30 research outputs found

    L-cysteine transporter-PCR to detect hydrogen sulfide-producing Campylobacter fetus

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    Phenotypic differences between Campylobacter fetus fetus and C. fetus venerealis subspecies allow the differential diagnosis of bovine genital campylobacteriosis. The hydrogensulfide production,for example,is atrait exclusive toC.fetus fetus and C. fetus venerealis biovar intermedius. This gas that can be biochemically tested can be produced from L-cysteine (L-Cys). Herein, we report a novel multiplex-PCR to differentiate C. fetus based on the evaluation of a deletion of an ATP-binding cassette-type L-Cys transporter that could be involved in hydrogen sulfide production, as previously described. A wet lab approach combined with an in silico whole genome data analysis showed complete agreement between this L-Cys transporter-PCR and the hydrogen sulfide production biochemical test. This multiplex-PCR may complement the tests currently employed for the differential diagnosis of C. fetus.Instituto de BiotecnologíaFil: Farace, Pablo Daniel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET); ArgentinaFil: Morsella, Claudia Graciela. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Unidad Integrada. Laboratorio de Bacteriología-Grupo de Sanidad Animal. Universidad Nacional de Mar del Plata; ArgentinaFil: Cravero, Silvio Lorenzo Pedro. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET); ArgentinaFil: Sioya, Bernardo Arturo. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET); ArgentinaFil: Amadio, Ariel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina. Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET); ArgentinaFil: Paolicchi, Fernando. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Unidad Integrada. Laboratorio de Bacteriología-Grupo de Sanidad Animal. Universidad Nacional de Mar del Plata; ArgentinaFil: Gioffre, Andrea. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET); Argentin

    The use of functional tests and planned coronary angiography after percutaneous coronary revascularization in clinical practice. Results from the AFTER multicenter study

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    Background: The follow-up strategies after percutaneous coronary intervention (PCI) have relevant clinical and economic implications. The purpose of this prospective observational multicenter study was to evaluate the effect of clinical, procedural and organizational variables on the execution of functional testing (FT) and planned coronary angiography (CA) after PCI, and to assess the impact of American College of Cardiology (ACC)/American Heart Association (AHA) guidelines on clinical practice. Methods: Four hundred twenty consecutive patients undergoing PCI were categorized as class I, IIB and III indications for follow-up FT according to ACC/AHA guidelines recommendations. Furthermore, all patients were grouped according to the presence or absence of FT and/or planned CA over 12 months after PCI. Multivariable analysis was used to assess the potential predictors of test execution. Results: During the 12-month follow-up at least one test was performed in 72% of patients with class I indication, 63% of patients with class IIB indication and 75% of patients with class III indication (p=ns). A total of 283 patients (67%) underwent testing. The use of tests was associated with younger age (R. R. 0.94, C. I. 0.91 +/- 0.97, p<0.001), a lower number of diseased vessels (R.R. 0.60, C.I. 0.43 +/- 0.84, p=0.003), follow-up by the center performing PCI (R. R. 2.64, C. I. 1.43 +/- 4.86, p=0.002), and the specific center at which PCI was performed. Most asymptomatic patients completed their testing prematurely with respect to the risk period for restenosis. Conclusions: The use of FT and planned CA after PCI is unrelated to patient's symptom status, and depends on patient's age and logistics. ACC/AHA guidelines have no influence in clinical practice, and test timing is not tailored to the risk period for restenosis. (C) 2008 Elsevier Ireland Ltd. All rights reserved

    Desarrollo de nuevas técnicas biotecnológicas para el diagnóstico y prevención de enfermedades bacterianas de rumiantes

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    El objetivo del siguiente trabajo es caracterizar antígenos de Mycobacterium bovis y M. paratuberculosis para diseñar un macroarreglo de proteínas, que será utilizado para analizar rodeos infectados con estas micobacterias.Trabajo galardonado con el Premio Fundación Pérez Companc, versión 2005Academia Nacional de Agronomía y Veterinaria (ANAV

    Search for Mycobacterium avium subspecies paratuberculosis antigens for the diagnosis of paratuberculosis

