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Citation: Joffrin, L.; Cooreman, T.;
Verheyen, E.; Vercammen, F.; Mariën, J.;
Leirs, H.; Gryseels, S. SARS-CoV-2
Surveillance between 2020 and 2021
of All Mammalian Species in Two
Flemish Zoos (Antwerp Zoo and
Planckendael Zoo). Vet. Sci. 2023,10,
382. https://doi.org/10.3390/
vetsci10060382
Academic Editors: Marieke Stammes,
Fuus Thate and Norbert Stockhofe
Received: 14 April 2023
Revised: 18 May 2023
Accepted: 25 May 2023
Published: 31 May 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
veterinary
sciences
Article
SARS-CoV-2 Surveillance between 2020 and 2021 of All
Mammalian Species in Two Flemish Zoos (Antwerp Zoo
and Planckendael Zoo)
Léa Joffrin 1,†, Tine Cooreman 1, *,† , Erik Verheyen 1,2 , Francis Vercammen 3, Joachim Mariën 1, Herwig Leirs 1
and Sophie Gryseels 1,2
1Evolutionary Ecology Group, Department of Biology, University of Antwerp, 2610 Antwerp, Belgium
2OD Taxonomy and Phylogeny, Royal Belgian Institute of Natural Sciences, 1000 Brussels, Belgium
3Centre for Research and Conservation, Antwerp Zoo Society, 2018 Antwerp, Belgium
*Correspondence: tine.cooreman@uantwerpen.be
† These authors contributed equally to this work.
Simple Summary:
COVID-19 emerged in China in 2019. It is caused by an until-then unknown
coronavirus that causes severe acute respiratory syndrome (SARS-CoV-2). Through experimental
infections in the search for a suitable animal model and reported infections in pets early in the
pandemic, it became clear that several animal species may be susceptible to SARS-CoV-2 infection.
According to the open access dataset of reported SARS-CoV-2 events in animals, about 119 zoo
animals have been reported with a SARS-CoV-2 infection. However, the detection of
SARS-CoV-2
infections in zoo animals has relied on the observation of symptoms (cough, nasal discharge),
behaviour changes (reduced appetite, lethargy), or death of these captive animals. SARS-CoV-2
infections may therefore remain undetected if animals do not show obvious symptoms. In this study,
we investigated the potential circulation of SARS-CoV-2 in zoo mammal species by sampling and
screening faecal samples from all the mammals in two zoos in Belgium between September 2020 and
July 2021 using molecular biology techniques. This study is the first to our knowledge to conduct
active SARS-CoV-2 surveillance for several months in all mammal species in a zoo. We conclude that
at the time of our investigation, none of the screened animals were excreting SARS-CoV-2.
Abstract:
The COVID-19 pandemic has led to millions of human infections and deaths worldwide.
Several other mammal species are also susceptible to SARS-CoV-2, and multiple instances of transmis-
sion from humans to pets, farmed mink, wildlife and zoo animals have been recorded. We conducted
a systematic surveillance of SARS-CoV-2 in all mammal species in two zoos in Belgium between
September and December 2020 and July 2021, in four sessions, and a targeted surveillance of selected
mammal enclosures following SARS-CoV-2 infection in hippopotamuses in December 2021. A total
of 1523 faecal samples from 103 mammal species were tested for SARS-CoV-2 via real-time PCR.
None of the samples tested positive for SARS-CoV-2. Additional surrogate virus neutralisation tests
conducted on 50 routinely collected serum samples from 26 mammal species were all negative. This
study is the first to our knowledge to conduct active SARS-CoV-2 surveillance for several months in
all mammal species of a zoo. We conclude that at the time of our investigation, none of the screened
animals were excreting SARS-CoV-2.
