Identifying Bacterial and Host Factors Involved in the Interaction of Mycobacterium bovis with the Bovine Innate Immune Cells

Bovine tuberculosis is an important animal and zoonotic disease caused by Mycobacterium bovis. The innate immune response is the first line of defense against pathogens and is also crucial for the development of an efficient adaptive immune response. In this study we used an in vitro co-culture model of antigen presenting cells (APC) and autologous lymphocytes derived from peripheral blood mononuclear cells to identify the cell populations and immune mediators that participate in the development of an efficient innate response capable of controlling the intracellular replication of M. bovis. After M. bovis infection, bovine immune cell cultures displayed upregulated levels of iNOS, IL-22 and IFN-γ and the induction of the innate immune response was dependent on the presence of differentiated APC. Among the analyzed M. bovis isolates, only a live virulent M. bovis isolate induced an efficient innate immune response, which was increased upon stimulation of cell co-cultures with the M. bovis culture supernatant. Moreover, we demonstrated that an allelic variation of the early secreted protein ESAT-6 (ESAT6 T63A) expressed in the virulent strain is involved in this increased innate immune response. These results highlight the relevance of the compounds secreted by live M. bovis as well as the variability among the assessed M. bovis strains to induce an efficient innate immune response.Fil: Blanco, Federico Carlos. 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: Gravisaco, María José. 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: Bigi, María Mercedes. Universidad de Buenos Aires; ArgentinaFil: Garcia, Elizabeth Andrea. 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: Marquez, Cecilia. No especifíca;Fil: McNeil, Mike. State University of Colorado - Fort Collins; Estados UnidosFil: Jackson, Mary. State University of Colorado - Fort Collins; Estados UnidosFil: Bigi, Fabiana. 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

Transcriptional response of bovine monocyte-derived macrophages after the infection with different argentinean mycobacterium bovis Isolates

Infection of bovines with Mycobacterium bovis causes important financial hardship in many countries presenting also a risk for humans. M. bovis is known to be adapted to survive and thrive within the intramacrophage environment. In spite of its relevance, at present the information about macrophage expression patterns is scarce, particularly regarding the bovine host. In this study, transcriptomic analysis was used to detect genes differentially expressed in macrophages derived from peripheral blood mononuclear cells at early stages of infection with two Argentinean strains of M. bovis, a virulent and an attenuated strains. The results showed that the number of differentially expressed genes in the cells infected with the virulent strain (5) was significantly lower than those in the cells infected with the attenuated strain (172). Several genes were more strongly expressed in infected macrophages. Among them, we detected encoding transcription factors, anthrax toxin receptor, cell division and apoptosis regulator, ankyrin proteins, cytoskeleton proteins, protein of cell differentiation, and regulators of endocytic traffic of membrane. Quantitative real-time PCR of a selected group of differentially expressed genes confirmed the microarrays results. Altogether, the present results contribute to understanding the mechanisms involved in the early interaction of M. bovis with the bovine macrophage.Fil: Caimi, Karina Cynthia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Blanco, Federico Carlos. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Soria, Marcelo Abel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; ArgentinaFil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Mycobacterium bovis deleted in mce2 and phoP loci protects C57BL/6 mice against tuberculosis

Here we confirm the efficacy of MbΔmce2-phoP to protect mice against bTB challenge and we demonstrated that this new attenuated strain is safer than our previous bTB vaccine MbΔmce2. Altogether these results support the further assessment of this candidate in cattle model of bTB.Experiments with mice were performed in compliance with the regulations of the Institutional Animal Care and Use Committee (CICUAE) of INTA (61/2014)..Inst. de BiotecnologíaFil: Garcia, Elizabeth Andrea. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Bianco, Marí­a Veronica. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Fisiología y Recursos Genéticos Vegetales; ArgentinaFil: Blanco, Federico Carlos. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentin

Evaluación de algunas variables fisiológicas en caninos sometidos a esterilización quirúrgica / Assessment of some physiological variables in canines subjected to surgical sterilization

In the present paper the possible association between body temperature, glycemia and lacticaemia with the surgical act of sterilization of male and female canines is studied. These variables were chosen since they are known to be modified in the face of stressful events. It is expect d to observed changes in metabolic substrates used by the patient during surgery. The variables of interest were studied in three times (pre and intra-surgical) in male and female canines. Significant decreases in lactate and rectal temperature and a significant increase in blood glucose were detectedEn el presente trabajo se estudió la posible asociación entre la temperatura corporal, glucemia y lactacidemia con el acto quirúrgico de esterilización de caninos machos y hembras. Estosparámetros fueron elegidos ya que se sabe que sufren modificaciones ante eventos estresantes(1) y es de esperar que dichos cambios se produzcan como consecuencia del acto quirúrgico. Se estudiaron las variables de interés en tres tiempos a lo largodel proceso quirúrgico en caninos macho y hembra. Los resultados demostraron disminuciones significativas del lactato y la temperatura rectal y un aumento significativo de la glucemi

