Abstract
Shiga toxin‐producing
Escherichia coli
(STEC) colonizes the bovine recto‐anal junction (RAJ), a key step in STEC pathogenesis in cattle, associated with animal disease, persistence, fecal shedding, carcass contamination, and zoonotic transmission. However, early adhesion mechanisms at the bovine RAJ remain insufficiently characterized, particularly among serotypes lacking the Locus of Enterocyte Effacement (LEE), referred to as LEE‐negative strains. This study investigated strain‐dependent epithelial interaction patterns at the bovine RAJ using complementary
in vitro
,
ex vivo
, and transcriptional approaches. Four representative strains were selected for functional assays, including two LEE‐positive strains (O157:H7 and O26:H11) and two LEE‐negative strains (O113:H21 and O130:H11). All strains adhered to primary bovine RAJ squamous epithelial cells, although no significant quantitative differences were detected among strains. In RAJ explants, however, LEE‐positive strains showed epithelial association extending beyond the superficial layer, with immunolabeling distributed across the epithelial regions, whereas LEE‐negative strains showed a predominantly superficial distribution. This pattern, not previously described in bovine RAJ explants, expands the current view of STEC colonization as a uniformly superficial epithelial process. Analysis of selected adhesion‐related genes revealed strain‐dependent transcriptional responses to the RAJ environment. LEE‐positive strains showed higher expression of
eae
, together with increased expression of
iha
,
csgD
, and
pgaA
. In contrast, LEE‐negative strains showed lower expression of these genes and consistent expression of
saa
, supporting the existence of distinct epithelial interaction strategies that may reflect strain‐specific behaviors among circulating LEE‐positive and LEE‐negative isolates. Overall, these findings demonstrate distinct epithelial interaction and transcriptional profiles among STEC strains and show that physiologically relevant tissue models can reveal serotype‐dependent colonization patterns not fully captured in simplified cell systems and may support the identification of potential targets for vaccine‐based intervention strategies in cattle.