Direct Real-Time PCR Quantification of Campylobacter jejuni in Chicken Fecal and Cecal Samples by Integrated Cell Concentration and DNA Purification

Abstract

Campylobacter jejuni is a major cause of diarrheal disease and food-borne gastroenteritis. The main reservoir of C. jejuni in poultry is the cecum, with an estimated content of 6 to 8 log 10 CFU/g. If a flock is infected with C. jejuni, the majority of the birds in that flock will harbor the bacterium. Diagnostics at the flock level could thus be an important control point. The aim of the work presented here was to develop a complete quantitative PCR-based detection assay for C. jejuni obtained directly from cecal contents and fecal samples. We applied an approach in which the same paramagnetic beads were used both for cell isolation and for DNA purification. This integrated approach enabled both fully automated and quantitative sample preparation and a DNA extraction method. We developed a complete quantitative diagnostic assay through the combination of the sample preparation approach and real-time 5-nuclease PCR. The assay was evaluated both by spiking the samples with C. jejuni and through the detection of C. jejuni in naturally colonized chickens. Detection limits between 2 and 25 CFU per PCR and a quantitative range of >4 log 10 were obtained for spiked fecal and cecal samples. Thirty-one different poultry flocks were screened for naturally colonized chickens. A total of 262 (204 fecal and 58 cecal) samples were analyzed. Nineteen of the flocks were Campylobacter positive, whereas 12 were negative. Two of the flocks contained Campylobacter species other than C. jejuni. There was a large difference in the C. jejuni content, ranging from 4 to 8 log 10 CFU/g of fecal or cecal material, for the different flocks tested. Some issues that have not yet promoted much attention are the prequantitative differences in the ability of C. jejuni to colonize poultry and the importance of these differences for causing human disease through food contamination. Understanding the colonization kinetics in poultry is therefore of great importance for controlling human infections by this bacterium.

The sensitivity and specificity of fecal and cecal culture for the detection of Campylobacter in Dutch broiler flocks quantified by Bayesian analysis

“…The lower sensitivity of the fecal compared to cecal culture could be explained by previous findings that Campylobacter mainly colonizes the ceca (Rudi et al, 2004). Another explanation is that fecal samples were collected by the farmer and the interval between sample collection and laboratory testing, and the possible mishandling might have exposed the bacterium to sub-optimal conditions resulting in less culturable bacteria (Havaei et al, 2006).…”

“…Generally flocks are tested for the presence of Campylobacter by culturing fecal and/or cecal samples (Corry et al, 1995;Musgrove et al, 2001;Payne et al, 1999;Berndston et al, 1996). Fecal samples are easier to gather, but it is generally assumed that cecal samples are more appropriate, since Campylobacter mainly colonizes the cecum and, consequently, fecal samples often contain a lower number of bacteria per gram than cecal samples (Rudi et al, 2004).…”

Campylobacter

“…Specific and sensitive methods are required to separate the target cells away from the sample matrix in a form amenable for PCRbased detection. In a recent study, paramagnetic beads were utilised as a method for isolating Campylobacter from chicken cecal contents and faecal samples, prior to PCR [139]. The beads initially bound to the cells in the liquid sample matrix and then lysis buffer was added to lyse the cells releasing the DNA, which then also bound to the beads.…”

Improved Detection of Five Major Gastrointestinal Pathogens by Use of a Molecular Screening Approach

“…The resultant misidentification increases hands-on time and delay in reporting of a definite negative result. Other problems are the viable but nonculturable state of Campylobacter jejuni (24,29) and the limited viability of shigellae outside the human body (35). These may compromise the sensitivity of culture.…”