Comparación de la cinética de la infección de ovas de trucha arcoíris (Oncorhynchus mykiss) con dos cepas de Piscirickettsia salmonis detectada mediante dot-blot#

En un estudio previo, se comprobó mediante microscopía de barrido, que la cepa LF-89 se adhiere a la pared de la ovamediante prolongaciones de su membrana externa, estructuras que han sido denominadas "Complejo de AdhesiónPiscirickettsial" o CAP, lo que facilitaría la posterior penetración de la bacteria al interior de la ova. Sin embargo,existen otras cepas aisladas, como la SLGO-95, que es más virulenta y resistente a antibióticos que la LF-89 y que no seha estudiado la posibilidad de unión a ovas. Por lo anterior, en el presente trabajo se comparó la cinética de infección deovas de trucha arcoíris, entre ambas cepas, utilizando como metodología la técnica de dot-blot. El método de "dot-blot"se realizó mediante la retención de proteínas en una membrana de polivinildifluoruro (PVDF), incubación conanticuerpos oligoclonales anti-P. salmonis y posteriormente con un segundo anticuerpo anti inmunoglobulina G deratón conjugado con peroxidasa. La reacción antígeno-anticuerpo se evidenció mediante un sustratoquimioluminiscente, utilizando una película autorradiográfica. La evaluación de la reacción se realizó mediantedensitometría utilizando un "software" computacional. Para estandarizar el método se realizaron diluciones seriadas dela suspensión de P. salmonis, desde 0,02 a 56 μg de proteína total. Para aumentar la sensibilidad se centrifugaron lasmuestras a 11.000 x g por 60 min. Además, se probaron diferentes concentraciones de anticuerpo primario, 1:1000,1:5000 y 1:10.000 y muestras de ovas y bacteria fueron sometidas a desnaturalización por ebullición. Se utilizaron ovasde reproductores libres de infecciones virales, Renibacterium salmoninarum y P. salmonis. Las ovas fueron incubadas(en duplicado) con 500 μL de una suspensión de P. salmonis, ya sea con la cepa LF-89 o SLGO-95 por 1,3, 10 y 60min. Luego, cada ova fue congelada a -70°C hasta ser procesada para la técnica de "dot blot". La muestra nocentrifugada de P. salmonis fue detectada como positiva sólo hasta 0,4 μg de proteína total. Cuando fue centrifugada a11.000 g por 1 h, la bacteria fue detectada hasta la última dilución estudiada (0,02 μg). Por otra parte, no hubo grandesdiferencias en la positividad obtenida mediante las tres diluciones de anticuerpos primarios. De acuerdo, al"background" obtenido y nitidez, se consideró como una mejor dilución de trabajo 1:5000. Cuando se realizó la cinéticade infección con la cepa LF-89, los resultados indicaron la aparición de señal positiva desde 1 min de exposición de lasovas a la bacteria. La reacción positiva se mantuvo hasta los 60 min. Con respecto a la cepa SLGO-95, los resultadosfueron similares, sin embargo, la cepa SLGO-95 demostró un mayor número de píxeles, lo que indica que esta cepa seune a la ova en mayor cantidad. Los resultados apoyan los estudios anteriores que indican que P. salmonis es capaz deinfectar verticalmente las ovas y que esta podría ser una de las formas de transmisión del agente en condiciones decultivo. Además, se comprueba que el grado de infección depende del tipo de cepa actuante.  

The jigsaw of PRRSV virulence

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of the, probably, most economically important disease for the pig industry worldwide. This disease, characterised by producing reproductive failure in sows and respiratory problems in growing pigs, appeared in the late 1980s in the United States and Canada. Since its appearance, strains capable of producing higher mortality rates as well as greater severity in clinical signs and lesions than classical strains have been identified. However, since the first reports of these “virulent” PRRSV outbreaks, no homogeneity and consensus in their description have been established. Moreover, to the authors’ knowledge, there is no published information related to the criteria that a PRRSV strain should fulfil to be considered as a “virulent” strain. In this review, we revise the terminology used and gather the information related to the main characteristics and differences in clinical signs, lesions, viral replication and tropism as well as immunological parameters between virulent and classical PRRSV strains and propose a first approximation to the criteria to define a virulent PRRSV strain

Activation of T-bet, FOXP3, and EOMES in Target Organs From Piglets Infected With the Virulent PRRSV-1 Lena Strain