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    The aim of this study was to evaluate a wide panel of antigens of Mycobacterium avium subsp. paratuberculosis (MAP) to select candidates for the diagnosis of paratuberculosis (PTB). A total of 54 recombinant proteins were spotted onto nitrocellulose membranes and exposed to sera from animals with PTB (n=25), healthy animals (n=10), and animals experimentally infected with M. bovis (n=8). This initial screening allowed us to select seven antigens: MAP 2513, MAP 1693, MAP 2020, MAP 0038, MAP 1272, MAP 0209c, and MAP 0210c, which reacted with sera from animals with PTB and showed little cross-reactivity with sera from healthy animals and animals experimentally infected with M. bovis. The second step was to evaluate the antigen cocktail of these seven antigens by ELISA. For this evaluation, we used sera from animals with PTB (n=25), healthy animals (n=26), and animals experimentally infected with M. bovis (n=17). Using ELISA, the cocktail of the seven selected MAP antigens reacted with sera from 18 of the 25 animals with PTB and did not exhibit cross-reactivity with healthy animals and only low reactivity with animals with bovine tuberculosis. The combined application of these antigens could form part of a test which may help in the diagnosis of PTB.Facultad de Ciencias Veterinaria

    Search for Mycobacterium avium subspecies paratuberculosis antigens for the diagnosis of paratuberculosis

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    The aim of this study was to evaluate a wide panel of antigens of Mycobacterium avium subsp. paratuberculosis (MAP) to select candidates for the diagnosis of paratuberculosis (PTB). A total of 54 recombinant proteins were spotted onto nitrocellulose membranes and exposed to sera from animals with PTB (n=25), healthy animals (n=10), and animals experimentally infected with M. bovis (n=8). This initial screening allowed us to select seven antigens: MAP 2513, MAP 1693, MAP 2020, MAP 0038, MAP 1272, MAP 0209c, and MAP 0210c, which reacted with sera from animals with PTB and showed little cross-reactivity with sera from healthy animals and animals experimentally infected with M. bovis. The second step was to evaluate the antigen cocktail of these seven antigens by ELISA. For this evaluation, we used sera from animals with PTB (n=25), healthy animals (n=26), and animals experimentally infected with M. bovis (n=17). Using ELISA, the cocktail of the seven selected MAP antigens reacted with sera from 18 of the 25 animals with PTB and did not exhibit cross-reactivity with healthy animals and only low reactivity with animals with bovine tuberculosis. The combined application of these antigens could form part of a test which may help in the diagnosis of PTB.Facultad de Ciencias Veterinaria

    Utilization of molecular and conventional methods for the identification of nontuberculous mycobacteria isolated from different water sources

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    Background: The environment is the nontuberculous mycobacteria (NTM) reservoir, opportunistic pathogens of great diversity and ubiquity, which is observed in the constant description of new species capable of causing infection. Since its introduction, molecular methods are essential for species identification. Most comparative studies between molecular and conventional methods, have used isolated strains from clinical samples. Methods: The aim of this study was to evaluate the usefulness of molecular methods, especially the hsp65-PRA (PCR-Restriction Enzyme Analysis), and biochemical tests in the identification of NTM recovered from water of different origins, using the sequencing of 16S rRNA and hsp 65 genes as assessment methods of the previous ones. Species identification was performed for all 56 NTM isolates what were recovered from 32 (42.1%) positive water samples, using conventional phenotypic methods, hsp65-PRA, partial sequencing of 16S rRNA and sequencing of hsp 65 genes. Results: Phenotypic evaluation and hsp65-PRA were concordant with 23 (41.1%) isolates. Also, the PRA was concordant with 16 (28.6%) and 27 (48.2%) isolates, with the partial sequencing of 16S rRNA and sequencing of hsp 65 genes, respectively. It is considered that the 19.6% (n = 11) could not be identified. Conclusion: Identification of NTM environmental isolates to the species level, especially when they are pigmented and fast-growing, both the analysis of the restriction patterns obtained by PRA and the sequencing of the 16S rRNA and hsp 65 genes are insufficient by themselves. Although they are demanding and time-consuming, biochemical tests are very useful to support data obtained by molecular methods.Instituto de BiotecnologíaFil: Tortone, Claudia Andrea. Universidad Nacional de La Pampa. Facultad de Ciencias Veterinarias; ArgentinaFil: Zumarraga, Martin Jose. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Gioffre, Andrea Karina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Oriani, Delia Susana. Universidad Nacional de La Pampa. Facultad de Ciencias Veterinarias; Argentin

