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The purpose of this systematic review was to produce a best evidence synthesis of the effects of Kinesiology Tape (KT) in the treatment of tendinopathies. An electronic search on five databases (PubMed, Cumulative Index to Nursing and Allied Health Literature (CINAHL), Cochrane Library, SportDiscus, and Physiotherapy Evidence Database (PEDro)) was conducted. Studies were included if (1) patients suffered from tendinopathy; (2) isolated KT was applied in at least one group; (3) comparisons between other techniques were developed; (4) outcomes based on pain, function, disability, or quality of life were analysed. Two reviewers independently extracted data and assessed methodological quality using the Physiotherapy Evidence Database (PEDro) scale. A total of 13 articles met the eligibility criteria, involving 454 participants. Nine of these studies presented upper extremity tendinopathies, and four explored lower extremity tendinopathies. Selected papers ranged from low to high quality, with an average score of 5 on the PEDro scale. According to our findings, there is limited evidence to support KT alone for the treatment of tendinopathies beyond the short-term. Due to the mixed methodological quality and the insufficient number of clinical trials, larger, long-term, high-quality studies are needed to support the theory that tendinopathies can benefit from KT applications.

The effectiveness of platelet-rich plasma (PRP) injections for osteoarthritis (OA) is still controversial. Previous research supports the use of intra-articular PRP injections to promote a favorable environment for joint tissue healing and to delay the progression of OA. The purpose of this review is to investigate the effectiveness of PRP in the management of hip osteoarthritis (HOA). Five electronic databases were searched from inception to May 2019: Medline (via PubMed), SportDiscus via EBSCO, ProQuest Health & Medical Complete, CINAHL, and Cochrane. Risk of bias was assessed with the Cochrane risk of bias tool. The GRADE method was used to assess the level of evidence for the studies included in this review. Clinical trials evaluate PRP injections among adult patients diagnosed with HOA according to the American College of Rheumatology criteria. At least one outcome measure for pain or function must have been reported. A total of 4 trials (334 participants, 340 hips) were included, all marked as “moderate risk of bias”. Pain and function were assessed throughout the studies with visual analogue scale (VAS), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), and Harris Hip Score (HHS) tools. Intra-articular PRP injections were more effective at stages earlier than 3 months for both treatment groups with the exception of WOMAC score in one study. The superiority of PRP against comparative treatments was only reported in one study; longer-term evaluations from 4 to 12 months showed diverse results, with only one study reporting significantly better results for PRP. PRP may be beneficial and safe for patients with HOA at mid-term follow-up. However, its superiority over other procedures such as hyaluronic acid remains unclear. Further researches with high-quality designs and larger samples become imperative.

The Neospora caninum Calcium-dependent protein kinase 1 (NcCDPK1) inhibitor BKI-1294 had demonstrated excellent efficacy in a pregnant mouse model of neosporosis, and was also highly efficacious in a pregnant sheep model of toxoplasmosis. In this work, we present the efficacy of BKI-1294 treatment (dosed 5 times orally every 48 h) starting 48 h after intravenous infection of sheep with 105 Nc-Spain7 tachyzoites at mid-pregnancy. In the dams, BKI-1294 plasma concentrations were above the IC50 for N. caninum for 12–15 days. In treated sheep, when they were compared to untreated ones, we observed a minor increase in rectal temperature, higher IFNγ levels after blood stimulation in vitro, and a minor increase of IgG levels against N. caninum soluble antigens through day 28 post-infection. Additionally, the anti-NcSAG1 and anti-NcSAG4 IgGs were lower in treated dams on days 21 and 42 post-infection. However, BKI-1294 did not protect against abortion (87% foetal mortality in both infected groups, treated and untreated) and did not reduce transplacental transmission, parasite load or lesions in placentomes and foetal brain. The lack of foetal protection was likely caused by short systemic exposure in the dams and suboptimal foetal exposure to this parasitostatic drug, which was unable to reduce replication of the likely established N. caninum tachyzoites in the foetus at the moment of treatment. New BKIs with a very low plasma clearance and good ability to cross the blood-brain and placental barriers need to be developed., Graphical abstract Image 1, Highlights • BKI-1294 triggers a transiently decrease in anti-N. caninum IgG levels in sheep. • BKI-1294 does not protect against abortion and vertical transmission of N. caninum. • The efficacy of BKI-1294 is different in pregnant mice and sheep models of neosporosis.

In this study, we describe SARS-CoV-2 infection dynamics in one cat and three dogs from households with confirmed human cases of COVID-19 living in the Madrid Community (Spain) at the time of expansion (December 2020 through June 2021) of the alpha variant (lineage B.1.1.7). A thorough physical exam and nasopharyngeal, oropharyngeal, and rectal swabs were collected for real-time reverse-transcription PCR (RT-qPCR) SARS-CoV-2 testing on day 0 and in successive samplings on days 7, 14, 21, and 47 during monitoring. Blood was also drawn to determine complete blood counts, biochemical profiles, and serology of the IgG response against SARS-CoV-2. On day 0, the cat case 1 presented with dyspnea and fever associated with a mild bronchoalveolar pattern. The dog cases 2, 3, and 4 were healthy, but case 2 presented with coughing, dyspnea, and weakness, and case 4 exhibited coughing and bilateral nasal discharge 3 and 6 days before the clinical exam. Case 3 (from the same household as case 2) remained asymptomatic. SARS-CoV-2 detection by RT-qPCR showed that the cat case 1 and the dog case 2 exhibited the lowest cycle threshold (Ct) (Ct < 30) when they presented clinical signs. Viral detection failed in successive samplings. Serological analyses revealed a positive IgG response in cat case 1 and dog cases 3 and 4 shortly after or simultaneously to virus shedding. Dog case 2 was seronegative, but seroconverted 21 days after SARS-CoV-2 detection. SARS-CoV-2 genome sequencing was attempted, and genomes were classified as belonging to the B.1.1.7 lineage.

The quinolone decoquinate (DCQ) is widely used in veterinary practice for the treatment of bacte-rial and parasitic infections, most notably coccidiosis in poultry and in ruminants. We have in-vestigated the effects of treatment of Toxoplasma gondii in infected human foreskin fibroblasts (HFF) with DCQ. It induced distinct alterations in the parasite mitochondrion within 24h, which persisted even after long-term (500 nM, 52 days) treatment, although there was no parasiticidal effect. Based on the low half-maximal effective concentration (IC50) of 1.1 nM and the high selec-tivity index of >5000, the efficacy of oral treatment of pregnant mice experimentally infected with T. gondii oocysts with DCQ at 10 mg/kg/day for 5 days was assessed. However, the treatment had detrimental effects, induced higher neonatal mortality than T. gondii infection alone, and did not prevent vertical transmission. Thus, three quinoline-O-carbamate derivatives of DCQ antici-pated to have better physicochemical properties than DCQ were assessed in vitro. One such com-pound RMB060 displayed an exceedingly low IC50 of 0.07 nM when applied concomitantly with infection of host cells and had no impact on HFF viability at 10 µM. As was the case for DCQ, RMB060 treatment resulted in alteration of the mitochondrial matrix and loss of cristae, but the changes became apparent at just 6h after commencement of treatment. After 48h, RMB060 induced the expression of the bradyzoite antigen BAG1, but TEM did not reveal any other features remi-niscent of bradyzoites. Exposure of infected cultures to 300 nM RMB060 for 52 days did not re-sult in complete killing of all tachyzoites, although mitochondria remained ultrastructurally damaged and there was a slower proliferation rate. Treatment of mice infected with T. gondii oocysts with RMB060 did reduce parasite burden in non-pregnant mice and dams, but vertical transmission to pups could not be prevented.

In this work, an experimental model for chronic besnoitiosis in bovine was developed and characterized. Using a previously established calf model, two new variables (parasite stage and inoculation route) were combined and used. Twelve Holstein Friesian 3-month-old male calves were randomly divided into four groups of three animals each. Bradyzoites were obtained from a chronically infected bull and used for inoculation via three different inoculation routes. Three groups were inoculated with 106 bradyzoites by intravenous (G1), subcutaneous (G2) and intradermal (G3) routes, and a non-infected control group (G4) was inoculated with PBS. The trial lasted for 90 days and included daily clinical monitoring as well as weekly skin biopsies and blood sampling. Sera were obtained to analyse both cellular and humoral responses. Once the calves were euthanized, tissues from the skin, eyes, respiratory and reproductive tracts, among others, were collected to study presence of the parasite. Clinically, the infection was classified as mild to moderate for the acute stage since all infected calves showed lymphadenopathy from four days post-infection (pi) and fever from one week pi until 24 days pi. However, the most relevant results were achieved during the chronic stage that was classified as moderate to severe. In fact, pathognomonic conjunctival cysts were observed in all infected calves from 40 days pi onwards and were more abundant in G3. Moreover, one calf from this group developed skin lesions (49 days pi). The microscopic tissue cysts and Besnoitia DNA were detected primarily in skin, reproductive tract and respiratory tissue samples, and parasite load was higher in G3. In conclusion, the parasite stage (bradyzoite) and the inoculation route are key factors that influence the outcome of an infection. In particular, the intradermal route led to more severe clinical signs of the chronic phase in the inoculated calves.

