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Glaría Ezquer, Idoia

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Glaría Ezquer

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Idoia

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Producción Agraria

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Open Access
Detection of PrPSc in lung and mammary gland is favored by the presence of Visna/maedi virus lesions in naturally coinfected sheep
(EDP Sciences, 2010) Salazar, Eider; Monleón, Eva; Bolea, Rosa; Acín, Cristina; Pérez, Marta María; Álvarez, Neila; Leginagoikoa, Iratxe; Juste, Ramón; Minguijón, Esmeralda; Reina Arias, Ramsés; Glaría Ezquer, Idoia; Berriatua, Eduardo; Andrés Cara, Damián de; Badiola, Juan José; Amorena Zabalza, Beatriz; Luján, Lluís; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
There are few reports on the pathogenesis of scrapie (Sc) and Visna/maedi virus (VMV) coinfections. The aim of this work was to study in vivo as well as post mortem both diseases in 91 sheep. Diagnosis of Sc and VMV infections allowed the distribution of animals into five groups according to the presence (+) or absence ( ) of infection by Sc and VMV: Sc /VMV , Sc /VMV+, Sc+/VMV and Sc+/ VMV+. The latter was divided into two subgroups, with and without VMV-induced lymphoid follicle hyperplasia (LFH), respectively. In both the lung and mammary gland, PrPSc deposits were found in the germinal center of hyperplasic lymphoid follicles in the subgroup of Sc+/VMV+ having VMV-induced LFH. This detection was always associated with (and likely preceded by) PrPSc observation in the corresponding lymph nodes. No PrPSc was found in other VMV-associated lesions. Animals suffering from scrapie had a statistically significantly lower mean age than the scrapie free animals at the time of death, with no apparent VMV influence. ARQ/ARQ genotype was the most abundant among the 91 ewes and the most frequent in scrapie-affected sheep. VMV infection does not seem to influence the scrapie risk group distribution among animals from the five groups established in this work. Altogether, these data indicate that certain VMVinduced lesions can favor PrPSc deposits in Sc non-target organs such as the lung and the mammary gland, making this coinfection an interesting field that warrants further research for a better comprehension of the pathogenesis of both diseases.
Open Access
Lentinula edodes β-glucan enriched diet induces pro- and anti-inflammatory macrophages in rabbit
(Taylor & Francis, 2017) Crespo, Helena; Guillén, Hugo; Pablo Maiso, Lorena de; Gómez Arrebola, Carmen; Rodríguez, Gregorio; Glaría Ezquer, Idoia; Andrés Cara, Damián de; Reina Arias, Ramsés; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
β-glucans exhibited in cell walls of several pathogens as bacteria or fungi are sensed by pathogen recognition receptors such as scavenger receptors present in antigen presenting cells, i.e., macrophages. β-glucans obtained from Shiitake mushrooms were chemically characterized. A β-glucan supplemented diet was assayed for 30 days in rabbits aiming to characterize the immune response elicited in blood-derived macrophages. M1 and M2 profiles of macrophage differentiation were confirmed in rabbits by in vitro stimulation with IFN-γ and IL-4 and marker quantification of each differentiation pathway. Blood derived macrophages from rabbits administered in vivo with the β-glucan supplemented diet showed higher IL-4, IFN-γ and RAGE together with lower IL-10 relative expression, indicative of an ongoing immune response. Differences in IL-1β, IL-13 and IL-4 expression were also found in rabbit sera by ELISA suggesting further stimulation of the adaptive response. Recent challenges in the rabbit industry include the search of diet supplements able to elicit an immune stimulation with particular interest in facing pathogens such as viruses or bacteria. β–glucans from fungi may contribute to maintain an immune steady state favouring protection and thus reducing antibiotic treatment.
Open Access
Ovine TRIM5α can restrict visna/maedi virus
(American Society for Microbiology, 2012) Jauregui, Paula; Crespo, Helena; Glaría Ezquer, Idoia; Luján, Lluís; Contreras, A.; Rosati, Sergio; Andrés Cara, Damián de; Amorena Zabalza, Beatriz; Towers, G. J.; Reina Arias, Ramsés; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua, IIQ14064.RI1
The restrictive properties of tripartite motif-containing 5 alpha (TRIM5α) from small ruminant species have not been explored. Here, we identify highly similar TRIM5α sequences in sheep and goats. Cells transduced with ovine TRIM5α effectively restricted the lentivirus visna/maedi virus DNA synthesis. Proteasome inhibition in cells transduced with ovine TRIM5α restored restricted viral DNA synthesis, suggesting a conserved mechanism of restriction. Identification of TRIM5α active molecular species may open new prophylactic strategies against lentiviral infections.
