Transforming growth factor (TGF)-ß1 and interferon (IFN)-γ differentially regulate ICAM-1 expression and adhesion of Toxoplasma gondii to human trophoblast (BeWo) and uterine cervical (HeLa) cells.

Toxoplasma gondii is a parasite able to infect various cell types, including trophoblast cells. Studies have demonstrated that interleukin (IL)-10, transforming growth factor (TGF)-ß1 and interferon (IFN)-γ are involved in the susceptibility of BeWo trophoblast cells to T. gondii infection. Furthermore, T. gondii is able to adhere to the plasma membrane of host cells through intercellular adhesion molecule (ICAM)-1. Thus, the present study aimed to assess the role of IL-10, TGF-ß1 and IFN-γ in the expression of ICAM-1 in BeWo and HeLa cells and to analyze the role of ICAM-1 in the adhesion and invasion of T. gondii to these cells under the influence of these cytokines. For this purpose, BeWo and HeLa cells were treated or not, before and after T. gondii infection, with rIL-10, rTGF-ß1 or rIFN-γ. For the BeWo cells, rIL-10 and rTGF-ß1 favored susceptibility to infection, but only rTGF-ß1 and rIFN-γ increased ICAM-1 expression, and TNF-α release. On the other hand, rIFN-γ downregulated the expression of ICAM-1 triggered by T. gondii in HeLa cells, leading to control of the infection. Moreover, we observed that upregulation of ICAM-1, mediated by cytokine's stimulation, in BeWo and HeLa cells resulted in a high number rate of both parasite adhesion and invasion to these cells, which were strongly reduced after ICAM-1 neutralization. Likewise, the blockage of ICAM-1 molecule also impaired T. gondii infection in human villous explants. Taken together, these findings demonstrate that TGF-ß1 and IFN-γ differentially regulate ICAM-1 expression, which may interfere in the adhesion/invasion of T. gondii to BeWo and HeLa cells for modulating susceptibility to infection.

ERK1/2 phosphorylation and IL-6 production are involved in the differential susceptibility to Toxoplasma gondii infection in three types of human (cyto/ syncytio/ extravillous) trophoblast cells.

During pregnancy, Toxoplasma gondii can triggers serious manifestations and potentially affect the fetal development. In this scenario, differences in susceptibility of trophoblast cells to T. gondii infection might be evaluated in order to establish new therapeutic approaches capable of interfering in the control of fetal infection by T. gondii. This study aimed to evaluate the susceptibility of cytotrophoblast, syncytiotrophoblast and extravillous trophoblast cells to T. gondii infection. Our data demonstrate that HTR-8/SVneo cells (extravillous trophoblast cells) present higher susceptibility to T. gondii infection when compared to syncytiotrophoblast and cytotrophoblast cells, whereas syncytiotrophoblast was the cell type more resistant to the parasite infection. Also, cytotrophoblast and syncytiotrophoblast cells produced significantly more IL-6 than HTR-8/SVneo cells. On the other hand, HTR-8/SVneo cells showed higher ERK1/2 phosphorylation than cytotrophoblast and syncytiotrophoblast cells. ERK1/2 inhibition reduced T. gondii infection and increased IL-6 production in HTR-8/SVneo cells. Thus, it is plausible to conclude that the greater susceptibility of HTR-8/SVneo cells to infection by T. gondii is related to a higher ERK1/2 phosphorylation and lower levels of IL-6 in these cells compared to other cells, suggesting that these mediators may be important to favor the parasite infection in this type of trophoblastic population.

A novel peptide-based sensor platform for detection of anti-Toxoplasma gondii immunoglobulins.