Although it is known that gestation could influence the clinical course of ovine toxoplasmosis, the precise effect of the term of gestation when sheep are infected are yet mostly unknown. The aim of this study was to evaluate the peripheral and placental immune responses developed in pregnant sheep after experimental infection with Toxoplasma gondii at different times of gestation. Thirty-six pregnant sheep were allocated in different groups, orally inoculated with sporulated oocysts of T. gondii at early, mid and late gestation and culled within 30 days post-infection. The peripheral humoral and cytokine responses were evaluated, as well as the transcription of cytokines at the placenta. Serological analysis revealed that, regardless the term of gestation when infected, specific IgG against T. gondii were detected from day 8 post-infection and there was an early peripheral release of IFN-γ at the first week post-infection followed by a short peak of IL10 and TNF-α at the second week post-infection. There were no significant differences in this response between infected groups. At the placenta, a similar increase in transcription of IFN-γ, and TNF-α was found at the three terms of gestation, while IL-4 increased mainly at the first and second terms and IL-10 transcription was higher at the last term. While these findings show that both Th1 and Th2 cytokines play a key role in the pathogenesis of ovine toxoplasmosis and that placental and peripheral immune responses do not closely correlate, there seems to be no clear modulation of these responses along the gestation., The authors wish also to thank to Dr Frank Katzer, from Moredun Research Institute for kindly providing the tachyzoites from M4 isolate, all the staff of the IGM (CSIC-ULE) responsible for handling the experimental animals, and the technical assistance of Mr. Panero, Mrs. Espiniella, Mrs. González and Mrs. Agudín. P. Castaño is founded by a predoctoral contract from the “Junta de Castilla y León”. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). This work was supported by Grants AGL2011-30205 and AGL2016-75935-C2-2-R and of the Spanish Ministry of Science and Innovation, and LE253U13, from the Junta de Castilla y León, Spain.

18 páginas, 3 figuras, 4 tablas., Breeding bulls infected with Besnoitia besnoiti may develop sterility during either acute or chronic infection. The aim of this study was to investigate the molecular pathogenesis of B. besnoiti infection with prognosis value in bull sterility. Accordingly, fve well-characterized groups of naturally and experimentally infected males were selected for the study based on clinical signs and lesions compatible with B. besnoiti infection, serological results and parasite detection. A broad panel of molecular markers representative of endothelial activation and fbrosis was investigated and comple‑ mented with a histopathological approach that included conventional histology and immunohistochemistry. The results indicated the predominance of an intense infammatory infltrate composed mainly of resident and recruited circulating macrophages and to a lesser extent of CD3+cells in infected bulls. In addition, a few biomarkers were associated with acute, chronic or subclinical bovine besnoitiosis. The testicular parenchyma showed a higher number of diferentially expressed genes in natural infections (acute and chronic infections) versus scrotal skin in experimental infections (subclinical infection). In subclinical infections, most genes were downregulated except for the CCL24 and CXCL2 genes, which were upregulated. In contrast, the acute phase was mainly characterized by the upregulation of IL-1α, IL-6 and TIMP1, whereas in the chronic phase, the upregulation of ICAM and the downregulation of MMP13, PLAT and IL-1α were the most relevant fndings. Macrophages could be responsible for the highest level of gene regulation in the testicular parenchyma of severely afected and sterile bulls, and all these genes could be prognostic markers of sterility., This study was fnanced by the Spanish Ministry of Economy and Competi‑ tiveness (AGL-2016-75202-R), the Spanish Ministry of Science and Innovation (PID2019-103960RB-I00) and by the Community of Madrid (PLATESA P2018/ BAA-4370). DG-B was funded by the Spanish Ministry of Science through a Juan de la Cierva postdoctoral fellowship (FJCI-2016-27875). CD-D was fnancially supported through a grant from the Spanish Ministry of Economy and Competitiveness (BES-2014-069839) and AJ-M through a grant from the Spanish Ministry of Education, Culture and Sports (FPU, Grant Number FPU13/05481). I. Ferre, L.M. Ortega Mora y G. Álvarez-García are part of the TOXOSOURCES consortium, supported by funding from the European Union’s Horizon 2020 Research and Innovation programme under grant agreement No 773830: One Health European Joint Programme. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Bovine neosporosis is currently considered one of the main causes of abortion in cattle worldwide and the outcome of the infection is, in part, determined byNeospora caninumisolate virulence. However, the dam and foetal immune responses associated with this factor are largely unknown. We used a model of bovine infection at day 110 of gestation to study the early infection dynamics (10- and 20-days post-infection, dpi) after experimental challenge with high- and low-virulence isolates ofN. caninum(Nc-Spain7 and Nc-Spain1H, respectively). In the present work, dam peripheral cellular immune responses were monitored twice a week from -1 to 20 dpi. At different time points, IFN-γ and IL-4 production was investigated in stimulated dam blood and the percentage of monocytes, NK cells, B cells and T cells (CD4+, CD8+ and γδ) in peripheral blood mononuclear cells (PBMC) were determined by flow cytometry. In addition, maternal iliofemoral lymph nodes and foetal spleen and thymus were collected at 10 and 20 dpi for the study of the same cell subpopulations. Peripheral immune response dynamics were similar after the infection with both isolates, with a significant increase in the percentage of CD4+ T cells at 6 and 9 dpi in PBMC, coincident with the higher levels of IFN-γ and IL-4 release. However, the levels of IFN-γ were significantly higher and an increase in CD8+ T cells at 9, 13 and 20 dpi was observed in the dams infected with Nc-Spain7. Nc-Spain1H infection induced higher IL4 levels in stimulated blood and a higher CD4+/CD8+ ratio in PBMC. The analysis of the maternal iliofemoral lymph node showed a significant enhancement in the percentage of NK, CD4+ and CD8+ T cells for the animals infected with the highly virulent isolate and euthanized at 20 dpi. Regarding the foetal responses, the most remarkable result was an increase in the percentage of monocytes at 20 dpi in the spleen of foetuses from both infected groups, which suggests that foetuses were able to respond toN. caninuminfection at mid gestation. This work provides insights into how isolate virulence affects the maternal and foetal immune responses generated againstN. caninum, which may influence the course of infection.

Neospora caninum and Toxoplasma gondii are one of the main concerns of the livestock sector as they cause important economic losses in ruminants due to the reproductive failure. It is well-known that the interaction of these parasites with the placenta determines the course of infection, leading to fetal death or parasite transmission to the offspring. However, to advance the development of effective vaccines and treatments, there are still important gaps on knowledge on the placental host-parasite interactions that need to be addressed. Ruminant animal models are still an indispensable tool for providing a global view of the pathogenesis, lesions, and immune responses, but their utilization embraces important economic and ethics restrictions. Alternative in vitro systems based on caruncular and trophoblast cells, the key cellular components of placentomes, have emerged in the last years, but their use can only offer a partial view of the processes triggered after infection as they cannot mimic the complex placental architecture and neglect the activity of resident immune cells. These drawbacks could be solved using placental explants, broadly employed in human medicine, and able to preserve its cellular architecture and function. Despite the availability of such materials is constrained by their short shelf-life, the development of adequate cryopreservation protocols could expand their use for research purposes. Herein, we review and discuss existing (and potential) in vivo, in vitro, and ex vivo ruminant placental models that have proven useful to unravel the pathogenic mechanisms and the host immune responses responsible for fetal death (or protection) caused by neosporosis and toxoplasmosis.

The COVID-19 pandemic recently challenges worldwide health system. Many countries have been establishing several rules in order to fight against the expected exponentially growth of affected people. After some weeks hospital bed occupancy started to grow because of infected people and this demand can potentially overlap hospital bed capacity. In this scenario, doctors would have to decide who should be treated and patients without adequate treatment can increase number of deaths. Thus, in order to avoid that patient demand overlap hospital bed capacity, this research propose a forecast and simulation approach in order to provide the number of intensive care beds and health care professionals that could be available in a short-term of 21 days ahead taking into account 3 patient demand scenarios. This research is applied to Rio de Janeiro city (Brazil) and all records were retrieved from Rio de Janeiro public database. Hospital length of stay (LOS) records were obtained with Rio de Janeiro City Health Department (RJHD). We concluded that to open the number of new intensive care unit (UCI) beds proposed by the government is lower than expected admission on the worst scenario.

RESUMEN Presentamos el caso de un paciente con foseta del disco óptico, quien presentó una disminución de la agudeza visual secundaria a desprendimiento seroso de retina. Se decidió realizar una inyección intravítrea de 0.3ml de gas C3F8 (100%), seguida de fotocoagulación con láser de argón en el borde temporal de la foseta, logrando reaplicación total de la retina, con reabsorción de todo el líquido subretiniano visible en la tomografía de coherencia optica (OCT) luego de 400 días. Además hubo una mejoría significativa en la agudeza visual.

This is a review of the development of bumped-kinase inhibitors (BKIs) for the therapy of One Health parasitic apicomplexan diseases. Many apicomplexan infections are shared between humans and livestock, such as cryptosporidiosis and toxoplasmosis, as well as livestock only diseases such as neosporosis. We have demonstrated proof-of-concept for BKI therapy in livestock models of cryptosporidiosis (newborn calves infected with Cryptosporidium parvum), toxoplasmosis (pregnant sheep infected with Toxoplasma gondii), and neosporosis (pregnant sheep infected with Neospora caninum). We discuss the potential uses of BKIs for the treatment of diseases caused by apicomplexan parasites in animals and humans, and the improvements that need to be made to further develop BKIs.

