Bovine Gastrointestinal Tract Research Articles

Host-Pathogen Interactions during Shiga Toxin-Producing Escherichia coli Adherence and Colonization in the Bovine Gut: A Comprehensive Review.

Shiga toxin-producing Escherichia coli (STEC) is a significant public health threat due to its ability to cause severe gastrointestinal diseases in humans, ranging from diarrhea to life-threatening conditions such as hemorrhagic colitis and hemolytic uremic syndrome (HUS). As the primary reservoir of STEC, cattle play a crucial role in its transmission through contaminated food and water, posing a considerable risk to human health. This comprehensive review explores host-pathogen interactions during STEC colonization of the bovine gut, focusing on the role of gut microbiota in modulating these interactions and influencing disease outcomes. We integrated findings from published transcriptomics, proteomics, and genomics studies to provide a thorough understanding of how STEC adheres to and colonizes the bovine gastrointestinal tract. The insights from this review offer potential avenues for the development of novel preventative and therapeutic strategies aimed at controlling STEC colonization in cattle, thereby reducing the risk of zoonotic transmission.

Immunoexpression of Spexin in Selected Segments of the Bovine (Bos taurus taurus) Gastrointestinal Tract.

In the expansive domain of neuropeptide investigation, spexin (SPX) has emerged as a captivating subject, exerting a significant impact on diverse physiological processes. Initially identified in mice, SPX's distribution transcends various organs, suggesting its potential regulatory roles. Despite extensive research in smaller species, a notable gap exists in our comprehension of SPX in larger mammals, particularly ruminants. Our study meticulously explores the immunolocalization of SPX within the gastrointestinal organs of bovines, with a specific focus on the abomasum, jejunum, and colon. Tissue samples from Holstein-Friesian cattle underwent careful processing, and gene mRNA expression levels, particularly GALR2 and SPX, were assessed. Intriguingly, our findings revealed that GALR2 expression was highest in the jejunum, signifying a potentially critical role in this digestive segment. Immunohistochemistry further unveiled distinct patterns of SPX immunoreactivity in each examined region-abomasum, jejunum, and colon-highlighting nuanced, region-specific responses. Notably, the abomasum and jejunum predominantly exhibited positive immunoreactivity in the submucosal plexus, while the colon, in contrast, demonstrated a higher degree of immunoreactivity in myenteric plexus neurons. Our investigation, grounded in the hypothesis of ubiquitous SPX distribution in ruminants, delves deeper into the intricate role of SPX within the enteric nervous system. This study meticulously explores the spatial distribution of SPX within the myenteric and submucosal plexuses, integral components of the enteric nervous system. These findings significantly enhance our understanding of SPX's potential roles in gastrointestinal regulation in bovines, providing a unique perspective on larger mammals and enriching our comprehension of this intriguing neuropeptide's significance in various physiological processes.

Prolonged survival of venereal Tritrichomonas foetus parasite in the gastrointestinal tract, bovine fecal extract, and water.

Bovine tritrichomonosis is a venereal disease that causes economic losses around the world. Over the last 100 years, the life cycle of the protozoan Tritrichomonas foetus in bovines has only considered venereal transmission, almost exclusively through natural mating from an infected animal to a healthy animal. Here, we provide the first direct evidence that T. foetus can survive the passage through the gastrointestinal tract in bovines. We demonstrated that the parasite can be discharged by feces in orally infected animals and contaminate the cow's reproductive tract. We showed that the parasite is capable of surviving at pH conditions usually found along the bovine gastrointestinal tract, and our findings suggest that the parasites could survive in bovine feces and water for several days, probably through the formation of resistant pseudocysts or cyst-like structures. Taken together, our results demonstrate that T. foetus is more resistant to adverse external conditions than previously considered, which could be relevant for the life cycle of this parasite in bovines. IMPORTANCE Nowadays, the routine herd diagnosis is usually performed exclusively on bulls, as they remain permanently infected, and prevention and control of Tritrichomonas foetus transmission are based on identifying infected animals and culling practices. The existence of other forms of transmission and the possible role of pseudocysts or cyst-like structures as resistant forms requires rethinking the current management and control of this parasitic disease in the future in some livestock regions of the world.

