Effect of vaccination on protection and immune response against paratuberculosis using small ruminant in vitro and in vivo experimental models

219 p.[EN] La paratuberculosis es una enfermedad crónica de los rumiantes causada por la bacteria intracelular Mycobacterium avium subespecie paratuberculosis (Map). Hasta el momento, la vacunación ha sido considerada el método de control con una mejor relación coste-beneficio, al reducir la aparición de nuevos casos clínicos y la excreción fecal de bacilos. No obstante, su efecto protector es incompleto y aún no se conoce en profundidad cómo influye sobre la patogenia de la enfermedad y la respuesta inmunitaria del hospedador. Además, la vacunación frente a paratuberculosis se ha asociado con un efecto protector inespecífico frente a otras infecciones, relacionadas o no con micobacterias, un fenómeno que podría ser causado por el establecimiento de lo que se conoce como respuesta inmunitaria entrenada. Esta respuesta esta mediada por células de la respuesta inmunitaria innata, como los macrófagos, células dianas de Map, o los neutrófilos. Teniendo en cuenta el número limitado de trabajos destinados a estudiar las características de la respuesta protectora tras la vacunación, el objetivo general de esta Tesis Doctoral consiste en investigar su influencia en la respuesta inmunitaria del hospedador, tratando de esclarecer aquellos factores relacionados con la protección. Para poder realizar posteriormente estudios in vitro sobre el efecto de la vacunación en la actividad de neutrófilos y macrófagos, el primer estudio de esta Tesis consistió en establecer un protocolo estandarizado y efectivo para el aislamiento simultáneo de células mononucleares a partir de sangre periférica (PBMCs) y neutrófilos, así como la purificación de monocitos y la obtención de macrófagos derivados de monocitos (MDMs), a partir de sangre periférica de ovejas y de cabras. Para ello, se compararon distintas técnicas de aislamiento o purificación, así como diversas condiciones de cultivo para la maduración de monocitos. La estimación del rendimiento de cada protocolo, la pureza y las características fenotípicas de las poblaciones celulares obtenidas se determinaron mediante recuento celular en cámara de Neubauer, microscopía y citometría de flujo. La mezcla de las capas de leucocitos y de células rojas durante la separación con Lymphoprep™ permitió aislar un gran número de PBMCs y neutrófilos con una elevada pureza a partir de la misma muestra de sangre. Por otro lado, se observó que las columnas inmunomagnéticas proporcionan un buen rendimiento (nº monocitos/nº inicial de PBMCs) en la obtención de monocitos con una elevada pureza, determinado también mediante el porcentaje de células CD14+ , CMH-II+ y CD11b+ . Estos valores fueron superiores a los encontrados en los monocitos purificados mediante adherencia, debido a la presencia de un mayor número de linfocitos contaminantes empleando esta última técnica que, sin embargo, no interfirió en la maduración de monocitos a macrófagos, ya que el número final de MDMs fue superior en los monocitos aislados mediante adherencia. Posteriormente, la suplementación de los cultivos de monocitos con concentraciones crecientes de GM-CSF incrementó de manera proporcional el número de MDMs en comparación con el suero autólogo. Por lo tanto, siguiendo el protocolo establecido en este estudio es posible realizar el aislamiento simultáneo de PBMCs y neutrófilos con una elevada eficiencia y pureza. Así mismo, estos PBMCs son aptos para la purificación de monocitos y la obtención de MDMs con cualquiera de las técnicas analizadas, según las necesidades del experimento y siguiendo las condiciones de maduración establecidas. En este caso, se eligió la técnica de adherencia para la purificación de monocitos por ser la que proporcionó un mayor número de MDMs. El segundo estudio de la Tesis se centró en estudiar el efecto de la vacuna inactivada Silirum® sobre la respuesta inmunitaria generada en los macrófagos un mes después de la vacunación. Se emplearon CaMØs (MDMs caprinos) procedentes de cabras vacunadas con Silirum® y sin vacunar, e infectadas in vitro con Map, tras lo cual se determinó la viabilidad de Map y su cuantificación mediante cultivo bacteriológico y reacción en cadena de la polimerasa cuantitativa (qPCR), respectivamente. Además, también se analizaron los niveles de expresión de distintas citoquinas pro y antiinflamatorias e iNOS mediante PCR con transcripción inversa (RT-qPCR) y se estimó el número de CaMØs infectados, así como el grado de infección, mediante inmunofluorescencia. Estas pruebas reflejaron una reducción significativa de la viabilidad de Map en los sobrenadantes y los CaMØs procedentes de las cabras vacunadas, que se acompañaba de un mayor porcentaje de