Keywords: SARS-CoV-2; surveillance; zoo; mammals; Belgium
1. Introduction
COVID-19 emerged in China in 2019. It is caused by an until-then unknown coronavirus
that causes severe acute respiratory syndrome (SARS-CoV-2). This infectious disease spread
to all continents in a few months and was declared a pandemic by the World Health Organ-
isation in March 2020. SARS-CoV-2 can be transmitted through three main routes: direct
Vet. Sci. 2023,10, 382. https://doi.org/10.3390/vetsci10060382 https://www.mdpi.com/journal/vetsci
Vet. Sci. 2023,10, 382 2 of 11
contact with infected secretions (saliva, respiratory secretions), droplet transmission (when
coughing or sneezing), and aerosol transmission [
1
]. Through experimental infections in the
search for a suitable animal model and reported infections in pets early in the pandemic, it
became clear that several animal species may be susceptible to SARS-CoV-2 infection [
2
–
8
].
Experimental
in vivo
and
in vitro
infections showed that SARS-CoV-2 can infect a broad
taxonomic range of mammals, including a.o. North American deer mice (Peromyscus man-
iculatus), macaques (Macaca mulatta and Macaca fascicularis), domestic cats (Felis catus),
ferrets (Mustela putorius furo), American mink (Neovison vison), raccoon dogs (Nyctereutes
procyonoides), Syrian hamsters (Mesocricetus auratus), and Egyptian fruit bats (Rousettus
aegyptiacus) [
3
,
9
–
18
]. Circulation of SARS-CoV-2 was reported in farmed American mink
(Neovison vison) in a multitude of farms around the world, and wild white-tailed deer
(Odocoileus virginianus) across North America [2–6].
In addition, functional, structural, and genetic analysis of viral receptor ACE2 or-
thologs reveals that many other species may be susceptible to SARS-CoV-2 [
19
,
20
]. While
these studies may appear helpful to estimate the potential host range of SARS-CoV-2,
the observed natural infections highlight that susceptibility based on the ACE2 receptor
alone is not a sufficient proxy to estimate potential spillover risk to other species [
21
]. For
example, mink or wild white-tailed deer are not considered highly susceptible based on
these in-silico analyses [5].
According to the open access dataset of reported SARS-CoV-2 events in animals (data
from January 2023), about 119 zoo animals have been reported with a SARS-CoV-2 infection,
representing 64 reported events and 17 species in 17 countries [
22
]. The most frequently
reported infected mammals in zoos are felines, followed by primates [
23
]. However, the
detection of SARS-CoV-2 infections in zoo animals has relied on the observation of symp-
toms (cough, nasal discharge), behaviour changes (reduced appetite, lethargy), or death in
these captive animals [
24
,
25
]. SARS-CoV-2 infections may therefore remain undetected if
animals do not show obvious symptoms.
Since infected animals have been found in zoos worldwide, and given the long-term
high incidence of the virus in humans, we deemed it prudent to monitor the presence of
SARS-CoV-2 in zoo animals. Furthermore, the high diversity of zoo animals, both regarding
taxonomy and geographical origin, makes zoos an ideal place to (i) contribute to unravelling
the potential host range of SARS-CoV-2 and (ii) evaluate the risk for the conservation of
wild animal populations in captivity and in situ. For this study, we investigated the
potential circulation of SARS-CoV-2 in zoo mammal species by sampling and screening
faecal samples from all the mammals in two zoos in Belgium in four sessions between
September 2020 and July 2021, via real-time polymerase chain reactions (PCR). Following
symptomatic SARS-CoV-2 infection in hippos in the Antwerp Zoo in December 2021 [
26
],
we additionally surveyed selected mammals deemed to be in potential indirect contact
with the hippos, or with expected relatively high SARS-CoV-2 susceptibility.
