MFLP-89 Procedure for the Detection of Verocytotoxin-Producing Escherichia coli in Food Samples
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Published by: POLYSCIENCE PUBLICATIONS, P.O. Box 1606, Station St-Martin, Laval, Quebec,Canada H7V 3P9. TEL.: 1-800-840-5870. FAX: (450) 688-1930.,Government of Canada Gouvernement du Canada,Laboratory Procedure MFLP-89,September 1996,HEALTH PROTECTION BRANCH,OTTAWA,PROCEDURE FOR THE DETECTION OF VEROCYTOTOXIN-PRODUCING,ESCHERICHIA COLI IN FOOD SAMPLES,Elroy D. Mann,Food Production and Inspection Branch,Health of Animals Laboratory,Guelph, Ontario, N1G 3W4,1. Application,This method is applicable to the screening of food, environmental and fecal samples for the presence of,Escherichia coli (E. coli) capable of producing verocytotoxins. Verocytotoxin (VT) -producing E. coli (VTEC),elaborate cytotoxins that are toxic for Vero cells, (African Green Monkey kidney cells). These organisms,have been implicated as the agents of hemolytic uremic syndrome (HUS) and hemorrhagic colitis (HC) in,humans (3.2, 3.6). E. coli O157:H7 has been the most common human pathogen, but many other serotypes,of VTEC have been associated with human disease (3.2, 3.5).,This method is primarily a reference method for laboratories that have facilities and the capability for,managing cell culture procedures. The reading of the test requires specialized training and experience and,this test should only be undertaken by laboratories that expect sufficient volume of testing to have personnel,able to maintain their skills (3.1). For more information about this test, please contact Dr. Elroy Mann at HAL,Guelph (1.1.). If such testing is required, contact one of the following laboratories:,1.1 Dr. Elroy Mann,Health of Animals Laboratory,110 Stone Rd. W.,Guelph, Ontario, N1G 3W4,Telephone: 519-822-3300,Fax: 519-822-2280,1.2 Dr. Marie-Josée Champagne,Laboratoire d'hygiène vétérinaire,3400, boul. Casvant Ouest,St. Hyacinthe, Québec, J2S 8E3,Telephone: 514-773-7730,Fax: 514-773-8152,1.3 Ms. Shirley Toms,Laboratory Services Division - Western Laboratory,3650 - 36th Street N.W.,Calgary, Alberta, T2L 2L1,MFLP-89,- 2 - September 1996,Telephone: (403) 299-7633,Fax: (403) 221-3293,2. Principal,This protocol describes the vero cell assay (VCA), a test that detects VTEC in samples by demonstrating the,effects of their toxin(s) on monolayer cultures of Vero cells (3.3, 3.4, 3.7). This sensitive method, involving,the use of freshly trypsinized Vero cells, may however have some day-to-day variation in it's sensitivity.,Clarified supernatants from enrichment broth cultures are diluted five fold (1/5 to 1/125) in microtiter plates,and a known number of Vero cells are added to the microplate wells. After incubation for two days to allow,growth of the Vero cells the microtiter plates are examined microscopically. Killing of the Vero cells is,considered presumptive evidence of the presence of verocytotoxins (VTs) in the broth culture supernatants.,Cytotoxic activity of positive samples is graded according to the highest dilution at which the cytotoxic effect,is evident. Positive samples are then processed for isolation of VTEC. VT positive isolates are then,biotyped, serotyped and genotyped. This method is based on the procedure used by Read et al. (3.3) and,Clarke et al. (3.7).,Other organisms may produce toxins unrelated to VTs but toxic to Vero cells. Consequently, the cytotoxicity,evident in some samples (up to 20%) in the screening test may not be due to VTs (3.3, 3.7). To avoid,unnecessary processing of false positive samples, an optional step can be included to distinguish these from,true VT positive samples. In the screening tests, an additional 1/5 dilution of each sample is prepared in,diluent containing antibodies which neutralize both antigenic types of verocytotoxins (VT1 and VT2). When,the plates are read, the two 1/5 dilutions for each sample are compared. A sample is considered positive for,VTs if its cytotoxicity is substantially or completely neutralized by the antiVT antibodies. Toxicity due to other,toxins will not be affected by these antibodies.,3. REFERENCES:,3.1 Mann ED. 1995. Procedure for the Detection of Verocytotoxin producing Escherichia coli in raw and,cooked meat samples. IN: Food Safety Procedures Manual Version 2.1, March 1996, Animal and,Plant Health Directorate Official Protocol.,3.2 Karmali MA. 1989. Infection by Verocytotoxin-producing Escherichia coli. Clin Microbiol Rev 2:15-,38.,3.3 Read SC, Gyles CL, Clarke RC, Lior H, and McEwen S. 1990. Prevalence of verocytotoxigenic,Escherichia coli in ground beef, pork, and chicken in southwestern Ontario. Epidimiol Infect 105:11-,20.,3.4 Konowalchuk J, Speirs JI, Stravric S. 1977. Vero response to a cytotoxin of Escherichia coli. Infect,Immun 18:775-7……
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