19 páginas, 9 figuras, 1 tabla., Neospora caninum is an apicomplexan cyst-forming parasite that is considered one of the main causes of abortion. The pathogenic mechanisms associated with parasite virulence at the maternal-foetal interface that are responsible for the outcome of infection are largely unknown. Here, utilizing placentomes from cattle experimentally infected with high-virulence (Nc-Spain7) and low-virulence (Nc-Spain1H) isolates, we studied key elements of the innate and adaptive immune responses, as well as components of the extracellular matrix (ECM), at 10 and 20 days post-infection (dpi). The low-virulence isolate elicited a robust immune response characterized by upregulation of genes involved in pathogen recognition, chemokines and pro-inflammatory cytokines, crucial for its adequate control. In addition, Nc-Spain1H triggered the expression of anti-inflammatory cytokines and other mechanisms implicated in the maintenance of ECM integrity to ensure foetal survival. In contrast, local immune responses were initially (10 dpi) impaired by Nc-Spain7, allowing parasite multiplication. Subsequently (20 dpi), a predominantly pro-inflammatory Th1-based response and an increase in leucocyte infiltration were observed. Moreover, Nc-Spain7-infected placentomes from animals carrying non-viable foetuses exhibited higher expression of the IL-8, TNF-α, iNOS and SERP-1 genes and lower expression of the metalloproteases and their inhibitors than Nc-Spain7-infected placentomes from animals carrying viable foetuses. In addition, profound placental damage characterized by an alteration in the ECM organization in necrotic foci, which could contribute to foetal death, was found. Two different host-parasite interaction patterns were observed at the bovine placenta as representative examples of different evolutionary strategies used by this parasite for transmission to offspring., This work was supported by the Spanish Ministry of Economy and Competi‑ tiveness (AGL2013-44694-R and AGL2016-75935-C2-1-R) and the Community of Madrid (PLATESA2-CM P2018/BAA-4370). Laura Jiménez-Pelayo was fnan‑ cially supported by a fellowship from the University Complutense of Madrid (including two research stays in 2017 and 2018) and Marta García-Sánchez was fnancially supported through a grant from the Spanish Ministry of Economy and Competitiveness (BES-2014-070723). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

© The Author(s) 2020., [Background]: Toxoplasma gondii is a major cause of abortion in small ruminants and presents a zoonotic risk when undercooked meat containing cysts is consumed. The aim of the present study was to investigate the genetic diversity among the T. gondii strains circulating in ovine livestock in Spain., [Methods]: Selected samples collected from abortion outbreaks due to toxoplasmosis (n = 31) and from chronically infected adult sheep at slaughterhouses (n = 50) in different Spanish regions were bioassayed in mice, aiming at parasite isolation. In addition, all original clinical samples and the resulting isolates were genotyped by multi-nested PCR-RFLP analysis of 11 molecular markers and by PCR-DNA sequencing of portions of the SAG3, GRA6 and GRA7 genes., [Results]: As a result, 30 isolates were obtained from 9 Spanish regions: 10 isolates from abortion-derived samples and 20 isolates from adult myocardial tissues. Overall, 3 genotypes were found: ToxoDB#3 (type II PRU variant) in 90% (27/30) of isolates, ToxoDB#2 (clonal type III) in 6.7% (2/30), and ToxoDB#1 (clonal type II) in 3.3% (1/30). When T. gondii-positive tissue samples (n = 151) were directly subjected to RFLP genotyping, complete restriction profiles were obtained for 33% of samples, and up to 98% of the specimens belonged to the type II PRU variant. A foetal brain showed a clonal type II pattern, and four specimens showed unexpected type I alleles at the SAG3 marker, including two foetal brains that showed I + II alleles as co-infection events. Amplicons of SAG3, GRA6 and GRA7 obtained from isolates and clinical samples were subjected to sequencing, allowing us to confirm RFLP results and to detect different single-nucleotide polymorphisms., [Conclusions]: The present study informed the existence of a predominant type II PRU variant genotype (ToxoDB#3) infecting domestic sheep in Spain, in both abortion cases and chronic infections in adults, coexisting with other clonal (ToxoDB#1 and ToxoDB#2), much less frequent genotypes, as well as polymorphic strains as revealed by clinical sample genotyping. The use of multilocus sequence typing aided in accurately estimating T. gondii intragenotype diversity., This research was supported by projects funded by the Spanish Ministry of Science and Innovation (AGL2016-75935-C2-R) and the Community of Madrid (PLATESA2-CM-P2018/BAA-4370). MF and RC were funded by UCM-Santander/2017 pre-doctoral grants, and PLATESA2 post-doctoral grants, respectively. CG was funded by DGAPA, National Autonomous University of Mexico (UNAM). RC, EC and LO are part of the TOXOSOURCES consortium, supported by funding from the European Union’s Horizon 2020 Research and Innovation programme under grant agreement No. 773830: One Health European Joint Programme.

The histological findings associated to Besnoitia besnoiti infection were exhaustively studied in target tissues from experimentally and chronically infected calves. Calves were inoculated with 106 bradyzoites via intravenous, subcutaneous and intradermal route. Visible pathognomonic sclera cysts were observed in all infected animals. Tissue cysts were more abundant and lesions were more frequent in calves inoculated via intradermal. The most parasitized tissues were skin, including scrotum (40.81% of positive samples), nostril and nasal turbinate. Tissue cysts were already fully developed as the average tissue cyst diameter was 181.20 μm. Microscopic lesions were mainly detected in skin samples, followed by reproductive and upper respiratory tracts. Mild lesions compatible with both acute (thrombus, oedema and inflammation) and chronic besnoitiosis (skin lesions, hyperkeratosis and dilated sweat glands) coexisted. Vascular damage and inflammation were more frequently observed in skin (including scrotum) followed by testicular parenchyma, epididymis and pampiniform plexus. Histological findings evidenced a subclinical chronic besnoitiosis.

Toxoplasmosis and neosporosis are closely related protozoan diseases that lead to important economic impacts in farm ruminants. Toxoplasma gondii infection mainly causes reproductive failure in small ruminants and is a widespread zoonosis, whereas Neospora caninum infection is one of the most important causes of abortion in cattle worldwide. Vaccination has been considered the most economic measure for controlling these diseases. However, despite vaccine development efforts, only a liveattenuated T. gondii vaccine has been licensed for veterinary use, and no promising vaccines against neosporosis have been developed; therefore, vaccine development remains a key goal. Additionally, drug therapy could be a valuable strategy for disease control in farm ruminants, as several drugs that limit T. gondii and N. caninum proliferation and dissemination have been evaluated. This approach may also be relevant to performing an initial drug screening for potential human therapy for zoonotic parasites. Treatments can be applied against infections in adult ruminants to minimize the outcomes of a primo-infection or the reactivation of a chronic infection during gestation or in newborn ruminants to avoid infection chronification. In this review, the current status of drug development against toxoplasmosis and neosporosis in farm ruminants is presented, and in an effort to promote additional treatment options, prospective drugs that have shown efficacy in vitro and in laboratory animal models of toxoplasmosis and neosporosis are examined.

15 páginas, 2 tablas, 7 figuras., Experimental infections in pregnant sheep have been focused on studying the effect of the time of challenge on the outcome of N. caninum infection, whereas the impact of the dose and route of challenge has not been studied in depth. Therefore, clinical outcome, immune responses, parasite detection and burden, and lesion severity in placental tissues and foetal brains were investigated in 90-day-pregnant sheep inoculated intravenously with 10(5) (G1), 10(4) (G2), 10(3) (G3), or 10(2) (G4) tachyzoites or subcutaneously with 10(4) (G5) tachyzoites of the virulent Nc-Spain7 isolate and an uninfected group (G6). Comparing challenge doses, G1 was the only group that had 100% abortion. Likewise, IFN gamma levels in G1 increased earlier than those in other intravenously infected groups, and IgG levels on day 21 post-infection (pi) were higher in G1 than those in other intravenously infected groups. Concerning vertical transmission, G1 shows a higher parasite burden in the foetal brain than did G2 and G3. Comparing routes of administration, no differences in foetal survival rate or parasite load in the foetal brain were found. Although G2 had higher IFN gamma levels than G5 on day 10 pi, no differences were found in humoral immune responses. Because the outcome after intravenous infection with 10(5) tachyzoites was similar to that observed after intravenous infection with 10(6) tachyzoites used in a previous work (100% abortion and vertical transmission), we conclude that it may be reasonable to use 10(5) tachyzoites administered by the intravenous route in further experiments when assessing drugs or vaccine candidates., We gratefully acknowledge Neus Callizo Esteve from Clinical Veterinary Hospital (Complutense University of Madrid, Spain) and Jose Maria Gonzalez (University of Zaragoza, Spain) for their excellent technical assistance. The Animal Experimentation Service (SEA) at the University of Zaragoza is acknowledged for using their facilities to carry out the reproduction programme. Roberto Sanchez Sanchez is supported by a fellowship from the Spanish Ministry of Education, Culture and Sports (MECD) as a part of the Programme of Training of University Teaching Staff (FPU, Grant Number FPU13/03438). This work was supported by the Ministry of Economy and Competitiveness (AGL2016-75935-C2) and Community of Madrid, Spain (PLATESA, S2013/ABI2906).

