Campylobacter cultivation and detection methods to recover injured or stressed cells need to be greatly improved to allow for the development and implementation of more effective intervention strategies for poultry.

Campylobacter spp. are a leading etiological agent for acute bacterial gastroenteritis in the human population. Mishandled raw poultry or under-cooked meats are considered to be important vehicles of human infection. Because of the difficulties in controlling bacterial spread in the kitchen and abattoir, control on the farm may be an effective means of reducing Campylobacter prevalence at the processing plant. A better understanding of Campylobacter epidemiology and ecology in poultry confinement rearing facilities is needed before more effective intervention strategies can be developed and implemented.

Detection of viable but non-culturable Campylobacter

Cultivation and detection methods to recover injured or stressed Campylobacter cells need to be greatly improved. Live Campylobacter can become non-culturable (viable but non-culturable, VBNC) when the environment is unfavorable. The VBNC state arises from exposure to sub-lethal adverse environmental conditions and ultimately leads to erroneous conclusions concerning the ecology of this microorganism in the poultry industry. For example, when Campylobacter are not detected in dry hatchery samples (eggshell, fluff, chick pads, etc.), the assumption may be made that they are not present, when in fact they may be present but not culturable.

Campylobacter overlooked in breeder flocks, fertile eggs

Breeder flocks and fertile eggs are indeed a source of Campylobacter to the young chick, but this is often overlooked, disregarded and misunderstood due to the non-culturable state of this microorganism in many hatchery samples. When a microbiologist fails to detect a particular organism with current laboratory procedures, the accuracy or sensitivity of the methodology is seldom, if ever, questioned. For Campylobacter, we know the fragile nature of the microorganism can prevent recovery even from pure cultures or stocks if conditions are unfavorable.

Comparison of recovery methods for VBNC

In order to better understand the ecology of this organism, we have approached the problem in a different manner. First, we compared commonly used enrichment broths to recover injured or stressed Campylobacter to determine the most effective protocol for revival. There were no significant differences (P>0.05) between Bolton’s or Tecra (TB) enrichment broths with or without delaying the addition of antibiotics.

However, the best recovery was noted when TB was pre-incubated for five hours at a reduced temperature (37 C) before adding the selective antibiotics and increasing the temperature to the standard 42 C for 48 hours in a microaerobic atmosphere. This method allowed for the recovery of VBNC forms of Campylobacter from these samples (Cox et al., 2015).

However, even with the best methods, Campylobacter spp. can become VBNC and not consistently detected in samples with low water activity. Therefore, in order to demonstrate the presence of viable Campylobacter in these types of samples, we use a chick assay.

Intracloacal route to detect VBNC Campylobacter

With inoculated dry samples that were culturally negative by our best recovery procedure, we inoculated ground, liquefied samples into one-day-old broiler chicks either orally or intracloacally. Seven days post inoculation, chicks were sacrificed and the ceca were aseptically removed and analyzed; the inoculated Campylobacter were recovered from these samples (Cox, et al., 2015). This was the first time anyone used the intracloacal route (IC) to detect VBNC Campylobacter. The IC route bypasses the hostile acidity of the gizzard and improves the chances of detecting the target organism.

Better resuscitation broths and methods are vital in order to accurately detect Campylobacter in a variety of poultry samples. Our hope is that studies such as ours will convince poultry scientists to question laboratory results and ultimately lead to a better understanding of this elusive organism. As long as the presence of Campylobacter goes undetected in a variety of dry samples, the ecology will be misunderstood and erroneous conclusions made.

 

 

N.A. Cox, USDA, ARS, U.S. National Poultry Research Center; D.E. Cosby, U.S. National Poultry Research Center ; M.E. Berrang, U.S. National Poultry Research Center ; R. Biggs, Tegel Foods, Ltd, Auckland, New Zealand; A.M. Lammerding, AML Consulting, Guelph, Canada; J.L. Wilson, University of Georgia, Department of Poultry Science; and L.J. Richardson, Coca-Cola Company, Atlanta, Ga.