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Citation: Fitzner, A.; Niedbalski, W.;
K˛esy, A.; Rataj, B.; Flis, M. European
Brown Hare Syndrome in Poland:
Current Epidemiological Situation.
Viruses 2022,14, 2423. https://
doi.org/10.3390/v14112423
Academic Editors: Jacek Ku ´zmak
and Magdalena Materniak-Kornas
Received: 21 September 2022
Accepted: 27 October 2022
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viruses
Article
European Brown Hare Syndrome in Poland: Current
Epidemiological Situation
Andrzej Fitzner 1, *, Wiesław Niedbalski 1, Andrzej K˛esy 1, Bogusław Rataj 2, * and Marian Flis 3, *
1Department of Foot and Mouth Disease, National Veterinary Research Institute, ul. Wodna 7,
98–220 Zdu´nska Wola, Poland
2Polish Hunting Association, District Board Nowy S ˛acz, 33–300 Nowy S ˛acz, Poland
3Department of Animal Ethology and Wildlife Management, Faculty of Animal Sciences and Bioeconomy,
University of Life Sciences in Lublin, ul. Akademicka 15, 20–950 Lublin, Poland
*
Correspondence: andrzej.fitzner@piwzp.pl (A.F.); brataj66@gmail.com (B.R.); marian.flis@up.lublin.pl (M.F.);
Tel.: +48-43-823-5134 (A.F.); +48-81-445-6038 (M.F.)
Abstract:
European brown hare syndrome (EBHS) is one of the main causes of mortality in brown
hares (Lepus europaeus) and mountain hares (Lepus timidus) in Europe. Since the mid-1990s, this highly
lethal and contagious plague has been widespread in many European countries, contributing to a
drastic decline in the number of free-living and farmed hares. A second lagovirus, able to infect some
species of hares is rabbit haemorrhagic disease virus 2 (RHDV2; GI.2) recognised in 2010, a new viral
emergence of RHDV (GI.1) which is known to be responsible for haemorrhagic disease in rabbits—
RHD. The aim of this study was to evaluate the current EBHS epidemiological situation on the basis
of the presence of antibodies to European brown hare syndrome virus (EBHSV) and anti-RHDV2
antibodies in sera collected from free-ranging hares in Central and Southeastern Poland in 2020–2021.
Additionally, studies on the presence of EBHSV and RHDV2 antigens or their genetic material in the
blood and internal organs taken from brown hares between 2014–2021 have been carried out. The
results of the serological examination showed nearly 88% of tested blood samples were positive for
EBHSV antibodies. No EBHSV was identified in the examined hares using virological and molecular
tests. The positive results of EBHS serological studies confirmed the circulation and maintenance
of EBHSV in free-living brown hares in Poland. However, no serological, virological or molecular
evidence was obtained indicating that the brown hares tested had been in contact with RHDV2.
Keywords: EBHS; European hare; lagoviruses; serology; epidemiology
1. Introduction
European brown hare syndrome (EBHS) isa highly contagious and fatal viral disease of
the European brown hare (Lepus europaeus) and mountain hare (Lepus timidus) affecting free-
ranging and farmed hares in Europe. The disease was first recognised in 1980 in Sweden,
although there are many indications of its earlier occurrence [
1
–
6
]. In the
mid-1990s,
as a
result of the intensive spread of the highly lethal and contagious EBHSV in many European
countries, a drastic decline in the number of free-living hare populations was noted [
7
–
14
].
EBHS shares clinical, pathological and epizootic similarities with another fatal viral disease
occurring in Leporidae—rabbit haemorrhagic disease (RHD)—recognised in 1984 [
15
].
Both aetiological agents, EBHSV (GII.1) and RHDV (GI.1–2) are members of the family
Caliciviridae, genus Lagovirus [
16
]. The first EBHSV isolate in Poland was confirmed in
1992 [
17
,
18
]. At that time, the presence of EBHS infections in the native hare population
was also proven on the basis of serological investigations [19,20].
Diagnostic methods of EBHS include serological tests, such as ELISA tests using
monoclonal antibodies (e.g., IZSLER kits, Brescia, Italy) to detect the presence of EBHSV-
specific antibodies, as well as virological tests for direct recognition of the antigen formed
Viruses 2022,14, 2423. https://doi.org/10.3390/v14112423 https://www.mdpi.com/journal/viruses
Viruses 2022,14, 2423 2 of 13
by the major structural protein of the capsid (VP60). Both conventional and real-time
RT-PCR tests are available for the detection of EBHSV RNA [21,22].