Open Access
Diagnosing infection with small ruminant lentiviruses of genotypes A and B by combining synthetic peptides in ELISA
(Elsevier, 2015) Sanjosé, Leticia; Crespo, Helena; Glaría Ezquer, Idoia; Andrés Cara, Damián de; Reina Arias, Ramsés; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
The major challenges in diagnosing small ruminant lentivirus (SRLV) infection include early detection and genotyping of strains of epidemiological interest. A longitudinal study was carried out in Rasa Aragonesa sheep experimentally infected with viral strains of genotypes A or B from Spanish neurological and arthritic SRLV outbreaks, respectively. Sera were tested with two commercial ELISAs, three based on specific peptides and a novel combined peptide ELISA. Three different PCR assays were used to further assess infection status. The kinetics of anti-viral antibody responses were variable, with early diagnosis dependent on the type of ELISA used. Peptide epitopes of SRLV genotypes A and B combined in the same ELISA well enhanced the overall detection rate, whereas single peptides were useful for genotyping the infecting strain (A vs. B). The results of the study suggest that a combined peptide ELISA can be used for serological diagnosis of SRLV infection, with single peptide ELISAs useful for subsequent serotyping.
Open Access
Post-entry blockade of small ruminant lentiviruses by wild ruminants
(BioMed Central, 2016) Sanjosé, Leticia; Crespo, Helena; Blatti-Cardinaux, Laure; Glaría Ezquer, Idoia; Martínez Carrasco, Carlos; Berriatua, Eduardo; Amorena Zabalza, Beatriz; Andrés Cara, Damián de; Bertoni, Giuseppe; Reina Arias, Ramsés; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua: IIQ010449.RI1; Gobierno de Navarra / Nafarroako Gobernua: IIQ14064.RI1; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Small ruminant lentivirus (SRLV) infection causes losses in the small ruminant industry due to reduced animal production and increased replacement rates. Infection of wild ruminants in close contact with infected domestic animals has been proposed to play a role in SRLV epidemiology, but studies are limited and mostly involve hybrids between wild and domestic animals. In this study, SRLV seropositive red deer, roe deer and mouflon were detected through modified ELISA tests, but virus was not successfully amplified using a set of different PCRs. Apparent restriction of SRLV infection in cervids was not related to the presence of neutralizing antibodies. In vitro cultured skin fibroblastic cells from red deer and fallow deer were permissive to the SRLV entry and integration, but produced low quantities of virus. SRLV got rapidly adapted in vitro to blood-derived macrophages and skin fibroblastic cells from red deer but not from fallow deer. Thus, although direct detection of virus was not successfully achieved in vivo, these findings show the potential susceptibility of wild ruminants to SRLV infection in the case of red deer and, on the other hand, an in vivo SRLV restriction in fallow deer. Altogether these results may highlight the importance of surveilling and controlling SRLV infection in domestic as well as in wild ruminants sharing pasture areas, and may provide new natural tools to control SRLV spread in sheep and goats.
Open Access
Multi-platform detection of small ruminant lentivirus antibodies and provirus as biomarkers of production losses
(Frontiers Media, 2020) Echeverría Garín, Irache; Miguel, Ricardo de; Pablo Maiso, Lorena de; Glaría Ezquer, Idoia; Benito, Alfredo A.; Blas, Ignacio de; Andrés Cara, Damián de; Luján, Lluís; Reina Arias, Ramsés; Agronomía, Biotecnología y Alimentación; Agronomia, Bioteknologia eta Elikadura
Small ruminant lentiviruses (SRLVs) are endemic in most areas of Europe, causing a chronic infection and a multisystemic disease affecting the udder, carpal joints, lungs, and central nervous system. Due to the lack of treatments and protective vaccination strategies, infection control is focused on the identification of infected animals through serological or molecular techniques. However, antigenic and genetic heterogeneity of SRLVs represent a clear drawback for diagnosis. Infected animals may present lower animal production parameters such as birth weight or milk production and quality, depending on productive systems considered and, likely, to the diagnostic method applied. In this study, four sheep flocks dedicated to dairy or meat production were evaluated using three different ELISA and two PCR strategies to classify animal population according to SRLV infection status. Productive parameters were recorded along one whole lactation or reproductive period and compared between positive and negative animals. SRLV was present in 19% of the total population, being unequally distributed in the different flocks. Less than half of the infected animals were detected by a single diagnostic method, highlighting the importance of combining different diagnostic techniques. Statistical analysis employing animal classification using all the diagnostic methods associated lambing size, lamb weight at birth, and daily weight gain with SRLV infection status in meat flocks. Milk production, somatic cell count, fat, and protein content in the milk were associated with SRLV infection in dairy flocks, to a greater extent in the flock showing higher seroprevalence. A multi-platform SRLV diagnostic strategy was useful for ensuring correct animal classification, thus validating downstream studies investigating production traits.
