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Fasciola hepatica causes liver fluke disease, a worldwide neglected and re-emerging zoonotic disease, leading to hepatitis in humans and livestock. In the pathogenesis, flukes actively migrate through liver parenchyma provoking tissue damage. Here, parasites must confront leukocytes of the innate immune system in vivo. Polymorphonuclear neutrophils (PMN) are the most abundant granulocytes and first ones arriving at infection sites. PMN may display neutrophil extracellular traps (NETs), consisting of nuclear DNA, decorated with histones, enzymes, and antimicrobial peptides. We investigated for the first time whether F. hepatica soluble antigens (FhAg) can also trigger NETosis and innate immune reactions in exposed ovine PMN. Thus, isolated PMN were co-cultured with FhAg and NET formation was visualized by immunofluorescence and scanning electron microscopy analyses resulting in various phenotypes with spread NETs being the most detected in vitro. In line, NETs quantification via Picogreen®-fluorometric measurements revealed induction of anchored- and cell free NETs phenotypes. Live cell 3D-holotomographic microscopy revealed degranulation of stimulated PMN at 30 min exposure to FhAg. Functional PMN chemotaxis assays showed a significant increase of PMN migration (p = 0.010) and intracellular ROS production significantly increased throughout time (p = 0.028). Contrary, metabolic activities profiles of FhAg-exposed PMN did not significantly increase. Finally, in vivo histopathological analysis on F. hepatica-parasitized liver tissue sections of sheep showed multifocal infiltration of inflammatory cells within liver parenchyma, and further fluorescence microscopy analyses confirmed NETs formation in vivo. Overall, we hypothesized that NET-formation is a relevant host defence mechanism that might have a role in the pathogenesis of fasciolosis in vivo.

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

Marine mammals, regarded as sentinels of aquatic ecosystem health, are exposed to different pathogens and parasites under natural conditions. We surveyed live South American fur seals Arctocephalus australis and South American sea lions Otaria flavescens in Uruguay for Leptospira spp., canine distemper virus (CDV), Mycobacterium spp., Toxoplasma gondii, and Neospora caninum. Samples were collected from 2007 to 2013. The seroprevalence of Leptospira spp. was 37.6% positive, 50.9% negative, and 11.5% suspect for A. australis (n = 61) while for O. flavescens (n = 12) it was 67% positive, 25% negative, and 8% suspect. CDV RNA was not detected in any of the analyzed samples. Most animals tested seropositive to tuberculosis antigens by WiZo ELISA (A. australis: 29/30; O. flavescens: 20/20); reactivity varied with a novel ELISA test (antigens MPB70, MPB83, ESAT6 and MPB59). Seroprevalence against N. caninum and T. gondii was 6.7 and 13.3% positive for O. flavescens and 0 and 2.2% positive for A. australis respectively. To evaluate possible sources of infection for pinnipeds, wild rats Rattus rattus and semi-feral cats Felis catus were also tested for Leptospira spp. and T. gondii respectively. Water samples tested for Leptospira revealed saprofitic L. bioflexa. Pathogenic Leptospira were detected in the kidneys of 2 rats, and cats tested positive for T. gondii (100%). These results represent a substantial contribution to the study of the health status of wild pinnipeds in Uruguay.

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.

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

The recognition of lesions of the mammary gland in small ruminants is a useful diagnostic procedure that can aid in the identification of several udder diseases. This article reviews the main pathological lesions in this organ in sheep and goats. Mastitis is, by far, the most commonly diagnosed change. Acute clinical mastitis is associated with bacterial infections, mainly Staphylococcus aureus or Mannheimia haemolytica. Lesions related to subclinical and chronic mastitis are also described, either as localized cases or as a part of systemic diseases such as contagious agalactia, maedi-visna or tuberculosis. Neoplasia is rare in the mammary gland of sheep and goats with sporadic mammary adenocarcinomas most commonly reported. Teat lesions, including those due to trauma, orf virus infection or papillomas, are predisposing factors for the subsequent development of mastitis.

Neospora 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%.

This study estimates the economic losses due to outbreaks of toxoplasma abortions in a dairy (1928 sheep) and a meat (700 sheep) flock in Spain raised under intensive and semi-extensive management conditions, respectively. In both flocks, sheep were divided into multiple groups to synchronise reproduction. The outbreaks resulted in abortion rates in individual lots of 12.6% (30/239) in the dairy flock and 33.3% (70/210) in the meat flock. Toxoplasma gondii was definitively diagnosed in most submitted cases and the only abortifacient pathogen identified despite extensive investigation. Upon completion of lambing and lactation, veterinarians and farmers completed a questionnaire to gather the data to determine the direct economic impact. The calculated total direct economic losses were €5154.5 (€171.8/abortion) in the dairy flock and €4456 (€63.6/abortion) in the meat flock. Results suggest that flock size, production system, abortion rate and control measures are the key factors influencing economic losses, which vary greatly between individual flocks.

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 T

Neosporosis and besnoitiosis, caused by cyst-forming protozoa Neospora caninum and Besnoitia besnoiti, respectively, are parasitic infestations of livestock in Israel. These parasites cause significant economic losses in cattle due to reproductive and productive disorders. Both parasites have been detected in several wild ruminant species throughout other regions of the world, while the existence of a sylvatic life cycle in Israel remains uncertain. Thus, a wide panel of 871 sera from two wild carnivores and nine wild ruminant species were tested. All sera were first analysed by MAT for an initial screening and positive samples were confirmed a posteriori by Western blot. Additionally, a complementary IFAT was used for the detection of antibodies against N. caninum. Neospora antibodies were present in six out of the 11 species investigated, whereas Besnoitia antibodies were undetected. Golden jackal, red fox, addax, Arabian oryx, Persian fallow deer, mouflon, mountain gazelle, Nubian ibex, scimitar horned oryx and water buffalo were seropositive against N. caninum infection by IFAT and/or MAT. Moreover, the presence of Neospora spp.-specific antibodies was confirmed by Western blot in golden jackal (6/189; 3.2%), red fox (1/75; 1.3%), Persian fallow deer (13/232; 5.6%), mouflon (1/15; 16.7%), Nubian ibex (22/ 55; 40%) and water buffalo (12/18; 66.7%). Addax (1/49) and water buffalo (1/18) were MAT-seropositive against B. besnoiti but were seronegative by Western blot. Hence, Neospora sylvatic cycle is present in Israel and may cross over to a domestic life cycle. In contrast, wildlife species investigated are unlikely to present a risk of transmitting Besnoitia to livestock in Israel.