Transcription factors (TFs) modulate genes involved in cell-type-specific proliferative and migratory properties, metabolic features, and effector functions. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogen agents in the porcine industry; however, TFs have been poorly studied during the course of this disease. Therefore, we aimed to evaluate the expressions of the TFs T-bet, GATA3, FOXP3, and Eomesodermin (EOMES) in target organs (the lung, tracheobronchial lymph node, and thymus) and those of different effector cytokines (IFNG, TNFA, and IL10) and the Fas ligand (FASL) during the early phase of infection with PRRSV-1 strains of different virulence. Target organs from mock-, virulent Lena-, and low virulent 3249-infected animals humanely euthanized at 1, 3, 6, 8, and 13 days post-infection (dpi) were collected to analyze the PRRSV viral load, histopathological lesions, and relative quantification through reverse transcription quantitative PCR (RT-qPCR) of the TFs and cytokines. Animals belonging to both infected groups, but mainly those infected with the virulent Lena strain, showed upregulation of the TFs T-bet, EOMES, and FOXP3, together with an increase of the cytokine IFN-g in target organs at the end of the study (approximately 2 weeks post-infection). These results are suggestive of a stronger polarization to Th1 cells and regulatory T cells (Tregs), but also CD4+ cytotoxic T lymphocytes (CTLs), effector CD8+ T cells, and gdT cells in virulent PRRSV-1-infected animals; however, their biological functionality should be the object of further studies

Activation of pro- and anti-inflammatory responses in lung tissue injury during the acute phase of PRRSV-1 infection with the virulent strain Lena

Porcine reproductive and respiratory syndrome virus (PRRSV) plays a key role in porcine respiratory disease complex modulating the host immune response and favouring secondary bacterial infections. Pulmonary alveolar macrophages (PAMs) are the main cells supporting PRRSV replication, with CD163 as the essential receptor for viral infection. Although interstitial pneumonia is by far the representative lung lesion, suppurative bronchopneumonia is described for PRRSV virulent strains. This research explores the role of several immune markers potentially involved in the regulation of the inflammatory response and sensitisation of lung to secondary bacterial infections by PRRSV-1 strains of different virulence. Conventional pigs were intranasally inoculated with the virulent subtype 3 Lena strain or the low virulent subtype 1 3249 strain and euthanised at 1, 3, 6 and 8 dpi. Lena-infected pigs exhibited more severe clinical signs, macroscopic lung score and viraemia associated with an increase of IL-6 and IFN-γ in sera compared to 3249-infected pigs. Extensive areas of lung consolidation corresponding with suppurative bronchopneumonia were observed in Lena-infected pigs. Lung viral load and PRRSV-N-protein+ cells were always higher in Lena-infected animals. PRRSV-N-protein+ cells were linked to a marked drop of CD163+ macrophages. The number of CD14+ and iNOS+ cells gradually increased along PRRSV-1 infection, being more evident in Lena-infected pigs. The frequency of CD200R1+ and FoxP3+ cells peaked late in both PRRSV-1 strains, with a strong correlation between CD200R1+ cells and lung injury in Lena-infected pigs. These results highlight the role of molecules involved in the earlier and higher extent of lung lesions in piglets infected with the virulent Lena strain, pointing out the activation of routes potentially involved in the restraint of the local inflammatory response.info:eu-repo/semantics/acceptedVersio

Antimicrobial and antibiofilm capacity of chitosan nanoparticles against wild type strain of pseudomonas sp. Isolated from milk of cows diagnosed with bovine mastitis

Indexación; Scopus.Bovine mastitis (BM) is the most prevalent bacterial infection in the livestock sector, affecting the dairy industry greatly. The prevention and treatment of this disease is mainly made via antibiotics, but the increasing antimicrobial resistance of pathogens has affected the efficiency of conventional drugs. Pseudomonas sp. is one of the pathogens involved in this infection. The therapeutic rate of cure for this environmental mastitis-causing pathogen is practically zero, regardless of treatment. Biofilm formation has been one of the main virulence mechanisms of Pseudomonas hence presenting resistance to antibiotic therapy. We have manufactured chitosan nanoparticles (NQo) with tripolyphosphate (TPP) using ionotropic gelation. These NQo were confronted against a Pseudomonas sp. strain isolated from milk samples of cows diagnosed with BM, to evaluate their antimicrobial and antibiofilm capacity. The NQo showed great antibacterial effect in the minimum inhibitory concentrations (MIC), minimum bactericidal concentration (MBC) and disk diffusion assays. Using sub lethal concentrations, NQo were tested for inhibition of biofilm formation. The results show that the nanoparticles exhibited biofilm inhibition and were capable of eradicate pre-existing mature biofilm. These findings indicate that the NQo could act as a potential alternative to antibiotic treatment of BM. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.https://www.mdpi.com/2079-6382/9/9/55

