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Research Article
High Mortality of Wild European Rabbits during a Natural
Outbreak of Rabbit Haemorrhagic Disease GI.2 Revealed by
a Capture-Mark-Recapture Study
Sa ´
ul Jim´
enez-Ruiz ,
1
,
2
,
3
Marta Rafael,
1
,
2
Joana Coelho,
1
,
4
Henrique Pacheco,
1
,
4
Manuel Fernandes,
5
Paulo C´
elio Alves,
1
,
2
,
6
,
7
and Nuno Santos
1
,
2
,
7
1
Research Centre in Biodiversity and Genetic Resources (CIBIO), Associated Laboratory (InBIO), Campus de Vairão,
University of Porto, Vairão 4485-661, Portugal
2
Program in Genomics, Biodiversity and Land Planning (BIOPOLIS), Campus de Vairão, Vairão 4485-661, Portugal
3
Animal Health and Zoonoses Research Group (GISAZ),
Competitive Research Unit on Zoonoses and Emerging Diseases (ENZOEM), University of Cordoba, Cordoba 14014, Spain
4
Interdisciplinary Research Centre in Animal Health (CIISA), Faculty of Veterinary Medicine, University of Lisbon,
Lisbon 1300-477, Portugal
5
Noudar Nature Park, Empresa de Desenvolvimento e Infra-Estruturas do Alqueva (EDIA), Barrancos 7230-031, Portugal
6
Department of Biology, Faculty of Sciences, University of Porto, Porto 4099-002, Portugal
7
EBM, M´
ertola Biological Station, Praça Lu´
ıs de Camões, M´
ertola 7750-329, Portugal
Correspondence should be addressed to Nuno Santos; nuno.santos@cibio.up.pt
Received 13 March 2023; Revised 22 May 2023; Accepted 7 June 2023; Published 19 June 2023
Academic Editor: Andrew Byrne
Copyright ©2023 Sa´ul Jim´
enez-Ruiz et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Rabbit haemorrhagic disease virus (RHDV) GI.2 has caused signicant declines in the abundance of wild European rabbits
(Oryctolagus cuniculus), contributing to the species being recently classied as “endangered” in its native range. e epidemiology
of this virus is still poorly understood despite its relevance for domestic and wild rabbits. During a longitudinal capture-mark-
recapture (CMR) study of wild Iberian rabbits, O. c. algirus, in a semiextensive breeding enclosure, an outbreak of RHDV GI.2
took place in January-February 2022, allowing us to estimate key epidemiological parameters of a natural outbreak. From April
2021 to July 2022, 340 rabbits were captured 466 times and individually identied, and some were vaccinated against myxoma
virus (MYXV) and/or RHDV GI.2. Sera were collected and tested for IgG specic for MYXV and RHDV GI.2, and data were
analyzed using multievent CMR models. During six weeks in January-February 2022, an estimated 81.0% (CI
95
77.1–84.3%) of the
population died. Intensive aboveground searches could recover 189 carcasses (50.5% of the estimated mortality, CI
95
41.8–63.4%),
with RHDV GI.2 detected in 6/7 tested. Apparent RHDV GI.2 seroprevalence rose from 15.4% (CI
95
8.0–27.5%) in January 2022
to 87.9% (CI
95
72.7–95.2%) in February 2022. e apparent mortality of RHDV GI.2-seropositive rabbits during the outbreak was
estimated as null, while for seronegative rabbits, it was 76.0% (CI
95
53.8–90.3%). Among the seronegative rabbits, mortality was
higher in unvaccinated (100%) than in recently vaccinated (60.0±16.6%) and in females (100%) than in males (52.0 ±17.1%).
Infected carcasses in the burrows might explain the medium-term disease persistence in the population following the outbreak.
Rabbits with antibodies at the cuto for seropositivity were fully protected from fatal infection. Females had a higher fatality rate
than males, underscoring the impact of RHDV GI.2 on the population dynamics of this endangered species.
Hindawi
Transboundary and Emerging Diseases
Volume 2023, Article ID 3451338, 9 pages
https://doi.org/10.1155/2023/3451338
1. Introduction
Capture-mark-recapture (CMR) studies are based on the
capture of animals using invasive (e.g., trapping) or non-
invasive methods (e.g., faecal samples) and their individual
identication using natural (e.g., phenotype or genotype) or
articial marks (e.g., tags). e animals are subsequently
released and eventually recaptured [1]. Such studies were
initially developed to understand ecological processes in
wildlife, including population demography, recruitment,
and survival rates [2, 3]. is approach was then extended to
epidemiological studies [4]. Inferences from CMR studies
have contributed to the management of wild species and
their pathogens [5, 6].