2. Materials and Methods
2.1. Samples Collection
We conducted this study at the Antwerp Zoo and Planckendael Zoo in, respectively,
Antwerp and Mechelen, Belgium. For the systematic surveillance, we collected the samples
during four periods (early September 2020, mid-October 2020, mid-December 2020 and
July 2021), with sampling following enclosure cleaning planning. During the first sampling
period, both zoos were still open to the public; during the second sampling series, both were
closed to the public and remained closed until after the third sampling due to government
regulations. The zoos reopened in February 2021, and the fourth sampling session was
conducted in July 2021. During the first three sampling sessions, the original Wuhan-
Hu1 variant was dominant in the human population in Belgium; during the fourth, the
delta variant, considered more contagious than the previous alpha, beta, and gamma
variants [
27
], was dominant in Belgium. Faecal samples were collected by zookeepers
in a 16.5 mL tube filled with RNAlater and then stored at
−
20
◦
C at the zoo for a few
Vet. Sci. 2023,10, 382 3 of 11
days before transport to the lab, where the samples were stored at
−
80
◦
C. RNAlater is a
suitable conservation medium widely used for microbiological studies [
28
,
29
]. The date
and freshness of each sample were documented (maximum two hours old, or no more than
twelve hours old), after which the samples were stored. A maximum of five samples per
species per zoo were collected at each sampling session. A total of 1417 faeces samples
were collected from 103 different mammal species (Antwerp n= 48 and Planckendael
n= 67) (Table 1). In Antwerp Zoo, the largest sampled taxonomic group was the Primates,
followed by the order of the Cetartiodactyla. In Planckendael, Cetartiodactyla was sampled
most often, followed by the order of the Carnivora.
Table 1.
Number of faecal samples collected and tested for SARS-CoV-2 RNA per order, family
and species in the two zoos for each sampling session. Session 1: early September 2020, Session 2:
mid-October 2020, Session 3: mid-December 2020, Session 4: July 2021, Session 5: December 2021.
Antwerp Planckendael
Order Family Species
Session 1
Session 2
Session 3
Session 4
Session 5
Total
Session 1
Session 2
Session 3
Session 4
Session 5
Total
Cetartiodactyla Bovidae Addax nasomaculatus 5554 19
Bison bison 5555 20
Bison bonasus 3333 12
Bos taurus 3333 12
Budorcas taxicolor 2222 8
Capra hircus 5555 20
Cephalophus natalensis 2222 8
Gazella leptoceros 3331 10
Madoqua kirkii 444 12 5553 18
Nanger dama 3335 14
Oryx dammah 1111 4
Oryx leucoryx 1111 4
Ovis aries 3332 11 5553 18
Ovis aries laticaudatus 222 6
Syncerus caffer 5555 20
Tragelaphus eurycerus 3333416 3331 10
Camelidae Camelus bactrianus 5555 20
Lama guanicoe 5553 18
Vicugna pacos 5 5 5 10 25
Vicugna vicugna 5554 19
Cervidae Cervus canadensis 5555 20
Muntiacus reevesi 5555 20
Equidae Equus asinus 2222 8
Equus caballus 444 12
Equus ferus przewalskii 4 4
Equus grevyi 5555 20
Equus zebra 4444 16
Giraffidae Giraffa camelopardalis 3333 12 5555 20
Okapia johnstoni 5554 19
Hippopotamidae
Hippopotamus amphibius 2222 8
Suidae Sus cebifrons 4444 16
Sus scrofa 3333 12
Tayassuidae Catagonus wagneri 5555 20
Total 33 33 33 27 4 130 102 102 102 95 401
Carnivora Canidae Crocuta crocuta 2223 9
Speothos venaticus 5555 20
Felidae Acinonyx jubatus 2222210
Panthera leo 3333618 3333315
Panthera onca 111126
Panthera pardus 111 3
Panthera uncia 2222 8
Herpestidae Cynictis penicillata 5555 20
Mungos mungo 5555 20
Suricata suricatta 5553 18
Vet. Sci. 2023,10, 382 4 of 11
Table 1. Cont.