Infectivity of a rickettsia isolated from coho salmon Oncorhynchus kisutch

Two species of salmonids were tested for their susceptibility to infection by a rickettsia isolated in cell culture from diseased coho salmon Oncorhynchus kisutch in Chile. Mortality approached 100% in coho and Atlantic salmon Salmo salar injected with 10-fold dilutions of cell culture medium containing the rickettsia. Typical disease signs were present in the coho salmon but were not observed in inoculated Atlantic salmon. However, the rickettsia was recovered in pure culture from moribund fish in each of the injected groups of either species. The rickettsia was thereby demonstrated to be pathogenic and the cause of the ongoing epizootic affecting salmonids cultured in Chile. Horizontal transmission was not demonstrated in a group of uninoculated coho salmon held in the same tank with experimentally infected fish.Keywords: Salmo salar, Rickettsia, Oncorhynchus kisutch, pathogensKeywords: Salmo salar, Rickettsia, Oncorhynchus kisutch, pathogen

Improving the Thermal Stability of Cellobiohydrolase Cel7A from \u3cem\u3eHypocrea jecorina\u3c/em\u3e by Directed Evolution

Secreted mixtures of Hypocrea jecorina cellulases are able to efficiently degrade cellulosic biomass to fermentable sugars at large, commercially relevant scales. H. jecorina Cel7A, cellobiohydrolase I, from glycoside hydrolase family 7, is the workhorse enzyme of the process. However, the thermal stability of Cel7A limits its use to processes where temperatures are no higher than 50 °C. Enhanced thermal stability is desirable to enable the use of higher processing temperatures and to improve the economic feasibility of industrial biomass conversion. Here, we enhanced the thermal stability of Cel7A through directed evolution. Sites with increased thermal stability properties were combined, and a Cel7A variant (FCA398) was obtained, which exhibited a 10.4 °C increase in Tm and a 44-fold greater half-life compared with the wild-type enzyme. This Cel7A variant contains 18 mutated sites and is active under application conditions up to at least 75 °C. The X-ray crystal structure of the catalytic domain was determined at 2.1 Å resolution and showed that the effects of the mutations are local and do not introduce major backbone conformational changes. Molecular dynamics simulations revealed that the catalytic domain of wild-type Cel7A and the FCA398 variant exhibit similar behavior at 300 K, whereas at elevated temperature (475 and 525 K), the FCA398 variant fluctuates less and maintains more native contacts over time. Combining the structural and dynamic investigations, rationales were developed for the stabilizing effect at many of the mutated sites

The First Galaxy Cluster Discovered by the VISTA Variables in the Vía Láctea Survey