Open Access
Base genética del tropismo de lentivirus de pequeños rumiantes y estudio de la resistencia innata por APOBEC3
(2015) Glaría Ezquer, Idoia; Reina Arias, Ramsés; Andrés Cara, Damián de; Producción Agraria; Nekazaritza Ekoizpena
Los lentivirus de pequeños rumiantes (SRLV), que incluyen al virus Visna/Maedi (VMV) y al de la artritis encefalitis caprina (CAEV), infectan ovejas y cabras distribuidas por todo el mundo causando un cuadro multisistémico que afecta articulaciones, pulmones, glándula mamaria y sistema nervioso central. Las pérdidas derivadas de la infección van desde el aumento en la tasa de reposición, a un descenso en las producciones animales o en el valor comercial del rebaño. Aunque la enfermedad causada por los SRLV es de carácter lento y generalmente afecta a pulmones y glándula mamaria en nuestro país, se han descrito brotes epidemiológicos causantes de artritis y encefalitis en ovinos que afectan un gran número de animales, causando pérdidas directas. En la actualidad existen 5 genotipos descritos (A‐E) que presentan una alta variabilidad genética y biológica. Los genotipos A y B a los que pertenecen las estirpes clásicas de VMV y CAEV respectivamente, están distribuidos mundialmente, mientras que los genotipos C y E están restringidos a zonas geográficas concretas. En ausencia de tratamientos o vacunas totalmente protectoras, las medidas de control se basan en la detección temprana de animales infectados y su posterior eliminación del rebaño. Tras la infección, los animales infectados desarrollan una respuesta de anticuerpos que, si bien no es capaz de eliminar el virus, es indicadora de la infección. Así, empleando métodos de detección serológica podemos diagnosticar la infección indirectamente. Los métodos más eficaces hasta el momento son los ELISAs basados en proteínas recombinantes y péptidos sintéticos. Sin embargo, los métodos disponibles en el comercio están diseñados teniendo en cuenta una única estirpe viral, que sumado a la alta variabilidad antigénica de los SRLV, hace que las medidas disponibles fallen a la hora de controlar todo el espectro antigénico presente. La organización genómica de los lentivirus está bastante conservada entre los miembros del género. Así, a los genes estructurales gag, pol y env encargados de codificar proteínas de la cápside, proteínas para la replicación del material genético e integración y las proteínas de la envoltura, respectivamente, hay que sumarles los accesorios vpr, rev y vif en el caso de los SRLV, todos ellos flanqueados por el regulador de la transcripción viral, la región LTR. En la patogénesis de las enfermedades lentivirales es esencial conocer las interacciones entre el virus y el hospedador que determinan el tropismo y el desarrollo de los síntomas. Los factores virales incluyen las proteínas de la envoltura, encargadas de unirse al receptor celular y la región LTR encargada de regular la actividad transcripcional. Los factores del hospedador son más diversos ya que pueden incluir todo el fondo genético de una raza o una población determinada capaz de restringir la replicación viral de manera efectiva. Uno de los pasos clave en el establecimiento de la infección es la superación de las barreras de la inmunidad innata, que reconocen directamente determinantes virales e inducen la eliminación del patógeno mediante factores de restricción como APOBEC3. La caracterización genética y virológica de las estirpes implicadas en los brotes epidemiológicos de artritis y encefalitis, puede aportar hallazgos esenciales en el conocimiento de la relación entre la secuencia genética de los aislados y la capacidad para establecer la infección en un tejido determinado. Por otro lado, componentes de la inmunidad innata del hospedador capaces de inhibir la replicación viral pueden ser también determinantes del tropismo. Por todo ello en esta tesis nos planteamos los siguientes estudios: a) Aislamiento y caracterización genética de estirpes implicadas en el brote artrítico. b) Aislamiento y caracterización genética de estirpes implicadas en el brote de encefalitis. c) Caracterización de la restricción de los SRLV por APOBEC3.