European rabbits (Oryctolagus cuniculus) were highly
abundant in their native range in the Iberian Peninsula and
Southern France until the introduction of the myxoma virus
(MYXV) in the 1950s [7]. e European rabbit is a keystone
species in its native range as it is a fundamental part of the
diet of many predators, ecosystem engineers, and seed
dispersers providing habitat for other species [8]. Rabbit
populations have undergone a decades-long decline cul-
minating in the recent reclassication by the International
Union for Conservation of Nature as “endangered” in its
native range [9]. Pathogens, particularly MYXV and rabbit
haemorrhagic disease virus (RHDV), are signicant drivers
of this decline [10–13].
Rabbit haemorrhagic disease virus is a Lagovirus that
causes fatal haemorrhagic hepatitis in domestic and wild
European rabbits, whose GI.1 variant has been present in the
Iberian Peninsula since 1988 [11]. A new variant RHDV
GI.2, rst detected in France in 2010 [14], rapidly spread
throughout Europe, replacing the previous GI.1 strain
[15–17]. e GI.2 variant is antigenically and epidemio-
logically distinct from the GI.1, notably showing high
pathogenicity in juvenile rabbits [18–20]. e RHDV GI.2
caused signicant declines in European rabbit abundance
following its emergence in the Iberian Peninsula and else-
where [21, 22]. While the pathogenicity of RHDV GI.2 in
European rabbits was thoroughly studied through experi-
mental infections [19, 23], its epidemiology in wild pop-
ulations in its native range is still largely unknown,
particularly for the southwestern Iberian subspecies
(Oryctolagus cuniculus algirus).
A comprehensive understanding of the epidemiological
processes that inuence pathogen dynamics in host pop-
ulations is critical for preventing and controlling diseases
[24]. Capture-mark-recapture studies could provide in-
formation on the epidemiology of RHDV GI.2 in wild
European rabbit populations and oer potential applications
in disease control [25]. e demography and epidemiology
of a population of the southwestern Iberian subspecies of the
European rabbit have been studied since 2021 through
a longitudinal CMR. e present study aims to describe key
epidemiological parameters of an outbreak of RHDV GI.2
during January and February 2022, providing useful in-
formation for disease control in wild and domestic
populations.
2. Materials and Methods
2.1. Study Area. A longitudinal CMR study of European
rabbits was performed in a 2.3-hectare semiextensive
breeding enclosure at Parque Natureza Noudar (PNN),
a 1,000 ha private estate in southeastern Portugal
(38°11′03.7″N, 7°02′24.4″W). e landscape at the breeding
enclosure and surroundings consist of a mosaic of scrub
(mostly Cistus sp.) sparsely forested with holm oak (Quercus
ilex). A perimetral 2 m-high rabbit-proof fence with elec-
trical wire prevented access by terrestrial carnivores and
rabbit emigration. Water and commercial feed were pro-
vided ad libitum year-round in the enclosure. Our team is
performing a long-term longitudinal study at several sites in
Portugal; the results obtained from 2019 to 2020 at another
enclosure in the same estate were previously reported [26].
2.2. Study Design and Sampling. Fifteen cage traps were
permanently placed near the pasture and feeding troughs in
the enclosure. ey were set 2 hours before sunset, baited
with vegetables, checked 2 hours after sunset, and again
1 hour after sunrise. Cage traps were closed during the day.
Between April 2021 and July 2022, nine trapping sessions of
two or three occasions (nights) each were performed in
April, June, July, and September 2021, and January, Feb-
ruary, May, June, and July 2022 (see X-axis in Figure 1), in
which 340 rabbits were captured 466 times.
Each trapped rabbit was individually identied with
a subcutaneous microchip when rst captured. A volume of
0.5–1.5 ml of whole blood (<0.25% body weight) was col-
lected by venipuncture of the saphenous vein, placed into
sterile clotting tubes, and centrifuged, and sera were col-
lected and stored at −20°C until analysis. Blood samples were
collected whenever the previous sampling of the individual
rabbit had occurred >1 month before. Rabbits were released
at the capture site, except for 200 rabbits translocated to
restock other breeding enclosures or free-ranging pop-
ulations within PNN.