Toxoplasma gondii is an intracellular protozoan parasite responsible for toxoplasmosis, which affects humans and animals. Serologic detection of anti-T. gondii immunoglobulins plays a crucial role in the clinical diagnosis of toxoplasmosis. In this work, a novel electrochemical immunosensor for detecting anti-T. gondii immunoglobulins is reported, based on immobilization of an in silico predicted peptide (PepB3), obtained from membrane protein of T. gondii, on the graphite electrode modified with poly(3-hydroxybenzoic acid). Indirect ELISA confirmed infection and binding specificity of peptide PepB3. Molecular modelling and simulations show this peptide binds to the T. gondii human Fab antibody in the surface antigen 1 (SAG1) binding site, remaining a stable complex during the molecular dynamic simulations, especially by hydrogen bonds and hydrophobic interactions. This electrochemical immunosensor was able to discriminate different periods of infection, using infected mouse serum samples, showing selectivity and discriminating infected and uninfected mouse serum.

Corrigendum: Neospora Caninum Activates p38 MAPK as an Evasion Mechanism against Innate Immunity.

[This corrects the article DOI: 10.3389/fmicb.2016.01456.].

Randomized Controlled Trial of Oropharyngeal Colostrum Administration in Very-low-birth-weight Preterm Infants.

OBJECTIVE: The purpose of this study was to evaluate the effects of oropharyngeal colostrum administration in the incidence of late-onset clinical and proven sepsis and in concentrations of immunoglobulin A (IgA) in very-low-birth-weight (VLBW) infants. METHODS: We conducted a double-blinded, randomized, placebo-controlled trial and assigned 113 VLBW infants to receive 0.2 mL of maternal colostrum or sterile water (placebo) via oropharyngeal route every 2 hours for 48 hours, beginning in the first 48 to 72 hours of life. Neonates of both groups were fed breast milk from the first 3 days of life until a volume of at least 100 mL ·â€Škg ·â€Šday. IgA was measured in serum and urine before and after treatment. Clinical data during hospitalization were collected. RESULTS: We found no statistically significant differences between colostrum and placebo groups in the incidence of late-onset clinical sepsis (odds ratio 0.7602; CI 95% 0.3-1.6) and proven sepsis (odds ratio 0.7028; CI 95% 0.3-1.6). The measurement of IgA was similar in serum before (P value 0.87) and after treatment (P value 0.26 day 4 and 0.77 day 18). No differences were also observed in IgA in urine before (P value 0.8) and after treatment (P value 0.73 day 4 and 0.52). CONCLUSIONS: This study could not confirm the hypothesis that oropharyngeal administration of maternal colostrum to VLBW could reduce the incidence of late-onset sepsis and increase the levels of IgA. We believe that this finding can be justified by the practice of feeding VLBW infants exclusively with breast milk in the first days of life and reinforces the prior knowledge of the importance of early nutrition, especially, with human milk. It also suggests that oropharyngeal administration of colostrum should be reserved for neonates who cannot be fed in first few days of life.

Macrophage Migration Inhibitory Factor (MIF) Prevents Maternal Death, but Contributes to Poor Fetal Outcome During Congenital Toxoplasmosis.

Migration inhibitory factor (MIF) is a pro-inflammatory cytokine that plays important roles in physiology, pathology, immunology and parasitology, including the control of infection by protozoa parasites such as Toxoplasma gondii. As the MIF function in congenital toxoplasmosis is not fully elucidated yet, the present study brings new insights for T. gondii infection in the absence of MIF based on pregnant C57BL/6MIF-/- mouse models. Pregnant C57BL/6MIF-/- and C57BL/6WT mice were infected with 05 cysts of T. gondii (ME49 strain) on the first day of pregnancy (dop) and were euthanized at 8 dop. Non-pregnant and non-infected females were used as control. Our results demonstrated that MIF-/- mice have more accentuated change in body weight and succumbed to infection first than their WT counterparts. Otherwise, pregnancy outcome was less destructive in MIF-/- mice compared to WT ones, and the former had an increase in the mast cell recruitment and IDO expression and consequently presented less inflammatory cytokine production. Also, MIF receptor (CD74) was upregulated in MIF-/- mice, indicating that a compensatory mechanism may be required in this model of study. The global absence of MIF was associated with attenuation of pathology in congenital toxoplasmosis, but resulted in female death probably because of uncontrolled infection. Altogether, ours results demonstrated that part of the immune response that protects a pregnant female from T. gondii infection, favors fetal damage.