Management of Cutaneous Squamous Cell Carcinoma (Clear Cell Variant) in Bovine - A Case Report

Tumors of the bovine gastrointestinal tract are rare, possibly because of the low age of the sample population (Bertone, 1990). Neoplasia in cattle carries a poor prognosis because of early adenocarcinoma metastasis and the high fatality of lymphosarcoma. Squamous cell carcinoma is the second most rude type of skin cancer, with basal cell carcinoma being the most common (Rinker et al., 2001). Squamous cell carcinoma has been documented in bovine (Musser et al., 1993). Clear-cell squamous cell carcinoma is a rare variant of squamous cell carcinoma, first described by Kuo (1980) as a skin tumor. The exact etiology is unclear, but ultraviolet radiation has been suggested as a possible cause in humans (Lawal et al., 2013). This report documents a case of cutaneous squamous cell carcinoma (clear cell variant) in perianal region of a cow and its successful surgical management.

Diversity and probiotic characterisation of yeast isolates in the bovine gastrointestinal tract.

The use of yeasts as a feed supplement for cattle can promote animal development and performance. However, for the positive results to be consistent, strains with probiotic properties must be selected. The objective of this study was to isolate and identify yeasts present in the bovine feces and evaluate their probiotic potential together with strains previously isolated from the rumen (preliminary study). A total of 193 isolates were studied, including 139 isolates (19 species) from fecal samples from 11 different animals (Bos taurus and Bos indicus) and 54 strains previously isolated from rumen fluid (Bos taurus). The yeast population in the feces ranged from 3.51 to 4.99 log CFU/g, with Candida pararugosa being the most abundant (isolated from the feces of six samples analysed). Isolates were selected that had negative results in the safety tests (hemolytic activity, DNAse, and gelatinase) and had percentages greater than 35 and 70% for hydrophobicity and auto-aggregation, respectively. In addition, selected isolates had percentages greater than 77.7 and 74.7% for coaggregation with pathogenic strains of Escherichia coli and Clostridium perfringens, respectively. The isolates with percentage growth at 39°C greater than 64.6% and viability greater than 96.7% were selected for survival testing under bovine gastrointestinal conditions. After the tests, the seven best isolates were selected, belonging to the species Candida pararugosa (L60, CCMA 928 and CCMA 930) and Pichia kudriavzevii (L97, L100, CCMA904, CCMA 907). The selected isolates were exopolysaccharide producers. Based on the results of the evaluated properties, the seven selected isolates were classified as potential probiotics for cattle.

The Lipidome of the Gastrointestinal Tract in Lactating Holstein Cows

The lipidome is a key determinant of structural and functional characteristics of tissues, contributing to optimal gut function and efficiency of nutrient use in the gastrointestinal tract (GIT). Our objective was to study lipidomic profiles in different sections of the GIT in lactating dairy cows and to link them with biological functions. We studied the lipid species in ruminal papillae and epithelium from duodenum, jejunum, and ileum harvested after slaughter from five lactating Holstein cows. Extracted lipids were identified by LC/MS/MS and analyzed via Lipidsearch, Metaboanalyst 5.0, and lipid ontology (LION). Of 1259 lipid species identified across the GIT, 387, 565, 193, and 86 were neutral lipids, phospholipids, sphingolipids, and derivatized lipids, respectively. Among the 1223 lipid species common to the GIT, a PLS-DA analysis revealed similar profiles for jejunum and ileum and discriminated them from rumen and duodenum. The content of 12 out of 28 lipid classes differed (p < 0.05) among GIT sections. The average fatty acid chain length in lipid species spanned from 9 to 37 carbons, and the average degree of unsaturation ranged from 0 to 6. The term ‘membrane component’ from LION analysis differed markedly between the rumen and the small intestine. Future studies will help better understand what factors (function or cellular component) in a given section of the GIT are related to the different lipid species. This is the first description of the lipidome profiles across sections of the GIT in lactating dairy cows. The unique lipidome profiles uncovered distinct structural and functional properties across the bovine GIT, which may impact the efficiency of nutrient use.

