Brucella abortus: Determination of survival times and evaluation of methods for detection in several matrices

Abstract

Background: Brucella abortus is a highly pathogenic zoonotic agent, tempting for the development of a rapid diagnostic method to enable adequate treatment and prevent further spread. Enrichment of the bacteria is often used as a first step in diagnostics to increase the bacterial number above the detection limit of the real-time PCR. The enrichment of Brucella spp. takes at least 3days, which might be avoidable if sensitive PCR methods can be used. Since many matrices contain PCR inhibitors, the limit of detection (LOD) must be determined for each separate matrix. Another aim of this study was the determination of survival of Brucella abortus in the analyzed matrices. Methods: The LOD for the detection of B. abortus in 14 matrices, relevant for human medicine, veterinary medicine and food and feed safety, was determined to evaluate the need of a pre-enrichment step prior to real-time PCR. The survival of B. abortus in the spiked matrices was tested by plate count in a 7-day interval for 132days. Results: The limit of detection for B. abortus in most matrices was in the range of 10 3 -10 4 CFU/g for cultivation and 10 4 -10 5 CFU/g for direct real-time PCR. The survival time of B. abortus was less than 21days in apple purée and stomach content and 28days in water while B. abortus remained viable at day 132 in milk, blood, spinach and minced meat. Conclusions: A direct PCR analysis without enrichment of bacteria saves at least 3days. However, the limit of detection between direct PCR and plate count differs in a 10 fold range. We conclude that this lower sensitivity is acceptable in most cases especially if quick analysis are required.