Shiga toxin 2eB-transgenic lettuce vaccine: N-glycosylation is important for protecting against porcine edema disease

Porcine edema disease (ED) is a life-threatening toxemia caused by enteric infection with Shiga toxin 2e (Stx2e)-producing Escherichia coli (STEC) in weaned piglets. We previously reported that the stx2eB-transgenic lettuce 2BH strain shows potential for use as an oral vaccine candidate against ED. However, the 2BH strain expressed a hemagglutinin (HA)-tag together with Stx2eB and contained non-canonical N-glycosylation. Therefore, we developed two Stx2eB-lettuce strains, the 3 (G+) strain in which the HA-tag was removed from 2BH, and the 3 (G-) lettuce strain, in which the 73rd Asn was replaced with Ser to prevent non-canonical N-glycosylation of Stx2eB from the 3 (G+) strain. We examined the protective effect of these newly developed two strains compared with the previous 2BH strain against ED using a colostrum-deprived piglet STEC infection model. We found that the N-glycosylated 2BH and 3 (G+) strains relieved the pathogenic symptoms of ED in STEC-challenged piglets, whereas the non-glycosylated 3 (G-) strain did not. N-Glycosylation of the Stx2eB product in lettuce may be involved in the immune response in piglets.     

Improved porcine model for Shiga toxin‐producing Escherichia coli infection by deprivation of colostrum feeding in newborn piglets

Porcine edema disease (ED) is a toxemia caused by enteric infection with Shiga toxin 2e (Stx2e)‐producing Escherichia coli (STEC). ED occurs most frequently during the weaning period and is manifested as emaciation associated with high mortality. In our experimental infection with a specific STEC strain, we failed to cause the suppression of weight gain in piglets, which is a typical symptom of ED, in two consecutive experiments. Therefore, we examined the effects of deprivation of colostrum on the sensitivity of newborn piglets to STEC infection. Neonatal pigs were categorized into two groups: one fed artificial milk instead of colostrum in the first 24 h after birth and then returned to the care of their mother, the other breastfed by a surrogate mother until weaning. The oral challenge with 10¹¹ colony‐forming units of virulent STEC strain on days 25, 26 and 27 caused suppression of weight gain and other ED symptoms in both groups, suggesting that colostrum deprivation from piglets was effective in enhancing susceptibility to STEC. Two successive STEC infection experiments using colostrum‐deprived piglets reproduced this result, leading us to conclude that this improved ED piglet model is more sensitive to STEC infection than the previously established models.     

The preventive effect of Bacillus subtilus strain DB9011 against experimental infection with enterotoxcemic Escherichia coli in weaning piglets

Porcine edema disease (ED) is caused by Shiga toxin 2e‐producing Escherichia coli (STEC). Post‐weaned piglets often suffer from ED as a result of intestinal infection with STEC, which causes impaired growth performance and high mortality. Antimicrobial therapy is a curative treatment for piglets infected with STEC, but the emergence of antimicrobial‐resistant STEC has become a serious problem for Japanese pig farmers. Therefore, an alternative strategy other than antimicrobial therapy is needed for the prevention or treatment of ED. In this study, we evaluated the effect of oral administration of Bacillus subtilis DB9011 (DB9011) to prevent the experimental infection of STEC in weaning piglets. Eight 21‐day‐old piglets were divided into two groups: STEC challenge with the basal diet, and STEC challenge with DB9011 supplemented diet. The challenge was carried out when the animals were 25, 26 and 27 days old using STEC contained in capsules resistant against gastric digestion. All pigs were euthanized at 36 days of age. DB9011 improved the symptoms of ED and decreased the number of STEC in the ileal digesta and feces. Accordingly, oral administration of DB9011 in weaned piglets prevents ED through the suppression of the growth of STEC in the ileum.     

