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\begin{document}

\title{Tick-borne encephalitis virus in an Alsatian forest, eastern France:
seasonal and annual variation of epidemiological parameters related to
nymph-to-larva transmission and exposure of small mammals}



\author[1]{Laure Bournez}%
\author[1]{Gérald Umhang}%
\author[1]{Marie Moinet}%
\author[1]{Céline RICHOMME}%
\author[1]{Jean-Michel Demerson}%
\author[1]{Christophe Caillot}%
\author[2]{Elodie Devillers}%
\author[1]{Jean-Marc Boucher}%
\author[3]{Yves Hansmann}%
\author[1]{Franck Boue}%
\author[2]{Sara Moutailler}%
\affil[1]{Anses}%
\affil[2]{UMR BIPAR, Animal Health Laboratory}%
\affil[3]{University Hospitals Strasbourg}%


\vspace{-1em}



  
  \date{May 08, 2024}


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\selectlanguage{english}
\begin{abstract}
Tick-borne encephalitis virus (TBEV) is responsible for the most serious
human viral tick-borne disease in Europe. A greater knowledge of the
natural foci of TBEV and their fluctuating characteristics is essential
to better assess the temporal variations of TBEV cycle's epidemiological
risks. To describe the seasonal and inter-annual variations of the
TBEV-cycle epidemiological parameters related to TBEV nymph-to-larva
transmission, exposure of small mammals to TBEV and tick aggregation on
small mammals, a longitudinal survey was conducted over a 3-year period
to monitor a TBEV focus in a mountain forest in Alsace, eastern France.
We trapped small mammals and collected questing ticks five times per
year from 2012 to 2014. TBEV prevalence in questing nymphs was lower in
2013 than in 2012 and 2014, probably because small mammals (Myodes
glaerolus and Apodemus flavicollis) were more abundant in 2012, reducing
tick aggregation and co-feeding transmission between ticks. This
prevalence was higher in autumn than spring, which suggests that
nymph-to-larva transmission of TBEV mainly occurs in spring. Despite
these variations in prevalence, the density of infected questing nymphs
was constant over time, leading to a constant risk for humans. The
seroprevalence of small mammals was also constant over time although the
proportion of small mammals harbouring ticks varied between years and
seasons. Our results draw attention to the importance of considering the
complex relationship between small mammal densities, tick aggregation on
small mammals, density of infected questing nymphs and prevalence of
infected nymphs in order to forecast the risk of TBEV for humans.%
\end{abstract}%



\sloppy


\textbf{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 (S\selectlanguage{ngerman}üss, 2011). TBEV-Eur is mainly
transmitted to a host by a tick bite and is maintained in nature by a
cycle involving ticks --- mainly \emph{Ixodes ricinus---} and small
mammals, especially those belonging to the genus \emph{Apodemus}
and\emph{Myodes} (Süss, 2011). \emph{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 (\emph{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 \emph{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 \emph{Apodemus} and \emph{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,
J\selectlanguage{polish}ę\selectlanguage{english}drzejewski, J\selectlanguage{polish}ę\selectlanguage{english}drzejewska, \& Pucek, 1993; Stenseth, Viljugrein,
J\selectlanguage{polish}ę\selectlanguage{english}drzejewski, 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
genus\emph{Apodemus} and \emph{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;
Ros\selectlanguage{ngerman}à 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 \emph{Apodemus flavicollis}
and\emph{Myodes 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.

\textbf{Materials and Methods}

\textbf{Study area}

The study focused on a 4.0-hectare area at Murbach (47°55'03 N, 07°08'46
E; average altitude of 630 m) in Guebwiller valley, Alsace, which is the
region where most of the human cases of TBEV have been reported in the
last two decades (Hansmann et al., 2006) and where the virus was
isolated from questing ticks in 2010 (unpublished data). The site is
covered by mixed forests classified as \emph{Asperulo-Fagetum} beech
forests, with a predominance of \emph{Fagus sylvatica} beech
and\emph{Abies alba} European silver fir.

\textbf{Questing tick sampling}

Questing tick were counted and collected by dragging a 1-m² white
blanket over the same area as the rodent trapping grid (see below). The
trapping grid was divided into 16 smaller grids and the blanket was
dragged over three different 10-metre-long transects within these 16
grids. In all, 48 different transects totalling a surface area of 480 m²
were investigated within the 4.0-hectare area. Ticks were collected
during the first week of the month, once a month from April to October
in 2011 and from May to October in 2012, 2013 and 2014, with the
exception of July and August 2011, August 2013 and October 2014 when no
ticks were sampled. At the laboratory, ticks were first identified at
species level based on their morphology using appropriate keys and
descriptions (Pérez\selectlanguage{english}-Eid, 2007). All questing nymphs and adults were
washed in 70\% ethanol, rinsed twice in distilled water, dried, and
stored at -~80degC until tested for TBEV.