infección y un mayor número de bacterias internalizadas en los CaMØs de este mismo grupo en comparación con los no vacunados. Además, los CaMØs procedentes de animales vacunados mostraban una mayor expresión de iNOS (acción proinflamatoria) e IL-10 (antiinflamatoria), mientras que solo la expresión MIP-β (proinflamatoria) se encontraba elevada en los animales no vacunados. Estos resultados sugieren que la vacunación frente a paratuberculosis modifica la respuesta de los CaMØs, de forma que aumentan su capacidad fagocítica y antimicrobiana frente a Map, lo que aparece asociado a una mayor producción de citoquinas con acción tanto pro como antiinflamatoria. El tercer estudio de la Tesis tuvo como fin valorar el efecto de la administración de Silirum® sobre la generación de trampas extracelulares de neutrófilos (NETs) en respuesta a la infección in vitro con Map y otros patógenos no relacionados con micobacterias. Para ello, se utilizaron ovejas vacunadas y sin vacunar, a partir de las cuales se aislaron neutrófilos sanguíneos que fueron infectados in vitro con Map, Staphylococcus aureus y Escherichia coli. Se analizó la formación de NETs por medio de la visualización mediante inmunofluorescencia del ADN, la mieloperoxidasa y la elastasa, y se llevó a cabo una cuantificación del ADN extracelular (PicoGreen™) mediante fluorimetría. Estas pruebas demostraron que la infección con Map es capaz de estimular la liberación de NETs, aunque en menor medida que S. aureus y E. coli. No obstante, no se detectaron diferencias entre las ovejas vacunadas y las no vacunadas, independientemente de la infección y del tiempo post-vacunación. Estos resultados sugieren que la liberación de NETs no jugaría un papel importante en la respuesta inmune protectora tras la vacunación frente a paratuberculosis, al menos durante el primer mes post-vacunación. No obstante, es posible que otros mecanismos antimicrobianos desarrollados por estas células y no analizados en este estudio, puedan estar implicados en esta respuesta. El cuarto y último estudio consistió en valorar el efecto que tiene la vacunación homóloga (frente a paratuberculosis) o heteróloga (frente a tuberculosis) sobre la respuesta inmunitaria y la protección en cabritos infectados de forma experimental con Map, y conocer el papel que puede jugar cada uno de los componentes de estas vacunas. Con este fin, se emplearon cabritos de un mes de edad divididos en varios grupos en función de la vacunación (Silirum®, HIMB-vacuna inactivada frente a Mbv o no vacunados), inmunización (bacterias inactivadas o adyuvantes) y/o infección. A los 45 días post-vacunación, los animales fueron infectados oralmente con Map y, posteriormente, sacrificados a los 190 días post-vacunación. Mensualmente, se recogieron muestras de sangre para la determinación de la respuesta inmunitaria periférica y la proporción de subpoblaciones linfocitarias mediante la prueba de liberación de IFN-γ (IGRA) o la detección de anticuerpos (ELISA), y citometría de flujo, respectivamente. Además, tras el sacrificio, se recogieron muestras de heces (cultivo bacteriológico) y de intestino y nódulos linfáticos asociados para la valoración de la producción local de IFN-γ (IGRA), la detección de Map (qPCR y cultivo bacteriológico), el estudio de las subpoblaciones linfocitarias (citometría de flujo) y el examen histológico. En todos los grupos de animales infectados se observaron lesiones granulomatosas, independientemente de la vacunación o la inmunización. Sin embargo, los grupos vacunados con Silirum® (frente a Map) o HIMB (frente a Mbv) mostraron una considerable reducción del número y la gravedad de dichas lesiones, que se acompañaba de una elevada respuesta celular y humoral periféricas. También se observó una disminución del número de granulomas en los animales inmunizados con las cepas vacunales y los adyuvantes en comparación con el grupo no vacunado e infectado. Cabe destacar que esta reducción fue incluso mayor en las cabras a las que se les administró el adyuvante, en comparación con aquellas que fueron inmunizadas con cepas vacunales. Por otro lado, no se observaron diferencias en la proporción periférica o local de las diferentes subpoblaciones de linfocitos, ni en la respuesta inmunitaria local, entre los grupos establecidos. A la luz de estos resultados, tanto la vacunación frente a paratuberculosis como a tuberculosis ofreció una protección homóloga y heteróloga, respectivamente, contra la infección por Map. Además, los animales inmunizados con el adyuvante experimentaron una reducción del número de lesiones, no asociada a la presencia de una respuesta inmunitaria específica frente a Map detectable, que podría estar mediada por un mecanismo de inmunidad inespecífica que necesita ser esclarecido.Ministerio de Ciencia e Innovació

