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

DA VILTE; M LARZÁBAL; S GARBACCIO; M GAMELLA; BC RABINOVITZ; F DELGADO; L VAGNONI; V MEIKLE; UB MAYR; A CATALDI; P LUBITZ; W LUBITZ; EC MERCADO

Buenos Aires

Simposio; 7th International Symposium on Shiga Toxin (verocytotoxin)- producing Escherichi coli infections; 2009

Cattle are the main reservoir of enterohemorrhagic Escherichia coli (EHEC) O157: H7, a bacterium that in humans causes hemolytic uremic syndrome (HUS). Therefore, the development of vaccines preventing colonization of cattle by EHEC O157: H7 could be a main tool of programs of control of SUH. This study evaluated bacterial ghosts of E. coli O157: H7, as bovine vaccine against this pathogen. Six months Holstein-Fresian calves confirmed to be negative for EHEC O157:H7 were selected. These animals showed low IgG serum titres against bacterial ghosts, as determined by an enzyme-linked immunosorbent assay (ELISA). Five calves received 2 doses of 10 mg of freeze-dried bacterial ghost derived from strain CIP 105282 (corresponding to 4.8 x 1010 bacterial ghosts) in 5 ml of phosphate buffered saline (PBS) through SC route with an interval of 21 days. Other 5 calves were immunized with 1 ml of PBS, at the same intervals and used as a placebo group. Sera and fecal samples were collected weekly for specific antibody determination. Presence of bacterial ghost specific IgG and IgA in serum and specific IgA in feces and saliva against both EHEC bacterial ghost vaccine and EHEC-proteins gamma-Intimin and EspB was determined by ELISA. On day 35 calves were inoculated via oral with 109 colony forming units (CFU) of a nalidixic acid-resistant EHEC O157:H7 strain. The presence of EHEC O157:H7 in feces was monitored at two days interval over the following two weeks. EHEC O157:H7 counts were performed by direct plating of serially diluted fecal samples onto sorbitol-MacConkey agar containing tellurite and nalidixic acid (SMAC-TN). EHEC O157:H7 shedding was also determinated by enrichment of recto-anal swabs in Triptone-Soy-nalidixic acid broth followed by immunomagnetic separation with anti-O157 beads, plating onto SMAC-TN and PCR for stx2, eae and rfbO157 genes from the confluence zone. Sorbitol-negative colonies were confirmed to be E. coli O157 by PCR. On day 49, animals were euthanized with the ethical approval of the Committee on Animal Welfare of INTA. Segments of recto-anal junction, ileum, cecum and colon were collected to determine the presence of E. coli O157:H7 NalR by IMS. A significant difference was observed in the frequency of E. coli O157:H7 NalR excretion between vaccinated and control groups, whereas no reduction in total CFU/g occurred. The inoculated strain was recovered from recto-anal junction and ileum segments from one animal belonging to the placebo group. The serum bacterial ghost-specific IgG antibody titre increased significantly in the vaccinated animals after the second immunization, compared to non vaccinated animals. Non significant anti-bacterial ghost IgA response was observed in serum, feces or saliva. EHEC O157 antigens alpha- Intimin and EspB were not significantly recognized by vaccinated animals. These results suggest that protection could be based on IgG response. In conclusion, we showed that systemic immunization of cattle with EHEC O157 bacterial ghosts could reduce the duration of EHEC O157 fecal shedding.