17 research outputs found
Besnoitia besnoiti lytic cycle in vitro and differences in invasion and intracellular proliferation among isolates
© 2016 Frey et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.[EN]Background: Bovine besnoitiosis, caused by the protozoan Besnoitia besnoiti, reduces productivity and fertility of affected herds. Besnoitiosis continues to expand in Europe and no effective control tools are currently available. Experimental models are urgently needed. Herein, we describe for the first time the kinetics of standardised in vitro models for the B. besnoiti lytic cycle. This will aid to study the pathogenesis of the disease, in the screening for vaccine targets and drugs potentially useful for the treatment of besnoitiosis. Methods: We compared invasion and proliferation of one B. tarandi (from Finland) and seven B. besnoiti isolates (Bb-Spain1, Bb-Spain2, Bb-Israel, Bb-Evora03, Bb-Ger1, Bb-France, Bb-Italy2) in MARC-145 cell culture. Host cell invasion was studied at 4, 6, 8 and 24 h post infection (hpi), and proliferation characteristics were compared at 24, 48, 72, 96, 120, and 144 hpi. Results: In Besnoitia spp., the key parameters that determine the sequential adhesion-invasion, proliferation and egress steps are clearly distinct from those in the related apicomplexans Toxoplasma gondii and Neospora caninum. Besnoitia spp. host cell invasion is a rather slow process, since only 50 % of parasites were found intracellular after 3-6 h of exposure to host cells, and invasion still took place after 24 h. Invasion efficacy was significantly higher for Bb-France, Bb-Evora03 and Bb-Israel. In addition, the time span for endodyogeny to take place was as long as 18-35 h. Bb-Israel and B. tarandi isolates were most prolific, as determined by the tachyzoite yield at 72 hpi. The total tachyzoite yield could not be predicted neither by invasion-related parameters (velocity and half time invasion) nor by proliferation parameters (lag phase and doubling time (dT)). The lytic cycle of Besnoitia was asynchronous as evidenced by the presence of three different plaque-forming tachyzoite categories (lysis plaques, large and small parasitophorous vacuoles). Conclusions: This study provides first insights into the lytic cycle of B. besnoiti isolates and a standardised in vitro model that allows screening of drug candidates for the treatment of besnoitiosis.SIThis study was funded by the Spanish Ministry of Economy and Competitiveness (AGL2013-46442-R) and CYTED (Thematic Network 113RT0469 Protozoovac). CFF and NM are supported by the Swiss National Science Foundation (grants no. PBBEP3_141435 and PBBEP3_139398, respectively). We gratefully acknowledge Vanessa Navarro for her excellent technical assistance, and Andrew Hemphill for critical reading of the manuscript
Parasitología interactiva: Protozoos y afines
Desarrollo de una guía interactiva donde se muestran los ciclos biológicos, imágenes y dibujos de los estadios evolutivos de los principales géneros y especies de parásitos protozoos relevantes en al ámbito veterinario
Parasitología Interactiva
Desarrollo de una guía interactiva donde se muestran los ciclos biológicos,imágenes y dibujos de los estadios evolutivos de los principales géneros y especies de parásitos protozoos relevantes en al ámbito veterinario
Besnoitia besnoiti lytic cycle in vitro and differences in invasion and intracellular proliferation among isolates
Strategies for the improvement of the serological diagnosis of bovine besnoitiosis
Tesis inédita de la Universidad Complutense de Madrid, Facultad de Veterinaria, Departamento de Sanidad Animal, leída el 20/01/2016. Tesis formato europeo (compendio de artículos)Besnoitia besnoiti es un protozoo apicomplejo formador de quistes responsable de la besnoitiosis bovina, una enfermedad crónica y debilitante que origina importantes pérdidas económicas en el ganado bovino a nivel mundial. En la actualidad, no existen tratamientos ni vacunas disponibles y, por tanto, las medidas de control se deben basar en la detección de animales infectados para reducir la prevalencia en zonas endémicas y evitar la entrada de la enfermedad en zonas libres a través de la compra de animales infectados. Los principales retos en la investigación sobre este parásito son, por un lado, la mejora de las técnicas serológicas para realizar un diagnóstico preciso y, por otro lado, el empleo de las nuevas pruebas diagnósticas desarrolladas para determinar el impacto de la enfermedad mediante estudios de prevalencia e incidencia. Dada la rápida diseminación de la besnoitiosis bovina en Europa, se han desarrollado numerosas técnicas de diagnóstico serológico. Sin embargo, éstas no han sido validadas en estudios comparativos y, por ello, los datos epidemiológicos descritos por los diferentes laboratorios, no son comparables. Además, no se ha establecido una prueba de referencia, lo cual es de vital importancia para el establecimiento de un protocolo de