    In vivo short-term exposure to residual oil fly ash impairs pulmonary innate immune response against environmental mycobacterium infection

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    Epidemiological studies have shown that pollution derived from industrial and vehicular transportation induces adverse health effects causing broad ambient respiratory diseases. Therefore, air pollution should be taken into account when microbial diseases are evaluated. Environmental mycobacteria (EM) are opportunist pathogens that can affect a variety of immune compromised patients, which impacts significantly on human morbidity and mortality. The aim of this study was to evaluate the effect of residual oil fly ash (ROFA) pre-exposure on the pulmonary response after challenge with opportunistic mycobacteria by means of an acute short-term in vivo experimental animal model. We exposed BALB/c mice to ROFA and observed a significant reduction on bacterial clearance at 24 h post infection. To study the basis of this impaired response four groups of animals were instilled with (a) saline solution (Control), (b) ROFA (1 mg kg21 BW), (c) ROFA and EM-infected (Mycobacterium phlei, 8 3 106 CFU), and (d) EMinfected. Animals were sacrificed 24 h postinfection and biomarkers of lung injury and proinflammatory madiators were examined in the bronchoalveolar lavage. Our results indicate that ROFA was able to produce an acute pulmonary injury characterized by an increase in bronchoalveolar polymorphonuclear (PMN) cells influx and a rise in O2 2 generation. Exposure to ROFA before M. phlei infection reduced total cell number and caused a significant decline in PMN cells recruitment (p<0.05), O2 2 generation, TNFa (p<0.001), and IL-6 (p<0.001) levels. Hence, our results suggest that, in this animal model, the acute short-term pre-exposure to ROFA reduces early lung response to EM infectionInstituto de BiotecnologíaFil: Delfosse, Verónica Cecilia. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; Argentina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Tasat, Deborah Ruth. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; Argentina. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Histología y Embriología; ArgentinaFil: Gioffre, Andrea. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; Argentina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentin

    A closed-tube loop-mediated isothermal amplification assay for the visual endpoint detection of Brucella spp. and Mycobacterium avium subsp. paratuberculosis

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    LAMP (loop-mediated isothermal amplification) is an isothermal nucleic acid amplification technique that is characterized by its efficiency, rapidity, high yield of final product, robustness, sensitivity, and specificity, with the blueprint that it can be implemented in laboratories of low technological complexity. Despite the conceptual complexity underlying the mechanistic basis for the nucleic acid amplification, the technique is simple to use and the amplification and detection can be carried out in just one step. In this chapter, we present a protocol based on LAMP for the rapid identification of isolates of Brucella spp. and Mycobacterium avium subsp. paratuberculosis, two major bacterial pathogens in veterinary medicine.Instituto de BiotecnologíaFil: Trangoni, Marcos David. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Gioffre, Andrea Karina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cravero, Silvio Lorenzo Pedro. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentin

    Liver toxicity due to 1,2-dichloropropane in the rat.

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    The effect of 1,2-dichloropropane on rat liver was studied after short (5 days) and long term (4 weeks) i.p. administration. Animals were injected daily with 10-500 mg/kg body wt 1,2-dichloropropane and biochemical and histological changes of liver were investigated. Treatment was monitored by measuring urinary mercapturic acid excretion. A significant increase of mercapturate excretion was observed at all dose levels, with no further increase during the treatment; at lower doses a return to baseline values occurred within 48 h after the end of treatment. Mercapturate excretion at the end of weeks 2, 3 and 4 of treatment was significantly lower than that observed at the end of week 1. The liver reduced glutathione content was different after single or repeated injections. A dose-dependent decrease of liver reduced glutathione was observed after a single injection and a dose-dependent increase after 4 weeks. The liver biochemical pattern after 4 weeks of treatment (characterized by a decrease of cytochrome P-450 and by an increase of reduced glutathione and glutathione S-transferase activity) suggests a hyperplastic evolution of the liver cells, probably a repair mechanism induced by early depletion of reduced glutathione. Light microscopy confirms that the prevalent alterations are regenerative in type (atypical mitosis and hyperplastic nodules). Areas of focal necrosis are isolated, and trend to disappear after long term treatment
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