Comparable stx2a expression and phage production levels between Shiga toxin‐producing Escherichia coli strains from human and bovine origin

Shiga toxin‐producing Escherichia coli (STEC) can cause diarrhoea and severe diseases in humans, such as haemolytic uraemic syndrome. STEC virulence is considered to correlate with the amount of Shiga toxins (Stx) produced, especially Stx2, whose subtype Stx2a is most frequently associated with high virulence. Stx are encoded in prophages, which play an important role in STEC pathogenesis. The aim of this study was to evaluate stx_2a expression levels and Stx2a phage production using qPCR and the double‐agar‐layer method in 29 STEC strains, corresponding to serotypes O26:H11 (6), O91:H21 (1), O145:H‐ (11) and O157:H7 (11), isolated from cattle and humans. Results were then tested for possible associations with serotype, origin or some genetic features. We observed heterogeneous levels of stx_2a expression and Stx2a phage production. However, statistical comparisons identified a higher stx_2a expression in response to mitomycin C in strains isolated from cattle than in those from humans. At the same time, compared to stx_2a/stx_2c strains, stx_2a strains showed a higher increase in phage production under induced conditions. Notably, most of the strains studied, regardless of serotype and origin, carried inducible Stx2a phages and evidenced expression of stx_2a that increased along with phage production levels under induced conditions.     

Multiple‐Aetiology Enteric Infections Involving Non‐O157 Shiga Toxin‐Producing Escherichia coli – FoodNet, 2001–2010

We describe multiple‐aetiology infections involving non‐O157 Shiga toxin‐producing Escherichia coli (STEC) identified through laboratory‐based surveillance in nine FoodNet sites from 2001 to 2010. A multiple‐aetiology infection (MEI) was defined as isolation of non‐O157 STEC and laboratory evidence of any of the other nine pathogens under surveillance or isolation of >1 non‐O157 STEC serogroup from the same person within a 7‐day period. We compared exposures of patients with MEI during 2001–2010 with those of patients with single‐aetiology non‐O157 STEC infections (SEI) during 2008–2009 and with those of the FoodNet population from a survey conducted during 2006–2007. In total, 1870 non‐O157 STEC infections were reported; 68 (3.6%) were MEI; 60 included pathogens other than non‐O157 STEC; and eight involved >1 serogroup of non‐O157 STEC. Of the 68 MEI, 21 (31%) were part of six outbreaks. STEC O111 was isolated in 44% of all MEI. Of patients with MEI, 50% had contact with farm animals compared with 29% (P < 0.01) of persons with SEI; this difference was driven by infections involving STEC O111. More patients with non‐outbreak‐associated MEI reported drinking well water (62%) than respondents in a population survey (19%) (P < 0.01). Drinking well water and having contact with animals may be important exposures for MEI, especially those involving STEC O111.     

The immunogenicity of fusion protein linking the carboxyl terminus of the B subunit of Shiga toxin 2 to the B subunit of E. coli heat-labile enterotoxin

To augment the immunogenicity of the subunit B of Shiga toxin (Stx2e B) produced by Escherichia coli and protect piglets from edema disease in china, a fusion gene was constructed consisting of Stx2e B genetically linked at the N-terminus of the B subunit of heat-labile enterotoxin (LTB) in a translational fusion. After being induced with IPTG, the expressed fusion protein of Stx2e B-LTB was about 8.8% of total proteins, approximately 13 microg/ml of the bacteria culture. The Stx2e B-LTB fusion protein was found to be nontoxic to Vero cells at the dose higher than 1 microg/ml and to mice less than 100 microg/ml. Antibody titer against the fusion protein Stx2e B-LTB was 1:76,800, much higher than that of the recombinant Stx2e B protein (1:12,800) alone. All of the mice immunized with the Stx2e B-LTB fusion protein survived when challenged with a lethal dose (LD) of Stx2e toxin. The results showed that the poor immunogenicity of Stx2e B was overcome by conjugating the stx2e B to ltB. The immunogenicity of the constructed fusion protein Stx2e B-LTB in the present study was highly qualified to protect animals against Shiga toxin produced from Shiga toxin-producing Escherichia coli (STEC). The fusion protein of Stx2e B-LTB could be a candidate for a vaccine against edema disease and post-weaning diarrhea simultaneously in piglets.     

Shiga/verocytotoxins and Shiga/verotoxigenic Escherichia coli in animals.