diagnóstico común entre los países afectados. Por otra parte, se ha puesto de manifiesto la necesidad de mejorar la sensibilidad (Se) de las pruebas para detectar a los animales infectados durante la fase aguda, así como los infectados durante la fase crónica que presentan niveles bajos de anticuerpos, los cuales en diversas ocasiones se encuentran por debajo del punto de corte de las técnicas diagnósticas. Además, también es necesario mejorar su especificidad (Esp), ya que las actuales pruebas ELISA, que se emplean de forma rutinaria en el diagnóstico de la infección, pueden dar lugar a un elevado número de resultados falsos-positivos que pueden repercutir de forma negativa en la eficacia de los planes de control...Besnoitia besnoiti is a cyst forming intracellular parasite that causes bovine besnoitiosis, a chronic and debilitating disease in cattle responsible for severe economic losses. Unfortunately, there are no vaccines or treatments available and disease control relies solely on management measures coupled with diagnosis. The two main challenges in the field of B. besnoiti research nowadays are: the improvement of serological tools for an accurate diagnosis of bovine besnoitiosis, and the use of these techniques in prevalence and incidence studies to determine the impact of the disease in affected countries. Due to the rapid spread of bovine besnoitiosis in Europe, many serological assays have been developed to date. Unfortunately, these tests have never been validated in comparative studies. Thus, the epidemiological data reported in different laboratories are not comparable. In addition, a gold standard test for bovine besnoitiosis, which is mandatory for establishing common control procedures among affected countries, has never been accepted. Moreover, recent studies have shown that the serological assays developed so far may show low sensitivity (Se) for detecting B. besnoiti acutely infected as well as B. besnoiti chronically infected cattle showing low anti-B. besnoiti antibody levels. Furthermore, the existence of false-positive reactors has been also reported using ELISA and IFAT tests, which may be due to the presence of cross-reactive antigens between B. besnoiti and other Sarcocystidae parasites. In the present Doctoral Thesis, the techniques that are routinely employed for the diagnosis of bovine besnoitiosis in Europe were standardized and a common diagnostic procedure among affected countries was established for the first time (Objective 1). Based on the results obtained in this study, all ELISAs evaluated performed well and are useful tests for both diagnosis and epidemiological studies. However, Western blot performed better and was considered as the gold standard. Therefore, it may be recommended as a confirmatory assay under certain conditions, such as ambiguous results, cattle prior to entry to herds free of the disease and valuable animals prior to a selective culling...Depto. de Sanidad AnimalFac. de VeterinariaTRUEunpu
The tandemly repeated NTPase (NTPDase) from Neospora caninum is a canonical dense granule protein whose RNA expression, protein secretion and phosphorylation coincides with the tachyzoite egress.
BACKGROUND
NTPases (also NTPDases) are enzymes with apyrase activity. They are widely distributed among eukaryotes, and also among members of the family Sarcocystidae. In Toxoplasma gondii, the TgNTPase accumulates in the dense granules, and has been commonly associated with the strain virulence. In the closely related Neospora caninum, the NcNTPase lacks nucleoside diphosphate hydrolase activity and appears to be more abundant in virulent isolates, indicating that it may contribute to the pathogenicity of neosporosis. However, so far no additional information on NcNTPase has been provided.
METHODS
Herein, the NcNTPase coding sequences were analysed by different in silico and de novo sequencing approaches. A comparative analysis of NcNTPase and NcGRA7 in terms of protein dynamics, secretion, phosphorylation, and mRNA expression profiles during the tachyzoite lytic cycle was also carried out. Moreover, NcNTPase immunolocalization was analysed by confocal microscopy techniques over a set number of time-points.
RESULTS
We describe the presence of three different loci containing three copies of the NcNTPase within the Nc-Liv genome, and report the existence of up to four different NcNTPase alleles in Nc-Liv. We also provide evidence for the occurrence of diverse protein species of the NcNTPase by two-dimensional gel electrophoresis. Both NcNTPase and NcGRA7 were similarly up-regulated and secreted during the egress and/or early invasion phases, and were phosphorylated. However, its secretion was not affected by the addition of calcium modulators such as A23187 and ethanol. NcNTPase and NcGRA7 localized in dense granules and parasitophorous vacuole membrane throughout the lytic cycle, although differed in their inmunolocalization during early invasion and egress.