97 Immunology and the gut: The key to Animal Health

Abstract The gut immune system consists of three lines of defense systems: barriers, innate immunity and adaptive or acquired immunity that work together to give ruminant gastrointestinal tract (GIT) protection from disease. The gastrointestinal tract is the largest immune organ of the body. The mucosal barrier- the tight junctions and the “kill zone” along with the gut mucosa and maintaining an “anti-inflammatory” state are essential for “good gut health”. The microbiome- the microorganisms in the GIT, which has more cells than the entire animal’s body, is essential for immune development, immune response, and maximizing ruminant productivity. Homeostasis is an important function of the gut immune response to maintain health. Management of bovine GIT immune system is not a simple process. Stressors along with the intake of feed and hydration affect the microbiome and the intestinal epithelial cells resulting in important immune interactions. Nutraceuticals (i.e., probiotics, prebiotics, hen yolk IgY, essential oils, organic acids) aid in both microbiome stability “homeostasis” and immune function.

Effect of Protracted Free-Choice Chlortetracycline-Medicated Mineral for Anaplasmosis Control on Escherichia coli Chlortetracycline Resistance Profile from Pastured Beef Cattle.

Anaplasmosis is an economically-significant, hemolytic, tick-borne disease of cattle caused by Anaplasma marginale which can cause clinical anemia and death. Current control options are limited, and FDA-approved antimicrobial control options do not have a defined duration of use. A practical and routinely used anaplasmosis control method involves feeding free-choice chlortetracycline (CTC)-medicated mineral to pastured cattle for several months. Constant antimicrobial use poses the risk of expediting the development and dissemination of antimicrobial resistance in off-target commensal bacteria in the bovine gastrointestinal tract. The objective of this study was to determine the CTC-susceptibility of Escherichia coli isolated from anaplasmosis endemic beef cattle herds provided different FDA-approved free-choice CTC-medicated mineral formulations, all intended to provide cattle a dosage of 0.5 to 2.0 mg CTC/lb bodyweight per day. A closed-herd, comprised of Hereford-Angus cows, naturally endemic for anaplasmosis, were grazed in five different pastures with one herd serving as an untreated control group. The other cattle herds were randomly assigned one of four FDA-approved CTC-medicated mineral formulations (700, 5000, 6000, and 8000 g CTC/ton) labeled for “the control of active anaplasmosis” and provided their respective CTC-medicated mineral formulation for five consecutive months. Fecal samples were collected monthly from a subset of cows (n = 6 or 10) per pasture. Fecal samples were cultured for E. coli isolates and the minimal inhibitory concentration of CTC was determined. Baseline CTC-susceptibility of E. coli was variable among all treatment and control groups. The susceptibility of E. coli isolates was significantly different between study herds over the treatment period (p = 0.0037 across time and 0.009 at the final sampling time). The interaction between study herds and treatment period was not significant (p = 0.075).

Comparative genomic analysis of Escherichia coli isolates from cases of bovine clinical mastitis identifies nine specific pathotype marker genes.