Assessment of Paratuberculosis Vaccination Effect on In Vitro Formation of Neutrophil Extracellular Traps in a Sheep Model

[EN] Vaccination of domestic ruminants against paratuberculosis has been related to homologous and heterologous protective effects that have been attributed to the establishment of a trained immune response. Recent evidence suggests that neutrophils could play a role in its development. Therefore, we propose an in vitro model for the study of the effect of paratuberculosis vaccination on the release of neutrophil extracellular traps (NETs) in sheep. Ovine neutrophils were obtained from non-vaccinated (n = 5) and vaccinated sheep (n = 5) at different times post-vaccination and infected in vitro with Mycobacterium avium subsp. paratuberculosis (Map), Staphylococcus aureus (SA), and Escherichia coli (EC). NETs release was quantified by fluorimetry and visualized by immunofluorescence microscopy. Typical NETs components (DNA, neutrophil elastase, and myeloperoxidase) were visualized extracellularly in all infected neutrophils; however, no significant percentage of extracellular DNA was detected in Map-infected neutrophils compared with SA- and EC-infected. In addition, no significant effect was detected in relation to paratuberculosis vaccination. Further assays to study NETs release in ovine neutrophils are needed. Preliminary results suggest no implication of NETs formation in the early immune response after vaccination, although other neutrophil functions should be evaluated.SIThis work was financially supported by the Spanish Ministry of Science and Innovation (projects AGL2015-66540-C2-1-R and RTI2018-099496-B-I00), and Junta de Castilla y León (project LE259P18). Noive Arteche-Villasol was the recipient of a predoctoral contract (BES-2016-076513) from the Spanish Ministry of Science and Innovation

Immunohistochemical Characterization of Tumor-Associated Macrophages in Canine Lymphomas

[EN] Macrophages have been confirmed to play a significant role in the behavior of human lymphomas, albeit no consistent data are so far available in canine lymphomas. The present study characterizes the macrophages present in cases of canine nodal lymphoma and their relationship with the histological grade and the immunophenotype. Samples from the lymph nodes of 25 dogs diagnosed with lymphoma were selected. Immunohistochemistry was used to determine the tumor immunophenotype (CD3 and CD20 antibodies) and macrophage characterization (Iba1, MAC387, CD204, CD163 and iNOS antibodies). Macrophage counting was performed in 10 randomly selected, high-power fields per sample. Generalized linear models with Poisson distribution were used for statistical analysis. A significantly greater number of macrophages (Iba1+) were detected in high-grade and B-cell lymphomas. The highest amount of both M1 (iNOS+) and M2 (CD204+ and CD163+) subtypes were observed in B-cell lymphomas. High-grade lymphomas showed a greater number of CD204+ and CD163+ cells and recently recruited MAC387+ macrophages. The latter were most abundant in T than in B-cell lymphomas. In conclusion, a significant population of macrophages is present in canine lymphomas, which constitute a heterogeneous population that shows variations in the amount and immunohistochemical profile according to the histological grade and immunophenotypeSIThis research received no external fundin