\textbf{Small mammal trapping and sampling}

Small mammals were trapped from 2012 to 2014 five times per year, in
mid-April and in each first week of June, July, September and October.
The trapping grid consisted of 196 live-traps (14 X 14 Uggland special
nr. 3, Grahnab, Sweden) set at 15 m intervals, covering a total area of
3.2 ha. For each session, traps were set for three consecutive nights
and baited with carrots and sunflower seeds. Trapped rodents were
individually marked with a transponder (Veterissimo Mini RWI-I, Vethica,
France). Blood samples were taken once per session through the
retro-orbital sinus. Species, body mass, sex and tick presence were
recorded and then the animals were released at the point of capture. We
used the minimum value of weight observed during a second capture
session to define the threshold value below which an individual was
considered juvenile. Accordingly, individuals weighting less than 14 g
for \emph{M. glareolus} and 16 g for \emph{A. flavicollis} were
considered juveniles. From April 2012 to July 2013, all the ticks found
on rodents were collected. In June and July 2012, given the high number
of small rodents captured, although all the animals were subject to
blood sampling and examined for tick presence, we only counted and
collected the ticks of a number of randomly-chosen animals infested by
ticks. All the ticks and blood samples were kept at +4degC until brought
back to the laboratory. There, the blood clot was gently detached from
the bottom of the Eppendorf tube before being centrifuged at 5000 rpm
for 5 min and the serum obtained was stored at -20degC until tested for
the presence of TBEV antibodies. Larvae were identified at genus level
and nymphs/adults at species level based on their morphology using
appropriate keys and descriptions (Perez\selectlanguage{english}-Eid, 2007). All the ticks were
washed in 70\% ethanol, rinsed twice in distilled water, dried, and
stored at -80degC until tested for TBEV

\textbf{Laboratory analysis}

TBEV antibody detection in rodents

Serum samples were screened for the presence of TBEV antibodies, using
an in-house indirect immunofluorescence assay developed to capture
specific IgG antibodies. ELISA plates were coated (2\selectlanguage{greek}µ\selectlanguage{english}g /well) with a
purified SNAP-TBEV EDIII recombinant protein or a non-relevant SNAP. The
recombinant SNAP-TBEV EDIII protein was produced on S2 cell lines from
the synthetic gene encoding the soluble ecto-domain (residues E293 to
E399) from TBEV strain Kumlinge A52 cloned into a pMT/BiP/V5-HisA vector
(Life Technologies, France) according to the manufacturer's
recommendations (Beck et al., 2015). For the non-relevant SNAP, we chose
a production with a pMT/BiP/V5-HisA vector alone without an added coding
gene. After overnight coating with antigen the plates were saturated
with 1\% BSA in PBS solution. Assays were performed in duplicate with
100\selectlanguage{greek}µ\selectlanguage{english}l of serum samples (diluted 1:500 in PBS), tested on SNAP-TBEV EDIII
recombinant protein and on the SNAP production on the same plate.
Following incubation for 1 hour at 37\selectlanguage{ngerman}°C the plates were washed three
times in PBS-Tween 0.05\%. The second antibody was a polyclonal goat
anti-mouse conjugated with horseradish peroxidase (Dako), diluted to
1:500. After 1 hour at 37°C the wells were washed four times with 300\selectlanguage{greek}μ\selectlanguage{english}l
of washing buffer. The bound peroxidase was revealed with 100\selectlanguage{greek}µ\selectlanguage{english}l 1.8mM of
o-Phenylenediamine (Invitrogen) in PBS and 0.02\%
H\textsubscript{2}O\textsubscript{2} according to the manufacturer's
instructions. After 40min in the dark at room temperature, the enzyme
reaction was stopped with 0.5M H\textsubscript{2}SO\textsubscript{4},
and the optical density (OD) was measured at 492nm on an automatic plate
reader.

The positive controls used to validate the ELISA plate protocol
consisted of serum from two groups of three Swiss mice that had
previously been immunised twice with the recombinant SNAP-TBEV EDIII
protein and used at a dilution of 1:5000. The negative controls were
three Swiss mice that had not been immunised twice with the recombinant
SNAP-TBEV EDIII protein. The samples were considered positive when the
duplicate mean OD values read for SNAP-TBEV EDIII minus the mean OD
values read for SNAP was higher than 0.100 OD.

TBEV detection in questing and fed ticks

Only ticks collected from 2012 to 2014 were analysed. For questing
ticks, adults were analysed individually and nymphs were analysed in a
pool of one to five ticks. For fed ticks (collected on small mammals),
larvae/nymphs were pooled per stage, engorgement status (fed, unfed),
animals and month of capture with one to ten ticks per pool, and adults
were analysed individually. Ticks were homogenised using 2.8 mm
stainless steel beads in a Precellys 24 lyser/homogeniser (Bertin,
France) at 5500 rpm for 20s. RNA was extracted using the Nucleospin RNA
II kit (Macherey Nagel, Germany) according to the manufacturer's
instructions. Purified RNA was eluted into 50 \selectlanguage{greek}µ\selectlanguage{english}l RNase-free water and
stored at -80\selectlanguage{ngerman}°C until use. RNA samples were screened for TBEV by
real-time RT-PCR targeting a 3\selectlanguage{english}' non-coding region of the TBEV genome
with specific primers and probes (Schwaiger \& Cassinotti, 2003).
Real-time RT-PCR Taqman assays were performed in a final volume of 20 \selectlanguage{greek}µ\selectlanguage{english}l
using the LightCycler 480 RNA Master Hydrolysis Probes master mix (Roche
Applied Science, Germany) according to the manufacturer's instructions
using 2 \selectlanguage{greek}µ\selectlanguage{english}l of RNA template. Positive and negative (water) controls were
included in each run. Real-time RT-PCR thermal cycling conditions were
as follows: 63\selectlanguage{ngerman}°C for 3 min, 95°C for 30 s, 45 cycles at 95°C for 10 s,
then 60°C for 30 s, followed by cooling at 40°C for 10 s. Conventional
RT-PCR followed by nested PCR using primers targeting the non-structural
protein gene NS5 (Puchhammer-Stöckl, Kunz, Mandl, \& Heinz, 1995) were
used to confirm the presence of TBEV in positive samples. Amplicons were
sequenced by Eurofins MWG Operon (Germany), and then assembled using
BioEdit software (Ibis Biosciences, Carlsbad). The online BLAST tool
(National Center for Biotechnology Information) was used to compare
results with published sequences listed in the GenBank sequence
databases.