Development of direct assays for Toxoplasma gondii and its use in genomic DNA sample.

This work describes an approach for the selection and detection of specific DNA probes related to Toxoplasma gondii, a protozoan parasite responsible for toxoplasmosis. The detection system was developed on graphite carbon electrode modified with poly(3-hydroxybenzoic acid) sensitized with ToxG1 probe. The hybridization of the specific genomic DNA related to T. gondii showed good response by direct detection of guanine residue oxidation using differential pulse voltammetry (DPV). The biosensor was able to distinguish both the complementary and non-complementary targets and detect up to 100ngµL-1 of the T. gondii genomic DNA. The hybridization (ToxG1: T. gondii genomic DNA) was confirmed by optical measurement. Optical assays using gold nanoparticles:ToxG1 probe showed a significant change in the absorbance peak in the presence of the T. gondii genomic DNA according to the electrochemical results. This novel biosensor shows potential as electrochemical transducer and was successfully applied in the biological sample.

Enrofloxacin and Toltrazuril Are Able to Reduce Toxoplasma gondii Growth in Human BeWo Trophoblastic Cells and Villous Explants from Human Third Trimester Pregnancy.

Classical treatment for congenital toxoplasmosis is based on combination of sulfadiazine and pyrimethamine plus folinic acid. Due to teratogenic effects and bone marrow suppression caused by pyrimethamine, the establishment of new therapeutic strategies is indispensable to minimize the side effects and improve the control of infection. Previous studies demonstrated that enrofloxacin and toltrazuril reduced the incidence of Neospora caninum and Toxoplasma gondii infection. The aim of the present study was to evaluate the efficacy of enrofloxacin and toltrazuril in the control of T. gondii infection in human trophoblast cells (BeWo line) and in human villous explants from the third trimester. BeWo cells and villous were treated with several concentrations of enrofloxacin, toltrazuril, sulfadiazine, pyrimethamine, or combination of sulfadiazine+pyrimethamine, and the cellular or tissue viability was verified. Next, BeWo cells were infected by T. gondii (2F1 clone or the ME49 strain), whereas villous samples were only infected by the 2F1 clone. Then, infected cells and villous were treated with all antibiotics and the T. gondii intracellular proliferation as well as the cytokine production were analyzed. Finally, we evaluated the direct effect of enrofloxacin and toltrazuril in tachyzoites to verify possible changes in parasite structure. Enrofloxacin and toltrazuril did not decrease the viability of cells and villous in lower concentrations. Both drugs were able to significantly reduce the parasite intracellular proliferation in BeWo cells and villous explants when compared to untreated conditions. Regardless of the T. gondii strain, BeWo cells infected and treated with enrofloxacin or toltrazuril induced high levels of IL-6 and MIF. In villous explants, enrofloxacin induced high MIF production. Finally, the drugs increased the number of unviable parasites and triggered damage to tachyzoite structure. Taken together, it can be concluded that enrofloxacin and toltrazuril are able to control T. gondii infection in BeWo cells and villous explants, probably by a direct action on the host cells and parasites, which leads to modifications of cytokine release and tachyzoite structure.

Importance of serological cross-reactivity among Toxoplasma gondii, Hammondia spp., Neospora spp., Sarcocystis spp. and Besnoitia besnoiti.