Local assessment of the immunohistochemical expression of Foxp3+ regulatory T lymphocytes in the different pathological forms associated with bovine paratuberculosis

[EN] Background: Mycobacterium avium subsp. paratuberculosis infected animals show a variety of granulomatous lesions, from focal forms with well-demarcated granulomas restricted to the gut-associated lymphoid tissue (GALT), that are seen in the initial phases or latency stages, to a diffuse granulomatous enteritis, with abundant (multibacillary) or scant (paucibacillary) bacteria, seen in clinical stages. Factors that determine the response to the infection, responsible for the occurrence of the different types of lesion, are still not fully determined. It has been seen that regulatory T cells (Treg) play an important role in various diseases where they act on the limitation of the immunopathology associated with the immune response. In the case of paratuberculosis (PTB) the role of Treg lymphocytes in the immunity against Map is far away to be completely understood; therefore, several studies addressing this subject have appeared recently. The aim of this work was to assess, by immunohistochemical methods, the presence of Foxp3+ T lymphocytes in intestinal samples with different types of lesions seen in cows with PTB. Methods: Intestinal samples of twenty cows showing the different pathological forms of PTB were evaluated: uninfected controls (n = 5), focal lesions (n = 5), diffuse paucibacillary (n = 5) and diffuse multibacillary (n = 5) forms. Foxp3+ lymphocyte distribution was assessed by differential cell count in intestinal lamina propria (LP), gut-associated lymphoid tissue (GALT) and mesenteric lymph node (MLN). Results: A significant increase in the number of Foxp3+ T cells was observed in infected animals with respect to control group, regardless of the type of lesion. However, when the different categories of lesion were analyzed independently, all individuals with PTB lesions showed an increase in the amount of Foxp3+ T lymphocytes compared to the control group but this increase was only significant in cows with focal lesions and, to a lesser extent, in animals with diffuse paucibacillary forms. The former showed the highest numbers, significantly different from those found in cows with diffuse lesions, where no differences were noted between the two forms. No specific distribution pattern was observed within the granulomatous lesions in any of the groups. Conclusions: The increase of Foxp3+ T cells in focal forms, that have been associated with latency or resistance to infection, suggest an anti-inflammatory action of these cells at these stages, helping to prevent exacerbation of the inflammatory response, as occurs in diffuse forms, responsible for the appearance of clinical signsSIThis research was funded by grants RTI2018-099496-B-I00 from the Spanish Ministry of Science and Innovation and LE259/P18 from “Junta de Castilla y León”. D. Zapico, M. Criado and N. Arteche-Villasol are supported by a predoctoral contract from the University of León (D. Zapico) or Spanish Ministry of Science and Innovation. J. Espinosa is recipient of a postdoctoral contract of “Juan de la Cierva-Formación (FJC2019-042422-I)” of the Spanish Ministry of Science and Innovatio

Peripheral IFN-ɣ Production after Blood Stimulation with Different Mycobacterial Antigens in Goats Vaccinated against Paratuberculosis