Antwerp Planckendael
Order Family Species
Session 1
Session 2
Session 3
Session 4
Session 5
Total
Session 1
Session 2
Session 3
Session 4
Session 5
Total
Mustelidae Aonyx cinereus 111 3444 12
Meles meles 1111 4
Otariidae Phoca vitulina 7772 23
Zalophus californianus 4442 14
Procyonidae Nasua narica 1112 5
Nasua nasua 333 9
Procyon lotor 222 6
Ursidae Ailurus fulgens 2222 8
Tremarctos ornatus 2222 8
Total 25 25 25 15 6 96 36 36 36 28 7 143
Chiroptera Pteropodidae Rousettus aegyptiacus 5552 17
Total 5 5 5 2 17
Dasyuromorphia Dasyuridae Sarcophilus harrisii 3333 12
Total 3 3 3 3 12
Diprotodontia Macropodidae Dendrolagus goodfellowi 111 3
Macropus giganteus 4443 15
Macropus parma 333 9
Macropus rufus 1 1 2 4
Thylogale brunii 111 31111 4
Wallabia bicolor 5555 20
Phascolarctidae Phascolarctos cinereus 1112 52221 7
Potoroidae Bettongia penicillata 1 1
Total 10 10 10 6 36 9 9 8 9 35
Lagomorpha Leporidae Oryctolagus cuniculus 5555 20
Total 5 5 5 5 20
Macroscelidea Macroscelididae Rhynchocyon petersi 3332 11
Total 3 3 3 2 11
Monotremata Tachyglossidae Tachyglossus aculeatus 2222 8
Total 2222 8
Perissodactyla Rhinocerotidae Ceratotherium simum
simum 2222 8
Rhinoceros unicornis 2223 9
Tapiridae Tapirus indicus 3332415
Total 5 5 5 4 4 23 2 2 2 3 9
Pilosa
Myrmecophagidae
Myrmecophaga tridactyla 2222210
Tamandua tetradactyla 111 3
Total 111 32222210
Primates Aotidae Aotus trivirgatus 111 3
Atelidae Ateles fusciceps 5555626
Callitrichidae Callimico goeldii 5555222
Callithrix geoffroyi 5555222
Cebuella pygmaea 111126
Leontopithecus chrysomelas
333 94443520
Saguinus imperator 222219
Cebidae Saimiri boliviensis 333 9
Cercopithecidae Cercopithecus hamlyni 3334316
Colobus guereza 4444218
Macaca nigra 5555323
Macaca sylvanus 5555323
Mandrillus sphinx 5555424
Hominidae Gorilla beringei 111115
Gorilla gorilla 5555525
Vet. Sci. 2023,10, 382 5 of 11
Table 1. Cont.
Antwerp Planckendael
Order Family Species
Session 1
Session 2
Session 3
Session 4
Session 5
Total
Session 1
Session 2
Session 3
Session 4
Session 5
Total
Pan paniscus 5 5 5 5 18 38
Pan troglodytes 5 5 5 5 11 31
Hylobatidae Nomascus leucogenys 2222210
Lemuridae Eulemur macaco 2223211
Lemur catta 2222 85555525
Varecia rubra 111 3
Loridae Loris lydekkerianus 5555222
Lorisidae Nycticebus pygmaeus 5552 17
Total 53 53 53 46 39 244 36 36 36 33 40 181
Proboscidea Elephantidae Elephas maximus 222 65555 20
Total 222 65555 20
Rodentia Castoridae Castor fiber 111 3
Caviidae Dolichotis patagonum 5555 20
Hydrochoerus hydrochaeris
3332 11
Dasyproctidae Dasyprocta prymnolopha 1111 4
Echimidae Myocastor coypus 5 3 8
Erethizontidae Erethizon dorsatum 4444 16
Hystricidae Hystrix africaeaustralis 333335 14
Murinae Lemniscomys barbarus 5555 20
Phleomys padillus 5553119
Total 14 14 14 20 4 66 13 13 13 13 52
Total 146 146 146 120 57 615 220 220 219 200 49 908
After our systematic surveillance, two female hippopotamuses in Antwerp Zoo showed
evidence of nasal discharge in late November 2021 for a few days [
26
,
30
].
SARS-CoV-2
was
detected by immunocytochemistry in nasal swab samples and by PCR in nasal swab samples,
faeces, and pool water [26]. Serological tests also detected antibodies against SARS-CoV-2.