Seventy-nine of the 146 rabbits released in the same
enclosure were vaccinated with a homologous inactivated
RHDV GI.2 vaccine (ERAVAC®, HIPRA, Spain), and 61 of
those 79 were also vaccinated with a heterologous live
MYXV vaccine employing Shope broma virus (MIX-
OHIPRA-FSA®, HIPRA, Spain), using the subcutaneous
dosages recommended by the manufacturers (0.5 ml). Until
January 2022, before the observed epidemic outbreak of
RHDV, only 36 RHDV GI.2-vaccinated rabbits were re-
leased back in the same enclosure; 33 of those 36 were also
vaccinated for MYXV.
To prevent pathogen transmission between individuals
and study sites, rabbits were kept in individual cleaned cloth
bags and handled with latex gloves over disposable pads, and
the equipment was sanitized with disinfectant wipes after
each trapping occasion. Live trapping and sample collection
were conducted under permits 23/2021 and 574/2022,
according to European Union directives on the protection of
animals used for scientic purposes (Directive 2010/63/EU)
and international wildlife standards [27, 28].
2Transboundary and Emerging Diseases
2.3. Rabbit Haemorrhagic Disease Virus Outbreak. Five
freshly dead rabbits were found in the breeding enclosure at
PNN during the nal trapping occasion in the January 2022
session (January 20
th
). After that, rabbit carcasses were
thoroughly searched aboveground in the enclosure two or
three times a week. Carcasses were collected under bio-
security measures, buried outside the enclosure and covered
in potassium hydroxide. Eorts to locate carcasses inside
burrows were not taken to minimize the risk of disturbing
rabbit litters during the peak birth season. A subset of the
recovered carcasses (n�7, including the ve initial ones)
were frozen until necropsy. Liver samples were collected and
tested for RHDV GI.2 using a chromatographic lateral ow
assay (INGEZIM®RHDV1/2 DIF CROM, EUROFINS,
Spain). is assay for RHDV GI.1 and GI.2 was recently
validated in liver samples of European rabbits, showing
a sensitivity of 94.4% and specicity of 100% compared to
molecular methods [29]. RHDV GI.2 was detected in 6 out
of the 7 necropsied rabbits.
2.4. Serological Analyses. An in-house indirect ELISA
(iELISA) to detect IgG specic against RHDV GI.2 [30] was
performed with minor modications, as previously de-
scribed by Pacheco et al. [26]. Briey, GI.2-derived virus-like
particles (VLP), expressed in a baculovirus expression sys-
tem and puried according to Almanza et al. [31], were
diluted in carbonate/bicarbonate buer (pH �9.5) and
absorbed to Nunc Maxisorp 96 wells ELISA plates (100ng/
well) by overnight incubation at 4°C. e plates were blocked
with phosphate-buered saline (PBS)-5% skim milk solution
and washed three times. Sera samples were tested at a 1/200
dilution in PBS-5% skim milk solution. e conjugate goat
anti-rabbit IgG/HRP (Bio-Rad) was added at 1/4,000 di-
lution, followed by the substrate (3.3′, 5.5′-tetrame-
thylbenzidine) (Abcam), reactions stopped with 100 µl of
1 M phosphoric acid, and the optical density at 450 nm
(OD
450 nm
) recorded within 15 min. Positive controls con-
sisted of pooled sera of rabbits with high iELISA readings
[26]. Negative controls were pooled sera of unvaccinated
domestic European rabbits raised indoors without a history
of clinical disease. To detect specic IgG against MYXV,
a commercial iELISA kit (INGEZIM®17.MIX.K1, EURO-
FINS, Spain) was performed according to the manufacturer’s
instructions.
All serum samples, positive and negative controls, were
tested in duplicate. e tests were valid if the average
OD
450 nm
of the two replicates of the positive control was >5x
the average OD
450 nm
of the two replicates of the negative
control. e iELISA results were standardized by the nor-
malized absorption ratios (NAR) according to the following
equation:
NAR �average OD450nm sample
2 x (average OD450nm negative control).(1)
e cuto for seropositivity was set at NAR �2.0 for
RHDV GI.2 and NAR �2.4 for MYXV, as previously de-
scribed [26]. ese tests using these cutos achieved 100%
diagnostic sensitivity and specicity [26].
2.5. Multievent Capture-Mark-Recapture Models.
Multievent CMR (MECMR) models were applied to sero-
logical data for each trapping session [32]. A single-state
goodness-of-t test was performed in U-CARE [33]. Test
3.SR indicated signicant transience (χ
2
�3.814, P<0.001,
7 df ). Transience can be dened as individuals captured for
the rst time having a lower probability of being recaptured
when compared with individuals previously captured [34].
