Recto-anal Junction Research Articles

Recto-Anal Junction (RAJ) and Fecal Microbiomes of Cattle Experimentally Challenged With Escherichia coli O157:H7.

Cattle are the asymptomatic reservoirs of Escherichia coli O157:H7 (O157) that preferentially colonizes the bovine recto-anal junction (RAJ). Understanding the influence of O157 on the diversity of the RAJ microbiota could give insights into its persistence at the RAJ in cattle. Hence, we compared changes in bovine RAJ and fecal microbiota following O157 challenge under experimental conditions. Cattle were either orally challenged (n = 4) with1010 CFU of a streptomycin-resistant O157 strain 86–24, or mock-challenged (n = 4) with phosphate buffered saline. Rectoanal mucosal swab (RAMS) and fecal samples were collected at different time points for analysis. Alpha diversity measures (Chao1 species richness and Shannon diversity index) were found to be significantly different between RAMS and fecal samples but not influenced by O157 challenge. The Firmicutes to Bacteroidetes (F: B) ratio was higher in RAMS samples from O157 colonized animals and this may have influenced the consistent yet decreased O157 colonization at the RAJ. Specific bacterial genera that were present in relative low abundance in fecal and RAMS microbiota did not affect overall microbial diversity but were associated with O157 colonization. Differential abundance analysis (DAA) of genera in samples from O157 shedding cattle indicated significantly higher relative abundance of Paenibacillus and Fusobacterium in RAMS, and Tyzzerella in fecal samples. Mock-challenged cattle showed higher relative abundance of Intestinimonas and Citrobacter in RAMS samples, and Succinivibrio, and Prevotella 1 in fecal samples. These results suggest that O157 challenge exerts transient influence on the intestinal microbial community which in turn might promote O157 colonization in a site-specific manner.

Supershed Escherichia coli O157:H7 Has Potential for Increased Persistence on the Rectoanal Junction Squamous Epithelial Cells and Antibiotic Resistance.

Supershedding cattle shed Escherichia coli O157:H7 (O157) at ≥ 104 colony-forming units/g feces. We recently demonstrated that a supershed O157 (SS-O157) strain, SS-17, hyperadheres to the rectoanal junction (RAJ) squamous epithelial (RSE) cells which may contribute to SS-O157 persistence at this site in greater numbers, thereby increasing the fecal O157 load characterizing the supershedding phenomenon. In order to verify if this would be the signature adherence profile of any SS-O157, we tested additional SS-O157 isolates (n = 101; each from a different animal) in the RSE cell adherence assay. Similar to SS-17, all 101 SS-O157 exhibited aggregative adherence on RSE cells, with 56% attaching strongly (>10 bacteria/cell; hyperadherent) and 44% attaching moderately (1–10 bacteria/cells). Strain typing using Polymorphic Amplified Typing Sequences (PATS) analysis assigned the 101 SS-O157 into 5 major clades but not to any predominant genotype. Interestingly, 69% of SS-O157 isolates were identical to human O157 outbreak strains based on pulsed field gel electrophoresis profiles (CDC PulseNet Database), grouped into two clades by PATS distinguishing them from remaining SS-O157, and were hyperadherent on RSE cells. A subset of SS-O157 isolates (n = 53) representing different PATS and RSE cell adherence profiles were analyzed for antibiotic resistance (AR). Several SS-O157 (30/53) showed resistance to sulfisoxazole, and one isolate was resistant to both sulfisoxazole and tetracycline. Minimum inhibitory concentration (MIC) tests confirmed some of the resistance observed using the Kirby–Bauer disk diffusion test. Each SS-O157 isolate carried at least 10 genes associated with AR. However, genes directly associated with AR were rarely amplified: aac (3)-IV in 2 isolates, sul2 in 3 isolates, and tetB in one isolate. The integrase gene, int, linked with integron-based AR acquisition/transmission, was identified in 92% of SS-O157 isolates. Our results indicate that SS-O157 isolates could potentially persist longer at the bovine RAJ but exhibit limited resistance towards clinical antibiotics.

Rheology of human faeces and pathophysiology of defaecation.

