The interaction of Escherichia coli O157 :H7 and Salmonella Typhimurium flagella with host cell membranes and cytoskeletal components.

Bacterial flagella have many established roles beyond swimming motility. Despite clear evidence of flagella-dependent adherence, the specificity of the ligands and mechanisms of binding are still debated. In this study, the molecular basis of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium flagella binding to epithelial cell cultures was investigated. Flagella interactions with host cell surfaces were intimate and crossed cellular boundaries as demarcated by actin and membrane labelling. Scanning electron microscopy revealed flagella disappearing into cellular surfaces and transmission electron microscopy of S. Typhiumurium indicated host membrane deformation and disruption in proximity to flagella. Motor mutants of E. coli O157:H7 and S. Typhimurium caused reduced haemolysis compared to wild-type, indicating that membrane disruption was in part due to flagella rotation. Flagella from E. coli O157 (H7), EPEC O127 (H6) and S. Typhimurium (P1 and P2 flagella) were shown to bind to purified intracellular components of the actin cytoskeleton and directly increase in vitro actin polymerization rates. We propose that flagella interactions with host cell membranes and cytoskeletal components may help prime intimate attachment and invasion for E. coli O157:H7 and S. Typhimurium, respectively.

Functional analysis of bovine TLR5 and association with IgA responses of cattle following systemic immunisation with H7 flagella.

Flagellin subunits are important inducers of host immune responses through activation of TLR5 when extracellular and the inflammasome if cytosolic. Our previous work demonstrated that systemic immunization of cattle with flagella generates systemic and mucosal IgA responses. The IgA response in mice is TLR5-dependent and TLR5 can impact on the general magnitude of the adaptive response. However, due to sequence differences between bovine and human/murine TLR5 sequences, it is not clear whether bovine TLR5 (bTLR5) is able to stimulate an inflammatory response following interaction with flagellin. To address this we have examined the innate responses of both human and bovine cells containing bTLR5 to H7 flagellin from E. coli O157:H7. Both HEK293 (human origin) and embryonic bovine lung (EBL) cells transfected with bTLR5 responded to addition of H7 flagellin compared to non-transfected controls. Responses were significantly reduced when mutations were introduced into the TLR5-binding regions of H7 flagellin, including an R90T substitution. In bovine primary macrophages, flagellin-stimulated CXCL8 mRNA and secreted protein levels were significantly reduced when TLR5 transcript levels were suppressed by specific siRNAs and stimulation was reduced with the R90T-H7 variant. While these results indicate that the bTLR5 sequence produces a functional flagellin-recognition receptor, cattle immunized with R90T-H7 flagella also demonstrated systemic IgA responses to the flagellin in comparison to adjuvant only controls. This presumably either reflects our findings that R90T-H7 still activates bTLR5, albeit with reduced efficiency compared to WT H7 flagellin, or that other flagellin recognition pathways may play a role in this mucosal response.

Flagella interact with ionic plant lipids to mediate adherence of pathogenic Escherichia coli to fresh produce plants.

Bacterial attachment to plant and animal surfaces is generally thought to constitute the initial step in colonization, requiring adherence factors such as flagella and fimbriae. We describe the molecular mechanism underpinning flagella-mediated adherence to plant tissue for the foodborne pathogen, enterohaemorrhagic Escherichia coli. Escherichia coli H7 flagella interacted with a sulphated carbohydrate (carrageenan) on a glycan array, which occurred in a dose-dependent manner. Adherence of E. coli O157 : H-expressing flagella of serotype H7, H6 or H48 to plants associated with outbreaks from fresh produce and to Arabidopsis thaliana, was dependent on flagella interactions with phospholipids and sulpholipids in plasma membranes. Adherence of purified H7 and H48 flagella to carrageenan was reduced at higher concentrations of KH2 PO4 or KCl, showing an ionic basis to the interactions. Purified H7 flagella were observed to physically interact with plasma membranes in spinach plants and in A.thaliana. The results show a specific interaction between E. coli H7, H6 and H48 flagella and ionic lipids in plant plasma membranes. The work extends our understanding of the molecular mechanisms underpinning E.coli flagella targeting of plant hosts and suggests a generic mechanism of recognition common in eukaryotic hosts belonging to different biological kingdoms.

