Discussion
This study describes the combined use of population genetics and
serological analyses to investigate the infection dynamics of a
rabies-related lyssavirus (RRLV) in the greater mouse-eared bat in South
Tyrol, the northernmost region in Italy. In particular, serological
tests were specifically directed towards EBLV-1, even if the circulation
of a highly related virus cannot be completely ruled out due to the lack
of virus identification in this study and, more in general, in this
species.
In this context, our aim was to define the spread of EBLV-1 (or its
related RRLV) in the area and to interpret serological data from
different locations using the connectivity between roosts derived from
genetic analyses as a proximate measure for the likelihood of viral
transmission. In addition, phylogenetic analyses were performed to
address the possible movement of individuals across the Alps, which
could be related with the dispersal of lyssaviruses that, despite being
widely circulating in Europe, have never been described in Italy
(Leopardi et al., 2018; Shipley et al., 2019). Our study has focused on
maternity roosts, which are easier to locate compared to groups of adult
males, and provide higher chances for virus amplification as a
consequence of a bigger colony size, more frequent and closer
interactions among individuals and due to the yearly replenish of
susceptible individuals after the birth pulse (Drexler et al., 2011).
The detection of antibodies against EBLV-1 in South Tyrolean maternity
colonies of greater mouse-eared bats dates back to 2012, despite no
mortality has ever been related to LYSV circulation so far (Leopardi et
al., 2018). The presence of antibodies in healthy bat colonies is
peculiar in the ecology of LYSVs, and has been extensively described
elsewhere (Amengual, Bourhy, López-Roíg, & Serra-Cobo, 2007; David T S
Hayman et al., 2010; Robardet et al., 2017; Suu-Ire et al., 2017).
Indeed, LYSVs are widely known for their pathogenicity in non-flying
mammals, invariably lethal when symptoms develop (Fisher et al., 2018).
In this study, we confirmed that all the investigated colonies are
exposed to the virus, as they show highly specific neutralizing
antibodies. Antibody titer were lower compared to other mammals; similar
pattern has been already described in bats for other pathogens, both in
the field and after experimental infection, and might be related with
the high efficiency of their innate, non-antibody-mediated pathway for
viral control (Baker, Schountz, & Wang, 2013). In addition, we detected
a significant variation of sero-positivity across a single reproductive
season, supporting seasonal variation in the circulation of LYSVs in all
the locations investigated and, overall, an increasing sero-prevalence
in late summer compared to spring. These data are consistent with
similar findings in Myotis and serotine bats (Amengual et al.,
2007; Robardet et al., 2017; Serra-Cobo et al., 2013), and could relate
to an increased virus transmission after the birth pulse, as suggested
for other viruses as a consequence of the large uptake of susceptible
individuals (Drexler et al., 2011). Indeed, higher antibody titers were
found during late summer, which could be a sign of seroconversion due to
viral exposure. This result differs from recent evidences determined for
EBLV-2 in Myotis daubentonii, for which it was suggested a peak
in virus transmission later during the autumnal swarming, when males and
females aggregate for mating (Horton et al., 2020). However, this is
rather predictable, considering that bats are highly variable in their
ecology, so that differences in life history traits including
hibernation, colony formation, parturition and mating are likely to
determine peculiarities in virus dynamics as well (D. T S Hayman et al.,
2013). Despite peaks in transmission might depend upon host ecology,
most species follow strict seasonal patterns which might affect
prevalence of viruses (D. T S Hayman et al., 2013); thus, we suggest
that comparison between data of sero-prevalence detected from different
years or locations should always account for differences in the sampling
period. In this context, 100% peak of sero-prevalence recorded in this
study is exceptional compared to previous evidences in the same host
species (Amengual et al., 2007; Schatz et al., 2014; Serra-Cobo et al.,
2002; Šimić et al., 2018) and is likely related to a very late sampling,
when adults were already dispersing for mating. In addition, it is wise
to consider to what extent the strong effect of seasonality might hide
the influence of other factors, including the geographical,
demographical or climatic data. Indeed, although preliminary analyses
suggested that the youngest animals were more likely to be serologically
positive compared to adults, the effect of age lost significance when
accounting for the dependence on seasonality. This is likely related to
non-homogeneous sampling across age classes of different sampling
campaigns.