13 páginas, 2 tablas, 7 figuras., Neospora caninum is one of the main causes of abortion in cattle, and recent studies have highlighted its relevance as an abortifacient in small ruminants. Vaccines or drugs for the control of neosporosis are lacking. Bumped kinase inhibitors (BKIs), which are ATP-competitive inhibitors of calcium dependent protein kinase 1 (CDPK1), were shown to be highly efficacious against several apicomplexan parasites in vitro and in laboratory animal models. We here present the pharmacokinetics, safety and efficacy of BKI-1553 in pregnant ewes and foetuses using a pregnant sheep model of N. caninum infection. BKI-1553 showed exposure in pregnant ewes with trough concentrations of approximately 4 mu M, and of 1 mu M in foetuses. Subcutaneous BKI-1553 administration increased rectal temperatures shortly after treatment, and resulted in dermal nodules triggering a slight monocytosis after repeated doses at short intervals. BKI-1553 treatment decreased fever in infected pregnant ewes already after two applications, resulted in a 37-50% reduction in foetal mortality, and modulated immune responses; IFN. levels were increased early after infection and IgG levels were reduced subsequently. N. caninum was abundantly found in placental tissues; however, parasite detection in foetal brain tissue decreased from 94% in the infected/untreated group to 69-71% in the treated groups. In summary, BKI-1553 confers partial protection against abortion in a ruminant experimental model of N. caninum infection during pregnancy. In addition, reduced parasite detection, parasite load and lesions in foetal brains were observed., We gratefully acknowledge Jacobo Sampere from the SALUVETgroup (Complutense University of Madrid, Spain), Teresa Navarro andJosé María González (University of Zaragoza, Spain), Isabel Ayala,Lucas Troya, Clara Colmenero, Dolores González, Natalia Peral,Verónica Pérez, Ignacio Gómez and Isabel Santiago from the Clinical Veterinary Hospital (Complutense University of Madrid, Spain) for their excellent technical assistance, and also Rama Subba Rao Vidadala and Dustin Maly (Department of Chemistry, University of Washington) fortheir assistance in further purification of the study drug. The AnimalExperimentation Service (SEA) at the University of Zaragoza is ac-knowledged for providing their facilities to carry out the reproduction programme. Roberto Sánchez Sánchez is supported by a fellowship from the Spanish Ministry of Education, Culture and Sports (MECD), asa part of the Program of Training of University Teaching Staff (FPU,grant number FPU13/03438). Patricia Vázquez has a Juan de la Cierva-Formación post-doctoral contract (FJCI-2014-20982) from the Spanish Ministry of Economy and Competitiveness (MINECO). Andrew Hemphill is supported by the Swiss National Science Foundation grant(No. 310030 165782). This work was supported by the Public Health Service, National Institutes of Health, Bethesda, MD (grants R01 AI111341 and R01 HD 080670), the U.S. Department of Agriculture(grant 2014-67015-22106) and the Community of Madrid, Spain(PLATESA, S2013/ABI2906).

9 páginas, 4 figuras, 1 tabla., Bovine besnoitiosis, caused by the apicomplexan Besnoitia besnoiti, is a chronic and debilitating disease characterized by cutaneous and systemic manifestations that primarily affects adult beef cattle. Previous studies have reported that clinical besnoitiosisis is rare in calves. However, we isolated B. besnoiti from a chronically infected calf for the first time. The identity of the Besnoitia species was determined after parasite isolation and molecular genotyping. According to the results obtained in vitro the new isolate, named as Bb-Spain3, was characterized in a reproducible in vitro model and was categorized as a low invader and low prolific isolate with a slower lytic cycle compared to Bb-Spain 1 isolate. Specific traits that differentiate isolates obtained from adult animals from those infecting calves were not found. Next, we described the first case report of chronic besnoitiosis in a female calf less than 6 months-old with a low body condition. The disease was confirmed by the presence of specific anti-B. besnoiti antibodies and parasite detection in the skin. At post-mortem examination, tissue samples were collected for histological, immunohistochemical and molecular analyses. DNA-parasite was detected in 31 different calf's tissues, being the most highly parasitized tissues the skin and the respiratory and reproductive tracts. In addition, the parasite was also present in heart, eyes, lymph nodes and brain. The high parasite load, a wide intra-organic parasite distribution and the presence of both viable and degenerated cysts, were indicative of a rapid progression of the disease. This case report underlines the need to include the inspection of young animals in besnoitiosis control., This study was financed by the Spanish Ministry of Economy and Competitiveness (AGL2013-46442-R), CYTED (The-matic Network 113RT0469 Protozoovac) and by the Community of Madrid (PLATESA S20137ABI-2906). Carlos Diezma-Díaz was financially supported through a grant from the Spanish Ministry of Economy and Competitiveness (BES-2014-069839), and Alejandro Jiménez-Meléndez was supported through a grant from the Spanish Ministry of Education, Culture and Sports (grant no. FPU 13/05481). M. Fernández is the recipient of a predoctoral contract from the “Junta de Castilla y León”, partially funded by the European Social Fund (European Union).We acknowledge the veterinarians Félix J. Díaz Gómez and Arturo Francisco González and the farmer for the help in samplings and data collection. Finally, we wish also to acknowledge Javier Moreno Gonzalo and Vanessa Navarro Lozano for their excellent technical assistance.

Bovine besnoitiosis, which is caused by the tissue cyst-forming intracellular parasite Besnoitia besnoiti, is a chronic and debilitating disease that is responsible for severe economic losses in the cattle raised under extensive husbandry systems. The absence of vaccines, treatments or a health scheme at local, national and international levels has led to a rapid spread of bovine besnoitiosis from western Europe towards eastern countries and northwards. Moreover, this parasitic disease is widely present in many sub-Saharan countries. Thus, bovine besnoitiosis should be included in the animal health scheme of beef cattle herds. Accurate diagnostic tools and common diagnostic procedures are mandatory in any control programme. Relevant advances have been made in this field during the last decade. Succeeding with accurate diagnosis relies on the technique employed and the antibody and parasite kinetics of the infection stage, which may notably influence control programmes and surveillance. Moreover, control programmes should be adapted to the epidemiological status of the disease, as the disease presentation in a herd has important implications for prospective control. Herein, we review the clinical disease presentation of bovine besnoitiosis and the correlation between its clinical course and laboratory parameters. We also provide an update on the available diagnostic tools, discuss their strengths and pitfalls, and provide guidelines for their use in control, surveillance and epidemiological studies. A rational control strategy is also recommended.

Neospora caninum is one of the most efficient transplacentally transmitted pathogens in cattle and is a cause of abortion in this domestic species. The invasion and proliferation of Neospora caninum in the placenta and its dissemination to the foetus are crucial events in the outcome of an infection. In the bovine placenta, the placentomes are formed by maternal caruncles, which are delimited by a maternal epithelium and foetal cotyledons, which are delimited by an epithelial layer named the trophoblast. These epithelia form a physical barrier against foetal infection. Furthermore, trophoblast cells act as an innate immune defence at the foetal-maternal interface. Neospora caninum invades and proliferates in trophoblast cells in vitro, but it is unknown whether host cell modulation events, which affect the immune response and other processes in the trophoblast, occur. In this work, we investigated the transcriptomic modulation by Neospora caninum infection in the bovine trophoblast cell line F3. In addition, two Neospora caninum isolates with marked differences in virulence, Nc-Spain1H and the Nc-Spain7, were used in this study to investigate the influence of these isolates in F3 modulation. The results showed a clear influence on extracellular matrix reorganisation, cholesterol biosynthesis and the transcription factor AP-1 network. Interestingly, although differences in the transcriptome profiles induced by each isolate were observed, specific isolate-modulated processes were not identified, suggesting very similar regulation in both isolates. Differential expression of the N. caninum genes between both isolates was also investigated. Genes involved in host cell attachment and invasion (SAG-related and microneme proteins), glideosome, rhoptries, metabolic processes, cell cycle and stress response were differentially expressed between the isolates, which could explain their variability. This study provides a global view of Neospora caninum interactions with bovine trophoblast cells and of the intra-specific differences between two Neospora caninum isolates with biological differences.

Objectives Establishment of a mouse model for congenital toxoplasmosis based on oral infection with oocysts from Toxoplasma gondii ME49 and its application for investigating chemotherapeutic options against congenital toxoplasmosis. Methods CD1 mice were mated, orally infected with 5, 25, 100, 500 or 2000 oocysts and monitored for clinical signs and survival of dams and pups until 4 weeks post partum . The parasite burden in infected mice was quantified by real-time PCR in lungs, brains and, in the case of surviving pups, also in eyes. Seroconversion was assessed by ELISA. T. gondii cysts in brain were identified by immunofluorescence. In a second experiment, pregnant CD1 mice challenged with 20 oocysts/mouse were treated with buparvaquone or the calcium-dependent protein kinase 1 inhibitor bumped kinase inhibitor (BKI)-1294 and the outcome of infection was analysed. Results T. gondii DNA was detected in the brain of all infected animals, irrespective of the infection dose. Seroconversion occurred at 3 weeks post-infection. Most pups born to infected dams died within 1 week post partum , but a small fraction survived until the end of the experiment. T. gondii DNA was detected in the brain of all survivors and half of them exhibited ocular infection. Chemotherapy with both compounds led to dramatically increased numbers of surviving pups and reduced cerebral infection. Most efficient were treatments with BKI-1294, with 100% survivors and only 7% brain-positive pups. Conclusions BKI-1294 and buparvaquone exert excellent activities against transplacental transmission in pregnant mice.

Bovine besnoitiosis has been deemed a re-emerging disease in Western Europe and considered endemic in Spain, Portugal, France and in some areas of Northern Italy. This report refers to an infection outbreak in a purebred beef herd from Northern Italy involving a large number of bulls. In October 2013, 544 animals were serologically tested with an in-house ELISA followed by a confirmatory Western blot to evaluate Besnoitia besnoiti seroprevalence. A year later, 461 animals were then serologically re-tested together with imported animals (n = 268). Overall, 812 animals were involved in the study. Histology and immunohistochemistry were performed on skin biopsies of suspected animals and several tissue samples from a slaughtered bull. In the first sampling, 100 animals were seropositive (18.4%); in the second sampling, prevalence increased up to 36.5%, with incidence calculated at 39.6%. The risk factor analysis revealed that the infection was associated with age (OR = 1.007) and sex, with males presenting a greater risk (OR = 2.006). In fact, prevalence values in bulls increased from 29.6 to 56.7%, with an incidence of infection of 53.3%. Moreover, mating with a seropositive bull enhanced infection risk for a seronegative cow (OR = 1.678). Clinical signs typical of bovine besnoitiosis were found in seven seropositive animals, with confirmation of B. besnoiti through histology and immunohistochemistry. The study outcomes confirm that bovine besnoitiosis is a disease with serious economic impact on beef cattle breeding, particularly on bulls in service. Good management practises such as clinical monitoring and serological testing of imported animals should be implemented to control its occurrence.

