Reduced faecal shedding of Escherichia coli O157:H7 in cattle following systemic vaccination with γ-intimin C280 and EspB proteins
Introduction
Enterohaemorragic Escherichia coli (EHEC) O157:H7 is a major aetiological agent of diseases in humans, whose clinical spectrum includes diarrhoea, haemorrhagic colitis and haemolytic uremic syndrome (HUS), the leading cause of chronic renal failure in children in Argentina and several other countries [1], [2]. This bacterium produces Shiga toxins types 1 and/or 2 [3], [4], [5], which are responsible for systemic damage, particularly the vascular endothelium of the kidney and brain, with severe renal and neurological sequelae in children and elder people. Current treatment is largely limited to supportive care, as no specific regimen against an E. coli O157:H7 infection exists and the use of antibiotics is not recommended because they can cause the release of Shiga toxins from the bacterium, which can worsen the clinical course [6].
The main reservoir for E. coli O157:H7 is cattle, which harbour the bacteria in their intestinal tract [7], [8], especially in the lymphoid follicle-dense mucosa at the terminal rectum [9]. The bacteria are usually isolated from healthy animals, although episodes of diarrhoea have been observed in young animals [10], [11]. Faecal contamination of meat during slaughter, the use of faeces as fertilizers, and the contamination of drinking water are major ways by which this organism can enter the human food chain [7], [12].
Besides producing Shiga toxins, E. coli O157:H7 is characterized by other virulence-associated traits, which enable it to colonize the intestinal mucosa of humans and animals with a histopathological lesion known as “attaching and effacing” (A/E) [13]. A large chromosomal pathogenicity island called the locus of enterocyte effacement (LEE) is associated with A/E activity [14], [15]. The LEE encodes for a type three secretion system (TTSS) that translocates into the host cell effector proteins responsible for the A/E lesion. The A/E lesion is also characteristic of enteropathogenic E. coli (EPEC), another category of E. coli strains associated with diarrhoea in children [13]. The TTSS forms EspA, a filamentous structure through which effector proteins are translocated into the host cell [16]. Intimin and its bacterially expressed receptor Tir are translocated by the TTSS in the host cell membrane, leading to the formation of the A/E lesion. EspB is translocated into the host cell and contributes, in turn, to the creation of a pore in the eukaryotic cell membrane [17].
Many virulence factors of E. coli O157:H7 induce an immune response during the course of natural or experimental infections in animals and patients with HUS. Oral inoculation of calves and steers with E. coli O157:H7 promotes an increase in serum antibody titres against the O157 lipopolysaccharide and neutralizing antibodies to Shiga toxins [18]. Recently, Bretschneider et al. [19] demonstrated that cattle respond serologically to intimin and EspB of E. coli O157:H7 during the course of experimental infection. Antibodies against these proteins have also been detected in serum during both human EHEC [20] and EPEC infections [21] and in colostrum and milk from healthy women [22], [23], [24], [25] and cows [26]. In addition, mice infected with Citrobacter rodentium, a bacterium that shows virulence determinants and pathological effects in mice highly similar to those of EPEC in humans, develop an immune response against LEE-encoded proteins, which makes them resistant to bacterial re-infection [27].
Vaccination with bacterial colonization factors has been proposed as a strategy to prevent E. coli O157:H7 infection. Various vaccine formulations have been assayed, both in cattle [28], [29], [30], [31], [32], [33] and in other animal models [34], [35], [36], [37], [38], [39], [40], with variable results. In the present study, we evaluated the efficacy of a systemic vaccine composed of γ-intimin C280 and EspB, to reduce E. coli O157:H7 colonization in cattle.
Section snippets
Animals
All animal experiments were performed with the ethical approval of the Instituto Nacional de Tecnología Agropecuaria (INTA) Animal Welfare Committee. Eight 6–8-month-old conventionally reared male Holstein-Friesian calves were obtained from a farm in Buenos Aires province, Argentina, and housed at the INTA Experimental Station. Prior to the first immunization and challenge, calves were confirmed twice to be negative for E. coli O157:H7 by enrichment of faecal samples followed by immunomagnetic
Purification of recombinant proteins
EspB and γ-intimin C280 proteins were analyzed by SDS-PAGE and Coomassie blue staining, and single bands of the predicted molecular weight were observed (Fig. 1).
Immune response elicited after intramuscular immunization with EspB and γ-intimin C280
The systemic immunization of 6–8-month-old calves with two doses of EspB and γ-intimin C280 antigens formulated with ISA206-D3 induced a strong and significant increase in serum IgG titres against both proteins after the first immunization (P < 0.05) but not IgA antibodies (data not shown). IgG-antibody titres showed a 13-fold increase
Discussion
Many efforts have been directed to the development of vaccines against enteric pathogens that are able to induce strong mucosal immune responses capable of preventing intestinal colonization. In this sense, immunization of cattle to reduce E. coli O157:H7 shedding should be aimed to block the adhesion and colonization process, thereby reducing the risk of microbial transmission to other susceptible hosts. As reported, intimin is a major antigen of bacteria producing attaching and effacing
Conclusions
Cattle vaccination would be a feasible pre-slaughter intervention key to reduce E. coli O157:H7 faecal shedding and a good strategy to lower the risk of contamination for humans. Our results in a cattle infection model suggest that systemic immunization with two doses of a vaccine containing recombinant intimin and EspB could reduce E. coli O157:H7 colonization and shedding in cattle. Further field trials are needed to evaluate the efficacy of the vaccine to reduce bovine E. coli O157:H7
Acknowledgements
This study was supported by grant PICT 05 32687 of the Agencia Nacional de Promoción Científica y Tecnológica, Ministerio de Ciencia y Tecnología. A. Cataldi is a fellow of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). We are indebted to DVM Lucas Vagnoni for expert care and handling of animals and Laura González for her invaluable technical assistance.
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