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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

Toxoplasma gondii is an important zoonotic agent with high genetic diversity, complex epidemiology, and variable clinical outcomes in animals and humans. In veterinary medicine, this apicomplexan parasite is considered one of the main infectious agents responsible for reproductive failure in small ruminants worldwide. The aim of this study was to phenotypically characterize 10 Spanish T. gondii isolates recently obtained from sheep in a normalized mouse model and in an ovine trophoblast cell line (AH-1) as infection target cells. The panel of isolates met selection criteria regarding such parameters as genetic diversity [types II (ToxoDB #1 and #3) and III (#2)], geographical location, and sample of origin (aborted foetal brain tissues or adult sheep myocardium). Evaluations of in vivo mortality, morbidity, parasite burden and histopathology were performed. Important variations between isolates were observed, although all isolates were classifed as “nonvirulent” (<30% cumulative mortality). The isolates TgShSp16 (#3) and TgShSp24 (#2) presented higher degrees of virulence. Signifcant diferences were found in terms of in vitro invasion rates and tachyzoite yield at 72 h post-inoculation (hpi) between TgShSp1 and TgShSp24 isolates, which exhibited the lowest and highest rates, respectively. The study of the CS3, ROP18 and ROP5 loci allelic profles revealed only type III alleles in ToxoDB #2 isolates and type II alleles in the #1 and #3 isolates included. We concluded that there are relevant intraand inter-genotype virulence diferences in Spanish T. gondii isolates, which could not be inferred by genetic charac‑terization using currently described molecular markers.

The information generated from the official eradication programmes of caprine tuberculosis (TB) in Castilla y León, Spain, during 2018, has been used to assess the effect of vaccination against paratuberculosis (PTB) and the presence of this infection, on the single intradermal tuberculin (SIT) test results. Data from 121,665 goats belonging to 1936 different herds were analysed using generalized linear models. An epidemiological survey was conducted to know the herd immunization status against PTB and the date of last vaccination. All SIT test-positive animals were further investigated in order to confirm the diagnosis of TB, through bacterial culture, and PTB, by histopathological and qPCR analyses. SIT positivity was found in 39 (2.01%) herds and 507 (0.41%) goats. TB was confirmed by M. caprae or M. bovis isolation in 10 (0.51%) herds and 46 (0.038%) goats. PTB was diagnosed in 13 (33.33%) and 55 (10.84%) of the SIT test-positive herds and goats, respectively. Vaccination against PTB showed a significant influence on the results of the SIT test at herd level, with higher positivity detected among those herds vaccinated. However, this effect was not observed when the total number of animals was considered, where the highest positivity was found in unvaccinated goats. The time elapsed between vaccination and SIT test performance also influenced the results. The strongest effect was found when less than eight months elapsed between performing both activities, and to a lesser extent between 8 and 12 months. Conversely, no positive herds or animals were found when the time elapsed was higher than one year. No significant effect of the presence of PTB was observed. These findings demonstrate that the use of PTB vaccine does not result in false positives to a SIT test at individual level, provided that the time elapsed between the performance of both practices is higher than 12 months.

The experimental vaccine for bovine malignant catarrhal fever consists of viable attenuated alcelaphine herpesvirus 1 (AlHV-1) derived‘ by extensive culture passage, combined with an oil-in-water adjuvant, delivered intramuscularly. This immunisation strategy was over 80% effective in previous experimental and field trials and protection appeared to be associated with induction of virus-neutralising antibodies. Whether the vaccine virus is required to be viable at the point of immunisation and whether adjuvant is required to induce the appropriate immune responses remains unclear. To address these issues two studies were performed, firstly to analyse immune responses in the presence and absence of adjuvant and secondly, to investigate immune responses to vaccines containing adjuvant plus viable or inactivated AlHV-1. The first study showed that viable attenuated AlHV-1 in the absence of adjuvant induced virus-specific antibodies but the titres of virus-neutralising antibodies were significantly lower than those induced by vaccine containing viable virus and adjuvant, suggesting adjuvant was required for optimal responses. In contrast, the second study found that the vaccine containing inactivated (>99.9%) AlHV-1 induced similar levels of virus-neutralising antibody to the equivalent formulation containing viable AlHV-1. Together these studies suggest that the MCF vaccine acts as an antigen depot for induction of immune responses, requiring adjuvant and a suitable antigen source, which need not be viable virus. These obser- vations may help in directing the development of alternative MCF vaccine formulations for distribution in the absence of an extensive cold chain.