Rectal evacuation involves multiple mechanisms that are not completely understood. The aim of this study was to quantify the rheologic property, i.e., yield stress, which governs the ease of deformation of a range of faeces of differing consistency and understand its influence on the pathophysiology of defaecation. Yield stresses of faeces of differing consistencies and Bristol scores were determined by the Vane test. We then explored the effects of this property on ease of defecation using a simple static model of the recto-anal junction based on the laws of flow for yield stress pastes and checked the conclusions by X-ray defaecography experience. The yield stress of faeces increased exponentially with their solid content, from 20 to 8000Pa. The static model of the recto-anal junction showed that evacuation of faeces of normal consistency and yield stress is possible with moderate dilatation of the anal canal, whilst the evacuation of faeces with higher yield stress requires greater dilatation of the anal canal. X-ray defaecography showed that such increases occurred in vivo. The diameter of the recto-anal junction is increased to enable the passage of feces with high yield stress. The finite limits to such dilation likely contribute to fecal impaction. Hence, difficulties in defaecation may result either from unduly high yield stress or pathologies of reflex recto-anal dilatation or a combination of the two.

Lactoferrin translocates to the nucleus of bovine rectal epithelial cells in the presence of Escherichia coli O157:H7

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.

A quantitative real time PCR assay to detect and enumerate Escherichia coli O157 and O26 serogroups in sheep recto-anal swabs

A quantitative PCR method is described for the detection and quantification of E. coli O157 and O26 in sheep recto-anal junction swabs. The method incorporated a short enrichment step (5 h) and the use of a developed standard calibration curve relating the real time PCR cycle threshold (Ct) values to the initial concentration of pathogen in the sheep sample.

Surveillance of Shiga-toxin producing Escherichia coli in Irish sheep

Shiga-toxin producing Escherichia coli (STEC) is a foodborne zoonotic pathogen of significant public health concern. Ruminant animals are considered the primary reservoir of STEC. STEC predominantly colonises the lower gastro-intestinal tract, termed the recto-anal junction (RAJ). The number of STEC shed in the faeces of ruminants can vary widely with some animals, termed ‘super-shedders’ (>Log104 c.f.u. g−1 faeces), high risk carriers of the pathogen. The objective of this study was to sample a large cohort of Irish sheep, with quantitative and qualitative analysis of each sample for STEC. RAJ swab samples (N=410) were collected over a 9 month period from an ovine slaughtering facility. Each swab was enriched in 30 ml of modified Tryptone Soya Broth with Novobiocin at 41.5 °C for 5 h and subjected to a quantitative real-time PCR assay to detect and enumerate serogroups O157 and O26 in super-shedding animals. Incubation was allowed to continue for 24 h and shiga-toxin prevalence was assessed using a targeted qualitative real-time PCR assay. Eight O157 strains were isolated, of which six were super-shedding strains. The incidence of stx, O157 and O26 positive swabs was 49.3 %, 1.95 % and 0.24 % respectively. The prevalence of stx1, stx2 and stx1/stx2 virulence factors in isolated strains was 15.9 %, 8.8 % and 22.4 %. Additionally, the occurrence of stx1/stx2 in combination with eaeA in strains was found to be significant according to Pearson’s correlation and a paired T-test. In conclusion, these results underline the risk Irish sheep pose as a potential source of STEC infection.

High Prevalence of Cefotaxime Resistant Bacteria in Grazing Beef Cattle: A Cross Sectional Study.

Although the over-use of antibiotics during food animal production is a potential driver of antimicrobial resistant microorganisms (ARMs), a high prevalence of cefotaxime resistant bacteria (CRB) has been observed in grazing animals raised without antibiotic supplementation. In this cross-sectional study, the prevalence and concentration of CRB in beef cattle on grazing farms were investigated. Fecal samples from the recto-anal junction of cattle (n = 840) and environmental samples (n = 258) were collected from 17 farms in North and Central Florida in the United States, and a survey of farm characteristics, animal husbandry practices, and antibiotic usage was conducted. CRB were detected in fecal samples from 47.4% of all cattle, with the prevalence ranging from 21.1 to 87.5% on farms, and significantly higher (P < 0.001) in calves compared to adult cows (54.1 vs. 41.8%). Environmental samples had a higher prevalence than fecal samples (P < 0.001), with CRB detected in 88.6% of water, 98.7% of soil, and 95.7% of forage samples. Compared to the concentration (log CFU/g) of CRB in fecal samples (2.95, 95% CI: 2.89, 3.02), the concentration of CRB was higher (P < 0.001) in soil and forage samples (5.37, 95% CI: 5.16, 5.57) and lower (P < 0.001) in water samples (1.08, 95% CI: 0.82, 1.36). Soil microbiota from farms with high prevalence of CRB clustered closer together and the proportion of Phylum Proteobacteria was higher on farms with high prevalence of CRB resistance. Large farming operations were associated with a 58% higher likelihood of CRB detection in fecal samples. Regular cleaning of drinking troughs and the addition of ionophores to feed were associated with CRB reduction in fecal samples. Taken together, the widespread of CRB into both cattle seldom treated with cephalosporin antibiotics and the surrounding environment suggests the environment is a natural source of antimicrobial resistance in beef cattle.