\textbf{Descriptive and statistical analyses}

Definition of three seasons

We defined three seasons: season 1 for early spring (beginning of nymph
activity and of small mammal reproduction), season 2 for the end of
spring/early summer (peak of nymph activity and of small mammal
reproduction) and season 3 for end of summer/early autumn (decrease of
nymph and small mammal abundance levels). Given the small number of
positives, we grouped data per season.

For small mammals, season 1 corresponded to the captures of April;
season 2 grouped the captures of early June and July; and season 3
grouped the captures of early September and October. For each season, we
listed each small mammal individuals captured along with its tick
infestation status, TBEV seropositive status, and the number of captures
(one or two). An individual was considered to be infested by ticks and
TBEV seropositive in season 2 (respectively season 3) if it was found to
be infested by at least one tick and TBEV seropositive in June or/and
July (respectively in September or/and October). Similarly, an
individual found to be infested by ticks or TBEV seropositive in at
least one of the five trapping sessions of the year (from April to
October) was considered positive in the year.

For questing ticks, season 1 corresponded to data for early May; season
2 grouped data for early June and July; and season 3 grouped data for
early August, September and October.

Descriptive and statistical analyses of tick density and TBEV infection
prevalence in ticks

The monthly density of questing ticks was estimated for each tick stage
by the mean density of questing ticks along each sampling transect, and
was expressed as the mean number of ticks per 100 m² with a 95\%
confidence interval (95\% CI). The density of questing ticks was
estimated per season by the mean of the monthly density of questing
ticks in the corresponding months, and per year by summing the tick
density for each season (= annual cumulated DON). Tick questing density
per year was compared using the observed yearly peak density and the
annual cumulated DON with a Mann-Whitney \emph{U} test.

As TBEV infection prevalence in ticks is usually low, having been
described as lower than 1\% in Alsace (Bestehorn et al., 2018; Perez-Eid
et al., 1992), prevalence in ticks was expressed as the minimum
infection rate per 100 tested (MIR) based on the assumption that a
single tick was positive within a positive pool. Exact 95\% CIs were
calculated on the basis of binomial distribution. For the 2012-2014
period, MIR was calculated per season for each year. The inter-annual
and inter-seasonnal MIR of questing ticks was compared using Fisher's
exact test. If no TBEV was detected in a sample of ticks, we calculated
the minimum prevalence of TBEV that could be detected in ticks with a
probability of 95\% given the sampling size used by applying the formula
proposed by Cannon (Cannon, 2001).

We calculated the density of infected nymphs (DIN) per season by
multiplying the seasonal MIR divided by 100 by the seasonal density of
questing nymphs, and the DIN per year by multiplying the annual MIR
divided by 100 with the annual cumulated DON. We used the lower and
upper limits of the 95\% CI for MIR per season and year to estimate the
lower and upper bounds of the 95\% CI for DIN.

Descriptive and statistical analyses of rodent density, tick infestation
and TBEV seroprevalence in rodents

Rodent density was calculated per session and per species according to
the standardised closed population Schnabel method that takes into
account multiple marking occasions (Schnabel, 1938). Since the captures
were carried out on three consecutive days per session, we considered
that the population was closed for each session. Rodent density per
season was calculated using the mean of rodent density estimated for the
corresponding months.

The tick infestation prevalence for small mammals was defined as the
number of small mammals carrying at least one tick divided by the number
of small mammals inspected. We calculated tick infestation prevalence
per year, season, species and age class (juveniles vs. adults). Exact
95\% CIs were calculated on the basis of binomial distribution. We
described the infestation of small mammals by larvae and nymphs in
season 2 (the period of highest nymph density) of 2012 and 2013 by
calculating (1) the mean number of larvae and nymphs per individual
infested by ticks, (2) the proportion of small mammals infested by ticks
carrying nymphs only or nymphs and larvae, and (3) the proportion of
larvae feeding on small mammals that were also carrying nymphs. The
results for 2012 and 2013 were compared using a Mann-Whitney
\emph{U}test.

