Discussion
The present study aimed at providing a general picture of the exposure
to BoDV in horses in Spain. The aim was to collect a representative
sample of autonomous communities throughout the country. However, this
was only possible in 5 of the 9 communities sampled, most of them in
central Spain (a high risk area in terms of transmission of equine
infectious diseases). This might be attributable to the fact that horses
being exported to continental Europe or abroad often stop in central
Spain (Cruz et al., 2016b) and because there the majority of national
and international equestrian competitions are held (Deloitte, 2013).
Up to present BoDV causing clinical disease has never been reported in
horses in Spain and the only information published is a case of an
Appaloosa pony showing neurological signs and being seropositive to
EHV-1 and BoDV. However in the latter case BoDV was not isolated and no
molecular tests were carried out (Vitale et al., 2018). This is the
first study to define BoDV seroprevalence in breeding and sport horses
in Spain. Even though Spain was thought to be free from BoDV, our
results showed that the virus may be endemic in this country. The
determined seroprevalence (33.1%; 95% CI 29.0-37.3) was even higher
than the seroprevalence in horses from areas with clinical disease in
endemic countries like Germany (22.5%) (Richt and Rott, 2001) or other
countries like USA (2.7%) (Kao et al., 1993), Finland (2.4%) (Kinnunen
et al., 2007), France (8.9%) (Galabru et al., 2000) or Turkey (4.9%)
(Yesilbag et al., 2012). The high BoDV seroprevalence found in our study
contrasts with the fact that no clinical cases have been reported up to
date. This can be explained by subclinical infection (Richt, 2007) or
misdiagnosed disease due to the fact that BoDV is not considered as a
possible cause for neurological signs in horses in Spain. This report
would suggest the need to test for BoDV in horses showing neurological
signs, along with Equine Herpesvirus type 1 (EHV-1) or West Nile virus
(WNV).
The seropositive horses in this study showed moderate to high titres,
with up to 14.6% of seropositives presenting a titre of ≥1:2560. High
titres have been restricted to clinical disease, and some reports have
demonstrated that titres decrease relatively fast and are hardly
detectable in a subacute or chronic disease (Dauphin and Zientara,
2001). The high titres shown by the horses in our study would therefore
either suggest a recent (or frequent) exposure to BoDV or the fact that
antibody titres against BoDV could remain high for a long time, in
agreement with a study reported in Japan, where horses were found to be
continuously seropositive for a period of four years (Inoue et al.,
2002a). Further studies investigating possible hosts like shrews or
other rodents would be necessary to corroborate a high prevalence of
BoDV in them (Hilbe et al., 2006) which would enable a repeated exposure
to BoDV in Spanish horses.
Regarding BoDV seroprevalence by autonomous community, no significant
differences were found between the different sampled areas (p= 0.87).
The slight differences observed could be due to bias in sampling, and in
order to establish an influence of the area on BoDV seroprevalence, a
more extensive sampling covering all areas in Spain should be carried
out. Furthermore, the population of shrews and other rodents in Spain
should be investigated for the presence of the virus, in order to point
out risk areas in Spain for this disease (Encarnacao et al., 2013).
The results of our study show that BoDV is endemic and stable through
the years in Spain, since there were no significant differences between
the seroprevalence in the two sampling periods (2011-2013 and 2015-2016)
(p= 0.55). Breeding horses sampled in both periods showed a slight
increase in the seroprevalence in 2015-2016, although this increase was
not significant (p= 0.53) and could be a reflection of BoDV dynamics or
fluctuation in wild rodents (Richt, 2007, Encarnacao et al., 2013,
Kinnunen et al., 2013).
Similarly to other studies, our results determined that BoDV
seropositivity was not associated to sex (p= 0.85) (Inoue et al., 2002b,
Inoue et al., 2002a). The only study that found a higher seroprevalence
in female horses was carried out in Italy using circulating
immunocomplexes (CIC) ELISA tests, which are not considered the gold
standard test for BoDV diagnosis (Pisoni et al., 2007).
Even though we did not find statistically significant differences (p=
0.17), seroprevalence in breeding horses (which are often kept in the
field) was higher than in sport horses (kept in stables). Further
studies would be necessary to prove or exclude this association, since
several studies have found higher BoDV seroprevalence rates in stabled
horses, probably associated to a higher presence of reservoir hosts
(Richt, 2007, Inoue et al., 2002b). A higher presence of the virus in
breeding horses, already seen by Pisoni et al. (2007) in Italy would
entail the need to investigate this population and the reservoir hosts
present in the fields of Spain, since the risk of vertical transmission
of the virus and the occurrence of abortion has been documented in the
literature (Hagiwara et al., 2000).
In the population of breeding horses of our study, factors associated to
BoDV seropositivity were the climate, the age and the time of the year
when the horses were sampled (Table 2). Horses residing in an area with
a cold semi-arid climate (BSK climate from the Köppen-Geiger Climate
Classification) (Rubel and Kottek, 2010) were 3 times more likely to be
BoDV seropositive. This factor has already been reported elsewhere, and
is probably related to the living conditions for reservoir hosts, which
prefer a cold climate with low precipitations (Encarnacao et al., 2013).
Our results showed a lower seropositivity in adult horses (7 to 14 years
old) (OR= 0.31, 95% CI 0.16 to 0.63) and in older horses (15 to 24
years old) compared to young horses (1 to 6 years old), However the
decrease was not significant in the oldest horses (p = 0.69). In
contrast other viruses like Equine Viral Arteritis virus or EHV-1 showed
a higher seroprevalence in older individuals in the same population
(Cruz et al., 2016a, Cruz et al., 2016b). In addition, other BoDV
studies showed no association between age and seropositivity rate
(Pisoni et al., 2007, Inoue et al., 2002b). The decrease in the
seroprevalence in older individuals could be related to a decrease in
the antibody titres after infection, suggesting that SP breeding horses
would be infected in the early years, when they are in the field, and
not in stables.
The literature has shown that BoDV clinical disease peaks in the period
between March and June due to infection during the autumn-winter when
the horses are stabled (Richt, 2007, Kinnunen et al., 2013, Durrwald and
Ludwig, 1997), and that antibodies rise late in the course of infection,
along with clinical disease (Richt et al., 2000). However, in our study,
the breeding horses sampled from February to June (supposedly the period
associated to clinical disease and high antibody titres) were 2.7 times
less likely to be BoDV seropositive than horses sampled from July to
January (OR= 0.37, 95% CI 0.19 to 0.73). This is interesting and agrees
with the idea that, in Spain, breeding SP horses would be more likely to
encounter BoDV when they are young, and while they are out in the fields
during the spring-summer, showing a rise in their antibodies during the
autumn-winter. Therefore, further studies in order to characterise BoDV
reservoir hosts may help to elucidate BoDV transmission in horses in
Spain.
Given the recent association of BoDV-1 with eight cases of fatal
encephalitis in humans in areas of BoDV endemicity in Germany (Niller et
al., 2020), our results would rise the concern regarding the zoonotic
potential of this virus in Spain; therefore, BoDV should be included in
the differential diagnosis of encephalitis in humans.
In conclusion, the present study provides the first report on the
endemicity of BoDV in apparently asymptomatic breeding and sport horses
in Spain. We suggest that Borna Disease should be considered as a
differential diagnosis to Equine Herpesvirus Myeloencephalopathy or West
Nile of a horse suffering from neurological symptoms, and that human
cases of encephalitis should also be tested for BoDV. Epidemiological
studies at the local levels are paramount to forecast disease risks and
understand the transmission and the reservoir hosts involved, in order
to implement preventive measures.