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New 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.

Reproductive 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.

Toxoplasma gondii is a major cause of reproductive failure in small ruminants. Genotypic diversity of T. gondii strains has been associated with variations in phenotypic traits in in vitro and murine models. However, whether such diversity could influence the outcome of infection in small ruminants remains mostly unexplored. Here, we investigate the outcome of oral challenge in sheep at mid-pregnancy with 10 sporulated oocysts from three different T. gondii isolates belonging to archetypal II and III and selected according to their genetic and phenotypic variations shown in previous studies. Seventy-three pregnant sheep were divided in four groups: G1 infected with TgShSp1 isolate (type II, ToxoDB#3), G2 with TgShSp16 isolate (type II, ToxoDB#3), G3 with TgShSp24 isolate (type III, ToxoDB#2) and G4 of uninfected control sheep. Two different approaches were carried out within this study: (i) the outcome for the pregnancy after infection (n = 33) and (ii) the lesions and parasite tropism and burden at 14 and 28 days post infection (dpi) (n = 40). The onset of hyperthermia and seroconversion occurred one and two days later, respectively in G1 when compared to G2 and G3. However, sheep that suffered from reproductive failure, either by abortion, foetal dead at the time of euthanasia or stillbirth were similar among infected groups (50%, 40% and 47%, respectively). Histological lesions in placentomes and foetal tissues from euthanized animals from the second approach were only detected at 28 dpi and mainly in G1. At 14 dpi, T. gondii-DNA was only detected in G1 in the 11% of the placentomes. However, at 28 dpi the frequency of detection in placentomes was higher in G1 (96%) than in G2 and G3 (7% and 47%, respectively) besides in foetuses was lower in G2 (20%) than in G1 and G3 (100% and 87%, respectively). Regarding late abortions, stillbirths, and lambs of G1, G2 and G3, the frequency of microscopic lesions was similar between groups (79%, 78% and 67%, respectively) whe