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.

Bovine respiratory disease (BRD) is still a serious concern in feedlots, where it exerts a negative effect on farm productivity. There is a shortage of studies focused on the evaluation of BRD-associated lesions at the slaughterhouse in clinically healthy animals. The objective of this work was to investigate the prevalence and type of subclinical pneumonic lesions in slaughtered beef cattle, according to the age range and management system, and its impact on carcass weight. A total of 1101 beef cattle intended for human consumption were examined at slaughter. Information on age, sex, management system and carcass weight was recorded. The presence and type of pneumonia were evaluated according to gross and microscopic findings and etiological agents by PCR. Lung pneumonic lesions appeared in 17.9% of animals and were predominant among veal calves. According to the type, chronic catarrhal pneumonia prevailed in the majority of animals, and mixed and extensively reared cattle were more likely to suffer acute fibrinous pneumonia. The presence of pneumonic lesions was associated with a significant decrease in carcass weight that had more of an impact in veal male calves coming from intensive systems. Bacterial infections were the predominant infectious agent and the only cause of acute fibrinous pneumonia, while viruses were infrequent and only found in lesions with chronic catarrhal pneumonia. This study shows the importance of BRD in beef feedlots upon production values and points out the feasibility of slaughterhouse assessment of pneumonia as a method for the evaluation of BRD significance.

The present study aimed to isolate and perform molecular and phenotypic characterization of Toxoplasma gondii strains infecting Iberian pigs bred under semi-free conditions and destined for human consumption. Blood and heart tissue samples from 361 fattening pigs from 10 various herds selected in the main areas of Iberian pig production were collected at a slaughterhouse; the sera were tested for anti-T. gondii antibodies using a commercial indirect ELISA kit, and a mouse bioassay was carried out using heart muscle of seropositive individual representatives from each geographical location. Seventy-nine (21.9%) of the 361 animals tested positive for anti-T. gondii antibodies according to the serology test. Fifteen samples of myocardial tissue were subjected to bioassay and 5 isolates (TgPigSp1 to TgPigSp5) were obtained. The isolates were characterized by using 11 PCR-RFLP genetic markers; three isolates had a ToxoDB #3 genotype (3/5) and two isolates had a ToxoDB #2 genotype (2/5). The TgPigSp1 and TgPigSp4 isolates were selected for virulence in mice characterization as instances of each different RFLP-genotype found. The TgPigSp1 isolate (#2 genotype) was virulent in mice with notable cumulative mortality (87.5%) and morbidity rates (100%); the TgPigSp4 (#3) was nonvirulent and triggered mild clinical signs in 42.1% of seropositive mice. Infection dynamics and organ distribution of both isolates were analyzed; the data revealed significant differences, including substantially higher parasite load in the lung during the acute phase of infection, in mice infected with TgPigSp1 than in the case of TgPigSp4 (median parasite load 7.6 vs. 0 zoites/mg, respectively; p < 0.05). Furthermore, degrees of severity of detected histopathological lesions appeared to be related to higher parasite burdens. Taking into account the unexpectedly high mortality rate and parasite load associated with the clonal genotype III, which is traditionally considered nonvirulent in mice, the ne