Mucosal cellular immune responses following Escherichia coli O157:H7 bacterin vaccination associated with reduced fecal shedding

Abstract Parenterally-delivered, bacterin-based vaccines can significantly limit shedding of Shiga-toxin producing Escherichia coli (STEC) O157:H7 from cattle, providing a potential intervention strategy to limit foodborne contamination. While the peripheral antibody response to STEC bacterin has been characterized, induction of STEC-specific cellular immunity, particularly at the site of colonization, remains poorly described. As STEC-specific antibodies likely provide some protection, cellular immune responses likely contribute to limiting colonization also. Following vaccination and challenge, lymphocytes were detected in the rectoanal junction (RAJ), a preferred colonization site for E. coli O157:H7. T cells present in the RAJ were positive for interferon (IFN)-γ mRNA, including a subset of γδ+ CD8+ T cells. Specifically within the RAJ, γδ+ T cells expressing IFN-γ mRNA were greater in number compared to classical αβ+ T cells. Also, distinct lymphoid follicles were present in greater number at the RAJ in vaccinated/challenged animals and fecal STEC-specific IgA was detected. These vaccine -primed responses likely support localized immune responses upon challenge. Local T cell-produced IFN-γ may impact epithelial cells, subsequently limiting E. coli adherence. Thus, distinct immune changes were induced at the mucosa of vaccinated and challenged animals, providing insight on mechanisms associated with limiting shedding. Enhancing mucosal immunity may be critical in the further development of efficacious vaccines for controlling STEC O157:H7 in ruminants, thus limiting human infection.

Interactions of the Hindgut Mucosa-Associated Microbiome with Its Host Regulate Shedding of Escherichia coli O157:H7 by Cattle.

Cattle are the primary carrier of Escherichia coli O157:H7, a foodborne human pathogen, and those shedding >104 CFU/gram of feces of E. coli O157:H7 are defined as supershedders (SS). This study investigated the rectoanal junction (RAJ) mucosa-associated microbiota and its relationship with host gene expression in SS and in cattle from which E. coli O157:H7 was not detected (nonshedders [NS]), aiming to elucidate the mechanisms involved in supershedding. In total, 14 phyla, 66 families, and 101 genera of RAJ mucosa-associated bacteria were identified and Firmicutes (61.5 ± 7.5%), Bacteroidetes (27.9 ± 6.4%), and Proteobacteria (5.5 ± 2.1%) were the predominant phyla. Differential abundance analysis of operational taxonomic units (OTUs) identified 2 OTUs unique to SS which were members of Bacteroides and Clostridium and 7 OTUs unique to NS which were members of Coprococcus, Prevotella, Clostridium, and Paludibacter Differential abundance analysis of predicted microbial functions (using PICRUSt [phylogenetic investigation of communities by reconstruction of unobserved states]) revealed that 3 pathways had higher abundance (log2 fold change, 0.10 to 0.23) whereas 12 pathways had lower abundance (log2 fold change, -0.36 to -0.20) in SS. In addition, we identified significant correlations between expression of 19 differentially expressed genes and the relative abundance of predicted microbial functions, including nucleic acid polymerization and carbohydrate and amino acid metabolism. Our findings suggest that differences in RAJ microbiota at both the compositional and functional levels may be associated with E. coli O157:H7 supershedding and that certain microbial groups and microbial functions may influence RAJ physiology of SS by affecting host gene expression.IMPORTANCE Cattle with fecal E. coli O157:H7 at >104 CFU per gram of feces have been defined as the supershedders, and they are responsible for the most of the E. coli O157:H7 spread into farm environment. Currently, no method is available for beef producers to eliminate shedding of E. coli O157:H7 in cattle, and the lack of information about the mechanisms of supershedding greatly impedes the development of effective methods. This study investigated the role of the rectoanal junction (RAJ) mucosa-associated microbiome in E. coli O157:H7 shedding, and our results indicated that the compositions and functions of RAJ microbiota differed between supershedders and nonshedders. The identified relationship between the differentially abundant microbes and 19 previously identified differentially expressed genes suggests the role of host-microbial interactions involved in E. coli O157:H7 supershedding. Our findings provide a fundamental understanding of the supershedding phenomenon which is essential for the development of strategies, such as the use of directly fed microbials, to reduce E. coli O157:H7 shedding in cattle.