Vero/Shiga toxins (VT/Stx) have an A-B structure: the A subunit carries the enzymatic activity and the B subunit binds the toxin to the membrane receptor (Gb3 or Gb4). The VT/Stx inhibit protein synthesis in the target eucaryotic cells, mainly the endothelial cells of blood vessels. The VT/Stx are subdivided into two families. VT1/Stx1 is a homogeneous family of toxins identical to the Stx of Shigella dysenteriae. VT2/Stx2 is a more heterogeneous family of toxins more distantly related to this Stx toxin. The VT2/Stx2 variants can be distinguished by the polymerase chain reaction (PCR) and/or the reaction with monoclonal antibodies. The VT/Stx-producing Escherichia coli are also subdivided into two main groups on the basis of the presence or absence of additional properties: the enterohaemorrhagic E. coli (EHEC) induce the formation of attaching/effacing lesions and carry a 60 MD plasmid encoding a specific haemolysin (the enterohaemolysin); the vero/shiga-toxigenic E. coli (VTEC/STEC) do not show these properties. The EHEC are isolated from humans and ruminants, especially young calves. They are associated with haemorrhagic enterocolitis and its sequelae in humans, the haemolytic-uraemic syndrome (HUS). The VT/Stx play a role in the occurrence of blood in the faeces and in the HUS by their action on the endothelial cells of blood vessels in the intestinal submucosa and in the renal glomeruli, after resorption through the intestinal walls. The VTEC/STEC are isolated from piglets, calves and humans. In recently weaned piglets, they cause the oedema disease, an enterotoxaemia characterized by subcutaneous, mesenteric and cerebral oedemas, with nervous disorders as main clinical signs. The oedema disease is the consequence of the action of the VT/Stx on the endothelial cells of blood vessels in various organs. In calves and humans, the role in disease of VTEC/STEC is controversial, but they could be associated with some cases of diarrhoea and HUS. The case of the O157:H7 EHEC which are present in healthy cattle of various ages, but are highly virulent for humans is of special interest. The potential zoonotic aspect of VT/Stx-producing E. coli infections in animals is detailed chapter by chapter. Prophylaxis of these infections by vaccination is the subject of the discussion on the future of the research studies on these pathogenic bacteria.     

Effect of vitamin E supplementation in milk replacer and Shiga toxoid vaccination on serum α‐tocopherol,performance, haematology and blood chemistry in male Holstein calves

Vitamin E (vit E), an essential antioxidant for maintaining the stability of biological membranes and the function of the immune system, is considered to support adaptive immune responses and performance in cattle. The principal virulence factor of Shiga toxin (Stx)‐producing Escherichia coli (STEC), the eponymous Stx, modulates cellular immune responses in cattle, the primary STEC reservoir. Active and passive immunization of calves with Shiga toxoids (rStx_MUT) was recently shown to reduce the STEC shedding. Here, we examined the influence of vit E on calves' serum α‐tocopherol, performance, haematology, blood chemistry and its interaction with rStx_MUT immunization. Data from calves having received passive (colostrum from immunized cows) and active (intramuscularly at 5th and 8th weeks of life) vaccination with rStx_MUT (n = 24) were compared to unvaccinated controls (n = 24; fed with low anti‐Stx colostrum, placebo injected). For each vaccination group, data were analysed according to the level of vit E supplementation offered by milk replacer (188 IU all‐rac‐α‐tocopheryl acetate daily [VitE_M] vs. 354 IU [VitE_H]). An increase by 79% in daily vit E supplementation led to slightly higher serum α‐tocopherol level and earlier concentrate intake at the beginning of the experiment without significant differences in live weight gain, haematology, blood chemistry parameters and peripheral CD4⁺ and CD8⁺ T‐cell subpopulations. rStx_MUT vaccination modulated the CD4⁺/CD8⁺ ratio irrespective of vit E supplementation but decreased concentrate intake in VitE_H in a time‐dependent manner. Results of our study indicate that an increase in daily vit E supplementation vastly fails to exert effects on laboratory parameters and growth performance. However, observed interactive effects of vit E supply and vaccination on the regulation of feed intake deserves further attention.     

Potent immune responses induced by a Salmonella ghost delivery system that expresses the recombinant Stx2eB,FedF, and FedA proteins of the Escherichia coli-producing F18 and Shiga toxin in a murine model and evaluation of its protective effect as a porcine vaccine candidate

Background: In the pathogenicity of porcine edema disease (ED), which is caused by the Escherichia coli-producing F18 and Shiga toxin, F18⁺ fimbrial adhesins and Shiga toxin 2e (Stx2e) play pivotal roles in the colonization and enterotoxicity of this pathogen.

