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\begin{document}

\title{Susceptibility of turkeys, chickens and chicken embryos to SARS-CoV-2
virus}



\author[1]{Yohannes Berhane}%
\author[1]{Matthew Suderman}%
\author[2]{Shawn Babiuk}%
\author[1]{Brad Pickering}%
\affil[1]{Canadian Food Inspection Agency}%
\affil[2]{Affiliation not available}%


\vspace{-1em}



  
  \date{May 21, 2024}


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\selectlanguage{english}
\begin{abstract}
Susceptibility of turkeys, chickens and chicken embryos to SARS-CoV-2
virus was evaluated by experiment inoculation. Turkeys and chickens were
inoculated using a combination of intranasal, oral and ocular routes.
Both turkeys and chickens did not develop clinical disease or antibodies
to the virus following inoculation. Viral RNA was not detected in oral
and cloacal swabs and in tissues using quantitative real-time RT-PCR. In
addition, chicken embryos were inoculated using the yolk sac,
intravenous, chorioallantoic membrane and allantoic cavity routes did
not support replication of the virus. SARS-COV-2 virus does not affect
both turkeys and chickens in the current genetic state and does not pose
any potential risk to establish in both species of domestic poultry.%
\end{abstract}%



\sloppy


\textbf{Susceptibility of turkeys, chickens and chicken embryos to
SARS-CoV-2 virus}

\textbf{Yohannes Berhane\textsuperscript{1, 2 *}, Matthew
Suderman\textsuperscript{1}, Shawn Babiuk\textsuperscript{1, 3} and
Bradley Pickering\textsuperscript{1, 4}}

Running Title: Susceptibility of poultry to SARS-CoV-2 virus

\textsuperscript{1}National Centre for Foreign Animal Disease, Canadian
Food Inspection Agency, 1015 Arlington Street, Winnipeg, Manitoba, R3E
3R2, Canada

\textsuperscript{2}University of Manitoba, Department of Animal Science,
12 Dafoe Road , 201 Animal Science Bldg. Winnipeg, MB , R3T 2N2, Canada

\textsuperscript{3}Department of Immunology, University of Manitoba,
Winnipeg, Manitoba, Canada

\textsuperscript{4}Department of Medical Microbiology and Infectious
Diseases, University of Manitoba, Winnipeg, Manitoba, Canada

\textsuperscript{*}Corresponding Author

Abstract

Susceptibility of turkeys, chickens and chicken embryos to SARS-CoV-2
virus was evaluated by experiment inoculation. Turkeys and chickens were
inoculated using a combination of intranasal, oral and ocular routes.
Both turkeys and chickens did not develop clinical disease or antibodies
to the virus following inoculation. Viral RNA was not detected in oral
and cloacal swabs and in tissues using quantitative real-time RT-PCR. In
addition, chicken embryos were inoculated using the yolk sac,
intravenous, chorioallantoic membrane and allantoic cavity routes did
not support replication of the virus. SARS-COV-2 virus does not affect
both turkeys and chickens in the current genetic state and does not pose
any potential risk to establish in both species of domestic poultry.

\textbf{KEYWORDS}

SARS-CoV-2; Susceptibility; Chicken Embryos; Turkeys, Chickens; COVID-19

\textbf{1 Introduction}

The coronavirus disease (COVID-19) outbreak starting in 2019 was
declared as public health emergency by the World Health Organization
(WHO) on January 30, 2020, infecting more than 16.5 million people and
continuing to spread throughout the world (WHO 1a, 2020; WHO 1b, 2020).
The disease COVID-19 was first described in Wuhan, China, in patients
that had respiratory tract infection and pneumonia (Zhou et al., 2020;
Andersen et al., 2020). It is caused by a novel betacoronavirus known as
severe acute respiratory syndrome virus 2 (SARS-CoV-2) that is
genetically related to bat coronavirus RaTG13 and other
betacoronaviruses that are found in bats (Zhou et al., 2020; Andersen et
al., 2020; Lu et al., 2020).

