Person: Pastor Fernández, Iván
Loading...
First Name
Iván
Last Name
Pastor Fernández
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Veterinaria
Department
Sanidad Animal
Area
Identifiers
15 results
Search Results
Now showing 1 - 10 of 15
Item NcGRA7 and NcROP40 Play a Role in the Virulence of Neospora caninum in a Pregnant Mouse Model(Pathogens, 2022) Rico San Román, Laura; Amieva, Rafael; Regidor Cerrillo, Javier; García Sánchez, Marta Eulalia; Collantes Fernández, Esther; Pastor Fernández, Iván; Saeij, Jeroen P. J.; Ortega Mora, Luis Miguel; Horcajo Iglesias, María Del PilarThe intraspecific variability among Neospora caninum isolates in their in vitro behaviour and in vivo virulence has been widely studied. In particular, transcriptomic and proteomic analyses have shown a higher expression/abundance of specific genes/proteins in high-virulence isolates. Consequently, the dense granule protein NcGRA7 and the rhoptry protein NcROP40 were proposed as potential virulence factors. The objective of this study was to characterize the role of these proteins using CRISPR/Cas9 knockout (KO) parasites in a well-established pregnant BALB/c mouse model of N. caninum infection at midgestation. The deletion of NcGRA7 and NcROP40 was associated with a reduction of virulence, as infected dams displayed milder clinical signs, lower parasite burdens in the brain, and reduced mortality rates compared to those infected with the wild-type parasite (Nc-Spain7). Specifically, those infected with the NcGRA7 KO parasites displayed significantly milder clinical signs and a lower brain parasite burden. The median survival time of the pups from dams infected with the two KO parasites was significantly increased, but differences in neonatal mortality rates were not detected. Overall, the present study indicates that the disruption of NcGRA7 considerably impairs virulence in mice, while the impact of NcROP40 deletion was more modest. Further research is needed to understand the role of these virulence factors during N. caninum infection.- Project number: 290
Item Parasitología Interactiva(2016) Gómez Bautista, Mercedes; Ortega Mora, Luis Miguel; Álvarez García, Gema; Ferré Pérez, Ignacio; Moreno Gonzalez, Javier; Gutierrez Expósito, Daniel; Arranz-Solís, David; Pastor Fernández, Iván; Rojo Montejo, Silvia; Sánchez Sánchez, Roberto; García Lunar, Paula; Jiménez Meléndez, Alejandro; Jimenez Pelayo, Laura; García Sánchez, Marta; Diezma Díaz, Carlos; Horcajo Iglesias, María Del Pilar; Regidor Cerrillo, Javier; Pérez Villalobos, Natividad; Collantes Fernández, EstherDesarrollo 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. Item Serological diagnosis of bovine neosporosis: A comparative study of commercially available ELISA tests(Veterinary Parasitology, 2013) Álvarez García, Gema; García Culebras, Alicia; Gutiérrez-Expósito, Daniel; Navarro-Lozano, Vanesa; Pastor Fernández, Iván; Ortega Mora, Luis MiguelBovine neosporosis control programs are currently based on herd management and serodiagnosis because effective treatments and vaccines are unavailable. Although a wide variety of serological tools have been developed, enzyme-linked immunosorbent assays (ELISAs) are the most commonly commercialized tests. Partial comparative studies have been performed in the past, and the panel of available ELISAs has notably changed in the last few years. Therefore, diagnostic laboratories are requesting updated information about the performance of these tests. Accordingly, the aim of this study was to compare all of the commercially available ELISAs (n = 10) by evaluating their performance and to re-standardize them based on TG-ROC analyses when necessary. For this purpose, a well-characterized serum panel from experimentally and naturally infected bovines and non-infected bovines (n = 458) was used. Two different definitions of gold standard were considered: (i) the result of the majority of tests and (ii) pre-test information based on epidemiological, clinical and serological data. Most of the tests displayed high sensitivity (Se) and specificity (Sp) values when both gold standard criteria were considered. Furthermore, all the tests showed near perfect agreement, with the exception of the pair-wise comparisons that included the VMRD and SVANOVIR. The best-adjusted ELISAs were the HIPRA-CIVTEST, IDVET, BIOVET