Serological survey of SARS‐CoV‐2 for experimental, domestic, companion and wild animals excludes intermediate hosts of 35 different species of animals

Abstract

The pandemic SARS‐CoV‐2 has been reported in 123 countries with more than 5000 patients died from it. However, the original and intermediate hosts of the virus remain unknown. In this study, 1914 serum samples from 35 animal species were used for detection SARS‐CoV‐2 specific antibodies using double antigen sandwich ELISA after validating its specificity and sensitivity. The results showed that no SARS‐CoV‐2 specific antibodies were detected in above samples which excluded the possibility of 35 animal species as intermediate host for SARS‐CoV‐2. More importantly, companion animals including pet dogs (including one dog the SARS‐CoV‐2 patient kept and two dogs which had close contact with it) and cats, street dogs and cats also showed serological negative to SARS‐CoV‐2, which relieved the public concerns for the pets as SARS‐CoV‐2 carriers.

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DR. KEGONG TIAN (Orcid ID : 0000-0001-5362-1415)
Article type : Short Communication
Short Communication
Serological survey of SARS-CoV-2 for experimental, domestic, companion and wild animals
excludes intermediate hosts of 35 different species of animals
Junhua Deng1, Yipeng Jin3, Yuxiu Liu2, Jie Sun2, Liying Hao2, Jingjing Bai2,
Tian Huang2, Degui Lin3, Yaping Jin1*, Kegong Tian2*
1College of veterinary medicine, Northwest A&F University, Yangling 712100,
2National Research Center for Veterinary Medicine, Luoyang 471003
3College of Veterinary Medicine, China Agricultural University, Beijing, 100193
*Corresponding authors: Profs. Yaping Jin and Kegong Tian
E-mail: yapingjin@163.com, tiankg@263.net
Running tittle: SARS-CoV-2 serological survey in 35 animal species
Abstract: The pandemic SARS-CoV-2 has been reported in 123 countries with more than 5000
patients died from it. However, the original and intermediate hosts of the virus remain unknown.
In this study, 1914 serum samples from 35 animal species were used for detection SARS-CoV-2
specific antibodies using double antigen sandwich ELISA after validating its specificity and
sensitivity. The results showed that no SARS-CoV-2 specific antibodies were detected in above
samples which excluded the possibility of 35 animal species as intermediate host for
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SARS-CoV-2. More importantly, companion animals including pet dogs (including one dog the
SARS-CoV-2 patient kept and two dogs which had close contact with it) and cats, street dogs and
cats also showed serological negative to SARS-CoV-2, which relieved the public concerns for the
pets as SARS-CoV-2 carriers.
Key words: SARS-CoV-2, Intermediate hosts, Wild animals
Introduction
SARS-CoV-2, previously was named as COVID-2019 by the WHO, is now pandemic which
has been reported 5077 human death of 136895 confirmed cases in 123 countries (updated on
March 14, 2020 from WHO official website). The viruses have been successfully isolated, but the
pathogenesis mechanisms and effective vaccines are undergoing extensively study. SARS-CoV-2
belongs to Betacoronavirus genera in the subfamily Orthocoronavirinae of family Coronaviridae,
in which SARS-CoV and MERS-CoV are also in this group. The natural host of highly pathogenic
SARS and MERS coronaviruses was confirmed as bats, and bats are also thought to be the natural
hosts for SARS-CoV-2 based upon genomic sequence analysis (Wang et al., 2020). Coronaviruses
needed intermediate hosts before being able to infect humans. Masked palm civets and dromedary
camels were confirmed as intermediate hosts for SARS CoV and MERS CoV (Guarner, 2020), but
the intermediate hosts remain unknown for SARS-CoV-2 (Ward et al., 2020).