[EN] Vaccination can be an efficient method for the control of paratuberculosis in ruminants. However, the official tuberculosis control tests cross-interfere with the animals vaccinated against paratuberculosis. In order to test and compare new antigens that could solve this problem, the production of interferon-gamma (IFN-γ) in peripheral blood at different post-vaccination days in experimental kids and adult goats, in field conditions, using the avian and bovine purified protein derivative (PPD), the johnin, two peptide cocktails of Mycobacterium bovis (PC-EC and PC-HP) and the antigens VK 055 and VK 067 of Mycobacterium avium subspecies paratuberculosis (Map) has been analyzed in vitro. The non-specific production of IFN-γ was observed after blood stimulation with the PC-EC and PC-HP cocktail in any sample from vaccinated animals, whereas it was detected when bovine PPD was used. These results support the possible use of these new Mycobacterium bovis antigens in the in the differentiation of animals vaccinated against paratuberculosis or infected with tuberculosis by improving the specificity of bovine PPD. In contrast, the two Map antigens tested in this study did not improve the sensitivity of johnin or avian PPD in the detection of vaccinated or Map-infected goatsSIThis research was funded by Ministry of Economy, Industry and Competitiveness through the research project with reference: AGL2012-39818-C02-01 titled “Development of pathogenesis and immunization models in paratuberculosis”. Marcos Royo and Noive Arteche-Villasol were recipients of a predoctoral contract for training of doctors by the Ministry of Economy, Industry and Competitivenes

Effects of Paratuberculosis Vaccination at Different Ages in a Dairy Goat Herd: A 2-Year Follow-Up

[EN] Vaccination could be considered as an effective method for paratuberculosis control, although controversial, with a need for investigation in some aspects. The objective of this study was to evaluate the effect of vaccination, depending on the age of the animals, on their immune response, the reduction of paratuberculosis cases, mortality and culled animals in a commercial dairy herd. Goats from three different ages were immunized with the inactivated Gudair® vaccine. Peripheral antibody and IFN-γ output were evaluated for 21 months post-vaccination (mpv) and intradermal skin tests (IDSTs) for tuberculosis, with avian- and bovine-purified protein derivatives (PPD), were carried out at 6 and at 18 mpv to evaluate the humoral and cellular immune peripheral responses, respectively. The number of dead or culled animals, regardless of the reason, was also monitored and the causes of death determined by pathological examination. A significant increase in the production of IFN-γ was observed in all the vaccinated groups when the blood samples were stimulated with avian PPD, from 3 mpv to 18 mpv, and with bovine PPD, between 3 and 21 mpv. Moreover, serum antibody levels increased between 3 and 21 mpv in all vaccinated groups. The highest levels were found in animals vaccinated at 5 months, and the lowest in adult individuals. No positive reactants to tuberculosis were found by intradermal skin test. No animal losses associated with clinical paratuberculosis were detected in any of the groups. The number of total culled animals was significantly lower in the vaccinated than in the unvaccinated groups, especially on 1.5-month-old vaccinated kids. These results suggest that vaccination of paratuberculosis, especially in young animals, could induce heterologous protectionSIThis research was funded by Ministry of Economy, Industry and Competitiveness through the research project with reference: AGL2012-39818-C02-01. Marcos Royo and Noive Arteche-Villasol were recipient of a predoctoral contract of the Ministry of Economy, Industry and Competitivenes

Effects of Ovine Monocyte-Derived Macrophage Infection by Recently Isolated Toxoplasma gondii Strains Showing Different Phenotypic Traits

[EN] Ovine toxoplasmosis is one the most relevant reproductive diseases in sheep. The genetic variability among different Toxoplasma gondii isolates is known to be related to different degrees of virulence in mice and humans, but little is known regarding its potential effects in sheep. The aim of this study was to investigate the effect of genetic variability (types II (ToxoDB #1 and #3) and III (#2)) of six recently isolated strains that showed different phenotypic traits both in a normalized mouse model and in ovine trophoblasts, in ovine monocyte-derived macrophages and the subsequent transcript expression of cytokines and iNOS (inducible nitric oxide synthase). The type III isolate (TgShSp24) showed the highest rate of internalization, followed by the type II clonal isolate (TgShSp2), while the type II PRU isolates (TgShSp1, TgShSp3, TgShSp11 and TgShSp16) showed the lowest rates. The type II PRU strains, isolated from abortions, exhibited higher levels of anti-inflammatory cytokines and iNOS than those obtained from the myocardium of chronically infected sheep (type II PRU strains and type III), which had higher levels of pro-inflammatory cytokines. The present results show the existence of significant intra- and inter-genotypic differences in the parasite-macrophage relationship that need to be confirmed in in vivo experimentsSIThis research was funded by projects of the “Junta de Castilla y León” (LE080U16) and the Spanish Ministry of Sciences (AGL2016-75935-C2-1-R and AGL2016-75935-C2-2-R). R.V. and N.A.-V. were recipients of a predoctoral contract from the Ministry of Science and Innovation (grant nos. FPU-17/05346 and BES-2016076513, respectively