Viruses in the coronavirus family are enveloped, single stranded
positive sense RNA viruses with genomes ranging between 26 to 32 kb (Su
et al., 2016; Fehr et al., 2015). The coronavirus family contains a wide
range of viruses that are pathogenic to a variety of animal species
including humans. However, many of these viruses tend to have limited
host range and can cause severe diseases only in domestic animals,
resulting in large production losses to producers. Some animal
coronaviruses such as transmissible gastroenteritis virus (TGEV), bovine
coronavirus (BCoV), feline coronavirus, canine coronavirus and turkey
coronavirus cause enteritis in their respective hosts (Su et al., 2016).
Others such as avian infectious bronchitis and canine respiratory
coronavirus cause respiratory diseases in their respective hosts (Su et
al., 2016).

Prior to 2003, viruses of the coronavirus family were not known to cause
severe disease in humans. However, since the beginning of the
21\textsuperscript{st} century three viruses from the betacoronavirus
subfamily, Severe Acute Respiratory Syndrome virus (SARS), Middle
Eastern Respiratory Syndrome virus (MERS) and SARS-Cov-2 have crossed
the species barrier to infect humans (Kin et al., 2015; Peiris et al.,
2003; Zhou et al., 2020).

When a novel virus results in an outbreak or pandemic, assessing the
risk of the novel agent to infect other species is of paramount
importance in order to control the disease. Since the emergence of
SARS-CoV-2, a number of studies have been conducted as part of the risk
assessment to determine if domestic animals that are used as companion
animals in households or as source of food are susceptible to the virus.
Additional studies were also conducted in laboratory animals such as
mice, ferrets, non-human primates and others in search of animal models
that could be used to study virus pathogenesis, vaccine efficacy studies
and prophylaxis of the disease.

The purpose of this study was to determine if domestic poultry such as
turkeys and chickens are susceptible to SARS-CoV-2 virus and play any
potential role in the dissemination of the virus. In addition, the
susceptibly of chicken embryos to SARS-CoV-2 was evaluated to determine
if eggs could potentially be used for the production of potential
SARS-CoV-2 vaccines.

\textbf{2 Materials and Methods}

\textbf{2.1 Animal Experiment}

The animal experiments were conducted under the approval of the Canadian
Science Center for Human and Animal Health Animal Care Committee which
follows the guidelines of the Canadian Council on Animal Care. To
determine if domestic poultry are susceptible to SARS-CoV-2 infection,
fourteen 4- to 6-week-old leghorn chickens (\emph{Gallus gallus
domesticus} ) were acquired from the CFIA SPF flock in Nepean, Ottawa
and fourteen 4-to 6-week-old turkey (\emph{Melleagris gallopavo} )
pullets were purchased from a local turkey breeder in Manitoba. Both
turkeys and chickens were kept for a week in biosafety level 3 animal
cubicles for acclimatization for a week. After a week, both turkeys and
chickens were split into 2 groups (SARS-CoV-2 and control). Prior to
infection, blood, oropharyngeal and cloacal swab samples were collected
from SARS-COV-2 and control group chickens. Ten chickens and 10 turkeys
were inoculated using a combination of natural routes of infection
(combination of oral, nasal and ocular) with one ml containing
10\textsuperscript{6} plaque forming units per ml (PFU/ml) of SARS-COV-2
virus. Control groups were mock inoculated using same routes with PBS.
Oropharyngeal and cloacal swabs were collected from infected and control
groups at 2, 4, 6, 9 and 12 days post-inoculation. Two chickens and two
turkeys were euthanized on 3, 5, and 7 days post-inoculation and tissues
samples were collected (brain, thymus, trachea, lungs, spleen, duodenum,
cecal tonsil, liver, kidneys, bursa and heart).

\textbf{2.2 Chicken Embryo Susceptibility Study}

To determine if chicken embryos were susceptible to SARS-CoV-2 virus
infection, chicken embryos were inoculated by different routes commonly
used to virus isolation. Six day old chicken embryos were inoculated
using the yolk sac route; 9 day old chicken embryos inoculated in the
chorionallantoic membrane (CAM) and the allantoic cavity; and 11 day old
chicken embryos inoculated by the intravenous route. The embryos were
candled twice daily to monitor for mortality. Two successive blind
passages were performed and after the 1\textsuperscript{st} and
2\textsuperscript{nd} passages, total RNA was extracted from the amion
allantoic fluid (AAF), embryo and CAM and checked for the presence of
SARS-CoV-2 genomic material using the E-gene specific RT-qPCR.