Following these hippo infections, we conducted a targeted surveillance for
SARS-CoV-2
in December 2021, collecting samples from mammals that could have been in indirect con-
tact with the hippo individuals (i.e., if they were managed by the same caretakers) or
that were of special interest due to their known increased susceptibility and conservation
status, namely, primates and large felines. We screened these samples with the CDC 2019-
Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel, specifically targeting
SARS-CoV-2 genes, and this was also used for the diagnosis of SARS-CoV-2 in the infected
hippopotamuses’ faecal samples [
31
]. Details on the 106 samples collected and tested in
December 2021 are also available in the table.
2.2. Sample Preparation, Extraction and PCR Testing
After thawing, the samples were processed under a Biosafety cabinet class II. Around
1 cm
3
of the faeces was cut off, rinsed with 200
µ
L of phosphate-buffered saline (PBS), and
mixed in a 1.5 mL Eppendorf tube filled with 800
µ
L of PBS. The tubes were briefly vortexed
and centrifuged (1500 g for 15 min), and for each collection date/enclosure/species, samples
were pooled to extract faecal RNA using the QIAGEN QIAamp viral RNA kit (Qiagen,
Valencia, CA, USA) following the manufacturer recommendations. Overall, 420 pools were
extracted. Reverse transcription was performed on 8
µ
L of RNA extract using the Maxima
Reverse Transcriptase and Random Hexamer Primers (Thermo Fisher Scientific, Waltham,
MA, USA) on a Biometra T3000 thermocycler (Biometra, Westburg, The Netherlands). A
pan-coronavirus system suitable for the detection of alpha-, beta-, gamma- and delta-CoVs
real-time PCR adapted version of the Muradrasoli et al. (2009) protocol [
32
,
33
] was used
Vet. Sci. 2023,10, 382 6 of 11
on a StepOne
™
Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) to screen
the samples for all potential coronaviruses that may occur in zoo animals.
2.3. Validation of the PCR System for the Detection of SARS-CoV-2
We conducted assays to validate the use of the Pan-CoV system for the detection of
SARS-CoV-2 in our samples. We compared the limit of detection of the Pan-CoV system
targeting the RNA-dependent RNA-polymerase (RdRp) gene to the CDC 2019-nCoV Real-
Time RT-PCR Diagnostic Panel, specifically targeting the SARS-CoV-2 nucleocapsid (N)
gene in two multiplex reactions (N1 and N2) [
31
]. The limit of detection was determined to
be the lowest dilution that still resulted with a Ct value. RNA from a SARS-CoV-2-positive
clinical sample was used to conduct this assay. The CDC Real-Time RT PCR was performed
on a serial dilution of the positive sample RNA ranging from 10
−1
to 10
−8
. The same 8-fold
dilution series was reverse-transcribed to cDNA (using the Maxima RT protocol described
above), which was then run on the Pan-CoV Real-Time PCR.
In addition, a synthetic N1 and N2 gene positive control (2019-nCoV_N_Positive
Control, Integrated DNA Technologies) with a known copy number was used in the CDC
panel at three concentrations: 2000 copies/
µ
L, 200 copies/
µ
L and 20 copies/
µ
L alongside
the dilution series of the clinical sample. The Pan-CoV Real-Time PCR was used on the
cDNA synthesised following the reverse-transcription protocol described above from the
SARS-CoV-2-positive clinical sample RNA ranging from 10
−1
to 10
−8
. Each dilution was
tested in triplicates.
For each system, the standard curve of the positive clinical sample was calculated by
plotting the PCR cycle threshold (Ct) to the dilution number of the positive clinical sample,
from which the logarithmic function (y =
−
a ln(x) + b) was calculated. If the R
2
value was
less than 0.96, all serially diluted RNA and cDNA was remade and retested.
The copy number concentration of SARS-CoV-2 N gene RNA in the SARS-CoV-2
clinical sample was inferred via the standard curve of the 2019-nCoV_N_Positive Control
dilution series used in the CDC system.
