Shiga Toxin-producing Escherichia Coli Shedding Research Articles

Evaluation of the Humoral Response after Immunization with a Chimeric Subunit Vaccine against Shiga Toxin-Producing Escherichia coli in Pregnant Sows and Their Offspring.

Shiga toxin-producing Escherichia coli (STEC) poses a significant public health risk due to its zoonotic potential and association with severe human diseases, such as hemorrhagic colitis and hemolytic uremic syndrome. Ruminants are recognized as primary reservoirs for STEC, but swine also contribute to the epidemiology of this pathogen, highlighting the need for effective prevention strategies across species. Notably, a subgroup of STEC that produces Shiga toxin type 2e (Stx2e) causes edema disease (ED) in newborn piglets, economically affecting pig production. This study evaluates the immunogenicity of a chimeric protein-based vaccine candidate against STEC in pregnant sows and the subsequent transfer of immunity to their offspring. This vaccine candidate, which includes chimeric proteins displaying selected epitopes from the proteins Cah, OmpT, and Hes, was previously proven to be immunogenic in pregnant cows. Our analysis revealed a broad diversity of STEC serotypes within swine populations, with the cah and ompT genes being prevalent, validating them as suitable antigens for vaccine development. Although the hes gene was detected less frequently, the presence of at least one of these three genes in a significant proportion of STEC suggests the potential of this vaccine to target a wide range of strains. The vaccination of pregnant sows led to an increase in specific IgG and IgA antibodies against the chimeric proteins, indicating successful immunization. Additionally, our results demonstrated the effective passive transfer of maternal antibodies to piglets, providing them with immediate, albeit temporary, humoral immunity against STEC. These humoral responses demonstrate the immunogenicity of the vaccine candidate and are preliminary indicators of its potential efficacy. However, further research is needed to conclusively evaluate its impact on STEC colonization and shedding. This study highlights the potential of maternal vaccination to protect piglets from ED and contributes to the development of vaccination strategies to reduce the prevalence of STEC in various animal reservoirs.

Prevalence and Implications of Shiga Toxin-Producing E. coli in Farm and Wild Ruminants

Shiga-toxin-producing Escherichia coli (STEC) is a food-borne pathogen that causes human gastrointestinal infections across the globe, leading to kidney failure or even death in severe cases. E. coli are commensal members of humans and animals' (cattle, bison, and pigs) guts, however, may acquire Shiga-toxin-encoded phages. This acquisition or colonization by STEC may lead to dysbiosis in the intestinal microbial community of the host. Wildlife and livestock animals can be asymptomatically colonized by STEC, leading to pathogen shedding and transmission. Furthermore, there has been a steady uptick in new STEC variants representing various serotypes. These, along with hybrids of other pathogenic E. coli (UPEC and ExPEC), are of serious concern, especially when they possess enhanced antimicrobial resistance, biofilm formation, etc. Recent studies have reported these in the livestock and food industry with minimal focus on wildlife. Disturbed natural habitats and changing climates are increasingly creating wildlife reservoirs of these pathogens, leading to a rise in zoonotic infections. Therefore, this review comprehensively surveyed studies on STEC prevalence in livestock and wildlife hosts. We further present important microbial and environmental factors contributing to STEC spread as well as infections. Finally, we delve into potential strategies for limiting STEC shedding and transmission.

Prevalence and patterns of fecal shedding of Shiga toxin–producing Escherichia coli by cattle at a commercial feedlot in South Africa

