Evaluation of a QIAamp DNA stool purification kit for Shiga-toxigenic Escherichia coli detection in bovine fecal swabs by PCR Evaluación del kit QIAamp DNA stool purification para la detección de Escherichia coli productor de toxina Shiga en hisopados de materia fecal bovina por PCR[

A commercial kit intended for Taq polymerase inhibitor removal was tested to detect Shiga-toxigenic Escherichia coli (STEC) by polymersase chain reaction (PCR) directly from cattle fecal samples. Forty-five samples were analysed for the presence of stx genes. Results were compared to those obtained by two other methods: amplification of DNA purified by a non-commercial procedure (heat lysis protocol), and amplification of DNA from samples cultured in solid media, commonly used in our lab. Identical numbers of positive samples (33/45, 73 %) were obtained with the QIAamp DNA stool purification kit and the culturing procedure, suggesting an adequate removal of inhibitors that interfere in PCR amplification from the feces. Besides, the number of positive samples detected using DNA purified by the non-commercial protocol was lower, 25/39 (64%) than that achieved by using the kit. In conclusion, the use of the QIAamp DNA stool purification kit provided a rapid stx gene detection by PCR in bovine fecal samples.Un kit comercial diseñado para la eliminación de inhibidores de la polimerasa Taq fue ensayado para la detección de STEC por PCR en muestras fecales de bovinos. Cuarenta y cinco muestras fueron evaluadas por la presencia de genes stx. Los resultados fueron comparados con aquéllos obtenidos por otros dos métodos: amplificación de ADN purificado por un procedimiento no comercial (protocolo de lisis por calor), y amplificación de ADN de muestras cultivadas en medio sólido, comúnmente usado en nuestro laboratorio. El mismo número de muestras positivas (33/45, 73 %), fueron obtenidas con el QIAamp DNA stool purification kit y el procedimiento de cultivo, sugiriendo una eliminación adecuada de inhibidores que interfieren con la amplificación en materia fecal. Por otro lado, el número de muestras positivas detectadas usando ADN purificado por el protocolo no comercial fue menor, 25/39 (64%). En conclusión, el uso del kit QIAamp DNA stool purification permitió una detección rápida de genes stx por PCR en muestras fecales bovinas

Soy protein isolate added to vacuum-packaged chorizos: effect on drip loss, quality characteristics and stability during refrigerated storage

Chorizo is a raw sausage, which is manufactured with beef, pork meat and pork fat, additives and spices. In Argentina, the expenditure of chorizo is through butchery and supermarkets where the product can be found packaged in both polyethylene films and vacuum sealed pouches. In the latter type of packaging an appearance problem has been detected in relation to drip loss. The aim of the work was to solve such problem through the incorporation of soy protein isolate (SPI). The sensory, microbiological and chemical stability of the product and its drip loss during a storage period of 14 days were studied. By adding a 2.5% SPI, the drip loss was prevented without introducing any change in the flavour, aroma and juiciness characteristics of the product. These sensory attributes were kept stable during the storage period studied. Chemical composition, oxidative and microbiological stability were not affected by the addition of SPI during the storage period, being similar for added and non-added SPI samples. Finally, SPI can be used in chorizos to improve their overall appearance during refrigerated storage while the product quality characteristics are not altered.Fil. Porcella, Marcela I. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Tecnología de Alimentos; ArgentinaFil: Sanchez, Guillermo. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Tecnología de Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Vaudagna, Sergio Ramon. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Tecnología de Alimentos; Argentina.Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Zanelli, Marta L. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Economía y Sociología; Argentina.Fil: Descalzo, Adriana Maria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Tecnología de Alimentos; ArgentinaFil: Meichtri, Lelis Hemil. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Tecnología de Alimentos; ArgentinaFil: Gallinger, Maria M. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Tecnología de Alimentos; Argentina.Fil: Lasta, Jorge Augusto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Tecnología de Alimentos; Argentina