Influence of Heterologous and Homologous Vaccines, and Their Components, on the Host Immune Response and Protection Against Experimental Caprine Paratuberculosis

[EN] Vaccination against paratuberculosis, a chronic disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map), has been considered as the most effective control method. However, protection is incomplete, and the mechanisms operating in the response of the animals to vaccination are not fully understood. Therefore, this study analyzed the immune response and the effects on protection against Map infection, elicited by paratuberculosis (Silirum®) and tuberculosis (heat-inactivated M. bovis [HIMB]) vaccines and their components in a caprine experimental model. Fifty goat kids were divided into 10 groups (n = 5) according to their vaccination (Silirum®, HIMB and nonvaccinated), immunization (inactivated bacteria or adjuvant), and/or infection. Oral challenge with Map was performed 45 days postvaccination/immunization (dpv), and animals were euthanized at 190 dpv. Peripheral immune response and proportion of lymphocyte subpopulations were assessed monthly by enzyme-linked immunosorbent assay and flow cytometry analysis, respectively. Local immune response, proportion of tissue lymphocyte subpopulations, Map detection (polymerase chain reaction), and histological examination were conducted in gut-associated lymphoid tissues. All infected groups developed paratuberculosis granulomatous lesions despite vaccination or immunization. The Silirum® and HIMB-vaccinated groups showed a considerable lesion reduction consistent with a significant peripheral cellular and humoral immune response. Besides, a lower number of granulomas were observed in groups immunized with inactivated bacteria and adjuvants in comparison to nonvaccinated and infected group. However, despite not being significant, this reduction was even higher in adjuvant immunized groups, which developed milder granulomatous lesion with no detectable peripheral immune responses associated with immunization. No changes in the peripheral and local proportion of lymphocyte subsets or local immune response were detected in relation to either vaccination/immunization or infection. Despite that paratuberculosis and tuberculosis vaccination showed a partial and cross-protection against Map infection, respectively, only histological examination could assess the progression of infection in these animals. In addition, the pattern observed in the reduction of the lesions in adjuvant immunized groups suggests the possible involvement of a nonspecific immune response that reduces the development of granulomatous lesionsSIThis work was financially supported by the Spanish Ministry of Science and Innovation (projects AGL2015- 66540-C2-1-R and RTI2018-099496-B-I00), Junta de Castilla y León (project LE259P18), and National Institute for Agronomic Research (project RTA 2017- 00089-00-00). NA-V was the recipient of a predoctoral contract (BES-2016-076513) from the Spanish Ministry of Science and Innovation and DG-E and JE of a postdoctoral contract from the Ministry of Science and Innovation (grants nos. FJCI-2017-32020 and FJC2019-042422-I respectively

Assessment of Acute-Phase Protein Response Associated with the Different Pathological Forms of Bovine Paratuberculosis