In order to find the intermediate host of SARS-CoV-2, a commercial double antigen sandwich
ELISA, which could be applied for different species of animals, was used to detect SARS-CoV-2
specific antibodies in different species of animals. Before applied to clinical serum samples, the
sensitivity and specificity of kit was initially confirmed using SARS-CoV-2 positive and negative
sera from experimental animals including rabbit, mouse, pig, and ferret. SARS-CoV-2 negative
sera from other species of experimental animals were also used which included chicken, duck, rat,
guinea pig, beagle dog, and rhesus monkey. After that, the kit was used to detect SARS-CoV-2
specific antibodies in domestic livestock (pig, cow, sheep, horse), poultry (chicken, duck, goose),
experimental animals (mice, rat, guinea pig, rabbit, and monkey), companion animal (dog and cat),
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and wild animals (camel, fox, mink, alpaca, ferret, bamboo rat, peacock, eagle, tiger rhinoceros,
pangolin, leopard cat, jackal, giant panda, masked civet, porcupine, bear, yellow-throated marten,
weasel, red pandas, and wild boar). The results showed that no SARS-CoV-2 specific antibodies
were detected in above species of animals including pangolin which has been reported as an
intermediate host of SARS-CoV-2 (Kangpeng Xiao, 2020). More importantly, we found
companion animals including dogs and cats were serologically negative to SARS-CoV-2 including
one dog kept by the SARS-CoV-2 patient and two dogs with close contact with it during the
quarantine.
Materials and methods
The SARS-CoV-2 double antigen sandwich ELISA were purchased from Luoyang Putai
Biotechnology Co., Ltd (Luoyang, China). The coating was based on S1 protein of SARS-CoV-2.
The same antigen was linked to horseradish peroxidase (HRP) to function as conjugate. The serum
samples were test as the manufacture manual instructions. Briefly, 100µl serum sample was added
into each well of ELISA plate and incubated at 37°C for 30 min. After washing the plate with
washing buffer for 5 times, HRP-labeled antigen was added into the wells at 37°C for 30 min
before 100μl of the substrate solution was added to each well and incubated at 37°C for 10 min to
stop the reaction. The optical density (OD) was measured at 450 nm. The final value of OD450 of
sample = the value of OD450 readout of sample - the value of OD450 of blank control. The cutoff
was set as 0.26 + the mean value of OD450 of negative controls.
The serum samples of chicken, duck, mouse, rat and pig were preserved in our laboratory. The
ferret SARS-CoV-2 positive and negative serum samples were provided by Harbin Veterinary
Research Institute, Chinese Academy of Agricultural Sciences. The ferrets (3-month old) were
infected with 1.2 mL SARS-CoV-2 (1×105TCID50/ml) by intranasal infection in ABSL-3 facility.
The infected ferrets were bled at 0, 7, 12, 17 and 22 dpi and euthanized at 22dpi. The serum
samples were collected and inactivated before use. The positive sera for other different
coronaviruses were also used in this study. The positive serum samples for porcine epidemic
diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine
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deltacoronavirus (PDCoV) were made by immunization of SPF pigs with the corresponding virus,
respectively. The positive serum samples for infectious bronchitis virus (IBV) was immunization
of SPF chickens with the virus. Positive sera for mouse hepatitis virus (MHV) and rat corona virus
(RCV) were made by infection of SPF mice and rats by MHV and RCV, respectively. The rest of
serum samples from different species were collected from November 2019 to March 2020 and
kept in our laboratory. All samples were collected in compliance with fundamental ethical
principles. The numbers of different animal species used in this study were shown in the brackets
below.