and IDEXX Rum (Se and Sp > 95%). After the TG-ROC analyses, higher Se and Sp values were obtained for the BIO-X, LSI Bov, LSI Rum and IDEXX Bov, though the increases were more significant for the SVANOVIR and VMRD. The Kappa values also increased with the new adjusted cut-offs. This is the first study that offers updated performance evaluations of commercially available ELISAs. Such analyses are essential for diagnostic laboratories and are valuable to the companies that develop and distribute these tests.Item Modeling the Ruminant Placenta-Pathogen Interactions in Apicomplexan Parasites: Current and Future Perspectives(Frontiers in Veterinary Science, 2021) Jiménez-Pelayo, Laura; Pastor Fernández, Iván; Collantes Fernández, Esther; Ortega Mora, Luis Miguel; Horcajo Iglesias, María Del PilarNeospora caninum and Toxoplasma gondii are one of the main concerns of the livestock sector as they cause important economic losses in ruminants due to the reproductive failure. It is well-known that the interaction of these parasites with the placenta determines the course of infection, leading to fetal death or parasite transmission to the offspring. However, to advance the development of effective vaccines and treatments, there are still important gaps on knowledge on the placental host-parasite interactions that need to be addressed. Ruminant animal models are still an indispensable tool for providing a global view of the pathogenesis, lesions, and immune responses, but their utilization embraces important economic and ethics restrictions. Alternative in vitro systems based on caruncular and trophoblast cells, the key cellular components of placentomes, have emerged in the last years, but their use can only offer a partial view of the processes triggered after infection as they cannot mimic the complex placental architecture and neglect the activity of resident immune cells. These drawbacks could be solved using placental explants, broadly employed in human medicine, and able to preserve its cellular architecture and function. Despite the availability of such materials is constrained by their short shelf-life, the development of adequate cryopreservation protocols could expand their use for research purposes. Herein, we review and discuss existing (and potential) in vivo, in vitro, and ex vivo ruminant placental models that have proven useful to unravel the pathogenic mechanisms and the host immune responses responsible for fetal death (or protection) caused by neosporosis and toxoplasmosis.Item Proteome expression changes among virulent and attenuated Neospora caninum isolates(Journal of Proteomics, 2012) Regidor-Cerrillo, Javier; Álvarez García, Gema; Pastor Fernández, Iván; Marugán Hernández, Virginia; Gómez Bautista, Mercedes; Ortega Mora, Luis MiguelNeospora caninum is a cyst-forming parasite that has been recognised worldwide as a cause of cattle abortion and neuromuscular disease in dogs. Variations in genetic profiles, behaviour in vitro, and pathogenicity have been established among N. caninum isolates. However, it is unclear which parasite factors are implicated in this intra-specific diversity. Comparative analysis of protein expression patterns may define the determinants of biological diversity in N. caninum. Using DIGE and MALDI-TOF MS techniques, we quantified and identified differentially expressed proteins in the tachyzoite stage across three N. caninum isolates: the virulent Nc-Liv and Nc-Spain 7 isolates, and the attenuated Nc-Spain 1H isolate. Comparison between Nc-Spain 7 and Nc-Spain 1H extracts revealed 39 protein spots that were more abundant in Nc-Spain 7 and 21 in Nc-Spain 1H. Twenty-four spots were also increased in Nc-Spain 7 and 12 in Nc-Liv. Three protein spots were more abundant in the Nc-Liv extracts than in the Nc-Spain 1H extracts. MS analysis identified 11 proteins differentially expressed that are potentially involved in gliding motility and the lytic cycle of the parasite, and oxidative stress. These differences could help to explain variations in behaviour between isolates and provide a better knowledge of mechanisms associated with virulence.- Project number: 25
