Introduction
Tick-borne encephalitis virus (TBEV) is responsible for the most serious human viral tick-borne disease in Europe. This flavivirus affects the human central nervous system causing meningitis or meningoencephalitis with long-term sequelae, the more severe forms progressing into a loss of consciousness, coma and even death (Bogovic & Strle, Franc, 2015). In Western Europe, only the European subtype (TBEV-Eur), the least virulent subtype, is present (Süss, 2011). TBEV-Eur is mainly transmitted to a host by a tick bite and is maintained in nature by a cycle involving ticks — mainly Ixodes ricinus— and small mammals, especially those belonging to the genus Apodemus andMyodes (Süss, 2011). I. ricinus require three blood meals during their lifecycle. The larvae mainly feed on small mammals, while nymphs feed on medium-sized mammals, birds and reptiles, and adults on large animals such as ungulates (Mannelli, Bertolotti, Gern, & Gray, 2012). Each stage of the life cycle takes from several months to around one year to develop to the next, so the entire life cycle is generally completed in 2 or 3 years, although this can vary from 2 to 6 years depending upon the geographical location (Gray, Kahl, Lane, Levin, & Tsao, 2016; Perret, Rais, & Gern, 2004).
The virus is transmitted transstadially from one life stage of the tick to the next after the moult (e.g. from larva to nymph), and on rare occasions can be transmitted vertically from a female tick to its eggs (Süss, 2011). Ticks can also become infected while feeding on a host during a viraemic phase. However, the duration of viraemia among small mammals and thus their infectivity to ticks are commonly considered short (2-9 days) (Chunikhin & Kurenkov, 1979; Ernek, Kozuch, Lichard, Nosek, & Albrecht, 1963; Heigl & Von Zeipel, 1966; Kozuch et al., 1981). A recent experimental study in bank voles (Myodes glareolus ) suggests that viraemia might last up to 28 days, and therefore be longer than previously thought, but infectivity to ticks was not tested (Michelitsch et al., 2019). Therefore, co-feeding transmission between infected nymphs and uninfected larvae when they feed in spatiotemporal proximity to each other on the same host is thought to be the main transmission mode of TBEV-Eur and the most efficient way to maintain TBEV-Eur in a given area (Sarah E. Randolph, 2011). This transmission can even occur through immune hosts, although its efficiency is reduced (Milan Labuda & Randolph, 1999).
The generational transfer of TBEV from infected nymphs to uninfected larvae via the host upon which they are feeding is the critical life history event that defines the epidemiology of TBEV (Voordouw, 2015). Therefore, the main key fitness component of TBEV is defined by the number of infected larvae produced via co-feeding transmission and, to a lesser extent, via systemic transmission. Thus, an essential prerequisite for TBEV persistence in a given area is the synchronous activity of larvae and nymphs (S. E. Randolph, Green, Peacey, & Rogers, 2000). The substantial aggregation of ticks on a limited number of hosts promotes nymph-to-larva transmission of TBEV (i.e. the number of infected larvae produced) and TBEV-cycle persistence. Ticks follow the “20/80 Rule” (Woolhouse et al., 1997) whereby 20% of the reservoir hosts feed about 80% of the ticks (C. Burri, Bastic, Maeder, Patalas, & Gern, 2011; Mannelli et al., 2012; Perkins, Cattadori, Tagliapietra, Rizzoli, & Hudson, 2003; S. E. Randolph, Miklisová, Lysy, Rogers, & Labuda, 1999; Rosà, Pugliese, Ghosh, Perkins, & Rizzoli, 2007). The propensity of ticks to aggregate depends partly on the tick-host contact rate (activity of questing ticks, host and tick abundance, host community structure). For instance, it has been shown that small mammals are subject to greater tick infestation and higher individual loads of ticks when their population densities are low, given that each individual host has a higher probability of contact with ticks (Kiffner, Vor, Hagedorn, Niedrig, & Rühe, 2011; Perez et al., 2017; Rosà et al., 2007). Therefore, the intensity of tick aggregation on a host is a useful parameter to study because the number of larvae co-feeding with infected nymphs impacts nymph-to-larva transmission.
The density of TBEV-infected nymphs that quest for vertebrate hosts in the environment (DIN), the prevalence of infected questing nymphs (NIP), and the proportion of small mammals harbouring TBEV-specific antibodies (seroprevalence) are three more relevant ecological and epidemiological parameters to monitor in order to understand the TBEV cycle. As small mammals rarely harbour adult ticks, DIN is an indicator of the instantaneous risk of exposure to TBEV for small mammals and for larvae via the host upon which they are feeding. NIP is a measurement of the proportion of larvae that became infected while feeding on a competent host in the preceding months or year (i.e. the nymph-to-larva transmission rate) and moulted into nymphs. In turn, the nymph-to-larva transmission depends on the intensity of tick aggregation on a few hosts and on the propensity of infected nymphs to feed on small mammals, which can be indirectly measured by the TBEV seroprevalence of small mammals. The relationship between DIN, NIP and the seroprevalence of small mammals and the parameters that influenced these parameters are presented in Figure 1.