Lysogeny with Shiga toxin 2-encoding bacteriophages represses type III secretion in enterohemorrhagic Escherichia coli.

Lytic or lysogenic infections by bacteriophages drive the evolution of enteric bacteria. Enterohemorrhagic Escherichia coli (EHEC) have recently emerged as a significant zoonotic infection of humans with the main serotypes carried by ruminants. Typical EHEC strains are defined by the expression of a type III secretion (T3S) system, the production of Shiga toxins (Stx) and association with specific clinical symptoms. The genes for Stx are present on lambdoid bacteriophages integrated into the E. coli genome. Phage type (PT) 21/28 is the most prevalent strain type linked with human EHEC infections in the United Kingdom and is more likely to be associated with cattle shedding high levels of the organism than PT32 strains. In this study we have demonstrated that the majority (90%) of PT 21/28 strains contain both Stx2 and Stx2c phages, irrespective of source. This is in contrast to PT 32 strains for which only a minority of strains contain both Stx2 and 2c phages (28%). PT21/28 strains had a lower median level of T3S compared to PT32 strains and so the relationship between Stx phage lysogeny and T3S was investigated. Deletion of Stx2 phages from EHEC strains increased the level of T3S whereas lysogeny decreased T3S. This regulation was confirmed in an E. coli K12 background transduced with a marked Stx2 phage followed by measurement of a T3S reporter controlled by induced levels of the LEE-encoded regulator (Ler). The presence of an integrated Stx2 phage was shown to repress Ler induction of LEE1 and this regulation involved the CII phage regulator. This repression could be relieved by ectopic expression of a cognate CI regulator. A model is proposed in which Stx2-encoding bacteriophages regulate T3S to co-ordinate epithelial cell colonisation that is promoted by Stx and secreted effector proteins.

Identifying and overcoming COVID-19 vaccination impediments using Bayesian data mining techniques.

The COVID-19 pandemic has profoundly reshaped human life. The development of COVID-19 vaccines has offered a semblance of normalcy. However, obstacles to vaccination have led to substantial loss of life and economic burdens. In this study, we analyze data from a prominent health insurance provider in the United States to uncover the underlying reasons behind the inability, refusal, or hesitancy to receive vaccinations. Our research proposes a methodology for pinpointing affected population groups and suggests strategies to mitigate vaccination barriers and hesitations. Furthermore, we estimate potential cost savings resulting from the implementation of these strategies. To achieve our objectives, we employed Bayesian data mining methods to streamline data dimensions and identify significant variables (features) influencing vaccination decisions. Comparative analysis reveals that the Bayesian method outperforms cutting-edge alternatives, demonstrating superior performance.

Recruitment and membrane interactions of host cell proteins during attachment of enteropathogenic and enterohaemorrhagic Escherichia coli.

EPEC (enteropathogenic Escherichia coli) and EHEC (enterohaemorrhagic Escherichia coli) are attaching and effacing pathogens frequently associated with infectious diarrhoea. EPEC and EHEC use a T3SS (type III secretion system) to translocate effectors that subvert different cellular processes to sustain colonization and multiplication. The eukaryotic proteins NHERF2 (Na(+)/H(+) exchanger regulatory factor 2) and AnxA2 (annexin A2), which are involved in regulation of intestinal ion channels, are recruited to the bacterial attachment sites. Using a stable HeLa-NHERF2 cell line, we found partial co-localization of AnxA2 and NHERF2; in EPEC-infected cells, AnxA2 and NHERF2 were extensively recruited to the site of bacterial attachment. We confirmed that NHERF2 dimerizes and found that NHERF2 interacts with AnxA2. Moreover, we found that AnxA2 also binds both the N- and C-terminal domains of the bacterial effector Tir through its C-terminal domain. Immunofluorescence of HeLa cells infected with EPEC showed that AnxA2 is recruited to the site of bacterial attachment in a Tir-dependent manner, but independently of Tir-induced actin polymerization. Our results suggest that AnxA2 and NHERF2 form a scaffold complex that links adjacent Tir molecules at the plasma membrane forming a lattice that could be involved in retention and dissemination of other effectors at the bacterial attachment site.