\textbf{2.3 RNA Extraction}

Total RNA extraction from all clinical samples was conducted using the
MagMAX Express-96 Magnetic Particle Processor (Thermo-Fisher) using the
MagMax CORE Nucleic Acid Purification Kit (Thermo-Fisher) with some
modification. Samples were inactivated with tripure reagent (1:9 ratio).
Then, 650 \selectlanguage{greek}µ\selectlanguage{english}L of the inactivated sample, 30\selectlanguage{greek}µ\selectlanguage{english}L of binding beads, and 350
\selectlanguage{greek}µ\selectlanguage{english}L binding buffer spiked with enteroviral armoured RNA (ARM-ENTERO;
Asuragen) was used for extraction. The extracted RNA was eluted in 30 \selectlanguage{greek}μ\selectlanguage{english}L
elution buffer. The spiked enteroviral armoured RNA was used as an
exogenous RNA extraction control.

\textbf{2.4 Real-time RT-PCR}

Total RNA was extracted from all clinical samples and checked for the
presence of SARS-Cov-2 RNA by an E gene specific semi quantitative
real-time RT-PCR assay (RT-qPCR) that detects a broad range of human and
bat coronaviruses (Corman et al., 2019). For the detection of SARS-CoV-2
RNA by RT-qPCR, we used 4X TaqMan\selectlanguage{ngerman}® Fast Virus one step RT-PCR kit (Life
Tech., USA) according to manufacturer's recommendations. For each
RT-qPCR reaction, 0.4 \selectlanguage{greek}µ\selectlanguage{english}M of each E gene forward and reverse primers and
0.2 \selectlanguage{greek}µ\selectlanguage{english}M of probe were used. RT-qPCR runs were performed using a 7500 Fast
Real-Time PCR System (Applied Biosystem) using the following cycle
conditions: 50\selectlanguage{ngerman}°C for 5 min, 95°C for 20 sec, and 40 cycles of 95°C for
3s followed by 60°C for 30s. RT-qPCR semi-quantitative results were
calculated based on a gBlock (Integrated DNA Technologies, IDT) standard
curve for SARS-CoV-2 E gene.

\textbf{2.5 Virus Neutralization Assay}

To determine the presence of neutralizing SARS-CoV-2 antibodies in the
sera of chickens and turkeys, a plaque reduction neutralization test was
used. Serum samples collected at 0, 7, 14 and 21 days after infection
were heat inactivated at 56°C for half an hour. Serial five-fold
dilutions of the inactivate sera were incubated with virus for 1 hour at
37°C. Each virus-serum mixture was then added to duplicate wells of Vero
E6 cells in a 48-well format, incubated for 1 hour at 37°C, and overlaid
with 500 \selectlanguage{greek}μ\selectlanguage{english}l of 2.00\% carboxymethyl cellulose (Sigma) in Dulbecco's
Modified Eagle Medium (Thermo-Fisher) per well. Plates were then
incubated at 37\selectlanguage{ngerman}°C for 72 hours, fixed with 10\% buffered formalin and
stained with 0.5\% crystal violet. Serum dilutions resulting in
\textgreater{}70\% reduction of plaque counts compared to virus controls
were considered positive for virus neutralization.

\textbf{2.6 Histopathology}

Tissue samples (brain, thymus, trachea, lungs, heart, kidneys, pancreas,
gut, cecal tonsil and liver) were fixed in formalin. After 48 hrs, the
fixed tissues samples were embedded in paraffin, sectioned, stained with
H \& E and were examined for microscopic lesions as described previously
(Neufield et al., 2009).

\textbf{3 Results and Discussion}

Chickens and turkeys did not develop any clinical signs of the disease
for the entire interval of the experiment. No shedding of SARS-COV-2
genomic material was detected in cloacal and oropharyngeal swabs
collected from both turkeys and chickens at 2, 4, 6, 9 and 12 days
post-inoculation by RT-qPCR. In addition, no SARS-COV-2 genomic material
was detected in tissues (brain, thymus, trachea, lungs, heart, kidneys,
pancreas, gut, cecal tonsil and liver) collected from chickens or
turkeys at 3, 5 and 7 days after infection by RT-qPCR.

No gross pathological and histopathological changes were observed in the
brain, thymus, trachea, lungs, heart, kidneys, pancreas, gut, cecal
tonsil and liver. No SARS-COV-2 specific antibodies were detected in
serum samples collected from both chickens and turkeys at 7, 14 and 21
days after infection using the plaque reduction neutralization test.

