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United States Department of Agriculture

Agricultural Research Service

Title: Rapid Identification of Escherichia Coli O157:h7 in Bovine Feces Using the Antibody-Direct Epifluorescent Filter Technique (Ab-Deft)

Authors
item Tortorello, M - FDA, SUMMIT-ARGO, IL
item Stewart, D - FDA, SUMMIT-ARGO, IL
item Cray Jr, William

Submitted to: Veterinary Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 5, 1996
Publication Date: N/A

Interpretive Summary: Escherichia coli O157:H7 are bacteria which produce toxins that can cause disease in humans. These bacteria can be found in the intestines of some healthy cattle. Most of the reported outbreaks of human E. coli O157:H7 disease are linked to the consumption of undercooked meat, which at some point were probably contaminated with feces. Testing for E. coli O157:H7 in cattle feces by traditional laboratory culture methods requires 24 hour to complete. We have developed a direct microscopic detection method to identify E. coli O157:H7 in bovine feces in less than 1 hour. Although the test is less sensitive than culture methods, it can be automated and will be useful for rapidly identifying animals shedding high numbers of E. coli O175:H7 prior to slaughter. Use of this could help detect cattle infected with E. coli O157:H7 before they are processed for human consumption. This would provide a more wholesome food supply.

Technical Abstract: The antibody-direct epifluorescent filter technique (Ab-DEFT) was adapted for direct identification of Escherichia coli O157:H7 in bovine feces. The method involved suspension of bovine feces in buffer, centrifugation for 30 s, treatment of the supernatant with trypsin and Triton X-100 at 50 C for 10 min, prefiltration through 5- and 1.2-um pore filters, and filtration through a 0.4 um pore filter. The final filter was stained with fluorescein-labeled polyclonal antibody specific for the O157 antigen and examined by epifluorescence microscopy. The Ab-DEFT was correlated with viable plate counts for enumeration of the pathogen in artificially inoculated bovine feces (r=0.96), and its sensitivity was approximately 10**4 CFU per g of feces. The prefiltration steps of the procedure provided a very clean background for microscopic visualization of cells; however, depending on the filterability of the fecal specimen, cell loss and inaccurate quantitation sometimes resulted. E. coli O157:H7 was identified in feces of an inoculated calf for more than 3 weeks postinoculation, demonstrating the ability of the Ab-DEFT to directly detect the organism in the natural environment. The Ab-DEFT may be useful for rapid screening of cattle for the presence of E. coli O157:H7 prior to slaughter and as an analytical method in ecological studies of the pathogen.

Last Modified: 7/25/2014
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