Person: Collantes Fernández, Esther
Universidad Complutense de Madrid
Faculty / Institute
Now showing 1 - 10 of 21
PublicationNcGRA7 and NcROP40 Play a Role in the Virulence of Neospora caninum in a Pregnant Mouse Model(MPDI, 2022-08-31) 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, 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. PublicationPatogenia de la neosporosis en el feto bovino y en un modelo murino experimental(Universidad Complutense de Madrid, Servicio de Publicaciones, 2005) Collantes Fernández, Esther; Ortega Mora, Luis MiguelEl primer objetivo de esta tesis consistió en el desarrollo de una PCR en tiempo realutilizando SYBR Green I o sondas TaqMan para la cuantificación de "N. caninum" en los tejidos infectados. El método más sensible fue el SYBR Green 1 el cual se aplicó con éxito en muestras de encéfalo. El segundo objetivo fue el desarrollo de un modelo murino experimental para el estudio de la patogenia de la infección. Para su cumplimiento se propusieron dos subobjetivos. El primero fue el estudio del efecto de los factores dependientes del hospedador y del parásito en el desarrollo de la infección crónica. Los resultados obtenidos sugieren que la infección estuvo influenciada por: la estirpe murina, tratamiento inmunodepresor y la dosis infectiva. En los ratones BALB/c existió una asociación entre la gravedad de la lesión y la carga parasitaria en el cerebro. En el segundo subobjetivo se caracterizó la distribución temporal y la cinética de la carga parasitaria en la sangre y en diversos órganos durante el curso de la infección. Se diferenciaron tres fases. La primera caracterizada por una diseminación sistémica del parásito y su multiplicación en diferentes órganos. El segundo periodo el parásito se detectó en pulmón y cerebro. En la tercera fase el parásito permaneció en cerebro. Se observaron diferencias en la virulencia de los aislados NC-Liv y NC-1 y se demostró una asociación entre la carga parasitaria en cerebro y la presencia de sintomatología. El último objetivo fue el estudio de la infección por "N. caninum" en fetos bovinos abortados, investigando la influencia de la edad fetal y las características epidemiológicas del aborto. Se observó una mayor distribución y cargas parasitarias, asícomo lesiones más graves en los fetos correspondientes al primer y segundo periodo de gestación. Los fetos de abortos epidémicos mostraron una inayor diseminación y cargas del parásito, así como lesiones más graves. PublicationParasitología interactiva: Protozoos y afines(2018-09-11) Collantes Fernández, Esther; Gómez Bautista, Mercedes; Ortega Mora, Luis Miguel; Álvarez García, Gema; Ferré Pérez, Ignacio; García Lunar, Paula; Sánchez Sánchez, Roberto; Jiménez Meléndez, Alejandro; García Sánchez, Marta; Jiménez Pelayo, Laura; Diezma Díaz, Carlos; Regidor Cerrillo, Javier; Moreno González, Javier; Horcajo Iglesias, PilarDesarrollo 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. PublicationSystemic and local immune responses in sheep after Neospora caninum experimental infection at early, mid and late gestation(BMC, 2016-01-06) Arranz-Solís, David; Benavides, Julio; Regidor-Cerrillo, Javier; Horcajo Iglesias, María Del Pilar; Castaño, Pablo; Ferreras, María del Carmen; Jiménez-Pelayo, Laura; Collantes Fernández, Esther; Ferré Pérez, Ignacio; Hemphill, Andrew; Pérez, Valentín; Ortega Mora, Luis MiguelBesides its importance in cattle, Neospora caninum may also pose a high risk as abortifacient for small ruminants. We have recently demonstrated that the outcome of experimental infection of pregnant sheep with 106 Nc-Spain7 tachyzoites is strongly dependent on the time of gestation. In the current study, we assessed peripheral and local immune response in those animals. Serological analysis revealed earlier and higher IFN-γ and IgG responses in ewes infected at early (G1) and mid (G2) gestation, when abortion occurred. IL-4 was not detected in sera from any sheep. Inflammatory infiltrates in the placenta mainly consisted of CD8+ and, to a lesser extent, CD4+ T cells and macrophages (CD163+). The infiltrate was more intense in sheep infected at mid-gestation. In the foetal mesenchyme, mostly free tachyzoites were found in animals infected at G1, while those infected in G2 displayed predominantly particulate antigen, and parasitophorous vacuoles were detected in sheep infected at G3. A similar pattern of placental cytokine mRNA expression was found in all groups, displaying a strengthened upregulation of IFN-γ and IL-4 and milder increases of TNF-α and IL-10, reminiscent of a mixed Th1 and Th2 response. IL-12 and IL-6 were only slightly upregulated in G2, and TGF-β was downregulated in G1 and G2, suggestive of limited T regulatory (Treg) cell activity. No significant expression of TLR2 or TLR4 could be detected. In summary, this study confirms the pivotal role of systemic and local immune responses at different times of gestation during N. caninum infection in sheep. PublicationTritrichomonas foetus infection in cats with diarrhea from denselyhoused origins(Elsevier, 2016-05) Arranz-Solís, David; Pedraza-Díaz, Susana; Miró Corrales, Guadalupe; Rojo-Montejo, Silvia; Hernández, Leticia; Ortega Mora, Luis Miguel; Collantes Fernández, EstherTritrichomonas foetus is a protozoan parasite that has been recently identified as a causative agent of chronic diarrhea in domestic cats. Transmission of infection occurs by the fecal-oral route through direct contact among animals. Consequently, feline trichomonosis (FT) is more likely to be present in multi-cat environments. The objective of this work was to study the presence of T. foetus and some associated risk factors in cats from densely housed origins and with a reported history of chronic diarrhea. Animals enrolled in this study were family cats (n = 15) acquired from pet shops, shelters or breeding centers and cattery cats belonging to one breeding center (n = 28) and two cat shelters (A