Systemic immunization of cows with EspA, EspB, γ-Intimin and Stx2 proteins from Escherichia coli O157:H7 induces specific colostral antibodies that are efficiently transferred to newborn calves

RABINOVITZ BETTINA C , IBARRA C, MERCADO EC; TIRONI FARINATI C; ABDALA ANTONIO; GALARZA ROXANA; LARZÁBAL MARIANO; VILTE DANIEL A; CATALDI ANGEL; IBARRA CRISTINA; MERCADO ELSA C

Buenos Aires

Simposio; VTEC2009: 7th International Symposium on Shiga Toxin (Verocytotoxin)-producing Escherichia coli infections.; 2009

Asociación Argentina de Microbiología

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 are responsible for intestinal disease and hemolytic uremic syndrome (HUS), a serious systemic complication which particularly affects children. Cattle are an important reservoir and the main source of infection for humans. Vaccination of pregnant cows with EHEC virulence factors could induce specific colostral antibodies, a biological tool to protect against intestinal colonization by EHEC O157:H7 to calves and non- breastfed children. In this study we evaluated the ability of γ-Intimin, EspA, EspB and Shiga toxin 2 (Stx2) to induce specific antibodies in colostrum and sera of colostrum fed calves. Recombinants proteins EspA, EspB, the carboxyl-terminal fragment of 280 amino acids of γ-intimin (γ-Int1-280) and inactivated Stx2 were used as immunogens in combination with a water/oil/water adjuvant (ISA206, SEPPIC, France) supplemented with 1α,25(OH)2D3. We selected 25 Holstein pregnant cows confirmed to be negative for EHEC O157:H7 infection. Animals were immunized by intramuscular route at 60, 40 and 20 days prepartum. Separated groups of five cows received 100 μg/dose of each protein formulated in 1 ml of adjuvant. A control vaccine group received PBS in adjuvant alone. Colostrum was collected from cows at parturition. Blood samples were taken from cows at the beginning of the vaccination and at parturition. Calves were bled 48 h after the first colostrum administration. Milk samples were collected the day 40 postpartum. Specific IgG titres against recombinant proteins in colostrum, milk and sera were determined by an enzyme-linked immunosorbent assay (ELISA) and confirmed by western blot. Anti-Stx2 titres were determined by neutralization in Vero cell cultures. The possible inhibitory effect of colostrum on the hemolytic activity exhibited by type three secretion system encoded by EHEC strains was evaluated. All vaccinated cows mounted a significant serum and colostrum IgG response against γ-Int1-280, EspA and EspB. Colostrum fed calves also exhibited elevated serum IgG titres against EspA, EspB and γ-Int1-280. Antibodies to EspB and γ-Int1-280 were also detected in milk samples of vaccinated cows. Western blotting assays confirmed the specificity of the responses measured by ELISA. Colostrum from EspB-vaccinated cows showed a significant inhibitory effect on the hemolytic activity exhibited by the TTSS-encoding E. coli strain. Colostrum and sera from colostrum fed calves from Stx2-vaccinated cows also showed high Stx2-neutralizing titres compared with the control group. In conclusion, bovine colostrum with elevated levels of antibodies against EHEC O157:H7 was obtained by systemic immunization of cows, and specific antibodies were efficiently transferred to newborn calves by feeding colostrum. Immune colostrum and milk might be an alternative to protect calves from early colonization by E. coli O157: H7 and a key source of antibodies which could block colonization and toxic activity of this bacterium in human intestine. Key words: EHEC O157:H7, Vaccine, Colostrum, Calves.