The tick infestation status of small mammals was modelled using a
logistic GLM (generalised linear model) with a binomial distribution and
logit link, and a binary response variable (absence of ticks = 0;
presence of ticks = 1). The variables considered were the small mammal
species, sex, season, year, and the interactions between year and season
(to allow seasonal patterns to vary between years) and between season
and species (to mimic seasonal variation of small mammal community
structure and density). We included the number of captures within the
season as an offset term. Some animals were captured in several
different seasons and constituted temporal pseudoreplication of
individual hosts. We therefore ran logistic GLMs for those individuals
with the tick infestation status in season 2 (respectively in season 3)
as the response variable, the infestation status in season 1
(respectively season 2), species, sex and year as the explanatory
variables and the number of captures in season 2 (respectively season 3)
as an offset term. As the infestation status of the previous season had
no effect on the infestation status of the following season, we
considered that these pseudo-replications would not bias the results of
the model.

We calculated the TBEV seroprevalence of small mammals per year, season
and species, and its exact 95\% CI based on binomial distribution. The
seropositive status of captured animals was modelled using a logistic
GLM as a function of the small mammal species, season and year. As few
individuals were seropositive (see Results), we limited the number of
variables included in the model.

Collinearity was checked in the models ensuring a variance inflation
factor (VIF) \textless{} 10 (James, Written, Hastie, \& Tibshirani,
2017). The backward elimination of explanatory variables was used to
identify the most parsimonious model with the smallest Akaike
information criteria (AIC).

Statistical computations were performed in R 3.5.0 (R Development Core
team, 2018). For all statistical tests, a p-value\textless{}0.05 was
considered statistically significant.

\textbf{Ethical statement} . The experimental protocol with small
mammals complied with EU Directive 2010/63/EU and was submitted to and
approved by the French Ministry of Research (APAFIS no.
2015120215112678). All efforts were made to minimize animal suffering.
The species studied are not protected in France or included in the IUCN
Red List of threatened species in France. The animal trapping took place
with permission from the landowners.

\textbf{Results}

\textbf{Questing tick densities}

All the ticks collected were identified as \emph{Ixodes ricinus}species.
Overall, questing \emph{I. ricinus} nymphs and adults were found on
vegetation in all the sampled months with a unimodal pattern (Figure 2).
The main period of activity for both nymphs and adults occurred between
April/May and early July. Questing larvae were found from April to
October and in higher numbers from May to early July, suggesting a
synchronous activity with nymphs.

Questing nymph densities varied significantly between years
(Mann-Whitney \emph{U} test, p-value\textless{}0.001). The annual
cumulative DON was higher in 2011 and 2013 (respectively 500 and 575
nymphs/100 m² with a maximum density per month of 270 and 350 nymphs/100
m²) than in 2012 and 2014 (respectively 120 and 240 nymphs/100 m² with a
maximum density per month of 110 and 150 nymphs/100 m²) (Figures 2 and
3). The annual cumulative density of questing adults fluctuated in line
with the annual cumulative DON, although the annual variations were
lower than for the nymphs. It varied from 12-18 adults/100 m² in 2012
and 2014 to 25-28 adults/100 m² in 2011 and 2013.

\textbf{TBEV infection prevalence in questing ticks and DIN}

A total of 4,054 questing \emph{Ixodes ricinus} ticks (7,070 nymphs in
570 pools, 219 females and 199 males in 200 pools) were tested for TBEV
(Supplementary Table 1). An overall MIR of 0.11\% (95\% CI: 0.05 --
0.22) of positive questing nymphs was observed. MIR in nymphs was
significantly lower in 2013 (0.03\%, 95\% CI: 0 -- 0.15) than in 2012
and 2014 (0.21\%, 95\% CI: 0.08 -- 0.43, Fisher's exact test,
p-value=0.03) whereas there was no significant difference between 2012
and 2014 (Fisher's exact test, p-value = 0.7, Figure 3). In 2012 and
2014, the MIR in nymphs was significantly lower in season 1 and 2
(0.13\%, 95\% CI: 0.04 -- 0.34) than in season 3 (0.92\%, 95\% CI: 0.19
-- 2.67, Fisher's exact test, p-value = 0.02) whereas there was no
significant difference between seasons 1 and 2 (Fisher's exact test,
p-value = 1, Figure 3). No questing adult was found to be infected. The
minimum TBEV prevalence that could be detected in adult ticks with a
probability of 95\%, given the overall sampling size from 2012 to 2014
was 0.7\%. This suggests that the overall prevalence in adults was less
than 0.7\%.

The annual cumulated DIN appeared lower in 2013 than 2012 and 2014
(Figure 3) but the difference was not significant (2013: 0.14 infected
nymphs per 100 m ², 95\% CI: 0 -- 0.76; 2012: 0.42 infected nymphs per
100 m², 95\% CI: 0.10 -- 1.23; 2014: 0.30 infected nymphs per 100 m²,
95\% CI: 0.08 -- 0.74). In other words, around 43 to 136 infected nymphs
quested a host each year within the study area as a whole. The DIN was
not different per season (Figure 3), ranging from 0.09 {[}95\% CI: 0.02
-- 0.25{]} to 0.15 {[}95\% CI: 0.02 -- 0.54{]}.

The eight sequences obtained from questing ticks (collected in 2012,
2013 and 2014) had 100\% identity with each other. One sequence was
deposited in GenBank (accession number: MT109187) and showed 99\%
homology with reference sequences from European subtype strains isolated
in ticks (Germany, GenBank KX268728; Finland, GenBank MK801808), and
human cerebellum (Finland, GenBank MG589937).