In addition to the natural host, EBHSV genetic material was also detected in faecal
swabs of wolves [
23
] and in fox carcasses [
24
], indicating that sympatric hare predators
may play a role in the passive dissemination of the EBHSV.
In 2010, a new type of RHDV (GI.1), RHDV2 (GI.2) was diagnosed in France [
25
].
RHDV2, similar to RHDV and its antigenic subtype RHDVa (GI.1a) is responsible for a
fatal infection in wild and domesticated rabbits (Oryctolagus cuniculus). However, RHDV2,
unlike to the previous two forms of this pathogen, is also capable of overcoming the inter-
species barrier and causing a fatal disease with clinical symptoms similar to EBHS in various
hare species, including brown hares as well as infecting non-lagomorph species
[22,26–32].
The first Polish RHDV2 outbreak was confirmed in 2016 in Łód´z Voivodeship [
33
]. Recently,
the presence of lagoviruses recombinants of the non-structural part of the EBHSV genome
and structural genes of RHDV2, resulting in the origin of a novel form of RHDV2, was
found [
34
]. At present, only one serotype of EBHSV is known, whereas due to the signifi-
cant antigenic differences found between RHDV2 and RHDV/RHDVa, two serotypes of
RHD causative lagoviruses can be identified [25,35,36].
The purpose of this study was to evaluate the current EBHS situation in Poland based
on a serological survey as well as virological and molecular analysis of samples obtained
from a free-living population of brown hares. The main serological study was carried out
on the native brown hares hunted in Central and Southeast Poland, for two winter seasons,
from December 2020 to December 2021. The blood samples and organ specimens were
taken from hares of both sexes, adults and young, of different weights in hunting districts
managed by Polish Hunting Association. Moreover, the results of the study on the presence
of RHDV2 in native, free-living hares by the serological, virological and molecular methods
were presented.
2. Materials and Methods
2.1. Specimens
In total, the samples of sera and internal organs from 170 free-living brown hares
were tested for the presence of EBHSV and RHDV2. Of these, 113 sera from wild brown
hares shot off in three hunting districts located in Southeast and Central Poland between
December 2020 and December 2021 were used for serological examinations. For the sake of
clarity of the description, two neighbouring hunting districts of the Lublin Voivodeship
(units no 199 and 216) are further treated as a common territory (Figure 1). Of this district,
103 samples of sera (batches I—IV) were collected over two successive hunting seasons from
2020–2021 (n= 73) and 2021–2022 (n= 30). The remaining 10 sera (batch V) were obtained
in December 2021 from the Łód´z Voivodeship (hunting unit 252). Moreover, five reference
anti-EBHS hare sera were obtained in Poland in the period 1999–2000 (collection of the
Department of Food and Environmental Virology of the NVRI) and two sera anti-RHDV2
taken from convalescent rabbits from RHD outbreaks in 2016 and 2021 (FMD laboratory’s
own collection) were included for serological examinations.
2.2. The Characteristics of the Hunting Areas
The areas are suitable for hares. In hunting units No 199 and 216 (adjacent to each
other), hare hunting on a limited scale has been practised continuously for many years. This
region includes 13,600 ha of fertile soil with 25.2% of forest covering. It is considered that
many small farms (<1 ha) and the strong heterogeneity of the environment are conducive
to the maintenance and development of the hares’ population in this area, where in 2017,
their number was estimated at 10 hares/100 ha [
37
]. The hunting district no 252, situated
in Central Poland near the village Rozprza (51
◦
18
0
09
00
N; 19
◦
38
0
44
00
E), includes 9500 ha
encompassing 49% of forests and 51% of arable agricultural land, with participation of
grasslands, meadows and peat bogs. The collective hare hunt in the 2021/2022 hunting
season was carried out for the first time after more than ten years of interruption. The
Viruses 2022,14, 2423 3 of 13
decision was based on a noticeable increase in hare population density and the effectiveness
of the predator reduction program implemented, including the red fox.
Viruses 2022, 14, 2423 3 of 12
● – hunting districts identification number
■ – administration unit (voivodeship)
Figure 1. Location of the places of blood sampling used.