Toxoplasma gondii, Neospora spp., Sarcocystis spp., Hammondia spp. and Besnoitia besnoiti are genetically related cyst-forming coccidia. Serology is frequently used for the identification of T. gondii, Neospora spp. and B. besnoiti-exposed individuals. Serologic cross-reactions occur in different tests among animals infected with T. gondii and H. hammondi, as well as among animals infected by T. gondii and N. caninum. Infections caused by N. caninum and N. hughesi are almost indistinguishable by serology. Neospora caninum, B. besnoiti and Sarcocystis spp. infections in cattle show some degree of serologic cross-reactivity. Antibody cross-reactivity between Neospora spp. and H. heydorni-infected animals is suspected, but not proven to occur. We review serologic cross-reactivity among animals and/or humans infected with T. gondii, Neospora spp., Sarcocystis spp., Hammondia spp. and B. besnoiti. Emphasis is laid upon antigens and serological methods for N. caninum diagnosis which were tested for cross-reactivity with related protozoa. Species-specific antigens, as well as stage-specific proteins have been identified in some of these parasites and have promising use for diagnosis and epidemiological surveys.

Lectins from Synadenium carinatum (ScLL) and Artocarpus heterophyllus (ArtinM) Are Able to Induce Beneficial Immunomodulatory Effects in a Murine Model for Treatment of Toxoplasma gondii Infection.

Infection by Toxoplasma gondii affects around one-third of world population and the treatment for patients presenting toxoplasmosis clinically manifested disease is mainly based by a combination of sulfadiazine, pyrimethamine, and folinic acid. However, this therapeutic protocol is significantly toxic, causing relevant dose-related bone marrow damage. Thus, it is necessary to improve new approaches to investigate the usefulness of more effective and non-toxic agents for treatment of patients with toxoplasmosis. It has been described that lectins from plants can control parasite infections, when used as immunological adjuvants in vaccination procedures. This type of lectins, such as ArtinM and ScLL is able to induce immunostimulatory activities, including efficient immune response against parasites. The present study aimed to evaluate the potential immunostimulatory effect of ScLL and ArtinM for treatment of T. gondii infection during acute phase, considering that there is no study in the literature accomplishing this issue. For this purpose, bone marrow-derived macrophages (BMDMs) were treated with different concentrations from each lectin to determine the maximum concentration without or with lowest cytotoxic effect. After, it was also measured the cytokine levels produced by these cells when stimulated by the selected concentrations of lectins. We found that ScLL showed high capacity to induce of pro-inflammatory cytokine production, while ArtinM was able to induce especially an anti-inflammatory cytokines production. Furthermore, both lectins were able to increase NO levels. Next, we evaluated the treatment effect of ScLL and ArtinM in C57BL/6 mice infected by ME49 strain from T. gondii. The animals were infected and treated with ScLL, ArtinM, ArtinM plus ScLL, or sulfadiazine, and the following parameters analyzed: Cytokines production, brain parasite burden and survival rates. Our results demonstrated that the ScLL or ScLL plus ArtinM treatment induced production of pro-inflammatory and anti-inflammatory cytokines, showing differential but complementary profiles. Moreover, when compared with non-treated mice, the parasite burden was significantly lower and survival rates higher in mice treated with ScLL or ScLL plus ArtinM, similarly with sulfadiazine treatment. In conclusion, the results demonstrated the suitable potential immunotherapeutic effect of ScLL and ArtinM lectins to control acute toxoplasmosis in this experimental murine model.

Si-Accumulation In Artemisia annua Glandular Trichomes Increases Artemisinin Concentration, but Does Not Interfere In the Impairment of Toxoplasma gondii Growth.