Chicken embryos inoculated with SARS-COV-2 using the different routes
were alive 6 days after inoculation. Pools of embryo tissues, AAF and
CAM were harvested and checked for presence SARS-CoV-2 genomic material
by RT-qPCR. After the first passage, we were able to detect presence of
SARS-CoV-2 genomic material in the AAF, CAM and body of the infected
embryos in ECEs inoculated using different inoculation routes. However,
based on RT-qPCR results, the amount SARS-COV-2 genomic material in all
ECE samples analyzed was almost a log lower when compared to the RT-qPCR
results of the original inoculum indicating that this is residual
genomic material SARS-CoV-2 from the inoculum (Fig. 1A). In normal
situations, if the virus is replicating in embryos you will see increase
in the presence of virus compared to the inoculum. Embryo samples from
each inoculation group were used for conducting a
2\textsuperscript{nd}passage. After 6 days of incubation, no chicken
embryo mortality was observed. Amion allantoic fluid was harvested from
each group and evaluated for the presence SARS-CoV-2 RNA using the
E-gene specific RT-qPCR (Fig. 1B). Only traces of genomic material of
the virus were detected in all samples collected from chicken embryos
inoculated using the different routes of inoculation indicating that
chicken embryos are not susceptible to SARS-CoV-2.

To determine if the SARS-CoV-2 that was collected from 4 different
embryo inoculation groups after 1\textsuperscript{st} and
2\textsuperscript{nd} passages was still viable, Vero E-6 cells grown to
90-95\% confluence were inoculated with chicken embryo samples that had
lower CT values based on the RT-qPCR. No cytopathic (CPE) was observed
in Vero E-76 cells after 2 consecutive cell passages. This demonstrates
that the virus that was detected in the embryo samples by RT-qPCR was
not viable and was residual genomic material of the virus inoculum used
to infect the embryos.

SARS-COV-2 virus does not affect both turkeys and chickens in the
current genetic state and does not pose any potential risk to establish
in these of species of domestic poultry. Previous study has shown that
chickens infected with SARS-CoV-2 using the intranasal route were not
susceptible to clinical infection (Shi et al., 2020). However, in the
current study, we also demonstrate that turkeys, chickens and chicken
embryos inoculated using different inoculation routes are not
susceptible to SARS-CoV-2 infection. Unlike \emph{Gammacoronavirus}
and\emph{Deltacoronavirus} genera of the Coronavirus family, viruses in
betacoronavirus genus do not seem to infect birds (Woo et al., 2012).
These findings are very important for risk analysis as chickens, turkeys
and eggs are an important component of the human diet and are also found
in large numbers in farms as well as in backyards of suburban
population.

\textbf{DATA AVAILABILITY STATEMENT}

The data that support the findings of this study are available from the
corresponding author upon reasonable request.

\textbf{ACKNOWLEDGEMENTS}

The authors gratefully acknowledge Dr. Valerie Smid and the Animal Care
unit for the support provided with the animal experiments. Funds for the
project were provided by the CFIA emergency funding for COVID-19
response.

\textbf{CONFLICT OF INTEREST}

The authors declare no conflict of interest.

\textbf{ETHICAL APPROVAL}

The authors confirm that the ethical policies of the Transboundary and
Emerging Diseases Journal have been adhered to and that the animal
experiments were conducted under the approval of the Canadian Science
Center for Human and Animal Health Animal Care Committee which follows
the guidelines of the Canadian Council on Animal Care.

\textbf{References}

Andersen, K. G., Rambaut, A., Lipkin, W. I., Holmes, E. C., \& Garry, R.
F. (2020). The proximal origin of SARS-CoV-2. Nature medicine, 26(4),
450--452. https://doi.org/10.1038/s41591-020-0820-9

Corman, V. M., Landt, O., Kaiser, M., Molenkamp, R., Meijer, A., Chu, D.
K., \ldots{} Drosten, C. (2020). Detection of 2019 novel coronavirus
(2019-nCoV) by real-time RT-PCR. Euro surveillance : bulletin Europeen
sur les maladies transmissibles = European communicable disease
bulletin, 25(3), 2000045.
https://doi.org/10.2807/1560-7917.ES.2020.25.3.2000045