2.2. The Characteristics of the Hunting Areas
The areas are suitable for hares. In hunting units No 199 and 216 (adjacent to each
other), hare hunting on a limited scale has been practised continuously for many years.
This region includes 13,600 ha of fertile soil with 25.2% of forest covering. It is considered
that many small farms (< 1 ha) and the strong heterogeneity of the environment are con-
ducive to the maintenance and development of the hares' population in this area, where
in 2017, their number was estimated at 10 hares /100 ha [37]. The hunting district no 252,
situated in Central Poland near the village Rozprza (51° 18´ 09” N; 19° 38´ 44” E), includes
9500 ha encompassing 49% of forests and 51% of arable agricultural land, with participa-
tion of grasslands, meadows and peat bogs. The collective hare hunt in the 2021/2022 hunt-
ing season was carried out for the first time after more than ten years of interruption. The
decision was based on a noticeable increase in hare population density and the effective-
ness of the predator reduction program implemented, including the red fox.
2.3. Hares Body Weight, Sex and Age
The body weight of the animals was determined with an accuracy of 0.1 kg immedi-
ately after the shooting. The sex of the hares was also determined directly in the field,
immediately after weighing, on the basis of the inspection of secondary sex characteristics.
The age of the hares was initially assessed on the basis of palpation detection of cartilage
thickening on the outer part of the ulna, which occurs in young hares. This made it possi-
ble to divide the material into young (up to 1 year old) and adult (over 1 year old) hares
[38,39]. Additionally, in order to verify the results obtained with the field method, the left
eyeball was dissected from each hunted hare and preserved in 10% buffered formalin. In
laboratory conditions, lenses were prepared from the secured material and dried in a la-
boratory dryer at a temperature of 100° C for 24 h to a constant weight [40]. After drying,
the lenses were weighed on a laboratory balance with an accuracy of 0.001 g and divided
into those weighing up to 290 mg (juveniles) and those weighing over 290 g (adults)
[37,40–43].
Additionally, 57 animals from seven voivodeships, collected over the period 2014–
2021 from hare deceased due to traffic incidents (n = 22), found dead in forest and rural
environments (n = 5), sent from the animal shelters (n = 8), and shot for hunting purposes
Figure 1. Location of the places of blood sampling used.
2.3. Hares Body Weight, Sex and Age
The body weight of the animals was determined with an accuracy of 0.1 kg imme-
diately after the shooting. The sex of the hares was also determined directly in the field,
immediately after weighing, on the basis of the inspection of secondary sex characteristics.
The age of the hares was initially assessed on the basis of palpation detection of cartilage
thickening on the outer part of the ulna, which occurs in young hares. This made it possible
to divide the material into young (up to 1 year old) and adult (over 1 year old) hares [
38
,
39
].
Additionally, in order to verify the results obtained with the field method, the left eyeball
was dissected from each hunted hare and preserved in 10% buffered formalin. In laboratory
conditions, lenses were prepared from the secured material and dried in a laboratory dryer
at a temperature of 100
◦
C for 24 h to a constant weight [
40
]. After drying, the lenses
were weighed on a laboratory balance with an accuracy of 0.001 g and divided into those
weighing up to 290 mg (juveniles) and those weighing over 290 g (adults) [37,40–43].
Additionally, 57 animals from seven voivodeships, collected over the period
2014–2021
from hare deceased due to traffic incidents (n= 22), found dead in forest and rural environ-
ments (n= 5), sent from the animal shelters (n= 8), and shot for hunting purposes
(n= 22)
,
were used for examinations (Table 1). The samples are archival biological material stored
frozen (
−
20
◦
C). Almost all of them were collected and made available by another labo-
ratory that had previously tested them for parasites and bacterial pathogens, but not for
lagoviruses. Some of the samples (n= 8) came from young hares, about 2 months old, who
died shortly after being placed in the animal shelter. These hares did not come from the
breeding sites and were not used for reintroduction.
Viruses 2022,14, 2423 4 of 13
Table 1. The type and origin of hare samples used in the study.