Background Bovine besnoitiosis, caused by the apicomplexan parasite Besnoitia besnoiti, is a chronic and debilitating cattle disease that notably impairs fertility. Acutely infected bulls may develop respiratory signs and orchitis, and sterility has been reported in chronic infections. However, the pathogenesis of acute disease and its impact on reproductive function remain unknown. Methods Herein, we studied the microscopic lesions as well as parasite presence and load in the testis (pampiniform plexus, testicular parenchyma and scrotal skin) of seven bulls with an acute B. besnoiti infection. Acute infection was confirmed by serological techniques (IgM seropositive results and IgG seronegative results) and subsequent parasite detection by PCR and histological techniques. Results The most parasitized tissue was the scrotal skin. Moreover, the presence of tachyzoites, as shown by immunohistochemistry, was associated with vasculitis, and three bulls had already developed juvenile tissue cysts. In all animals, severe endothelial injury was evidenced by marked congestion, thrombosis, necrotizing vasculitis and angiogenesis, among others, in the pampiniform plexus, testicular parenchyma and scrotal skin. Vascular lesions coexisted with lesions characteristic of a chronic infection in the majority of bulls: hyperkeratosis, acanthosis and a marked diffuse fibroplasia in the dermis of the scrotum. An intense inflammatory infiltrate was also observed in the testicular parenchyma accompanied by different degrees of germline atrophy in the seminiferous tubules with the disappearance of various strata of germ cells in four bulls. Conclusions This study confirmed that severe acute besnoitiosis leads to early sterility that might be permanent, which is supported by the severe lesions observed. Consequently, we hypothesized that testicular degeneration might be a consequence of (i) thermoregulation failure induced by vascular lesions in pampiniform plexus and scrotal skin lesions; (ii) severe vascular wall injury induced by the inflammatory response in the testis; and (iii) blood-testis barrier damage and alteration of spermatogenesis by immunoresponse.

Dogs play a potential role as reservoirs for zoonotic parasites, being especially problematic uncontrolled dog populations such as stray and farm dogs with access to populated areas. In order to investigate the prevalence of canine intestinal parasites in at-risk dog populations, we tested a total of 233 faecal samples shed by stray and dairy farm dogs from northern Spain. Telemann method was used to detect the presence of eggs and (oo)cysts of common dog intestinal parasites and Cryptosporidium was detected by PCR. One hundred and forty eight out of 233 samples (63.5%) were positive for at least one intestinal parasite, being Ancylostomidae (35.6%; 83/233) and Trichuris (35.2%; 82/233) the parasites most frequently identified. Cryptosporidium DNA was not detected in any of the faecal samples analysed. The overall prevalence was significantly higher in stray dogs than in farm dogs (72.5% vs 58.8%). Specifically, stray dogs had a significantly higher prevalence of Ancylostomatidae, Toxocara, Toxascaris and Taenidae. These dog populations are an important source of environmental contamination with intestinal parasite forms, which could be of significance to animal and human health. This work was supported by project PR1/06-14467-B (Complutense University of Madrid) Sí

Exposure of Neospora caninum tachyzoites to BKI-1294 in vitro results in the formation of long-lived multinucleated complexes (MNCs). However, in vivo treatment of BALB/c mice with BKI-1294 shortly after N. caninum infection during pregnancy was safe and profoundly reduced pup mortality and vertical transmission. We hypothesized that the formation of MNCs could trigger immune responses that contribute to BKI efficacy in vivo. In this study, mice were first vaccinated with a sublethal dose of N. caninum tachyzoites and were treated with BKI-1294. We then investigated the effects of these treatments after mating and re-infection during pregnancy. Effects on fertility, pup survival, vertical transmission, and parasite load in dams were evaluated. Cytokines in sera or splenocyte culture supernatants were assessed by either ELISA or the Luminex™ 200 system, and humoral immune responses against tachyzoite and MNC antigens were compared by ELISA, Western blotting and immunoproteomics. Our results showed that BKI-1294 treatment of live-vaccinated mice reduced the cerebral parasite load in the dams, but resulted in higher neonatal pup mortality and vertical transmission. In live-vaccinated mice, cytokine levels, most notably IFN-y, IL-10, and IL-12, were consistently lower in BKI-1294 treated animals compared to non-treated mice. In addition, comparative Western blotting identified two protein bands in MNC extracts that were only recognized by sera of live-vaccinated mice treated with BKI-1294, and were not found in tachyzoite extracts. We conclude that treatment of live-vaccinated mice with BKI-1294 influenced the cellular and humoral immune responses against infection, affected the safety of the live-vaccine, and decreased protection against re-infection and vertical transmission during pregnancy.

10 páginas, 2 tablas, 9 figuras., There is an unacknowledged clinical presentation of ovine toxoplasmosis characterized by early abortions and lesions of fetal leukoencephalomalacia. To investigate the pathogenesis of this condition, the extent and distribution of leukomalacia and the variations in the cell populations associated with it were characterized in 32 fetal brains from 2 previously published experimental studies of Toxoplasma gondii infection in pregnant sheep. Immunohistochemical labeling of βAPP allowed for the detection of leukomalacia in 100/110 (91%) studied samples. There was no clear influence of the challenge dose or the area of the brain (frontal lobe, corpus callosum, midbrain, and cerebellum). In tissues with leukomalacia, there was loss of oligodendrocytes and increased number of astrocytes and microglia both in the areas of necrosis but also in the surrounding area. These findings were similar to those described in ovine experimental models (inflammation syndrome and hypoxic models) of periventricular leukomalacia in humans. Thus, a fetal inflammatory syndrome may be involved in the pathogenesis of early abortion in ovine toxoplasmosis. However, further studies are needed to determine the pathogenesis of this clinical presentation because placental thrombosis and resulting hypoxia could also be responsible for the leukomalacia., This work was supported by research projects from the “Junta de Castilla y León” (LE080U16) and Spanish Ministry of Sciences (AGL2016-75935-C2- 1-R and AGL2016-75935-C2-2-R). Daniel Gutiérrez-Expósito is the recipient of a postdoctoral contract from the Ministry of Science and Innovation (Grant No. FJCI-2017-32020).

Background Bovine besnoitiosis, caused by the cyst-forming apicomplexan parasite Besnoitia besnoiti, is a chronic and debilitating cattle disease that continues to spread in Europe in the absence of control tools. In this scenario, in vitro culture systems are valuable tools to carry out drug screenings and to unravel host-parasite interactions. However, studies performed in bovine target cells are scarce. Methods The objective of the present study was to obtain primary bovine aortic endothelial cells (BAECs) and fibroblast cell cultures, target cells during the acute and the chronic stage of the disease, respectively, from healthy bovine donors. Afterwards, expression of surface (CD31, CD34 and CD44) and intracellular markers (vimentin and cytokeratin) was studied to characterize cell populations by flow cytometry. Next, the lytic cycle of B. besnoiti tachyzoites was studied in both target cells. Invasion rates (IRs) were determined by immunofluorescence at several time points post-infection, and proliferation kinetics were studied by quantitative PCR (qPCR). Finally, the influence of bovine viral diarrhea virus (BVDV) co-infection on the host cell machinery, and consequently on B. besnoiti invasion and proliferation, was investigated in BAECs. Results Morphology and cytometry results confirmed the endothelial and fibroblast origins. CD31 was the surface marker that best discriminated between BAECs and fibroblasts, since fibroblasts lacked CD31 labelling. Expression of CD34 was weak in low-passage BAECs and absent in high-passage BAECs and fibroblasts. Positive labelling for CD44, vimentin and cytokeratin was observed in both BAECs and fibroblasts. Regarding the lytic cycle of the parasite, although low invasion rates (approximately 3–4%) were found in both cell culture systems, more invasion was observed in BAECs at 24 and 72 hpi. The proliferation kinetics did not differ between BAECs and fibroblasts. BVDV infection favoured early Besnoitia invasion but there was no difference in tachyzoite yields observed in BVDV-BAECs compared to BAECs. Conclusions We have generated and characterized two novel standardized in vitro models for Besnoitia besnoiti infection based on bovine primary target BAECs and fibroblasts, and have shown the relevance of BVDV coinfections, which should be considered in further studies with other cattle pathogens.

Early Neospora caninum infection dynamics were investigated in pregnant heifers intravenously inoculated with PBS (G-Control) or 107 tachyzoites of high (G-NcSpain7)- or low (G-NcSpain1H)-virulence isolates at 110 days of gestation. Serial culling at 10 and 20 days post-infection (dpi) was performed. Fever was detected at 1 dpi in both infected groups (P, This work was supported by the Spanish Ministry of Economy and Competitiveness (AGL2013-44694-R) and the Community of Madrid (PLATESA2-CM P2018/BAA-4370). Laura Jiménez-Pelayo was financially supported by a fellowship from the Complutense University of Madrid and Marta García-Sánchez was financially supported through a grant from the Spanish Ministry of Economy and Competitiveness (BES-2014-070723). Patricia Vázquez had a Juan de la Cierva-Formación post-doctoral contract (FJCI-2014-20982) from the Spanish Ministry of Economy and Competitiveness (MINECO). Alicia Román-Trufero was supported by a FPI-INIA fellowship from the Spanish National Institute for Agriculture and Food Research and Technology (INIA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

