Salmonella detection in poultry samples^[*]

Summary

Study: The efficiency of two commercial PCR methods based on real-time technology, the foodproof^® Salmonella detection system and the BAX^® PCR Assay Salmonella system was compared to standardized culture methods (EN ISO 6579:2002 – Annex D) for the detection of Salmonella spp. in poultry samples. Material and methods: Four sample matrices (feed, dust, boot swabs, feces) obtained directly from poultry flocks, as well as artificially spiked samples of the same matrices, were used. All samples were tested for Salmonella spp. using culture methods first as the gold standard. In addition samples spiked with Salmonella Enteridis were tested to evaluate the sensitivity of both PCR methods. Furthermore all methods were evaluated in an annual ring-trial of the National Salmonella Reference Laboratory of Germany. Results: Salmonella detection in the matrices feed, dust and boot swabs were comparable in both PCR systems whereas the results from feces differed markedly. The quality, especially the freshness, of the fecal samples had an influence on the sensitivity of the real-time PCR and the results of the culture methods. In fresh fecal samples an initial spiking level of 100 cfu/25 g Salmonella Enteritidis was detected. Two-days-dried fecal samples allowed the detection of 14 cfu/25 g. Both real-time PCR protocols appear to be suitable for the detection of Salmonella spp. in all four matrices. The foodproof^® system detected eight samples more to be positive compared to the BAX^® system, but had a potential false positive result in one case. In 7-days-dried samples none of the methods was able to detect Salmonella likely through letal cell damage. Clinical relevance: In general the advantage of PCR analyses over the culture method is the reduction of working time from 4–5 days to only 2 days. However, especially for the analysis of fecal samples official validation should be conducted according to the requirement of EN ISO 6579:2002 – Annex D.

Zusammenfassung

Gegenstand: Prüfung der Eignung zweier kommerziell erhältlicher Real-Time-PCR-Systeme [foodproof^® Salmonella Detection Kit (Merck) und BAX^®-System Q7 Screening Salmonella (DuPont Qualicon/Oxoid)] im Vergleich zur gesetzlich vorgeschriebenen kulturellen Anzucht (EN ISO 6579:2002 – Annex D) für den Nachweis von Salmonellen in Proben von Geflügel. Material und Methoden: Zum Einsatz kamen vier Probenmatrizes aus Legehennenbeständen: Futter, Staub, Sockentupfer und Kot. Die Proben waren zuvor in der Routinediagnostik der Klinik mittels kultureller Anzucht als Goldstandard als nativ positiv bzw. negativ auf Salmonellen bewertet worden. Artifiziell mit Salmonella Enteritidis dotierte Proben wurden im Anschluss untersucht, um die Sensiti vität der Methoden zu ermitteln. Zudem erfolgte eine Evaluierung aller Methoden während eines Ringversuchs des deutschen nationalen Salmonella-Referenzlabors. Ergebnisse: Die Untersuchung von Futter, Staub und Sockentupfern ergab bei beiden PCR-Systemen vergleichbare Resultate. Als problematisch erwies sich der Nachweis von Salmo nellen im Kot mittels Real-Time-PCR-Methoden. Der Feuchtigkeits- bzw. Frischegrad der Kotproben beeinflusst das Detektionsvermögen der PCRs. In frischfeuchtem Kot ließ sich eine Einmischkonzentration von 100 KbE/25 g und in 2 Tage getrockneten Kotproben bereits ab 14 KbE/25 g nachweisen. Das foodproof^®-System detektierte insge samt acht Kotproben mehr als positiv, die mit dem BAX^®-System als negativ bewertet wurden. In einem Fall zeigte das foodproof^®-System eine Probe möglicherweise als falsch positiv an. In den über 7 Tage getrockneten Kotproben ließen sich Salmonellen weder mittels PCR nachweisen noch anzüchten, da sie vermutlich letal geschädigt wurden. Klinische Relevanz: Mit der PCR-Technik kann die Untersuchungs dauer im Vergleich zur kulturellen Anzucht von 4–5 auf 2 Tage verkürzt werden. Beide geprüften Real-Time-PCR-Systeme eignen sich für die Detektion von Salmonella spp., doch sollten offizielle Probenvalidierungen nach den Maßgaben der EN ISO 6579:2002 – Annex D erfolgen.

Key words

Poultry - Salmonella detection - culture - commercial real-time PCR

Schlüsselwörter

Geflügel - Salmonellennachweis - kulturelle Anzucht - kommerzielle Real-Time-PCR

^* Dedicated to Prof. Dr. H. M. Hafez to his 65th birthday.

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