 0.022) (Fig. 1). In 2002, the in Poland. The Mazury Lake District is a highly endemic seroprevalence rate of TBEV was lowest in age class 1 region for TBE in Poland (in 2010–2016: the mean inci- bank voles, but much higher or similar in age classes 2 dence was 3.17 cases/100,000 people) , and the average and 3. A similar pattern was recorded in 2010, but in 2006 percentage of seropositive patients with neurological the pattern was slightly different, with the seroprevalence symptoms was found to be 15.5% (12.9–18.9) in this rate being low and similar in age classes 1 and 2, but much region . Our results show high overall seroprevalence higher in age class 3 (Fig. 1). rate of TBEV antibodies (14.8%) in M. glareolus, one of The differences in the seroprevalence rates between the most common rodent species in the locality, sug- sites were also confounded by a significant interaction gesting an important role for this species as a reservoir with host age (SITE × AGE × PRESENCE/ABSENCE of host of TBEV in this region. These findings are not only of TBEV antibodies; χ = 13.05; P = 0.011) (Fig. 2). There considerable relevance to public health in the region but was a progressive increase in TBEV seroprevalence rates could also be important for other European regions with the increase in host age among bank voles from populated by M. glareolus. They complement earlier Pilchy. The highest seroprevalence rate among bank voles reports from Poland and other European countries sug- from Talty was also recorded in the oldest voles; but, in gesting frequent infection of Myodes (Clethrionomys) spp. 13,14,23–25 Urwitałt, no clear trend was apparent. We also observed a with TBEV . Grzybek et al. Emerging Microbes & Infections (2018) 7:145 Page 4 of 8 Once infected after feeding on viremic rodents, ticks We originally hypothesized that the dependence of TBEV transmit TBEV to new susceptible hosts during sub- on tick vectors and that the widespread distribution of sequent feeds, and continue to harbor the virus until they ticks in Polish forests where their final deer hosts are also 26 42,43 die . The prevalence of the virus in questing ticks is present , would essentially tend to negate any differ- generally low when compared to the reported ser- ences in these parameters between sites. However, as our oprevalence rate in rodents, e.g., 0.28% in Scandinavia , data revealed, this turned out not to be the case. Spatial 28,29 0.24% in Lithuania and 0.11–0.96% in Poland . How- differences in the seroprevalence of TBEV, combined with ever, TBEV infection in ticks enhances their questing temporal changes as discussed above, added another level activity . Our previous studies, carried out at the same of complexity to the epidemiology of TBEV infection in sites as the current study, showed a very high prevalence rodents. Thus, temporal changes in the prevalence were rate (80–100%) of tick infestations in woodland and fallow not consistent across sites, and for example, a sharp land rodents . Paziewska et al. (2010) also reported a increase in the prevalence of seropositivity was recorded high prevalence rate (81%) of juvenile stages of I. ricinus among bank voles from Pilchy between 2002 and 2006, on bank voles and heavy infestations with a high ratio of I. whereas the seroprevalence rates remained low among ricinus larvae to nymphs in forest rodents in contiguous bank voles from the other two sites. Therefore, short-term sites. Although M. glareolus may develop resistance to monitoring may be insufficient to fully understand the feeding ticks after repeated infestations, these rodent circulation of the virus within rodent populations. Based hosts still play a significant role as TBEV reservoirs, on our results, a reliable picture of how a given pathogen alongside Apodemus spp ., because the virus can persist is distributed spatially and how it fluctuates temporally in 13,14 in bank voles as a latent infection . The high pre- its host population can only be derived from studies uti- valence rate of antibodies against TBEV that we detected lizing a multi-site approach for monitoring microparasites in bank voles reinforces the idea that they play a role as and macroparasites in a chosen geographical region, reservoir hosts for TBEV, and thereby are a source of applied over many years. Such a long-term approach, with infection for human communities in the region, and regular sampling of wild rodent populations over a should not be underestimated. lengthy period of years, is more likely to capture crucial Year-to-year fluctuations in the prevalence and abun- unidirectional as well cyclical changes in prevalence and dance of other pathogens have been well documented in that will improve our understanding of the epidemiology 33–37 bank voles sampled from our study sites in the past . of TBEV in its rodent reservoirs. Our present data show that host age also plays an While some pathogen species have fluctuated markedly (e.g., some helminths and hemoparasites) or have even important role, significantly affecting the seroprevalence become locally extinct in our study sites, others have shown rate of