Ovine enzootic abortion (OEA), caused by Chlamydia abortus, is an economically important disease in many countries. Inactivated vaccines have been used for many years as they induce immunity in sheep, although outbreaks of abortions have been described in vaccinated flocks. In addition, there is a commercially available live attenuated vaccine that provides good protective results. Recently however, reports question the attenuation of this vaccine and associate it with the appearance of outbreaks of OEA in vaccinated flocks. In the present study, a recently commercialized inactivated vaccine (INMEVA®; Laboratorios Hipra S.A., Amer, Spain) has been evaluated using mouse and sheep experimental models. In the mouse models (non-pregnant and pregnant models), the efficacy of INMEVA vaccine has been compared to an unvaccinated control group and to an experimental inactivated vaccine considered as a positive protection control (UMU vaccine). In the non- pregnant model, the UMU vaccine was more effective than the INMEVA vaccine regarding the impact on body weight or the presence of C. abortus in the liver, but both vaccinated groups (UMU and INMEVA) had significantly lower C. abortus in the liver compared to the control group. In the pregnant model in terms of reproductive failures, pups per mouse or the presence of C. abortus in the liver or uterus, no significant differences were found between both vaccines, inducing protection compared to the control group. In the ovine pregnant model, where INMEVA vaccine was compared only to an unvaccinated group, the results indicate that this new commercial vaccine is safe and provides a suitable level of protection against an experimental challenge with C. abortus. A 75% reduction in reproductive disorders, 55% reduction in animals with C. abortus shedding on day of parturition/abortion, and a significant reduction of the average amount of chlamydial shedding from parturition/abortion over the next 21 days was observed, in relation

Peripheral blood from healthy sheep (n = 3) and goats (n = 3) were employed to establish an efficient method for simultaneous isolation of peripheral blood mononuclear cells (PBMCs) and neutrophils and to standardize protocols for monocyte purification and generation of monocyte-derived macrophages (MDMs). In both species, a significantly enriched population of PBMCs, with higher purity and number of cells determined by flow cytometry, was achieved when processing through a density gradient a mixture of buffy-coat and red blood cell layer (RBC) in comparison to the use of just the buffy-coat (p < 0.05). Neutrophils could be subsequently isolated from the layer, located underneath PBMCs fraction with significant higher purity rates, higher than 85 % determined by flow cytometry, than those obtained with protocols without density gradients (< 60 %) (p < 0.05). This technique would allow the isolation of both cell populations from the same sample of blood. A pure cell population of monocytes, CD14+ cells, was purified from PBMCs when using immunomagnetic columns, which allow for 17 % (nº monocytes/nº PBMCs) of yield and high percentages of expression of CD14+ (88 %), MHC-II+ (91.5 %) and CD11b+ (94 %) established by flow cytometry. On the other hand, the classical and nonexpensive purification of monocytes from PBMCs based on the adherence capacity of the former, allowed significantly lower yield of monocytes (4.6 %), with percentages of surface markers expression that dropped to 35 %, 65 % and 55 %, respectively (p < 0.001), suggesting the isolation of a mixed population of cells. The addition of GM-CSF to the culture, at concentration from 25 to 125 ng/mL, enhanced proportionally the number of MDMs generated compared to the absence of supplementation or the use of autologous serum from 5% to 20 %. However, purification of monocytes through the adherence method achieved higher yields of MDMs than those isolated through immunomagnetic columns in both species (p < 0.001

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 lesions.