Item Parasit`Xpert, un blog de Parasitología Veterinaria(2023) Ferré Pérez, Ignacio; Ortega Mora, Luis Miguel; Álvarez García, Gema; Collantes Fernández, Esther; Horcajo Iglesias, María Del Pilar; Calero Bernal, Rafael; Pastor Fernández, Iván; Arranz-Solís, David; Sánchez Sánchez, Roberto; Regidor Cerrillo, Javier; Hecker, Yanina PaolaParasit’Xpert es un blog sobre enfermedades parasitarias de los rumiantes domésticos para el veterinario (https://parasitxpert.es/). Su objetivo es ser un punto de encuentro y referencia para los profesionales del sector y estudiantes de veterinaria que quieran conocer y profundizar en el campo de las enfermedades parasitarias. La plataforma se inició en enero de 2021 y se estructura en diversas secciones prácticas: parásito del mes, ¿sabías qué?, diagnóstico, control, zoonosis y avances. El grupo SALUVET y la empresa de transferencia del conocimiento universitario SALUVET-Innova han puesto en marcha la web Parasit’xpert junto a la empresa farmacéutica Boehringer Ingelheim Animal Health España. El objetivo general del presente proyecto de innovación docente fue desarrollar el blog Parasit’Xpert (https://parasitxpert.es/) ya en activo e implementar un plan de difusión promocional para incrementar el número de usuarios, especialmente en países hispanohablantes. Item Characterization of Neospora caninum virulence factors NcGRA7 and NcROP40 in bovine target cells(Veterinary Parasitology, 2023) Rico San Román, Laura; Amieva, Rafael; Horcajo Iglesias, María Del Pilar; García Sánchez, Marta Eulalia; Pastor Fernández, Iván; Ortega Mora, Luis Miguel; Collantes Fernández, EstherBovine neosporosis is one of the major causes of reproductive failure in cattle worldwide, and differences in virulence between isolates have been widely shown. However, the molecular basis and mechanisms underlying virulence in Neospora caninum are mostly unknown. Recently, we demonstrated the involvement of NcGRA7 and NcROP40 in the virulence of N. caninum in a pregnant murine model using single knockout mutants in these genes generated by CRISR/Cas9 technology. In this study, the role of these proteins was investigated in two in vitro models using bovine target cells: trophoblast (F3 cell line) and monocyte-derived macrophages (BoMØ). The proliferation capacity of the single knockout mutant parasites was compared to the wild-type strain, the Nc-Spain7 isolate, using both cell populations. For the bovine trophoblast, no differences were observed in the growth of the defective parasites compared to the wild-type strain, neither in the proliferation kinetics nor in the competition assay. However, in naïve BoMØ, a significant decrease in the proliferation capacity of the mutant parasites was observed from 48 h pi onwards. Stimulation of BoMØ with IFN-γ showed a similar inhibition of tachyzoite growth in defective and wild-type strains in a dose-dependent manner. Finally, BoMØ infected with knockout parasites showed higher expression levels of TLR3, which is involved in pathogen recognition. These results suggest that NcGRA7 and NcROP40 may be involved in the manipulation of innate immune defense mechanisms against neosporosis and confirm the usefulness of the BoMØ model for the evaluation of N. caninum virulence mechanisms. However, the specific functions of these proteins remain unknown, opening the way for future research.Item Identificación y caracterización de las proteínas NcROP40 y NcNTPasa, y evaluación de su utilidad vacunal frente a la neosporosis(2016) Pastor Fernández, Iván; Ortega Mora, Luis Miguel; Álvarez García, Gema; Regidor Cerrillo, JavierNeospora caninum es un parásito formador de quistes reconocido a nivel mundial como la principal causa de aborto en ganado vacuno, donde produce importantes pérdidas económicas. A pesar de que la vacunación se ha descrito como la estrategia de control más eficiente frente a la neosporosis bovina, hasta la fecha no existen formulaciones eficaces que prevengan la transmisión vertical y el aborto. En la actualidad, las medidas de control de la enfermedad dependen de un diagnóstico adecuado asociado a unas medidas de manejo concretas, por lo que el desarrollo de una vacuna frente a la neosporosis bovina es una tarea urgente. En este sentido, las vacunas de subunidades se presentan como una buena alternativa al uso de vacunas vivas, debido a su mayor seguridad y menor coste de producción; además, dichas vacunas pueden ser específicamente diseñadas frente a proteínas determinadas con el fin de bloquear procesos esenciales