TBEV. The seroprevalence rate of the virus was relative stability from year to year. The temporal dynamics higher among mature bank voles compared with younger of TBEV infection clearly placethisinfectiousagent among individuals. In the case of pathogens that cause chronic the former group since we found marked temporal varia- infections, the likelihood of being infected and the tions in seroprevalence rates of TBEV in bank voles. abundance of parasite burdens increase with the age of Populations of many rodent species, including bank voles, the host . The current work was based on the presence/ are also known to fluctuate markedly, exhibiting regular and absence of specific antibodies against TBEV, and hence predictable cycles over several years but can often fluc- positivity in our assay reflected the history of previous tuate without a predictable period between peak den- infections and not necessarily current infections. There- 33,39,40 sities . Similar and concurrent fluctuations have been fore, it was not unexpected to find that older animals were 40,41 observed in their ectoparasite populations . more likely to have experienced infection than juveniles. We also found significant differences in the ser- Bank voles are born in nests and spend most of their time 44,45 oprevalence TBEV between voles from different sites in the nests until they are capable of foraging outside , despite the documented similarity in the ecological so questing ticks are unlikely to be encountered until they structure and relative proximity of our three study sites become more mobile and spend more time in the vege- (Fig. 4). Our current results therefore, complement those tation surrounding nests. that we have previously reported on other pathogens Finally, the results presented in this paper provide a sig- (helminths and hemoparasites), and clearly establish that nificant and novel contribution to our understanding of the the site from which host populations are sampled is the seroprevalence rate of TBEV within bank vole populations. most important factor influencing prevalence and abun- Our data show that the dynamics of TBEV transmission dance of infection. Both parameters can vary markedly change markedly with time but not always to the same when derived from host populations living in different degree in sites in close proximity to one another. Future sites within the same geographical region, even when studies should establish exactly how peaks of infection can those sites are considered to be ecologically very similar. be accurately predicted locally. The patchy distribution of Grzybek et al. Emerging Microbes & Infections (2018) 7:145 Page 5 of 8 Fig. 4 Localizations of the study sites in the Mazury Lake District in N.E. Poland (Google Maps, 2018). Sites are approximately 10 km from one another in a NW-SE transect seropositivity among bank voles from local subpopulations, Materials and methods as reflected in the between-site differences in the ser- Ethical approval oprevalence rates that we found, is of fundamental interest, This study was carried out in accordance with the and sample coverage over a wider geographical range would recommendations found in the Guidelines for the Care and be more informative. Likewise, the relatively low prevalence Use of Laboratory Animals of the Polish National Ethics of the virus in the I. ricinus population compared to some Committee for Animal Experimentation. Formal permits other tick-transmitted pathogens (i.e., Anaplasma phago- were obtained, allowing for trapping of animals in the field cytophilum and Borrelia burgdorferi) (Stanczak et al. 2004) and for subsequent laboratory analysis of sampled materials. is puzzling, given the prevalence of TBEV in mammalian Our project was approved by the First Warsaw Local Ethics hosts. Future research should focus on resolving the enigma Committee for Animal Experimentation. of how the TBEV is maintained for long periods of time despite such a low prevalence in I. ricinus. Our study sites Study sites are located in a region of Poland that is extremely popular Our three study sites are located in the Mazury Lake with the tourists and thousands of summer holidaymakers District region in the northeastern corner of Poland who visit the Mazury Lake District each year. Therefore, the (Fig. 4). They are separated by natural barriers, i.e., lakes, high seroprevalence rate of TBEV in bank voles presents a and therefore are isolated from one another in ecological significant threat to public health, and a capacity to predict time. The host species is panmictic across the region, and peak-years and high risk sites may help to prevent human genetic studies have revealed that some gene flow exists cases of TBE and thereby contribute significantly to the between the three populations . The sites have been public health of local populations and visitors to the region. described comprehensively in our earlier paper . Grzybek et al. Emerging Microbes & Infections (2018) 7:145 Page 6 of 8 Collection of bank voles likelihood techniques based on log-linear analysis of Bank voles were sampled from mid-August to mid- contingency