Person: Ortega Mora, Luis Miguel
Universidad Complutense de Madrid
Faculty / Institute
Now showing 1 - 10 of 13
PublicationProteome expression changes among virulent and attenuated Neospora caninum isolates(Elsevier, 2012-04-18) 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. 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. PublicationToxoplasma gondii Genetic Diversity in Mediterranean Dolphins(MPDI, 2022-08-12) Fernández Escobar, Mercedes; Giorda, Federica; Mattioda, Virgina; Audino, Tania; Di Nocera, Fabio; Lucifora, Giuseppe; Varello, Katia; Grattarola, Carla; Ortega Mora, Luis Miguel; Casalone, Cristina; Calero Bernal, RafaelToxoplasma gondii constitutes a major zoonotic agent but also has been frequently identified as an important cause of clinical disease (e.g., abortion, pneumonia, encephalitis) in wildlife; specifically, T. gondii has been associated with neurological disease in cetaceans. This study investigated the genetic diversity of T. gondii strains involved in infections in dolphins found stranded in the Mediterranean coastlines of Italy. Tissue samples from 16 dolphins (Stenella coeruleoalba and Tursiops truncatus species) positive for T. gondii-DNA presence by PCR were examined by histology and subjected to further genetic characterization of strains detected by PCR-RFLP and multilocus PCR-sequencing assays. According to fully genotyped samples, the genotypes ToxoDB#3 (67%) and #2 (22%) were detected, the latter being reported for the first time in cetaceans, along with a mixed infection (11%). Subtyping by PCR-seq procedures provided evidence of common point mutations in strains from southwestern Europe. Despite evidence of T. gondii as a cause of neurological disease in dolphins, sources of infections are difficult to identify since they are long-living animals and some species have vast migration areas with multiple chances of infection. Finally, the genetic diversity of T. gondii found in the dolphins studied in the Mediterranean coastlines of Italy reflects the main genotypes circulating inland in the European continent. PublicationNeospora caninum: Structure and Fate of Multinucleated Complexes Induced by the Bumped Kinase Inhibitor BKI-1294(MDPI, 2020-05-16) Winzer, Pablo; Anghel, Nicoleta; Imhof, Dennis; Balmer, Vreni; Ortega Mora, Luis Miguel; Ojo, Kayode K.; Van Voorhis, Wesley C.; Müller, Joachim; Hemphill, AndrewBackground: Bumped kinase inhibitors (BKIs) are potential drugs for neosporosis treatment in farm animals. BKI-1294 exposure results in the formation of multinucleated complexes (MNCs), which remain viable in vitro under constant drug pressure. We investigated the formation of BKI-1294 induced MNCs, the re-emergence of viable tachyzoites following drug removal, and the localization of CDPK1, the molecular target of BKIs. Methods: N. caninum tachyzoites and MNCs were studied by TEM and immunofluorescence using antibodies directed against CDPK1, and against NcSAG1 and IMC1 as markers for tachyzoites and newly formed zoites, respectively. Results: After six days of drug exposure, MNCs lacked SAG1 surface expression but remained intracellular, and formed numerous zoites incapable of disjoining from each other. Following drug removal, proliferation continued, and zoites lacking NcSAG1 emerged from the periphery of these complexes, forming infective tachyzoites after 10 days. In intracellular tachyzoites, CDPK1 was evenly distributed but shifted towards the apical part once parasites were extracellular. This shift was not affected by BKI-1294. Conclusions: CDPK1 has a dynamic distribution depending on whether parasites are located within a host cell or outside. During MNC-to-tachyzoite reconversion newly formed tachyzoites are generated directly from MNCs through zoites of unknown surface antigen composition. Further in vivo studies are needed to determine if MNCs could lead to a persistent reservoir of infection after BKI treatment. PublicationImmunization with a Multivalent Listeria monocytogenes Vaccine Leads to a Strong Reduction in Vertical Transmission and Cerebral Parasite Burden in Pregnant and Non-Pregnant Mice Infected with Neospora caninum(MPDI, 2023-01-11) Imhof, Dennis; Pownall, William; Hänggeli, Kai