In this study we determined the causes of mortality and disease in a total of 325 lagomorphs (rabbits and hares) in northern Spain between 2000 and 2018. Risk factors such as the species, age, sex, time of year and origin were also considered. Clinical signs, gross and histopathological findings and ancillary test results were the basis for the final diagnoses that were reviewed to classify and identify the different disorders. A total of 26 different conditions were identified. A single cause of death or illness was detected in 267 animals. They were grouped into parasitic conditions (n= 65; 24.34%) represented by encephalitozoonosis, hepatic coccidiosis, hepatoperitoneal cysticercosis, intestinal coccidiosis, parasitic gastritis and cutaneous ectoparasitosis; bacterial diseases (n = 56; 20.97%) including pseudotuberculosis, blue breast, skin abscesses, tularemia, pneumonic pasteurellosis and staphylococcal infections; nutritional and metabolic diseases (n = 48; 17.97%) with epizootic rabbit enteropathy, hepatic steatosis and pregnancy toxemia as prominent diseases; viral infections (n= 31; 11.61%) comprising rabbit hemorrhagic disease and myxomatosis and miscellaneous causes (n = 31; 11.61%) where rabbit enteritis complex, renal conditions (nephrosis), heat stroke, and arterial bone metaplasia were included; neoplasms (n = 12; 4.49%) represented by uterine adenocarcinoma, mammary adenocarcinoma, cutaneous fibroma, intestinal lymphoma and hepatic cholangiocarcinoma; toxicoses (n = 11; 4.11%); trauma-related injuries (n = 9; 3.37%) and finally congenital diseases (n = 4; 1.49%). In 58 animals of the study, some of these conditions were presented jointly. We discuss the detection frequency, possible causes or associated factors of the different pathologies as well as the importance of the different variables considered.

Rift Valley fever (RVF) and bluetongue (BT) are two important ruminant diseases transmitted by arthropods. Both viruses have shown important geographic spread leading to endemicity of BT virus (BTV) in Africa and Europe. In this work, we report a dual vaccine that simultaneously induces protective immune responses against BTV and RVFV based on modified vaccinia Ankara virus (MVA) expressing BTV proteins VP2, NS1, or a truncated form of NS1 (NS1-Nt), and RVFV Gn and Gc glycoproteins. IFNAR mice immunized with two doses of MVA-GnGc-VP2 developed a significant neutralizing antibody response against BTV-4 and RVFV. Furthermore, the homologous prime-boost immunization with MVA-GnGc-NS1 or MVA-GnGc-NS1-Nt triggered neutralizing antibodies against RVFV and NS1-specific cytotoxic CD8+ T cells in mice. Moreover, all mice immunized with MVA-GnGc-NS1 or MVA-GnGc-NS1-Nt remained healthy after lethal challenge with RVFV or BTV-4. The homologous prime-boost vaccination with MVA-GnGc-NS1, which was the best immunization strategy observed in mice, was assayed in sheep. Clinical signs and viremia were absent or highly reduced in vaccinated sheep after challenge with BTV-4 or RVFV. These results indicate that MVA-GnGc-NS1 vaccination elicits immune protection against RVFV and BTV in sheep.

Ovine herpesvirus 2 (OvHV-2) is one of the gammaherpesviruses in the genus Macavirus that can cause malignant catarrhal fever (MCF) in ungulates. Sheep are the adapted host for OvHV-2 and it is generally assumed that infection is not associated with disease in this species. However, cases of "polyarteritis nodosa" or idiopathic systemic necrotizing vasculitis reported in sheep are similar to vascular lesions in clinically susceptible species with MCF. Using a recently developed in situ hybridization (ISH) method, we were able to identify OvHV-2 nucleic acids within lesions and correlate the viral distribution with systemic necrotizing vasculitis in 9 sheep, including both naturally and experimentally OvHV-2-infected animals. ISH, combined with polymerase chain reaction and histology, identify OvHV-2 as the likely agent responsible for sporadic, MCF-like vascular disease in sheep.

