title: Rapid real-time PCR methods to distinguish Salmonella Enteritidis wildtype field isolates from vaccine strains Salmovac SE/Gallivac SE and AviPro SALMONELLA VAC E.
abstract: Salmonella enterica serovar Enteritidis is a major non-typhoid Salmonella serovar causing human salmonellosis mainly associated with the consumption of poultry and products thereof. To reduce infections in poultry, S. Enteritidis live vaccine strains AviPro SALMONELLA VAC E and Salmovac SE/Gallivac SE have been licensed and used in several countries worldwide. To definitively diagnose a S. Enteritidis contamination in vaccinated herds a reliable and fast method for the differentiation between vaccine and wildtype field isolates is required. In this study, we developed and validated real-time PCR (qPCR) assays to distinguish those variants genetically. Suitable target sequences were identified by whole genome sequencing (WGS) using the Illumina MiSeq system. SNP regions in kdpA and nhaA proved to be most useful for differentiation of AviPro SALMONELLA VAC E and Salmovac SE/Gallivac SE, respectively, from wildtype strains. For each vaccine strain one TaqMan-qPCR assay and one alternative approach using High Resolution Melting (HRM) analysis was designed. All 30 Salmovac SE and 7 AviPro SALMONELLA VAC E vaccine strain reisolates tested were correctly identified by both approaches (100% inclusivity). Furthermore, all 137 (TaqMan) and 97 (HRM) Salmonella non-vaccine and related Enterobacteriaceae strains tested were excluded (100% exclusivity). The analytical detection limits were determined to be approx. 10(2) genome copies/reaction for the TaqMan and 10(4) genome copies/reaction for the HRM approach. The real-time PCR assays proved to be a reliable and fast alternative to the cultural vaccine strain identification tests helping decision makers in control measurements to take action within a shorter period of time.
authors: Federal Institute for Risk Assessment, Unit Molecular Microbiology and Genome Analysis, National Salmonella Reference Laboratory, Max-Dohrn Str. 8-10, D-10589 Berlin, Germany.
authors: Sven
authors: S
authors: Maurischat
authors: Federal Institute for Risk Assessment, Unit Molecular Microbiology and Genome Analysis, National Salmonella Reference Laboratory, Max-Dohrn Str. 8-10, D-10589 Berlin, Germany.
authors: Istvan
authors: I
authors: Szabo
authors: Federal Institute for Risk Assessment, Unit Molecular Microbiology and Genome Analysis, National Salmonella Reference Laboratory, Max-Dohrn Str. 8-10, D-10589 Berlin, Germany.
authors: Beatrice
authors: B
authors: Baumann
authors: Federal Institute for Risk Assessment, Unit Molecular Microbiology and Genome Analysis, National Salmonella Reference Laboratory, Max-Dohrn Str. 8-10, D-10589 Berlin, Germany. Electronic address: burkhard.malorny@bfr.bund.de.
authors: Burkhard
authors: B
authors: Malorny
mesh_terms: D000818:Animals; D003937:Diagnosis, Differential; D008828:Microbiological Techniques / Q000379:methods*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D011200:Poultry; D011201:Poultry Diseases / Q000175:diagnosis* / Q000382:microbiology / Q000517:prevention & control; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012481:Salmonella Infections, Animal / Q000175:diagnosis* / Q000382:microbiology / Q000517:prevention & control; D022562:Salmonella Vaccines / Q000235:genetics*; D012477:Salmonella enteritidis / Q000235:genetics* / Q000302:isolation & purification; D012680:Sensitivity and Specificity
chemical_list: D022562:Salmonella Vaccines
keywords: Diagnostic; Poultry; Real-time PCR; Salmonella Enteritidis; Vaccine