None of the five roosts investigated was more likely to be infected,
suggesting that EBLV-1 or a related cross-reacting virus is circulating
across the area with no geographical restriction. Indeed, genetic data
derived from South Tyrolean Myotis myotis are consistent with a
single meta-colony, with movement of individuals between colonies likely
favoring viral transmission. Despite several studies already documented
regional panmixia of M. myotis at the nuclear level, we also
found lack of structuring based on mitochondrial DNA, strengthening the
hypothesis that population across the whole South Tyrol are highly
connected. In addition, FST values inferred from nuclear
and mitochondrial markers were similar, suggesting a much lower
philopatric behaviour than previously described in Germany, France and
Switzerland despite a similar spatial scale (Castella et al., 2001;
Petri, Pääbo, Von Haeseler, & Tautz, 1997), and supporting regular
movements of individuals between colonies, similarly to what observed in
the serotine bat (Eptesicus serotinus ) (Moussy et al., 2015). The
potential use of several roosts in the area was also supported by
occasional recapture in the big colony two of adult females which were
tagged elsewhere, despite a structured capture-recapture study has still
to be performed to quantify inter-colonial movements. Interestingly,
colony two showed the highest diversity in the area, also supporting the
mixing of bats in this spot, characterized by the biggest population
size and by a central position within the study area. However, such a
higher diversity might also be explained by the historical aggregation
of bats in this territory from different colonies already existing in
neighbouring zones; indeed, the church was completed only in the early
19th century, well after the remaining roosts included
in the study.
A meta-population structure for Myotis myotis is described here
for the first time, providing critical information to unravel dynamics
of LYSVs in this bat species. Indeed, previous mathematical models
suggested this bat was unable to sustain the circulation of EBLV-1
alone, supporting that sero-positivity in the Balearic Islands was
likely associated with inter-species transmission from the highly vagile
species Miniopterus scheibersii (Colombi et al., 2019;
Pons-Salort et al., 2014). In South Tyrol, the greater
mouse-eared bats form very large maternity colonies within churches,
where the interaction with other species is strongly limited compared to
the cave dwelling populations typical of the Mediterranean area. In the
area target of our study, colonies mostly consist of M. myotis,with M. blythii being sporadically found throughout the season
and a small population of M. emarginatus recorded in a single
roost from May to July. No co-roosting with Miniopterus
schreibersii have ever been recorded in the studied colonies (Drescher,
2004). However, the yearly detection of seropositive individuals highly
supports maintenance of the virus within the population (Leopardi et
al., 2018). In this context, the meta-population structure seen in our
sampling area as opposed to the close population model in the Balearic
Islands could explain virus maintenance in M. myotis even in the
absence of M. schreibersii . Indeed, several studies suggest that
the exchange of individuals between roosts may be one of the main
factors favoring the persistence of viruses across the whole population,
sometimes allowing for local fade out of infection with subsequent
reintroduction (Blackwood, Streicker, Altizer, & Rohani, 2013; Colombi
et al., 2019; Horton et al., 2020; Pons-Salort et al., 2014).
Despite being largely described across Europe, EBLV-1 has never been
detected in Italy (Leopardi et al., 2018; Shipley et al., 2019). Indeed,
the large majority of individuals included in our sample carried a
mitochondrial signature typical of the Italian peninsula, confirming the
Alps as major barrier for bats, including M. myotis (Ruedi &
Castella, 2003; Ruedi et al., 2008). However, our data also confirmed
that individuals belonging to the major European clade A and to the
clade C, previously reported only across the border between Switzerland
and North-Western Italy, are present in South Tyrol, suggesting that
transboundary admixture of animals is possible. Indeed, haplotypes from
all three clades were found in the small colony three, located just on
the border with Austria, which also showed a weak but significant sign
of nuclear fixation. While it is possible that these results are due to
continental individuals reaching this location during dispersal,
coexistence of several divergent matrilines in this population may also
result from the persistence of ancestral polymorphism during
coalescence. As colony three is located in the oldest church in the
area, dating back to 1337, it is likely that the current genetic pattern
is shaped by both present- time and recent- past demographic events.
