Preservation of protective capacity of hyperimmune anti-Stx2 bovine colostrum against enterohemorrhagic Escherichia coli O157:H7 pathogenicity after pasteurization and spray-drying processes.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a major etiologic agent that causes bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). Shiga toxin (Stx) is the main virulence factor of EHEC responsible for the progression to HUS. Although many laboratories have made efforts to develop an effective treatment for Stx-mediated HUS, a specific therapy has not been found yet. Human consumption of bovine colostrum is known to have therapeutic effects against several gastrointestinal infections because of the peptide and proteins (including antibodies) with direct antimicrobial and endotoxin-neutralizing effects contained in this fluid. We have previously demonstrated that colostrum from Stx type 2 (Stx2)-immunized pregnant cows effectively prevents Stx2 cytotoxicity and EHEC O157:H7 pathogenicity. In this study we evaluated the preservation of the protective properties of hyperimmune colostrum against Stx2 (HIC-Stx2) after pasteurization and spray-drying processes by performing in vitro and in vivo assays. Our results showed that reconstituted HIC-Stx2 colostrum after pasteurization at 60°C for 60 min and spray-dried under optimized conditions preserved specific IgG that successfully neutralized Stx2 cytotoxicity on Vero cells. Furthermore, this pasteurized/dehydrated and reconstituted HIC-Stx2 preserved the protective capacity against EHEC infection in a weaned mice model. The consumption of hyperimmune HIC-Stx2 bovine colostrum could be effective for HUS prevention in humans as well as in EHEC control in calves. However, further studies need to be done to consider its use for controlling EHEC infections.

Characterization of non-Shiga-toxin-producing Escherichia coli O157 strains isolated from dogs.

Shiga toxin-negative Escherichia coli O157 strains of various H types have been associated with diarrhea in children and are considered potentially pathogenic for humans. In this study, we describe non-Shiga toxin-producing E. coli O157 E. coli strains previously obtained from dogs in Argentina. Different E. coli phylogenetic lineages corresponding to flagellar types H16, H29 and H45 were identified. E. coli serotypes O157:H16 and O157:H45 contained intimin subtypes epsilon and alpha 1, respectively. Serotype O157:H45 carried the bfp gene encoding the bundle-forming pilus. Localized adherence-like patterns to HEp-2 cells were observed in O157:H16 strains, while O157:H45 adhered in a typical localized pattern. A total of eight different XbaI-pulse field electrophoresis patterns with more than 74 % similarity were identified among the nine E. coli O157:H16 strains. Our data emphasized the fact that dogs may harbor human pathogenic E. coli O157 which do not correspond to Shiga toxin-producing strains and whose potential human health hazard should not be underestimated.

A one-year longitudinal study of enterohemorrhagic Escherichia coli O157 fecal shedding in a beef cattle herd.

Bovines are the primary reservoir of enterohemorrhagic Escherichia coli (EHEC) O157:H7 and the main source of its transmission to humans. Here, we present a one-year longitudinal study of fecal shedding of E. coli O157. E. coli O157 obtained from recto-anal mucosal samples were characterized by multiplex PCR. The E. coli O157 prevalence ranged from 0.84% in July to 15.25% in November. The confinement within pens resulted in prevalence of 11%. Most animals (61.86%; 75/118) shed E. coli O157 at least in one sampling occasion. Of the positive animals, 82.19%, 16.44%, and 1.37% were stx positive on one, two and three sampling occasions, respectively. All the E. coli O157 isolated strains carried the genes eae and rfbO157, whereas 11%, 33% and 56% contained stx1, stx2 and stx1/stx2, respectively. The stx1/stx2 and stx2 types were significantly higher during the grazing and finishing periods, respectively, in comparison with the rearing and grazing periods. The presence of stx2a subtype was evident in four isolates, whereas stx2c was present in at least seven. However, both subtypes were present simultaneously in two isolates. The stx1/stx2c, stx1/stx2d and stx1/stx2NT genotypes occurred in 24, 2 and 15 isolates, respectively. The simultaneous occurrence of stx1 and stx2c significantly increased during grazing. Some cases of within-pen and between-pen transmission occurred throughout the study. Contagion levels during in-field grazing were higher than during permanent confinement in the pens. Thus, the individual patterns of shedding varied depending on the proportion of animals shedding the bacteria within pens and the time of shedding.

The intranasal vaccination of pregnant dams with Intimin and EspB confers protection in neonatal mice from Escherichia coli (EHEC) O157:H7 infection.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for intestinal disease and hemolytic uremic syndrome (HUS), a serious systemic complication which particularly affects children. In this study, we evaluated whether passive immunization protects from EHEC O157:H7 colonization and renal damage, by using a weaned BALB/c mouse model of infection. Recombinant proteins EspB and the carboxyl-terminal fragment of 280 amino acids of γ-intimin (γ-IntC280) were used in combination with a macrophage-activating lipopeptide-2 (MALP) adjuvant to immunize pregnant mice by the intranasal route. Neonatal mice were allowed to suckle vaccinated or sham-vaccinated dams until weaning when they were challenged by the oral route with a suspension of an E. coli O157:H7 Stx2+ strain. The excretion of the inoculated strain was followed for 72h. All vaccinated dams exhibited elevated serum IgG response against both γ-Int C280 and EspB. Passive immunization of newborn mice resulted in a significant increase in serum IgG titers against γ-Int C280 and a slight increase in EspB-specific antibodies. The neonates from vaccinated dams showed a significant reduction in EHEC O157:H7 colonization 48h post challenge. In addition, the level of plasma urea concentration, a marker of renal failure, was significantly higher in offsprings of sham-vaccinated mice. In conclusion, vaccination of pregnant dams with γ-Int C280 and EspB could reduce colonization and systemic toxicity of EHEC O157:H7 in their suckling offsprings.