The natural foci of TBEV are fluctuating ecosystems having characteristics that vary both intra- and inter-annually. A greater knowledge of these natural foci and their fluctuating characteristics are essential to better assess temporal variations of the epidemiological risks of TBEV-Eur. However, very few studies have addressed this issue (Perez-Eid, Hannoun, & Rodhain, 1992; Rosà et al., 2019; Zöldi, Papp, Rigó, Farkas, & Egyed, 2015). Both tick (larvae and nymphs) and small mammal densities show large inter-annual and intra-annual fluctuations. The activity of I. ricinus depends on humidity and temperature, and is therefore seasonal. Depending upon meteorological and climatic conditions and host availability, the abundance peak for questing larvae is either in late spring-early summer (in northern and central Europe, including our study area in eastern France) or in autumn (in western Europe) (Gray et al., 2016; Perez et al., 2017; S. E. Randolph et al., 2000). In western and central Europe, the peak for questing nymphs usually occurs in spring and early summer followed by comparatively low-level activity in mid-summer. In many areas, a second and minor abundance peak is observed in early autumn (Gray et al., 2016). This second peak has been partly attributed in Switzerland to the early emergence of larvae that have fed in spring (Perret et al., 2004). In temperate European forests, most small mammal species of the genus Apodemus and Myodes start breeding in spring and their population size reaches a peak in summer or autumn before decreasing during the winter (Crespin et al., 2002; Pucek, Jędrzejewski, Jędrzejewska, & Pucek, 1993; Stenseth, Viljugrein, Jędrzejewski, Mysterud, & Pucek, 2002). Therefore, TBEV nymph-to-larva transmission and exposure of small mammals to TBEV may vary seasonally as tick densities, TBEV-tick prevalence and their aggregation on host varies. Early spring, late spring/early summer and late summer/early autumn are seasons of particular interest for studying the TBEV epidemiological cycle.
In temperate forests, small mammal populations of the genusApodemus and Myodes are also subject to irregular multiannual oscillations, with a year of peak abundance occurring after a year of a heavy seed crop of oak and beech, and followed by a year of crashed abundance (Crespin et al., 2002; Jensen, 1982; Pucek et al., 1993; Stenseth et al., 2002). By the annual fluctuation in the number of larvae they feed, the temporal variation in the small mammal population may lead to an annual fluctuation in the density of questing nymphs, with a higher nymph density the year after a peak in rodent density (Brugger, Walter, Chitimia-Dobler, Dobler, & Rubel, 2018; Krawczyk et al., 2020; Ostfeld, Levi, Keesing, Oggenfuss, & Canham, 2018). The effects of these variations on TBEV nymph-to-larva transmission and on the exposure of small mammals to TBEV are not straightforward, as the intensity of tick aggregation also varies annually (Perez et al., 2017; Rosà et al., 2007). Another factor that can influence the temporal variation of TBEV nymph-to-larva transmission is the variation over time of the community structure of small mammals (the relative density and the proportion of each small mammal species). Indeed, tick burden and the transmission-competence of the host — i.e. the ability of the host species to foster and amplify nymph-to-larva transmission through co-feeding or systemic (viraemic) transmission —, vary from one small mammal species to another, such as Apodemus flavicollis andMyodes glareolus (Dizij & Kurtenbach, 1995; Kurtenbach et al., 1995; Milan Labuda & Randolph, 1999; Pérez, Kneubühler, Rais, & Gern, 2012).
France is located on the western border of the known distribution of TBEV, with about ten cases reported each year since its discovery in 1968. Most human clinical cases of TBE have been reported in Alsace, a region in the extreme East of France bordering Germany and Switzerland (Hansmann et al., 2006; Velay et al., 2019). Contrary to the endemic area of TBEV in these neighbouring countries, the incidence rate in Alsace is low, with a yearly incidence of 0.5/100,000 inhabitants on average. The epidemiological characteristics of TBEV’s natural foci, such as the intensity of virus circulation and nymph-to-larva transmission over time, has not been thoroughly investigated in Alsace. The only study of TBEV in ticks and small mammals in France was conducted from 1970 to 1974 in a closed peri-urban forest (Neuhof forest) near Strasbourg, an Alsatian city (Perez-Eid et al., 1992). We therefore conducted a longitudinal study of a TBEV focus in an Alsatian mountain forest over a 3-year period. The aims of the present study were to characterise the intensity of virus circulation and to describe both seasonal and inter-annual variations of the TBEV cycle’s epidemiological parameters: (i) the density of TBEV-infected questing nymphs (DIN) and the prevalence of TBEV in questing nymphs (NIP) related to TBEV nymph-to-larva transmission, (ii) the TBEV seroprevalence of small mammals related to their exposure to TBEV; (iii) the prevalence of tick infestations of small mammals as a proxy for the intensity of aggregation on hosts, since these parameters are well correlated. The seasonal and inter-annual results were then discussed in relation to the variation over time of the density of ticks, the density of small mammals and the community structure of small mammals.