Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

The liver is the largest solid organ in the body, yet it remains incompletely characterized. Here we present a spatial proteogenomic atlas of the healthy and obese human and murine liver combining single-cell CITE-seq, single-nuclei sequencing, spatial transcriptomics, and spatial proteomics. By integrating these multi-omic datasets, we provide validated strategies to reliably discriminate and localize all hepatic cells, including a population of lipid-associated macrophages (LAMs) at the bile ducts. We then align this atlas across seven species, revealing the conserved program of bona fide Kupffer cells and LAMs. We also uncover the respective spatially resolved cellular niches of these macrophages and the microenvironmental circuits driving their unique transcriptomic identities. We demonstrate that LAMs are induced by local lipid exposure, leading to their induction in steatotic regions of the murine and human liver, while Kupffer cell development crucially depends on their cross-talk with hepatic stellate cells via the evolutionarily conserved ALK1-BMP9/10 axis.

A joint NCBI and EMBL-EBI transcript set for clinical genomics and research.

Comprehensive genome annotation is essential to understand the impact of clinically relevant variants. However, the absence of a standard for clinical reporting and browser display complicates the process of consistent interpretation and reporting. To address these challenges, Ensembl/GENCODE1 and RefSeq2 launched a joint initiative, the Matched Annotation from NCBI and EMBL-EBI (MANE) collaboration, to converge on human gene and transcript annotation and to jointly define a high-value set of transcripts and corresponding proteins. Here, we describe the MANE transcript sets for use as universal standards for variant reporting and browser display. The MANE Select set identifies a representative transcript for each human protein-coding gene, whereas the MANE Plus Clinical set provides additional transcripts at loci where the Select transcripts alone are not sufficient to report all currently known clinical variants. Each MANE transcript represents an exact match between the exonic sequences of an Ensembl/GENCODE transcript and its counterpart in RefSeq such that the identifiers can be used synonymously. We have now released MANE Select transcripts for 97% of human protein-coding genes, including all American College of Medical Genetics and Genomics Secondary Findings list v3.0 (ref. 3) genes. MANE transcripts are accessible from major genome browsers and key resources. Widespread adoption of these transcript sets will increase the consistency of reporting, facilitate the exchange of data regardless of the annotation source and help to streamline clinical interpretation.

Integrated gut metabolome and microbiome fingerprinting reveals that dysbiosis precedes allergic inflammation in IgE-mediated pediatric cow's milk allergy.

BACKGROUND: IgE-mediated cow's milk allergy (IgE-CMA) is one of the first allergies to arise in early childhood and may result from exposure to various milk allergens, of which ß-lactoglobulin (BLG) and casein are the most important. Understanding the underlying mechanisms behind IgE-CMA is imperative for the discovery of novel biomarkers and the design of innovative treatment and prevention strategies. METHODS: We report a longitudinal in vivo murine model, in which two mice strains (BALB/c and C57Bl/6) were sensitized to BLG using either cholera toxin or an oil emulsion (n = 6 per group). After sensitization, mice were challenged orally, their clinical signs monitored, antibody (IgE and IgG1) and cytokine levels (IL-4 and IFN-γ) measured, and fecal samples subjected to metabolomics. The results of the murine models were further extrapolated to fecal microbiome-metabolome data from our population of IgE-CMA (n = 22) and healthy (n = 23) children (Trial: NCT04249973), on which polar metabolomics, lipidomics and 16S rRNA metasequencing were performed. In vitro gastrointestinal digestions and multi-omics corroborated the microbial origin of proposed metabolic changes. RESULTS: During mice sensitization, we observed multiple microbially derived metabolic alterations, most importantly bile acid, energy and tryptophan metabolites, that preceded allergic inflammation. We confirmed microbial dysbiosis, and its associated effect on metabolic alterations in our patient cohort, through in vitro digestions and multi-omics, which was accompanied by metabolic signatures of low-grade inflammation. CONCLUSION: Our results indicate that gut dysbiosis precedes allergic inflammation and nurtures a chronic low-grade inflammation in children on elimination diets, opening important new opportunities for future prevention and treatment strategies.

