|BIERNBAUM, ERICKA - Oak Ridge Institute For Science And Education (ORISE)|
Submitted to: International Symposium and Workshop on Shiga Toxin ... Escherichia coli
Publication Type: Abstract Only
Publication Acceptance Date: 1/30/2023
Publication Date: N/A
Technical Abstract: Cattle are the primary reservoirs for Shiga toxin-producing Escherichia coli (STEC) and principal source of human infection. Developing effective preharvest control methods that limit STEC in cattle requires better understanding of STEC interactions with the animal. Towards this end, we developed two model systems that would permit such evaluations while limiting animal usage through reuse or reduced use. I. In vivo model: The bovine rumen is the initial transit site for STEC before progressing through rest of the ruminant gastrointestinal tract (GIT). We developed an economical, novel, non-terminal and reusable rumen-fistulated animal model that permits in vivo evaluation of multiple STEC strains in the bovine rumen. Using this model system, we successfully evaluated STEC survival and determined proteins expressed in vivo in the rumen. Animals were fed contrasting maintenance (high fiber) and lactation (high energy-protein) diets to study dietary impacts. STEC were contained within dialysis cartridges to prevent animal GIT contamination. To demonstrate the uniqueness of in vivo evaluations, parallel studies were conducted using extracted rumen fluid in vitro in flasks maintained under anaerobic conditions. Strain/serotype-dependent differences in O157 and non-O157 STEC recovery from the rumen fluid of cattle fed either diet was observed. Analysis of the STEC genes expressed in the rumen demonstrated a preference to survival over virulence. Differences in the adaptive responses to rumen fluid was influenced by the animal’s diet and growth conditions. These new insights into the STEC responses could help formulate modalities to control STEC colonization of cattle. II. In vitro model: The bovine GIT site at which O157 persists is the recto-anal (RAJ) junction. An in-depth understanding of O157-RAJ tissue interactions could help develop novel modalities to limit O157 in cattle. Here, we report the development of a complete RAJ-in vitro organ culture (RAJ-IVOC) system that represents both cell types of the RAJ and permits adherence studies with results as observed in vivo. Pieces of RAJ tissue, collected at cattle necropsies from unrelated studies, were assembled and tested variously to derive optimal conditions for assaying bacterial adherence on a viable IVOC. O157 strain EDL933 and E. coli K12 with known adherence differences were used to standardize the RAJ-IVOC adherence assay. Cell viability, structural cell markers and histopathology were used to determine tissue integrity, and adherence assessed via microscopy and culture. DNA fingerprinting verified recovered bacteria against the inoculum. Appropriately assembled RAJ-IVOC, in Dulbecco’s Modified Eagle Medium, at 39oC, with 5% CO2 and gentle shaking for 3-4 hours, maintained tissue integrity and reproduced the expected adherence phenotype of the test bacteria. The RAJ-IVOC model system provides a convenient method to pre-screen multiple bacteria-RAJ interactions prior to in vivo experiments, thereby reducing animal usage.