2.4. Serological Screening
Additionally, 50 blood samples from 26 mammal species were available from routine
collection by the zoo veterinary service, both before (14 samples/12 species) and after
(36 samples/26 species) 2020, for animals that either moved between zoos or for those
requiring a veterinary follow-up (pregnancy, injury, illness). Serum samples were tested for
the presence of antibodies against SARS-CoV-2 with the L00847 surrogate virus neutralisa-
tion test (sVNT) (GenScript cPass
™
, Piscataway, NJ, USA) as described in
Mariën et al. [34]
.
The percentage inhibition was calculated as: ((1
−
OD value of sample)/OD value of Neg-
ative control)
×
100%. If inhibition values were greater than 20%, serum samples were
considered SARS-CoV-2-positive. Two negative serum samples, two positive serum sam-
ples and two positive serum samples from SARS-CoV-2-infected humans were used as
controls. Details on the samples tested are available in Supplementary material Table S1.
3. Results and Discussion
None of the 1523 faecal samples across the five collection periods tested positive with
the pan-coronavirus screening system. Furthermore, all serum samples were seronegative
for neutralising antibodies, suggesting that the tested mammals had not experienced
SARS-CoV-2
infection at the time of sample collection (Supplementary material Figure S1).
As such, apart from the infection in two hippos in December 2021 that was discovered
because of clinical symptoms and not through our active surveillance study, there was no
evidence of SARS-CoV-2 or other coronavirus infection among the mammals residing in
the Antwerp and Planckendael Zoos during the period of the study.
We compared the Pan-CoV PCR system used to test faecal samples collected during
the first four sampling sessions between September 2020 and July 2021 with a golden
standard test for SARS-CoV-2 detection (CDC N1/N2), to ensure that the sensitivity of
Vet. Sci. 2023,10, 382 7 of 11
the detection system was not an issue. We inferred the copy number per
µ
L of a positive
control SARS-CoV-2 RNA from a sample by comparing it with the known copy numbers of
the N1 synthetic control of the CDC SARS-CoV-2 system. In both systems, the template was
detectable up to a 10
−5
dilution, corresponding to 2.42 N1-gene-copies/
µ
L with Ct values
of 34.76
±
0.12 (CDC) and 38.84
±
2.48 (Pan-CoV) (Supplementary Material Figure S2).
Hence, the detection limit and sensitivity of the CDC and the Pan-CoV system were very
comparable, making it unlikely that the choice of a pan-coronavirus RT-PCR system instead
of a SARS-CoV-2-specific detection system caused false-negative results. The advantage
of the Pan-CoV system is that we could also determine the possible presence of other
coronaviruses with one PCR test. While the entire range of coronaviruses may not be
detected with the same sensitivity with this Pan-CoV system, it has been validated to detect
the SARS-CoV-2 RNA-dependent RNA-polymerase (RdRp) gene.
Virus survival or the successful detection of viral RNA depends on the virus variant,
the medium in which it is present and environmental conditions (temperature, pH, moisture
content, organic matter, light, etc.) [
35
]. Although the SARS-CoV-2 virus is stable on most
indoor surfaces [
36
–
38
], other factors in outdoor environments may reduce its survival [
39
].
Studies on the effect of temperature on SARS-CoV-2 survival showed that it might survive
from 5 to 10 days at 20
◦
C and from 1 to 4 days at 30
◦
C, depending on the surface type [
40
].
Even if no studies on SARS-CoV-2 stability in faeces in outdoor environments have been
conducted, a comprehensive study on the survival of several other coronaviruses in faeces
concluded that SARS-CoV-2 could survive from 1 h to 4 days in human faeces, depending
on the type and pH of the stool samples [
35
]. In our study, the delay between excretion
and collection, and other environmental factors, might have influenced the quality of the
samples. However, we tried to limit these issues by collecting samples that were as fresh
as possible (less than 12 h after excretion). Finally, the mean temperature ranged from
0 to 22 ◦C
during the whole sampling campaign. We therefore assume that the impact of
temperature on the preservation of faeces on the ground of the enclosure will be minimal.