No trap dependence was identied (test 2.CT χ
2
� −1.411,
P�0.158, 6 df).
Models were implemented using the software E-SURGE
[35]. e model included the matrices: initial state, survival
(time-varying survival probability), transitions between
seropositive and seronegative states (time-varying sero-
conversion probability; constant seroreversion probability),
detection (time-varying capture probability), probability of
being tested, and uncertainty in state assignment (corre-
sponding to the diagnostic sensitivity and specicity; see
[26]) (Supplementary material, Appendix S1). A quasi-
Newton nonlinear solver was used to obtain the maxi-
mum likelihood estimator, and 50 models run using dif-
ferent sets of random initial values were applied to avoid
local minima.
e following nonobservable states were considered in
the models: seronegative (S−), seropositive (S+), and dead
(D). In any sampling session, an individual rabbit may be
alive in classes S−, S+, or may be dead. In each sampling
session, the possible observations were as follows: not de-
tected (0), detected S−(1), detected S+ (2), or detected but
not tested (3). e probability of being assigned the event
“detected but not tested” was xed as the proportion of
detections where no blood was collected and no iELISA
result was obtained (38.3% of the captures). Models were
selected under an information-theoretical approach by their
Akaike information criterion corrected for small sample size
(AICc) [36]. Unequal time intervals between sampling oc-
casions were dened, with an interval 1 corresponding to
30 days, meaning the estimated parameters are monthly
probabilities. e rabbits removed from the study pop-
ulation were right-censored, so they did not contribute to the
demographic or epidemiological parameters after their last
capture.
Rabbit abundance was estimated for each trapping
session using nonspatial Jolly–Seber–Schwarz–Arnason
(JSSA) CMR models. ey were implemented using the
package “openCR” v. 2.2.4 [37] in R 3.6.1 [38] through the
interface Rstudio 2022.07.1 [39].
3. Results
3.1. During the Outbreak. e rabbit population in the
enclosure was estimated at 462 ±93 (mean ±standard error)
rabbits on January 18–20
th
, while it was 88 ±17 on February
22–24
th
(Figure 1(a)). Assuming a closed population, the
total mortality during the rst ve weeks of the outbreak was
estimated at 374 rabbits (CI
95
298–452), corresponding to
81.0% (CI
95
77.1–84.3%; 374/462) of the population before
the outbreak. From January 20
th
to March 2
nd
, 189 rabbit
carcasses were collected aboveground, most of them (73.0%)
Transboundary and Emerging Diseases 3
in the rst two weeks of the outbreak (Figure 1(b)). e
recovered carcasses correspond to 50.5% (CI
95
41.8–63.4%,
189/374) of the estimated total mortality during the rst ve
weeks of the outbreak.
Apparent mortality during the January-February 2022
outbreak peaked at 76.7% (CI
95
53.8–90.3%) for RHDV-
seronegative rabbits, while it was estimated as null for the
seropositive ones (Table 1 and Figure 2(a)). A previous
minor peak in the apparent mortality of RHDV-seronegative
rabbits occurred in June-July 2021 (35.0%, CI
95
12.2–67.5%),
without carcasses being detected (Figure 2(a)). e apparent
mortality of RHDV-seropositive rabbits rose slightly in June
2022 (Figure 2(a)).
From April 2021 to January 2022, the apparent sero-
prevalence of RHDV GI.2 was stable and low, ranging from
12.5% (CI
95
4.3–31%) to 15.4% (CI
95
8.0–27.5%). In Feb-
ruary 2022, the RHDV seroprevalence sharply increased to
87.9% (CI
95
72.7–95.2%), remaining high until June of the
same year (Figure 2(b)). e same pattern was observed for
MYXV, except that seroprevalence rose gradually from
22.5% (CI
95
13.0–35.9%) in January 2022 to 92.3% (CI
95
79.7–97.4%) in June 2022 (Figure 2(b)).
e most supported MECMR model included the eect
of vaccination during the January 2022 trapping session on
rabbit survival during the RHDV GI.2 outbreak (Model 1,
Table S1). Vaccination at the start of the outbreak decreased
the mortality of previously seronegative rabbits from 100%
to 60.0% ±16.6% (Table 1 and Figure 3(a)). e monthly
probability of seroreversion was estimated as null (Model 1,
Table S1).