AbstractHealthy colonized cattle are the major reservoir of Shiga toxin–producing Escherichia coli (STEC) and play a key role in the entry point of the pathogen into the beef chain. Excretion rates and the concentration of the pathogen in feces influence the epidemiology and transmission of the pathogen within herds and to humans. This study evaluated the prevalence and dynamics of fecal shedding of STEC by cattle in a commercial feedlot in Gauteng, South Africa. An initial cross‐sectional survey was conducted; fecal samples were obtained from 106 randomly selected weaned beef calves on arrival at the feedlot using polymerase chain reaction (PCR) to screen by detecting stx1 and stx2 genes. Subsequently, a longitudinal study was conducted, and 15 STEC‐positive and 11 STEC‐negative cattle were sampled monthly and followed to slaughter. STEC O157 and non‐O157 were enumerated in samples using commercial chromogenic agar. Initial prevalence of STEC shedding was 27% (29/106; 95% CI [19, 37%]). All 26 cattle shed STEC intermittently or continuously during the study period, all except one were super‐shedders (≥4 log10 CFU/g) at one or more samplings, and 19 (73%) were persistent or intermittent super‐shedders. Of the 38 STEC isolates recovered, 15 (39%) were serotypeable, representing 11 non‐O157 serogroups, including O101, O168, O178, and O68. The most frequent virulence combination profile was stx1 + eaeA + ehxA (n = 12; 32%). This study confirms the occurrence and variability of STEC super‐shedding in feedlot cattle and highlights that super‐shedding is not limited to STEC O157. It also shows their public health significance.

Characterizing the Cattle Gut Microbiome in Farms with a High and Low Prevalence of Shiga Toxin Producing Escherichia coli.

Cattle are the main reservoirs of Shiga toxin producing Escherichia coli (STEC), a major foodborne pathogen associated with acute enteric disease and hemolytic–uremic syndrome in humans. A total of 397 beef and dairy cattle from 5 farms were included in this study, of which 660 samples were collected for 16S rRNA gene sequencing. The microbiota of farms with a high-STEC prevalence (HSP) had greater richness compared to those of farms with a low-STEC prevalence (LSP). Longitudinal analyses showed STEC-shedders from LSP farms had higher microbiome diversity; meanwhile, changes in the microbiome composition in HSP farms were independent of the STEC shedding status. Most of the bacterial genera associated with STEC shedding in dairy farms were also correlated with differences in the percentage of forage in diet and risk factors of STEC carriage such as days in milk, number of lactations, and warm temperatures. Identifying factors that alter the gut microbiota and enable STEC colonization in livestock could lead to novel strategies to prevent fecal shedding and the subsequent transmission to humans.

Duration of fecal shedding of Shiga toxin-producing Escherichia coli among children with hemolytic uremic syndrome.

Knowing the duration of fecal shedding of Shiga toxin-producing Escherichia coli (STEC) among patients with hemolytic uremic syndrome would be useful to control disease transmission. 1) To analyze the characteristics of STEC shedding duration. 2) To assess the association with sex, age, need of dialysis, antibiotics, and STEC serotypes. Prospective, observational, longitudinal, and analytical study in the 2013-2019 period. Stool cultures were done upon admission and every 5-7 days until 2 negative results were obtained. Shedding duration was defined as the period from diarrhea onset to the first negative result. STEC was confirmed with polymerase chain reaction detection of stx1, stx2, and rfbO157 genes. The mean (95 % CI) and percentile values of the STEC shedding duration were estimated, and the studied outcome measures were compared using the t test. A total of 43 patients were included. The mean duration of shedding was 10.2 days (95 % CI: 8.92-11.59), range: 3-22 days. After 15 days, 90 % of patients had a negative stool culture. There were no differences in terms of sex (p = 0.419), age (p = 0.937), need of dialysis (p = 0.917), antibiotics (p = 0.147) or serotype (p = 0.231). Fifteen days after the onset of diarrhea, 90 % of patients had a negative stool culture, and all patients had one after 22 days. No association was observed between the duration of shedding and studied outcome measures.

Genetic and Phenotypic Factors Associated with Persistent Shedding of Shiga Toxin-Producing Escherichia coli by Beef Cattle