\textbf{Small mammal abundance and tick infestation prevalence}

Over the 15 capture sessions (totalling 8,820 trapping nights), 1,371
captures were recorded in total, corresponding to 906 different rodents
captured per session over the 15 sessions (hereinafter called
rodent-sessions) and 564 different individuals over the same 15
sessions: 276 \emph{Myodes glareolus} , 287 \emph{Apodemus flavicollis}
, 1 \emph{Microtus} \emph{agrestis} . Overall, 342 rodents were captured
during two or more trapping sessions. Data on each individual's weight
and sex were completed for more than 90\% of individuals for each
trapping session, except in September and October 2012 when 33
individuals died in traps due to the predation of a stone marten.

The populations of bank vole (\emph{M. glareolus} ) and yellow-necked
mice (\emph{A. flavicollis} ) peaked in 2012 (453 individuals trapped),
dropped dramatically in 2013 (32 individuals) and was intermediate in
2014 (85 individuals) (Figure 4). Every year, the density of small
mammals peaked during season 2 (June and July). Both species were
similarly abundant in 2012 whereas in 2013 and 2014 ---during the years
of the lowest small mammal abundances---, \emph{A. flavicollis} was
captured slightly more often than \emph{M. glareolus} (Figure 4). In
2012, bank voles were more abundant than yellow-necked mice in April and
in September-October. Overall, 74 individuals were captured as
juveniles, of which nine (12\%), 41 (55\%) and 19 (26\%) were
respectively captured in April, June and July.

The proportion of rodents infested at least once by ticks significantly
varied between years and seasons (Table 1). For all years, the
prevalence of tick infestation on rodents was the highest in season 2
compared to the two other seasons (Chi², 2012, p-value \textless{}
0.001, 2013, p-value = 0.001, 2014, p-value \textless{} 0.001). The
prevalence of tick infestation was lower in 2012 than 2013 and 2014
except in season 1. In season 2, this prevalence varied from 44\% (95\%
CI: 38.8 -- 49.3) in 2012 to 94.2\% (95\% CI: 85.8 -- 98.4) in 2013 and
100\% (95\% CI: 71.5 -- 100) in 2014.

The best GLM for tick infestation prevalence on rodents included the
small mammal species, season, year, the interaction between season and
year and the interaction between season and small mammal species (Figure
5). Small mammals were significantly more likely to be infested by ticks
in season 2 than in season 1 and 3. Yellow-necked mice were about twice
as more likely to be infested than bank voles in season 2 of 2012. In
other seasons and years, there was no significant difference in the
prevalence of tick infestation between species.

A total of 349 larvae of \emph{Ixodes sp} ., 17 nymphs of \emph{I.
ricinus} and 4 females of \emph{I. trianguliceps} were collected on 152
rodent-sessions in 2012 and 2013. During June and July, the mean number
of larvae and nymphs per infested rodent-session (the intensity of tick
infestation) was higher in 2013 (5.9 ticks +/- 4.8) than in 2012 (2.2
ticks +/- 2.2) (Mann-Whitney \emph{U} test, p-value = 0.01, Table 2) and
was not significantly different between species (Mann-Whitney
\emph{U}test, 2012: p-value = 0.64, 2013: p-value = 0.45, Table 2). The
proportion of rodent-sessions infested by nymphs and co-infested by
larvae and nymphs was higher in 2013 (6/12 and 5/12 respectively) than
in 2012 (3/67 and 2/67 respectively, Table 2, Fisher's exact test,
p-value\textless{}0.001). As a consequence, only 2.7\% (4/127) of larvae
fed simultaneously with nymphs in 2012 vs. 57.8\% (37/64) in 2013.

\textbf{Detection of TBEV antibodies in small rodents}

We analysed 448 serum samples from 249 \emph{M. glareolus} and 381 from
274 \emph{A. flavicollis} . Overall, 4.2\% {[}95\% CI: 2.6 -- 6.3{]} of
individuals and 2.8\% {[}95\% CI: 1.8 -- 4.1{]} of rodent-sessions were
seropositive for TBEV. This proportion did not differ significantly
between years (Fisher's exact test, p-value =0.43), varying from 1.3 \%
{[}95\% CI: 0.0 -- 7.1{]} in 2014 (n=76 individuals) to 4.7\% {[}95\%
CI: 2.9 -- 7.2{]} in 2012 (n=422 individuals). Seroprevalence levels
were similar between seasons in 2012 (3.2-5.2\%, Fisher's exact test,
p-value =0.53) (Table 1), with TBEV antibodies detected from April to
October. In 2013 and 2014, TBEV antibodies were inconsistently detected
throughout the year (Table 1).

Among the 22 seropositive rodents, three and one rodents were
re-captured respectively one and two months after the detection of their
seroconversion. TBEV antibodies were detected in two consecutive months
for only one individual. Two juvenile yellow-necked mice (out of 61
juveniles of both species tested) with a body mass of 11 g and 15 g
tested seropositive.

None of the variables included in the logistic model --- species, year
and season --- had a significant effect on the probability that a rodent
would be seropositive.