Artemisia annua is used as a source of artemisinin, a potent therapeutic agent used for the treatment of infectious diseases, chiefly malaria. However, the low concentration (from 0.01 to 1.4% of dried leaf matter) of artemisinin in the plant obtained with the traditional cropping system makes it a relatively expensive drug, especially in developing countries. Considering that artemisinin and silicon (Si) are both stored in A. annua glandular trichomes, and that Si accumulation has never been investigated, this study aimed to look into Si effects on A. annua trichome artemisinin concentration, and whether leaf infusion from Si-treated A. annua plants is able to control Toxoplasma gondii growth. T. gondii is the etiologic agent of toxoplasmosis, a zoonotic parasitic disease whose traditional treatment shows significant side effects. The experimental design consisted of A. annua seedlings randomly planted in soil treated with different doses of calcium/magnesium silicate (0, 200, 400, 800, and 1600 kg ha-1). Analysis of foliar macronutrients showed significant increases of nitrogen content only at the highest dose of silicate. Foliar micronutrients, Si concentrations, and plant height were not affected by any of the silicate doses. However, the dose of 400 kg ha-1 of silicate increased the trichome size, which in turn raised artemisinin concentration in leaves and the infusion. In contrast, the 800 and 1600 kg ha-1 doses dramatically decreased artemisinin concentration. HeLa cell treatment with the infusion of A. annua grown in soil treated with 400 kg ha-1 of silicate decreased parasite proliferation in a dose-dependent manner when the treatment was carried out after or along with T. gondii infection. However, this effect was similar to A. annua grown in soil without silicate treatment. Thus, it can be concluded that, even though Si applied to the soil at 400 kg ha-1 has a positive effect on the A. annua glandular trichome size and the artemisinin concentration, this outcome cannot be directly associated with the efficiency of A. annua infusion on T. gondii growth, suggesting that other components from A. annua leaves could be acting in synergy with artemisinin.

Neospora caninum Activates p38 MAPK as an Evasion Mechanism against Innate Immunity.

Due to the high prevalence and economic impact of neosporosis, the development of safe and effective vaccines and therapies against this parasite has been a priority in the field and is crucial to limit horizontal and vertical transmission in natural hosts. Limited data is available regarding factors that regulate the immune response against this parasite and such knowledge is essential in order to understand Neospora caninum induced pathogenesis. Mitogen-activated protein kinases (MAPKs) govern diverse cellular processes, including growth, differentiation, apoptosis, and immune-mediated responses. In that sense, our goal was to understand the role of MAPKs during the infection by N. caninum. We found that p38 phosphorylation was quickly triggered in macrophages stimulated by live tachyzoites and antigen extracts, while its chemical inhibition resulted in upregulation of IL-12p40 production and augmented B7/MHC expression. In vivo blockade of p38 resulted in an amplified production of cytokines, which preceded a reduction in latent parasite burden and enhanced survival against the infection. Additionally, the experiments indicate that the p38 activation is induced by a mechanism that depends on GPCR, PI3K and AKT signaling pathways, and that the phenomena here observed is distinct that those induced by Toxoplasma gondii's GRA24 protein. Altogether, these results showed that N. caninum manipulates p38 phosphorylation in its favor, in order to downregulate the host's innate immune responses. Additionally, those results infer that active interference in this signaling pathway may be useful for the development of a new therapeutic strategy against neosporosis.

Toxoplasma gondii-Derived Synthetic Peptides Containing B- and T-Cell Epitopes from GRA2 Protein Are Able to Enhance Mice Survival in a Model of Experimental Toxoplasmosis.

Toxoplasmosis is a zoonosis distributed all over the world, which the etiologic agent is an intracellular protozoan parasite, Toxoplasma gondii. This disease may cause abortions and severe diseases in many warm-blood hosts, including humans, particularly the immunocompromised patients. The parasite specialized secretory organelles, as micronemes, rhoptries and dense granules, are critical for the successful parasitism. The dense granule protein 2 (GRA2) is a parasite immunogenic protein secreted during infections and previous studies have been shown that this parasite component is crucial for the formation of intravacuolar membranous nanotubular network (MNN), as well as for secretion into the vacuole and spatial organization of the parasites within the vacuole. In the present study, we produced a monoclonal antibody to GRA2 (C3C5 mAb, isotype IgG2b), mapped the immunodominant epitope of the protein by phage display and built GRA2 synthetic epitopes to evaluate their ability to protect mice in a model of experimental infection. Our results showed that synthetic peptides for B- and T-cell epitopes are able to improve survival of immunized animals. In contrast with non-immunized animals, the immunized mice with both B- and T-cell epitopes had a better balance of cytokines and demonstrated higher levels of IL-10, IL-4 and IL-17 production, though similar levels of TNF-α and IL-6 were observed. The immunization with both B- and T-cell epitopes resulted in survival rate higher than 85% of the challenged mice. Overall, these results demonstrate that immunization with synthetic epitopes for both B- and T-cells from GRA2 protein can be more effective to protect against infection by T. gondii.