e models including the eect on rabbit survival during
the outbreak of the serological status for MYXV
(ΔAICc �2.22, Model 2, Table S1) and sex (ΔAICc �2.25,
Model 3, Table S1) were almost equally supported. Rabbits
seronegative for both RHDV GI.2 and MYXV showed
higher apparent mortality (72.9% ±14.2%) than those that
were seronegative to RHDV GI.2 but seropositive to MYXV
(41.5% ±23.5%) (Table 1 and Figure 3(a)). Among the
RHDV GI.2-seronegative rabbits, females showed higher
mortality (100% ±0%) than males (52.0% ±17.1%) (Table 1
and Figure 3(a)).
e model that included the eect of age on rabbit
survival during the outbreak was weakly supported
(ΔAICc �4.77, Model 5, Table S1) and estimated higher
mortality in juveniles, both RHDV GI.2-seronegative
(100% ±0% vs. 72.1% ±10.2% for adults) and seropositive
(50.0% vs null for adults) (Table 1 and Figure 3(a)).
3.2. Postoutbreak. e same MECMR analysis was per-
formed for May–June 2022 to assess the postoutbreak ep-
idemiological scenario. After the January-February
outbreak, the apparent mortality increased in RHDV GI.2-
seropositive rabbits, and it decreased in RHDV GI.2-sero-
negative ones (Figure 3(b)). e mortality of RHDV GI.2-
seropositive/MYXV-seronegative rabbits rose from 0%
during the outbreak to 16.2% ±9.1% postoutbreak
(Figure 3(b)).
3.3. Minimum Protective Humoral Immunity. e most
supported model (Model 1, Table S1) was further explored to
assess the minimum level of humoral immunity that fully
protects rabbits from fatal RHDV GI.2 infection. e ap-
parent mortality of RHDV GI.2-seropositive rabbits in
January-February 2022 was estimated across a range of
cuto thresholds for seropositivity (Figure 4). For rabbits
with iELISA normalized absorbance ratios slightly below the
diagnostic threshold (NAR ≥1.9), it showed increased
mortality during the outbreak, compared to the null mor-
tality estimated for those at or above that threshold (NAR
≥2.0).
4. Discussion
An epidemic of RHDV GI.2 in a European rabbit population
undergoing a detailed longitudinal study allowed us to es-
timate some critical epidemiological parameters of this
pathogen of signicant relevance for the conservation of this
800
600
400
200
100
50
0
Apr 2021
Jun
Jul
Jun
Sep
Jan 2022
Feb
Jul
May
Abundance (number of rabbits)
(a)
Jan
2022
Feb
2022
100
50
Carcasses re covered
0
(b)
Figure 1: Rabbit abundance and carcasses recovered during the outbreak. (a) Estimated abundance of rabbits in each trapping session with
standard error. Trapping sessions in the X-axis, light grey rectangle highlights the rabbit haemorrhagic disease virus GI.2 outbreak duration.
(b) Weekly number of rabbit carcasses recovered during the outbreak.
4Transboundary and Emerging Diseases
Table 1: Monthly apparent mortality during the outbreak of rabbit haemorrhagic disease virus GI.2. Estimated mortality between January
20
th
and February 24
th
, 2022, from the models detailed in Table S1.
Variables Classes RHDV GI.2 seropositive RHDV GI.2 seronegative Model
Mean ±standard error (%) Mean ±standard error (%)
n. a. All rabbits 0 ±0 76.7 ±9.5 Model 4
Vaccine Vaccinated
(1)
0±0 60.0 ±16.6 Model 1
Unvaccinated 0 ±0 100 ±0
Myxoma virus (MYXV) Seropositive to MYXV 0 ±0 41.5 ±23.5 Model 2
Seronegative to MYXV 0 ±0 72.9 ±14.2
Sex Females 0 ±0 100 ±0Model 3
Males 0 ±0 52.0 ±17.1
Age Adults 0 ±0 72.1 ±10.2 Model 5
Juveniles 50.0 ±0 100 ±0
n. a., not applicable.
(1)
Vaccinated during the January 2022 trapping session.
100
80
60
40
20
0
Apparent mortality (%)
Apr 2021
Jun
Jul
Sep
Jan 2022
Feb
May
Jun
(a)
100
80
60
40
20
0
Apparent seroprevalence (%)
Apr 2021
Jun
Jul
Sep
Jan 2022
Feb
May
Jun
(b)
Figure 2: Apparent mortality and seroprevalence during the study. (a) Monthly apparent mortality of rabbits seropositive (blue) and
seronegative (red) for rabbit haemorrhagic disease virus (RHDV GI.2). (b) Apparent seroprevalence of RHDV GI.2 (dark orange) and
myxoma virus (green) with 95% condence intervals. Trapping sessions in the X-axis, light grey rectangle highlights the rabbit haemorrhagic
disease virus GI.2 outbreak duration.