Shiga toxin-producing Escherichia coli (STEC) is a leading cause of foodborne infections. Cattle are an important STEC reservoir, although little is known about specific pathogen traits that impact persistence in the farm environment. Hence, we sought to evaluate STEC isolates recovered from beef cattle in a single herd in Michigan. To do this, we collected fecal grabs from 26 cattle and resampled 13 of these animals at 3 additional visits over a 3-month period. In all, 66 STEC isolates were recovered for genomics and biofilm quantification using crystal violet assays. The STEC population was diverse, representing seven serotypes, including O157:H7, O26:H11, and O103:H2, which are commonly associated with human infections. Although a core genome analysis of 2,933 genes grouped isolates into clusters based on serogroups, some isolates within each cluster had variable biofilm levels and virulence gene profiles. Most (77.8%; n = 49) isolates harbored stx 2a, while 38 (57.5%) isolates formed strong biofilms. Isolates belonging to the predominant serogroup O6 (n = 36; 54.5%) were more likely to form strong biofilms, persistently colonize multiple cattle, and be acquired over time. A high-quality single nucleotide polymorphism (SNP) analysis of 33 O6 isolates detected between 0 and 13 single nucleotide polymorphism (SNP) differences between strains, indicating that highly similar strain types were persisting in this herd. Similar findings were observed for other persistent serogroups, although key genes were found to differ among strong and weak biofilm producers. Together, these data highlight the diversity and persistent nature of some STEC types in this important food animal reservoir.IMPORTANCE Food animal reservoirs contribute to Shiga toxin-producing Escherichia coli (STEC) evolution via the acquisition of horizontally acquired elements like Shiga toxin bacteriophages that enhance pathogenicity. In cattle, persistent fecal shedding of STEC contributes to contamination of beef and dairy products and to crops being exposed to contaminated water systems. Hence, identifying factors important for STEC persistence is critical. This longitudinal study enhances our understanding of the genetic diversity of STEC types circulating in a cattle herd and identifies genotypic and phenotypic traits associated with persistence. Key findings demonstrate that multiple STEC types readily persist in and are transmitted across cattle in a shared environment. These dynamics also enhance the persistence of virulence genes that can be transferred between bacterial hosts, resulting in the emergence of novel STEC strain types. Understanding how pathogens persist and diversify in reservoirs is important for guiding new preharvest prevention strategies aimed at reducing foodborne transmission to humans.

The Role of Escherichia coli Shiga Toxins in STEC Colonization of Cattle.

Many cattle are persistently colonized with Shiga toxin-producing Escherichia coli (STEC) and represent a major source of human infections with human-pathogenic STEC strains (syn. enterohemorrhagic E. coli (EHEC)). Intervention strategies most effectively protecting humans best aim at the limitation of bovine STEC shedding. Mechanisms enabling STEC to persist in cattle are only partialy understood. Cattle were long believed to resist the detrimental effects of Shiga toxins (Stxs), potent cytotoxins acting as principal virulence factors in the pathogenesis of human EHEC-associated diseases. However, work by different groups, summarized in this review, has provided substantial evidence that different types of target cells for Stxs exist in cattle. Peripheral and intestinal lymphocytes express the Stx receptor globotriaosylceramide (Gb3 syn. CD77) in vitro and in vivo in an activation-dependent fashion with Stx-binding isoforms expressed predominantly at early stages of the activation process. Subpopulations of colonic epithelial cells and macrophage-like cells, residing in the bovine mucosa in proximity to STEC colonies, are also targeted by Stxs. STEC-inoculated calves are depressed in mounting appropriate cellular immune responses which can be overcome by vaccination of the animals against Stxs early in life before encountering STEC. Considering Stx target cells and the resulting effects of Stxs in cattle, which significantly differ from effects implicated in human disease, may open promising opportunities to improve existing yet insufficient measures to limit STEC carriage and shedding by the principal reservoir host.

Shiga toxin-producing Escherichia coli (STEC) shedding in a wild roe deer population

Worldwide infections by Shiga toxin-producing Escherichia coli (STEC) in humans have been reported after consumption of mainly beef, but also deer meat. Not only the consumption of contaminated deer meat represents a risk, but also the transmission of STEC between deer and domestic animals should be considered. Within the framework of a telemetry study of roe deer (Capreolus capreolus) the aim was to analyse the occurrence of STEC. Due to the chance to sample some animals several times it was possible to obtain data on the repeated shedding of STEC in roe deer. In total 124 faeces or rectal swabs of 77 live trapped roe deer were collected. The isolates obtained were characterized for stx subtypes, different virulence genes, the so-called top-five serogroups, phylogenetic groups, PFGE-types and antimicrobial susceptibilities.The majority of roe deer were stx-positive whenever sampled. Twenty-eight animals were sampled more than once and were used to examine the duration of shedding STEC. The time interval of 6 persistently stx-negative tested animals was between 6 and 440d (median 49d, interquartile range (IQR) 17-258d). Ten animals excreted undistinguishable STEC strains in intervals between 4 and 778d (median 42d, IQR 22-79d).Most of the isolates were stx2b-positive, eae-negative and frequently ehlyA-positive. None of the isolates belonged to serogroup O26, O103, O111, O145 and O157, respectively. All isolates were sensitive to the antimicrobial substances tested. Although the duration of each shedding event could not be determined the results indicate long-term excretion of STEC in roe deer. This is an important consideration for the observance of good hygiene practice while field dressing of deer and preparing deer meat.

