Rapid identification of Escherichia coli O157:H7 in bovine feces using the antibody-direct epifluorescent filter technique (Ab-DEFT)

Abstract

The antibody-direct epifluorescent filter technique (Ab-DEFT) was adapted for direct detection 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, pre-filtration through 5 and 1.2 μm pore filters, and filtration through a 0.4 μm 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). The limit of detection was approximately 10 4 – 10 5 CFU/g feces. The procedure provided a clean background for microscopic visualization of cells; however, cell loss and inaccurate quantitation sometimes resulted. E. coli O157:H7 was detected in feces of an inoculated calf for more than 3 weeks post-inoculation. The Ab-DEFT may be useful for rapid screening of cattle for the presence of the E. coli O157:H7 and as an analytical method in ecological studies of the pathogen.