RHDV-unvaccinated
RHDV-vaccinated
RHDV-juvenile
RHDV-adult
RHDV-male
RHDV-female
RHDV-MYXV-
RHDV-MYXV+
RHDV-
RHDV+
0 25 50 75 100
Mortality (%)
(a)
0 25 50 75 100
Mortality (%)
RHDV+unvaccinated
RHDV+vaccinated
RHDV+juvenile
RHDV+adult
RHDV+male
RHDV+female
RHDV+MYXV-
RHDV+MYXV+
RHDV-unvaccinated
RHDV-vaccinated
RHDV-juvenile
RHDV-adult
RHDV-male
RHDV-female
RHDV-MYXV-
RHDV-MYXV+
RHDV-
RHDV+
(b)
Figure 3: Determinants of apparent monthly mortality during and after the outbreak. Eects of the serological status for myxoma and rabbit
haemorrhagic disease virus (RHDV GI.2), sex, age, and prior vaccination on the apparent monthly mortality, with standard errors. (a)
During the outbreak: parameters estimated from January 20
th
to February 24
th
, 2022. (b) Postoutbreak: parameters estimated from February
24
th
to June 16
th
, 2022. Light grey bars highlight the apparent mortality of RHDV GI.2-seropositive and RHDV GI.2-seronegative rabbits.
Transboundary and Emerging Diseases 5
keystone species [8, 12, 13]. While the epidemiology of
RHDV GI.1 in wild populations was studied soon after its
emergence (e.g., [40, 41]), that of RHDV GI.2 has only been
addressed through experimental infections (e.g.,
[18, 23, 42, 43]).
e reported case fatality of RHDV GI.2 was variable
[14, 42], with higher values associated with recent strains,
suggesting viral evolution towards higher pathogenicity [18].
Our results support the high pathogenicity of the strains
circulating in the Iberian Peninsula based on the overall
mortality of seronegative rabbits (76.7 ±9.5%) over ve
weeks of the outbreak (Table 1). Although our study does not
allow us to estimate the case fatality of RHDV GI.2 directly,
the large scale of the outbreak suggests it was extremely high.
Given an estimated rabbit abundance of 88 ±17 and sero-
prevalence of 87.9% (CI
95
72.7–95.2%) after the outbreak,
only 11 rabbits (CI
95
9–13) were not infected. In such
a scenario, where the timeframe of the outbreak was short,
almost all individuals were infected, and emigration is
impossible, the apparent mortality should be a close proxy of
the case fatality [44].
e RHDV GI.2 outbreak occurred during the breeding
season, between January and February, according to the
phenology described in wild populations of European
rabbits [43, 45]. Declines of 60–80% in the rabbit pop-
ulations over several years have been reported following the
emergence of RHDV GI.2 in na¨
ıve rabbit populations in the
Iberian Peninsula and Australia [21, 22]. Such drastic de-
clines can unbalance ecosystems, e.g., by reducing the fe-
cundity of rabbit predators [22]. 14 hours study documented
a reduction of 81% in rabbit abundance after a large out-
break. Abundance increased in the following spring
(353 ±99 rabbits in July 2022), although it did not attain the
values of the previous year (Figure 1(a)). It should be noted
that the density of the study population was unnaturally high
(>200 rabbits/hectare) as the apparent mortality was <20%
before the outbreak, as expected for an enclosure with
supplemental feeding and no terrestrial predators [46]. e
seroprevalence before the outbreak was extremely low
(12.5%–15.6%), which suggests that our model system could
be close to a na¨
ıve population [47].
e detected mortality spanned six weeks, concentrated
in the rst two weeks of the outbreak (Figure 1(b)). Only
approximately half of the estimated number of dead rabbits
could be recovered despite intensive aboveground searches
for carcasses. Given the perimetral rabbit- and carnivore-
proof fence, two hypotheses can explain this observation:
rst, some of the carcasses could have been removed and
consumed by avian scavengers, which are abundant in this
estate because of the conservation-oriented management;
second, it could be due to many rabbits dying inside the
burrows, thus contributing to the persistence of RHDV GI.2
[20]. is hypothesis was supported by the evidence of the
continuing circulation of RHDV GI.2 in the months fol-
lowing the detected outbreak, as seronegative rabbits con-
tinued to exhibit higher mortality than seropositive ones
(Figures 2(a) and 3(b)).