GITR Activation Positively Regulates Immune Responses against Toxoplasma gondii.

Toxoplasma gondii is a widespread parasite responsible for causing clinical diseases especially in pregnant and immunosuppressed individuals. Glucocorticoid-induced TNF receptor (GITR), which is also known as TNFRS18 and belongs to the TNF receptor superfamily, is found to be expressed in various cell types of the immune system and provides an important costimulatory signal for T cells and myeloid cells. However, the precise role of this receptor in the context of T. gondii infection remains elusive. Therefore, the current study investigated the role of GITR activation in the immunoregulation mechanisms induced during the experimental infection of mice with T. gondii. Our data show that T. gondii infection slightly upregulates GITR expression in Treg cells and B cells, but the most robust increment in expression was observed in macrophages and dendritic cells. Interestingly, mice infected and treated with an agonistic antibody anti-GITR (DTA-1) presented a robust increase in pro-inflammatory cytokine production at preferential sites of parasite replication, which was associated with the decrease in latent brain parasitism of mice under treatment with DTA-1. Several in vivo and in vitro analysis were performed to identify the cellular mechanisms involved in GITR activation upon infection, however no clear alterations were detected in the phenotype/function of macrophages, Tregs and B cells under treatment with DTA-1. Therefore, GITR appears as a potential target for intervention during infection by the parasite Toxoplasma gondii, even though further studies are still necessary to better characterize the immune response triggered by GITR activation during T. gondii infection.

Strength and Aerobic Physical Exercises Are Able to Increase Survival of Toxoplasma gondii-Infected C57BL/6 Mice by Interfering in the IFN-γ Expression.

Physical exercise has been implicated in several immunophysiological improvements, particularly during the aging process, when an immunocompromised status could be established. Toxoplasma gondii is a protozoan parasite that causes a widespread opportunistic infection, which may present severe consequences, mainly to the fetus and immunocompromised patients. It is estimated that one-third of the human population worldwide has been infected by this parasite, being the reactivation during immunesenescence an unexplored public health issue. The major purpose of the present study was to observe the immunophysiological differences between exercised vs. sedentary C57BL/6 male mice that have been experimentally infected by T. gondii. In the first set of experiments, the animals were infected after exercising and three groups were set up: experimental groups-infected sedentary (IS, n = 6); infected exercised (IEx, n = 6) and control group-non-infected sedentary (NIS, n = 6). When stimulated in vitro by T. gondii-soluble tachyzoite antigen, it was found that splenocytes from exercised group produced higher levels of IFN-γ, as well as of IFN-γ/IL-10 ratios in comparison with splenocytes from sedentary animals (P < 0.001). However, it was not found significant differences concerning quantification of T. gondii genomic DNA by qRT-PCR and immunohistochemistry analysis in brain cysts from both group of animals (P > 0.05). In order to further investigate the consequences of these data for the host, a second set of experiments was performed, when the animals were infected before exercising and four groups of animals were established for comparison purpose, as follows: experimental groups-infected sedentary (IS, n = 7); infected exercised (IEx, n = 6) and control groups-non-infected sedentary (NIS, n = 6) and non-infected exercised (NIEx, n = 6). It was found significant differences in the survival rates of the exercised group the animals, as they survived longer than sedentary groups (P = 0.0005). In both sets of experiments, mice have been submitted to moderate exercises: aerobic (14 m/min; 3 x/week) and strength (60-80% of one maximum repetition; 2 x/week). Overall, our findings are showing that the aerobic and strength exercises are able to modulate immune response against T. gondii infection, being these immunological features beneficial to the host.