Identification of bacterial target proteins for the salicylidene acylhydrazide class of virulence-blocking compounds.

A class of anti-virulence compounds, the salicylidene acylhydrazides, has been widely reported to block the function of the type three secretion system of several Gram-negative pathogens by a previously unknown mechanism. In this work we provide the first identification of bacterial proteins that are targeted by this group of compounds. We provide evidence that their mode of action is likely to result from a synergistic effect arising from a perturbation of the function of several conserved proteins. We also examine the contribution of selected target proteins to the pathogenicity of Yersinia pseudotuberculosis and to expression of virulence genes in Escherichia coli O157.

Transcriptional regulators of the GAD acid stress island are carried by effector protein-encoding prophages and indirectly control type III secretion in enterohemorrhagic Escherichia coli O157:H7.

Type III secretion (T3S) plays a pivotal role in the colonization of ruminant hosts by Enterohemorrhagic Escherichia coli (EHEC). The T3S system translocates effector proteins into host cells to promote bacterial attachment and persistence. The repertoire and variation in prophage regions underpins differences in the pathogenesis and epidemiology of EHEC strains. In this study, we have used a collection of deletions in cryptic prophages and EHEC O157 O-islands to screen for novel regulators of T3S. Using this approach we have identified a family of homologous AraC-like regulators that indirectly repress T3S. These prophage-encoded secretion regulator genes (psr) are found exclusively on prophages and are associated with effector loci and the T3S activating Pch family of regulators. Transcriptional profiling, mutagenesis and DNA binding studies were used to show that these regulators usurp the conserved GAD acid stress resistance system to regulate T3S by increasing the expression of GadE (YhiE) and YhiF and that this regulation follows attachment to bovine epithelial cells. We further demonstrate that PsrA and effectors encoded within cryptic prophage CP933-N are required for persistence in a ruminant model of colonization.

Growth promotion and antibiotic induced metabolic shifts in the chicken gut microbiome.

Antimicrobial growth promoters (AGP) have played a decisive role in animal agriculture for over half a century. Despite mounting concerns about antimicrobial resistance and demand for antibiotic alternatives, a thorough understanding of how these compounds drive performance is missing. Here we investigate the functional footprint of microbial communities in the cecum of chickens fed four distinct AGP. We find relatively few taxa, metabolic or antimicrobial resistance genes similarly altered across treatments, with those changes often driven by the abundances of core microbiome members. Constraints-based modeling of 25 core bacterial genera associated increased performance with fewer metabolite demands for microbial growth, pointing to altered nitrogen utilization as a potential mechanism of narasin, the AGP with the largest performance increase in our study. Untargeted metabolomics of narasin treated birds aligned with model predictions, suggesting that the core cecum microbiome might be targeted for enhanced performance via its contribution to host-microbiota metabolic crosstalk.

In silico, in vitro and in vivo characterization of host-associated Latilactobacillus curvatus strains for potential probiotic applications in farmed Atlantic salmon (Salmo salar).

