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

Jun-Feng Zhang,Ke Shang,Hyung-Kwan Jang,Bai Wei,Se-Yeoun Cha,Min Kang,Grzegorz Woźniakowski

doi:10.1371/journal.pone.0238630

Abstract

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 (ELD50), isolates considered to be virulent had a Log10ELD50 of ≤ 4.0, moderately virulent strains had a Log10ELD50 of 4.0−6.1, and avirulent isolates had a Log10ELD50 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.

Highlights

Gallinarum 287/91 (NCTC 13346), A17-DW-005, and A18-GCVP-014 were selected as representative virulent strains and the vaccine Nobilis SG9R (Intervet International, Boxmeer, The Netherlands), A17-DW-005ΔspiC and A17-DW-005ΔwaaJΔspiC were selected as representative avirulent strains [24, 25]
All 6-day-old chicken embryos died when inoculated by yolk sac (YS), which indicated that virulent and avirulent strains could not be distinguished through the YS route
When the 13-day-old chicken embryos were inoculated by the allantoic cavity (AC), the most significant difference between the ELD50 values of the virulent and avirulent strains was manifested

Summary

Introduction

Gallinarum) is the causative agent of fowl typhoid (FT), a serious systemic disease that causes huge economic losses to the commercial poultry industry [1]. Gallinarum in embryonic chicken eggs (716002-7, 120005-2) 2. Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry(IPET) through Animal Disease Management Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs(MAFRA)(119059-2) 3. The selection of research-oriented professor of Jeonbuk National University in 2020 The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Methods

Results

Conclusion