16 páginas, 3 tablas, 6 figuras., Previous studies on drug efficacy showed low protection against abortion and vertical transmission of Toxoplasma gondii in pregnant sheep. Bumped kinase inhibitors (BKIs), which are ATP-competitive inhibitors of calcium-dependent protein kinase 1 (CDPK1), were shown to be highly efficacious against several apicomplexan parasites in vitro and in laboratory animal models. Here, we present the safety and efficacy of BKI-1294 treatment (dosed orally at 100 mg/kg of body weight 5 times every 48 h) initiated 48 h after oral infection of sheep at midpregnancy with 1,000 TgShSp1 oocysts. BKI-1294 demonstrated systemic exposure in pregnant ewes, with maximum plasma concentrations of 2 to 3 M and trough concentrations of 0.4 M at 48 h after each dose. Oral administration of BKI-1294 in uninfected sheep at midpregnancy was deemed safe, since there were no changes in behavior, fecal consistency, rectal temperatures, hematological and biochemical parameters, or fetal mortality/morbidity. In ewes infected with a T. gondii oocyst dose lethal for fetuses, BKI-1294 treatment led to a minor rectal temperature increase after infection and a decrease in fetal/lamb mortality of 71%. None of the lambs born alive in the treated group exhibited congenital encephalitis lesions, and vertical transmission was prevented in 53% of them. BKI-1294 treatment during infection led to strong interferon gamma production after cell stimulation in vitro and a low humoral immune response to soluble tachyzoite antigens but high levels of anti-SAG1 antibodies. The results demonstrate a proof of concept for the therapeutic use of BKI-1294 to protect ovine fetuses from T. gondii infection during pregnancy., We gratefully acknowledge the SALUVET group (Complutense University of Madrid, Spain), Luis Miguel Ferrer, Jose Ventura, José Calasanz Jiménez, Francisco Saura, Teresa Navarro, and José María González from the University of Zaragoza (Spain), and Victor Herrero and Javier Blanco from Clinical Veterinary Hospital (Complutense University of Madrid, Spain) for their excellent technical assistance, Dale Kempf and Kennan Marsh, from AbbVie, Inc., for their suggestion of the Phosal vehicle used in the experiments reported here, Rama S. R. Vidadala and Dustin J. Maly, Department of Chemistry, University of Washington, for characterization and further purification of BKI-1294, and advice from Robert Choy and Eugenio L. de Hostos from the PATH Drug Development Program. The Animal Experimentation Service (SEA) at the University of Zaragoza is acknowledged for providing their facilities to carry out the reproduction program. R.S.-S. is supported by a fellowship from the Spanish Ministry of Education, Culture and Sports (MECD), as a part of the Program of Training of University Teaching Staff (FPU; grant number FPU13/03438). A.H. is supported by a Swiss National Science Foundation grant (no. 310030-165782). This work was supported by the Public Health Service, National Institutes of Health, Bethesda, MD (grants R01 AI 111341 and R01 HD 080670), the U.S. Department of Agriculture (grant 2014-67015-22106), and the Community of Madrid, Spain (PLATESA2, P2018/BAA-4370). I.F., A.H., K.K.O., W.C.V.V., and L.M.O.-M. conceived the study and participated in its design. R.S.-S. wrote the manuscript, with results interpretation and discussion inputs from I.F., J.R.-C., A.H., M.A.H., L.K.B., W.C.V.V., and L.M.O.-M. J.J.R. selected the animals and executed the reproductive program. R.S.-S., I.F., and M.R. carried out oocyst infection and drug administration. R.S.-S., I.F., M.R., M.P.D., M.G.-H., E.T., and J.B. participated in clinical examination and sampling of animals and performed necropsies and hematological, biochemical, and histopathological analyses. M.H., L.K.B., R.C., G.R.W., and K.K.O. determined the pharmacokinetics of the compound. R.S.-S. performed peripheral blood stimulation assay, serological assays, PCR analyses, statistical analysis, and interpretation of the results. All authors read and approved the final manuscript. W.C.V.V. is the president and co-owner of ParaTheraTech, Inc., a company that is developing BKIs for animal health. W.C.V.V. did not perform the experiments or interpret the results of the experiments, but he did edit the paper and helped plan the experiments. The other authors have no competing interests to declare.

Background Bovine neosporosis, one of the main causes of reproductive failure in cattle worldwide, poses a challenge for the immune system of pregnant cows. Changes in the Th-1/Th-2 balance in the placenta during gestation have been associated with abortion. Cotyledon and caruncle cell layers form the maternal-foetal interface in the placenta and are able to recognize and induce immune responses against Neospora caninum among other pathogens. The objective of the present work was to elucidate the immunomodulation produced by high- (Nc-Spain7) and low-virulence (Nc-Spain1H) isolates of N. caninum in bovine trophoblast (F3) and caruncular cells (BCEC-1) at early and late points after infection. Variations in the mRNA expression levels of toll-like receptor-2 (TLR-2), Th1 and Th2 cytokines (IL-4, IL-10, IL-8, IL-6, IL-12p40, IL-17, IFN-γ, TGF-β1, TNF-α), and endothelial adhesion molecules (ICAM-1 and VCAM-1) were investigated by RT-qPCR, and protein variations in culture supernatants were investigated by ELISA. Results A similar pattern of modulation was found in both cell lines. The most upregulated cytokines in infected cells were pro-inflammatory TNF-α (P

Neospora caninum, a protozoan parasite closely related to Toxoplasma gondii, represents one of the main causes of abortion in cattle. Macrophages (MØs) are mediators of the innate immune response against infection and likely one of the first cells encountered by the parasite during the host infection process. In this study, we investigated in vitro how high or low virulent isolates of N. caninum (Nc-Spain7 and Nc-Spain1H, respectively) interact with bovine monocyte-derived MØs and the influence of the isolate virulence on the subsequent cellular response. Both isolates actively invaded, survived and replicated in the MØs. However, Nc-Spain7 showed a higher invasion rate and a replication significantly faster, following an exponential growth model, whereas Nc-Spain1H presented a delayed replication and a lower growth rate without an exponential pattern. N. caninum infection induced a hypermigratory phenotype in bovine MØs that was characterized by enhanced motility and transmigration in vitro and was accompanied by morphological changes and abrogated extracellular matrix degradation. A significantly higher hypermotility was observed with the highly virulent isolate Nc-Spain7. Nc-Spain1H-infected MØs showed elevated reactive oxygen species (ROS) production and IL12p40 expression, which also resulted in increased IFN-γ release by lymphocytes, compared to cells infected with Nc-Spain7. Furthermore, IL-10 was upregulated in MØs infected with both isolates. Infected MØs exhibited lower expression of MHC Class II, CD86, and CD1b molecules than uninfected MØs, with non-significant differences between isolates. This work characterizes for the first time N. caninum replication in bovine monocyte-derived MØs and details isolate-dependent differences in host cell responses to the parasite.

Neospora caninum is worldwide recognized as one of the major abortive pathogens in cattle. Although cases of abortion are also registered in sheep and goats, information and epidemiological data on neosporosis in small ruminants are usually scant. In Italy, but few exceptions, data are limited to spare reports of abortions. This survey was aimed to i) update information on N. caninum infection in sheep and goats from north-eastern Italy; ii) analyze associated individual and flock risk factors and iii) compare N. caninum positivity with previously obtained data on Toxoplasma gondii. Four hundred and fourteen goats and 428 sheep from 39 flocks in Lombardy region were enrolled. Blood samples were collected and analyzed by an in-house ELISA followed by a confirmatory Western Blot. The test resulted positive for 5.7% of goats and 19.3% of sheep. As to farms, 32.1% and 89.4% of caprine and ovine flocks showed positive, respectively. Managerial variables, including rearing system and farm size, affected N. caninum infection. Small, family-run farms were at higher risk rather than intensive caprine farms or transhumant sheep flocks. Outcomes from the survey mainly indicate that these farms should implement their care about proper sanitary measures and monitor the spread of the infection among small ruminants.

Herein, we report the first incidence of systemic besnoitiosis in a male juvenile roe deer Capreolus capreolus. The animal was found dead in an area where bovine besnoitiosis is endemic and showed cachexia and multiple skin erosions in the metacarpal and metatarsal areas. Moreover, round and elevated white structures suggestive of Besnoitia spp. tissue cysts were also present. Twenty-eight tissue samples from different anatomical locations were collected for microscopic lesion and parasite detection through histopathology and PCR. Immunohistochemistry was performed to confirm Besnoitia-positive reaction in the tissue cysts. In addition, the identity of Besnoitia spp. in PCR-positive tissue samples was also investigated using microsatellite (MS) markers, and the comparison of protein disulphide isomerase gene sequences (BbPDI) of B. besnoiti and B. tarandi isolated from cattle and reindeer, respectively. Besnoitia cysts were detected in the skin (several parts), respiratory and upper digestive tracts, eyes, kidney, liver, testicle, cardiac muscle and lymphoid tissue. Remarkably, the presence of tissue cysts in the brain confirmed the capacity of Besnoitia spp. to form tissue cysts in the central nervous system (CNS). Finally, the Besnoitia species detected showed the same MS genotype as B. besnoiti, and BbPDI sequences from roe deer and two B. besnoiti isolates were genetically identical throughout multiple sequence alignment. Thus, for the first time, there is evidence that roe deer might act as an intermediate host of B. besnoiti. Further molecular analyses and parasite isolations are needed to corroborate these findings.

Toxoplasma gondii and Neospora caninum are two major abortifacient protozoans in domestic small ruminants and cattle, respectively, and they also parasitize a wide range of wildlife. Numerous serosurveys have been conducted in wild ruminants worldwide. However, the potential effect of different ecosystems and management practices on these infections has not been investigated. We studied the prevalence of antibodies to T. gondii and N. caninum in wild ruminants between 2007 and 2012 from four national wildlife reserves: three open space reserves in northwest Spain (Ancares, Mampodre, and Riaño) and a fenced reserve in central Spain (Quintos de Mora). Sera from roe deer ( Capreolus capreolus ) and chamois ( Rupicapra rupicapra ) were collected in Ancares (roe deer), Mampodre (both species), and Riaño (both species), whereas red deer ( Cervus elaphus ) sera were collected only in Quintos de Mora. The results of immunofluorescence antibody tests showed a T. gondii antibody prevalence significantly higher in red deer (13%; 17/131) than in roe deer (2%; 5/228) and chamois (4%; 6/149) (P0.05, Fisher's exact test). Moreover, N. caninum -specific antibodies were only detected in 1% of animals (2/131 red deer, 2/228 roe deer, and 2/149 chamois). Management measures were implemented in the Quintos de Mora reserve and T. gondii antibody prevalence in red deer decreased from 13% to 2% after 5 yr. In contrast, N. caninum antibody prevalences were very low (2%) over the years. The results suggest a low frequency of sylvatic life cycles in the hunting reservations studied, so interconnection between sylvatic and domestic life cycles is unlikely. Regardless, a sustainable exploitation of natural resources in wildlife reserves may help to reduce the prevalence of T. gondii infection.

