Rapid real-time PCR methods to distinguish Salmonella Enteritidis wildtype field isolates from vaccine strains Salmovac SE/Gallivac SE and AviPro SALMONELLA VAC E

Sven Maurischat; Istvan Szabo; Beatrice Baumann; Burkhard Malorny

doi:10.1016/j.mimet.2015.03.015

Rapid real-time PCR methods to distinguish Salmonella Enteritidis wildtype field isolates from vaccine strains Salmovac SE/Gallivac SE and AviPro SALMONELLA VAC E

J Microbiol Methods. 2015 May:112:92-8. doi: 10.1016/j.mimet.2015.03.015. Epub 2015 Mar 18.

Authors

Sven Maurischat¹, Istvan Szabo¹, Beatrice Baumann¹, Burkhard Malorny²

Affiliations

¹ Federal Institute for Risk Assessment, Unit Molecular Microbiology and Genome Analysis, National Salmonella Reference Laboratory, Max-Dohrn Str. 8-10, D-10589 Berlin, Germany.
² 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.

PMID: 25794902
DOI: 10.1016/j.mimet.2015.03.015

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.

Keywords: Diagnostic; Poultry; Real-time PCR; Salmonella Enteritidis; Vaccine.

Publication types

Research Support, Non-U.S. Gov't
Validation Study

MeSH terms

Animals
Diagnosis, Differential
Microbiological Techniques / methods*
Molecular Diagnostic Techniques / methods*
Poultry
Poultry Diseases / diagnosis*
Poultry Diseases / microbiology
Poultry Diseases / prevention & control
Real-Time Polymerase Chain Reaction / methods*
Salmonella Infections, Animal / diagnosis*
Salmonella Infections, Animal / microbiology
Salmonella Infections, Animal / prevention & control
Salmonella Vaccines / genetics*
Salmonella enteritidis / genetics*
Salmonella enteritidis / isolation & purification
Sensitivity and Specificity

Substances

Salmonella Vaccines