Enhancing Food Safety Through Control of Food-Borne Disease Agents

Recipient Organization
AUBURN UNIVERSITY
108 M. WHITE SMITH HALL
AUBURN,AL 36849

Performing Department
POULTRY SCIENCE

Non Technical Summary
In poultry feces, campylobacters are usually found at high numbers and the use of microwell dilution methods may provide a simpler, more inexpensive alternative for direct enumeration. The purpose of our studies is to evaluate the effectiveness of different agar plates, or agar plate combination, for the isolation of Campylobacter from poultry fecal samples, and to determine the efficacy of microwell dilution methods for direct enumeration of Campylobacter spp. from fecal samples.

Animal Health Component

40%

Research Effort Categories

Basic

20%

Applied

40%

Developmental

40%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	3220	1100	100%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
3220 - Meat-type chicken, live animal;

Field Of Science
1100 - Bacteriology;

Keywords

poultry

campylobacteer

dillution method

Goals / Objectives
The main objectives of this work are: To determine the most successful plate or plate combination for the isolation of Campylobacter spp. from poultry feces. To test a microwell dilution method for the enumeration of Campylobacter from poultry fecal samples. Determine the genotype (fingerprinting) of different isolates collected from the same sample but on different plates to determine the genomic variations of Campylobacter spp. present in feces, and if different genotypes are isolated with different plates.

Project Methods
Ten fecal samples will be collected from each commercial broilers house. Each house will represent a single farm. Each sample will be enriched in Preston broth (1:9 ratio) for detection of low numbers of Campylobacter. Samples will then be pooled in three groups (3, 3 and 4), serially diluted in phosphate buffer solution and plated on triplicates on mCCDA, Campy-Cefex agar, and Campy-Line agar agar for isolation and enumeration of Campylobacter-like colonies. All plates and enrichment broth samples will be incubated at 42C under microaerophilia for 48 hours. Direct colony counts on plates will be converted to log10 CFU per gram. Colonies will be confirmed using a described multiplex polymerase chain reaction technique. A fingerprinting profile will be performed on each isolate using pulsed filed gel electrophoresis. All isolates will be stored in Brucella broth with 30% glycerol at -80C. The 96-microwell plate dilution method is an adaptation of a most probable number described for the enumeration of Campylobacter jejuni, Listeria monocytogenes and E. coli.

Progress 10/01/05 to 09/30/06

Outputs
A comparison among modified Charcoal Cefoperazone Deoxycholate Agar (mCCDA), Campy-Cefex (mCC) agar, Campy-Line (CL) agar and Campy BAP (CB) agar plates showed that mCCDA, mCC and CB performed similarly to detect and isolated Campylobacter spp. from cecal samples of chickens artificially inoculated with a strain of C. jejuni or broiler chickens from commercial farms with natural contamination. However, CL was consistently lower (p < 0.05) than the other plates. Thirty-nine isolated were kept for hippurate test, PCR and PFGE analysis. From these isolates, 36 (92%) were C. jejuni and the rest (8%) were a mix of C. jejuni and C. coli. The use of sterile membrane helped obtain isolated colonies that resulted in C. coli isolates. PFGE analysis showed that there was a predominant isolate for each farm, although the isolates from different farms had unique restriction patters. However, in few samples more than one C. jejuni strains were isolated from the same farm. No correlation was observed between the restriction pattern of the isolates and any particular plate medium.

Impacts
Direct plating can be used successfully for isolation of Campylobacter from broiler samples, bu the choice of plate medium may influence the results. The medium chosen may affect the recovery of Campylobacter spp. Considering performance and cost, mCC and mCCDA appear to be the media of choice for isolation of Campylobacter from fecal and cecal samples from broiler chickens. In addition, sampling of commercial flocks on day 35 may allow for the analysis of the broilers closer to at market-age, which would in turn be the most appropriate time to identify if the flock will be positive of not for Campylobacter before processing. In the future, this information may be of value to the industry if a logistic scheduling of process is incorporated to reduce the chances of contamination of free flock during processing.

Publications

Potturi-Venkata, L.-P., S. Backert, S. L. Vieira, R. A. Norton, R. S. Miller, S. Pierce1, and O. A. Oyarzabal. 2007. Evaluation of different plate media for direct isolation of Campylobacter spp. from live broilers. In preparation.