\textbf{Detection of TBEV in feeding ticks}

All the ticks collected on rodents in 2012 and 2013 were tested for
TBEV. In 2012, a MIR of 1.1\% {[}95\% CI : 0.2 -- 3.1{]} was found on
larvae (n=283) and no nymphs (n=9) nor females (n=4) were found to be
infected. No larvae were found positive in season 1. The MIR in larvae
did not differ significantly between seasons 2 and 3 (season 2: 0.7\%
{[}95\% CI: 0.0 -- 3.7{]}, season 3: 4.2\% {[}95\% CI:0.5 -- 14.2{]},
Fisher's exact test, p-value=0.15). TBEV was detected in larvae feeding
on three rodent-sessions (2.2\% of rodent-sessions infested by ticks,
n=137): one seronegative \emph{A. flavicollis} captured in June (1/67
rodents infested by ticks during the June session) and two
seronegative\emph{M. glareolus} captured in September (2/22 rodents
infested by ticks during the September session). These animals were not
re-captured later. None of the feeding ticks collected in 2013 were
found to be infected. Overall, 31 larvae and one nymph feeding on eight
TBEV-seropositive rodents were negative for TBEV.

The three sequences obtained from feeding ticks (collected in 2012) had
100\% identity with each other. One sequence was deposited in GenBank
(accession number: MT109186) and showed 100\% homology with sequences
obtained from questing ticks during our study, and 99\% homology with
reference sequences from European subtype strains isolated in ticks
(Germany, GenBank KX268728 ; Finland, GenBank MK801808), and human
cerebellum (Finland, GenBank MG589937).

\textbf{Discussion}

Knowledge about the seasonal and inter-annual variation of TBEV
nymph-to-larva transmission and small mammal exposure to TBEV is still
sparse, especially in France where only one study was conducted prior to
this one. Our study describes and monitors the parameters related to
nymph-to-larvae transmission and small mammal exposure during a 3-year
period in a forest in Alsace, eastern France.

\textbf{Low circulation of TBEV in the studied site}

TBEV was detected in nymph ticks from 2012 to 2014 with a very low
prevalence varying annually from 0.03\% {[}95\% CI: 0 -- 0.15{]}) to
0.24\% {[}95\% CI : 0.07 -- 0.62{]}. Although adult ticks are generally
more frequently infected than nymphs (Carpi, Bertolotti, Rosati, \&
Rizzoli, 2009; Gaumann, Muhlemann, Strasser, \& Beuret, 2010; Perez-Eid
et al., 1992; Rieille et al., 2014), our study did not detect the virus
in adult ticks. This could nonetheless be explained by our small sample
size. The method of collecting ticks by blanket dragging is more
suitable for collecting nymph ticks than for adult ticks of \emph{I.
ricinus} . Considering the number of adults tested for TBEV, its
prevalence in adult ticks was estimated to be lower than 0.7\%. These
estimations fit within the lower range values found in other studies in
Europe, ranging from \textless{}0.1 to 5\% (Süss, 2011), and are similar
to those observed by Perez-Eid et al. (Perez-Eid et al., 1992) in the
Neuhof forest in Alsace between 1970 to 1974 with a maximum prevalence
of 0.12\% {[}95\% CI : 0.04 -- 0.44{]} in nymphs and 0.77\% {[}95\% CI :
0.37 -- 1.42{]} in adults.

In rodents, anti-TBEV antibodies were detected from 2012 to 2014 in 0\%
- 5.2\% of individuals per season. It is worth noting that TBEV
antibodies can only be detected during a short period between the
5\textsuperscript{th} and the 13\textsuperscript{th} day up to the
42\textsuperscript{nd} day post-infection (Achazi et al., 2011; Milan
Labuda et al., 1997). This is consistent with the observed disappearance
of antibodies on seropositive animals captured a second time in our
study and the finding of seronegative animals carrying infected larvae.
These animals may have been recently infected and not yet seroconverted.
Therefore, TBEV seroprevalence in small mammals is a good proxy for the
occurrence of new infections in a month, although it is impossible to
distinguish antibodies due to maternal transfer from those developed
after a recent infection. In small mammals, maternal antibodies
generally last from 6 to 10 weeks (Gomez-Chamorro et al., 2019;
Grindstaff, Brodie, \& Ketterson, 2003; Kallio et al., 2006) but this
duration is unknown for TBEV. We captured two juveniles aged 3-4 weeks
according to their weight that were seropositive. Considering the time
needed for seroconversion after an infection, these antibodies were
probably maternal antibodies. Although serological tests and sampling
design vary according to studies, the seroprevalence we observed in
2012-2015 is similar to that found by Perez-Eid et al. in Alsace
(Perez-Eid et al., 1992) (2.4\%) and also fits within the lower values
found in western and central Europe (prevalence ranging from 1.6\% to
23\% (Achazi et al., 2011; Bakhvalova, Potapova, Panov, \& Morozova,
2009; Caroline Burri et al., 2012; Cagnacci et al., 2012; Knap et al.,
2012; Kozuch, Gresíková, Nosek, Lichard, \& Sekeyová, 1967; Tonteri et
al., 2011)). In 2012 and 2013, we also observed a small proportion of
rodents from which feeding ticks acquired the virus (2\%) associated
with a low prevalence in feeding ticks (1.6\%).