Open Access
Characterization of ovine A3Z1 restriction properties against small ruminant lentiviruses (SRLVs)
(MDPI, 2017) Pablo Maiso, Lorena de; Glaría Ezquer, Idoia; Crespo, Helena; Nistal Villán, Estanislao; Andrésdóttir, Valgerdur; Andrés Cara, Damián de; Amorena Zabalza, Beatriz; Reina Arias, Ramsés; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Intrinsic factors of the innate immune system include the apolipoprotein B editing enzyme catalytic polypeptide-like 3 (APOBEC3) protein family. APOBEC3 inhibits replication of different virus families by cytosine deamination of viral DNA and a not fully characterized cytosine deamination-independent mechanism. Sheep are susceptible to small ruminant lentivirus (SRLVs) infection and contain three APOBEC3 genes encoding four proteins (A3Z1, Z2, Z3 and Z2-Z3) with yet not deeply described antiviral properties. Using sheep blood monocytes and in vitro-derived macrophages, we found that A3Z1 expression is associated with lower viral replication in this cellular type. A3Z1 transcripts may also contain spliced variants (A3Z1Tr) lacking the cytidine deaminase motif. A3Z1 exogenous expression in fully permissive fibroblast-like cells restricted SRLVs infection while A3Z1Tr allowed infection. A3Z1Tr was induced after SRLVs infection or stimulation of blood-derived macrophages with interferon gamma (IFN- ). Interaction between truncated isoform and native A3Z1 protein was detected as well as incorporation of both proteins into virions. A3Z1 and A3Z1Tr interacted with SRLVs Vif, but this interaction was not associated with degradative properties. Similar A3Z1 truncated isoforms were also present in human and monkey cells suggesting a conserved alternative splicing regulation in primates. A3Z1-mediated retroviral restriction could be constrained by different means, including gene expression and specific alternative splicing regulation, leading to truncated protein isoforms lacking a cytidine-deaminase motif.
Open Access
Identification of the ovine mannose receptor and its possible role in Visna/Maedi virus infection
(BioMed Central, 2011) Crespo, Helena; Reina Arias, Ramsés; Glaría Ezquer, Idoia; Ramírez, Hugo; Andrés, Ximena de; Jauregui, Paula; Luján, Lluís; Martínez Pomares, Luisa; Amorena Zabalza, Beatriz; Andrés Cara, Damián de; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
This study aims to characterize the mannose receptor (MR) gene in sheep and its role in ovine visna/maedi virus (VMV) infection. The deduced amino acid sequence of ovine MR was compatible with a transmembrane protein having a cysteine-rich ricin-type amino-terminal region, a fibronectin type II repeat, eight tandem C-type lectin carbohydrate-recognition domains (CRD), a transmembrane region, and a cytoplasmic carboxy-terminal tail. The ovine and bovine MR sequences were closer to each other compared to human or swine MR. Concanavalin A (ConA) inhibited VMV productive infection, which was restored by mannan totally in ovine skin fibroblasts (OSF) and partially in blood monocyte-derived macrophages (BMDM), suggesting the involvement of mannosylated residues of the VMV ENV protein in the process. ConA impaired also syncytium formation in OSF transfected with an ENV-encoding pN3-plasmid. MR transcripts were found in two common SRLV targets, BMDM and synovial membrane (GSM) cells, but not in OSF. Viral infection of BMDM and especially GSM cells was inhibited by mannan, strongly suggesting that in these cells the MR is an important route of infection involving VMV Env mannosylated residues. Thus, at least three patterns of viral entry into SRLV-target cells can be proposed, involving mainly MR in GSM cells (target in SRLV-induced arthritis), MR in addition to an alternative route in BMDM (target in SRLV infections), and an alternative route excluding MR in OSF (target in cell culture). Different routes of SRLV infection may thus coexist related to the involvement of MR differential expression.
Open Access
Mannose receptor may be involved in small ruminant lentivirus pathogenesis
(BioMed Central, 2012) Crespo, Helena; Jauregui, Paula; Glaría Ezquer, Idoia; Sanjosé, Leticia; Polledo, Laura; García Marín, Juan F.; Luján, Lluís; Andrés Cara, Damián de; Amorena Zabalza, Beatriz; Reina Arias, Ramsés; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua: IIQ14064.RI1; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Thirty-one sheep naturally infected with small ruminant lentiviruses (SRLV) of known genotype (A or B), and clinically affected with neurological disease, pneumonia or arthritis were used to analyse mannose receptor (MR) expression (transcript levels) and proviral load in virus target tissues (lung, mammary gland, CNS and carpal joints). Control sheep were SRLV-seropositive asymptomatic (n = 3), seronegative (n = 3) or with chronic listeriosis, pseudotuberculosis or parasitic cysts (n = 1 in each case). MR expression and proviral load increased with the severity of lesions in most analyzed organs of the SRLV infected sheep and was detected in the affected tissue involved in the corresponding clinical disease (CNS, lung and carpal joint in neurological disease, pneumonia and arthritis animal groups, respectively). The increased MR expression appeared to be SRLV specific and may have a role in lentiviral pathogenesis.