Shiga toxin-producing Escherichia coli in healthy young beef steers from Argentina: prevalence and virulence properties

Between July 1999 and December 2000, the prevalence of Shiga toxin-producing Escherichia coli (STEC) was established in 200 Argentine healthy young beef steers (14-16 months old) grown under local production systems with a feed grain period of 3-4 months, and the STEC strains isolated were examined in regard to their phenotypic and genotypic characteristics.Stool samples (n=70) and rectal swabs (n=130) were taken at the slaughterhouse level.By polymerase chain reaction (PCR), Shiga toxin (stx) gene sequences were detected in 69% of the samples. Eighty-six STEC strains were isolated from 39% of the animals. Serogroups identified, in order of frequency, were: O8 (16 strains), O113 (14), O103 (5), O91 (4), O171 (3), O174 (3), O25 (2), O112 (2), O145 (2), O2, O11, O104, O121, O128, O143, O146, O157. the most frequent serotype isolated was O8:H19 (12.9%). A total of 17 serotypes, including E. coli O157:H7 found in one animal (0.5%), have been previously associated with hemolytic uremic syndrome (HUS), bloody and non-bloody diarrhea in different countries, including Argentina.The prevalent genotype isolated was stx2 (51 of 86, 59.3%). Subtyping of stx2 variants showed the prevalence of stx2vh-b (25.6%) and stx2vh-a types (24.4%), and revealed the presence of an atypical stx2-v. Only 7.0% of STEC strains carried eae, and 33.7% harbored EHEC-hlyA gene. the full virulent genotype (stx/eae/EHEC-hlyA) was found to be present in 4 of the 86 (4.7%) STEC strains isolated.This research indicates that young steers from the main beef-producing area of Argentina are an important reservoir of STEC strains; however, its importance as agents of human diseases in our country has still to be established. (C) 2004 Elsevier B.V. All rights reserved.Inst Nacl Tecnol Agropecuaria, Ctr Invest Agroind, Inst Tecnol Alimentos, RA-1708 Buenos Aires, DF, ArgentinaMinist Salud, Inst Nacl Enfermedades Infecciosas ANLIS Dr Carlo, Serv Fisiopatogenia, RA-1281 Buenos Aires, DF, ArgentinaInst Nacl Tecnol Agropecuaria, Ctr Invest Ciencias Vet & Agron, Inst Biotecnol, RA-1712 Buenos Aires, DF, ArgentinaUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, BrazilWeb of Scienc

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

A Translational Murine Model of Sub-Lethal Intoxication with Shiga Toxin 2 Reveals Novel Ultrastructural Findings in the Brain Striatum

Infection by Shiga toxin-producing Escherichia coli causes hemorrhagic colitis, hemolytic uremic syndrome (HUS), acute renal failure, and also central nervous system complications in around 30% of the children affected. Besides, neurological deficits are one of the most unrepairable and untreatable outcomes of HUS. Study of the striatum is relevant because basal ganglia are one of the brain areas most commonly affected in patients that have suffered from HUS and since the deleterious effects of a sub-lethal dose of Shiga toxin have never been studied in the striatum, the purpose of this study was to attempt to simulate an infection by Shiga toxin-producing E. coli in a murine model. To this end, intravenous administration of a sub-lethal dose of Shiga toxin 2 (0.5 ηg per mouse) was used and the correlation between neurological manifestations and ultrastructural changes in striatal brain cells was studied in detail. Neurological manifestations included significant motor behavior abnormalities in spontaneous motor activity, gait, pelvic elevation and hind limb activity eight days after administration of the toxin. Transmission electron microscopy revealed that the toxin caused early perivascular edema two days after administration, as well as significant damage in astrocytes four days after administration and significant damage in neurons and oligodendrocytes eight days after administration. Interrupted synapses and mast cell extravasation were also found eight days after administration of the toxin. We thus conclude that the chronological order of events observed in the striatum could explain the neurological disorders found eight days after administration of the toxin