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.