CCp5A Protein from Toxoplasma gondii as a Serological Marker of Oocyst-driven Infections in Humans and Domestic Animals.

Considering that the current immunoassays are not able to distinguish the infective forms that cause Toxoplasma gondii infection, the present study was carried out to evaluate the reactivity of two recombinant proteins (CCp5A and OWP1) from oocyst/sporozoite, in order to differentiate infections occurring by ingestion of oocysts or tissue cysts. The reactivity of the recombinant proteins was assessed against panels of serum samples from animals (chickens, pigs, and mice) that were naturally or experimentally infected by different infective stages of the parasite. Also, we tested sera from humans who have been infected by oocysts during a well-characterized toxoplasmosis outbreak, as well as sera from pregnant women tested IgM(+)/IgG(+) for T. gondii, which source of infection was unknown. Only the sporozoite-specific CCp5A protein was able to differentiate the parasite stage that infected chickens, pigs and mice, with specific reactivity for oocyst-infected animals. Furthermore, the CCp5A showed preferential reactivity for recent infection by oocyst/sporozoite in pigs and mice. In humans, CCp5A showed higher reactivity with serum samples from the outbreak, compared with serum from pregnant women. Altogether, these findings demonstrate the usefulness of the CCp5A protein as a new tool to identify the parasite stage of T. gondii infection, allowing its application for diagnosis and epidemiological investigations in animals and humans. The identification of parasite infective stage can help to design effective strategies to minimize severe complications in immunocompromised people and, particularly, in pregnant women to prevent congenital infection.

Calomys callosus chronically infected by Toxoplasma gondii clonal type II strain and reinfected by Brazilian strains is not able to prevent vertical transmission.

Considering that Toxoplasma gondii has shown high genetic diversity in Brazil, the aim of this study was to determine whether Calomys callosus chronically infected by the ME-49 strain might be susceptible to reinfection by these Brazilian strains, including vertical transmission of the parasite. Survival curves were analyzed in non-pregnant females chronically infected with ME-49 and reinfected with the TgChBrUD1 or TgChBrUD2 strain, and vertical transmission was analyzed after reinfection of pregnant females with these same strains. On the 19th day of pregnancy (dop), placentas, uteri, fetuses, liver, spleen, and lung were processed for detection of the parasite. Blood samples were collected for humoral and cellular immune response analyses. All non-pregnant females survived after reinfection and no changes were observed in body weight and morbidity scores. In pregnant females, parasites were detected in the placentas of ME-49 chronically infected females and reinfected females, but were only detected in the fetuses of reinfected females. TgChBrUD2 reinfected females showed more impaired pregnancy outcomes, presenting higher numbers of animals with fetal loss and a higher resorption rate, in parallel with higher levels of pro-inflammatory cytokines and IgG2a subclass antibodies. Vertical transmission resulting from chronic infection of immunocompetent C. callosus is considered a rare event, being attributed instead to either reactivation or reinfection. That is, the pregnancy may be responsible for reactivation of the latent infection or the reinfection may promote T. gondii vertical transmission. Our results clearly demonstrate that, during pregnancy, protection against T. gondii can be breached after reinfection with parasites belonging to different genotypes, particularly when non-clonal strains are involved in this process and in this case the reinfection promoted vertical transmission of both type II and Brazilian T. gondii strains.

Fluorescent ester dye-based assays for the in vitro measurement of Neospora caninum proliferation.