A case of haemolytic uraemic syndrome (HUS) revealed an outbreak of Shiga toxin-2-producing Escherichia coli O26:H11 infection in a nursery, with long-lasting shedders and person-to-person transmission, Italy 2015.

Purpose. Shiga toxin-producing Escherichia coli (STEC) represents a major issue for public health because of the severity of the associated illnesses, including haemolytic uraemic syndrome (HUS). In 2015, investigation of a case of HUS revealed an outbreak of Shiga toxin-2-producing E. coli O26 : H11 infection in a nursery in Italy. The investigation showed that the infection was transmitted to cases' contacts via person to person.Methods. The case finding was performed by testing for STEC stool samples of the HUS case's contacts within the family and the nursery. STEC O26 isolates were characterized by whole genome sequencing. Confirmed cases were repeatedly tested to monitor the duration of STEC shedding.Results. Eleven STEC O26 cases were identified, including adults and asymptomatic patients. Clinical illness was only observed in children. Strain characterization revealed that a single clone harbouring the stx2a and eae genes and the complete array of STEC-associated virulence genes, belonging to ST(21), was implicated in the outbreak. To reduce bacterial shedding, patients were treated with cefixime following clinical recovery. This antibiotic was well tolerated and did not induce any apparent consequences on patients' health.Conclusions. This study confirms that Stx2-producing E. coli O26 represents an emerging public health problem. The occurrence of outbreaks of infection by Stx2-producing E. coli O26 in nurseries is of particular concern, given the high probability of infection progressing in severity and resulting in secondary cases.

Prevalence and characteristics of Shiga toxin-producing Escherichia coli in finishing pigs: Implications on public health

Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens, which can cause serious illnesses, including hemorrhagic colitis and hemolytic uremic syndrome. To study the epidemiology of STEC in finishing pigs and examine the potential risks they pose for human STEC infections, we conducted a longitudinal cohort study in three finishing sites. Six cohorts of pigs (2 cohorts/site, 20 pigs/cohort) were randomly selected, and fecal samples (n=898) were collected every two weeks through their finishing period. Eighty-two pigs (68.3%) shed STEC at least once, and the proportion of STEC-positive pigs varied across sites (50–97.5%) and cohorts (15–100%). Clinically important serotypes, O157:H7 (stx2c, eae) and O26:H11 (stx1a, eae), were recovered from two pigs at sites C and A, respectively. The most common serotype isolated was O59:H21 (stx2e), which was particularly prevalent in site B as it was recovered from all STEC positive pigs (n=39). Each cohort showed different patterns of STEC shedding, which were associated with the prevalent serotype. The median shedding duration of STEC in pigs was 28days, consistent with our prior study. However, among pigs shedding O59:H21 at least once, pigs in cohort B2 had a significantly longer shedding duration of 42days (P<0.05) compared to other cohorts. Stx2e was the most commonly observed stx variant in finishing pigs (93.9%), in accordance with the previous studies. Stx2e has been reported to be significantly associated with edema disease in pigs, however, the pathogenicity in humans warrants further investigations. Nonetheless, our findings affirm that pigs are an important reservoir for human STEC infections, and that the circulating serotypes in a cohort and site management factors may significantly affect the prevalence of STEC. Molecular characterization of STEC isolates and epidemiological studies to identify risk factors for shedding in pigs are strongly warranted to further address the significance to public health and to develop mitigation strategies.

Effect of severe weather events on the shedding of Shiga toxigenic Escherichia coli in slaughter cattle and phenotype of serogroup O157 isolates.