Date of
Collection/Additional
Information
Geographical Region of
Poland
/Voivodeship
No. of Hares
Sex
M/ F Weight Specimens Collected
Age
J/A (kg) Blood Liver Duod. Lungs Kidneys Spleen Faeces (f)/Heart
(h)
Hunting season
2021–2022 Central/Łód´z 10
serol. study batch V 2MJ, 2MA,
2FJ, 4FA 3.3–4.8 10 10 7 0 0 0 0
Hunting season
2021–2022
Southeastern/
Lublin
30
serol. study batch IV 5MJ, 9MA, 8FJ, 8FA 3.2–5.0 30 30 20 0 0 0 0
Hunting season
2020–2021
20
serol. study batch III 4MJ, 6MA,
4FJ, 6FA 3.5–5.0 20 20 5 0 0 0 0
20
serol. study batch II 2MJ, 5MA, 7FJ, 6FA 3.0–5.0 20 20 10 0 0 0 0
33
serol. study batch I 7MJ, 8MA,
6FJ, 12FA 3.6–5.0 33 32 32 0 0 0 0
2021/traffic incident North-Central/
Kuyavian-Pomeranian
(Bydgoszcz)
1 A - 0 1 1 1 1 0 0
2021 /animals shelter 3 J (8 weeks) - 0 3 3 0 0 0 0
2020 /animals shelter 1 J (4 weeks) - 0 1 0 0 0 0 0
2020/traffic incident Southeastern/
Lublin
1 A - 0 1 0 1 1 1 0
2019/group hunting 5 A - 0 5 0 0 0 0 1 (h)
2019/animals shelter
North-Central/
Kuyavian-Pomeranian
(Bydgoszcz)
1 J (4 months) - 0 1 1 0 1 1 0
2019/traffic incident 3 A - 0 3 0 3 3 0 3 (h)
2018/animals shelter 3 J (10 weeks) - 0 3 0 3 3 0 3 (f)
2016/traffic incident 1 J (11 weeks) - 0 1 0 0 0 0 0
2 A - 0 2 0 2 2 0 0
2016/traffic incident North/
Pomeranian
(Gda´nsk)
1 A - 0 1 0 1 0 1 1 (h)
2016/found in the forest 1 A - 0 1 0 0 1 0 1 (h)
Viruses 2022,14, 2423 5 of 13
Table 1. Cont.
Date of
Collection/Additional
Information
Geographical Region of
Poland
/Voivodeship
No. of Hares
Sex
M/ F Weight Specimens Collected
Age
J/A (kg) Blood Liver Duod. Lungs Kidneys Spleen Faeces (f)/Heart
(h)
2015/traffic incident Southwestern/
Lower Silesia (Wrocław)
1 A - 0 1 0 0 0 1 0
2015/found in the forest 1 A - 0 1 0 1 1 0
2015/traffic incident North-Central/
Kuyavian-Pomeranian
(Bydgoszcz)
2 A - 0 2 0 1 0 2 0
8 A - 0 8 0 0 0 2 0
2015/found in a rural
environment 2 A - 0 0 0 0 0 1 0
2015/group hunting Central/Łód´z 12 A - 0 12 0 0 0 0 0
2014/found in a rural
environmnet Central/Masovia
(Warszawa) 1 A - 0 1 0 1 1 0 0
2014/traffic incident Southwestern/Opole 2 A - 0 2 0 0 0 0 0
2014/group hunting Southeastern/
Lublin 5 A - 0 5 0 0 0 0 0
A—adult (>1 year); J—juvenile (<1 year); M—male; F—female.
Viruses 2022,14, 2423 6 of 13
2.4. Serological Methods
Antibodies to EBHSV and anti-RHDV2 in sera from 113 hares were detected using two
inhibition ELISA kits commercially available from the Istituto Zooprofilattico Sperimentale
della Lombardia e dell’ Emilia (IZSLER), Brescia, Italy, in accordance with the manufac-
turer’s instructions. Each ELISA kit contains the appropriate negative and positive control
sera, viral antigen, and specific Mabs conjugated to horseradish peroxidase, enabling highly
sensitive detection of EBHSV or RHDV2 antibodies. The serum titre corresponds to the
dilution of the serum tested, which inhibits the absorbance (492 nm) value of negative
control serum by 50%.