Parameters such as pathogen dose and inoculation route are paramount in animal models when studying disease pathogenesis. Here, clinical findings, including foetal mortality, parasite transmission rates and lesion severity, and immune responses were evaluated in Asturiana pregnant heifers at day 110 of gestation challenged with a virulent (Nc-Spain7) Neospora caninum isolate. Four different doses of parasite tachyzoites were inoculated intravenously (IV1, 107 parasites, n = 6; IV2, 105, n = 6; IV3, 103, n = 6; and IV4, 102, n = 5), and the subcutaneous (SC) inoculation route was also assessed for the dose of 105 tachyzoites (SC, n = 6). In addition, a control group (n = 4 pregnant heifers) was evaluated. Foetal death was observed in all infected groups from 25 to 62 days post-infection, varying with the dose (IV1:4/6, IV2:3/6; IV4:2/5, IV3:1/6), and was three times less frequently associated with the SC route than IV inoculation (1/6 vs. 3/6). A dose-dependent effect for parasite loads in placental and foetal brain tissues was also detected. After SC challenge, a reduced number of tachyzoites were able to reach foetal brain tissues, and no lesions were observed. In calves, specific IgG responses in precolostral sera were mainly associated with high-dose groups (IV1 [100.0%] and IV2 [66.7%]), and cerebral parasite DNA detection was scarce (3/18). In dams, IFN-γ production and the dynamics of anti-N. caninum IgG antibodies varied with the dose, and the cell-mediated immune response was also found to be route-dependent. Our results confirm the influence of parasite dose and inoculation route on the outcome and dynamics of bovine neosporosis at mid-gestation., Patricia Vázquez had a Juan de la Cierva-Formación post-doctoral contract (FJCI-2014-20982) from the Spanish Ministry of Economy and Competitiveness (MINECO). Laura Jiménez-Pelayo was founded by UCM-Santander and Marta García-Sánchez was founded by MINECO (BES-2014-070723). Alicia Román-Trufero was supported by a 4-year FPI-INIA fellowship from the Spanish National Institute for Agriculture and Food Research and Technology (INIA).

The protozoan parasite Toxoplasma gondii is a zoonotic parasite that can be transmitted from animals to humans. Felids, including domestic cats, are definitive hosts that can shed oocysts with their feces. In addition to infections that occur by accidental oral uptake of food or water contaminated with oocysts, it is assumed that a large proportion of affected humans may have become infected by consuming meat or other animal products that contained infective parasitic stages of T. gondii. Since farm animals represent a direct source of infection for humans, but also a possible reservoir for the parasite, it is important to control T. gondii infections in livestock. Moreover, T. gondii may also be pathogenic to livestock where it could be responsible for considerable economic losses in some regions and particular farming systems, e.g. in areas where the small ruminant industry is relevant. This review aims to summarize actual knowledge on the prevalence and effects of infections with T. gondii in the most important livestock species and on the effects of toxoplasmosis on livestock. It also provides an overview on potential risk factors favoring infections of livestock with T. gondii. Knowledge on potential risk factors is prerequisite to implement effective biosecurity measures on farms to prevent T. gondii infections. Risk factors identified by many studies are cat-related, but also those associated with a potential contamination of fodder or water, and with access to a potentially contaminated environment. Published information on the costs T. gondii infections cause in livestock production, is scarce. The most recent peer reviewed reports from Great Britain and Uruguay suggest annual cost of about 5–15 million US $ per country. Since these estimates are outdated, future studies are needed to estimate the present costs due to toxoplasmosis in livestock. Further, the fact that T. gondii infections in livestock may affect human health needs to be considered and the respective costs should also be estimated, but this is beyond the scope of this article., Graphical abstract Unlabelled Image, Highlights • Brief overview on T. gondii prevalence in livestock • Natural and experimental T. gondii infections • Risk factors for T. gondii infection of livestock • Economic impact of toxoplasmosis in livestock

18 páginas, 6 figuras, 3 tablas., Toxoplasma gondii is an apicomplexan parasite that infects almost all warm-blooded animals. Little is known about how the parasite virulence in mice extrapolates to other relevant hosts. In the current study, in vitro phenotype and in vivo behavior in mice and sheep of a type II T. gondii isolate (TgShSp1) were compared with the reference type II T. gondii isolate (TgME49). The results of in vitro assays and the intraperitoneal inoculation of tachyzoites in mice indicated an enhanced virulence for the laboratory isolate, TgME49, compared to the recently obtained TgShSp1 isolate. TgShSp1 proliferated at a slower rate and had delayed lysis plaque formation compared to TgME49, but it formed more cyst-like structures in vitro. No mortality was observed in adult mice after infection with 1–105 tachyzoites intraperitoneally or with 25–2,000 oocysts orally of TgShSp1. In sheep orally challenged with oocysts, TgME49 infection resulted in sporadically higher rectal temperatures and higher parasite load in cotyledons from ewes that gave birth and brain tissues of the respective lambs, but no differences between these two isolates were found on fetal/lamb mortality or lesions and number of T. gondii-positive lambs. The congenital infection after challenge at mid-pregnancy with TgShSp1, measured as offspring mortality and vertical transmission, was different depending on the challenged host. In mice, mortality in 50% of the pups was observed when a dam was challenged with a high oocyst dose (500 TgShSp1 oocysts), whereas in sheep infected with the same dose of oocysts, mortality occurred in all fetuses. Likewise, mortality of 9 and 27% of the pups was observed in mice after infection with 100 and 25 TgShSp1 oocysts, respectively, while in sheep, infection with 50 and 10 TgShSp1 oocysts triggered mortality in 68 and 66% of the fetuses/lambs. Differences in vertical transmission in the surviving offspring were only found with the lower oocyst doses (100% after infection with 10 TgShSp1 oocysts in sheep and only 37% in mice after infection with 25 TgShSp1 oocysts). In conclusion, virulence in mice of T. gondii type II isolates may not be a good indicator to predict the outcome of infection in pregnant sheep., RS-S is supported by a fellowship from the Spanish Ministry of Education, Culture, and Sports (MECD), as a part of the Program of Training of University Teaching Staff (FPU, grant number FPU13/03438) and a mobility grant for predoctoral short stays in R+D centers (EST16/0719). DG-E is the recipient of a postdoctoral contract from the Junta de Castilla y León, partially funded by the European Social Fund (European Union). NA-V is the recipient of a predoctoral contract from the Ministerio de Economía, Industria y Competitividad (Ref. BES-2016-076513). This work was supported by the Ministry of Economy and Competitiveness (AGL2016-75935-C2-1-R and C2-2-R), the Community of Madrid, Spain (PLATESA, S2013/ABI2906), Junta de Castilla y León (LE080U16), and a grant of the Swiss National Science Foundation to AH (project No. 310030_165782).

Methods based around statistics and linear algebra have been increasingly used in attempts to address emerging questions in microarray literature. Microarray technology is a long-used tool in the global analysis of gene expression, allowing for the simultaneous investigation of hundreds or thousands of genes in a sample. It is characterized by a low sample size and a large feature number created a non-square matrix, and by the incomplete rank, that can generate countless more solution in classifiers. To avoid the problem of the ‘curse of dimensionality’ many authors have performed feature selection or reduced the size of data matrix. In this work, we introduce a new logistic regression-based model to classify breast cancer tumor samples based on microarray expression data, including all features of gene expression and without reducing the microarray data matrix. If the user still deems it necessary to perform feature reduction, it can be done after the application of the methodology, still maintaining a good classification. This methodology allowed the correct classification of breast cancer sample data sets from Gene Expression Omnibus (GEO) data series GSE65194, GSE20711, and GSE25055, which contain the microarray data of said breast cancer samples. Classification had a minimum performance of 80% (sensitivity and specificity), and explored all possible data combinations, including breast cancer subtypes. This methodology highlighted genes not yet studied in breast cancer, some of which have been observed in Gene Regulatory Networks (GRNs). In this work we examine the patterns and features of a GRN composed of transcription factors (TFs) in MCF-7 breast cancer cell lines, providing valuable information regarding breast cancer. In particular, some genes whose αi ∗ associated parameter values revealed extreme positive and negative values, and, as such, can be identified as breast cancer prediction genes. We indicate that the PKN2, MKL1, MED23, CUL5 and GLI genes demonstrate a tumor suppressor profile, and that the MTR, ITGA2B, TELO2, MRPL9, MTTL1, WIPI1, KLHL20, PI4KB, FOLR1 and SHC1 genes demonstrate an oncogenic profile. We propose that these may serve as potential breast cancer prediction genes, and should be prioritized for further clinical studies on breast cancer. This new model allows for the assignment of values to the αi ∗ parameters associated with gene expression. It was noted that some αi ∗ parameters are associated with genes previously described as breast cancer biomarkers, as well as other genes not yet studied in relation to this disease.