High-event periods (HEPs) occur sporadically when beef carcasses and meat have episodes of acute contamination with Shiga toxin-producing Escherichia coli (STEC). In this study, severe weather events were investigated as catalysts for HEPs based on PCR and isolate prevalence of seven E. coli serogroups in slaughter cattle feces. Winter ambient temperatures with daily means 10.5oC warmer or 12.3°C colder than seasonal norms (-10.4°C) most altered STEC shedding. Fecal samples yielded increased proportions (P<0.05) of O26 and O157 isolates during winter warm periods, and reduced (P<0.05) O45 isolates during cold periods compared to samplings during seasonal norms. Based on changing PCR prevalence and isolates collected, O157 was the serogroup most responsive to severe weather events. Consequently, O157 isolates (n =219) were evaluated for heat resistance, biofilm-forming potential and virulence gene subtypes. Two isolates had heat-resistant phenotypes with thermal death time at 60°C (D60) >10 min and one also had strong biofilm-forming potential. However, this isolate lacked eae and stx genes. Severe weather can influence STEC shedding, particularly of O157, and could possibly trigger HEPs. However, our data suggest that it is unlikely for isolates to carry virulence genes and possess phenotypes capable of evading post-harvest microbiological interventions.

Nationwide investigation of Shiga toxin-producing Escherichia coli among cattle in Japan revealed the risk factors and potentially virulent subgroups.

A nationwide study of Shiga toxin-producing Escherichia coli (STEC) was performed to determine the prevalence, characteristics and risk factors for fecal shedding of STEC among cattle in Japan. Information on rearing practices was also collected to identify risk factors for fecal shedding of STEC. STEC was isolated from 24·1% of samples (133/551) collected from 59·1% of farms (65/110). Bayesian clustering using the virulence marker profiles of the isolates subdivided the isolates into four genetically distinct groups, two of which corresponded to eae- or saa-positive STEC, which can cause severe disease in human. Both STEC groups exhibited characteristic phylogeny and virulence marker profiles. It is noteworthy that the tellurite resistance gene was not detected in all saa-positive STEC isolates, suggesting that the standard isolation method using tellurite might lead to an underestimation of the prevalence of saa-positive STEC. A multivariate logistic regression model using epidemiological information revealed a significantly (P < 0·01) high odds ratio on STEC fecal shedding in tie-stall housing and a low odds ratio in flat feed box and mechanical ventilation. Information on isolate characteristics of the two major pathotypes and risk factors in rearing practices will facilitate the development of preventative measures for STEC fecal shedding from cattle.

Immune Response in Calves Vaccinated with Type Three Secretion System Antigens and Shiga Toxin 2B Subunit of Escherichia coli O157:H7.

Ruminants are the primary reservoir of Shiga-toxin producing Escherichia coli (STEC) O157:H7 and the main source of infection for humans. The aim of this study was to assess the immunogenic properties of a candidate vaccine consisting on the recombinant proteins of E. coli O157:H7 IntiminC280, the carboxy-terminal fraction of Intimin γ, EspB and the fusion protein between the B subunit of Stx2 and Brucella Lumazine Synthase (BLS)(BLS-Stx2B), in Holstein Fresian calves.To accomplish this goal we vaccinated calves with two doses of different vaccine formulations: 2 antigens (IntiminC280, EspB), 3 antigens (IntiminC280, EspB, BLS-Stx2B), BLS-Stx2B alone and a control non-vaccinated group. All antigens were expressed as recombinant proteins in E. coli. Specific IgG titres increased in vaccinated calves and the inclusion of BLS-Stx2B in the formulation seems to have a stimulatory effect on the humoral response to IntiminC280 and EspB after the booster. The neutralizing activity of antibodies against these two antigens was assessed in Red Blood Cell lysis assays and adherence to Hep-2 cells as a correlate of T3SS activity. Both sera from animals vaccinated with 2 or 3 antigens inhibited both virulence properties. Serological response to Stx2 was observed in animals vaccinated only with BLS-Stx2B and with 3 antigens and neutralization of Stx2 cytotoxicity was also observed in both groups. In conclusion, immunization of calves with BLS-Stx2B, IntiminC280 and EspB elicited a potent humoral response able to neutralize Shiga toxin 2 cytotoxity and the T3SS virulence properties in vitro. These results suggest that this formulation is a good candidate vaccine to reduce STEC shedding in cattle and needs to be further assessed in vivo.