2.5. Virological and Molecular Tests EBHSV and RHDV2 Detection
The internal organs liver (n= 167), lungs (n= 14), kidneys (n=13), spleen (n= 10),
duodenum (n= 67), heart (n= 6), faeces (n= 3), and blood samples (n= 113) of 170 brown
hares were tested for the presence of EBHSV and RHDV antigens using RHD-EBHS ELISA
kit (IZSLER, Brescia, Italy) according to the manufacturer’s instruction, as previously
described [
44
]. For the detection of the genetic material of the relevant lagoviruses, the
real-time RT-qPCR with TaqMan primer and probe sets, which had been established for
RHDV2 [
21
] and EBHSV [
22
] were used. Amplification was carried out using the QuantiTect
Probe RT-PCR Kit (QIAGEN) and AB 7500 thermocycler.
3. Results
3.1. Seroprevalence
The total seroprevalence of EBHS virus antibodies in 113 free-living hares from two
different geographical regions of Poland, collected from December 2020 to December 2021,
has been estimated at 87.6% with a titre range of >10
≤
2560. In individual batches of the
samples, the percentage of seropositive hares was 91%, 75%, and 90% in five lots tested.
No EBHS antibodies were detected in 12.4% of the animals (Table 2).
Table 2.
Prevalence of antibodies to EBHSV in brown hares (Lepus europaeus) in the southeastern and
central region of Poland over the period December 2020—December 2021.
Batch/
Hunting Season
No.
of
Hares
Tested
Positive Negative
No. Percentage 1
Anti-EBHSV Antibody Titre Distribution 2
Titre (Pab ELISA/Mab ELISA) No. Percentage 1
%
>10–80 160–640 >640 <10
No. % No. % No. % No. %
I/2020–2021 33 30 90.9 18 60 11 36.66 1 3.33 3 9.1
II/2020–2021 20 15 75 5 33.33 8 53.33 2 13.33 5 25
III/2020–2021 20 18 90 11 61.1 6 33.33 1 5.6 2 10
IV/2021–2022 30 27 90 13 48.1 13 48.1 1 3.7 3 10
V/2021–2022 10 9 90 4 44.4 5 55.6 0 0 0 10
Total 113 99 87.6 51 51.5 43 43.4 5 5.1 14 12.4
Males 49 44 38.9 23 23.2 20 20.2 1 1 5 4.4
J 20 19 16.8 10 10.1 8 8.1 1 1 1 0.9
A 29 25 22.1 13 13.1 12 12.1 0 0 4 3.5
Females 64 55 48.7 28 28.3 23 23.2 4 4 9 8
J 28 23 20.4 13 13.1 9 9.1 1 1 5 4.44
A 36 32 28.3 15 15.2 14 14.1 3 3 4 3.5
1
Percentage of positive/negative in relation to the number of hares tested (in groups/total);
2
percentage of posi-
tive sera with the specified titre in relation to the total number of positives. A: adult (>1 year); J: juvenile
(<1 year).
The distribution of the EBHSV antibody in 113 sera showed 51.5%, 43.4%, and 5.1%
positive results with titres ranging from 10–80, 160–640, and >640, respectively. The highest
antibody titre (2560) was found in one adult female hare from Lublin voivodeship (batch III
from hunting season 2020/2021). In the two groups tested in this province, the percentage
of sera with low titres (10–80) was higher than sera with medium titres (160–640) and
ranged from 61% to 33%. In one of the two remaining groups of the region, the percentage
Viruses 2022,14, 2423 7 of 13
of hares with low and medium titres were evenly distributed (about 48%), while the other
showed an advantage of medium titres over low titres in the ratio of 53% to 33%. In the
hunting district located in the central part of Poland, the percentage of hares with low and
medium titres ranged from 55.6% to 44.4%. The overall proportion of EBHS seropositive
females (48.7%) was higher than that of males (38.9%) (Figure 2). The relationships in
individual batches of animals were similar or the percentage of positive results for females
was slightly higher than for males, both in the range of low and medium titres. Depending
on age, a generally higher percentage of EBHS-positive animals was found in adult hares
(50.4%) compared to young hares (37.2%). Similar relationships were observed in young
and adult hares in five lots tested (Figure 3).