Results and discussion
To test the specificity of ELISA kit, the serum samples of SPF chicken (28), duck (25), mouse
(23), rat (20) and pig (20) were applied. The final value of OD450 of samples ranged from 0.005 to
0.103 (median 0.007), 0.004 to 0.008 (median 0.006), 0.005 to 0.190 (median 0.007), 0.004 to
0.050 (median 0.007), 0.005 to 0.134 (median 0.007) for chicken, duck, mouse, rat and pig,
respectively. Serum samples from other species of experimental animals including guinea pig (30),
rabbits (34), beagle dogs (130), rhesus monkeys (38) were also tested. There were no
SARS-CoV-2 antibodies detected in above animals (data not shown). Next, the potential
cross-reaction with other coronavirus including IBV (26), PEDV (24), TGEV (20), PDCoV (20),
MHV (20) and RCV (20) were tested with corresponding positive serum samples. The final value
of OD450 of samples ranged from 0.004 to 0.134 (median 0.007), 0.005 to 0.022 (median 0.007),
0.007 to 0.071(median 0.018), 0.005 to 0.040 (median 0.010), 0.004 to 0.061(median 0.006),
0.005 to 0.064 (median 0.009) for IBV, PEDV, TGEV, PDCoV, MHV and RCV, respectively.
The above results showed that the SARS-CoV-2 ELISA has good specificity without
cross-reaction with other coronaviruses from different animal species.
We next tested the sensitivity of ELISA kit. The SARS-CoV-2 experimental-infected ferret
positive sera were tested. As shown in Table1, the neutralizing antibody titers of 5 infected ferret
(F1-F5) were between 1:128 and 1:256 at 22 days post-infection (dpi). By contrast, the
neutralizing antibody titers of 5 placebo ferrets (C1-C5) were all negative at 22 dpi. In the line
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with the results of neutralizing antibodies, the final OD450 of 5 positive sera detected by ELISA
were all above 3, which indicated strongly serological positive to SARS-CoV-2. To further test the
dynamic changes of ELISA titer of infected ferret, serum samples from one ferret were collected
from 0, 7, 12, 17, 22dpi, respectively. The positive ELISA results were shown at 7dpi and lasted
until 22dpi when the ferrets were humanely euthanized (Table1). The above results showed that
the ELISA has good specificity and sensitivity and suitable for different species of animals.
After confirming the specificity, sensitivity and suitability of SARS-CoV-2 ELISA kit for
different species of experimental animals, clinical serum samples from domestic livestock (pig,
cow, sheep, horse), poultry (chicken, duck, goose), experimental animal (mice, rat, and rhesus
monkey), companion animal (dog and cat), and wild animals (camel, fox, mink, alpaca, ferret,
bamboo rat, peacock, eagle, tiger rhinoceros, pangolin, leopard cat, jackal, giant panda, masked
civet, porcupine, bear, yellow-throated marten, weasel, red pandas, and wild boar). were used for
antibody detection. As shown in Table2, all serum samples had negative results which excludes
the above animal species as intermediate host of SARS-CoV-2. Real-time PCR with specific
primers and probe for SARS-CoV-2 recommended by Chinese Center for Disease Control and
Prevention was also performed for parts of serum samples including the dog kept by confirmed
SARS-CoV-2 patient and two dogs with close contact with it, and the results were negative (data
not shown). Of note, no SARS CoV-2 specific antibodies were detected in all dogs and cats, even
for the street dogs and cats in Wuhan City and the pet dog raised by SARS CoV-2 patient.
So far, seven coronaviruses were confirmed infection of human including SARS CoV, MERS
CoV, HCoV NL63, HCoV 229E, HCoV OC43, HKU1 and SARS-CoV-2. Bat was deemed to be
the natural host for SARS CoV, MERS CoV, HCoV NL63 and HCoV 229E, and rodents for
HCoV OC43 and HKU1(Khan et al., 2020). The intermediate hosts for SARS CoV, MERS CoV,
HCoV 229E, and HCoV OC43 were found to be palm civets, dromedary camels, alpacas and
cattle, respectively. However, the natural and intermediate hosts SARS-CoV-2 remain unknown.
