P1-16 FDA Campylobacter jejuni and Campylobacter coli Detection Method from Raw Silo Milk

Monday, July 23, 2012
Exhibit Hall (Rhode Island Convention Center)
Qian Wang, Illinois Institute of Technology, Bedford Park, IL
Lacey Guillen, Illinois Institute of Technology, Summit-Argo, IL
Don Bark, U.S. Food and Drug Administration, Bothell, WA
Carlos Abeyta, U.S. Food and Drug Administration, Bothell, WA
Greg Gharst, U.S. Food and Drug Administration, Bedford Park, IL
Introduction:  Campylobacter is recognized as one of the leading foodborne sources of gastroenteritis. The current FDA BAM Campylobacter detection method has not been updated since 2001 despite advances made by the scientific community.

Purpose: The purpose of this study was to design and then validate a new detection protocol using current media and molecular techniques against the reference FDA method.

Methods:  Pure cultures of ten different strains of Campylobacter (jejuni & coli) and five cultures of non-Campylobacter strains were serially diluted and inoculated at different levels (5, 50,125 CFU/25g) into separate 25g samples of raw silo milk.  Each sample was then enriched in 100 ml of Bolton Broth without blood and incubated in microaerophilic conditions at 41-42°C for 24 hrs. For each sample, 10 μl was streaked in duplicate onto R & F® Campylobacter Chromogenic Plating Medium (CCPM) and modified Cefoperazone Charcoal Deoxycholate Agar (mCCDA) which was then incubated in microaerophilic conditions at 41-42°C for 48 hrs. Confirmation was conducted by real-time PCR (qPCR) with a Cepheid Smartcycler utilizing primers and probes (FAM-cj and TxR-cc) within the ceuE gene of Campylobacter.

Results:  At the lowest spiking level (5 cfu/25g), 100% cells were recovered with CCPM compared to 0% with mCCDA. At the low spiking level (50 cfu/25g), the results showed 100% recovery with CCPM compared to 20% with mCCDA. At the high spiking level (125 cfu/25g), both CCPM and mCCDA recovered 100% cells. Confirmation, using a qPCR, verified all the positive isolates as either C. jejuni or coli.  

Significance: In conclusion, CCPM is a more viable choice for detection at low levels and our qPCR protocol is a demonstrated confirmation step for C. jejuni and coli. More research with a larger sample size is needed to determine the significant differences for recovering low levels of Campylobacter from complex food matrices.