Salmon aquaculture is the fastest growing animal protein production system in the world; however, intensive farming leads to poor weight gain, stress, and disease outbreaks. Probiotics offer the potential to enhance growth performance and feed efficiency in Atlantic salmon, as well as immunostimulate fish against common pathogens, benefitting farmers and consumers with more efficient production. Here, we isolated and identified 900 native microbial isolates including 18 Lactobacilli from the farmed salmon intestines. Based on whole-genome sequencing and phylogenetic analysis, the Lactobacillus candidates belonged to Latilactobacillus curvatus (L. curvatus) species and formed two distinct phylogenetic groups. Using bioinformatics and in vitro analyses, we selected two candidates L. curvatus ATCC PTA-127116 and L. curvatus ATCC PTA-127117, which showed desirable safety and probiotic properties. The two L. curvatus candidates were evaluated for safety and efficacy (higher final weight) in Atlantic salmon alongside spore-forming Bacilli isolated from salmon, poultry, and swine. All the tested candidates were safe to salmon with no adverse effects. While we did not see efficacy in any Bacillus supplemented groups, compared to untreated group, the group administered with the two L. curvatus strains consortium in feed for seven weeks in freshwater showed indicators of improvement in final body weight by 4.2%. Similarly, the two L. curvatus candidates were also evaluated for safety and efficacy in Atlantic salmon in saltwater; the group administered with the two L. curvatus strains consortium in feed for 11 weeks showed indicators of improvement in final body weight by 4.7%. Comprehensive metabolomics analyses in the presence of different prebiotics and/or additives identified galactooligosaccharide as a potential prebiotic to enhance the efficacy of two L. curvatus candidates. All together, these data provide comprehensive genomic, phenotypic and metabolomic evidence of safety and desirable probiotic properties as well as indicators of in vivo efficacy of two novel endogenous L. curvatus candidates for potential probiotic applications in Atlantic salmon. The in vivo findings need to be confirmed in larger performance studies, including field trials.

Comparative analysis of EspF variants in inhibition of Escherichia coli phagocytosis by macrophages and inhibition of E. coli translocation through human- and bovine-derived M cells.

The EspF protein is secreted by the type III secretion system of enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively). EspF sequences differ between EHEC O157:H7, EHEC O26:H11, and EPEC O127:H6 in terms of the number of SH3-binding polyproline-rich repeats and specific residues in these regions, as well as residues in the amino domain involved in cellular localization. EspF(O127) is important for the inhibition of phagocytosis by EPEC and also limits EPEC translocation through antigen-sampling cells (M cells). EspF(O127) has been shown to have effects on cellular organelle function and interacts with several host proteins, including N-WASP and sorting nexin 9 (SNX9). In this study, we compared the capacities of different espF alleles to inhibit (i) bacterial phagocytosis by macrophages, (ii) translocation through an M-cell coculture system, and (iii) uptake by and translocation through cultured bovine epithelial cells. The espF gene from E. coli serotype O157 (espF(O157)) allele was significantly less effective at inhibiting phagocytosis and also had reduced capacity to inhibit E. coli translocation through a human-derived in vitro M-cell coculture system in comparison to espF(O127) and espF(O26). In contrast, espF(O157) was the most effective allele at restricting bacterial uptake into and translocation through primary epithelial cells cultured from the bovine terminal rectum, the predominant colonization site of EHEC O157 in cattle and a site containing M-like cells. Although LUMIER binding assays demonstrated differences in the interactions of the EspF variants with SNX9 and N-WASP, we propose that other, as-yet-uncharacterized interactions contribute to the host-based variation in EspF activity demonstrated here.

Mitotic Arrest-Deficient 2 Like 2 (MAD2L2) Interacts with Escherichia coli Effector Protein EspF.

Enteropathogenic (EPEC) and Enterohemorrhagic (EHEC) Escherichia coli are considered emerging zoonotic pathogens of worldwide distribution. The pathogenicity of the bacteria is conferred by multiple virulence determinants, including the locus of enterocyte effacement (LEE) pathogenicity island, which encodes a type III secretion system (T3SS) and effector proteins, including the multifunctional secreted effector protein (EspF). EspF sequences differ between EPEC and EHEC serotypes in terms of the number and residues of SH3-binding polyproline-rich repeats and N-terminal localization sequence. The aim of this study was to discover additional cellular interactions of EspF that may play important roles in E. coli colonization using the Yeast two-hybrid screening system (Y2H). Y2H screening identified the anaphase-promoting complex inhibitor Mitotic Arrest-Deficient 2 Like 2 (MAD2L2) as a host protein that interacts with EspF. Using LUMIER assays, MAD2L2 was shown to interact with EspF variants from EHEC O157:H7 and O26:H11 as well as EPEC O127:H6. MAD2L2 is targeted by the non-homologous Shigella effector protein invasion plasmid antigen B (IpaB) to halt the cell cycle and limit epithelial cell turnover. Therefore, we postulate that interactions between EspF and MAD2L2 serve a similar function in promoting EPEC and EHEC colonization, since cellular turnover is a key method for bacteria removal from the epithelium. Future work should investigate the biological importance of this interaction that could promote the colonization of EPEC and EHEC E. coli in the host.

