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Salmonella  Enteritidis is a major cause of salmonellosis worldwide and more than 80% of outbreaks investigated in Europe have been associated with the consumption of poorly cooked eggs or foods containing raw eggs. Vaccination has been proven to be one of the most important measures to control  Salmonella  Enteritidis infections in poultry farms as it can decrease colonization of the reproductive organs and intestinal tract of laying hens, thereby reducing egg contamination. Differentiation of live vaccine from field or wild type  S . Enteritidis isolates in poultry is essential for monitoring of veterinary isolates and targetting control actions. Due to decreasing costs, whole genome sequencing (WGS) is becoming a key tool for characterization of  Salmonella  isolates, including vaccine strains. Using WGS we described the genetic changes in the live attenuated Salmovac 440 and AviPro SALMONELLA VAC E vaccine strains and developed a method for differentiation from the wildtype  S . Enteritidis strains. SNP analysis confirmed that streptomycin resistance was associated with a Lys43Arg missense mutation in the  rps L gene whilst 3 missense mutations in  acrB  and 1 missense mutation in  acrA  confer erythromycin sensitivity in AviPro SALMONELLA VAC E. Further mutations Arg242His in  purK  and Gly236Arg in the  hisB  gene were related to adenine and histidine dependencies in Salmovac 440. Unique SNPs were used to construct a database of variants for differentiation of vaccine from the wildtype isolates. Two fragments from each vaccine were represented in the database to ensure high accuracy. Each of the two selected Salmovac 440 fragments differed by 6 SNPs from the wildtype and the AviPro SALMONELLA VAC E fragments differed by 4 and 6 SNPs, respectively. CD-hit software was applied to cluster similar fragments that produced the best fit output when searched with SRST2. The developed vaccine differentiation method was tested with 1,253 genome samples including field isolates of Salmovac 440 ( n  = 51), field isolates of AviPro SALMONELLA VAC E ( n  = 13),  S . Gallinarum ( n  = 19),  S . Pullorum ( n  = 116),  S . Enteritidis ( n  = 244),  S . Typhimurium ( n  = 810) and achieved 100% sensitivity and specificity.

frontiers in veterinary science

Identification of Genetic Features for Attenuation of Two Salmonella Enteritidis Vaccine Strains and Differentiation of These From Wildtype Isolates Using Whole Genome Sequencing