Since SARS-CoV-2 is genetically close to SARS-CoV, it has been proposed that bat could be the
natural host (Phan, 2020). Snake is also presumed as wildlife animal reservoir for SARS-CoV-2
based on the virus relative synonymous codon usage (RSCU) bias (Ji et al., 2020). However, there
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is no report of SARS-CoV-2 isolation or molecular and serological confirmation of infection from
snake samples. Pangolins recently was suggested to be direct animal source of SARS-CoV-2 for
humans since the SARS-CoV-2 related coronaviruses were isolated from Malayan pangolins
which shared 97.4% similarity with SARS-CoV-2 in virus receptor binding domain in S gene
(Kangpeng Xiao, 2020). In our study, we did not detect SARS-CoV-2 antibodies in 17 pangolin
serum samples. Consistent with our results, Li X, et al reported the coronavirus carried by
pangolins did not have the RRAR motif, a unique peptide insertion in the human SARS-CoV-2
virus (Li et al., 2020). The RRAR motif may be involved in the proteolytic cleavage of spike
protein and host range and transmissibility which suggests human SARS-CoV-2 virus did not
come directly from pangolins (Li et al., 2020). Masked civet and camel were confirmed to be
natural hosts for SARS-CoV and MERS-CoV, no specific SARS-CoV-2 antibodies were detected
in 10 masked civets and 31 camels in this study.
The susceptibility of companion animals including cats and dogs to the SARS-CoV-2 has been
major concern for the public. One pet dog was reported to be SARS-CoV-2 positive detected by
RT-PCR in Hongkong (https://www.news.gov.hk/eng/2020/02/
20200228/20200228_093205_796.html). Later, the serological result of the dog showed negative
after quarantine of 14 days. In our study, 87 cats including 66 pet cats and 21 street cats showed
serological negative to SARS-CoV-2 (Table2). At the same time, 487 dogs including 90 beagle
dogs, 147 pet dogs and 250 street dogs during the outbreak of SARS-CoV-2 were also tested
serological negative. Among them, 15 pet dog and 99 street dog sera were collected from Wuhan
City. It should be noted that, one pet dog from confirmed SARS-CoV-2 infected patient showed
serologically negative, and other two dogs which had close contact with this dog also tested to be
negative. However, we cannot rule out of susceptibility of cats and dogs to SARS-CoV-2, which
need to be tested by experimental infections.
Molecular techniques such as reverse-transcriptase PCR tests and viral genome sequencing are
widely used for the confirmation of human infection. These techniques are also used to explore the
potential hosts of SARS-CoV-2 (Pfefferle et al., 2020). Compared to these molecular methods,
serological test such as ELISA has several advantages. First, the host generates SARS-CoV-2
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specific antibodies after infection which could last longer than the viremia. It provides a wider
detection window for ELISA than RT-PCR. Second, RNA extraction from susceptive infected
samples has to be performed in a BSL-3 laboratory. By contrast, ELISA can be performed in a
safety level 2 laboratory and does not require high containment facilities after the serum samples
were inactivated at 56°C for 30 min. Third, double antigen sandwich ELISA based on
recombinant S1 protein could detect both IgM and IgG antibodies and is not limited to species. To
find the host of SARS-CoV-2, the screening of other wild animals using ELISA in undergoing in
our laboratory.
Acknowledgments
We want to thank Dr. Zhigao Bu, Director of Harbin Veterinary Research Institute, Chinese
Academy of Agricultural Sciences for providing inactivated SARS-CoV-2 negative and positive
ferret serum samples. This study was supported by Luoyang Heluo Talent Plan (Kegong Tian).
Conflict of interest statement
There was no conflict of interest with others.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon
reasonable request.
Ethical Statement
We declare that Ethical Statement is not applicable.
Reference
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identified coronavirus 2019-nCoV. Journal of medical virology, 92, 433-440.
Kangpeng Xiao, J. Z., Yaoyu Feng, Niu Zhou, Xu Zhang, Jie-Jian Zou, Na Li, Yaqiong Guo,
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Table Legends
Table1. ELISA and neutralizing antibody titer results of ferret sera.