An investigation of the expression and adhesin function of H7 flagella in the interaction of Escherichia coli O157 : H7 with bovine intestinal epithelium.

Enterohaemorrhagic Escherichia coli O157 : H7 is a bacterial pathogen that can cause haemorrhagic colitis and haemolytic uremic syndrome. In the primary reservoir host, cattle, the terminal rectum is the principal site of E. coli O157 colonization. In this study, bovine terminal rectal primary epithelial cells were used to examine the role of H7 flagella in epithelial adherence. Binding of a fliC(H7) mutant O157 strain to rectal epithelium was significantly reduced as was binding of the flagellated wild-type strain following incubation with H7-specific antibodies. Complementation of fliC(H7) mutant O157 strain with fliC(H7) restored the adherence to wild-type levels; however, complementation with fliC(H6) did not restore it. High-resolution ultrastructural and imunofluorescence studies demonstrated the presence of abundant flagella forming physical contact points with the rectal epithelium. Binding to terminal rectal epithelium was specific to H7 by comparison with other flagellin types tested. In-cell Western assays confirmed temporal expression of flagella during O157 interaction with epithelium, early expression was suppressed during the later stages of microcolony and attaching and effacing lesion formation. H7 flagella are expressed in vivo by individual bacteria in contact with rectal mucosa. Our data demonstrate that the H7 flagellum acts as an adhesin to bovine intestinal epithelium and its involvement in this crucial initiating step for colonization indicates that H7 flagella could be an important target in intervention strategies.

Characterization of the effects of salicylidene acylhydrazide compounds on type III secretion in Escherichia coli O157:H7.

Recent work has highlighted a number of compounds that target bacterial virulence by affecting gene regulation. In this work, we show that small-molecule inhibitors affect the expression of the type III secretion system (T3SS) of Escherichia coli O157:H7 in liquid culture and when this bacterium is attached to bovine epithelial cells. Inhibition of T3SS expression resulted in a reduction in the capacity of the bacteria to form attaching and effacing lesions. Our results show that there is marked variation in the abilities of four structurally related compounds to inhibit the T3SS of a panel of isolates. Using transcriptomics, we performed a comprehensive analysis of the conserved and inhibitor-specific transcriptional responses to these four compounds. These analyses of gene expression show that numerous virulence genes, located on horizontally acquired DNA elements, are affected by the compounds, but the number of genes significantly affected varied markedly for the different compounds. Overall, we highlight the importance of assessing the effect of such "antivirulence" agents on a range of isolates and discuss the possible mechanisms which may lead to the coordinate downregulation of horizontally acquired virulence genes.

The Escherichia coli O157:H7 EhaB autotransporter protein binds to laminin and collagen I and induces a serum IgA response in O157:H7 challenged cattle.

Enterohaemorrhagic Escherichia coli (EHEC) are a subgroup of Shiga toxin-producing E. coli that cause gastrointestinal disease with the potential for life-threatening sequelae. Cattle serve as the natural reservoir for EHEC and outbreaks occur sporadically as a result of contaminated beef and other farming products. While certain EHEC virulence mechanisms have been extensively studied, the factors that mediate host colonization are poorly defined. Previously, we identified four proteins (EhaA,B,C,D) from the prototypic EHEC strain EDL933 that belong to the autotransporter (AT) family. Here we characterize the EhaB AT protein. EhaB was shown to be located at the cell surface and overexpression in E. coli K-12 resulted in significant biofilm formation under continuous flow conditions. Overexpression of EhaB in E. coli K12 and EDL933 backgrounds also promoted adhesion to the extracellular matrix proteins collagen I and laminin. An EhaB-specific antibody revealed that EhaB is expressed in E. coli EDL933 following in vitro growth. EhaB also cross-reacted with serum IgA from cattle challenged with E. coli O157:H7, indicating that EhaB is expressed in vivo and elicits a host IgA immune response.