Randomized trial of bilateral gene therapy injection for m.11778G>A MT-ND4 Leber optic neuropathy.

Leber hereditary optic neuropathy (LHON) is an important example of mitochondrial blindness with the m.11778G>A mutation in the MT-ND4 gene being the most common disease-causing mtDNA variant worldwide. The REFLECT phase 3 pivotal study is a randomized, double-masked, placebo-controlled trial investigating the efficacy and safety of bilateral intravitreal injection of lenadogene nolparvovec in patients with a confirmed m.11778G>A mutation, using a recombinant adeno-associated virus vector 2, serotype 2 (rAAV2/2-ND4). The first-affected eye received gene therapy; the fellow (affected/not-yet-affected) eye was randomly injected with gene therapy or placebo. The primary end point was the difference in change from baseline of best-corrected visual acuity (BCVA) in second-affected/not-yet-affected eyes treated with lenadogene nolparvovec versus placebo at 1.5 years post-treatment, expressed in logarithm of the minimal angle of resolution (LogMAR). Forty-eight patients were treated bilaterally and 50 unilaterally. At 1.5 years, the change from baseline in BCVA was not statistically different between second-affected/not-yet-affected eyes receiving lenadogene nolparvovec and placebo (primary end point). A statistically significant improvement in BCVA was reported from baseline to 1.5 years in lenadogene nolparvovec-treated eyes: -0.23 LogMAR for the first-affected eyes of bilaterally treated patients (P < 0.01); and -0.15 LogMAR for second-affected/not-yet-affected eyes of bilaterally treated patients and the first-affected eyes of unilaterally treated patients (P < 0.05). The mean improvement in BCVA from nadir to 1.5 years was -0.38 (0.052) LogMAR and -0.33 (0.052) LogMAR in first-affected and second-affected/not-yet-affected eyes treated with lenadogene nolparvovec, respectively (bilateral treatment group). A mean improvement of -0.33 (0.051) LogMAR and -0.26 (0.051) LogMAR was observed in first-affected lenadogene nolparvovec-treated eyes and second-affected/not-yet-affected placebo-treated eyes, respectively (unilateral treatment group). The proportion of patients with one or both eyes on-chart at 1.5 years was 85.4% and 72.0% for bilaterally and unilaterally treated patients, respectively. The gene therapy was well tolerated, with no systemic issues. Intraocular inflammation, which was mostly mild and well controlled with topical corticosteroids, occurred in 70.7% of lenadogene nolparvovec-treated eyes versus 10.2% of placebo-treated eyes. Among eyes treated with lenadogene nolparvovec, there was no difference in the incidence of intraocular inflammation between bilaterally and unilaterally treated patients. Overall, the REFLECT trial demonstrated an improvement of BCVA in LHON eyes carrying the m.11778G>A mtDNA mutation treated with lenadogene nolparvovec or placebo to a degree not reported in natural history studies and supports an improved benefit/risk profile for bilateral injections of lenadogene nolparvovec relative to unilateral injections.

Improved reference genome of Aedes aegypti informs arbovirus vector control.

Female Aedes aegypti mosquitoes infect more than 400 million people each year with dangerous viral pathogens including dengue, yellow fever, Zika and chikungunya. Progress in understanding the biology of mosquitoes and developing the tools to fight them has been slowed by the lack of a high-quality genome assembly. Here we combine diverse technologies to produce the markedly improved, fully re-annotated AaegL5 genome assembly, and demonstrate how it accelerates mosquito science. We anchored physical and cytogenetic maps, doubled the number of known chemosensory ionotropic receptors that guide mosquitoes to human hosts and egg-laying sites, provided further insight into the size and composition of the sex-determining M locus, and revealed copy-number variation among glutathione S-transferase genes that are important for insecticide resistance. Using high-resolution quantitative trait locus and population genomic analyses, we mapped new candidates for dengue vector competence and insecticide resistance. AaegL5 will catalyse new biological insights and intervention strategies to fight this deadly disease vector.