Viruses 2022, 14, 2423 6 of 12
IV/2021–2022
30
27
90
13
48.1
13
48.1
1
3.7
3
10
V/2021–2022
10
9
90
4
44.4
5
55.6
0
0
0
10
Total
113
99
87.6
51
51.5
43
43.4
5
5.1
14
12.4
Males
49
44
38.9
23
23.2
20
20.2
1
1
5
4.4
J
20
19
16.8
10
10.1
8
8.1
1
1
1
0.9
A
29
25
22.1
13
13.1
12
12.1
0
0
4
3.5
Females
64
55
48.7
28
28.3
23
23.2
4
4
9
8
J
28
23
20.4
13
13.1
9
9.1
1
1
5
4.44
A
36
32
28.3
15
15.2
14
14.1
3
3
4
3.5
1 Percentage of positive/negative in relation to the number of hares tested (in groups/total); 2 percent-
age of positive sera with the specified titre in relation to the total number of positives.A: adult (> 1
year); J: juvenile (< 1 year).
The distribution of the EBHSV antibody in 113 sera showed 51.5%, 43.4%, and 5.1%
positive results with titres ranging from 10–80, 160–640, and >640, respectively. The high-
est antibody titre (2560) was found in one adult female hare from Lublin voivodeship
(batch III from hunting season 2020/2021). In the two groups tested in this province, the
percentage of sera with low titres (10–80) was higher than sera with medium titres (160–
640) and ranged from 61% to 33%. In one of the two remaining groups of the region, the
percentage of hares with low and medium titres were evenly distributed (about 48%),
while the other showed an advantage of medium titres over low titres in the ratio of 53%
to 33%. In the hunting district located in the central part of Poland, the percentage of hares
with low and medium titres ranged from 55.6% to 44.4%. The overall proportion of EBHS
seropositive females (48.7%) was higher than that of males (38.9%) (Figure 2). The rela-
tionships in individual batches of animals were similar or the percentage of positive re-
sults for females was slightly higher than for males, both in the range of low and medium
titres. Depending on age, a generally higher percentage of EBHS-positive animals was
found in adult hares (50.4%) compared to young hares (37.2%). Similar relationships were
observed in young and adult hares in five lots tested (Figure 3).
Figure 2. Distribution of EBHSV antibodies in seropositive hares, related to sex, place and period of
sampling.
0
10
20
30
40
50
60
70
80
M F M F M F M F M F M F
I/2020-21 II/2020-21 III/2020-21 IV/2021-22 V/2021-22 Total
>10-80 160-640 >640 positive in groups overall positive
Prevalence (%)
Sex: Male (M), Female (F)
Titres
Figure 2.
Distribution of EBHSV antibodies in seropositive hares, related to sex, place and period of
sampling.
Figure 3.
Distribution of EBHSV antibodies in seropositive hares, related to age, place and period of
sampling.
3.2. RHDV2 Serology
No RHDV2-specific antibodies were detected in the brown hares’ sera tested.
Viruses 2022,14, 2423 8 of 13
3.3. EBHSV and RHDV2 Detection
No EBHSV and RHDV2 antigens or their genetic material were detected in the internal
organs and blood samples of 170 brown hares tested (killed for hunting purposes, found
dead following traffic incidents in rural and forest areas, as well as in hares showing
nonspecific symptoms that died in an animal shelter) using the RHDV-EBHSV CR ELISA
kit and two RT-qPCR methods specific for each virus. RHDV2 and EBHSV virus RNA
was not detected by real-time RT-PCR also in blood samples of seropositive EBHSV hares,
including the hare with the highest anti-EBHSV antibody titres (2560).
4. Discussion
Data available from some European countries from the last 50 years show a drastic
decline in the abundance of free-living brown (Lepus europaeus) and mountain
(Lepus timidus) hares due to environmental, and demographic reasons, as well as the effect
of diseases [
1
,
2
,
45
–
49
]. In Poland, a constant trend in the decline of the abundance of brown
hares and grey partridges accelerated dramatically at the beginning of the 1990s [
50
,
51
]. At
the end of the 20th century, the average autumn hare density decreased twice as compared
to the 1970s and remained at a low level, ranging from 2 to 20 hares/100 ha, over the next
two decades. Unfavourable trends in the density of the hares’ population influenced the
safety of hunting exploitation of this species. As a result, in many hunting districts shooting
was discontinued or suspended. In the years 2014–2017, only 13–18 thousand hares per
year were obtained from hunting [
50
,
52
,
53
]. Among the main reasons for the decrease in
the population of hares in Poland, apart from the increase in the number of natural preda-
tors, such as foxes, the chemicalisation of agricultural crops, and far-reaching urbanisation
the emergence of the viral, deadly plague disease, EBHS, should be indicated [
2
,
50
]. The
attempts to increase the native population of hares by introducing captive-bred animals
bring limited results due to the low survival rate [
54
]. However, the latest official data
show a slight, systematic increase in the total number of hares in Poland, from about
560,000 animals
in 2010 to almost 710,000 in 2015 and 870,000 in 2021. The most hares were
recorded in voivodeships of central and eastern parts of the country: Masovia, Lublin, and
Łód´z voivodeships [
55
]. Accordingly, serological studies on EBHS presented in this paper
were carried out in hunting districts located in two of the aforementioned provinces, with
the highest population density of hares (Figure 1). In terms of the structure of agricultural
crops and intensification of agricultural production, the hunting areas from which samples
for serological tests were obtained can be defined as areas of low of agricultural production
intensity, with a predominance of small farms. These can be compared to one of the hunting
districts in Central Poland, where recently, hare density from 16.4 to 37.5 hares/100 ha
was found [45].
