Evaluación de vacunas a subunidades recombinantes contra la colonización del bovino por Escherichia coli O157:H7, agente causal del Síndrome Urémico Hemolítico : análisis de parámetros inmunológicos

Escherichia coli O157:H7 is a zoonotic pathogen and the major etiologic agent of Haemolytic Uremic Syndrome (HUS). Cattle are the main reservoir of these and other Shiga toxin-producing E. coli (STEC).\nDuring this work, protection conferred after immunization of calves with recombinant type-three secreted proteins, the C-terminal fraction of ?-Intimin (?-IntiminC280) and EspB, as well as the B subunit of Shiga toxin 2 fused to Brucella Lumazine Synthase (BLS-Stx2B) was evaluated upon experimental infection. \nImmunization with these antigens induced a significant increase in specific antibodies in sera as well as mucosa, able to neutralize several virulence properties of the bacteria in vitro. After experimental infection, a significant reduction in shedding was observed between vaccinated and control animals. However, no difference was observed between animals vaccinates only with ?-IntiminC280 and EspB, and those vaccinated with these two antigens and BLS-Stx2B.\nThese results suggest that the inclusion of BLS-Stx2B does not confer further protection that the already provided by ?-IntiminC280 and EspB, at least after the first weeks after experimental infection. Further studies are required to elucidate if the addition of this antigen influences E. coli O157:H7 under natural conditions of exposure.Fil: Martorelli, Luisina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Buenos Aires, ArgentinaEscherichiacoli O157:H7 es un patógeno zoonótico de importancia mundial y el agente causal delSindrome Urémico Hemolítico (SUH). El ganado bovino es el principal reservorio de esta y otras Escherichiacoli productoras de toxinas Shiga (STEC).\nDurante este trabajo se evaluó la protección conferida por la vacunación con proteínas recombinantes del Sistema de Secreción de Tipo III, la fracción carboxilo-terminal de Intimina? (Intimina?C280) y EspB; y la subunidad B de la toxina Shiga 2 fusionada a BrucellaLumazinaSintasa (BLS-Stx2B), frente al desafío experimental en terneros.\nLa inmunización con estos antígenos generó anticuerpos específicos tanto en suero como en mucosas, que eficientemente neutralizaron diversas propiedades de virulencia de la bacteria in vitro. Frente al desafío experimental, se observó una reducción significativa en la excreción de E. coli O157:H7 en los animales vacunados respecto de los controles. Sin embargo, no se observaron diferencias entre los grupos inmunizados con Intimina?C280 + EspB respecto del grupo que, además de estos dos antígenos, incluyó a BLS-Stx2B.\nEstos resultados sugieren que la adición de este antígeno no confiere protección adicional respecto de la observada para Intimina?C280 y EspB, al menos en las primeras semanas luego del desafío experimental. Aún queda por determinarse si la inclusión de este antígeno altera los patrones deexcreción frente a condiciones de exposición naturales

Shiga toxin-producing Escherichia coli (STEC) O22:H8 isolated from cattle reduces E. coli O157:H7 adherence in vitro and in vivo

Problem addressed Shiga toxin-producing Escherichia coli (STEC) are a group of bacteria responsible for food-associated diseases. Clinical features include a wide range of symptoms such as diarrhea, hemorrhagic colitis and the hemolytic uremic syndrome (HUS), a life-threatening condition. Objective Our group has observed that animals naturally colonized with STEC strains of unknown serotype were not efficiently colonized with E. coli O157:H7 after experimental infection. In order to assess the basis of the interference, three STEC strains were isolated from STEC persistently-colonized healthy cattle from a dairy farm in Buenos Aires, Argentina. Methods and results The three isolated strains are E. coli O22:H8 and carry the stx1 and stx2d genes. The activatable activity of Stx2d was demonstrated in vitro. The three strains carry the adhesins iha, ehaA and lpfO113. E. coli O22:H8 formed stronger biofilms in abiotic surface than E. coli O157:H7 (eae+, stx2+) and displayed a more adherent phenotype in vitro towards HeLa cells. Furthermore, when both serotypes were cultured together O22:H8 could reduce O157:H7 adherence in vitro. When calves were intragastrically pre-challenged with 108 CFU of a mixture of the three STEC strains and two days later challenged with the same dose of the strain E. coli O157:H7 438/99, the shedding of the pathogen was significantly reduced. Conclusions These results suggest that E. coli O22:H8, a serotype rarely associated with human illness, might compete with O157:H7 at the bovine recto-anal junction, making non-O157 carrying-calves less susceptible to O157:H7 colonization and shedding of the bacteria to the environment.Fil: Martorelli, Luisina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; ArgentinaFil: Albanese, Adriana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Vilte, Daniel. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; ArgentinaFil: Cantet, Rodolfo Juan Carlos. Universidad de Buenos Aires. Facultad de Agronomía; ArgentinaFil: Bentancor, Adriana Beatriz. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Zolezzi, G.. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; ArgentinaFil: Chinen, I.. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; ArgentinaFil: Ibarra, Cristina Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Rivas, M.. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; ArgentinaFil: Mercado, Elsa Cristina. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; ArgentinaFil: Cataldi, Ángel Adrián. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentin

Whole-genome sequencing analysis of Shiga toxin-producing Escherichia coli O22:H8 isolated from cattle prediction pathogenesis and colonization factors and position in STEC universe phylogeny

Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen capable of causing illness in humans. In a previous study, our group showed that a STEC isolate belonging to O22:H8 serotype (strain 154) can interfere with STEC O157:H7 colonization both in vitro and in vivo. Using whole-genome sequencing and genomic comparative, we predicted a subset of genes acquired by O22:H8 strain 154 through horizontal gene transfer that might be responsible for the phenotype previously described by our group. Among them were identified genes related to the pathogenesis of non-LEE (locus of enterocyte effacement) STEC, specific metabolic processes, antibiotic resistance and genes encoding for the T6SS-1 that is related to inter-bacterial competition. In addition, we showed that this strain carries stx1c and stx2dact, a mucus-inducible variant. The results obtained in this study provide insights into STEC genomic plasticity and the importance of genomic islands in the adaptation and pathogenesis of this pathogen.Fil: Marques Da Silva, Wanderson. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Larzabal, Mariano. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Aburjaile, Flavia Figueira. Universidade Federal de Minas Gerais; BrasilFil: Riviere, Nahuel Agustín. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Martorelli, Luisina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Patobiología Veterinaria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Patobiología Veterinaria; ArgentinaFil: Bono, James. Agricultural Research Service; Estados UnidosFil: Amadio, Ariel Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Investigación de la Cadena Láctea. - Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela. Instituto de Investigación de la Cadena Láctea; ArgentinaFil: Cataldi, Ángel Adrián. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentin

Oropharyngeal meningococcal carriage in children and adolescents, a single center study in Buenos Aires, Argentina.

BackgroundNeisseria meningitidis (Nm) pharyngeal carriage is a necessary condition for invasive disease. We present the first carriage study in children in Buenos Aires, Argentina, considering 2017 as a transition year. Aims: to assess the rate of Nm carriage, to determine genogroup, clonal complex and outer membrane protein distribution, to determine carriage risk factors by age.MethodsCross-sectional study including children 1-17 yrs, at Ricardo Gutiérrez Children's Hospital in Buenos Aires 2017. Oro-pharyngeal swabs were taken and cultured within a short time after collection. Genogroup was determined by PCR and clonal complex by MLST. Categorical variables were analyzed.ResultsA total of 1,751 children were included. Group 1: 943 children 1-9 yrs, 38 Nm were isolated; overall carriage 4.0%. Genogroup distribution: B 26.3%, W 5.3%, Y 2.6%, Z 5.3%, other groups 7.9% and capsule null (cnl) 52.6%. Participating in extracurricular activities was the only independent predictor of Nm carriage. Group 2: 808 children 10-17 yrs, 76 Nm were isolated; overall carriage 9.4%. Genogroup distribution: B 19.7%, C 5.3%, W 7.9%, Y 9.2%, Z 5.3%, other groups 7.9% and cnl 44.7%. Independent predictors of carriage: attending pubs/night clubs and passive smoking (adjusted OR: 0.55, 95%CI = 0.32-0.93; p = 0.025).ConclusionsOverall carriage was higher in 10-17 yrs. The isolates presenting the cnl locus were prevalent in both age groups and genogroup B was the second most frequent

Immune Response in Calves Vaccinated with Type Three Secretion System Antigens and Shiga Toxin 2B Subunit of Escherichia coli O157:H7.

Ruminants are the primary reservoir of Shiga-toxin producing Escherichia coli (STEC) O157:H7 and the main source of infection for humans. The aim of this study was to assess the immunogenic properties of a candidate vaccine consisting on the recombinant proteins of E. coli O157:H7 IntiminC280, the carboxy-terminal fraction of Intimin γ, EspB and the fusion protein between the B subunit of Stx2 and Brucella Lumazine Synthase (BLS)(BLS-Stx2B), in Holstein Fresian calves.To accomplish this goal we vaccinated calves with two doses of different vaccine formulations: 2 antigens (IntiminC280, EspB), 3 antigens (IntiminC280, EspB, BLS-Stx2B), BLS-Stx2B alone and a control non-vaccinated group. All antigens were expressed as recombinant proteins in E. coli. Specific IgG titres increased in vaccinated calves and the inclusion of BLS-Stx2B in the formulation seems to have a stimulatory effect on the humoral response to IntiminC280 and EspB after the booster. The neutralizing activity of antibodies against these two antigens was assessed in Red Blood Cell lysis assays and adherence to Hep-2 cells as a correlate of T3SS activity. Both sera from animals vaccinated with 2 or 3 antigens inhibited both virulence properties. Serological response to Stx2 was observed in animals vaccinated only with BLS-Stx2B and with 3 antigens and neutralization of Stx2 cytotoxicity was also observed in both groups. In conclusion, immunization of calves with BLS-Stx2B, IntiminC280 and EspB elicited a potent humoral response able to neutralize Shiga toxin 2 cytotoxity and the T3SS virulence properties in vitro. These results suggest that this formulation is a good candidate vaccine to reduce STEC shedding in cattle and needs to be further assessed in vivo