Conditional Function of Autoaggregative Protein Cah and Common cah Mutations in Shiga Toxin-Producing Escherichia coli.

Cah is a calcium-binding autotransporter protein involved in autoaggregation and biofilm formation. Although cah is widespread in Shiga toxin-producing Escherichia coli (STEC), we detected mutations in cah at a frequency of 31.3% in this pathogen. In STEC O157:H7 supershedder strain SS17, a large deletion results in a smaller coding sequence, encoding a protein lacking the C-terminal 71 amino acids compared with Cah in STEC O157:H7 strain EDL933. We examined the function of Cah in biofilm formation and host colonization to better understand the selective pressures for cah mutations. EDL933-Cah played a conditional role in biofilm formation in vitro: it enhanced E. coli DH5α biofilm formation on glass surfaces under agitated culture conditions that prevented autoaggregation but inhibited biofilm formation under hydrostatic conditions that facilitated autoaggregation. This function appeared to be strain dependent since Cah-mediated biofilm formation was diminished when an EDL933 cah gene was expressed in SS17. Deletion of cah in EDL933 enhanced bacterial attachment to spinach leaves and altered the adherence pattern of EDL933 to bovine recto-anal junction squamous epithelial (RSE) cells. In contrast, in trans expression of EDL933 cah in SS17 increased its attachment to leaf surfaces, and in DH5α, it enhanced its adherence to RSE cells. Hence, the ecological function of Cah appears to be modulated by environmental conditions and other bacterial strain-specific properties. Considering the prevalence of cah in STEC and its role in attachment and biofilm formation, cah mutations might be selected in ecological niches in which inactivation of Cah would result in an increased fitness in STEC during colonization of plants or animal hosts.IMPORTANCE Shiga toxin-producing Escherichia coli (STEC) harbors genes encoding diverse adhesins, and many of these are known to play an important role in bacterial attachment and host colonization. We demonstrated here that the autotransporter protein Cah confers on E. coli DH5α cells a strong autoaggregative phenotype that is inversely correlated with its ability to form biofilms and plays a strain-specific role in plant and animal colonization by STEC. Although cah is widespread in the STEC population, we detected a mutation rate of 31.3% in cah, which is similar to that reported for rpoS and fimH The formation of cell aggregates due to increased bacterium-to-bacterium interactions may be disadvantageous to bacterial populations under conditions that favor a planktonic state in STEC. Therefore, a loss-of-function mutation in cah is likely a selective trait in STEC when autoaggregative properties become detrimental to bacterial cells and may contribute to the adaptability of STEC to fluctuating environments.

Prevalence and antibiotic susceptibility pattern of Escherichia coli in cattle on Bathan and intensive rearing system

The aim of this longitudinal study was to verify the prevalence and antibiotic susceptibility pattern of Escherichia coli (E. coli) in cattle reared on Bathan and intensive farming system in Bangladesh. Fecal materials originated from recto anal junction (RAJ) of 100 cattle used for primary screening on MacConkey agar. The diversities among the pink color colony producing isolates on MacConkey agar were verified by conventional cultural methods and biochemical tests. Phenotypically positive E. coli isolates were further investigated for the variations in the antimicrobial resistance profiles to 10 selected antibiotics, by the disk-diffusion method. This study revealed that the overall prevalence of E. coli was 70% of in the rectal swab sample of cattle. However, the prevalence of E. coli was found significantly higher (p= 0.002) in cattle under intensive farming (84%) than cattle on Bathan (56%). Antibiotic susceptibility pattern shows that among the tested isolates 83%, 73%, 68% and 64% were sensitive to chloramphenicol, gentamicin, ciprofloxacin and ampicillin, respectively. On the other hand, all the 70 (100%) E. coli isolates were found resistant to tetracycline and sulfamethoxazole. A high antibiotic resistance profile was also found against amoxicillin (90%), ampicillin (87%), nalidixic acid (86%) and erythromycin (83%). In total, 24 (34%) isolates were resistant against ?2 antimicrobials. The result clearly shows that antibiotic resistant E. coli isolates are commonly present in cattle of different management systems (intensive and Bathan). Therefore, careful selection of appropriate antibiotics with optimal doses might be ensured to prevent the emergence of antibiotic resistance bacteria.Microbes and Health, June 2017, 6(1): 1-4

Comparative genomics of two super-shedder isolates of Escherichia coli O157:H7.