Escherichia coli is a major causative agent of environmental bovine mastitis and this disease causes significant economic losses for the dairy industry. There is still debate in the literature as to whether mammary pathogenic E. coli (MPEC) is indeed a unique E. coli pathotype, or whether this infection is merely an opportunistic infection caused by any E. coli isolate being displaced from the bovine gastrointestinal tract to the environment and, then, into the udder. In this study, we conducted a thorough genomic analysis of 113 novel MPEC isolates from clinical mastitis cases and 100 bovine commensal E. coli isolates. A phylogenomic analysis indicated that MPEC and commensal E. coli isolates formed clades based on common sequence types and O antigens, but did not cluster based on mammary pathogenicity. A comparative genomic analysis of MPEC and commensal isolates led to the identification of nine genes that were part of either the core or the soft-core MPEC genome, but were not found in any bovine commensal isolates. These apparent MPEC marker genes were genes involved with nutrient intake and metabolism [adeQ, adenine permease; nifJ, pyruvate-flavodoxin oxidoreductase; and yhjX, putative major facilitator superfamily (MFS)-type transporter], included fitness and virulence factors commonly seen in uropathogenic E. coli (pqqL, zinc metallopeptidase, and fdeC, intimin-like adhesin, respectively), and putative proteins [yfiE, uncharacterized helix-turn-helix-type transcriptional activator; ygjI, putative inner membrane transporter; and ygjJ, putative periplasmic protein]. Further characterization of these highly conserved MPEC genes may be critical to understanding the pathobiology of MPEC.

Characterization of Listeria monocytogenes isolates from lactating dairy cows in a Wisconsin farm: Antibiotic resistance, mammalian cell infection, and effects on the fecal microbiota

Listeria monocytogenes is an invasive foodborne pathogen that is ubiquitously present in the dairy farm environment. Although cattle are a reservoir of L. monocytogenes, most adult animals do not exhibit clinical symptoms, suggesting a homeostasis between this pathogen and the bovine gastrointestinal ecosystem. Nevertheless, substantial prevalence of L. monocytogenes fecal shedding by dairy cattle has been reported in many studies, posing threats of transmission within the herd and contamination of the human food supply. Accordingly, understanding the L. monocytogenes ecology within the bovine gastrointestinal tract is important to prevent clinical illness in the animal host, reduce transmission, and guide intervention strategies. In this study, we conducted a longitudinal sampling of fecal samples from 20 lactating dairy cows in one Wisconsin farm over a 29-d period and found a strikingly high incidence of L. monocytogenes shedding, in 90% of sampled animals. The L. monocytogenes isolates were genetically diverse, representing all common serotypes previously identified from cattle. Additionally, most tested isolates were resistant to ampicillin, and a few were also resistant to gentamicin or trimethoprim/sulfamethoxazole. Most isolates effectively infected human epithelial cells (Caco-2) and murine fibroblasts (L2), suggesting that they are all capable of causing systemic infection if the intestinal barrier is breached. Finally, we investigated the effects of L. monocytogenes colonization on the gastrointestinal tract microbiota by analyzing the fecal bacterial communities of some shedding and nonshedding cows. Whereas L. monocytogenes did not affect the α and β diversity of tested animals, a subset of shedding cows exhibited different abundances of certain operational taxonomic units within the Bacteroidetes and Firmicutes phyla compared with nonshedding cows. Overall, our findings highlight the threat of antibiotic resistance among some L. monocytogenes isolates, emphasize the need for a strain-specific approach in listeriosis treatment, and suggest the potential negative influence of subclinical L. monocytogenes carriage on animal gut health.

Arthrobotrys cladodes and Pochonia chlamydosporia: Nematicidal effects of single and combined use after passage through cattle gastrointestinal tract

Nematodiosis are responsible for financial losses in cattle production systems due to treatment costs, falling production rates and animal deaths. The incorporation of sodium alginate pellets containing nematophagous fungi in the bovine diet is a method for the control of nematodiosis. The aims of this study were to evaluate the viability of Arthrobotrys cladodes and Pochonia chlamydosporia contained in sodium alginate pellets after passage through the bovine gastrointestinal tract, as well as to evaluate the effects of single and combined use of these fungi against bovine parasitic nematodes. The reduction in parasitic nematode infective larvae of bovines by the combined use of Arthrobotrys cladodes and Pochonia chlamydosporia was more than the reduction in infective larvae by Arthrobotrys cladodes or Pochonia chlamydosporia alone in the collections 12, 24 and 36 h after giving the pellets to the animals. Arthrobotrys cladodes and Pochonia chlamydosporia combined promoted maximum reduction of infective larvae of 86.3%. Therefore, the combination of Arthrobotrys cladodes and Pochonia chlamydosporia may be an effective method to control gastrointestinal nematodiosis affecting grazing cattle.

