• Dr. Daniel Engeljohn, Working Group Chair
• Dr. Gary Ades
• Dr. Peggy Cook
• Dr. Patricia Griffin
• Dr. Walt Hill
• MAJ Robin King
• Dr. Ann Marie McNamara
• Dr. Jianghong Meng
• Dr. Dale Morse
• Dr. Eli Perencevich
• Dr. Irene Wesley
• Dr. Donald Zink

Background and Work Charge
Campylobacter spp. has been characterized as among the top bacterial agents of human foodborne gastroenteritis. Poultry is a primary reservoir of Campylobacter spp. and studies show that prevalence may be more than 80% in commercial chicken carcasses. Data show that 95% of human illnesses associated with campylobacterosis are caused by C. jejuni, followed by C. coli comprising 4%, and other species comprising 1%. Currently, the Food Safety and Inspection Service (FSIS) has conducted but not published baselines focused on Campylobacter studies of poultry and there is no performance standard for Campylobacter spp. for any Agency-regulated products, including poultry.

Previously, at the August 28, 2002, National Advisory Committee on Microbiological Criteria for Foods (NACMCF) meeting, a work charge relating to Campylobacter was introduced. The presentation consisted of three talks outlining: the methods and resulting data from previous Campylobacter baseline studies performed in 1994-1995 and 1999-2000 by FSIS; available laboratory methods for Campylobacter spp.; and Campylobacter cell aggregation (transcript available at http://www.fsis.usda.gov/
ophs/nacmcf/2002/transcript082802.pdf; PDF Only).

The original charge included three elements:

• Review and compare the methodologies used for Campylobacter detection in the 1994-1995 and 1999-2000 baseline studies in young chickens.
• Evaluate them for the accuracy and precision that they provide in assessing the prevalence and quantity of Campylobacter on chicken carcasses.
• Compare the methodologies used in the two studies with recent methodological advances for their ability to provide data on the presence and quantity of Campylobacter for application in risk assessment and the establishment of baselines.

The subcommittee met and discussed the topic but no report was presented. The baseline data have never been released due to Campylobacter methodology concerns expressed by FSIS.

In the past, FSIS has used a labor intensive and resource consuming most probable number (MPN) method for the detection and enumeration of Campylobacter spp. Current literature indicates that when Campylobacter spp. are present, their numbers per ml of carcass rinse can vary from 1 to 3 log CFU and therefore direct enumeration on agar plates can be an alternative to MPN methods. The Food and Drug Administration (FDA), the Centers for Disease Control and Prevention (CDC), and industry constituents currently use methods incorporating selective plating to detect and/or enumerate Campylobacter spp. from clinical and food samples. FSIS currently, in cooperation with the Agricultural Research Service (ARS), is conducting a special project with Campylobacter spp. detection and enumeration as part of the project. The ARS method entails aseptic whole bird rinses with 100 ml of buffered peptone water followed by storage and overnight transport of rinses at 4°C ± 4° to the laboratory for analysis. (Note: FSIS Hazard Analysis Critical Control Points (HACCP) verification samples for Salmonella use 400 ml of buffered peptone water. Based on preliminary results from the ARS project, the higher volume of rinsate used in the FSIS HACCP verification program may contribute to a lower prevalence of Salmonella in broiler rinses as compared to the ARS project). The rinses are serially diluted and plated onto Campy-Cefex agar with Bolton broth enrichment and selective agar plate streaks as a backup for presence/absence using a customized atmosphere of 5% O₂, 10% CO₂, and 85% N₂ in sealed bags. Presumptive colonies are then examined microscopically and confirmed using a serological latex agglutination test confirming C. jejuni, C. coli, and C. lari. Confirmed cultures are then stored at -80°C in Brucella broth and glycerol for possible subtyping at a later date.

Current Charge to the Subcommittee
In the near future, the Agency will conduct a baseline study looking at prevalence and enumeration of Campylobacter spp. of known importance on poultry (possibly including carcasses, parts, and ground product).

It is currently proposed that the study will focus on thermophilic species, C. coli and C. jejuni, because these human pathogens account for the vast majority of laboratory confirmed Campylobacter infections. An additional justification for this focus is derived from the numerous microbiological studies of poultry products that indicate that these two species are the only species of Campylobacter that are routinely isolated from chickens. Although some of the other 16 named Campylobacter species are reported to cause occasional human illness, the burden of human illness is low and poultry have not been shown to be a reservoir of infection. Many of these other Campylobacter species require specialized growth conditions, such as atmospheres containing 5% H₂ (i.e., non-thermophilic Campylobacters) or growth media other than Campy-Cefex agar, which contains the antimicrobial cefoperazone that inhibits growth of C. upsaliensis.

The Agency seeks advice on the proposed Campylobacter methodology, as well as any other relevant methodology that may be of equal or greater value and should be considered, for the upcoming baseline study.

The questions to be addressed are:

1. What additional circumstances should be considered in order for FSIS to conclude that the poultry baseline study should address more than the two principal Campylobacter species of C. jejuni and C. coli?
   
   
2. How can the ARS method be most successfully used for high volume analysis in the conduct of a baseline study of Campylobacter presence and enumeration on poultry (chicken, turkey, goose, etc.) carcasses, parts, and ground product that may lead to a potential performance standard or guideline for the regulated industry? What if any modifications should be made as a result of discussing this method in comparison with others presented to the Committee? Please consider whether the above described atmospheric conditions, media, and pre-enrichment, and storage media are acceptable for the objective of this baseline study.
   
   
3. To utilize FSIS resources efficiently and effectively, FSIS expects to maintain as much continuity as possible between the current broiler rinse sampling for Salmonella and the proposed sampling for Campylobacter spp. What concerns regarding the Campylobacter spp. sampling method need to be attended to in order to properly address post-chill injured Campylobacter spp. cells as well as viable non-culturable (coccoid) cells?
   
   
4. What further subtyping methods should be performed on confirmed cultures (restriction fragment length polymorphism (RFLP), amplified fragment-length polymorphism (AFLP), pulsed-field gel electrophoresis (PFGE), ribosomal DNA sequencing, antibiotic susceptibility, etc.), and what, if any, limitations do any of these methods pose?
   
   
5. What effect would in situ Campylobacter spp. cell aggregation have on the accuracy and reproducibility of enumeration counts and is there any remedy to address this issue?
   
   
6. Occasionally non-thermophilic Campylobacter species cause human illness. It is unclear whether livestock and poultry are reservoirs for these species, or if they are present on meat and poultry products following slaughter and processing. Current methodologies use selective agents and incubation conditions which may reduce their detection. If a pilot study was conducted to ascertain the presence of these species on meat and poultry products, what methodologies would best detect these species?