Fehr, A. R., \& Perlman, S. (2015). Coronaviruses: an overview of their
replication and pathogenesis. Methods in molecular biology (Clifton,
N.J.), 1282, 1--23. https://doi.org/10.1007/978-1-4939-2438-7\_1

Kin, N., Miszczak, F., Lin, W., Gouilh, M. A., Vabret, A., \& EPICOREM
Consortium (2015). Genomic Analysis of 15 Human Coronaviruses OC43
(HCoV-OC43s) Circulating in France from 2001 to 2013 Reveals a High
Intra-Specific Diversity with New Recombinant Genotypes. Viruses, 7(5),
2358--2377. https://doi.org/10.3390/v7052358

Lu, R., Zhao, X., Li, J., Niu, P., Yang, B., Wu, H., \ldots{} Tan, W.
(2020). Genomic characterisation and epidemiology of 2019 novel
coronavirus: implications for virus origins and receptor binding. Lancet
(London, England), 395(10224), 565--574.
https://doi.org/10.1016/S0140-6736(20)30251-8

Peiris, J. S., Lai, S. T., Poon, L. L., Guan, Y., Yam, L. Y., Lim, W.,
\ldots{} SARS study group (2003). Coronavirus as a possible cause of
severe acute respiratory syndrome. Lancet (London, England), 361(9366),
1319--1325. https://doi.org/10.1016/s0140-6736(03)13077-2

Neufeld, J. L., Embury-Hyatt, C., Berhane, Y., Manning, L., Ganske, S.,
\& Pasick, J. (2009). Pathology of highly pathogenic avian influenza
virus (H5N1) infection in Canada geese (Branta canadensis): preliminary
studies. Veterinary pathology, 46(5), 966--970.
https://doi.org/10.1354/vp.08-VP-0168-E-FL

Shi, J., Wen, Z., Zhong, G., Yang, H., Wang, C., Huang, B., \ldots{} Bu,
Z. (2020). Susceptibility of ferrets, cats, dogs, and other domesticated
animals to SARS-coronavirus 2. Science (New York, N.Y.), 368(6494),
1016--1020. https://doi.org/10.1126/science.abb7015

Su, S., Wong, G., Shi, W., Liu, J., Lai, A., Zhou, J., \ldots{} Gao, G.
F. (2016). Epidemiology, Genetic Recombination, and Pathogenesis of
Coronaviruses. Trends in microbiology, 24(6), 490--502.
https://doi.org/10.1016/j.tim.2016.03.003

WHO. WHO Director-General's statement on IHR Emergency Committee on
Novel Coronavirus (2019-nCoV). 30 January 2020.
https://www.who.int/dg/speeches/detail/who-director-general-s-statement-on-ihr-emergency-committee-on-novel-coronavirus-(2019-ncov)

WHO. WHO Coronavirus Disease (COVID-19) Dashboard for 2020/07/29.
https://covid19.who.int/

Woo, P. C., Lau, S. K., Lam, C. S., Lau, C. C., Tsang, A. K., Lau, J.
H., \ldots{} Yuen, K. Y. (2012). Discovery of seven novel Mammalian and
avian coronaviruses in the genus deltacoronavirus supports bat
coronaviruses as the gene source of alphacoronavirus and betacoronavirus
and avian coronaviruses as the gene source of gammacoronavirus and
deltacoronavirus. Journal of virology, 86(7), 3995--4008.
https://doi.org/10.1128/JVI.06540-11

Zhou, P., Yang, X. L., Wang, X. G., Hu, B., Zhang, L., Zhang, W.,
\ldots{} Shi, Z. L. (2020). A pneumonia outbreak associated with a new
coronavirus of probable bat origin. Nature, 579(7798), 270--273.
https://doi.org/10.1038/s41586-020-2012-7

\textbf{Figure Legends}

Figure 1. Persistence of SARS-CoV-2 genomic material in AAF, CAM and
body of the chicken embryos inoculated using different inoculation
routes after 1 passage (Fig 1A) and 2nd passage (Fig 1B). RT-qPCR
semi-quantitative results were calculated based on a gBlock (Integrated
DNA Technologies, IDT) standard curve for SARS-CoV-2 E gene.\selectlanguage{english}
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