Bovine Leukemia Virus and Mycobacterium avium subsp. paratuberculosis Are Not Associated with Shiga Toxin–Producing Escherichia coli Shedding in Cattle

Bovine leukemia virus (BLV) is a retrovirus that causes enzootic bovine leukosis in cattle, and Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's disease in cattle. Both diseases are chronic in nature and can lead to the disruption of normal immunological or physiological processes. Cattle are the major reservoir of Shiga toxin-producing Escherichia coli (STEC), a cause of foodborne illness in humans. We tested the hypothesis that cattle infected with BLV or MAP are more likely to shed STEC. We conducted a cross-sectional study during the summers of 2011 and 2012 in 11 Michigan cattle herds. A fecal sample from each animal was collected for STEC culture, and multiplex PCR for stx1, stx2, and eaeA was used to screen suspect colonies for STEC confirmation. Antibody detection enzyme-linked immunosorbent assays for BLV and MAP were used to screen serum from each animal. Flow cytometry was used to quantify the percentage of lymphocytes, monocytes, and neutrophils in a subsample (n =497) of blood samples. Of the animals sampled, 34.9% were BLV positive, 2.7% were MAP positive, and 16% were shedding STEC. Cattle in the dairy herds had a higher frequency of BLV and MAP than did those in beef herds, but more cattle in beef herds were shedding STEC. Neither BLV nor MAP was associated with STEC shedding (P values of 0.6838 and 0.3341, respectively). We also observed no association between STEC status and the percentage of neutrophils (P value of 0.3565), lymphocytes (P value of 0.8422), or the lymphocyte-to-monocyte ratio (P value of 0.1800). Although controlling both BLV and MAP is important for overall herd health and productivity, we found no evidence that controlling BLV and MAP has an impact on STEC shedding in cattle.

Factors Associated with Shiga Toxin-Producing Escherichia coli Shedding by Dairy and Beef Cattle.

Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen that can cause hemorrhagic colitis and hemolytic-uremic syndrome. Cattle are the primary reservoir for STEC, and food or water contaminated with cattle feces is the most common source of infections in humans. Consequently, we conducted a cross-sectional study of 1,096 cattle in six dairy herds (n = 718 animals) and five beef herds (n = 378 animals) in the summers of 2011 and 2012 to identify epidemiological factors associated with shedding. Fecal samples were obtained from each animal and cultured for STEC. Multivariate analyses were performed to identify risk factors associated with STEC positivity. The prevalence of STEC was higher in beef cattle (21%) than dairy cattle (13%) (odds ratio [OR], 1.76; 95% confidence interval [CI], 1.25, 2.47), with considerable variation occurring across herds (range, 6% to 54%). Dairy cattle were significantly more likely to shed STEC when the average temperature was >28.9°C 1 to 5 days prior to sampling (OR, 2.5; 95% CI, 1.25, 4.91), during their first lactation (OR, 1.8; 95% CI, 1.1, 2.8), and when they were <30 days in milk (OR, 3.9; 95% CI, 2.1, 7.2). These data suggest that the stress or the negative energy balance associated with lactation may result in increased STEC shedding frequencies in Michigan during the warm summer months. Future prevention strategies aimed at reducing stress during lactation or isolating high-risk animals could be implemented to reduce herd-level shedding levels and avoid transmission of STEC to susceptible animals and people. STEC shedding frequencies vary considerably across cattle herds in Michigan, and the shedding frequency of strains belonging to non-O157 serotypes far exceeds the shedding frequency of O157 strains, which is congruent with human infections in the state. Dairy cattle sampled at higher temperatures, in their first lactation, and early in the milk production stage were significantly more likely to shed STEC, which could be due to stress or a negative energy balance. Future studies should focus on the isolation of high-risk animals to decrease herd shedding levels and the potential for contamination of the food supply.

Colonization of Beef Cattle by Shiga Toxin-Producing Escherichia coli during the First Year of Life: A Cohort Study.