In a previous attempt, an experimental model of bovine besnoitiosis was established in calves that were intravenously inoculated with different doses of Besnoitia besnoiti tachyzoites. Despite the fact that all infected calves developed the acute stage of disease, only microscopic findings characteristic of chronic besnoitiosis were reported. In the present study, calves were inoculated by subcutaneous and intradermal routes with B. besnoiti tachyzoites with the aim of developing clinical signs and macroscopic lesions characteristic of chronic besnoitiosis. Nine 3-month-old male calves were randomly distributed into three groups of three animals each. Next, 106 tachyzoites were inoculated by either the subcutaneous (G1) or intradermal route (G2). The negative control group (G3) was inoculated with PBS. Daily clinical monitoring and regular blood collection were performed. At 70 days post-infection (pi), animals were euthanized, and tissues were collected to investigate lesions and parasites. Infected animals developed mild-moderate acute besnoitiosis characterized by lymphadenopathy from four days to 47 days pi, and sporadic fever peaks were only observed in one calf from G2. However, other clinical signs and macroscopic lesions characteristic of chronic besnoitiosis were not detected. Only nine tissue samples were B. besnoiti-DNA-positive, eight of which belonged to reproductive and respiratory tracts tissues from G1. Finally, the kinetics of the immune responses were similar in both infected groups. However, delayed and lower cellular and humoral immune responses were observed in G1 followed by G2 and were compared with intravenously inoculated calves. The differences observed among the three inoculation routes could be due to different effector mechanisms of the host early innate immune response against B. besnoiti. Accordingly, the inoculation route of B. besnoiti tachyzoites does not significantly influence the clinical outcome of the infection in calves. Thus, a further refinement of this experimental model of bovine besnoitiosis is needed to reproduce macroscopic lesions characteristic of chronic stage disease.

This is the first report on the development and characterization of eight monoclonal antibodies (MABs) generated against whole- and membrane-enriched tachyzoite extracts of the apicomplexan parasiteBesnoitia besnoiti. Confocal laser scanning immunofluorescence microscopy was used to localize respective epitopes inB. besnoititachyzoites along the lytic cycle. A pattern compatible with dense granule staining was observed with MABs 2.A.12, 2.F.3 and 2.G.4, which could be confirmed by immunogold electron microscopy for MABs 2.A.12 and 2.F.3. In particular, MABs 2.F.3 and 2.G.4 were secreted during early invasion, proliferation and egress phases. MABs 3.10.8 and 5.5.11 labelled the tachyzoite surface, whilst MABs 1.17.8, 8.9.2 and 2.G.A recognized the apical tip, which is reminiscent for microneme localization. Besides, the epitopes recognized by the latter two (MABs 8.9.2 and 2.G.A) exhibited a redistribution from the anterior part across the parasite surface towards the posterior end during invasion. Most MABs developed were genus-specific. Indeed, the MABs cross-reacted neither withT. gondiinor withN. caninumtachyzoites. In summary, we have generated MABs that will be useful to study the key processes in the lytic cycle of the parasite and with additional promising diagnostic value. However, the molecular identity of the antigens recognized remains to be elucidated.

OprI is an outer membrane lipoprotein from Pseudomonas aeruginosa, and when fused to a recombinant antigen, will exert adjuvant properties by engaging Toll-like receptor 2, leading to dendritic cell activation. Previous studies have shown that the Neospora caninum (Nc) antigens NcPDI, NcROP2 and NcROP40 are implicated in host cell interactions and are promising vaccine candidates. In two independent experiments, the efficacy of a polyvalent vaccine formulation composed of OprI-NcPDI, OprI-NcROP2 and OprI-NcROP40 (collectively named O-Ags) was assessed in non-pregnant and pregnant Balb/c mouse models challenged with tachyzoites of the high-virulence isolate Nc-Spain7. Parameters that were investigated were clinical signs, fertility, parasite burden in adult mice, humoral and cellular immune responses at different time-points prior to and after challenge infection, vertical transmission and post-natal survival of offspring mice, all to explore potential correlations with efficacy. Vaccination of mice with O-Ags induced a mixed Th1/Th2 immune response in adult mice and led to significantly increased protection against cerebral infection. Vaccination with O-Ags also resulted in reduced vertical transmission, and postnatal disease in offspring was significantly inhibited at a rate not observed in mice infected with a high-virulence isolate to date. However, O-Ags mixed with TLR ligands targeting TLR3 and TLR7, which are known to induce clear Th1-biased responses, or vaccination with OprI fused to the non-N. caninum antigen ovalbumin (OprI-OVA) did not confer protection.

There is growing concern about the spreading of human microorganisms in relatively untouched ecosystems such as the Antarctic region. For this reason, three pinniped species (Leptonychotes weddellii, Mirounga leonina and Arctocephalus gazella) from the west coast of the Antartic Peninsula were analysed for the presence of Escherichia spp. with the recovery of 158 E. coli and three E. albertii isolates. From those, 23 harboured different eae variants (α1, β1, β2, ε1, θ1, κ, ο), including a bfpA-positive isolate (O49:H10-A-ST206, eae-k) classified as typical enteropathogenic E. coli. Noteworthy, 62 of the 158 E. coli isolates (39.2%) exhibited the ExPEC status and 27 (17.1%) belonged to sequence types (ST) frequently occurring among urinary/bacteremia ExPEC clones: ST12, ST73, ST95, ST131 and ST141. We found similarities >85% within the PFGE-macrorrestriction profiles of pinniped and human clinic O2:H6-B2-ST141 and O16:H5/O25b:H4-B2-ST131 isolates. The in silico analysis of ST131 Cplx genomes from the three pinnipeds (five O25:H4-ST131/PST43-fimH22-virotype D; one O16:H5-ST131/PST506-fimH41; one O25:H4-ST6252/PST9-fimH22-virotype D1) identified IncF and IncI1 plasmids and revealed high core-genome similarities between pinniped and human isolates (H22 and H41 subclones). This is the first study to demonstrate the worrisome presence of human-associated E. coli clonal groups, including ST131, in Antarctic pinnipeds. The sampling was funded by the Spanish Ministry of Science and Innovation (CGL-2005-25073-E/ANT and CTM2008-00570) and the Sea World & Bush Gardens Conservation Fund. Work at USC-LREC was supported by projects AGL2013-47852-R from the Ministerio de Economía y Competitividad (MINECO, Spain) and Fondo Europeo de Desarrollo Regional (FEDER); AGL2016-79343-R from the Agencia Estatal de Investigación (AEI, Spain) and FEDER; PI16/01477 from Plan Estatal de I + D + I 2013-2016, Instituto de Salud Carlos III (ISCIII), Subdirección General de Evaluación y Fomento de la Investigación, and FEDER; CN2012/303 and ED431C 2017/57 from the Consellería de Cultura, Educación e Ordenación Universitaria, (Xunta de Galicia) and FEDER. We would like to thank the military personnel at the Spanish Antarctic Base “Gabriel de Castilla” for their help and assistance and the Marine Technology Unit (CSIC), the Spanish Navy’s Oceanographic Research Ship “Las Palmas” and the Unidad de Tecnología Marítima (UTM, CSIC) for logistics and transport. We also express our gratitude to J. Castro-Urda, F.T. García-Moreno, C. Rengifo-Herrera, S. Rojo-Montejo, I. Ferre, V. Navarro, M. Gómez-Bautista, T. Alvaro-Alvarez and J. Coello-Pérez for their invaluable help in the sample collection. A. Mora acknowledges the Ministerio de Educación, Cultura y Deporte (Spain) for the mobility grant for teachers and researchers from the Programa Estatal de Promoción del Talento y su Empleabilidad, Plan Estatal de Investigación Científica y Técnica y de Innovación 2013–2016. Sí

Debido a la mejora constante de los servicios de neonatología, se logra una mayor sobrevida de los bebés prematuros; no obstante, su grado de inmadurez los coloca en una situación de fragilidad que los expone tanto a una mayor morbilidad, como a retrasos en el desarrollo, con repercusiones a corto, mediano y largo plazo. Los servicios de neonatología, en ocasiones de Unidad de Cuidados Intensivos Neonatales, son el primer contacto del recién nacido al momento del nacimiento, donde es sometido a múltiples estímulos ambientales que interfieren en su desarrollo. El objetivo de este estudio es analizar la mejor evidencia científica disponible respecto de las estrategias de estimulación temprana aplicadas por enfermería a los recién nacidos pretérmino, hospitalizados, que han sido útiles para reducir o evitar alteraciones leves o graves en el neurodesarrollo. Respecto de la metodología para desarrollar este protocolo se aplicará la práctica clínica basada en evidencia, que consta de cinco pasos: la pregunta clínica, búsqueda de evidencia científica, análisis crítico de la evidencia, transferencia de la evidencia y evaluación de la transferencia de la evidencia.

Low antibody titers to Neospora caninum have been reported in humans, but infection has not been confirmed. We used N. caninum-specific PCR to test 600 clinical samples from patients with toxoplasmosis signs but Toxoplasma gondii-negative PCR results. We did not detect N. caninum DNA, demonstrating it is an unlikely opportunistic zoonotic agent.

The existence of free continental shelf waves in the Western Mediterranean Sea is studied using a statistical approach. Hourly mean sea level at six locations extended along the northern and southern coast of the Alboran Sea were analyzed by standard correlation and spectral analysis techniques for two periods of 91 and 92 days, respectively. The mean sea level was adjusted for the effect of atmospheric pressure according to the hydrostatic approximation. The wind-coherent response of sea level was removed by calculating the transfer function between the input alongshore wind stress and the output adjusted sea level. The cross-correlation analyses indicate time lags that are consistent with the hypothesis of the propagation of the free shelf waves predicted by the continental shelf wave theory. The presence of a westward-travelling signal on the NCS and a west-to-east travelling signal on the SCS of the Alboran Sea is confirmed. The cross spectra between residual sea level at the six locations show significant coherence at long periods. For these periods, the phase difference spectra show that the directions of propagation of the free waves are consistent with the interpretation of the cross correlation analysis. There is evidence of a seasonal variation in the phase speed of the waves, which may be related to variations in the speeds of currents in the Alboran Sea. The observations suggest the presence of continental shelf waves on both coasts of the Alboran Sea.