Abstract
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a foodborne pathogen which causes illness in humans. Ruminants are the main reservoirs and EHEC predominantly colonizes the epithelium of the recto-anal junction of cattle. Immunosuppression by EHEC promotes re-infection of cattle. However, bovine lactoferrin (bLF) apparently can overrule the immunosuppression by inducing EHEC-specific IgA responses at the mucosal site. The IgA responses are significantly correlated with reduced EHEC shedding and the absence of colonization at the rectal mucosa following re-infection. Therefore, to examine the interaction between bLF and bovine rectal epithelial cells, we first developed a method to establish a primary cell culture of epithelial cells of the rectum of cattle. Furthermore, we used LC–MS/MS to demonstrate the presence of secreted lactoferrin in bovine milk and the absence of a “delta” isoform which is known to translocate to the nucleus of cells. Nevertheless, lactoferrin derived from bovine milk was internalized by rectal epithelial cells and translocated to the nuclei. Moreover, nuclear translocation of bLF was significantly enhanced when the epithelial cells were inoculated with EHEC, as demonstrated by confocal fluorescence microscopy and confirmed by Raman microscopy and 3D imaging.
Highlights
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a foodborne pathogen which colonizes the colon of humans and causes illness ranging from watery or bloody diarrhea and haemorrhagic colitis to acute renal failure and haemolytic uremic syndrome (HUS) [1]
Rectal crypts were obtained by enzymatic digestion (60 min, 100 rpm, 37 °C) carried out in Dulbecco’s Modified Eagle medium (DMEM) (Gibco, Grand Island, NY) supplemented with 1% (v/v) heat inactivated fetal calf serum (FCS), 100 U/mL penicillin (Sigma), 100 μg/mL streptomycin (Sigma), 25 μg/mL gentamicin (Gibco), 2.5 μg/mL amphotericin B (Sigma) and 100 U/mL collagenase (Sigma) until isolated crypts were observed under a microscope
The method was successful as confirmed by the staining of vimentin, which is known as fibroblast intermediate filament
Summary
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a foodborne pathogen which colonizes the colon of humans and causes illness ranging from watery or bloody diarrhea and haemorrhagic colitis to acute renal failure and haemolytic uremic syndrome (HUS) [1]. A number of approaches have been evaluated to prevent EHEC colonization and shedding by ruminants in order to diminish the risk of human infections [5]. During a previous study, we demonstrated that rectal administration of bovine lactoferrin (bLF) derived from milk cleared EHEC infections at the rectal mucosa of cattle. We Rybarczyk et al Vet Res (2019) 50:75 showed that bLF activated the mucosal immune system and induced protection against EHEC re-infection [6]. Rectal administration of bLF induced EspA- and EspB- specific mucosal IgA titers. EspA and EspB are part of the type III secretion system (TTSS) of EHEC. EspA and EspB-specific IgA responses at the mucosal site significantly correlated with reduced EHEC shedding and the absence of bacterial colonization at the rectal mucosa following re-infection. The specific IgA response was not detectable in the serum indicating the local nature of the protective response induced by bLF
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