Our results in rodents and questing ticks therefore suggest a very low
circulation of TBEV in our site. This strengthens the hypothesis that
the virus circulates at a very low level in the natural foci of the
Alsace region, located at the western boundary of TBEV distribution.
Randolph et al. (S. E. Randolph et al., 1999) theorised that the
strength of TBEV enzootic cycles was influenced by the number of larvae
co-feeding with nymphs on small mammals. Like the results of Perez-Eid
et al. (Perez-Eid, 1990) in Alsace in the 1970s, we observed a very low
number of co-feeding ticks in June-July during the period of highest
tick activity, especially in 2012. We only captured 4.5\% of rodents
infested by nymphs in 2012 when rodent abundance was high, whereas 17\%
to 33\% of rodents were reported to be infested by nymphs in
TBEV-infected sites in Switzerland and Slovakia (C. Burri et al., 2011;
S. E. Randolph et al., 1999). In addition, the mean infestation of
larvae per rodent-session infested by ticks (2.2) and per rodent-session
infested by nymphs (2) in 2012 was also lower than that observed in
other TBEV-infected sites (from 10 to 80 larvae per infested rodent and
from 5 to 65 larvae per host infested by nymphs) (C. Burri et al., 2011;
S. E. Randolph et al., 1999; Rosà et al., 2007). However, the apparent
difference in the number of feeding ticks in our study compared to other
field observations might be slightly lower considering that our method
for counting feeding ticks is not directly comparable with the methods
employed in other field studies and may have increased the
underestimation of the number of feeding ticks.

\textbf{Seasonal and inter-annual variation of TBEV prevalence in ticks
and rodents in 2012-2014}

Although the variations were low, we detected an annual and seasonal
effect on NIP (the TBEV prevalence of questing nymphs) but neither on
DIN nor rodent seroprevalence. Our study site in 2012-2014 was
characterised by a high inter-annual and inter-seasonal variation in
small mammal and questing nymph densities. Annually, the peak of
activity for questing larvae and nymphs, of larvae feeding on small
mammals, and of small mammal density occurred during the same season,
i.e. in June-July. The density of bank voles and yellow-necked mice
started to decrease from September. This decrease was greater for
yellow-necked mice than for bank voles, only 13\% of yellow-necked mice
being captured in September-October. This seasonality is similar to
field observations in TBEV-infected sites in neighbouring countries (C.
Burri et al., 2011; S. E. Randolph et al., 1999; Rosà et al., 2007), but
differs from findings in Brittany, western France, where the peak of
small mammal densities and of feeding larvae were found to occur in
autumn (Perez et al., 2017). Our observations on inter-annual variations
comply with previous observations (Kiffner et al., 2011; Krawczyk et
al., 2020; Ostfeld et al., 2018; Perez et al., 2017; Rosà et al., 2007):
we found that the density of questing nymphs was higher the year
following a year of high rodent density and that the intensity of tick
aggregation on small mammals was higher when the density of small
mammals was low. Indeed, the prevalence of infestation by both stages
--- larvae and nymphs --- and the mean infestation was higher in 2013 (a
low-density year for small mammals) than in 2012 (a high-density for
small mammals).

NIP was higher in both 2012 and 2014 compared than 2013; this is
contrary to the density of questing nymphs, for which we found opposite
variations. However, DIN was near-constant over those years. Therefore,
our results suggest that the proportion of infected larvae produced was
higher in 2013 when more larvae and nymphs fed on the same very few
hosts because there were fewer hosts available. This led to a higher
proportion of infected questing nymphs the year after. This is
consistent with the fact that the proportion of larvae feeding on
rodents infested by nymphs was higher in 2013 than 2012. Therefore, the
inter-annual difference in NIP can be explained by the variation in
aggregation intensity of ticks on rodents along with rodent abundance.
However, the overall number of fed larvae produced was probably lower in
2013, the year of a lower abundance of small mammals. Consequently, the
DIN stayed constant over the years. Our study was only conducted over a
3-year period and the results cannot be generalised. Further
investigations over a longer period would be needed to better understand
the relationship between DON, DIN, NIP and small mammal density for
TBEV. Few studies have investigated these relationships for other
tick-borne pathogens amplified by rodents. In North America, Ostfeld et
al. (Ostfeld et al., 2018) studied for 19 years the relationship between
the \emph{Ixodes scapularis} tick, the \emph{Borrelia burgdorferi} sl.
bacteria and the white-footed mouse (\emph{Peromyscus leucopus} ). They
found no effect of white-footed mouse density on
NIP\textsubscript{\emph{Borrelia} \emph{burgdorferi} sl}. whereas DIN
NIP\textsubscript{\emph{Borrelia} \emph{burgdorferi} sl} was positively
but not linearly related to mouse density in the previous year. At low
mouse density values, DIN \textsubscript{\emph{Borreliaburgdorferi} sl}.
was almost constant. In a short-term study in the Netherlands, Krawczyk
et al. (Krawczyk et al., 2020) observed that the relationship between
NIP, DIN and the rodent density the previous year depended on the
transmission mode of the tick-borne pathogens. Contrary to most
tick-borne pathogens amplified by rodents, TBEV is mainly transmitted by
co-feeding and the nymph-to-larva transmission of TBEV strongly depends
on the intensity of tick aggregation on small mammals. These elements
probably induced a different relationship between DIN, NIP and rodent
density that it would be interesting to investigate more.