Physiopathological effects of Escherichia coli O157:H7 inoculation in weaned calves fed with colostrum containing antibodies to EspB and Intimin.

Escherichia coli O157:H7 is responsible for severe intestinal disease and hemolytic uremic syndrome (HUS), a serious systemic complication which particularly affects children. Cattle are the primary reservoir for E. coli O157:H7 and the main source of infection for humans. In this study, we evaluated the ability of transferred maternal colostral antibodies against γ-Intimin C280 and EspB, to protect young weaned calves from E. coli O157:H7 infection. Hyperimmune colostra were obtained by immunization of pregnant cows with a mix of the mentioned antigens. All vaccinated cows mounted a significant IgG response against γ-Intimin C280, and EspB in sera and colostra. Colostrum-fed calves also exhibited high serum IgG titers against γ-Intimin C280 and EspB along with a rise in mucosal γ-Intimin C280-specific IgG antibodies at recto-anal junction and ileum. Additionally, 70 day-old calves received a challenge with E. coli O157:H7 but no reduction in total bacterial shedding or frequency of E. coli O157:H7 excretion from these calves was observed. Most tissue samples showed granulocyte focal infiltrations of the lamina propria and enterocyte erosion. In conclusion, up to the 70th day, the passively acquired γ-Intimin-C280 and EspB-IgG antibodies present in sera and recto-anal mucosa reached a titer insufficient to reduce EHEC O157 shedding and damages of experimentally inoculated young calves.

Profile of Shiga toxin-producing Escherichia coli strains isolated from dogs and cats and genetic relationships with isolates from cattle, meat and humans.

Pets can be reservoirs of Shiga toxin-producing Escherichia coli (STEC) strains. The aim of this study was to examine nine strains belonging to several serotypes (O91:H21, O91:H16, O178:H19, O8:H19, O22:H8, O22:HNT, ONT:H8), previously recovered from cats or dogs. To this end, we assessed a set of additional virulence genes (stx(2) subtype, subAB, ehxA, eae and saa), cytotoxic activity, and genetic relationships with strains isolated from cattle, meat and humans using pulsed-field gel electrophoresis (PFGE). Most of the isolates carried the stx(2) and/or stx(2vh-b) sequences, while only the O91:H21 isolate presented the mucus-activatable stx(2d) variant, as confirmed by sequencing the genes of subunits A and B. All the strains showed cytotoxic activity in cultured cells. One of the two O178:H19, selected for its high level of cytotoxicity in Vero cells, showed the ability to cause functional alterations in the human colon mucosa in vitro. None of the strains possessed the subAB, eae or saa genes and only the strains belonging to serotype O8:H19 carried the ehxA gene. The isolates shared 90-100% similarity by PFGE to epidemiologically unrelated strains of the corresponding serotypes recovered from cattle, meat or humans. Our results demonstrate that dogs and cats may have a role in the infection of humans by STEC, probably serving as a vehicle for bovine strains in the cycle of human infection, and thus emphasize the health risks for owners and their families.

A systemic vaccine based on Escherichia coli O157:H7 bacterial ghosts (BGs) reduces the excretion of E. coli O157:H7 in calves.

Cattle are the main reservoir of enterohemorrhagic Escherichia coli O157:H7, a bacterium that, in humans, causes hemorrhagic colitis and hemolytic uremic syndrome (HUS), a life-threatening disease, especially in children and older people. Therefore, the development of vaccines preventing colonization of cattle by E. coli O157:H7 could be a main tool for an HUS control program. In the present study, we evaluated bacterial ghosts (BGs) of E. coli O157:H7 as an experimental vaccine against this pathogen. BGs are empty envelopes of Gram-negative bacteria, which retain the morphological surface make-up of their living counterparts and are produced by controlled expression of the cloned protein E, which causes loss of all the cytoplasm content. In this work, E. coli O157:H7 BGs were used for subcutaneous immunization of calves. The vaccinated animals elicited significant levels of BG-specific IgG but not IgA antibodies in serum. Low levels of IgA and IgG antibodies against BGs were detected in saliva from vaccinated animals. Following oral challenge with E. coli O157:H7, a significant reduction in both the duration and total bacterial shedding was observed in vaccinated calves compared to the nonimmunized group. We demonstrated that systemic vaccination with E. coli O157 BGs provides protection in a bovine experimental model. Further research is needed to reach a higher mucosal immune response leading to an optimal vaccine.