Objective: To develop a vaccine candidate against ED by combining three selected antigens of F18⁺ E. coli.

Methods: Genetically engineered Salmonella Typhimurium (ST) ghosts that express Stx2eB, FedF, and FedA were individually inserted in a ghost plasmid cassette, and the resultant plasmids were transformed into an attenuated ST (JOL912). The individual expression of Stx2eB, FedF, and FedA in JOL912 was validated by using an immunoblotting assay.

Results: Immunization of the ghosts in BALB/c mice led to a significant increase in antigen-specific secretory IgA and serum IgG. Significantly marked elevation of the CD3⁺CD4⁺ T cell subpopulation and lymphocyte proliferating activity in the primed splenocytes were also observed. Furthermore, mRNA of IL-4 and IFN-γ were highly upregulated in in vitro stimulated splenic T cells. Subsequently, the immunized mice showed significant protection efficacy against a lethal dose 50 of a virulent strain, resulting in approximately 85% and 92% survival rates in mice with a single- and double-dose immunization, respectively, compared to only 40% of the non-immunized controls.

Conclusion: A mixture of the ghosts expressing these three antigens is a potential vaccine candidate for protection against the porcine edema disease.     

A globotetraosylceramide (Gb4) receptor-based ELISA for quantitative detection of Shiga toxin 2e

Currently, no simple assays are available for routine quantitative detection of Escherichia coli-produced Shiga toxin 2e (Stx2e) that causes porcine edema disease. Here, we present a novel quantitative detection method for Stx2e based on the measurement of Stx2e binding to the specific globotetraosylceramide (Gb₄) receptor by ELISA (Gb₄-ELISA). No cross-reactivity was found with the other Shiga toxins Stx1 and Stx2, indicating high specificity. When the recombinant Stx2e B subunit (Stx2eB) was used, the absorbance measured by Gb₄-ELISA increased linearly with Stx2eB concentration in the range of 20–2,500 ng/ml. The Gb₄-ELISA method can be easily performed, suggesting that it would be a useful diagnostic tool for porcine edema disease.     

Stochastic Simulation Model Comparing Distributions of STEC O157 Faecal Shedding Prevalence Between Cattle Vaccinated With Type III Secreted Protein Vaccines and Non‐Vaccinated Cattle

Pens of cattle with high Escherichia coli O157:H7 (STEC O157) prevalence at harvest may present a greater risk to food safety than pens of lower prevalence. Vaccination of live cattle against STEC O157 has been proposed as an approach to reduce STEC O157 prevalence in live cattle. Our objective was to create a stochastic simulation model to evaluate the effectiveness of pre‐harvest interventions. We used the model to compare STEC O157 prevalence distributions for summer‐ and winter‐fed cattle to summer‐fed cattle immunized with a type III secreted protein (TTSP) vaccine. Model inputs were an estimate of vaccine efficacy, observed frequency distributions for number of animals within a pen, and pen‐level faecal shedding prevalence for summer and winter. Uncertainty about vaccine efficacy was simulated using a log‐normal distribution (mean = 58%, SE = 0.14). Model outputs were distributions of STEC O157 faecal pen prevalence of summer‐fed cattle unvaccinated and vaccinated, and winter‐fed cattle unvaccinated. The simulation was performed 5000 times. Summer faecal prevalence ranged from 0% to 80% (average = 30%). Thirty‐six per cent of summer‐fed pens had STEC O157 prevalence >40%. Winter faecal prevalence ranged from 0% to 60% (average = 10%). Seven per cent of winter‐fed pens had STEC O157 prevalence >40%. Faecal prevalence for summer‐fed pens vaccinated with a 58% efficacious vaccine product ranged from 0% to 52% (average = 13%). Less than one per cent of vaccinated pens had STEC O157 prevalence >40%. In this simulation, vaccination mitigated the risk of STEC O157 faecal shedding to levels comparable to winter, with the major effects being reduced average shedding prevalence, reduced variability in prevalence distribution, and a reduction in the occurrence of the highest prevalence pens. Food safety decision‐makers may find this modelling approach useful for evaluating the value of pre‐harvest interventions.     

