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 2ndpassage. 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 1st and
2nd 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 Gammacoronavirus andDeltacoronavirus 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.