*2.2.5 Vaccines based on attenuated Salmonella*

In 2010, Khare et al. [29] assessed a live attenuated recombinant *Salmonella enterica* serovar Dublin *aroA* expressing intimin. The recombinant *Salmonella* was inoculated three times by oral route, but this did not produce a significant increase of intimin-specific IgA in serum and feces. Interestingly, they observed a transient clearance of *E. coli* O157:H7 in feces from vaccinated calves that subsequently reduced colonization and shedding of bacteria after an experimental challenge.

## *2.2.6 Vaccines based on Shiga toxins*

*The Universe of Escherichia coli*

of Agriculture.

*2.2.2 Vaccines based on siderophor receptors (SRP) and porins proteins*

*2.2.3 Vaccines based on bacterins and bacterial envelopes*

shedding of bacteria in feces was also observed.

This proposal is based on reducing the ability of the bacterium to obtain iron from the environment to decrease the level of infection [22]. Thornton et al. [23] assessed the efficacy of an SRP-composed vaccine (Epitopix LLC) to reduce the prevalence and fecal excretion of EHEC O157:H7 in calves after an experimental infection. A significant response in serum anti-SRP antibody titers was detected, and they concluded that the vaccination tended to decrease the fecal prevalence and concentration of EHEC O157:H7. In other study [24], this group evaluated the vaccine to control the burden of *E. coli* O157:H7 in feedlot cattle in field conditions. Vaccination with SRP was associated with the reduction of fecal concentration of EHEC O157:H7 and suggested to reduce the burden of these bacteria on cattle. In a third assay, the vaccine was evaluated in feedlot cattle naturally shedding *E. coli* O157. There were two different inoculum volumes of vaccine, 2 and 3 ml. They concluded that SRP vaccine at the 3 ml dose reduced prevalence of *E. coli* O157. These results led to the commercial elaboration of a product known as *E. coli* bacterial extract vaccine with SRP® technology [25] and manufactured by Pfizer Animal Health (Now Zoetis Services LLC). It has conditional license of the U.S. Department

To evaluate the protection conferred by a bacterin of EHEC O157:H7, van Diemen et al. [17] prepared a formalin-inactivated bacterin from EDL933nalR strain that was inoculated in a combined schedule by intramuscular (with Alu-Oil) and intranasal (mixed with cholera toxin B subunit) routes. It elicited significant IgG responses against intimin and LPS from *E. coli* O157:H7, but did not confer protection against intestinal colonization by EHEC O157:H7 after challenge.

In 2011, Sharma et al. [26] evaluated three heat-inactivated bacterins to reduce the fecal shedding of *E. coli* O157:H7. They used a *hha* + strain of *E. coli* O157:H7 and constructed a *hha* and *hha sepB* deletion mutants. These deletions enhance the expression and intracellular accumulation of T3SS proteins, respectively. There was a significant increase in IgG against LEE-encoded proteins in calves vaccinated with *hha* or *hha sepB* mutant bacterins compared to wild strain, and a reduction in the numbers of animals shedding EHEC O157:H7 and in the duration of the fecal

An alternative to bacterins was assayed by Vilte et al. [27] by means of empty envelopes of EHEC O157:H7 known as bacterial ghosts (BGs). These envelopes retain all surface components in a nondenatured form. Animals were vaccinated with BGs (without adjuvants) by subcutaneous route and elicited significant levels of specific IgG in serum. Following oral challenge with *E. coli* O157:H7, a significant reduction in both the duration and total bacterial shedding was observed in vac-

In 2008, McNeilly et al. [28] assayed a systemic (intramuscular) and mucosal (intrarectal) immunization with purified H7 flagellin to evaluate its effects on the colonization of EHEC O157:H7 after a challenge. The vaccination induced high titers of anti-H7 IgG and IgA antibodies in both serum and nasal secretions by intramuscular injection, but the intrarectal route failed in generating any response against H7. With respect to colonization of EHEC O157:H7, they concluded that

**104**

cinated calves.

*2.2.4 Vaccines based on flagellin*

An attractive target to research in cattle constitutes the Shiga toxins (Stx), the more important virulence factor for human health. In fact, Stx modulates cellular immune responses in cattle [30–32]. For that, in 2018, Schmidt et al. [33] evaluated the response, in a calf cohort, to immunization with recombinant Shiga toxoids genetically inactivated (rStx1MUT/rStx2MUT). Calves were passively (colostrum from immunized cows) and actively (intramuscularly) vaccinated, and this generated a significant difference in serum antibody titers compared with a control group. There was no EHEC O157:H7 challenge, but the natural presence of fecal STEC was monitored, and they observed less fecal positive (by PCR) samples from calves vaccinated than those from control animals. It is interesting because this investigation was not restricted to a determined serotype of EHEC.

In other study, Martorelli et al. [34] combined recombinant intimin and EspB with the B subunit of Stx2 fused to *Brucella* lumazine synthase (BLS-Stx2B) in order to evaluate whether the presence of Stx was able to improve the effect of the vaccine on fecal shedding of EHEC O157:H7 following an experimental inoculation. The immunization generates antibodies against Stx2B in serum and intestinal mucosa, but a superior level of protection compared with the use of intimin and EspB alone was not observed.

As was seen, there were and there are numerous efforts looking for a solution to reduce the contamination of cattle and its environment for EHEC O157:H7 and other dangerous serotypes too. Even two commercial products have been achieved, one of which has unfortunately been removed from the market. However, the fact that this pathogen does not constitute a direct problem for farmers, and because EHEC are not a cause of severe illness in cattle, makes our work more challenging. We have not only to find an adequate immunogen or formulation or doses that have a good response, but it must also be attractive enough for farmers to take it as a possible and desirable alternative to collaborate with one health perspective.