Escherichia coli O157:H7 colonization in cattle following systemic and mucosal immunization with purified H7 flagellin.

Escherichia coli O157:H7 is an important pathogen of humans. Cattle are most frequently identified as the primary source of infection, and therefore, reduction in E. coli O157:H7 prevalence in cattle by vaccination represents an attractive strategy for reducing the incidence of human disease. H7 flagella have been implicated in intestinal-epithelial colonization of E. coli O157:H7 and may represent a useful target for vaccination. In this study, calves were immunized either systemically with H7 flagellin by intramuscular injection or mucosally via the rectum with either H7 or H7 incorporated into poly(DL-lactide-co-glycolide) microparticles (PLG:H7). Systemic immunization resulted in high levels of flagellin-specific immunoglobulin G (IgG) and IgA in both serum and nasal secretions and detectable levels of both antibody isotypes in rectal secretions. Rectal administration of flagellin resulted in levels of rectal IgA similar to those by the intramuscular route but failed to induce any other antibody response, whereas rectal immunization with PLG:H7 failed to induce any H7-specific antibodies. Following subsequent oral challenge with E. coli O157:H7, reduced colonization rates and delayed peak bacterial shedding were observed in the intramuscularly immunized group compared to nonvaccinated calves, but no reduction in total bacterial shedding occurred. Rectal immunization with either H7 or PLG:H7 had no effect on subsequent bacterial colonization or shedding. Furthermore, purified H7-specific IgA and IgG from intramuscularly immunized calves were shown to reduce intestinal-epithelial binding in vitro. These results indicate that H7 flagellin may be a useful component in a systemic vaccine to reduce E. coli O157:H7 colonization in cattle.

EhaA is a novel autotransporter protein of enterohemorrhagic Escherichia coli O157:H7 that contributes to adhesion and biofilm formation.

Autotransporter (AT) proteins have been identified in many Gram-negative pathogens and are unique in that their primary sequence is sufficient to direct their transport across the bacterial membrane system. Where characterized they are uniformly associated with virulence. Using conserved AT motifs as a search tool, four putative AT proteins were identified in the Enterohemorrhagic Escherichia coli O157:H7 EDL933 genome. The genes encoding these proteins (z0402/ehaA, z0469/ehaB, z3487/ehaC and z3948/ehaD) were PCR amplified, cloned and expressed in an E. coli K-12 MG1655flu background. Preliminary characterization revealed that ehaA, ehaB and ehaD encode proteins associated with increased biofilm formation. One of these genes (ehaA) resides on a genomic island in E. coli O157:H7 strains EDL933 and Sakai. Over-expression of EhaA in E. coli K-12 demonstrated it is located at the cell surface and resulted in the formation of large cell aggregates, promoted significant biofilm formation and mediated adhesion to primary epithelial cells of the bovine terminal rectum. The expression of ehaA was demonstrated in E. coli EDL933 by RT-PCR. An EhaA-specific antibody revealed the EhaA protein was expressed in 24/50 generic Shiga toxin-producing E. coli (STEC) strains of various serotypes including O157:H7. However, the deletion of ehaA from E. coli EDL933 and a STEC strain from serotype O111:H(-) did not affect biofilm growth. Our results suggest that EhaA may contribute to adhesion, colonization and biofilm formation by E. coli O157:H7 and possibly other STEC serotypes.

EspP, a Type V-secreted serine protease of enterohaemorrhagic Escherichia coli O157:H7, influences intestinal colonization of calves and adherence to bovine primary intestinal epithelial cells.