The non-detection of SARS-CoV-2 RNA in this study might be related to the study
sampling design. Faecal samples are suitable materials for the detection of SARS-CoV-2
RNA, even if there is no consensus about which sample type (i.e., nasopharyngeal swabs,
oropharyngeal swabs, faeces, or rectal swabs) is best suited to detect SARS-CoV-2 RNA,
especially in non-human animals [
41
–
43
]. Moreover, we cannot exclude that we missed
a potential SARS-CoV-2 infection in zoo mammals in our study because of the duration
of SARS-CoV-2 RNA in faeces after the acute infection. Zhang et al. (2021) conducted a
systematic review and meta-analysis on 14 studies on the faecal shedding of SARS-CoV-2
RNA in human patients (n= 620) with COVID-19 infection [
42
]. On average, viral RNA
could be detected up to 21.8 days after infection, while nasopharyngeal swabs could only
detect RNA 14.7 days after infection. The sampling sessions were, on average, six weeks
apart, with over six months between the two subsequent sessions. We therefore cannot
exclude that SARS-CoV-2 infections occurred between the sampled sessions. However, due
to logistical reasons, more frequent sampling was not feasible. Nevertheless, longitudinal
faecal screening of infected tigers and lions in the USA and hippos in Belgium showed
that SARS-CoV-2 RNA could be detected up to 35 days after symptom onset [
25
,
26
]. Viral
RNA shedding in these animals’ faeces may be more apparent than in humans, where only
about half of the patients have detectable SARS-CoV-2 RNA in faeces at any point during
infection [42]. If they do, viral RNA remains detectable for 3–4 weeks.
Additionally, a systematic blood sampling of all the animals to conduct serology testing
and look for past infection rather than ongoing infection could have helped unravel this
bias related to the time windows. In humans, IgG antibodies can be detected at least three
months after SARS-CoV-2 infection [
44
–
47
]. However, little is known about the persistence
of antibodies in non-human mammals after infection, though the few longitudinal studies
available revealed broad interspecific and intraspecific variations in the persistence of IgG
antibodies after SARS-CoV-2 [
48
–
50
]. For example, in captive Malaysian tigers (Panthera
tigris jacksoni), the antibody response was observed up to 3 months after the first clinical
Vet. Sci. 2023,10, 382 8 of 11
signs of infection [
48
]. In pets, some cats (Felis catus) and dogs (Canis lupus familiaris)
were seronegative less than three months post-symptoms, while in others, neutralising
SARS-CoV-2 antibodies have been detected up to 10 months post-symptoms [
49
]. In
captive white-tailed deer (Odocoileus virginianus), the duration of persistence of neutralising
antibodies was estimated to be at least 13 months [
51
]. Notably, some reports mentioning
the long persistence of antibody response after SARS-CoV-2 infection in animals have
qualified their results, as they were unsure if another infection occurred during their
survey [49–51].
Systematic blood sampling would have involved heavy logistic organisation and
animal stress in our case. We therefore relied on the 50 collected serum samples from
26 species that were collected for other purposes, representing about 10% of the mammals
from the zoo. Their seronegativity suggests that at least up until 2021, there has been no
widespread multi-species SARS-CoV-2 epidemic in the zoos.
Previously reported cases of SARS-CoV-2 infections in zoo animals have been traced
back to asymptomatic infected zookeepers that were in contact with these animals [
23
–
25
].
The close contact of zookeepers when preparing food, veterinary consultations of animals,
or enclosure cleaning represents an important risk of transmission. Since the summer of
2020 and throughout our study, face masks have been worn in the Antwerp and Planck-
endael Zoos by zookeepers and visitors, in addition to extensive hygiene measures when
preparing food and entering the facilities. Likely, the hygiene measures implemented in
Antwerp and Planckendael Zoos at the beginning of the pandemic have helped to avoid the
transmission of SARS-CoV-2 from humans to animals during most of the pandemic. The
origin of the infection of the two hippos at Antwerp Zoo in November 2021 is unknown.
The caretakers had no known infection, had no COVID-19 symptoms before the hippo’s
infection, and were wearing surgical masks during their work [
26
]. While several meters
of distance is kept from the visitors, as the hippos are housed indoors, aerosol transmis-
sion from an infected visitor without perfect masking could have occurred. The genome
sequence of the Delta variant with which the hippos were infected was closely related to
strains commonly circulating in Belgium at the time [26].
