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.