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F1 (0 dpi)
0.007
-
﹤2
-
F1 (7 dpi)
0.841
+
16
+
F1 (12 dpi)
1.301
+
32
+
F1 (17 dpi)
3.477
+
128
+
F1 (22 dpi)
3.234
+
128
+
F2 (22 dpi)
3.023
+
256
+
F3 (22 dpi)
3.444
+
256
+
F4 (22 dpi)
3.5
+
256
+
F5 (22 dpi)
3.332
+
256
+
C1 (0 dpi)
0.022
-
<2
-
C2 (0 dpi)
0.014
-
<2
-
C3 (0 dpi)
0.009
-
<2
-
C4 (0 dpi)
0.013
-
<2
-
C5 (0 dpi)
0.025
-
<2
-
C1 (22 dpi)
0.021
-
<2
-
C2 (22 dpi)
0.027
-
<2
-
C3 (22dpi)
0.018
-
<2
-
C4 (22 dpi)
0.005
-
<2
-
Ferret
C5 (22dpi)
0.011
-
<2
-
*The neutralizing antibody titer of positive samples were ≥4. dpi: days post-infection.
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Animal
species
Serum
sample ID
Final OD450
Results
Neutralizing
antibody titer *
Results

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Table2. Summary of ELISA antibody titer results of 35 animal species.
Animal species
Number
Minimum of
ELISA readout
Maximum of
ELISA
readout
Median of
ELISA
readout
Pig1
187
0.005
0.134
0.007
Cow
107
0.002
0.18
0.007
Sheep
133
0.002
0.169
0.01
Domestic
Animals (4)
Horse
18
0.002
0.189
0.011
Chicken2
153
0.005
0.134
0.006
Duck3
153
0.004
0.189
0.007
Poultry (3)
Goose
25
0.005
0.121
0.005
Mice4
81
0.004
0.19
0.006
Rat5
67
0.004
0.095
0.008
Guinea pig6
30
0.005
0.031
0.008
Rabbit7
34
0.005
0.029
0.006
Monkey8
39
0.001
0.141
0.011
Experimental
and
companion
animals (7)
Dog9
487
0.004
0.198
0.007
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Cat10
87
0.005
0.045
0.007
Camel
31
0.005
0.178
0.008
Fox
89
0.005
0.197
0.009
Mink
91
0.001
0.195
0.008
Alpaca
10
0.004
0.02
0.006
Ferret
2
0.036
0.038
0.037
Bamboo rat
8
0.005
0.008
0.006
Peacock
4
0.006
0.009
0.007
Eagle
1
0.0066
0.0066
0.0066
Tiger
8
0.004
0.077
0.005
Rhinoceros
4
0.005
0.006
0.005
Pangolin
17
0.004
0.156
0.006
Leopard cat
3
0.005
0.007
0.005
Jackal
1
0.01
0.01
0.01
Giant panda
14
0.005
0.05
0.007
Masked civet
10
0.004
0.014
0.006
Porcupine
2
0.007
0.007
0.007
Bear
9
0.005
0.006
0.006
Yellow-throate
d marten
4
0.005
0.095
0.008
Weasel
1
0.006
0.006
0.006
Red pandas
3
0.005
0.005
0.005
Wild animals
(21)
Wild boar
1
0.005
0.005
0.005
Note: All above results were negative. 1Including 20 SPF pigs. 2Including 28 SPF chickens. 3Including 25 SPF
ducks.4Including 23 SPF mice.5Including 20 SPF rats. 6Experimental animals. 7Experimental animals.8Including 38
rhesus monkey and one wild Ruffed lemur. 9Including 90 beagle dogs, 250 street dogs, and 147 pet dogs. Fifteen pet
dog and 99 street dog sera were collected from Wuhan City. 10Including 66 pet cats and 21 street cats.
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