猪水肿病大肠杆菌贵州分离株毒素Stx2e对Vero细胞的毒性作用

猪水肿病毒素即Ⅱ型志贺毒素变异体e亚型(Shiga toxin 2e,Stx2e)。采用吖啶橙/溴化乙锭(AO/EB)双荧光染色法和四甲基偶氮唑盐(MTT)比色法,比较了从患水肿病猪粪便样品中分离的28株大肠杆菌所产志贺毒素对Vero细胞的毒性作用。结果显示,MTT比色法检测的细胞比活力与AO/EB染色法检测的正常细胞与早期凋亡细胞比率之和呈正相关。将28株菌株产生的Stx2e作用于Vero细胞,均能抑制Vero细胞的生长,并诱导Vero细胞的凋亡;其中8株病原菌所产Stx2e毒素对Vero细胞的毒性作用强于O157∶H7。这些毒素的毒力差异可能与其A亚基基因的结构变异有关。结果提示,贵州分离的产Stx2e大肠杆菌的毒力存在一定差异,其中部分菌株的毒力作用较强,应加强对养殖场仔猪的保护。     

An investigation of shedding and super‐shedding of Shiga toxigenic Escherichia coli O157 and E. coli O26 in cattle presented for slaughter in the Republic of Ireland

Shiga toxigenic Escherichia coli (STEC) are an important group of pathogens and can be transmitted to humans from direct or indirect contact with cattle faeces. This study investigated the shedding of E. coli O157 and O26 in cattle at the time of slaughter and factors associated with super‐shedding (SS) animals. Rectoanal mucosal swab (RAMS) samples were collected from cattle (n = 1,317) at three large Irish commercial beef abattoirs over an 18 month period, and metadata were collected at the time of sampling regarding farm of origin, animal age, breed and gender. RAMS swabs were examined for the presence and numbers of E. coli O157 and O26 using a previously developed quantitative real‐time PCR protocol. Samples positive by PCR were culturally examined and isolates analysed for the presence of stx subtypes, eae and phylogroup. Any samples with counts >10⁴ CFU/swab of STEC O157 or O26 were deemed to be super‐shedders. Overall, 4.18% (55/1,317) of RAMS samples were positive for STEC O157, and 2.13% (28/1,317) were classified as STEC O157 SS. For STEC O26, 0.76% (10/1,317) of cattle were positive for STEC O26, and 0.23% (3/1,317) were classified as super‐shedders. Fewer STEC shedders and SS were noted among older animals (>37 months). There was a seasonal trend observed for STEC O157, with the highest prevalence of shedding and SS events in the autumn (August to October). The majority of E. coli O157 (50/55) isolates had stx2 and were eae positive, with no significant difference between SS and low shedders (LS). Interestingly, all STEC O26 (n = 10) were eae negative and had varied stx profiles. This study demonstrates that, while the overall shedding rates are relatively low in cattle at slaughter, among positive animals there is a high level of SS, which may pose a higher risk of cross‐contamination during slaughter.     

Antibiotic‐resistant Shiga toxin‐producing Escherichia coli: An overview of prevalence and intervention strategies

Shiga toxin‐producing Escherichia coli (STEC) are foodborne pathogens that can cause severe diseases, including bloody diarrhoea and kidney failure, in humans, while remaining harmless to its primary reservoir hosts, cattle. Antibiotics such as azithromycin, fosfomycin and meropenem are being used and recommended in the treatment of early‐stage STEC (mainly E. coli O157:H7) infections, as these are reportedly effective in preventing Shiga toxin release and kidney failure while eliminating the pathogen. However, antibiotic resistance among STEC isolates could negatively impact these and other similar treatment options while contributing towards the spread of antibiotic resistance genes especially if encoded on mobile genetic elements like plasmids. Antibiotic resistance among STEC isolates recovered from animals and patients is being reported globally. A comprehensive understanding of the prevalence of antibiotic‐resistant STEC (AR‐STEC) and the mechanisms promoting this resistance among these bacteria could help direct therapies and develop strategies to effectively reduce/eliminate these pathogens. Here, we have reviewed literature from the past three decades to gain insights on this prevalence and its impact on human infections. In addition, we have reviewed various strategies proposed by researchers to control STEC that in turn would be applicable to AR‐STEC as well.     