[EN] In this study, the concentrations of two acute-phase proteins (APPs), haptoglobin (Hp) and serum amyloid A (SAA), were quantitatively assessed in serum samples from cattle naturally infected with paratuberculosis (PTB). APP profiles were compared across 190 animals classified according to the different pathological forms associated with infection: uninfected (n = 59), with focal lesions (n = 73), multifocal lesions (n = 19), and diffuse paucibacillary (n = 11) and diffuse multibacillary lesions (n = 28). Our results showed a significant increase in both APPs in infected animals compared to the control group, with differences depending on the type of lesion. Hp and SAA levels were increased significantly in all infected animals, except in cows with diffuse multibacillary lesions that showed similar values to non-infected animals. The expression pattern of both APPs was similar and negatively correlated with the antibody levels against PTB. These results indicate that the release of Hp and SAA is related to the presence of PTB lesions associated with a high cell-mediated immune response and a lower bacterial load, suggesting that the pro-inflammatory cytokines that are associated with these forms are the main stimulus for their synthesis. These molecules could show some potential to be used as putative biomarkers of PTB infection, particularly for the identification of subclinical animals showing pathological forms related to latency or resistance to the development of advanced lesionsSIThis study was funded by grants RTI2018-099496-B-I00 from the Spanish Ministry of Science and Innovation and LE259/P18 from “Junta de Castilla y León

Virulence in Mice of a Toxoplasma gondii Type II Isolate Does Not Correlate With the Outcome of Experimental Infection in Pregnant Sheep

[EN] Toxoplasma gondii is an apicomplexan parasite that infects almost all warm-blooded animals. Little is known about how the parasite virulence in mice extrapolates to other relevant hosts. In the current study, in vitro phenotype and in vivo behavior in mice and sheep of a type II T. gondii isolate (TgShSp1) were compared with the reference type II T. gondii isolate (TgME49). The results of in vitro assays and the intraperitoneal inoculation of tachyzoites in mice indicated an enhanced virulence for the laboratory isolate, TgME49, compared to the recently obtained TgShSp1 isolate. TgShSp1 proliferated at a slower rate and had delayed lysis plaque formation compared to TgME49, but it formed more cyst-like structures in vitro. No mortality was observed in adult mice after infection with 1-105 tachyzoites intraperitoneally or with 25-2,000 oocysts orally of TgShSp1. In sheep orally challenged with oocysts, TgME49 infection resulted in sporadically higher rectal temperatures and higher parasite load in cotyledons from ewes that gave birth and brain tissues of the respective lambs, but no differences between these two isolates were found on fetal/lamb mortality or lesions and number of T. gondii-positive lambs. The congenital infection after challenge at mid-pregnancy with TgShSp1, measured as offspring mortality and vertical transmission, was different depending on the challenged host. In mice, mortality in 50% of the pups was observed when a dam was challenged with a high oocyst dose (500 TgShSp1 oocysts), whereas in sheep infected with the same dose of oocysts, mortality occurred in all fetuses. Likewise, mortality of 9 and 27% of the pups was observed in mice after infection with 100 and 25 TgShSp1 oocysts, respectively, while in sheep, infection with 50 and 10 TgShSp1 oocysts triggered mortality in 68 and 66% of the fetuses/lambs. Differences in vertical transmission in the surviving offspring were only found with the lower oocyst doses (100% after infection with 10 TgShSp1 oocysts in sheep and only 37% in mice after infection with 25 TgShSp1 oocysts). In conclusion, virulence in mice of T. gondii type II isolates may not be a good indicator to predict the outcome of infection in pregnant sheepSIRS-S is supported by a fellowship from the Spanish Ministry of Education, Culture, and Sports (MECD), as a part of the Program of Training of University Teaching Staff (FPU, grant number FPU13/03438) and a mobility grant for predoctoral short stays in R+D centers (EST16/0719). DG-E is the recipient of a postdoctoral contract from the Junta de Castilla y León, partially funded by the European Social Fund (European Union). NA-V is the recipient of a predoctoral contract from the Ministerio de Economía, Industria y Competitividad (Ref. BES-2016-076513). This work was supported by the Ministry of Economy and Competitiveness (AGL2016-75935-C2-1-R and C2-2-R), the Community of Madrid, Spain (PLATESA, S2013/ABI2906), Junta de Castilla y León (LE080U16), and a grant of the Swiss National Science Foundation to AH (project No. 310030_165782