Either way, these data support that South Tyrolean valleys might act as
narrow corridors across the Alps, allowing for the movement of
individuals from the north. In particular, the dispersal of bats across
the Alps could either provide or have provided in the recent past a
corridor for the introduction of LYSVs from continental Europe, where
they are widespread. Regarding EBLV-1, the origin of the current lineage
has been dated back to 1400 (Troupin et al., 2017), so that introduction
in Italy could be related to both ongoing or recent admixture of animals
from South Tyrol and other northern areas, where the virus has been
confirmed virologically. The spread of viruses across the home range of
bat populations has already been described for other pathogens,
including lyssaviruses, henipaviruses, coronaviruses and astroviruses
(Halczok et al., 2017; Leopardi et al., 2016; Olival et al., 2020; Peel,
Sargan, et al., 2013). Unfortunately, the lack of viral characterization
in this bat species prevented us to compare the geographical structuring
of LYSVs and Myotis myotis , which could help to explain the role
of this bat in the dispersal and evolution of EBLV-1 (Carver & Lunn,
2020; Olival et al., 2020). Challenges in the detection of bat viruses
are not peculiar to our study or to LYSVs, but are likely related with
low prevalence and short shedding period of viruses in these animals
and, more generally, in wildlife (Wilkinson & Hayman, 2017). In this
context, antibodies are easier to detect and persist longer, so that
serology is widely used as a proxy for virus circulation in wildlife to
model mechanisms of virus persistence within bat population even prior
to virus detection (Gilbert et al., 2013; Peel, McKinley, et al., 2013).
Regarding the genetic approach used in this study, we were able to
amplify 12 microsatellites described by Castella et al., (2001), and
developed a new protocol for the amplification of the complete control
region of the mitochondrion. This included sequencing of two
hypervariable regions (HVI and HVII), which were successfully amplified
and concatenated from most samples. Compared to previous studies which
had used the sole HVII, the additional presence of the HVI fragment
allowed to identify a higher variability, suggesting that our approach
might have a slightly better resolution for genetic analyses. South
Tyrolean Myotis bats showed variability in all but one
microsatellite (C113), confirming results already obtained by Berthier
et al (Berthier, Excoffier, & Ruedi, 2006). In general, mean diversity
within colonies was similar to what has already been described in other
studies, both at the nuclear and mitochondrial levels (Castella et al.,
2001, 2000; Ruedi & Castella, 2003; Ruedi et al., 2008). Our analyses
showed more heterogeneity for the parameter “nucleotide diversity”
compared to the “gene diversity” calculated within different colonies,
ranging between 0.001 to 0.013 and from 0.431 to 0.783, respectively
(Table 2). Indeed, gene diversity only considers the number of detected
haplotypes while the nucleotide diversity also takes into account the
degree of their divergence. Thus, the ten-fold higher nucleotide
diversity in colonies two and three is likely related to the admixture
of haplotypes that belong to different phylogenetic clades, as shown in
other studies (Castella et al., 2001; Ruedi & Castella, 2003; Ruedi et
al., 2008). Similarly to other studies, we determined genetic
structuring of M. myotis based on both nuclear and mitochondrial
markers, in order to test for female philopatry, which is generally
supported for this species (Castella et al., 2001). However, we found
comparable results from microsatellites and mitochondrial sequences,
thus disproving the expected pattern in our sample. These unexpected
results could be either related to peculiar ecology of South Tyrolean
populations or to lower spatial scale compared to other studies on the
same species.
In conclusion, we provide serological evidence for the circulation of a
RRLV in the South Tyrolean population of M. myotis, antigenically
related to EBLV-1. Unlike the Mediterranean populations, M.
myotis in northern Italy is a strictly house dwelling species, where
adult females aggregate in church roofs between April and September to
form large maternity colonies reaching over 2000 individuals, with a
remarkable chance for human encroachment (Zahn, 1999). As all LYSVs can
cause clinical rabies in humans, this study has a strong impact on
public health, regardless of the viral species actually circulating in
the study area. Fortunately, we founded a strong seasonal pattern,
suggesting the highest risk for human exposure might be limited in time,
easily predictable and likely associated with animal birth. In this
context, preventive measures to limit human exposure by limiting access
to the colony during most critical times could be beneficial to both
humans and animals, which is imperative considering the ecological
importance of this endangered species.
Genetic data supported animal movements between colonies, potentially
favoring viral dispersal across the host population home range and
likely allowing for viral maintenance in the area. In addition, genetic
data from this study provide important information on the ecology of the
species, suggesting that females might be more faithful to the natal
area rather than to the natal roost. Besides being crucial to inform
epidemiological modeling for disease dynamics, these results have
relevant implications for the conservation of this endangered species,
suggesting for example that monitoring and protection should be
performed at the regional rather than local level, as animal movements
between roosts might confound fluctuations in colony sizes.