We report the first confirmed detection of the galaxy cluster VVV-J144321-611754 at very low latitudes (l = 315.°836, b = -1.°650) located in the tile d015 of the VISTA Variables in the Vía Láctea (VVV) survey. We defined the region of 30 ×30 arcmin 2 centered in the brightest galaxy finding 25 galaxies. For these objects, extinction-corrected median colors of (H-K s ) = 0.34 ± 0.05 mag, (J-H) = 0.57 ± 0.08 mag, and (J-K s ) = 0.87 ± 0.06 mag; R 1/2 = 1.59 ± 0.″16; C = 3.01 ± 0.08; and Sérsic index n = 4.63 ± 0.39 were estimated. They were visually confirmed showing characteristics of early-type galaxies in the near-IR images. An automatic clustering analysis performed in the whole tile found that the concentration of galaxies VVV-J144321-611754 is a real, compact concentration of early-type galaxies. Assuming a typical galaxy cluster with low X-ray luminosity, the photometric redshift of the brightest galaxy is z = 0.196 ± 0.025. Follow-up near-IR spectroscopy with FLAMINGOS-2 at the Gemini-South telescope revealed that the two brighter cluster galaxies have typical spectra of early-type galaxies and the estimated redshift for the brightest galaxy VVV-J144321.06-611753.9 is z = 0.234 ± 0.022 and that for VVV-J144319.02-611746.1 is z = 0.232 ± 0.019. Finally, these galaxies clearly follow the cluster red sequence in the rest-frame near-IR color-magnitude diagram with a slope similar to a galaxy cluster at a redshift of 0.2. These results are consistent with the presence of a bona fide galaxy cluster beyond the Milky Way disk.Fil: Baravalle, Laura Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Astronomía Teórica y Experimental. Universidad Nacional de Córdoba. Observatorio Astronómico de Córdoba. Instituto de Astronomía Teórica y Experimental; ArgentinaFil: Nilo Castellón, José Luis. Universidad de La Serena; ChileFil: Alonso, Maria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Astronomía Teórica y Experimental. Universidad Nacional de Córdoba. Observatorio Astronómico de Córdoba. Instituto de Astronomía Teórica y Experimental; ArgentinaFil: Díaz Tello, J.. Pontificia Universidad Católica de Chile; Chile. Universidad Nacional Autónoma de México; MéxicoFil: Damke, G.. Universidad de La Serena; ChileFil: Valotto, Carlos Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Astronomía Teórica y Experimental. Universidad Nacional de Córdoba. Observatorio Astronómico de Córdoba. Instituto de Astronomía Teórica y Experimental; ArgentinaFil: Cuevas Larenas, H.. Universidad de La Serena; ChileFil: Sánchez, Bruno Orlando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Astronomía Teórica y Experimental. Universidad Nacional de Córdoba. Observatorio Astronómico de Córdoba. Instituto de Astronomía Teórica y Experimental; ArgentinaFil: Ríos, M. de los. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Astronomía Teórica y Experimental. Universidad Nacional de Córdoba. Observatorio Astronómico de Córdoba. Instituto de Astronomía Teórica y Experimental; ArgentinaFil: Minniti, D.. Universidad Andrés Bello; ChileFil: Domínguez, M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Astronomía Teórica y Experimental. Universidad Nacional de Córdoba. Observatorio Astronómico de Córdoba. Instituto de Astronomía Teórica y Experimental; ArgentinaFil: Gurovich, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Astronomía Teórica y Experimental. Universidad Nacional de Córdoba. Observatorio Astronómico de Córdoba. Instituto de Astronomía Teórica y Experimental; ArgentinaFil: Barbá, R.. Universidad de La Serena; ChileFil: Soto, M.. Universidad de Atacama; ChileFil: Castro, F. Milla. Universidad de La Serena; Chil

ANÁLISIS DE INSTRUMENTOS DE MEDICIÓN DEL PENSAMIENTO CRÍTICO

Critical thinking is a relevant ability nowadays in higher education, but there is a lack of consensus on its definitions and assessment instruments. This article offers a conceptual and methodological review about the instruments used to measure critical thinking, in order to generate a discussion that allows a better understanding and appreciation of the aspects that are considered in this skill. The methodology consisted of a systematic review of studies in databases, selecting 31 studies out of 97 founded, and 8 of them for deep analysis; atopic content analysis in definitions and test characteristics is also used. Results show the existence of many differences in definitions and assessments of critical thinking, with a variety of tests and little consensus on the measured components. The need of an integrative model of Critical Thinking, considering cognitive, metacognitive and dispositional skills is discussed.El pensamiento crítico es una competencia relevante hoy en día en la formación universitaria, con escaso consenso conceptual y metodológico en sus instrumentos de medición. En este artículo se ofrece una revisión acerca de los instrumentos que se han utilizado para medir el pensamiento crítico, con la finalidad de generar una discusión que permita una mejor comprensión y valoración de los aspectos que componen esta habilidad. La metodología utilizada consiste en una revisión sistemática de estudios en bases de datos, seleccionando 31 estudios de 97 encontrados, y analizando en profundidad ocho de ellos; se utilizó además, análisis de contenido temático para las definiciones y características de instrumentos. Los resultados señalan la existencia de divergencias a la hora de definir y evaluar el pensamiento crítico, con variedad de instrumentos y escaso consenso en los componentes medidos. Se discute la necesidad de lograr un modelo de Pensamiento Crítico integrado que considere habilidades cognitivas, metacognitivas y disposicionales