Development of a rapid and specific colony-lift immunoassay for detection and enumeration of Campylobacter jejuni, C. coli, and C. lari.
Contamination of retail poultry by Campylobacter spp. is a significant source of human diarrheal disease. We have developed a colony-lift immunoassay (CLI) for the detection of Campylobacter jejuni, C. coli, and C. lari isolated from such sources and grown on selective agar medium or on filter membranes. This technique has been successfully utilized to quantify Campylobacter colonies within 18 to 28 h after sampling. Hydrophobic, high-protein-binding membranes were prewet with methanol and used to imprint bacterial cells from the agar or filter membrane, while leaving colonies intact and viable. The membranes were air dried, peroxidase neutralized, blocked with bovine serum albumin in phosphate-buffered saline, and hybridized for 5 min with an affinity-purified, horseradish peroxidase-labeled goat anti-Campylobacter antibody preparation (Kirkegaard and Perry Laboratories). The membranes were washed briefly, exposed to a 3,'5,5'-tetramethylbenzidine membrane substrate, rinsed in deionized water, and allowed to dry. Lifted colonies of Campylobacter were identified by a blue color reaction on the membrane. Replicas of the membranes were made by marking the location of the Campylobacter colonies on clear transparencies, which were subsequently utilized to locate the original colony on the filter membrane or agar plate. The specificity of this antibody preparation has been evaluated against a wide range of Campylobacter spp., including American Type Culture Collection type and references strains, retail poultry isolates, and isolates obtained from cloacal swabs of live commercial broiler chickens. Specificity against numerous non-Campylobacter spp. obtained from the same sources was also evaluated. The CLI provided a rapid and simple means for detection and enumeration of enteropathogenic Campylobacter organisms. We have successfully combined this CLI procedure with methods recently developed in our laboratories for retail meat and poultry sampling. Potentially, broader applications for use of this technique include detection and enumeration of campylobacters from clinical, veterinary, and environmental samples.
Rice
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Enteric Diseases Program, Naval Medical Research Institute, Bethesda, Maryland 20889, USA. rice@mail2.nmri.nnmc.navy.mil
Lamichhane
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Joseph
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Rollins
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D000362:Agar; D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D017000:Campylobacter coli / Q000302:isolation & purification*; D016123:Campylobacter jejuni / Q000302:isolation & purification*; D002645:Chickens / Q000382:microbiology; D015169:Colony Count, Microbial / Q000379:methods*; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D007106:Immune Sera; D013379:Substrate Specificity
D003470:Culture Media; D007106:Immune Sera; D000362:Agar