Potential Role of SLC5A8 Expression in the Etiology of Subacute Ruminal Acidosis.

Rumen fluid of cows with subacute ruminal acidosis (SARA) has high concentrations of short chain fatty acids (SCFA). However, the mechanism of SCFA accumulation is unknown. The solute-linked carrier 5a8 (SLC5A8) plays a key role in the transportation and absorption of SCFA in the intestinal epithelium. The objective of the current study was to investigate (1) SLC5A8 gene and protein expression in various parts of the bovine gastrointestinal tract, (2) the effect of SCFA on SLC5A8 expression in rumen epithelial cells, and (3) SLC5A8 gene and protein expression in SARA and healthy cows. A total of 10 dairy cows, 84 ± 26 days in milk and in their second to fourth parity were allocated to control (n = 5) and SARA groups (n = 5). Three cows from the control group and three calves (1-day-old, female, 45–50 kg, healthy, fasting) were chosen to collect a total of 10 sections of digestive tract, from rumen to rectum, and then bovine ruminal epithelial cells were isolated from the three calves. Gene and protein expression of SLC5A8 was detected in all tested regions of the gastrointestinal tract in calves and adult cows by Western blot and quantitative real-time PCR and were both highest in the rumen. Gene and protein expression of SLC5A8 was more than 50% lower in the rumen epithelium of SARA vs. control cows and was partly restored after therapy of SARA cows. Compared with SCFA concentrations typical for control cows (60 mM acetate, 30 mM propionate, and 20 mM butyrate), gene and protein expression of SLC5A8 in rumen epithelium was lower at elevated SCFA concentrations typical for SARA cows (90 mM acetate, 40 mM propionate, and 30 mM butyrate), specifically for elevated concentrations of propionate or butyrate in contrast to elevated concentrations of acetate increased gene and protein expression of SLC5A8 in rumen epithelium. In conclusion, the elevated concentrations of propionate and butyrate inhibit ruminal absorption of SCFA via downregulation of SLC5A8 in SARA cows; the expression of SLC5A8 plays an important role in the etiology of SARA.

Anti-proliferative effect of pomegranate peel extracts on bovine peripheral blood mononuclear cells (PBMCs)

Pomegranate peel extracts prepared in our laboratories from a waste of juice fruit processing were tested on bovine peripheral blood mononuclear cells to evaluate the effects on viability, oxidative stress and proliferation. The (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay pointed out that the extracts were not cytotoxic at the tested concentrations (0.1, 1.0, and 10 µg/mL). A moderate protective effect against Reactive Oxygen Species production induced by hydrogen peroxide or lipopolysaccharide and a significant anti-proliferative activity against proliferation induced by concanavalin A were observed on cell lines treated with the extracts at 10 μg/mL. Based on these results, pomegranate peel extracts seem promising as feed supplement for dairy cattle, in particular around calving, when the animals are subjected to an increase of the metabolic activity, responsible for oxidative stress and diseases. However, in vivo studies are needed to investigate the stability of the extracts across the bovine gastrointestinal tract barrier.

A meta-analysis of the bovine gastrointestinal tract microbiota.