Immortalised canine buccal epithelial cells' CXCL8 secretion is affected by allergen extracts, Toll-like receptor ligands, IL-17A and calcitriol.

Epithelial cells are known to produce mediators which can influence the behaviour of neighbouring immune cells. Although the oral mucosa has gained increased interest as a route to induce allergy desensitisation and mucosal pathogen immunisation in dogs, there is only limited knowledge on the factors which impact mediator secretion by canine oral epithelial cells. The study's objective was to enlarge the knowledge on the stimuli that can influence the secretion of some pro- and anti-inflammatory cytokines and the chemokine CXCL8 by canine buccal epithelial cells. To investigate this, buccal epithelial cells were isolated from a biopsy of a dog and immortalised by lentiviral transduction of the SV40 large T antigen. The cells were stained with a CD49f and cytokeratin 3 antibody to confirm their epithelial origin. Cells were incubated with allergen extracts, Toll-like receptor ligands (TLRL), recombinant cytokines and vitamin A and D metabolites. Subsequently, the secretion of the cytokines interleukin (IL)-4, IL-6, IL-10, IL-17A, IFN-γ, TGF-ß1 and the chemokine CXCL8 was assayed by ELISA. Immortalised canine buccal epithelial cells stained positive for CD49f but not for cytokeratin 3. The cells produced detectable amounts of CXCL8 and TGF-ß1. A Dermatophagoides farinae extract, an Alternaria alternata extract, Pam3CSK4, heat-killed Listeria monocytogenes, FSL-1, flagellin and canine recombinant IL-17A significantly increased CXCL8 secretion, while the vitamin D metabolite calcitriol significantly suppressed the production of this chemokine. This study showed that certain allergens, TLRL, IL-17A and calcitriol modulate CXCL8 secretion in a cell line of canine buccal epithelial cells.

Intestinal Epithelial Cells Modulate the Production of Enterotoxins by Porcine Enterotoxigenic E. coli Strains.

Enterotoxigenic Escherichia coli (ETEC) strains are one of the most common etiological agents of diarrhea in both human and farm animals. In addition to encoding toxins that cause diarrhea, ETEC have evolved numerous strategies to interfere with host defenses. These strategies most likely depend on the sensing of host factors, such as molecules secreted by gut epithelial cells. The present study tested whether the exposure of ETEC to factors secreted by polarized IPEC-J2 cells resulted in transcriptional changes of ETEC-derived virulence factors. Following the addition of host-derived epithelial factors, genes encoding enterotoxins, secretion-system-associated proteins, and the key regulatory molecule cyclic AMP (cAMP) receptor protein (CRP) were substantially modulated, suggesting that ETEC recognize and respond to factors produced by gut epithelial cells. To determine whether these factors were heat sensitive, the IEC-conditioned medium was incubated at 56 °C for 30 min. In most ETEC strains, heat treatment of the IEC-conditioned medium resulted in a loss of transcriptional modulation. Taken together, these data suggest that secreted epithelial factors play a role in bacterial pathogenesis by modulating the transcription of genes encoding key ETEC virulence factors. Further research is warranted to identify these secreted epithelial factors and how ETEC sense these molecules to gain a competitive advantage in the early engagement of the gut epithelium.

Lactoferrin, a versatile natural antimicrobial glycoprotein that modulates the host's innate immunity.

Lactoferrin is a multifunctional protein found in the secretions of mammals. The antimicrobial activity of lactoferrin was the first to be discovered and was assumed to be solely dependent on its iron-chelating ability. However, lactoferrin has been reported to display proteolytic activity towards bacterial virulence factors and to modulate the host defence by stimulating the immune system and balancing pathogen-induced inflammation. Here, we review the current understandings of the antimicrobial effect, interaction with host cells, and innate immune modulation of lactoferrin, and put forward this moonlighting protein as a possible alternative for antibiotics.

Tyrosine phosphorylation and proteolytic cleavage of Notch are required for non-canonical Notch/Abl signaling in Drosophila axon guidance.

