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Salmonella enterica  serovar Gallinarum ( S . Gallinarum) can cause fowl typhoid, a severe systemic disease responsible for considerable economic losses. Chicken pathogenicity test is the traditional method for assessing the virulence of  S . Gallinarum. However, this method is limited by several factors, including ethical considerations, costs, and the need for specialized facilities. Hence, we established a chicken embryo lethality assay (ELA) model to determine the virulence of  S . Gallinarum. Three virulent and three avirulent representative strains, which were confirmed by the chicken pathogenicity test, were used to perform the ELA. The most significant difference between the virulent and avirulent strains could be observed when 13-day-old embryos were inoculated via the AC route and incubated for 5 days. Based on a 50% embryo lethal dose (ELD 50 ), isolates considered to be virulent had a Log 10 ELD 50  of ≤ 4.0, moderately virulent strains had a Log 10 ELD 50  of 4.0−6.1, and avirulent isolates had a Log 10 ELD 50  of ≥ 6.1. Different abilities to invade the liver of embryos were found between the virulent and avirulent strains by a growth curve experiment  in vitro . The maximum colony-forming units (CFU) of the virulent strain was about 10,000 times higher than that of the avirulent strain in the liver at 5 days post infection. The ELA results of 42 field strains showed that thirty-two strains (76.2%) were virulent, nine were moderately virulent (21.4%), and one strain was avirulent (2.4%). In conclusion, these results suggest that the ELA can be used as an alternative method to assess the virulence of  S . Gallinarum, which will contribute to the study of virulence genes, virulence evolution, pathogenic mechanisms and vaccine development.

The use of embryonic chicken eggs as an alternative model to evaluate the virulence of Salmonella enterica serovar Gallinarum