Each year Shiga toxin-producing Escherichia coli (STEC) are responsible for 2.8 million acute illnesses around the world and > 250,000 cases in the US. Lowering the prevalence of this pathogen in animal reservoirs has the potential to reduce STEC outbreaks in humans by controlling its entrance into the food chain. However, factors that modulate the colonization and persistence of STEC in beef cattle remain largely unidentified. This study evaluated if animal physiological factors such as age, breed, sex, and weight gain influenced the shedding of STEC in beef cattle. A cohort of beef calves (n = 260) from a multi-breed beef calf population was sampled every three months after birth to measure prevalence and concentration of STEC during the first year of life. Metagenomic analysis was also used to understand the association between the STEC colonization and the composition of gut microflora. This study identified that beef calves were more likely to shed STEC during the first 6 months and that STEC shedding decreased as the animal matured. Animal breed group, sex of the calf, and average weight gain were not significantly associated with STEC colonization. The metagenomic analysis revealed for the first time that STEC colonization was correlated with a lower diversity of gut microflora, which increases as the cattle matured. Given these findings, intervention strategies that segregate younger animals, more likely to be colonized by STEC from older animals that are ready to be harvested, could be investigated as a method to reduce zoonotic transmission of STEC from cattle to humans.

Use of probiotics to reduce faecal shedding of Shiga toxin-producing Escherichia coli in sheep.

Shiga toxin-producing Escherichia coli (STEC) are zoonotic, foodborne pathogens of humans. Ruminants, including sheep, are the primary reservoirs of STEC and there is a need to develop intervention strategies to reduce the entry of STEC into the food chain. The initiation of the majority of bacterial, enteric infections involves colonisation of the gut mucosal surface by the pathogen. However, probiotic bacteria can serve to decrease the severity of infection via a number of mechanisms including competition for receptors and nutrients, and/or the synthesis of organic acids and bacteriocins that create an environment unfavourable for pathogen development. The aim of the current study was to determine whether the administration of a probiotic mixture to sheep experimentally infected with a non-O157 STEC strain, carrying stx1, stx2 and eae genes, was able to decrease faecal shedding of the pathogen. The probiotic mixture contained Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus bulgaricus, Lactobacillus lactis, Streptococcus thermophilus and Enterococcus faecium. The numbers of non-O157 STEC in faecal samples collected from sheep receiving daily doses of the probiotic mixture were significantly lower at the 3rd, 5th and 6th week post-inoculation when compared to the levels recorded in untreated animals. It was concluded that administration of the probiotic mixture reduced faecal shedding of non-O157 STEC in sheep, and holds potential as a pre-harvest intervention method to reduce transmission to humans.

Isolation and Characterization of Shiga Toxin-Producing Escherichia coli Serogroups O26, O45, O103, O111, O113, O121, O145, and O157 Shed from Range and Feedlot Cattle from Postweaning to Slaughter

Cattle are the main reservoirs for Shiga toxin-producing Escherichia coli (STEC) strains. E. coli O26, O45, O103, O111, O121, O145, and O157 are among the STEC serogroups that cause severe foodborne illness and have been declared as adulterants by the U.S. Department of Agriculture, Food Safety and Inspection Service. The objectives of this study were (i) to estimate the prevalence of non-O157 STEC and E. coli O157 in naturally infected beef cows and in steer calves at postweaning, during finishing, and at slaughter and (ii) to test non-O157 STEC isolates for the presence of virulence genes stx1, stx2, eaeA, and ehlyA. Samples were collected from study animals during multiple sampling periods and included fecal grabs, rectal swabs, and midline sponge samples. Laboratory culture, PCR, and multiplex PCR were performed to recover and identify E. coli and the virulence genes. The prevalence of non-O157 STEC (serogroups O26, O45, O103, O111, O121, O113, and O145) fecal shedding ranged from 8% (4 of48 samples) to 39% (15 of 38 samples) in cows and 2% (1 of 47 samples) to 38% (9 of 24 samples) in steer calves. The prevalence of E. coli O157 fecal shedding ranged from 0% (0 of 38 samples) to 52% (25 of 48 samples) in cows and 2% (1 of 47 samples) to 31% (15 of 48 samples) in steer calves. In steer calves, the prevalence of non-O157 STEC and E. coli O157 was highest at postweaning, at 16% (15 of 96 samples) and 23% (22 of 96 samples), respectively. Among the 208 non-O157 STEC isolates, 79% (164 isolates) had stx1, 79% (165 isolates) had stx2, and 58% (121 isolates) had both stx1 and stx2 genes. The percentage of non-O157 STEC isolates encoding the eaeA gene was low; of the 165 isolates tested, 8 (5%) were positive for eaeA and 135 (82%) were positive for ehlyA. Findings from this study provide further evidence of non-O157 STEC shedding in beef cows and steer calves particularly at the stage of postweaning and before entry into the feedlot.