Notch signaling is required for the development and physiology of nearly every tissue in metazoans. Much of Notch signaling is mediated by transcriptional regulation of downstream target genes, but Notch controls axon patterning in Drosophila by local modulation of Abl tyrosine kinase signaling, via direct interactions with the Abl co-factors Disabled and Trio. Here, we show that Notch-Abl axonal signaling requires both of the proteolytic cleavage events that initiate canonical Notch signaling. We further show that some Notch protein is tyrosine phosphorylated in Drosophila, that this form of the protein is selectively associated with Disabled and Trio, and that relevant tyrosines are essential for Notch-dependent axon patterning but not for canonical Notch-dependent regulation of cell fate. Based on these data, we propose a model for the molecular mechanism by which Notch controls Abl signaling in Drosophila axons.

Porcine small intestinal organoids as a model to explore ETEC-host interactions in the gut.

Small intestinal organoids, or enteroids, represent a valuable model to study host-pathogen interactions at the intestinal epithelial surface. Much research has been done on murine and human enteroids, however only a handful studies evaluated the development of enteroids in other species. Porcine enteroid cultures have been described, but little is known about their functional responses to specific pathogens or their associated virulence factors. Here, we report that porcine enteroids respond in a similar manner as in vivo gut tissues to enterotoxins derived from enterotoxigenic Escherichia coli, an enteric pathogen causing postweaning diarrhoea in piglets. Upon enterotoxin stimulation, these enteroids not only display a dysregulated electrolyte and water balance as shown by their swelling, but also secrete inflammation markers. Porcine enteroids grown as a 2D-monolayer supported the adhesion of an F4+ ETEC strain. Hence, these enteroids closely mimic in vivo intestinal epithelial responses to gut pathogens and are a promising model to study host-pathogen interactions in the pig gut. Insights obtained with this model might accelerate the design of veterinary therapeutics aimed at improving gut health.

Porcine Enterotoxigenic Escherichia coli Strains Differ in Their Capacity To Secrete Enterotoxins through Varying YghG Levels.

Enterotoxigenic Escherichia coli (ETEC) strains are important pathogens for humans and farm animals such as pigs. Porcine ETEC strains induce diarrhea through the production of heat-labile enterotoxin (LT) and/or heat-stable enterotoxins (pSTa/STb). Although LT secretion levels differ between porcine ETEC strains, and this has been linked to virulence, it is unclear whether ST secretion levels also differ between porcine ETEC strains. In addition, the molecular mechanism underlying different LT secretion levels has not been elucidated. In this work, multiple porcine ETEC strains were assessed for their capacity to produce and secrete the enterotoxins LT, pSTa, and STb. The strains differed greatly in their capacity to secrete LT, pSTa, and STb. Remarkably, in some strains, periplasmic production did not correlate with their ability to secrete LT, resulting in high periplasmic production and low LT secretion levels. Furthermore, the results indicated that the type II secretion system (T2SS) protein YghG plays a regulatory role in controlling LT secretion levels. These findings highlight YghG as an important mediator of the secretion of the heat-labile enterotoxin LT by porcine ETEC strains and provide better insights into ETEC enterotoxin secretion.IMPORTANCE Enterotoxigenic E. coli strains are a major health concern. Enterotoxins secreted by enterotoxigenic E. coli are crucial for diarrhea induction. Enterotoxin secretion levels differ between strains; however, it is currently unclear what drives these differences. The discrepancy in the production and secretion capacities of enterotoxins in ETEC is important to clarify their function involved in diarrhea induction. Our results further deepen our understanding of how type II secretion system (T2SS) components of ETEC control enterotoxin secretion levels and may lay the foundation for a better understanding of ETEC molecular pathogenesis.

Porcine and Bovine Forms of Lactoferrin Inhibit Growth of Porcine Enterotoxigenic Escherichia coli and Degrade Its Virulence Factors.