Techniques for the measurement of parasite loads in different experimental models have evolved throughout the years. The quantification of stained slides using regular cytological stains is currently the most common technique. However, this modality of evaluation is labor-intensive, and the interpretation of the results is subjective because the successes of the assays mainly rely on the abilities of the professionals involved. Moreover, the novel genetic manipulation techniques that are commonly applied for closely related Toxoplasma gondii have not yet been developed for Neospora caninum. Thus, we aimed to develop a simple protocol for parasite quantification using pre-stained N. caninum tachyzoites and fluorescent probes based on ester compounds (i.e., CFSE and DDAO). For this purpose, we employed a quantification procedure based on flow cytometry analysis. Pre-stained parasites were also examined with a fluorescent microscope, which revealed that both dyes were detectable. Direct comparison of the numbers of CFSE+ and DDAO+ cells to the values obtained with classical cytology techniques yielded statistically comparable results that also accorded with genomic DNA amplification results. Although the fluorescence emitted by DDAO was more intense and provided better discrimination between the populations of parasitized cells, CFSE+ tachyzoites were detected for several days. In conclusion, this study describes a simple, fast, low-cost and reproducible protocol for N. caninum quantification that is based on parasite pre-staining with fluorescent ester-based probes.

Azithromycin is able to control Toxoplasma gondii infection in human villous explants.

BACKGROUND: Although Toxoplasma gondii infection is normally asymptomatic, severe cases of toxoplasmosis may occur in immunosuppressed patients or congenitally infected newborns. When a fetal infection is established, the recommended treatment is a combination of pyrimethamine, sulfadiazine and folinic acid (PSA). The aim of the present study was to evaluate the efficacy of azithromycin to control T. gondii infection in human villous explants. METHODS: Cultures of third trimester human villous explants were infected with T. gondii and simultaneously treated with either PSA or azithromycin. Proliferation of T. gondii, as well as production of cytokines and hormones by chorionic villous explants, was analyzed. RESULTS: Treatment with either azithromycin or PSA was able to control T. gondii infection in villous explants. After azithromycin or PSA treatment, TNF-α, IL-17A or TGF-ß1 levels secreted by infected villous explants did not present significant differences. However, PSA-treated villous explants had decreased levels of IL-10 and increased IL-12 levels, while treatment with azithromycin increased production of IL-6. Additionally, T. gondii-infected villous explants increased secretion of estradiol, progesterone and HCG+ß, while treatments with azithromycin or PSA reduced secretion of these hormones concurrently with decrease of parasite load. CONCLUSIONS: In conclusion, these results suggest that azithromycin may be defined as an effective alternative drug to control T. gondii infection at the fetal-maternal interface.

Toxoplasma gondii soluble tachyzoite antigen triggers protective mechanisms against fatal intestinal pathology in oral infection of C57BL/6 mice.

Toxoplasma gondii induces a potent IL-12 response early in infection that results in IFN-γ-dependent control of parasite growth. It was previously shown that T. gondii soluble tachyzoite antigen (STAg) injected 48 hr before intraperitoneal infection reduces lipoxin A4 and 5-lipoxygenase (5-LO)-dependent systemic IL-12 and IFN-γ production as well as hepatic immunopathology. This study investigated the ability of STAg-pretreatment to control the fatal intestinal pathology that develops in C57BL/6 mice orally infected with 100 T. gondii cysts. STAg-pretreatment prolonged the animals' survival by decreasing tissue parasitism and pathology, mainly in the ilea. Protection was associated with decreases in the systemic IFN-γ levels and IFN-γ and TNF message levels in the ilea and with increased TGF-ß production in this tissue, but protection was independent of 5-LO and IL-4. STAg-pretreatment decreased CD4(+) T cell, NK cell, CD11b(+) monocyte and CD11b(+)CD11c(+) dendritic cell numbers in the lamina propria and increased CD8(+) T cells in the intestinal epithelial compartment. In parallel, decreases were observed in iNOS and IL-17 expression in this organ. These results demonstrate that pretreatment with STAg can induce the recruitment of protective CD8(+) T cells to the intraepithelial compartment and decrease proinflammatory immune mechanisms that promote intestinal pathology in T. gondii infection.