It can be assumed that EBHSV antibodies detected in this study in both juvenile (J)
and adult (A) apparently healthy hares of both sexes were produced as a result of contact
of these animals with EBHSV, despite the fact that the last time EBHSV-RNA was detected
in Poland in 2012 [
56
]. No EBHSV-positivity in 57 hares from 2014–2021 (the specimens
not related with hunting seasons 2020/2021 and 2021/2022 described) may be explained
by the fact that only 8.8% (n= 5) of these samples comprised animals found dead in the
field. As a highly pathogenic virus, EBHSV probably causes death in a short period so the
window of opportunity to find positive hares is narrow.
On the other hand, since the first data on the occurrence of EBHS in Poland were
described in the early 1990s, it can also be assumed that the disease has been endemic here
since then [
17
,
19
]. The above assessment is reflected in the phylogenetic characteristics of
the EBHS strains isolated in Poland between 1992–2004, confirming the close relationship
of the EBHSV G104 strain from 2004 with younger European EBHSV strains [
44
]. New
strains of EBHSV, which have recently been detected in the eastern regions of Germany
adjacent to Poland, also point out the active circulation of EBHSV in this part of Europe.
Moreover, genomic recombination between the non-structural part of the EBHSV genome
Viruses 2022,14, 2423 9 of 13
and the genes of the RHDV2 structural proteins indicates new scenarios of genetic variation
of both lagoviruses with the perspective of antigenic changes [33].
RHD has been endemic in Poland since 1988 [
53
,
57
]. The first classic strains of RHDV
(currently classified in the genetic group GI.1c) were detected at the same time as in neigh-
bouring countries—in Slovakia, Germany and the Czech Republic. The outbreaks were
linked to the spread of the RHD pandemic wave, which began in China in 1984, then spread
through Asia, and Europe and reached Mexico in North America [
15
]. At this point, it
should be explained that the emergence and maintenance of RHD in Poland are related to
the presence of numerous small-scale breeding rabbit farms, having a significant quantita-
tive advantage over industrial breeding farms. In recent years, companion rabbits have
gradually been playing an increasingly important role in the epidemiology of RHD. RHDVa
strains appeared in Poland 7–8 years after their diagnosis in Italy and Germany [
58
–
61
].
In turn, the first RHDV2 strains were diagnosed in Poland in domestic rabbits (including
companion animals) 6 years after their detection in France [
25
,
33
]. Currently, RHD out-
breaks are still detected in domestic rabbit farms, caused by both RHDVa and RHDV2.
However, the severity of the disease is not as high as in the early 1990s or the first decade
of the 2000s. Contrary to many Western European countries, wild rabbits do not seem to
play a significant role in the epidemiology of RHD in Poland. Their general low numbers
and occurrence restricted to small enclaves may favour the preservation of the antigenic
and genetic stability of RHDV [
44
]. So far, only the presence of RHDVa has been confirmed
in these rabbits. As there were no wild rabbits in the study area and there was no direct
evidence of the presence of RHDV2 in breeding rabbits in close proximity, at the time of the
survey, the chance of detecting RHDV2 antigen or its RNA in hares was significantly lower
than in a case of EBHSV.
NoRHDV2 transmission to the brown hares examined was also confirmed by the sero-
logical results. Since the pathogenic RHDVa and RHDV2 lagoviruses appeared in Poland a
few years later than in many other European countries, it can be expected that in the near
future, RHDV2 infections will also occur in native hares. To verify this assumption, it will
be necessary to continue research and obtain samples from other regions of the country.