Mycobacterium bovis requires P27 (LprG) to arrest phagosome maturation and replicate within bovine macrophages

Mycobacterium bovis causes tuberculosis in a wide variety of mammals, with strong tropism for cattle and eventually humans. P27, also called LprG, is among the proteins involved in the mechanisms of the virulence and persistence of M. bovis and Mycobacterium tuberculosis. Here, we describe a novel function of P27 in the interaction of M. bovis with its natural host cell, the bovine macrophage. We found that a deletion in the p27-p55 operon impairs the replication of M. bovis in bovine macrophages. Importantly, we show for the first time that M. bovis arrests phagosome maturation in a process that depends on P27. This effect is P27 specific since complementation with wild-type p27 but not p55 fully restored the wild-type phenotype of the mutant strain; this indicates that P55 plays no important role during the early events of M. bovis infection. In addition, we also showed that the presence of P27 from M. smegmatis decreases the association of LAMP-3 with bead phagosomes, indicating that P27 itself blocks phagosome-lysosome fusion by modulating the traffic machinery in the cell host.Fil: Vázquez, Cristina Lourdes. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; ArgentinaFil: Bianco, María Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; ArgentinaFil: Blanco, Federico Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; ArgentinaFil: Forrellad, Marina Andrea. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gutierrez, Maximiliano Gabriel. The Francis Crick Institute; Reino Unido. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentin

Fluid structural analysis of urine flow in a stented ureter

Many urologists are currently studying new designs of ureteral stents to improve the quality of their operations and the subsequent recovery of the patient. In order to help during this design process, many computational models have been developed to simulate the behaviour of different biological tissues and provide a realistic computational environment to evaluate the stents. However, due to the high complexity of the involved tissues, they usually introduce simplifications to make these models less computationally demanding. In this study, the interaction between urine flow and a double-J stented ureter with a simplified geometry has been analysed.The Fluid-Structure Interaction (FSI) of urine and the ureteral wall was studied using three models for the solid domain: Mooney-Rivlin, Yeoh, and Ogden. The ureter was assumed to be quasi-incompressible and isotropic. Data obtained in previous studies fromex vivo and in vivo mechanical characterization of different ureters were used to fit thementioned models.The results show that the interaction between the stented ureter and urine is negligible. Therefore, we can conclude that this type of models does not need to include the FSI and could be solved quite accurately assuming that the ureter is a rigid body and, thus, using the more simple Computational Fluid Dynamics (CFD) approach

Bovine macrophages responses to the infection with virulent and attenuated Leptospira interrogans serovar Pomona

Leptospirosis is a zoonosis, caused by pathogenic spirochetes of the genus Leptospira. Although cattle are usually the maintenance hosts of serovar Hardjo, Pomona is the most frequent serovar circulating in Argentina. The understanding of bovine innate immune response and the virulence of this serovar is important for future control measures. This work compares infection of bovine macrophages with the virulent L. interrogans sv Pomona strain AKRFB (P1) and its attenuated counterpart (P19). First, we confirmed attenuation in the hamster model. Mortality and lung hemorrhages occurred after P1 inoculation, while the survival rate was 100% in P19-infected animals. Cells infected with both strains showed statistically upregulated gene expression of pro-inflammatory cytokines, IL-1β, IL-6 and TNFα. The level of expression of anti-inflammatory cytokine IL-10 was statistically different between strains. Increased expression of IL-10 was observed only in P1-infected cells. For the first time, we describe macrophages extracellular traps induced by infection of bovine macrophages (bMETs) with both, the virulent and attenuated Leptospira interrogans Pomona strains. P1 was found higher internalized when the phagocytosis was inhibited, suggesting a cell entrance of this strain also by an independent-phagocytosis pathway. Furthermore, P1 was higher colocalized with acidic and late endosomal compartments compared with P19. This data emphasizes the importance to deepen in Leptospira bovine macrophages particular invasion mechanisms and, furthermore, underline the value of studying the main hosts.Fil: Nagel, Ariel Gastón. 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: Vázquez, Cristina Lourdes. 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: Etulain, Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Blanco, Federico Carlos. 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: Gravisaco, Maria José. 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: Gómez, Ricardo Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Caimi, Karina Cynthia. 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