Pascal Alexander; Monney, Camille; Rico San Román, Laura; Ortega Mora, Luis Miguel; Forterre, Franck; Oevermann, Anna; Hemphill, AndrewNeospora caninum is an apicomplexan parasite that causes abortion and stillbirth in cattle. We employed the pregnant neosporosis mouse model to investigate the efficacy of a modified version of the attenuated Listeria monocytogenes vaccine vector Lm3Dx_NcSAG1, which expresses the major N. caninum surface antigen SAG1. Multivalent vaccines were generated by the insertion of gra7 and/or rop2 genes into Lm3Dx_NcSAG1, resulting in the double mutants, Lm3Dx_NcSAG1_NcGRA7 and Lm3Dx_NcSAG1_NcROP2, and the triple mutant, Lm3Dx_NcSAG1_NcGRA7_NcROP2. Six experimental groups of female BALB/c mice were inoculated intramuscularly three times at two-week intervals with 1 × 107 CFU of the respective vaccine strains. Seven days post-mating, mice were challenged by the subcutaneous injection of 1 × 105 N. caninum NcSpain-7 tachyzoites. Non-pregnant mice, dams and their offspring were observed daily until day 25 post-partum. Immunization with Lm3Dx_NcSAG1 and Lm3Dx_NcSAG1_NcGRA7_NcROP2 resulted in 70% postnatal pup survival, whereas only 50% and 58% of pups survived in the double mutant-vaccinated groups. Almost all pups had died at the end of the experiment in the infection control. The triple mutant was the most promising vaccine candidate, providing the highest rate of protection against vertical transmission (65%) and CNS infection. Overall, integrating multiple antigens into Lm3Dx_SAG1 resulted in lower vertical transmission and enhanced protection against cerebral infection in dams and in non-pregnant mice. PublicationReactivation and Foetal Infection in Pregnant Heifers Infected with Neospora caninum Live Tachyzoites at Prepubertal Age(MPDI, 2022-07-25) Hecker, Yanina P.; Burucúa, Mercedes M.; Fiorani, Franco; Maldonado Rivera, Jaime E.; Cirone, Karina M.; Dorsch, Matías A.; Cheuquepán, Felipe A.; Campero, Lucía M.; Cantón, Germán J.; Marín, Maia S.; Ortega Mora, Luis Miguel; Moore, Dadín P.Neospora caninum is recognised for causing cattle abortion, provoking severe economic losses in the livestock industry worldwide. The aim of the present study was to evaluate the reactivation and foetal infection in pregnant heifers inoculated with live N. caninum tachyzoites before puberty. A total of 15 30-month-old pregnant heifers were allocated into four groups: animals inoculated with live tachyzoites of NC-Argentina LP1 isolate before puberty and challenged with live tachyzoites of NC-1 strain at 210 days of gestation (DG) (Group A); animals mock inoculated before puberty and challenged with NC-1 strain at 210 DG (Group B), animals inoculated before puberty but not subsequently challenged (Group C); and noninfected and nonchallenged animals (Group D). The results of this study showed that 100% of animals infected before puberty (Groups A and C) suffered reactivation of the infection at the seventh month of gestation. In addition, in three and two calves from Groups A and C, respectively, congenital infection was confirmed. Interestingly, we provide evidence that the use of live N. caninum tachyzoites in young animals as a strategy to induce protection is neither safe nor effective. PublicationFatal toxoplasmosis in a captive squirrel monkey (Saimiri boliviensis) in Portugal(Springer Nature, 2023-07-21) Salas-Fajardo, Martha Ynés; Benavides, Julio; Azevedo, Alexandre; Figueiras, Paulo; Monteiro, Madalena; Orge, Leonor; Mendoça, Paula; Carvalho, Paulo; Waap, Helga; Ortega Mora, Luis Miguel; Calero Bernal, RafaelNew World monkeys are especially vulnerable to develop severe clinical manifestations and succumb to acute toxoplasmosis. This study aimed to describe the histopathological findings and genotypic characterization of the Toxoplasma gondii strain involved in a lethal case occurring in a zoo-housed black-capped squirrel monkey (Saimiri boliviensis) in Portugal. Cyst-like structures suggestive of Sarcocystidae parasites and acute injuries in liver and brain were observed by light microscopy examination. By immunohistochemistry, calprotectin, T. gondii antigen and Iba1 antigen had a positive signaling