We found evidence of the prolonged circulation of
MYXV, starting in January 2022, when MYXV seropreva-
lence rose from very low levels (6.3%), achieving 92.3% in
June 2022 (Figure 1(b)). Furthermore, the mortality of
RHDV GI.2-seropositive/MYXV-seronegative rabbits rose
from 0% in January-February to 17.7 ±8.6% in May–June
(Figure 3), supporting the circulation of MYXV in the
postoutbreak period. During the outbreak, the mortality of
RHDV-seronegative MYXV-seropositive rabbits
(41.5% ±23.5%) was lower than that of RHDV-seronegative
MYXV-seronegative rabbits (72.9% ±14.2%). While this
could suggest the co-circulation of both viruses, RHDV-
seropositive rabbits showed no dierence in mortality
according to the serological status for MYXV (0% for both
MYXV-seropositive and negative). Together, these results
support that humoral immunity to MYXV might reduce
RHDV GI.2 pathogenicity, in contrast to what was suggested
for RHDV GI.1 in Australia [48]. Unspecied dierences in
the immune response to RHDV GI.1 and GI.2 variants
[49, 50] might explain these dierences, was as well as the
complex interactions between other pathogens and the
immune response to MYXV and RHDV GI.2 [42, 51].
e apparent seroprevalence of RHDV GI.2 was stable
and low (12.5–15.4%) until January 2022, despite the vac-
cination against RHDV GI.2 of 36 rabbits released back into
this enclosure, which suggests that the virus was not cir-
culating before that date. A slight peak in the mortality of
RHDV-seronegative rabbits (35.0%; Figure 2(a)) occurred in
June-July 2021 without detected mortality. is observation
suggests a minor outbreak of RHDV in the summer of 2021,
without the persistence of viral circulation in the following
months.
Our study evidenced two relevant parameters related to
the immunology of RHDV GI.2. First, the probability of
seroreversion (shifting from seropositive to seronegative)
was estimated as null by MECRM models. ese results
support the presence of lifelong immunity against this agent.
Second, despite the high case fatality of RHDV GI.2 in-
fections, the humoral immunity developed against this virus
seems to be highly protective because the mortality of se-
ropositive rabbits was also estimated as null during the
≥ 2≥ 1.9≥ 1.8≥ 1.7≥ 1.6≥ 1.5≥ 1
0
20
40
60
80
100
Apparent mortality (%)
Figure 4: Apparent mortality of seropositive rabbits across a range
of thresholds for seropositivity. Estimated apparent mortality from
January 20 to February 24, 2022, during the rabbit haemorrhagic
disease virus GI.2 outbreak, across a range of normalized absor-
bance ratios cuto thresholds for seropositivity.
6Transboundary and Emerging Diseases
outbreak (Table 1 and Figure 2(a)). Furthermore, our results
suggest that the minimum level of humoral immunity that
fully protects from fatal infection coincides with the iELISA
cuto threshold for seropositivity of NAR �2 (Figure 4).
Strikingly, even a slightly lower NAR ≥1.9 yields an estimate
of 38.4% mortality in seropositive rabbits, compared to 0%
in rabbits with NAR ≥2.0. Single-dose subcutaneous vac-
cination with commercial inactivated RHDV GI.2 virus right
before the outbreak decreased the mortality of previously
seronegative rabbits from 100% to 60 ±16.6%. is obser-
vation suggests that vaccination at the outbreak’s start does
not fully protect from fatal infection, despite attenuating its
impact.
e MECMR models also showed higher mortality
during the January-February outbreak in female RHDV
GI.2-seronegative rabbits (100%) compared to males
(52.0 ±17.1%) (Table 1 and Figure 3). Experimental in-
fections did not report dierences in the susceptibility to
RHDV GI.2 between sexes [18, 19, 23, 42, 50, 52, 53], but it is
unclear whether this potential risk factor was included in
some of the analyses. Our data underscore the demographic
impact of RHDV GI.2 in wild European rabbits, highlighting
the eect of a single outbreak in drastically reducing the
number of breeding females in a population at the peak of
the breeding season, which could signicantly impair its
recovery. Interestingly, in the postoutbreak period where
RHDV GI.2 and MYXV cocirculated, mortality in males was
slightly higher than in females, irrespective of the serological
status for RHDV GI.2 (Figure 3).