The infection of precisely two hippopotamuses in the Antwerp Zoo was unexpected
in the sense that other mammal species have been predicted to be much more susceptible
to SARS-CoV-2 based on in silico models of the molecular interaction between the virus
Spike protein and the host receptor ACE2 [
19
,
20
]. The predictions of SARS-CoV-2
0
s binding
propensity to the hippopotamus viral receptor ACE2 classified the hippopotamus at only
medium risk of being infected with SARS-CoV-2, while other taxa, such as primates,
were classified as high-risk [
19
,
20
]. However, no primates have been reported infected
in Antwerp and Planckendael Zoos. The fact that the hippos were housed in an indoor
complex where visitors also enter could have contributed to an elevated infection risk
for these species. Other species, including bongo, tapir and nutria, that were kept in
the vicinity of the infected hippos were negative when sampled right after the reported
hippopotamus infections. Visitors did not have access to their indoor enclosure. The
hippopotamus infections emphasise that the structural analysis of the SARS-CoV-2 cellular
receptor alone is insufficient to estimate the relative spillover risk of SARS-CoV-2 to other
animal species [19–21].
Monitoring zoonotic infections remains the main key to controlling and limiting the
spread of zoonotic pathogens. The absence of SARS-CoV-2 in our samples prevented us
from expanding the list of potential hosts of SARS-CoV-2. We, however, assessed that
SARS-CoV-2 was not circulating in the mammals from Antwerp and Planckendael Zoos
between September 2020 and July 2021. The reinforcement of strict hygiene measures and
the zoo caretakers’ effective implementation of gloves and masks likely has contributed
to avoiding the transmission of SARS-CoV-2 from humans to animals. Therefore, these
measures seem efficient in limiting the spread of human pathogens to captive animal
populations, and we recommend that these measures be implemented again in case of a
new pandemic.
Vet. Sci. 2023,10, 382 9 of 11
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/vetsci10060382/s1, Figure S1: Serological screening of serum
samples from various mammal zoo species.; www.mdpi.com/xxx/s2 Figure S2. Graph of mean
PCR cycle threshold (Ct) vs. ten-fold dilutions of SARS-CoV-2 template for each PCR system target.
www.mdpi.com/xxx/t1, Table S1: Details of serum samples screened.
Author Contributions:
Conceptualisation, E.V., F.V., S.G. and H.L.; methodology, L.J., E.V., F.V., S.G.,
J.M. and H.L.; formal analysis, T.C. and L.J.; investigation, T.C. and L.J.; data curation, T.C. and L.J.;
writing—original draft preparation, L.J.; writing—review and editing, L.J., E.V., F.V., S.G., T.C., J.M.
and H.L.; visualisation, L.J.; supervision, S.G., H.L. and E.V.; project administration, T.C.; funding
acquisition, H.L. All authors have read and agreed to the published version of the manuscript.
Funding:
This research was funded by ERA-NET BiodivERsA—EC funding BIODIV-AFREID
(Research Foundation—Flanders: G0G2119N), ERA-NET BiodivERsA—EC funding BioRodDis (Re-
search Foundation—Flanders: G0G2219N and the ERA-NET BiodivERsA—EC funding
COVID-19
(Research Foundation—Flanders: G0G1221N), funded by the European Commission and the Research
Foundation–Flanders (FWO). SG is a FED-tWIN scholar funded by the Belgian federal government
(Prf-2019-prf004_OMEga). LJ is, and JM was a postdoctoral fellow of the Research Foundation–
Flanders (FWO) [Grant # 1271922N and #12ZJ721N].
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
All data generated or analysed in this study are presented within the
tables and figures of the manuscript.
Acknowledgments:
We thank the zookeepers from the Antwerp and Planckendael Zoos for collecting
the samples. We thank Valeria Colombo, Rianne van Vredendaal and Tanmay Dharmadhikari for
their assistance in the laboratory.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.
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