Postweaning diarrhea (PWD) is an economically important, multifactorial disease affecting pigs within the first 2 weeks after weaning. The most common agent associated with PWD is enterotoxigenic Escherichia coli (ETEC). Currently, antibiotics are used to control PWD, and this has most likely contributed to an increased prevalence of antibiotic-resistant strains. This puts pressure on veterinarians and farmers to decrease or even abandon the use of antibiotics, but these measures need to be supported by alternative strategies for controlling these infections. Naturally derived molecules, such as lactoferrin, could be potential candidates due to their antibacterial or immune-modulating activities. Here, we analyzed the ability of bovine lactoferrin (bLF), porcine lactoferrin (pLF), and ovotransferrin (ovoTF) to inhibit ETEC growth, degrade ETEC virulence factors, and inhibit adherence of these pathogens to porcine intestinal epithelial cells. Our results revealed that bLF and pLF, but not ovoTF, inhibit the growth of ETEC. Furthermore, bLF and pLF can degrade several virulence factors produced by ETEC strains, more specifically F4 fimbriae, F18 fimbriae, and flagellin. On the other hand, ovoTF degrades F18 fimbriae and flagellin but not F4 fimbriae. An in vitro adhesion assay showed that bLF, ovoTF, and pLF can decrease the number of bacteria adherent to epithelial cells. Our findings demonstrate that lactoferrin can directly affect porcine ETEC strains, which could allow lactoferrin to serve as an alternative to antimicrobials for the prevention of ETEC infections in piglets.IMPORTANCE Currently, postweaning F4+ and F18+Escherichia coli infections in piglets are controlled by the use of antibiotics and zinc oxide, but the use of these antimicrobial agents most likely contributes to an increase in antibiotic resistance. Our work demonstrates that bovine and porcine lactoferrin can inhibit the growth of porcine enterotoxigenic E. coli strains. In addition, we also show that lactoferrin can reduce the adherence of these strains to small intestinal epithelial cells, even at a concentration that does not inhibit bacterial growth. This research could allow us to develop lactoferrin as an alternative strategy to prevent enterotoxigenic E. coli (ETEC) infections in piglets.

The Intriguing Interaction of Escherichia coli with the Host Environment and Innovative Strategies To Interfere with Colonization: a Summary of the 2019 E. coli and the Mucosal Immune System Meeting.

The third E. coli and the Mucosal Immune System (ECMIS) meeting was held at Ghent University in Belgium from 2 to 5 June 2019. It brought together an international group of scientists interested in mechanisms of colonization, host response, and vaccine development. ECMIS distinguishes itself from related meetings on these enteropathogens by providing a greater emphasis on animal health and disease and covering a broad range of pathotypes, including enterohemorrhagic, enteropathogenic, enterotoxigenic, enteroaggregative, and extraintestinal pathogenic Escherichia coli As it is well established that the genus Shigella represents a subspecies of E. coli, these organisms along with related enteroinvasive E. coli are also included. In addition, Tannerella forsythia, a periodontal pathogen, was presented as an example of a pathogen which uses its surface glycans for mucosal interaction. This review summarizes several highlights from the 2019 meeting and major advances to our understanding of the biology of these pathogens and their impact on the host.

Beta-glucan's varying structure characteristics modulate survival and immune-related genes expression from Vibrio harveyi-infected Artemia franciscana in gnotobiotic conditions.

ß-Glucans have long been used as an immunostimulant in aquaculture. However, the relationship of its structure to its immunomodulatory properties are poorly understood. In this study, the particle size and chemical structure of ß-glucans extracted from wild-type strain of baker's yeast (Saccharomyces cerevisiae) and its null-mutant yeasts Gas1 were characterised. Using Sigma ß-glucan as a reference, the immunomodulatory properties of these polysaccharides in the germ-free Artemia franciscana model system in the presence of Vibrio harveyi bacterial challenge were investigated. The survival of the A. franciscana nauplii, upon challenge with V. harveyi, was significantly higher in all three glucan-treated groups compared to the control. The glucan Gas1 with a lower degree of branching and shorter side chain length had the most prominent V. harveyi-protective effects. The particle size did not affect the nauplii survival when challenged with V. harveyi. Results also showed that the salutary effect of the tested glucans was associated with the upregulation of innate immune genes such as lipopolysaccharide and ß-1,3-glucan-binding protein (lgbp), high mobility group box protein (hmgb), and prophenoloxidase (proPO). Interestingly, the up-regulation of superoxidase dismutase (sod) and glutathione-s-transferase (gst) was only observed in Gas1 treated group, indicating that Gas1 could function to induce higher reactive oxygen species and stronger immunomodulatory function in A. franciscana, and therefore higher survival rate. The expression of heat shock protein 70 (hsp70), peroxinectin (pxn), and down syndrome cell adhesion molecule (dscam) remain unaltered in response to glucan treatment. Taken together, this study provides insights into the structure-function relationship of ß-glucan and the results confirmed that ß-glucan can be an effective immunostimulant in aquaculture, especially the Gas1 glucan.