The bovine gastrointestinal (GI) tract microbiota has important influences on animal health and production. Presently, a large number of studies have used high-throughput sequencing of the archaeal and bacteria 16S rRNA gene to characterize these microbiota under various experimental parameters. By aggregating publically available archaeal and bacterial 16S rRNA gene datasets from 52 studies we were able to determine taxa that are common to nearly all microbiota samples from the bovine GI tract as well as taxa that are strongly linked to either the rumen or feces. The methanogenic genera Methanobrevibacter and Methanosphaera were identified in nearly all fecal and rumen samples (> 99.1%), as were the bacterial genera Prevotella and Ruminococcus (≥ 92.9%). Bacterial genera such as Alistipes, Bacteroides, Clostridium, Faecalibacterium and Escherichia/Shigella were associated with feces and Fibrobacter, Prevotella, Ruminococcus and Succiniclasticum with the rumen. As expected, individual study strongly affected the bacterial community structure, however, fecal and rumen samples did appear separated from each other. This meta-analysis provides the first comparison of high-throughput sequencing 16S rRNA gene datasets generated from the bovine GI tract by multiple studies and may serve as a foundation for improving future microbial community research with cattle.

Plant-Produced Chimeric VHH-sIgA Against Enterohemorrhagic E. coli Intimin Shows Cross-Serotype Inhibition of Bacterial Adhesion to Epithelial Cells.

Enterohemorrhagic Escherichia coli (EHEC) has consistently been one of the foremost foodborne pathogen threats worldwide based on the past 30 years of surveillance. EHEC primarily colonizes the bovine gastrointestinal (GI) tract from which it can be transmitted to nearby farm environments and remain viable for months. There is an urgent need for effective and easily implemented pre-harvest interventions to curtail EHEC contamination of the food and water supply. In an effort to address this problem, we isolated single-domain antibodies (VHHs) specific for intimin, an EHEC adhesin required for colonization, and designed chimeric VHH fusions with secretory IgA functionality intended for passive immunotherapy at the mucosal GI surface. The antibodies were produced in leaves of Nicotiana benthamiana with production levels ranging between 1 and 3% of total soluble protein. in vivo assembly of all subunits into a hetero-multimeric complex was verified by co-immunoprecipitation. Analysis of multivalent protection across the most prevalent EHEC strains identified one candidate antibody, VHH10-IgA, that binds O145:Hnm, O111:Hnm, O26:H11, and O157:H7. Fluorometric and microscopic analysis also indicated that VHH10-IgA completely neutralizes the capacity of the latter three strains to adhere to epithelial cells in vitro. This study provides proof of concept that a plant-produced chimeric secretory IgA can confer cross-serotype inhibition of bacterial adhesion to epithelial cells.

Transcriptomic analysis reveals specific metabolic pathways of enterohemorrhagic Escherichia coli O157:H7 in bovine digestive contents

BackgroundThe cattle gastrointestinal tract (GIT) is the main enterohemorrhagic Escherichia coli (EHEC) reservoir. In order to identify nutrients required for the survival or multiplication of EHEC in the bovine GIT, we compared the transcriptomes of the EHEC O157:H7 reference strain EDL933 cultured in vitro in bovine digestive contents (DCs) (rumen, small intestine and rectum) using RNA-sequencing.ResultsGene expression profiles showed that EHEC EDL933 activated common but also specific metabolic pathways to survive in the different bovine DCs. Mucus-derived carbohydrates seem important in EHEC nutrition in posterior DCs (small intestine and rectum) but not in rumen content. Additional carbohydrates (xylose, ribose, mannitol, galactitol) as well as gluconeogenic substrates (aspartate, serine, glycerol) would also be used by EHEC as carbon and/or nitrogen sources all along the bovine GIT including the rumen. However, xylose, GalNac, ribose and fucose transport and/or assimilation encoding genes were over-expressed during incubation in rectum content compared with rumen and intestine contents, and genes coding for maltose transport were only induced in rectum. This suggests a role for these carbohydrates in the colonization of the cattle rectum, considered as the major site for EHEC multiplication. In contrast, the transcription of the genes associated with the assimilation of ethanolamine, an important nitrogen source for EHEC, was poorly induced in EHEC growing in rectum content, suggesting that ethanolamine is mainly assimilated in the cattle rumen and small intestine. Respiratory flexibility would also be required for EHEC survival because of the redundancy of dehydrogenases and reductases simultaneously induced in the bovine DCs, probably in response to the availability of electron donors and acceptors.ConclusionEHEC EDL933 showed a high flexibility in the activation of genes involved in respiratory pathways and assimilation of carbon and nitrogen sources, most of them from animal origin. This may allow the bacterium to adapt and survive in the various bovine GIT compartments. Obtaining a better understanding of EHEC physiology in bovine GIT is a key step to ultimately propose strategies to limit EHEC carriage and shedding by cattle.

