Prevalence of Non- Prevalence of Non -O157 Shiga Toxin O157 Shiga Toxin- -Producing Producing Escherichia coli Escherichia coli in Live Animals and at Various Steps During Harvest in Live Animals and at Various Steps During Harvest Terry Arthur Terry Arthur USDA- USDA -ARS ARS- -MARC MARC
Outline Outline -Introduction Introduction - -Detection/Isolation of non Detection/Isolation of non- -O157 STEC O157 STEC - -Non Non- -O157 STEC prevalence in cattle O157 STEC prevalence in cattle - -Non - Non- -O157 STEC prevalence on beef carcasses O157 STEC prevalence on beef carcasses -Conclusion Conclusion -
Shiga higa t toxin oxin- -producing producing E Escherichia scherichia c coli oli (STEC) (STEC) S -Foodborne pathogens Foodborne pathogens - -Cause serious disease and death in humans Cause serious disease and death in humans - -Cattle serve as reservoir Cattle serve as reservoir - X X
Nomenclature Nomenclature - Shiga toxin initially discovered as a product of Shiga toxin initially discovered as a product of Shigella dysenteriae Shigella dysenteriae - - Antibodies against Shiga toxin shown to inhibit - Antibodies against Shiga toxin shown to inhibit cytotoxicity cytotoxicity of an of an E. E. coli strain strain coli - E. coli E. coli strain discovered to be toxic to vero cells strain discovered to be toxic to vero cells - Shiga- -like toxin (SLTEC) like toxin (SLTEC) Vero toxin (VTEC) Shiga Vero toxin (VTEC) Shiga toxin (STEC) Shiga toxin (STEC) Cause disease in humans Cause disease in humans Entero nteroh hemorrhagic emorrhagic E E. . c coli oli (EHEC (EHEC) ) E
Non- -O157 STEC O157 STEC Non Estimated to cause one- -third of the clinical EHEC cases, third of the clinical EHEC cases, Estimated to cause one however, this is probably an underestimation. however, this is probably an underestimation. Over 200 STEC serotypes have been isolated from cattle. Over 200 STEC serotypes have been isolated from cattle. The proportion of non- -O157 STEC able to cause disease O157 STEC able to cause disease The proportion of non in humans is unknown. in humans is unknown.
E. coli serotyping serotyping E. coli O157:H7 O157:H7 O111:H8 O111:H8 O26:H11 O26:H11 Lipopolysaccharide (LPS) Lipopolysaccharide (LPS) = O antigen = O antigen = H antigen Flagella = H antigen Flagella Y O1- -O173 O173 O1 Y H1- -H56 H56 H1
Non- -O157 disease outbreaks in U.S. O157 disease outbreaks in U.S. Non Approximately 35 E. coli E. coli O157 outbreaks occur each O157 outbreaks occur each Approximately 35 year, but only 3 non- -O157 outbreaks ever reported in O157 outbreaks ever reported in year, but only 3 non the U.S. the U.S. Montana O104:H21 11 persons milk? 1994 Montana O104:H21 11 persons milk? 1994 Connecticut O121:H19 11 persons swimming in lake 1999 Connecticut O121:H19 11 persons swimming in lake 1999 Texas O111:H8 58 persons ice? 1999 Texas O111:H8 58 persons ice? 1999
Non- -O157 STEC disease in US O157 STEC disease in US Non O2.H7 HUS O83.H1 HUS O121.H19 HUS/D O4.H- HC O85.H- D O125.H- BD/HUS O5.H- BD/HUS O88.H- D O126.H27 HUS O14.H- HUS O103.H2 BD/HUS O137.H41 HUS O22.H5 HUS O103.H2 HUS O145.H- HUS/BD O26.H11 BD/HUS O103.H25 D/BD O153.H2 BD/HUS O26.H2 D O103.H6 D O165.H25 HUS/BD O38.H21 D O104.H- BD/HUS O172.H- HUS/D O45.H2 BD/HUS O104.H2 BD O?.H11 BD/HUS O45.H2 HC O111.H- HUS OR.H- HUS O48.H21 BD O111.H8 BD/HUS OR.H9 BD/HUS O50.H7 BD/HUS O113.H21 BD/HUS OR:H2 D O68.H- BD/HUS O118.H16 BD OX3.H21 HUS O79.H7 HUS O119.H- D HUS – – hemolytic uremic syndrome hemolytic uremic syndrome HUS HC – – hemorrhagic colitis hemorrhagic colitis HC BD BD – – bloody diarrhea bloody diarrhea D – – diarrhea diarrhea D
STEC virulence factors STEC virulence factors Shiga toxins – – two types: two types: stx stx 1 and 1 and stx stx 2 2 Shiga toxins - ribosome inactivating proteins ribosome inactivating proteins - Intimin ( eae) eae) – – attachment to epithelial cells attachment to epithelial cells Intimin ( EHEC- -hemolysin hemolysin – – iron acquisition? iron acquisition? EHEC
E. coli E. coli Family: Enterobacteriaceae Family: Enterobacteriaceae Gram negative Gram negative Rod Rod- -shaped shaped Motile Motile Ferment: several sugars Ferment: several sugars including sorbitol including sorbitol β β - -glucuronidase positive glucuronidase positive