para la supervivencia del parásito. Desgraciadamente, el conocimiento de los mecanismos de invasión y proliferación de N. caninum es muy limitado a nivel molecular. Además, son pocos los estudios en los que se haya abarcado la identificación de factores de virulencia potenciales del parásito. Todo ello dificulta la selección de dianas apropiadas a la hora de diseñar nuevas formulaciones vacunales frente a la neosporosis...Item Characterization of the Neospora caninum NcROP40 and NcROP2Fam-1 rhoptry proteins during the tachyzoite lytic cycle(Parasitology, 2015) Regidor-Cerrillo, Javier; Jiménez-Ruiz, Elena; Marugán-Hernández, Virginia; Hemphill, Andrew; Pastor Fernández, Iván; Álvarez García, Gema; Ortega Mora, Luis MiguelVirulence factors from the ROP2-family have been extensively studied in Toxoplasma gondii, but in the closely related Neospora caninum only NcROP2Fam-1 has been partially characterized to date. NcROP40 is a member of this family and was found to be more abundantly expressed in virulent isolates. Both NcROP2Fam-1 and NcROP40 were evaluated as vaccine candidates and exerted a synergistic effect in terms of protection against vertical transmission in mouse models, which suggests that they may be relevant for parasite pathogenicity. NcROP40 is localized in the rhoptry bulbs of tachyzoites and bradyzoites, but in contrast to NcROP2Fam-1, the protein does not associate with the parasitophorous vacuole membrane due to the lack of arginine-rich amphipathic helix in its sequence. Similarly to NcROP2Fam-1, NcROP40 mRNA levels are highly increased during tachyzoite egress and invasion. However, NcROP40 up-regulation does not appear to be linked to the mechanisms triggering egress. In contrast to NcROP2Fam-1, phosphorylation of NcROP40 was not observed during egress. Besides, NcROP40 secretion into the host cell was not successfully detected by immunofluorescence techniques. These findings indicate that NcROP40 and NcROP2Fam-1 carry out different functions, and highlight the need to elucidate the role of NcROP40 within the lytic cycle and to explain its relative abundance in tachyzoites.Item A vaccine formulation combining rhoptry proteins NcROP40 and NcROP2 improves pup survival in a pregnant mouse model of neosporosis(Veterinary Parasitology, 2015) Pastor Fernández, Iván; Arranz-Solís, David; Regidor-Cerrillo, Javier; Álvarez García, Gema; Hemphill, Andrew; García Culebras, Alicia; Cuevas-Martín, Carmen; Ortega Mora, Luis MiguelCurrently there are no effective vaccines for the control of bovine neosporosis. During the last years several subunit vaccines based on immunodominant antigens and other proteins involved in adhesion, invasion and intracellular proliferation of Neospora caninum have been evaluated as targets for vaccine development in experimental mouse infection models. Among them, the rhoptry antigen NcROP2 and the immunodominant NcGRA7 protein have been assessed with varying results. Recent studies have shown that another rhoptry component, NcROP40, and NcNTPase, a putative dense granule antigen, exhibit higher expression levels in tachyzoites of virulent N. caninum isolates, suggesting that these could be potential vaccine candidates to limit the effects of infection. In the present work, the safety and efficacy of these recombinant antigens formulated in Quil-A adjuvant as monovalent vaccines or pair-wise combinations (rNcROP40 + rNcROP2 and rNcGRA7 + rNcNTPase) were evaluated in a pregnant mouse model of neosporosis. All the vaccine formulations elicited a specific immune response against their respective native proteins after immunization. Mice vaccinated with rNcROP40 and rNcROP2 alone or in combination produced the highest levels of IFN-γ and exhibited low parasite burdens and low IgG antibody levels after the challenge. In addition, most of the vaccine formulations were able to increase the median survival time in the offspring. However, pup survival only ensued in the groups vaccinated with rNcROP40 + rNcROP2 (16.2%) and rNcROP2 (6.3%). Interestingly, vertical transmission was not observed in those survivor pups immunized with rNcROP40 + rNcROP2, as shown by PCR analyses. These results show a partial protection against N. caninum infection after vaccination with rNcROP40 + rNcROP2, suggesting a synergistic effect of the two recombinant rhoptry antigens.