Shiga toxin-producing Escherichia coli O157:H7 (O157) are zoonotic foodborne pathogens and of major public health concern that cause considerable intestinal and extra-intestinal illnesses in humans. O157 colonize the recto-anal junction (RAJ) of asymptomatic cattle who shed the bacterium into the environment through fecal matter. A small subset of cattle, termed super-shedders (SS), excrete O157 at a rate (≥ 104 CFU/g of feces) that is several orders of magnitude greater than other colonized cattle and play a major role in the prevalence and transmission of O157. To better understand microbial factors contributing to super-shedding we have recently sequenced two SS isolates, SS17 (GenBank accession no. CP008805) and SS52 (GenBank accession no. CP010304) and shown that SS isolates display a distinctive strongly adherent phenotype on bovine rectal squamous epithelial cells. Here we present a detailed comparative genomics analysis of SS17 and SS52 with other previously characterized O157 strains (EC4115, EDL933, Sakai, TW14359). The results highlight specific polymorphisms and genomic features shared amongst SS strains, and reveal several SNPs that are shared amongst SS isolates, including in genes involved in motility, adherence, and metabolism. Finally, our analyses reveal distinctive patterns of distribution of phage-associated genes amongst the two SS and other isolates. Together, the results of our comparative genomics studies suggest that while SS17 and SS52 share genomic features with other lineage I/II isolates, they likely have distinct recent evolutionary histories. Future comparative and functional genomic studies are needed to decipher the precise molecular basis for super shedding in O157.

Shiga toxin-producing Escherichia coli (STEC) O22:H8 isolated from cattle reduces E. coli O157:H7 adherence in vitro and in vivo

Problem addressedShiga toxin-producing Escherichia coli (STEC) are a group of bacteria responsible for food-associated diseases. Clinical features include a wide range of symptoms such as diarrhea, hemorrhagic colitis and the hemolytic uremic syndrome (HUS), a life-threatening condition. ObjectiveOur group has observed that animals naturally colonized with STEC strains of unknown serotype were not efficiently colonized with E. coli O157:H7 after experimental infection. In order to assess the basis of the interference, three STEC strains were isolated from STEC persistently-colonized healthy cattle from a dairy farm in Buenos Aires, Argentina. Methods and resultsThe three isolated strains are E. coli O22:H8 and carry the stx1 and stx2d genes. The activatable activity of Stx2d was demonstrated in vitro. The three strains carry the adhesins iha, ehaA and lpfO113. E. coli O22:H8 formed stronger biofilms in abiotic surface than E. coli O157:H7 (eae+, stx2+) and displayed a more adherent phenotype in vitro towards HeLa cells. Furthermore, when both serotypes were cultured together O22:H8 could reduce O157:H7 adherence in vitro. When calves were intragastrically pre-challenged with 108 CFU of a mixture of the three STEC strains and two days later challenged with the same dose of the strain E. coli O157:H7 438/99, the shedding of the pathogen was significantly reduced. ConclusionsThese results suggest that E. coli O22:H8, a serotype rarely associated with human illness, might compete with O157:H7 at the bovine recto-anal junction, making non-O157 carrying-calves less susceptible to O157:H7 colonization and shedding of the bacteria to the environment.

Draft Genome Sequences of Escherichia coli Strains Isolated at Calving from the Uterus, Vagina, Vulva, and Rectoanal Junction of a Dairy Cow That Later Developed Metritis.

ABSTRACTEscherichia coli is involved in the pathogenicity of metritis in cows. We report here the genome sequences of E. coli strains isolated at calving from the uterus, vagina, vulva, and rectoanal junction of a dairy cow that later developed metritis. The genomic similarities will give an insight into phylogenetic relationships among strains.

Curli Temper Adherence of Escherichia coli O157:H7 to Squamous Epithelial Cells from the Bovine Recto-Anal Junction in a Strain-Dependent Manner.