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

En noviembre de 2017 se remitió para la necropsia una coneja, cruce industrial, adulta (3 años). La lesión macroscópica más significativa se observó en la arteria aorta, arteria pulmonar y carótidas que aparecían como tubos rígidos y de color blanquecino. También se encontraban afectadas las arterias de las extremidades posteriores y en los riñones se observaron pequeños focos blanquecinos en la unión córtico-medular. Microscópicamente en la aorta ascendente, aorta abdominal, carótidas y arteria pulmonar se observó calcificación de fibras elásticas, focos de metaplasia cartilaginosa, ósea y formación de médula ósea. Las arterias musculares (coronarias, arqueadas renales, hepáticas, mesentéricas, uterinas, ováricas y de la musculatura esquelética) mostraban calcificaciones en la capa media. También se observaron calcificaciones focales en túbulos renales, esclerótica y cartílagos bronquiales. En la mucosa gástrica las glándulas aparecían atróficas por la presencia de amiloide. En conejas reproductoras la calcificación de la aorta es una lesión relativamente frecuente asociada a hipervitaminosis D, administración excesiva de suplementos minerales o lesión renal crónica. La edad también parece ser un factor de riesgo, siendo más frecuente en conejas con más de 2 años. Sin embargo, la metaplasia cartilaginosa y ósea con formación de médula ósea de arterias elásticas son alteraciones menos frecuentes. Aunque su patogenia aún no es clara, se ha sugerido que tanto células indiferenciadas de la capa media del vaso o los propios miocitos, células con gran plasticidad fenotípica, favorecen la formación de estos tejidos conectivos, más como resultado de la lesión previa que como una respuesta adaptativa. La calcificación arterial provoca hipertensión e insuficiencia cardíaca que unida a la pérdida de función gástrica (por amiloidosis) favoreció probablemente el estado de anorexia y emaciación del animal.

Although the detection of Toxoplasma gondii in bovine tissues is rare, beef might be an important source of human infection. The use of molecular techniques, such as magnetic capture qPCR (MC-qPCR), in combination with the gold standard method for isolating the parasite (mouse bioassay), may increase the sensitivity of T. gondii detection in infected cattle. The risk of transmission of the parasite to humans from undercooked/raw beef is not fully known and further knowledge about the predilection sites of T. gondii within cattle is needed. In the current study, six Holstein Friesian calves (Bos taurus) were experimentally infected with 106 T. gondii oocysts of the M4 strain and, following euthanasia (42 dpi), pooled tissues were tested for presence of the parasite by mouse bioassay and MC-qPCR

Bovine besnoitiosis is continuing to spread in Europe. Therefore, the development ofruminant animal models of infection is urgently needed to evaluate therapeutic andprophylactic tools. Herein, we studied the effect of parasite dose and host age on theinfection dynamics withBesnoitia besnoititachyzoites in cattle in two independentexperimental infections. In experiment A, twelve 3‐month‐old male calves were inocu-lated intravenously with either three different doses of tachyzoites (G1: 108; G2: 107;G3: 106) or with PBS (G4). In experiment B, six 14‐month‐old bulls were inoculatedwith 106tachyzoites based on results obtained in experiment A. In both trials, clinicalsigns compatible with acute and chronic besnoitiosis were monitored daily; blood andskin samples were collected regularly for 70–115 days post‐infection (pi). Finally, ani-mals were killed, and tissues were collected for lesion and parasite detections. Infectedanimals developed mild–moderate signs compatible with acute besnoitiosis. Lym-phadenopathy and fever were observed in both calves (from 12 hr until 7 days pi) andbulls (from 6 days until 9 days pi). Seroconversion was detected at 16–19 days pi, andantibody levels remained high. Infected animals did not developed characteristic clini-cal signs and macroscopic lesions of chronic besnoitiosis. However, successfully, para-site‐DNA was detected in a reduced number of target tissues: conjunctiva, ocularsclera, epididymis, skin of the scrotum and carpus in calves (n= 10, 6 of whichbelonged to G3), and pampiniform plexus and testicular parenchyma in bulls. Remark-ably, one tissue cyst and mild microscopic lesions were also detected. In summary,inoculated animals developed the acute besnoitiosis and chronic infection was evi-denced by microscopic findings. However, our results suggest that tachyzoite doseand host age are not key variables for inducing clinical signs and macroscopic lesionscharacteristic of chronic besnoitiosis. Thus, a further refinement of t