An outbreak of Shiga toxin‐producing Escherichia coli O157:H7 linked to a mud‐based obstacle course,England, August 2018

In August 2018, Public Health England (PHE) was made aware of five probable cases of Shiga toxin‐producing Escherichia coli (STEC) O157:H7 among individuals reporting participation in a mud‐based obstacle race. An additional four cases, identified via routine whole‐genome sequencing, were subsequently linked to the same event. Two of the nine cases were due to secondary household transmission. Despite an agreement between the event organizers and the local authority, to ensure that all livestock were removed from the site 28 days before the event, sheep were observed grazing on some of the routes taken by the runners 2 days prior to the race taking place. A retrospective review of incidents reported to PHE between 2015 and 2018 identified 41 cases of gastroenteritis associated with muddy assault course events. Of these, 25 cases were due to infection with STEC O157:H7, of which all but one were associated with outbreaks. Due to the environment in which such events take place, it is impossible to entirely remove the risk of exposure to potentially pathogenic zoonoses. However, race organizers should ensure that livestock are removed from the course 28 days before the event. They should also ensure that participants are made aware of the risk of contracting gastrointestinal disease from the environment, and to stress the importance of hand hygiene post‐event and the risk of secondary transmission, particularly to children who are at risk of developing haemolytic uraemic syndrome.     

Molecular epidemiology of Shiga toxin‐producing O113:H21 isolates from cattle and meat

The serotype O113:H21 is considered one of the relevant non‐O157 STEC serotypes associated with severe human infections. Due to the increased detection of O113 strains and their relationship with clinical cases, which emphasizes the importance of this serogroup as an emerging pathogen, our aim was to determine the characteristics of STEC O113:H21 strains circulating in bovine cattle and retail meat from Argentina. For this purpose, we determined the presence and combinations of various virulence genes (and their variants) related to adhesion and toxicity in a collection of 34 isolates. Their genetic relatedness using multiple‐locus variable‐number tandem repeat analysis (MLVA) was also studied. Subtyping of stx genes indicated that O113:H21 strains circulating in Argentina mainly present stx_2a alone or together with stx_2c or, less frequent, with stx_2d, all of which are subtypes associated with human disease. We found plasmid markers, such as saa, ehxA and subA, in a higher proportion than previous studies, and five variants of saa, two of which were novel ones. In relation to MLVA subtyping, we detected a limited diversity among the isolates considering that several loci were not discriminative and, that in some farms, the same clone seemed to remain circulating throughout the year. The O113:H21 strains studied harbour several toxin and adhesion genes (saa, espP, fimCD, ehaA, iha, hcpA, elfA, lpfO113, ecpA, subA, cdt‐V) and Stx subtypes associated with human disease. Results also highlighted that subtyping of stx and saa is useful to discriminate O113:H21 strains that share virulence genes. In conclusion, this study shows that a number of O113:H21 strains that occur in foods and bovines could be pathogenic for humans. This situation calls for further attention in the prevention and control of foodborne disease caused by these strains.     

Evaluation of the ability of colistin,amoxicillin (components of Potencil®), and fluoroquinolones to attenuate bacterial endotoxin‐ and Shiga exotoxin‐mediated cytotoxicity—In vitro studies

Escherichia coli is one of the major pathogens in humans and animals causing localized and systemic infections, which often lead to acute inflammation, watery diarrhea, and hemorrhagic colitis. Bacterial lipopolysaccharide (LPS) and Shiga exotoxins (Stx) are mostly responsible for such clinical signs. Therefore, highly effective treatment of E. coli infections should include both eradication of bacteria and neutralization of their toxins. Here, for the first time, we compared the in vitro ability of common antibiotics to decrease LPS‐ and Stx‐mediated cytotoxicity: colistin, amoxicillin (used separately or combined), enrofloxacin, and its metabolite ciprofloxacin. Three experimental scenarios were realized as follows: (a) the direct effect of antibiotics on endotoxin, (b) the effect of antibiotic treatment on LPS‐mediated cytotoxicity in an experiment mimicking “natural infection,” (c) the effect of antibiotics to decrease Stx2e‐mediated cytotoxicity. Two cell lines, A549 and Vero cells, were used to perform cytotoxic assays with the methyl tetrazolium (MTT) and lactate dehydrogenase leakage (LDH) methods, respectively. Colistin and amoxicillin, especially used in combination, were able to attenuate LPS toxic effect, which was reflected by increase in A549 cell viability. In comparison with other antibiotics, the combination of colistin and amoxicillin exhibited the highest boster or additive effect in protecting cells against LPS‐ and Stx2e‐induced toxicity. In summary, in comparison with fluoroquinolones, the combination of colistin and amoxicillin at concentrations similar to those achieved in plasma of treated animals exhibited the highest ability to attenuate LPS‐ and Stx2e‐mediated cytotoxicity.     