in lung, liver and brain tissues. Toxoplasma gondii B1, ITS1 and 529 repetitive element fragments amplifications together with the genotyping of 13 microsatellite markers confirmed a systemic T. gondii infection linked to a non-clonal type II strain. This description is consistent to the majority T. gondii strains circulating in Europe. PublicationDynamics of Neospora caninum-Associated Abortions in a Dairy Sheep Flock and Results of a Test-and-Cull Control Programme(MPDI, 2021-11-20) Sánchez Sánchez, Roberto; Vázquez Calvo, Ángela; Fernández Escobar, Mercedes; Regidor Cerrillo, Javier; Benavides, Julio; Gutiérrez, Jorge; Gutierrez Expósito, Daniel; Crespo Ramos, Francisco José; Ortega Mora, Luis Miguel; Álvarez García, GemaNeospora caninum is an apicomplexan parasite that can cause abortions and perinatal mortality in sheep. Although ovine neosporosis has been described worldwide, there is a lack of information about the relationship between N. caninum serostatus and the reproductive performance. In this study, we described the infection dynamics in a dairy sheep flock with an abortion rate up to 25% and a N. caninum seroprevalence of 32%. Abortions were recorded in 36% and 9% of seropositive and seronegative sheep, respectively. Seropositive sheep were more likely to abort twice (OR = 4.44) or three or more times (OR = 10.13) than seronegative sheep. Endogenous transplacental transmission was the main route of transmission since 86% of seropositive sheep had seropositive offspring. Within dams that had any abortion, seropositive sheep were more likely than seronegative ones to have female descendants that aborted (OR = 8.12). The slight increase in seropositivity with the age, the low percentage of animals with postnatal seroconversion or with low avidity antibodies, and the seropositivity of one flock dog, indicated that horizontal transmission might have some relevance in this flock. A control programme based on selective culling of seropositive sheep and replacement with seronegative animals was effective in reducing the abortion rate to 7.2%. 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. PublicationA short-term treatment with BKI-1294 does not protect foetuses from sheep experimentally infected with Neospora caninum tachyzoites during pregnancy(Elsevier, 2021-10-08) Sánchez Sánchez, Roberto; Ferré Pérez, Ignacio; Re, Michela; Pérez Arroyo, Bárbara; Cleofé Resta, Darío; Herrero García, Victor; Pizarro Díaz, Manuel; Ferrer, Luis Miguel; Ruiz, Hector; Vallejo García, Raquel; Benavides, Julio; Hulverson, Matthew A.; Choi, Ryan; Whitman, Grant R.; Hemphill, Andrew; Van Voorhis, Wesley C.; Ortega Mora, Luis MiguelThe Neospora caninum Calcium-dependent protein kinase 1 (NcCDPK1) inhibitor BKI-1294 had demonstrated excellent efficacy in a pregnant mouse model of neosporosis, and was also highly efficacious in a pregnant sheep model of toxoplasmosis. In this work, we present the efficacy of BKI-1294 treatment (dosed 5 times orally every 48 h) starting 48 h after intravenous infection of sheep with 105 Nc-Spain7 tachyzoites at mid-pregnancy. In the dams, BKI-1294 plasma concentrations were above the IC50 for N. caninum for 12–15 days. In treated sheep, when they were compared to untreated ones, we observed a minor increase in rectal temperature, higher IFNγ levels after blood stimulation in vitro, and a minor increase of IgG levels against N. caninum soluble antigens through day 28 post-infection. Additionally, the anti-NcSAG1 and anti-NcSAG4 IgGs were lower in treated dams on days 21 and 42 post-infection. However, BKI-1294 did not protect against abortion (87% foetal mortality in both infected groups, treated and untreated) and did not reduce transplacental transmission, parasite load or lesions in placentomes and foetal brain. The lack of foetal protection was likely caused by short systemic exposure in the dams and suboptimal foetal exposure to this parasitostatic drug, which was unable to reduce replication of the likely established N. caninum tachyzoites in the foetus at the moment of treatment. New BKIs with a very low plasma clearance and good ability to cross the blood-brain and placental barriers need to be developed.