[EN] Toxoplasmosis is a protozoan infectious disease caused by the parasite Toxoplasma gondii. It is an important disease in both in Europe and globally due to its zoonotic potential and the reproductive failure it triggers in livestock, mainly in sheep, which results in heavy economic losses. Despite the fact that there are multiple methods for controlling this disease, such as management, vaccination or treatment; toxoplasmosis is still a very relevant animal health and public problem. There are still many unknown mechanisms of the pathogenesis of ovine toxoplasmosis, mainly regarding the occurrence of abortions. On the other hand, there are evidences that the variability of T. gondii isolates could influence the pathogenesis of the disease in murine models and humans’ observational studies. However, little is known regarding the influence of this variation in sheep. Bearing in mind that extrapolation of results between species is not always possible, it is necessary to study how the genotypic and phenotypic variability of T. gondii isolates could influence the development of the disease in pregnant sheep. For this, it is essential to use an experimental model of pregnant sheep, as the placenta and fetus are the main targets of the parasite. In order to clearly identified lesions caused by the disease, a detailed analysis of the morphological and physiological findings in both placenta and fetuses is also necessary. Thus, the aim of this PhD Thesis is to analyze the influence of T. gondii isolates variability on parasite/host interaction in ovine toxoplasmosis and the consequences it may have on pregnant sheep. For this purpose, four studies have been conducted, focused in four specific objectives and based on two different experiments (Experiment 1 and 2). An in vitro assay with ovine macrophages derived from peripheral blood monocytes (OvMØs) was developed for the evaluation of the influence of isolates variability on these cells (Experiment 1). Then an in vivo, Chapter I is based on Experiment 1 with the specific aim of developing an in vitro model of OvMØs and study the influence that genetic variability of different T. gondii isolates may have on their response (objective 1). In this study, OvMØs were infected with six T. gondii isolates, obtained from Spanish sheep. Afterwards, the subsequent cell infections rates and transcript expression of cytokines and iNOS were evaluated. TgShSp24 isolate (Type III, ToxoDB#2) had a higher internalization/infection rate, followed by TgShSp2 (Type II, ToxoDB#1); compared with the rest of type II isolates: TgShSp1, TgShSp3, TgShSp11 and TgShSp16 (Type II, ToxoDB#3). Moreover, these two isolates (i.e. TgShSp24 and TgShSp2) also exhibited increments in cytokines that favored inflammation by a M1 macrophages polarization. TgShSp1 and TgShSp3 (Type II, ToxoDB#3 isolated from abortions) exhibited highest levels of anti-inflammatory cytokines. Isolates coming from abortions (fetal brains) triggered a higher iNOS expression. These results show intra- and inter-genotypic differences in the parasite/macrophage relationship, proving that more genotypic and phenotypic traits must be evaluated for the study of the virulence of T. gondii isolates. Besides, macrophages polarization could be affected depending on the infecting isolate. Chapter II, III and IV are based in the results from Experiment 2, where seventy-three pregnant sheep were divided in four groups an orally inoculated at day 90 of gestation according to the following distribution: G1 infected with TgShSp1 isolate (type II, ToxoDB#3), G2 with TgShSp16 isolate (type II, ToxoDB#3), G3 with TgShSp24 isolate (type III, ToxoDB#2) and G4 of uninfected control sheep. Once inoculated clinical sings were evaluated and early and late abortions were noted. Five animals per group were culled at 14 and 28 dpi. The remaining sheep were left until delivery occurred (i.e. lambs or stillbirths). Specifically, Chapter II is based on the description of, Moreover, almost half of the placentomes evaluated showed hemorrhages, foci of cellular debris, protein extravasation and mineralization. Moderate levels of T cells and Iba1 positive macrophages, and scarce B cells were present in the interdigitating zone of the placentome. The most striking finding was the absence of NK cells in the placentome. Cellular debris and areas of hyalinization were not immunolabelled for caspase-3. In fetal organs the most common findings were hematopoiesis, lymphocyte infiltrates and vacuolation of the epithelial cells of the eye, esophagus, intestine and kidney. It is relevant to establish that all these macroscopic and histological findings are found under normal conditions, as they might lead to misinterpretations when analyzing samples from an experimental model of pregnant sheep. Chapter III is aimed to study the influence of the genetic and phenotypic variability of previously mentioned three T. gondii isolates on the development of toxoplasmosis in pregnant sheep at mid gestation (objective 3), and it is based on the examination of all the animals from Experiment 2. The isolates used in this experiment had shown intra- and inter-genotypic variations in previous in vitro and in vivo experimental studies. In this study, the clinical outcome of the infection up to the end of gestation was investigated, together with the occurrence of histological lesions, the parasite distribution and burden at 14 and 28 dpi. Reproductive failure including early or late abortions and stillbirths was similar for all the groups. However, the onset of fever and seroconversion occurred later for sheep infected with TgShSp1 Isolate compared with those infected with TgShSp16 isolate and TgShSp24 isolate. Regarding serial culling, T. gondii-DNA was only detected in few placentomes from TgShSp1 infected sheep at 14 dpi, while at 28 dpi, the parasite was detected in all the groups in at least one animal. However, at 28 dpi, parasite detection and burdens were, [ES] La Equinococosis quística (CE) o Hidatidosis es una zoonosis cosmopolita crónica causada por el metacéstodo de un complejo de especies crípticas pertenecientes a Echinococcus granulosus s.l., siendo Echinococcus granulosus s.s. la especie causante de la mayor parte de los casos de CE humana y se encuentra distribuida en todo el mundo. Esta cestodiasis es un importante problema de salud pública y económico para las poblaciones afectadas, principalmente en zonas rurales y ganaderas. En Sudamérica, Chile es uno de los países en donde la Equinococosis quística es endémica, a pesar de esto y a los programas de intervención para controlar esta parasitosis en regiones y provincias rurales en donde se practica la ganadería caprina y ovina, esta parasitosis sigue presente, siendo las regiones de Aysén, Magallanes, La Araucanía y Coquimbo, las que poseen los índices de prevalencia más altos. En este estudio se determinó la prevalencia de la equinococosis quística y sus posibles factores de riesgo en cuatro comunas de la provincia de Limarí, región de Coquimbo, entre los años 2016-2017 y se evaluó la diversidad genética de E. granulosus s.s. en tres regiones endémicas, Coquimbo, La Araucanía y Magallanes, mediante la secuencia de la subunidad 1 del gen mitocondrial de la de la citocromo oxidasa (cox1). Un total de 1221 personas entre 5 - 88 años fueron examinados con el método de ELISA. Los factores de riesgo fueron evaluados por medio de un cuestionario en 908 personas mayores de 18 años. Se utilizó el método de regresión logística para determinar la probabilidad de asociación entre la presencia de la infección equinococócica y los factores de riesgo; y la escolaridad y los factores de riesgo. La diversidad genética y la estructura poblacional de metacéstodos de E. granulosus s.s. fue evaluada en tres regiones endémicas de Chile, mediante la secuencia de la sub unidad 1 del gen mitocondrial de la citocromo oxidasa (cox1). Se secuenciaron 46 quistes, 6 de la región de Coq, De este modo se observó que la prevalencia general de CE en la población analizada fue de 7,9% (97/1221), 6,71% (21/313) de menores de 18 años y el 8,4% (76/908) de los adultos, no se observaron diferencias significativas entre los grupos etarios analizados. La comuna de Punitaqui presenta la mayor frecuencia relativa 13.33% (12/90) de CE. En el análisis de los factores de riesgo para la CE, el tener agua potable en las casas y el haber tenido equinococosis quística en alguna etapa de su vida se asociaron con la mayor probabilidad de presentar equinococosis quística. Los resultados de este estudio muestran una alta endemicidad de Equinococosis quística en la provincia de Limarí. Así también, todos los quistes hidatídicos analizados fueron identificados como E. granulosus s.s.. Predominó la cepa EG01, detectando 4 haplotipos, EG01, EG1A, EG1D, EgCLOM16, con una baja diversidad de haplotipos (Hd=0,461±0,00637) y una baja diversidad de nucleótidos (0,00181±0,00036). La red de parsimonia de cox1 en la población chilena mostró forma de estrella, en donde el haplotipo EG01 se encontró en el centro. Los índices de neutralidad Tajima`s D y F de Fu`s fueron negativos para las poblaciones de Coquimbo (D= -0,93302; Fs= -0,003) y Magallanes (D= -0,17406; Fs= -0,121), lo que indica un exceso de sitios polimórficos raros, expansión poblacional y una desviación significativa de la neutralidad. Los índices D de Tajima y Fs de Fu fueron positivos para la población de La Araucanía (D= 0,8695; Fs= 1,039), mostrando niveles bajos de alelos raros o falta de alelos raros. El índice de fijación de Fisher por pares (Fst), muestran valores negativos en las poblaciones Coquimbo- La Araucanía (-0,08761). El valor de Fst más alto y significativo fue entre las poblaciones de La Araucanía- Magallanes (0,10703), indicando diferencias entre estas poblaciones. Nuestros resultados muestran que la estructura genética poblacional de E. granulosus s.s es compleja, debido a la presencia de diferentes ha