Although the difference was small, TBEV prevalence in questing ticks was
higher in autumn than in spring. This finding had already been observed
in the previous study conducted in Alsace (Perez-Eid et al., 1992) and
in studies targeting other tick-and-rodent-borne pathogens in Germany
and Luxembourg (May, Jordan, Fingerle, \& Strube, 2015; Reye, Hubschen,
Sausy, \& Muller, 2010). There are no data on the timings of diapause
and moulting of \emph{I.ricinus} stages in the Alsace region. However,
if larvae that feed early in the season (in April-May) become nymphs
that quest later the same year (in July-October) as observed in
Switzerland (Perret et al., 2004), then this could be a potential
mechanism to explain the higher prevalence in questing nymphs in autumn.
Indeed, TBEV transmission to larvae might be higher in June-early July
since this period coincides in our study area with the peak abundance of
questing larvae and nymphs, of larvae feeding on small mammals and of
small mammals themselves, especially \emph{Apodemus sp} ., which has
been shown experimentally to be more efficient in fostering TBEV
transmission than \emph{M. glareolus} (M. Labuda et al., 1993; Milan
Labuda et al., 1997). Moreover, the virus titre may drop over time after
the moult and in unfed ticks undergoing a winter diapause, as observed
experimentally (Mishaeva and Erofeeva (Mishaeva \& Erofeeva, n.d.) cited
by Perez-Eid et al. (Perez-Eid, 1990)), which would also induce lower
prevalence in early spring.

Surprisingly, from 2012 to 2014 we did not detect any annual or seasonal
effects on rodents' TBEV infection probability in keeping with the
variation in the probability of a rodent being infested by ticks,
whereas DIN values were constant. A higher seroprevalence could be
expected in those years or seasons of low rodent abundance along with
the higher prevalence of tick (nymph) infestation on rodents, as
observed in other studies (Caroline Burri et al., 2012) and for other
tick-borne pathogens amplified by rodents (Perez et al., 2017).
Similarly, there was no species effect on the infection probability
despite \emph{A. flavicollis} being found to be more infested by ticks
in this study (especially in spring when \emph{A. flavicollis} is
abundant). This apparent contrast may result from a difference in the
immune response between species with a lower TBEV antibody titre and
persistence in \emph{A. flavicollis} compared with \emph{M.
glareolus}(Heigl \& Von Zeipel, 1966; Knap et al., 2012; Milan Labuda et
al., 1996, 1997; Zeipel \& Heigl, 1966). We could then have expected to
see an annual or seasonal difference in the infection probability per
species, but our sample size of rodents was probably too small to detect
a significant difference given the low seroprevalence of rodents, the
low persistence of the antibodies and the low DIN. Another explanation
for the absence of any annual effect on small mammal seroprevalence
could be a lower detection of seropositive small mammals the years when
they were scarce. Indeed, during those years, small mammals may acquire
the infection much earlier in the year given the high tick aggregation
level when fewer hosts are available. The exposure to this infection
would probably remain undetected since the anti-TBEV antibodies'
half-life is very short in small mammals.

In conclusion, this study shows that the virus was circulating at a very
low level in our study site. Despite this very low-level circulation, we
were able to observe a significant variation in the inter-annual and
inter-seasonal prevalence of TBEV in questing nymphs, indicating that
the nymph-to-larva transmission of TBEV varied over time. However, the
density of infected questing nymphs showed no detectable variations over
time, suggesting that the rate of exposure for humans was probably
relatively constant over the period of the study. The seroprevalence of
small mammals was constant over time although the prevalence of tick
infestation varied on an annual and seasonal basis. More studies are
needed to understand the relationship between the density of
TBEV-infected questing infected nymphs, the density of questing nymphs,
the prevalence of TBEV in questing nymphs, TBEV seroprevalence in small
mammals and small mammal density.

\textbf{Acknowledgements}

The authors are very grateful to all the volunteers who helped in the
capture of small mammals and to the French forestry agency, ONF, which
authorised trapping in Murbach forest. The authors would also like to
thank Philippe Desprès, Institut Pasteur, Paris, France for providing
recombinant proteins SNAP-TBEV EDIII and SNAP. We thank the ``Tiques et
Maladies a Tiques (TMT)'' group `of the ``Reseau Ecologie des
Interactions Durables'' (REID) for stimulating discussion and support.

\textbf{Funding}

This project was funded by ANSES through a transversal project grant
called ``PREV-TBE-TIQRO'' from 2010 to 2012.

\textbf{Conflicts of interest}

The authors declare that they have no conflicts of interest.

\textbf{Data availability statement}

The data that support the findings of this study will be available
online on a repository in zenodo after acceptance.

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\includegraphics[width=0.70\columnwidth]{figures/Figure1-schema-VF/Figure1-schema-VF}

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\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure2-questing-ticks-densities-per-100m2/Figure2-questing-ticks-densities-per-100m2}

\end{center}
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\includegraphics[width=0.70\columnwidth]{figures/Figure3-DIN-DON-MIR/Figure3-DIN-DON-MIR}

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\includegraphics[width=0.70\columnwidth]{figures/Figure4-density-small-mamals/Figure4-density-small-mamals}

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