Attribution of human infections with Shiga toxin‐producing Escherichia coli (STEC) to livestock sources and identification of source‐specific risk factors,The Netherlands (2010–2014)

Shiga toxin‐producing Escherichia coli (STEC) is a zoonotic pathogen of public health concern whose sources and transmission routes are difficult to trace. Using a combined source attribution and case–control analysis, we determined the relative contributions of four putative livestock sources (cattle, small ruminants, pigs, poultry) to human STEC infections and their associated dietary, animal contact, temporal and socio‐econo‐demographic risk factors in the Netherlands in 2010/2011–2014. Dutch source data were supplemented with those from other European countries with similar STEC epidemiology. Human STEC infections were attributed to sources using both the modified Dutch model (mDM) and the modified Hald model (mHM) supplied with the same O‐serotyping data. Cattle accounted for 48.6% (mDM) and 53.1% (mHM) of the 1,183 human cases attributed, followed by small ruminants (mDM: 23.5%; mHM: 25.4%), pigs (mDM: 12.5%; mHM: 5.7%) and poultry (mDM: 2.7%; mHM: 3.1%), whereas the sources of the remaining 12.8% of cases could not be attributed. Of the top five O‐serotypes infecting humans, O157, O26, O91 and O103 were mainly attributed to cattle (61%–75%) and O146 to small ruminants (71%–77%). Significant risk factors for human STEC infection as a whole were the consumption of beef, raw/undercooked meat or cured meat/cold cuts. For cattle‐attributed STEC infections, specific risk factors were consuming raw meat spreads and beef. Consuming raw/undercooked or minced meat were risk factors for STEC infections attributed to small ruminants. For STEC infections attributed to pigs, only consuming raw/undercooked meat was significant. Consuming minced meat, raw/undercooked meat or cured meat/cold cuts were associated with poultry‐attributed STEC infections. Consuming raw vegetables was protective for all STEC infections. We concluded that domestic ruminants account for approximately three‐quarters of reported human STEC infections, whereas pigs and poultry play a minor role and that risk factors for human STEC infection vary according to the attributed source.     

Non‐O157 Shiga Toxin‐producing Escherichia coli Isolated from Diarrhoeic Calves in Argentina

Faecal samples from 76 diarrhoeic calves belonging to 36 farms located in the Pampas plain, Argentina, were examined for Shiga toxin‐producing Escherichia coli (STEC). A total of 15 STEC strains were isolated from 12 (15.8%) calves which came from six different farms. All stx positive strains assayed by PCR were also positives in the Vero cell cytotoxicity test. The majority (60.0%) of the STEC strains carried the stx₁ gene. Twelve (80.0%) of the STEC isolates which belonged to serotypes O5:H‐ (n = 4), O26:H11 (n = 4), O26:H‐ (n = 1), O111:H‐ (n = 2), and O123:H38 (n = 1) were also enterohaemolysin (EHly) positive and carried the gene encoding for intimin (eae). All the stx positive strains were negative for the bfpA gene. Localized adherence to HEp‐2 cells were observed in 83.3% of the eae+ STEC strains. STEC belonging to serotype O5:H‐ showed atypical biochemical properties, including urease production. Urease was also produced by two strains belonging to serotypes O153:H? and non‐typeable, respectively. Resistance to three or more antibiotics was observed in 12 (80.0%) of the STEC isolates. Most of the serotypes of STEC recovered in this survey carried virulence traits that are associated with increased human and bovine pathogenicity. The present study shows that highly virulent STEC strains are being shed by diarrhoeic calves from farms located in a high incidence area of human STEC infections.