Salmonella isolates from food animals at slaughter (1997-present): carcass rinsates (chicken), carcass swabs (turkey, cattle and swine), and ground products (chicken, turkey, and beef) were collected through USDA-FSIS's Salmonella PR/HACCP verification testing programfrom all federally inspected plants throughout the United States.
Sampling methods used by FSIS for the PR/HACCP Salmonella verification testing program have changed since NARMS animal testing began. Before June of 2006, there were two phases of the FSIS regulatory program for Salmonella in raw products: non-targeted and targeted testing. Non-targeted samples were collected at establishments randomly selected from the population of eligible establishments, with a goal of scheduling every eligible establishment at least once a year. Targeted samples were collected from establishments that had a previously failed non-targeted sample set. Beginning in June of 2006, sampling was scheduled using risk-based criteria designed to focus FSIS resources on establishments with the most samples positive for Salmonella and the greatest number of samples with serotypes most frequently associated with human salmonellosis1,2.
Recovery of Campylobacter (1998-present), E. coli (2000-present), and Enterococcus (2003-present)was through culture of chicken carcass rinsates.
Salmonella isolates from Sentinel Sites (1998-2006): State veterinary laboratories from various states including: California, Colorado, Florida, Iowa, Indiana, Nebraska, New York, Ohio, Oklahoma, Pennsylvania, Tennessee, Washington and Wisconsin participated in the NARMS program at various times by submitting isolates recovered from ill animals with an attempt to provide a geographic representation that complemented states submitting human isolates to CDC.
Salmonella isolates from the National Veterinary Services Laboratories (1997-2005): To augment the numbers of diagnostic isolates, Salmonella isolates recovered from primary or secondary associated infections were obtained from the USDA-APHIS, National Veterinary Services Laboratories (NVSL) in Ames, IA.
Salmonella isolation from slaughter samples was conducted at all three FSIS Regulatory Field Services Laboratories [Eastern (Athens, GA), Midwestern (St. Louis, MO) and Western (Alameda, CA)] following the "Isolation and Identification of Salmonella from Meat, Poultry, and Egg" as described in the Microbiology Laboratory Guidebook, section 42. Positive isolates were forwarded by FSIS to the National Veterinary Services Laboratories (NVSL) for serotyping and were subsequently sent to the BEAR unit as serotyping results became available.
From 1998 to 2000, Campylobacter was isolated by FSIS using the method described in the FSIS Microbiology Laboratory Guidebook (MLG)3. For the first half of 2001, ARS tested several isolation methods until a new method was adopted in July whereby Campylobacter was isolated by ARS from FSIS' Eastern lab spent chicken carcass rinsates. Effective July 2011, FSIS implemented new Campylobacter performance standards for whole carcasses of young chickens (broilers) and began the isolation, identification and enumeration of Campylobacter as described in the FSIS MLG3. Campylobacter isolates were then given to ARS for susceptibility testing.
ARS started isolating E.coli and Enterococcus from these same rinsates in 2000 and 2003, respectively. Additionally, Enterococcus and Campylobacter speciation was performed as described below.
C. Enterococcus Speciation
A species-specific multiplex PCR was performed on presumptive Enterococcus isolates which provided a simultaneous genus and species identification of 23 species of enterococci4. Confirmed Enterococcus isolates of other species not identified with this procedure were labeled as 'Enterococcus species'.
D. Campylobacter Speciation
Final confirmation and speciation were obtained using the Campylobacter BAX? PCR (DuPont Qualicon; Wilmington, DE). This multiplex assay, specific for C. coli and C. jejuni, was performed according to manufacturer's directions as previously described6.
E. Antimicrobial Susceptibility
Salmonella,Campylobacter, E.coli, and Enterococcus were tested using a semi-automated system (Sensitire?, Trek Diagnostic Systems, Inc., Cleveland, Ohio). Resistance trends for Campylobacter include data from 1998-2004 which was obtained using Etest? (AB Biodisk). Antimicrobial resistance was determined using Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS) standards, when available7,8. For antimicrobial agents without CLSI approved standards, NARMS interpretive criteria as established by the NARMS working group were used. Antimicrobials tested and their breakpoints for Salmonella/E.coli, Campylobacter, and Enterococcus can be found on the following page: Antimicrobials tested: concentration ranges and breakpoints.
Quality control strains used for Salmonella and E. coli susceptibility testing included E. coli ATCC 25922, Enterococcusfaecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213. Campylobacter jejuni ATCC 33560 was used as a control for Campylobacter susceptibility testing. For Enterococcus testing, Enterococcus faecalis ATCC 29212 and ATCC 51299 were used.
Mention of trade names or commercial products is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.
1 USDA/FSIS. 2008. Serotypes Profile of Salmonella Isolates from Meat and Poultry Products. Available athttp://www.fsis.usda.gov/Science/Serotypes_Profile_Salmonella_Isolates/index.asp.
2 USDA/FSIS. FSIS Scheduling Criteria for Salmonella Sets in Raw Classes of Product. Available at http://www.fsis.usda.gov/PDF/Scheduling_Criteria_Salmonella_Sets.pdf.
3 USDA/FSIS. 2004. Isolation and Identification of Salmonella from Meat, Poultry, and Egg Products. Microbiological Lab Guidebook 4.03. Available at http://www.fsis.usda.gov/PDF/MLG_4_03.pdf
4 USDA/FSIS. 1998. Isolation, Identification, And Enumeration Of Campylobacter jejuni/coli From Meat And Poultry Products. Microbiology Laboratory Guidebook, chapter 6. Available at http://www.fsis.usda.gov/ophs/Microlab/Mlgchp6.pdf
5 Jackson, C. 2004. Use of a Genus- and Species-Specific Multiplex PCR for Identification of Enterococci. Journal of Clinical Microbiology, 42(8):3558-65.
6 Englen, M.D. and Paula J. Fedorka-Cray. 2002. Evaluation of a Commercial Diagnostic PCR for the Identification of Campylobacter jejuni and Campylobacter coli. Lett. Appl. Microbiol, 35:353-356.
7 NCCLS/CLSI. 2002. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals. Approved Standard, M31-A2. NCCLS, Wayne, PA.
8 CLSI. 2006. Performance Standards for Antimicrobial Susceptibility Testing; Sixteenth Informational Supplement (M100-S16). CLSI, Wayne, PA.