Global Identification of Small Ubiquitin-related Modifier (SUMO) Substrates Reveals Crosstalk between SUMOylation and Phosphorylation Promotes Cell Migration.

Proteomics studies have revealed that SUMOylation is a widely used post-translational modification (PTM) in eukaryotes. However, how SUMO E1/2/3 complexes use different SUMO isoforms and recognize substrates remains largely unknown. Using a human proteome microarray-based activity screen, we identified over 2500 proteins that undergo SUMO E3-dependent SUMOylation. We next constructed a SUMO isoform- and E3 ligase-dependent enzyme-substrate relationship network. Protein kinases were significantly enriched among SUMOylation substrates, suggesting crosstalk between phosphorylation and SUMOylation. Cell-based analyses of tyrosine kinase, PYK2, revealed that SUMOylation at four lysine residues promoted PYK2 autophosphorylation at tyrosine 402, which in turn enhanced its interaction with SRC and full activation of the SRC-PYK2 complex. SUMOylation on WT but not the 4KR mutant of PYK2 further elevated phosphorylation of the downstream components in the focal adhesion pathway, such as paxillin and Erk1/2, leading to significantly enhanced cell migration during wound healing. These studies illustrate how our SUMO E3 ligase-substrate network can be used to explore crosstalk between SUMOylation and other PTMs in many biological processes.

Consensus coding sequence (CCDS) database: a standardized set of human and mouse protein-coding regions supported by expert curation.

The Consensus Coding Sequence (CCDS) project provides a dataset of protein-coding regions that are identically annotated on the human and mouse reference genome assembly in genome annotations produced independently by NCBI and the Ensembl group at EMBL-EBI. This dataset is the product of an international collaboration that includes NCBI, Ensembl, HUGO Gene Nomenclature Committee, Mouse Genome Informatics and University of California, Santa Cruz. Identically annotated coding regions, which are generated using an automated pipeline and pass multiple quality assurance checks, are assigned a stable and tracked identifier (CCDS ID). Additionally, coordinated manual review by expert curators from the CCDS collaboration helps in maintaining the integrity and high quality of the dataset. The CCDS data are available through an interactive web page (https://www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi) and an FTP site (ftp://ftp.ncbi.nlm.nih.gov/pub/CCDS/). In this paper, we outline the ongoing work, growth and stability of the CCDS dataset and provide updates on new collaboration members and new features added to the CCDS user interface. We also present expert curation scenarios, with specific examples highlighting the importance of an accurate reference genome assembly and the crucial role played by input from the research community.

Molecular Study of Toxoplasma gondii Isolates Originating from Humans and Organic Pigs in Belgium.