One of the most notable characteristics of RHDV GI.2 is
its high pathogenicity in juvenile rabbits, with case fatality
upon experimental infection estimated at 21–100%
[18, 19, 23, 42, 50, 52, 53] and in adult rabbits at 0–89%
[19, 23, 52]. e MECMR model including the eect of age
was weakly supported but suggested higher mortality in
juvenile rabbits, either seropositive (50% vs. 0% in adults) or
seronegative (100% vs. 72.1 ±10.2% in adults) to RHDV GI.2
(Table 1).
e most notable aspect of the eld epidemiology of
RHDV GI.2 is the high pathogenicity in juveniles, even if
seropositive (Table 1), conrming the results of experimental
infections [19, 42]. By contrast, RHDV GI.1 was weakly
pathogenic for this age class [40]. Another relevant aspect of
RHDV GI.2 is the complete protection from fatal infection
provided by circulating antibodies (Table 1), in contrast to
the signicant mortality induced by RHDV GI.1 in sero-
positive rabbits [41, 54].
is study supports the high case fatality rate of RHDV
GI.2 in seronegative European rabbits during natural out-
breaks and the eective protection conferred by humoral
immunity. e initial abundance in the enclosure was ex-
tremely high, and despite a low seroprevalence (15.4%), the
number of survivors was large enough for the prompt re-
covery of the population. is study highlights the threat
posed by RHDV GI.2 to isolated low-abundance populations
of European rabbits and other threatened lagomorph taxa
where this pathogen recently emerged, such as the riparian
brush rabbit (Sylvilagus bachmani riparius) and pygmy
rabbit (Brachylagus idahoensis) in North America [55, 56].
Populations with high abundance and/or seroprevalence
should be a management goal for conserving endangered
lagomorphs in their native ranges.
5. Conclusions
e detailed longitudinal demographic and epidemiological
study allowed us to estimate some key epidemiological
parameters of a natural epidemic of RHDV GI.2 in a pop-
ulation of European rabbits. We estimated the apparent
mortality during and postoutbreak, highlighting the overall
high pathogenicity and demographic impact of circulating
RHDV GI.2 strains. e detectable epidemic had a short
course of six weeks. Still, the virus apparently kept circu-
lating in the following months, probably facilitated by the
environmental contamination arising from a proportion of
infected carcasses remaining inside burrows. Specic IgG is
fully protective against fatal infection by RHDV GI.2 at the
seropositivity threshold, but subsequent MYXV infection
might negatively impact the immune response. Vaccination
at the outbreak’s start is only partially protective against fatal
disease. e novel information provided by this study
contributes to a better understanding of the epidemiology of
RHDV GI.2, a pathogen of major relevance for conserving
endangered keystone species.
Data Availability
All data supporting the ndings of the present study are
available upon reasonable request from the corresponding
author.
Ethical Approval
Live trapping and sampling procedures were conducted
under permits 23/2021 and 574/2022, and according to
European Union directives on the protection of animals
used for scientic purposes (Directive 2010/63/EU), and
international standards on the use of wildlife in research.
Conflicts of Interest
e authors declare that there are no conicts of interest.
Acknowledgments
is work was funded by Fundação para a Ciˆ
encia e Tec-
nologia (Grant no. SFRH/BPD/116596/2016 to N. Santos)
and it was cofunded by Norte Portugal Regional Operational
Programme (NORTE2020) (Project no. NORTE-01-0246-
FEDER-000063), under the PORTUGAL 2020 Partnership
Agreement, through the European Regional Development
Fund (ERDF), and project LINX2020—Realização de Ações
Preparat´
orias da Reintrodução do Lince-Ib´
erico nos SIC
Malcata, S. Mamede e Moura-Barrancos (POSEUR-03-2215-
FC-000043). S. Jim´
enez-Ruiz was granted for two research
mobilities within the “Plan Propio” of the University of
Castilla-La Mancha (UCLM) for the years 2021 and 2022,
being currently supported by a postdoctoral contract
Margarita Salas (UCLM) from the Program of
Transboundary and Emerging Diseases 7
Requalication of the Spanish University System (Spanish
Ministry of Universities) nanced by the European Union-
NextGenerationEU. e authors also acknowledge the
support of Parque de Natureza de Noudar S. A. and Empresa
de Desenvolvimento e Infra-estruturas do Alqueva S.A., as
well as the students who assisted with eldwork.
Supplementary Materials
Appendix S.1: summary of the methodological approach for
the multievent capture-mark-recapture models. Table S1:
summary of the model selection. (Supplementary Materials)
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