Assessment of tuberculosis biomarkers in paratuberculosis-infected cattle

Introduction: Mycobacterium bovis and Mycobacterium avium subsp. paratuberculosis, respectively the causative agents of bovine tuberculosis (bTB) and bovine paratuberculosis (PTB), share a high number of antigenic proteins. This characteristics makes the differential diagnosis of the diseases difficult. The interferon gamma (IFN-γ), C-X-C motif chemokine ligand 10 (CXCL10), matrix metallopeptidase 9 (MMP9), interleukin 22 (IL-22) and thrombospondin 1 (THBS1) bovine genes have already been shown to be accurate transcriptional biomarkers of bTB. In order to improve the diagnosis of bTB and PTB, in the present study we evaluated the risk of false positivity of these bTB biomarkers in cattle with PTB. Material and methods: The transcription of these genes was studied in 13 PTB-infected cattle, using Mycobacterium avium subsp. paratuberculosis (MAP)-stimulated peripheral blood mononuclear cells (PBMC). Results: Overall, the levels of IFN-γ, CXCL10, MMP9 and IL-22 transcripts in MAP-stimulated PBMC failed to differentiate animals with PTB from healthy animals. However, as bTB-afflicted cattle do, the MAP-infected group also displayed a lower level of THBS1 transcription than the non-infected animals. Conclusion: The results of this study add new specificity attributes to the levels of transcription of IFN-γ, CXCL10, MMP9 and IL-22 as biomarkers for bTB.Instituto de BiotecnologíaFil: Klepp, Laura Ines. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); ArgentinaFil: Klepp, Laura Ines. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Colombatti Olivieri, Maria Alejandra. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Colombatti Olivieri, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Moyano, Roberto Damian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Moyano, Roberto Damian. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Romano, Maria Isabel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Romano, Maria Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Malovrh, Tadej. University of Ljubljana. Veterinary Faculty. Institute for Microbiology and Parasitology; EsloveniaFil: Ocepek, Matjaž. University of Ljubljana. Veterinary Faculty. Institute for Microbiology and Parasitology; EsloveniaFil: Blanco, Federico Carlos. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); ArgentinaFil: Blanco, Federico Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); ArgentinaFil: Bigi, Fabiana. Consejo Nacional de investigaciones Científicas y Tecnológicas; Argentin

Assessment of the Immune Responses Induced in Cattle after Inoculation of a Mycobacterium bovis Strain Deleted in Two mce2 Genes

The generation of efficient candidate vaccines against bovine tuberculosis will contribute to the control of this zoonotic disease. Rationally attenuated Mycobacterium bovis strains generated by knockout of virulence genes are promising candidate vaccines. However, to be effective, these candidate vaccines should at least maintain the immunological properties of their virulent parental M. bovis strains. Therefore, the aim of this study was to obtain an M. bovis strain deleted in the mce2 genes and evaluate the effect of the mutation on the immunological profile elicited by the bacteria in cattle. We showed that the activation of CD4+ T cells in cattle inoculated with the mutant strain was equivalent to that in animals inoculated with the parental strain. Moreover, after in vitro stimulation, peripheral blood mononuclear cells from animals inoculated with the mutant produced higher levels of mRNA Th-1 cytokines than the parental strain. Therefore, these results indicate that the mce2 mutant is a promising candidate vaccine against bovine tuberculosis

Puentes de madera con estructura recíproca. Análisis comparativo del puente tradicional chino y el puente de Leonardo

During the last decades reciprocal structures have found a new purpose linked to the execution of temporary and recyclable projects in relation with current trends in sustainable architecture. The objective of this work is to analyze the behaviour of timber bridges with reciprocal structure. A numerical model was made and the structural behavior of a traditional Chinese bridge and Leonardo’s removable bridge were compared. The model was verified by measuring the deformation of a real bridge built in a workshop. In addition to the mechanical behavior, the load transmission mechanism, the influence of friction, lateral stability and robustness were studied. The Chinese bridge works mainly in compression, getting closer to the behavior of a funicular arch and transmitting a horizontal load on the supports around three times greater than the Leonardo bridge. The latter works fundamentally on bending and subjected to stresses and deformations closer to its ultimate limits.En las últimas décadas las estructuras recíprocas han encontrado una nueva utilidad vinculada a la ejecución de obras temporales y reciclables en línea con las tendencias actuales en arquitectura sostenible. El objetivo de este trabajo consiste en analizar el comportamiento de puentes de madera con estructura recíproca. Se realizó un modelo numérico y se compararon las respuestas estructurales del puente tradicional chino y el puente desmontable de Leonardo. El modelo se verificó midiendo la deformación de un puente real construido en taller. Además del comportamiento mecánico se estudió el mecanismo de transmisión de cargas, la influencia del rozamiento, la estabilidad lateral y la robustez. El puente chino trabaja fundamentalmente a compresión, acercándose más al comportamiento de un arco funicular, y transmitiendo una carga horizontal en los apoyos del orden de tres veces mayor que el puente de Leonardo, que trabaja fundamentalmente a flexión y con niveles de agotamiento y deformación mayores