Aspartate metabolism is involved in the maintenance of enterohaemorrhagic Escherichia coli O157:H7 in bovine intestinal content

The gastrointestinal tract (GIT) of healthy cattle is the main reservoir of enterohaemorrhagic Escherichia coli (EHEC). Therefore, it is crucial to better understand the physiology of EHEC in the bovine GIT. In this study, we demonstrate that aspartate present in bovine small intestine content (BSIC), was exhausted after incubation of the reference EHEC strain EDL933 but was poorly assimilated by the endogenous microbiota. Furthermore, the bovine commensal E. coli strain BG1 appeared less efficient than EDL933 in aspartate assimilation suggesting a competitive ability of EHEC to assimilate this amino acid. Our results strongly suggest that aspartate, internalized via the DcuA aspartate: succinate antiporting system, is then converted to fumarate and carbamoyl-aspartate, the precursor for UMP biosynthesis. Aspartate assimilation by these two pathways conferred a competitive growth advantage to EHEC in BSIC. In summary, supply of intracellular fumarate due to aspartate deamination and used as an electron acceptor for anaerobic fumarate respiration, as well as de novo synthesis of pyrimidine from aspartate appear to be important pathways favouring EHEC persistence in the bovine gut. Aspartate probably represents an ecological niche for EHEC in the bovine small intestine.

Factors Involved in the Persistence of a Shiga Toxin-Producing Escherichia coli O157:H7 Strain in Bovine Feces and Gastro-Intestinal Content

Healthy cattle are the primary reservoir for O157:H7 Shiga toxin-producing E. coli responsible for human food-borne infections. Because farm environment acts as a source of cattle contamination, it is important to better understand the factors controlling the persistence of E. coli O157:H7 outside the bovine gut. The E. coli O157:H7 strain MC2, identified as a persistent strain in French farms, possessed the characteristics required to cause human infections and genetic markers associated with clinical O157:H7 isolates. Therefore, the capacity of E. coli MC2 to survive during its transit through the bovine gastro-intestinal tract (GIT) and to respond to stresses potentially encountered in extra-intestinal environments was analyzed. E. coli MC2 survived in rumen fluids, grew in the content of posterior digestive compartments and survived in bovine feces at 15°C predicting a successful transit of the bacteria along the bovine GIT and its persistence outside the bovine intestine. E. coli MC2 possessed the genetic information encoding 14 adherence systems including adhesins with properties related to colonization of the bovine intestine (F9 fimbriae, EhaA and EspP autotransporters, HCP pilus, FdeC adhesin) reflecting the capacity of the bacteria to colonize different segments of the bovine GIT. E. coli MC2 was also a strong biofilm producer when incubated in fecal samples at low temperature and had a greater ability to form biofilms than the bovine commensal E. coli strain BG1. Furthermore, in contrast to BG1, E. coli MC2 responded to temperature stresses by inducing the genes cspA and htrA during its survival in bovine feces at 15°C. E. coli MC2 also activated genes that are part of the GhoT/GhoS, HicA/HicB and EcnB/EcnA toxin/antitoxin systems involved in the response of E. coli to nutrient starvation and chemical stresses. In summary, the large number of colonization factors known to bind to intestinal epithelium and to biotic or abiotic surfaces, the capacity to produce biofilms and to activate stress fitness genes in bovine feces could explain the persistence of E. coli MC2 in the farm environment.

