Semi-Quantitative Method to Estimate Levels of Campylobacter

Introduction: Research projects utilizing live animals and/or animal systems often require reliable, accurate quantification of Campylobacter following treatments.  Even with marker strains, conventional methods designed to quantify are labor and material intensive requiring either serial dilutions or MPN procedures to estimate the numbers of Campylobacter in ceca.

Purpose: The objective of this study was to devise an economical method which could accurately estimate the colony forming units per gram (CFU/g) of Campylobacter in the ceca of chicks. 

Methods: Day old chicks, housed in isolation units, were gavaged with a marker Campylobacter coli (Cc^GR), euthanized at 7 or 14 days and the ceca aseptically removed. All cecal samples were diluted 1:3 (w:v) in Tecra Broth (TB) and stomached for 60 s. Cc^GR recovery was conducted on Campy Cefex agar plates w/ 200 ppm gentamicin using a semi-quantitative method (SQM) previously used for Salmonella and by enumeration.  Ceca were obtained from a local processing plant, divided into three aliquots and inoculated with a known CFU of the Cc^GR at three levels. The ceca and inoculum were weighed, 3 times the volume of TB added and then enumerated as described for the above cecal samples.

Results: The SQM was comparable to the conventional enumeration method in 20/20 7-day and 19/20 14-day old broiler chicks.  In addition to the bird study, a controlled experiment verified the reliability of the SQM.  With inoculated ceca using three levels of Cc^GR (10¹, 10³, and 10⁵ CFU/g), the SQM showed no statistical difference (P > 0.05 by paired t-test) from the CFU/g of inoculated cecal material and was more accurate than the standard method when predicting the inoculated number of cells.

Significance: This SQM provides researchers with an accurate, inexpensive and useful method to enumerate Campylobacter marker strains utilized in research studies.