Toxoplasma gondii is a worldwide prevalent, zoonotic parasite of major importance for public health, which can infect any warm-blooded animal species, including humans. Humans can get infected by consumption of meat from a chronically infected animal, by ingestion of sporulated oocysts (resulting from the sexual replication in felids), via contaminated water, soil, or vegetables, and by vertical transmission via the placenta. Infection through meat consumption is estimated to be one of the main sources of human toxoplasmosis cases in developed countries, and more specifically pork is considered to be responsible for 41% of foodborne human toxoplasmosis cases in the United States. To better assess the role of pork as a source of T. gondii infection in humans in Belgium, parasites were isolated from pigs to compare with human clinical isolates in a molecular epidemiological study. A positive result was obtained by magnetic capture-quantitative polymerase chain reaction for T. gondii in 14 out of the 92 hearts sampled during 2016 and 2017 from pigs raised in organic farms. From 9 of these 14 samples, parasites were isolated by mouse bioassay, demonstrating the presence of viable T. gondii in animals intended for human consumption. When genotyped and compared with 15 human isolates obtained during 2015 and 2016, a highly related structured population was demonstrated. Overall, these findings demonstrate the presence of infectious T. gondii in pigs intended for human consumption. Therefore, a potential transmission of T. gondii strains from pigs to humans could occur. However, both species could also be infected via a common source of infection such as oocysts. Furthermore, Belgium does not have an official surveillance program for T. gondii in human cases or food-producing animals; as a consequence, the detection of the infection source of a patient is very rare. Overall, this study reinforces the identification of pork as a potential risk for the consumers.

Escherichia coli O157:H7: distribution, molecular characterization, antimicrobial resistance patterns and source of contamination of sheep and goat carcasses at an export abattoir, Mojdo, Ethiopia.

BACKGROUND: Cattle have been identified as a major reservoir of E. coli O157:H7 for human infection; the ecology of the organism in sheep and goats is less understood. This study was carried out to determine prevalence, source of infection, antibiotic resistance and molecular characterization of Escherichia coli O157: H7 isolated from sheep and goat. METHODS: Systematic random sampling was carried out at Modjo export abattoir, Ethiopia, from November 2012 to April 2013 to collect 408 samples from 72 sheep and 32 goats. Samples collected were skin swabs, fecal samples, intestinal mucosal swabs and the inside and outside part of carcasses as well as carcass in contacts such as workers hands, knife, hook and carcass washing water. Then, samples were processed following standard bacteriological procedures. Non-Sorbitol fermenting colonies were tested on latex agglutination test and the positives are subjected to PCR for detection of attaching and effacing genes (eaeA) and shiga toxin producing genes (stx1 and stx2). All E. coli O157:H7 isolates were checked for their susceptibility pattern towards 15 selected antibiotics. RESULTS: E. coli O157:H7 were detected in only 20/408 samples (4.9%). Among these 20 positive samples, 70% (14/20), 25% (5/20) and 5% (1/20) were from sheep, goats and knife samples, respectively. No significant associations were found between carcasses and the assumed sources of contaminations. Of all the 20 isolates virulence genes were found in 10 (50%) of them; 3 (15%) with only the eaeA gene and 7(35%) expressing eaeA and stx2 genes. All the isolates were susceptible to Norfloxacin (NOR) (100%). CONCLUSIONS: The presence of virulence genes shows E. coli O157:H7 is a potential source of human infection in Ethiopia.

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.

Toll-like receptor 5-mediated IL-17C expression in intestinal epithelial cells enhances epithelial host defense against F4+ ETEC infection.

Enterotoxigenic Escherichia coli (ETEC) are an important cause of post-weaning diarrhea (PWD) in piglets. The IL-17 cytokine family is well known to play important roles in the host defense against bacterial infections at the mucosa. Previously, we reported the potential role of IL-17A in clearing an ETEC infection in piglets. IL-17C, another member of the IL-17 family, is highly expressed in the intestinal epithelium, however, its role during an ETEC infection is still unclear. In this study, we demonstrate that F4+ ETEC induce IL-17C mRNA and protein expression in intestinal tissues as well as in porcine intestinal epithelial cells (IPEC-J2). This IL-17C production is largely dependent on TLR5 signaling in IPEC-J2 cells. Both F4+ ETEC infection and exogenous IL-17C increased the expression of antimicrobial peptides and tight junction proteins, such as porcine beta-defensin (pBD)-2, claudin-1, claudin-2 and occludin in IPEC-J2 cells. Taken together, our data demonstrate that TLR5-mediated IL-17C expression in intestinal epithelial cells enhances mucosal host defense responses in a unique autocrine/paracrine manner in the intestinal epithelium against ETEC infection.