This study demonstrated that O157 strains interact with epithelial cells in a host-specific manner. The fimbriae/adhesins that are significant for adherence to human cell lines may not have a role or may have a modulating role in O157 adherence to bovine cells. Targeting such adhesins may not prevent O157 attachment to bovine cells but instead may result in improved adherence. Hence, conducting host-specific evaluations is critical when selecting targets for O157 control strategies.

Potential immunosuppressive effects of Escherichia coli O157:H7 experimental infection on the bovine host.

BackgroundEnterohaemorrhagic Escherichia coli (EHEC), like E. coli O157:H7 are frequently detected in bovine faecal samples at slaughter. Cattle do not show clinical symptoms upon infection, but for humans the consequences after consuming contaminated beef can be severe. The immune response against EHEC in cattle cannot always clear the infection as persistent colonization and shedding in infected animals over a period of months often occurs. In previous infection trials, we observed a primary immune response after infection which was unable to protect cattle from re-infection. These results may reflect a suppression of certain immune pathways, making cattle more prone to persistent colonization after re-infection. To test this, RNA-Seq was used for transcriptome analysis of recto-anal junction tissue and ileal Peyer’s patches in nine Holstein-Friesian calves in response to a primary and secondary Escherichia coli O157:H7 infection with the Shiga toxin (Stx) negative NCTC12900 strain. Non-infected calves served as controls.ResultsIn tissue of the recto-anal junction, only 15 genes were found to be significantly affected by a first infection compared to 1159 genes in the ileal Peyer’s patches. Whereas, re-infection significantly changed the expression of 10 and 17 genes in the recto-anal junction tissue and the Peyer’s patches, respectively. A significant downregulation of 69 immunostimulatory genes and a significant upregulation of seven immune suppressing genes was observed.ConclusionsAlthough the recto-anal junction is a major site of colonization, this area does not seem to be modulated upon infection to the same extent as ileal Peyer’s patches as the changes in gene expression were remarkably higher in the ileal Peyer’s patches than in the recto-anal junction during a primary but not a secondary infection. We can conclude that the main effect on the transcriptome was immunosuppression by E. coli O157:H7 (Stx−) due to an upregulation of immune suppressive effects (7/12 genes) or a downregulation of immunostimulatory effects (69/94 genes) in the ileal Peyer’s patches. These data might indicate that a primary infection promotes a re-infection with EHEC by suppressing the immune function.

Prevalence of sorbitol non-fermenting Shiga toxin-producing Escherichia coli in Black Bengal goats on smallholdings

A cross-sectional survey was carried out in Bangladesh with the sampling of 514 Black Bengal goats on smallholdings to determine the presence of sorbitol non-fermenting (SNF) Shiga toxin-producing E. coli (STEC). Swab samples collected from the recto-anal junction were plated onto cefixime and potassium tellurite added sorbitol MacConkey (CT-SMAC) agar, a selective medium for STEC O157 serogroup, where this serogroup and other SNF STEC produce colourless colonies. The SNF E. coli (SNF EC) isolates obtained from the survey were investigated by PCR for the presence of Shiga toxin-producing genes, stx1 and stx2, and two other virulence genes, eae and hlyA that code for adherence factor (intimin protein) and pore-forming cytolysin, respectively. The SNF EC isolates were also assessed for the presence of the rfbO157 gene to verify their identity to O157 serogroup. The results revealed that the proportions of goats carrying SNF EC isolates and stx1 and stx2 genes were 6·2% (32/514) [95% confidence interval (CI) 4·4-8·7)], 1·2% (95% CI 0·5-2·6) and 1·2% (95% CI 0·5-2·6), respectively. All the SNF STEC tested negative for rfbO157, hlyA and eae genes. The risk for transmission of STEC from Black Bengal goats to humans is low.

Standardized Escherichia coli O157:H7 Exposure Studies in Cattle Provide Evidence that Bovine Factors Do Not Drive Increased Summertime Colonization.