Enterohaemorrhagic Escherichia coli (EHEC) comprise a group of zoonotic diarrhoeal pathogens of worldwide importance. Cattle are a key reservoir; however the molecular mechanisms that promote persistent colonization of the bovine intestines by EHEC are ill-defined. The large plasmid of EHEC O157:H7 encodes several putative virulence factors. Here, it is reported that the pO157-encoded Type V-secreted serine protease EspP influences the intestinal colonization of calves. To dissect the basis of attenuation, a bovine primary rectal epithelial cell line was developed. Adherence of E. coli O157:H7 to such cells was significantly impaired by espP mutation but restored upon addition of highly purified exogenous EspP. Data of this study add to the growing body of evidence that cytotoxins facilitate intestinal colonization by EHEC.

Simple methods for measurement of bovine mucosal antibody responses in vivo.

The mucosal immune response serves as the first line of defence against many bacterial and viral diseases. Therefore, measurement of mucosal immune responses is important in evaluating mucosal immunisation protocols and understanding initial host/pathogen interactions. In this study we compare two methods for repeated sampling of bovine rectal mucosal secretions, namely rectal swabbing and rectal biopsies, and evaluate a simple swabbing method for sampling bovine nasal secretions. Both rectal swabs and rectal biopsies yielded similar quantities of total IgA (TIgA)/ml. However, rectal biopsies yielded five times more total IgG (TIgG)/ml than rectal swabs. Blood contamination was estimated to contribute approximately 7% of TIgG and <0.05% TIgA in rectal swab samples compared to 40% of TIgG and 4.5% of TIgA in rectal biopsy samples, indicating that rectal swabbing was more effective at sampling rectal mucosal secretions. Nasal swabs were effective at obtaining nasal secretion samples with only 1% of TIgG and <0.05% TIgA estimated to be blood derived. Furthermore, H7 flagellin-specific antibodies were detected in both nasal and rectal swab samples following either rectal immunisation with purified H7 flagellin or oral challenge with live E. coli O157:H7, indicating that both techniques are effective methods for monitoring mucosal antibody responses in cattle.

Optimizing the Protection of Cattle against Escherichia coli O157:H7 Colonization through Immunization with Different Combinations of H7 Flagellin, Tir, Intimin-531 or EspA.

Enterohemorrhagic Escherichia coli (EHEC) are important human pathogens, causing hemorrhagic colitis and hemolytic uraemic syndrome in humans. E. coli O157:H7 is the most common serotype associated with EHEC infections worldwide, although other non-O157 serotypes cause life-threatening infections. Cattle are a main reservoir of EHEC and intervention strategies aimed at limiting EHEC excretion from cattle are predicted to lower the risk of human infection. We have previously shown that immunization of calves with recombinant versions of the type III secretion system (T3SS)-associated proteins EspA, intimin and Tir from EHEC O157:H7 significantly reduced shedding of EHEC O157 from experimentally-colonized calves, and that protection could be augmented by the addition of H7 flagellin to the vaccine formulation. The main aim of the present study was to optimize our current EHEC O157 subunit vaccine formulations by identifying the key combinations of these antigens required for protection. A secondary aim was to determine if vaccine-induced antibody responses exhibited cross-reactive potential with antigens from other EHEC serotypes. Immunization with EspA, intimin and Tir resulted in a reduction in mean EHEC O157 shedding following challenge, but not the mean proportion of calves colonized. Removal of Tir resulted in more prolonged shedding compared with all other groups, whereas replacement of Tir with H7 flagellin resulted in the highest levels of protection, both in terms of reducing both mean EHEC O157 shedding and the proportion of colonized calves. Immunization of calves with recombinant EHEC O157 EspA, intimin and Tir resulted in the generation of antibodies capable of cross-reacting with antigens from non-O157 EHEC serotypes, suggesting that immunization with these antigens may provide a degree of cross-protection against other EHEC serotypes. Further studies are now required to test the efficacy of these vaccines in the field, and to formally test the cross-protective potential of the vaccines against other non-O157 EHEC.