E. coli O157 O157 E. coli Family: Enterobacteriaceae Family: Enterobacteriaceae Gram negative Gram negative Rod Rod- -shaped shaped Motile: flagella Motile: flagella Ferment: several sugars Ferment: several sugars not sorbitol not sorbitol β - β -glucuronidase glucuronidase negative negative Produce Shiga toxins Produce Shiga toxins Infectious dose <50 organisms Infectious dose <50 organisms
Non- -O157 STEC O157 STEC Non Family: Enterobacteriaceae Family: Enterobacteriaceae Gram negative Gram negative Rod Rod- -shaped shaped Motile: flagella Motile: flagella Ferment: several sugars Ferment: several sugars including sorbitol including sorbitol β - β -glucuronidase positive glucuronidase positive Produce Shiga toxins Produce Shiga toxins Infectious dose ~10 organisms Infectious dose ~10 organisms
Non- -O157 STEC Detection O157 STEC Detection Non -Sampling is the same as for O157 Sampling is the same as for O157 - -Enrichment is the same as for O157 Enrichment is the same as for O157 - -No universal No universal immunomagnetic immunomagnetic separation method available separation method available - -Cannot use sorbitol - Cannot use sorbitol- -based detection based detection β - Cannot use β -Cannot use -glucuronidase glucuronidase- -based detection based detection - Detection has focused on identification of strains carrying the Detection has focused on identification of strains carrying the Shiga toxin genes or expressing the toxin proteins Shiga toxin genes or expressing the toxin proteins PCR EIA PCR EIA Colony hybridization Vero cell assay Colony hybridization Vero cell assay
Vero Cell Assay (VCA) Vero Cell Assay (VCA) A cultured cell line, vero cells, is grown with the addition of culture culture A cultured cell line, vero cells, is grown with the addition of supernatant in microtiter microtiter plates. plates. supernatant in After incubation for 48 to 72 hours, the microtiter microtiter plates are examined plates are examined After incubation for 48 to 72 hours, the microscopically. microscopically. Killing of the Vero cells is considered presumptive evidence of the Killing of the Vero cells is considered presumptive evidence of the presence of Shiga toxins in the broth culture supernatants. presence of Shiga toxins in the broth culture supernatants. Other organisms may produce toxins unrelated to Shiga toxins but Other organisms may produce toxins unrelated to Shiga toxins but toxic to toxic to Vero cells. To avoid false positive samples, antibodies which neutralize eutralize Vero cells. To avoid false positive samples, antibodies which n both Stx1 and Stx2 are used. both Stx1 and Stx2 are used. All positive samples are streaked for isolated colonies, which are then re then All positive samples are streaked for isolated colonies, which a tested by the same assay or by PCR to isolate STEC strains. tested by the same assay or by PCR to isolate STEC strains.
Enzyme Enzyme- -Immunoassay (EIA) Immunoassay (EIA) Enzyme- -immunoassays use anti immunoassays use anti- -Shiga toxin antibodies to capture and Shiga toxin antibodies to capture and Enzyme detect the toxins in culture supernatants. detect the toxins in culture supernatants. 96- -well format well format 96 Commercially available Commercially available Test samples Test samples - - then individual isolates then individual isolates
PCR PCR DNA primers for specific virulence factor genes Can identify multiple genes in one reaction Detects viable and nonviable cells - Hemolysin Hemolysin - - Intimin Intimin - - Shiga toxin 2 - Shiga toxin 2 - - Shiga toxin 1 Shiga toxin 1
Colony hybridization Colony hybridization Grow colonies from sample enrichments on agar media Grow colonies from sample enrichments on agar media Transfer colonies to nylon membranes Transfer colonies to nylon membranes Lyse cells and fix DNA to the membrane cells and fix DNA to the membrane Lyse Hybridize with DNA probe Hybridize with DNA probe Detect bound probe Detect bound probe Identify target colonies Identify target colonies
STEC Plating STEC Plating -EC agar with 1% glucose
Before After
Confirmation of STEC Confirmation of STEC Biochemical tests are required to confirm isolates are E. coli Shigella dysenteriae, Citrobacter freundii, and Enterobacter cloacae have been found to produce Shiga toxins.
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