The increased summertime prevalence of cattle carriage of enterohemorrhagic Shiga toxin-producing Escherichia coli O157:H7 (STEC O157) is associated with the increased summertime incidence of human infection. The mechanism driving the seasonality of STEC O157 carriage among cattle is unknown. We conducted experimental challenge trials to distinguish whether factors extrinsic or intrinsic to cattle underlie the seasonality of STEC O157 colonization. Holstein steers (n = 20) exposed to ambient environmental conditions were challenged with a standardized pool of STEC O157 strains four times at 6-month intervals. The densities and durations of rectoanal junction mucosa (RAJ) colonization with STEC O157 were compared by season (winter versus summer), dose (10(9) CFU versus 10(7) CFU), and route of challenge (oral versus rectal). Following summer challenges, the RAJ STEC O157 colonization density was significantly lower (P = 0.016) and the duration was shorter (P = 0.052) than for winter challenges, a seasonal pattern opposite to that observed naturally. Colonization was unaffected by the challenge route, indicating that passage through the gastrointestinal microbiome did not significantly affect the infectious dose to the RAJ. A 2-log reduction of the challenge doses in the second-year trials was accompanied by similarly reduced RAJ colonization in both seasons (P < 0.001). These results refute the hypothesis that cattle are predisposed to STEC O157 colonization during the summer months, either due to intrinsic factors or indirectly due to gastrointestinal tract microbiome effects. Instead, the data support the hypothesis that the increased summertime STEC O157 colonization results from increased seasonal oral exposure to this pathogen.

Development of a quantitative real time PCR assay to detect and enumerate Escherichia coli O157 and O26 serogroups in bovine recto-anal swabs

Escherichia coli O157 and O26 shedding patterns in cattle are known to vary widely. To address gaps in the understanding of the underlying factors which impact on shedding dynamics, sensitive and rapid quantitative methods which can be applied in surveillance studies on cattle are required. Current approaches for enumeration of verocytotoxigenic E. coli (VTEC) in cattle faeces are based on direct plating onto selective agars, most probable number (MPN) or real time PCR applied directly to faecal samples, all of which have limitations in terms of the labour involved or their sensitivity. The objective of this study was to develop a sensitive real time quantitative PCR assay, to quantify O157 and O26 in bovine recto-anal junction (RAJ) swabs. The approach was to target serogroup specific genes rfbE and wzx, and to couple a short enrichment, with the use of a standard calibration curve relating real time PCR cycle threshold (Ct) values against the initial concentration of the pathogen in the sample. Following initial experiments in broth culture, a 5h enrichment in modified tryptone soya broth with novobiocin (20mg/l) (mTSBn) was found to be optimal, and a linear correlation between inocula (Log10 1 to 6CFUml−1) and the PCR Ct values for both E. coli O157 (R2=0.99, rsd=0.58) and E. coli O26 (R2=0.99, rsd=0.44) was confirmed. The developed method was then applied to bovine RAJ swab samples (n=153), which were inoculated with E. coli O157 or O26 (Log10 1 to 7CFUswab−1). Calibration curves yielded correlations for E. coli O157 of R2=0.86, rsd=0.72 and for O26 (R2=0.88, rsd=0.69). In conclusion, a sensitive method for detection and enumeration of two significant VTEC serogroups in bovine RAJ samples has been developed and validated, and will support studies on the bovine shedding dynamics of these pathogens in cattle.

The Escherichia coli O157:H7 cattle immunoproteome includes outer membrane protein A (OmpA), a modulator of adherence to bovine rectoanal junction squamous epithelial (RSE) cells.

Building on previous studies, we defined the repertoire of proteins comprising the immunoproteome (IP) of Escherichia coli O157:H7 (O157) cultured in DMEM supplemented with norepinephrine (O157 IP), a β-adrenergic hormone that regulates E. coli O157 gene expression in the gastrointestinal tract, using a variation of a novel proteomics-based platform proteome mining tool for antigen discovery, called "proteomics-based expression library screening" (PELS; Kudva et al., 2006). The E. coli O157 IP (O157-IP) comprised 91 proteins, and included those identified previously using proteomics-based expression library screening, and also proteins comprising DMEM and bovine rumen fluid proteomes. Outer membrane protein A (OmpA), a common component of the above proteomes, and reportedly a contributor to E. coli O157 adherence to cultured HEp-2 epithelial cells, was interestingly found to be a modulator rather than a contributor to E. coli O157 adherence to bovine rectoanal junction squamous epithelial cells. Our results point to a role for yet to be identified members of the O157-IP in E. coli O157 adherence to rectoanal junction squamous epithelial cells, and additionally implicate a possible role for the outer membrane protein A regulator, TdcA, in the expression of such adhesins. Our observations have implications for the development of efficacious vaccines for preventing E. coli O157 colonization of the bovine gastrointestinal tract.