Aflatoxin, Fumonisin and Shiga Toxin-Producing Escherichia coli Infections in Calves and the Effectiveness of Celmanax®/Dairyman’s Choice™ Applications to Eliminate Morbidity and Mortality Losses

Mycotoxin mixtures are associated with Shiga toxin-producing Escherichia coli (STEC) infections in mature cattle. STEC are considered commensal bacteria in mature cattle suggesting that mycotoxins provide a mechanism that converts this bacterium to an opportunistic pathogen. In this study, we assessed the mycotoxin content of hemorrhaged mucosa in dairy calves during natural disease outbreaks, compared the virulence genes of the STECs, evaluated the effect of the mucosal mycotoxins on STEC toxin expression and evaluated a Celmanax®/Dairyman’s Choice™ application to alleviate disease. As for human infections, the OI-122 encoded nleB gene was common to STEC genotypes eliciting serious disease. Low levels of aflatoxin (1–3 ppb) and fumonisin (50–350 ppb) were detected in the hemorrhaged mucosa. Growth of the STECs with the mycotoxins altered the secreted protein concentration with a corresponding increase in cytotoxicity. Changes in intracellular calcium indicated that the mycotoxins increased enterotoxin and pore-forming toxin activity. A prebiotic/probiotic application eliminated the morbidity and mortality losses associated with the STEC infections. Our study demonstrates: the same STEC disease complex exists for immature and mature cattle; the significance of the OI-122 pathogenicity island to virulence; the significance of mycotoxins to STEC toxin activity; and, finally, provides further evidence that prebiotic/probiotic applications alleviate STEC shedding and mycotoxin/STEC interactions that lead to disease.

Characterization of Shiga toxin – producing Escherichia coli infections in beef feeder calves and the effectiveness of a prebiotic in alleviating Shiga toxin - producing Escherichia coli infections

BackgroundIn the less-sensitive mouse model, Shiga toxin-producing Escherichia coli (STEC) challenges result in shedding that reflect the amount of infection and the expression of virulence factors such as Shiga toxins (Stx). The purpose of this study was to characterize the contribution of STEC diversity and Stx expression to shedding in beef feeder calves and to evaluate the effectiveness of a prebiotic, Celmanax®, to alleviate STEC shedding. Fecal samples were collected from calves at entry and after 35 days in the feedlot in spring and summer. STECs were evaluated using selective media, biochemical profile, serotyping and Stx detection. Statistical analysis was performed using repeated measures ANOVA and logistic regression.ResultsAt entry, non-O157 STEC were dominant in shedding calves. In spring, 21%, 14% and 14% of calves acquired O157, non-O157 and mixed STEC infections, respectively. In contrast, 45%, 48% and 46% of calves in summer acquired O157, non-O157 and mixed STEC infections, respectively. Treatment with a prebiotic, Celmanax®, in spring significantly reduced 50% of the O157 STEC infections, 50% of the non-O157 STEC infections and 36% of the STEC co-infections (P = 0.037). In summer, there was no significant effect of the prebiotic on STEC infections. The amount of shedding at entry was significantly related to the number and type of STECs present and Stx expression (r2 = 0.82). The same relationship was found for shedding at day 35 (r2 = 0.85), but it was also related to the number and type of STECs present at entry. Stx - producing STEC infections resulted in 100 to 1000 × higher shedding in calves compared with Stx-negative STECs.ConclusionsSTEC infections in beef feeder calves reflect the number and type of STECs involved in the infection and STEC expression of Stx. Application of Celmanax® reduced O157 and non-O157 STEC shedding by calves but further research is required to determine appropriate dosages to manage STEC infections.