Lactobacillus reuteri suppresses E. coli O157:H7 in bovine ruminal fluid: Toward a pre-slaughter strategy to improve food safety?

The bovine gastrointestinal tract (GIT) is the main reservoir for enterohaemorrhagic Escherichia coli (EHEC) responsible for food-borne infections. Therefore, it is crucial to develop strategies, such as EHEC suppression by antagonistic microorganisms, to reduce EHEC survival in the GIT of cattle and to limit shedding and food contamination. Most human-derived Lactobacillus reuteri strains produce hydroxypropionaldehyde (HPA), an antimicrobial compound, during anaerobic reduction of glycerol. The capacity of L. reuteri LB1-7, a strain isolated from raw bovine milk, to produce HPA and its antimicrobial activity against an O157:H7 EHEC strain (FCH6) were evaluated in bovine rumen fluid (RF) under strict anaerobiosis. EHEC was totally suppressed when incubated in RF inoculated with L. reuteri LB1-7 and supplemented with 80 mM glycerol (RF-Glyc80). The addition of LB1-7 or glycerol alone did not modify EHEC survival in RF. Glycerol was converted to HPA (up to 14 mM) by LB1-7 during incubation in RF-Glyc80, and HPA production appeared to be responsible for EHEC suppression. The bactericidal activity of L. reuteri LB1-7, the concentration of glycerol required and the level of HPA produced depended on physiological and ecological environments. In vitro experiments also showed that EHEC inoculated in rumen fluid and exposed to L. reuteri and glycerol had a very limited growth in rectal contents. However, L. reuteri exerted an antimicrobial activity against the rumen endogenous microbiota and perturbed feedstuff degradation in the presence of glycerol. The potential administration of L. reuteri and glycerol in view of application to finishing beef cattle at the time of slaughter is discussed. Further in vivo studies will be important to confirm the efficiency of L. reuteri and glycerol supplementation against EHEC shedding in ruminants.

No evidence for a bovine mastitis Escherichia coli pathotype

BackgroundEscherichia coli bovine mastitis is a disease of significant economic importance in the dairy industry. Molecular characterization of mastitis-associated E. coli (MAEC) did not result in the identification of common traits. Nevertheless, a mammary pathogenic E. coli (MPEC) pathotype has been proposed suggesting virulence traits that differentiate MAEC from commensal E. coli. The present study was designed to investigate the MPEC pathotype hypothesis by comparing the genomes of MAEC and commensal bovine E. coli.ResultsWe sequenced the genomes of eight E. coli isolated from bovine mastitis cases and six fecal commensal isolates from udder-healthy cows. We analyzed the phylogenetic history of bovine E. coli genomes by supplementing this strain panel with eleven bovine-associated E. coli from public databases. The majority of the isolates originate from phylogroups A and B1, but neither MAEC nor commensal strains could be unambiguously distinguished by phylogenetic lineage. The gene content of both MAEC and commensal strains is highly diverse and dominated by their phylogenetic background. Although individual strains carry some typical E. coli virulence-associated genes, no traits important for pathogenicity could be specifically attributed to MAEC. Instead, both commensal strains and MAEC have very few gene families enriched in either pathotype. Only the aerobactin siderophore gene cluster was enriched in commensal E. coli within our strain panel.ConclusionsThis is the first characterization of a phylogenetically diverse strain panel including several MAEC and commensal isolates. With our comparative genomics approach we could not confirm previous studies that argue for a positive selection of specific traits enabling MAEC to elicit bovine mastitis. Instead, MAEC are facultative and opportunistic pathogens recruited from the highly diverse bovine gastrointestinal microbiota. Virulence-associated genes implicated in mastitis are a by-product of commensalism with the primary function to enhance fitness in the bovine gastrointestinal tract. Therefore, we put the definition of the MPEC pathotype into question and suggest to designate corresponding isolates as MAEC.