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 experiments.

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.

In the past 20 years, Neospora caninum infection in sheep has been reported in at least 31 countries worldwide from all sheep-rearing continents (Europe, Asia, the Americas, Africa, and Oceania), and its role as an abortifacient agent is becoming more evident. Most studies of ovine neosporosis have focused on its epidemiology, based primarily on serological analysis, with only a few studies investigating the actual presence of the parasite by PCR and/or IHC. Individual seroprevalence rates were highly variable between countries, and even between regions within the same country, ranging from 0.0% to 67.4% positive. Furthermore, most of the studies were not directly comparable due to differences in experimental designs, sample sizes, husbandry systems, ecological factors, and serological tests (e.g., IFAT, ELISA, MAT, Western blot). The latter, along with the scarcity of studies on the relevance of N. caninum as an abortifacient agent, may bias the perception of the importance of this disease. This review summarizes the situation of N. caninum infection in sheep using all available published studies describing natural ovine neosporosis. The epidemiology shows that ovine neosporosis is found worldwide, and it poses a relevant risk to the sustainability of sheep flocks.

Bluetongue, caused by bluetongue virus (BTV), is a widespread arthropod-borne disease of ruminants that entails a recurrent threat to the primary sector of developed and developing countries. In this work, we report modified vaccinia virus Ankara (MVA) and ChAdOx1-vectored vaccines designed to simultaneously express the immunogenic NS1 protein and/or NS2-Nt, the N-terminal half of protein NS2 (NS21-180). A single dose of MVA or ChAdOx1 expressing NS1-NS2-Nt improved the protection conferred by NS1 alone in IFNAR(-/-) mice. Moreover, mice immunized with ChAdOx1/MVA-NS1, ChAdOx1/MVA-NS2-Nt, or ChAdOx1/MVA-NS1-NS2-Nt developed strong cytotoxic CD81 T-cell responses against NS1, NS2-Nt, or both proteins and were fully protected against a lethal infection with BTV serotypes 1, 4, and 8. Furthermore, although a single immunization with ChAdOx1-NS1-NS2-Nt partially protected sheep against BTV-4, the administration of a booster dose of MVA-NS1- NS2-Nt promoted a faster viral clearance, reduction of the period and level of viremia and also protected from the pathology produced by BTV infection. IMPORTANCE Current BTV vaccines are effective but they do not allow to distinguish between vaccinated and infected animals (DIVA strategy) and are serotype specific. In this work we have develop a DIVA multiserotype vaccination strategy based on adenoviral (ChAdOx1) and MVA vaccine vectors, the most widely used in current phase I and II clinical trials, and the conserved nonstructural BTV proteins NS1 and NS2. This immunization strategy solves the major drawbacks of the current marketed vaccines.

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 respo