The results of the EBHS serological survey presented in this paper are similar to the
data received in Italy in the early 1990s and previous serological investigations of Polish
free-living brown hares from 1992–1995, which showed 95% and 86% seroconversion,
respectively [
13
,
19
]. Italian data pointed out that the most numerous were the animals
with low (
≤
160) to average (1:320–1:640) antibody titres [
13
]. A study by Frölich and
colleagues performed in Poland in this period has shown a 38% prevalence of antibodies to
EBHSV in hares in the Czempin area (Greater Poland Province) [
20
]. At that time, the high
and mild seropositivity of EBHS in wild and captive hares was also recorded in Austria
(87.5%), Slovakia (73–90%), and Germany (29%) [
10
,
62
,
63
]. The observations in areas where
EBHS is endemic have shown that the circulation of the virus is linked to hare population
densities, the age and sex of the animals. According to this, the young hares, less than
2–3 months old, are naturally resistant to EBHSV infection. In the light of these data, a
high percentage of seropositive hares found in the presented research may result from a
higher population density in the studied areas than it would appear from official data. In
turn, early subclinical contact of juveniles with EBHSV would favour the development of
long-term protective immunity and a high percentage of seropositive hares, which is also
characteristic of areas with a high population density of hares and may explain their low
mortality [
36
,
64
–
66
]. The results of our research indicate that in the analysed populations
these key conditions, relating to a large percentage of seropositive young hares, were met
(Figure 3). The serological study of brown hares performed in two independent territories
located in Southeastern and Central Poland and used for hunting purposes confirmed the
high seroprevalence of anti-EBHSV seroreagents and, thus, the circulation and impact of
the virus on this species. The result of the presented serological study proves that EBHS is
still endemic in Poland. Based on our results, it can be assumed that the endemic presence
of EBHS, still one of the most important infectious diseases of hares, in addition to many
Viruses 2022,14, 2423 10 of 13
other pathological factors [
67
] does not have such a negative impact on the decline in the
population of these animals. At present, environmental changes are considered the main
cause of a deep decline in brown hare populations [
45
]. Moreover, it can be concluded that,
according to the data from other EBHS endemic infections [
36
], the high seroprevalence
in the hares tested was the result of early and regular contact of young hares with EBHSV.
This situation probably creates a certain state of equilibrium between the virus and the
susceptible hares, finally leading to low animal mortality. It can also be assumed that
hares living in freedom in the examined areas (possibly throughout the entire country) are
prepared for contact with the current antigenic and genetic form of EBHSV.
5. Conclusions
The results of this study confirmed the maintenance of EBHSV in the field and its
role as an important factor which can affect the mortality of the brown hare population in
Poland. The high percentage of seropositive hares shows that EBHS still has a significant
impact on hare density. This confirms the importance of diseases as an influencing factor
responsible for the survival of small game, a key element in maintaining the balance and
biodiversity in wildlife. The results obtained indirectly indicate the presence of a higher
density level of hares than expected in the native population of free-living brown hares and
the existence of a fairly high-level population immunity limiting the negative impact of
the EBHSV. The lack of evidence for the presence of rabbit haemorrhagic disease virus 2 in
the examined brown hares does not exclude the possibility of its appearance and supports
the continuation of research on both pathogenic lagoviruses, taking into account the other
areas of the existence of hares (a potential host of RHDV2) in the country.
Author Contributions:
Conceptualisation, A.F. and M.F. methodology, A.F. and W.N. investigation,
A.F., W.N. and M.F. formal analysis, A.F., W.N. and M.F. writing—original draft preparation, A.F., W.N.
and M.F. writing—review and editing, A.F., W.N., A.K., M.F. and B.R. visualisation, A.F. supervision,
A.F. and M.F. All authors have read and agreed to the published version of the manuscript.
Funding:
This research was funded by the Ministry of Agriculture and Rural Development of Poland.
Institutional Review Board Statement:
This study did not require the approval of the Ethics Com-
mittee. The study did not involve humans.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments:
The authors are grateful to members of hunting clubs leasing hunting districts
number 199, 216 and 252 for providing samples for research.
Conflicts of Interest: The authors declare no conflict of interest.
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