Efficacy of a recombinant Intimin, EspB and Shiga toxin 2B vaccine in calves experimentally challenged with Escherichia coli O157:H7

Escherichia coli O157:H7 is a zoonotic pathogen of global importance and the serotype of Shiga toxin-producing E. coli (STEC) most frequently associated with Hemolytic Uremic Syndrome (HUS) in humans. The main STEC reservoir is cattle. Vaccination of calves with the carboxy-terminal fraction of Intimin γ (IntC280) and EspB can reduce E. coli O157:H7 fecal shedding after experimental challenge. Shiga toxin (Stx) exerts local immunosuppressive effects in the bovine intestine and Stx2B fused to Brucella lumazine synthase (BLS-Stx2B) induces Stx2-neutralizing antibodies. To determine if an immune response against Stx could improve a vaccine's effect on fecal shedding, groups of calves were immunized with EspB + IntC280, with EspB + IntC280 + BLS-Stx2B, or kept as controls. At 24 days post vaccination calves were challenged with E. coli O157:H7. Shedding of E. coli O157:H7 was assessed in recto-anal mucosal swabs by direct plating and enrichment followed by immunomagnetic separation and multiplex PCR. Calves were euthanized 15 days after the challenge and intestinal segments were obtained to assess mucosal antibodies. Vaccination induced a significant increase of IntC280 and EspB specific antibodies in serum and intestinal mucosa in both vaccinated groups. Antibodies against Stx2B were detected in serum and intestinal mucosa of animals vaccinated with 3 antigens. Sera and intestinal homogenates were able to neutralize Stx2 verocytotoxicity compared to the control and the 2-antigens vaccinated group. Both vaccines reduced E. coli O157:H7 shedding compared to the control group. The addition of Stx2B to the vaccine formulation did not result in a superior level of protection compared to the one conferred by IntC280 and EspB alone. It remains to be determined if the inclusion of Stx2B in the vaccine alters E. coli O157:H7 shedding patterns in the long term and after recurrent low dose exposure as occurring in cattle herds.Fil: Martorelli, Luisina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; ArgentinaFil: Garimano, Nicolás Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Fiorentino, Gabriela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Vilte, Daniel A.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; ArgentinaFil: Garbaccio, Sergio G.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; ArgentinaFil: Barth, Stefanie A.. Friedrich Loeffler Institut; AlemaniaFil: Menge, Christian. Friedrich Loeffler Institut; AlemaniaFil: Ibarra, Cristina Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Palermo, Marina Sandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Cataldi, Ángel Adrián. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentin

Stx2 cytotoxicity neutralization in Vero cells.

<p>Results are expressed as percentage of cell viability and 100% represents cells incubated under identical conditions but without the toxin treatment. Data are shown as mean ± standard error of the mean of 3 experiments performed in quadruplicate (*P<0.05).</p

Inhibition of E. <i>coli</i> O157:H7 adherence to Hep-2 cells by sera from calves vaccinated with IntiminC₂₈₀ + EspB + BLS-Stx2B (3Ag); IntiminC₂₈₀ + EspB (2Ag); and PBS (control).

<p>Results are represented as % of inhibition of adherence respect to strain EHEC 438/99 without sera. Data are shown as mean ± standard error of the mean of 3 experiments performed (*P<0.05). * Indicates statistical significance compared to the control (p < 0.05).</p

Immunoglobulin G responses to Stx2B in calves vaccinated with IntiminC₂₈₀ + EspB (2Ag); IntiminC₂₈₀ + EspB + BLS-Stx2B (3Ag); BLS-Stx2B and PBS (control) measured by ELISA at different times post vaccination.

<p>Data are shown as OD492 ± standard error of the mean. * Indicates statistical significance (p < 0.05) compared to the control and † indicates statistical significance compared to the preimmune condition.</p

Immune Response in Calves Vaccinated with Type Three Secretion System Antigens and Shiga Toxin 2B Subunit of <i>Escherichia coli</i> O157:H7 - Fig 1

<p>Immunoglobulin G1 (A,C) and G2 (B,D) responses to IntiminC₂₈₀ (A, B) and EspB (C,D) in calves vaccinated with IntiminC₂₈₀ + EspB + BLS-Stx2B (3Ag); IntiminC₂₈₀ + EspB (2Ag); BLS-Stx2B and PBS (control) measured by ELISA at different times post vaccination. Data are shown as titers (as reciprocal of dilutions) ± standard error of the mean. * Indicates statistical significance (p < 0.05) compared to the control and † indicates statistical significance compared to the 2Ag group.</p