CONCLUSIONS
The present study reveals the complexity of the NcNTPase loci in N. caninum. We hypothesize that the expression of different isoforms of the NcNTPase protein could contribute to parasite virulence. Our findings showed regulation of expression, secretion and phosphorylation of NcNTPase suggesting a potential role for progression through the tachyzoites lytic cycle
Effect of pulmonary artery denervation in postcapillary pulmonary hypertension: results of a randomized controlled translational study
There is scarce evidence for pulmonary artery denervation (PADN) as a potential treatment for chronic postcapillary pulmonary hypertension (PH). We aimed to perform a proof-of-concept of PADN in a translational model of chronic PH. Nineteen pigs with chronic postcapillary PH (secondary to pulmonary vein banding) were randomized to surgical-PADN (using bipolar radiofrequency clamps) or sham procedure. Additionally, 6 healthy animals underwent percutaneous-PADN to compare the pulmonary artery (PA) lesion generated with both approaches. In the surgical-PADN arm, hemodynamic evaluation and cardiac magnetic resonance (CMR) were performed at baseline and at 2 and 3-month follow-up. Histological assessment was carried out at the completion of the protocol. Eighteen pigs (6 following surgical-PADN, 6 sham and 6 percutaneous-PADN) completed the protocol. A complete transmural PA lesion was demonstrated using surgical clamps, whereas only focal damage to adventitial fibers was observed after percutaneous-PADN. In the surgical-PADN arm, the hemodynamic profile did not significantly differ between groups neither at baseline [mean pulmonary artery pressure (mPAP) median values of 32.0 vs. 27.5 mmHg, P = 0.394 and indexed pulmonary vascular resistance (iPVR) 5.9 vs. 4.7 WU m2, P = 0.394 for PADN/sham groups, respectively] nor at any follow-up (mPAP of 35.0 vs. 35.0 mmHg, P = 0.236 and iPVR of 8.3 vs. 6.7 WU m2, P = 0.477 at third month in PADN/sham groups, respectively). Surgical-PADN was not associated with any benefit in RV anatomy or function on CMR/histology. In a large-animal model of chronic postcapillary PH, transmural PADN with surgical clamps was associated with a neutral pulmonary hemodynamic effect.This work has been partially funded by the grant “Translational research project from the Sociedad Española de Cardiología” (to Dr. García-Álvarez) and by an unrestricted grant from Medtronic. The CNIC is supported by the Ministerio de Ciencia, Innovación y Universidades and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). IDIBAPS belongs to the CERCA Programme and receives partial funding from the Generalitat de Catalunya.17.165 JCR (2020) Q1, 6/142 Cardiac & Cardiovascular SystemsUE
Effect of sildenafil on right ventricular performance in an experimental large-animal model of postcapillary pulmonary hypertension
Right ventricle (RV) dysfunction is a main determinant of morbidity and mortality in postcapillary pulmonary hypertension (PH). However, currently there are not available therapies. Since reduced nitric oxide (NO) availability and cyclic guanylate monophosphate (cGMP) levels are central in this disease, therapies targeting the NO pathway might have a beneficial effect on RV performance. In this regard, sildenafil has shown contradictory results. Our objective was to evaluate the effect of sildenafil on RV performance in an experimental pig model of postcapillary PH induced by a fixed banding of the venous pulmonary confluent. Animals were evaluated by right heart catheterization and cardiac magnetic resonance before randomization and after 8 weeks on sildenafil (n = 8) or placebo (n = 8), and myocardial tissues were analyzed with histology and molecular biology. At the end of the study, animals receiving sildenafil showed better RV performance as compared with those on placebo (improvement in RV ejection fraction of 7.3% ± 5.8% versus -0.6% ± 5.0%, P= 0.021) associated with less apoptotic cells and gene expression related with reduced oxidative stress and increased anti-inflammatory activity in the myocardium. No differences were observed in pulmonary hemodynamics. In conclusion, in a translational large animal model of chronic postcapillary PH, sildenafil improved RV systolic function independently of afterload. Further research with pharmacological approaches able to manipulate the NO-cGMP axis are needed to confirm this potential cardioprotective effect.Sin financiación7.012 JCR (2020) Q1, 2/29 Medical Laboratory Technology2.174 SJR (2021) Q1, 3/60 Biochemistry (medical)No data IDR 2020UE