tables in the software package IBM SPSS September in 2002, 2006 and 2010. Trapping was carried Statistics Version 21 (IBM Corporation). This approach is out for 3–4 consecutive days at a time at each site. The based on categorical values of the factors of interest, methods used for trapping rodents and for sampling and which are used to fit hierarchical log-linear models to processing trapped animals have been thoroughly multidimensional cross-tabulations using an iterative 36,37,47 proportional-fitting algorithm and detects associations described by Behnke et al. . Three age classes were established according to the methods of Behnke et al. between the factors, one of which may be presence/ and Grzybek et al. using principal components analysis absence of anti-TBE-IgG antibodies against the TBE virus. of a range of morphological measures including body Initially, full factorial models were fitted, incorporating as weight and dried eye lens weight as follows: class 1— factors sex (2 levels: males and females), age (3 levels), immature juvenile bank voles; class 2—mostly young year (3 levels: 2002, 2006, and 2010), and site (3 levels: adult bank voles; and class 3—breeding older animals. Urwitałt, Tałty, and Pilchy). The presence or absence of Blood samples were collected directly from the heart by anti-TBE-IgG antibodies against the TBE virus (ser- cardiac puncture using a sterile 1.5 mL syringe immedi- oprevalence rate) was considered as a binary factor. All ately after death from over-exposure to an anesthetic. these five factors were fitted initially to all models that Blood was allowed to clot at room temperature. After were evaluated. For each level of analysis, beginning with separation of the blood clot, samples were centrifuged at the most complex model involving all possible main 5000 rpm for 10 min using an MPW High-Speed Brush- effects and interactions, those combinations that did not less Centrifuge. Serum was collected and stored at −80 °C contribute significantly to explaining variation in the data until the samples could be analyzed upon completion of were eliminated stepwise beginning with the highest level the fieldwork. interaction (backward selection procedure). A minimum sufficient model was then obtained, for which the like- Immunochemical analysis by ELISA lihood ratio of χ was not significant, indicating that the We carried out ELISAs for the quantitative determina- model was sufficient in explaining the data. The impor- tion of anti-TBE-IgG antibodies using the IMMUNO- tance of each term in interactions involving ser- ZYM® FSME (TBE) IgG All Species Kit (PROGEN oprevalence in the final model was assessed by the Biotechnik GmbH, Germany) and according to the probability that its exclusion would alter the model sig- manufacturer’s instructions. In total, we analyzed 668 nificantly and these values are given in the text. The bank vole sera. The optical density was measured at a remaining terms in the final model that did not include wavelength of 450 nm (0.1 s) using a PerkinElmer Victor 3 seroprevalence (for example, variation among sites in the Multilabel Plate Counter. Calculation of anti-TBE-IgG number of animals of each sex sampled [site × sex]) are concentration was performed quantitatively using the not given but can be made available from the authors on reference curve. The optical density at 450 nm was request. transformed into Vienna units (VIEU). Samples were Acknowledgements scored as negative for anti-TBE-IgG antibodies if VIEU/ We thank the University of Nottingham, Warsaw University, University of Life mL was <63, as borderline if VIEU/mL was in the range of 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 63–126, and as positive if VIEU/mL was >126. All bor- Grabowski Fund. A.B. was supported by the Polish State Committee for derline samples were tested twice, and if the second test Scientific Research and the British Council’s Young Scientist Programme. M.G. confirmed a borderline score for a sample, it was was supported by the Ministry of Science and Higher Education in Poland, Fellowship for Outstanding Scientists (428/STYP/11/2016). treated subsequently as negative. Otherwise, previously borderline samples were considered to be positive or Author contributions negative according to the value of the score derived on The study was conceived and designed by M.G., B.B., J.M.B., and A.B. retesting. Supervision of the long-term monitoring of bank vole populations in the region was conducted by J.M.B. and A.B. Samples were collected in the field by J.M.B., A.B., M.A., K.T., J.B.B., and M.G. The immunological analysis and laboratory Statistical analysis work was conducted by M.G., A.S., K.S., L.G., and J.P. Data analysis was carried Seroprevalence values (percentage of seropositive ani- out by MG, AB, and JMB. The manuscript was written by M.G., B.B., A.B., J.S., and J.M.B. in consultation with all co-authors. M.G., A.B., J.S., J.P., and J.M.B. revised mals) are given with 95% confidence limits in parenthesis the manuscript. All authors accepted the final manuscript version. 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