and B, n = 25 each). In the catteries, a follow-up analysis for a period of up to 2 months was also performed to determine the parasite shedding pattern in feces and the incidence of infection. Fecal samples were analyzed using in vitro culture and a PCR technique. T. foetus was detected in a total of 38.7% (36/93) of the cats with chronic diarrhea. Parasite infection was similarly detected in family cats and cattery animals (40% versus 38.4%). In the catteries, the parasite was detected in 50%, 44% and 20% of the animals from the breeding center and shelters A and B, respectively. The follow-up analysis showed that 58.3% of infected cats intermittently shed trophozoites in their feces, with an incidence of 23.1%. Investigation of potential risk factors showed that cats ≤1 year old were more likely to be infected than older cats (57.1% versus 27.3%; P < 0.05). No significant differences were found when sex and breed factors were studied. These results confirm the importance of FT as a cause of chronic diarrhea in cats and highlight the relevance of close contact conditions for T. foetus transmission. PublicationMultilocus analysis reveals further genetic differences between Tritrichomonas foetus from cats and cattle(Elsevier, 2019-12) Pedraza-Díaz, Susana; Arranz-Solís, David; Gómez-Couso, Hipólito; Fuschs, Lumila; Fort, Marcelo; Rengifo-Herrera, Claudia; Navarro-Lozano, Vanesa; Ortega Mora, Luis Miguel; Collantes Fernández, EstherTritrichomonas foetus isolates from feline and bovine origin has been previously shown to carry a certain degree of genetic heterogeneity. Here, novel candidate molecular markers were developed by means of multilocus sequence typing of the gap2 gene (encoding for T. foetus glyceraldehyde-3-phosphate dehydrogenase), ITS region, the TR7/TR8 variable-length repeat and microsatellite genotyping. These markers were used to characterize T. foetus field isolates from bulls and domestic cats and to compare phylogenetically with the following ATCC isolates: T. foetus isolated from cattle and pig (syn. Tritrichomonas suis), Tritrichomonas mobilensis, Tetratrichomonas gallinarum and Pentatrichomonas hominis. Among them, TFMS10 and TFMS7 were found to be the most polymorphic markers. Moreover, an 809 bp fragment of the gap2 gene was successfully amplified from all the trichomonads included in this study and the sequence analysis revealed differences between T. foetus porcine and feline genotypes and T. mobilensis in comparison to the bovine T. foetus ATCC isolate. The TR7/TR8 repeat pattern was not reproducible, being only consistent the fragments of approximately 110 and 217 bp. Sequence analysis of the latter revealed the existence of 3 SNPs resulting in 98.6 % homology between bovine and feline isolates. A search for similar sequences was carried out to develop a Restriction Length Fragment Polymorphism analysis. A 503 bp region, named TF1, revealed the existence of two BbvI restriction enzyme sites that were able to generate different length fragments for T. foetus feline and bovine isolates. Finally, the neighbour-joining analyses showed that T. foetus porcine genotype clusters together with bovine genotype, whereas T. mobilensis and the feline genotype form a separate cluster. PublicationOvine placental explants: a new ex vivo model to study host‒pathogen interactions in reproductive pathogens(Elsevier, 2023-09-13) Horcajo Iglesias, María Del Pilar; Ortega Mora, Luis Miguel; Benavides, Julio; Sánchez Sánchez, Roberto; Ammieva, Rafael; Collantes Fernández, Esther; Pastor Fernández, IvánReproductive failure is one of the main performance constraints in ruminant livestock. Transmissible agents such as Toxoplasma gondii and Neospora caninum are commonly involved in the occurrence of abortion in ruminants, but little is known about the mechanisms involved. While in vivo models are optimal for the study of abortion pathogenesis, they have a high economic cost and come with ethical concerns. Unfortunately, alternative in vitro models fail to replicate the complex in vivo placental structure. To overcome the limitations of currently available models, we developed an ex vivo model based on the cultivation of fresh and cryopreserved sheep placental explants, enabling the biobanking of tissues. Reproducible and simple markers of tissue integrity (histology, RNA concentrations), viability (resazurin reduction), and functionality (synthesis of steroid hormones) were also investigated, allowing a clear quality assessment of the model. This work shows that, similar to fresh explants, tissues cryopreserved in ethylene glycol using slow freezing rates maintain not only their structure and function but also their receptivity to T. gondii and N. caninum infection. In addition, the findings demonstrate that explant lifespan is mainly limited by the culture method, with protocols requiring improvements to extend it beyond 2 days. These findings suggest that cryopreserved tissues can be exploited to study the initial host‒pathogen interactions taking place in the placenta, thus deepening the knowledge of the specific mechanisms that trigger reproductive failure in sheep. Importantly, this work paves the way for the development of similar models in related species and contributes to the reduction of experimental animal use in the future. PublicationCharacterization of Neospora caninum virulence factors NcGRA7 and NcROP40 in bovine target cells(Elsevier, 2023-06-14) 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. PublicationModeling the Ruminant Placenta-Pathogen Interactions in Apicomplexan Parasites: Current and Future Perspectives(Frontiers Media, 2021-01-21) 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. PublicationParasit`Xpert, un blog de Parasitología Veterinaria(2023-07-12) Ferre Pérez, Ignacio; Ortega Mora, Luis Miguel; Álvarez García, Gema; Collantes Fernández, Esther; Horcajo Iglesias, 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.