Search

Um teste imunoenzimático reverso foi padronizado utilizando-se como fase sólida, microplacas de polivinil sensibilizadas com anticorpos anti-IgM.7 Estas foram incubadas seqüencialmente com alíquotas de soros de pacientes com suspeita de toxoplasmose aguda, antígeno solúvel de Toxoplasma gondii, conjugado peroxidase F (ab')2 anti-toxoplasma e substrato enzimático. A atividade enzimática foi determinada por leitura espectrofotométrica, considerando-se como títulos dos soros a máxima diluição fornecendo valores de absorbância maiores que os obtidos com a menor diluição do soro padrão não-reativo. Em 69 amostras de soros de pacientes com toxoplasmose aguda, a média geométrica dos títulos no teste ELISA-Reverso IgM foi superior à de todos os outros testes para anticorpos IgM, não se observando resultados negativos falsos devidos a altos títulos de IgG específica. Não foi encontrada, também, reatividade cruzada em nenhuma das 104 amostras de soros de pacientes com outras patologias, inclusive em amostras contendo fator reumatóide IgM.

Toxoplasma gondii 's propensity to infect its host and cause disease is highly dependent on its ability to modulate host cell functions. One of the strategies the parasite uses to accomplish this is via the export of effector proteins from the secretory dense granules. Dense granule (GRA) proteins are known to play roles in nutrient acquisition, host cell cycle manipulation, and immune regulation. Here, we characterize a novel dense granule protein named GRA83, which localizes to the parasitophorous vacuole (PV) in tachyzoites and bradyzoites. Disruption of GRA83 results in increased virulence, weight loss, and parasitemia during the acute infection, as well as a marked increase in the cyst burden during the chronic infection. This increased parasitemia was associated with an accumulation of inflammatory infiltrates in tissues in both acute and chronic infections. Murine macrophages infected with ∆ gra83 tachyzoites produced less interleukin-12 (IL-12) in vitro , which was confirmed with reduced IL-12 and interferon-gamma in vivo . This dysregulation of cytokines correlates with reduced nuclear translocation of the p65 subunit of the nuclear factor-κB (NF-κB) complex. While GRA15 similarly regulates NF-κB, infection with ∆ gra83/ ∆ gra15 parasites did not further reduce p65 translocation to the host cell nucleus, suggesting these GRAs function in converging pathways. We also used proximity labeling experiments to reveal candidate GRA83 interacting T. gondii -derived partners. Taken together, this work reveals a novel effector that stimulates the innate immune response, enabling the host to limit the parasite burden. Importance Toxoplasma gondii poses a significant public health concern as it is recognized as one of the leading foodborne pathogens in the United States. Infection with the parasite can cause congenital defects in neonates, life-threatening complications in immunosuppressed patients, and ocular disease. Specialized secretory organelles, including the dense granules, play an important role in the parasite's ability to efficiently invade and regulate components of the host's infection response machinery to limit parasite clearance and establish an acute infection. Toxoplasma' s ability to avoid early clearance, while also successfully infecting the host long enough to establish a persistent chronic infection, is crucial in allowing for its transmission to a new host. While multiple GRAs directly modulate host signaling pathways, they do so in various ways highlighting the parasite's diverse arsenal of effectors that govern infection. Understanding how parasite-derived effectors harness host functions to evade defenses yet ensure a robust infection is important for understanding the complexity of the pathogen's tightly regulated infection. In this study, we characterize a novel secreted protein named GRA83 that stimulates the host cell's response to limit infection., Competing Interests: The authors declare no conflict of interest.

Trichomonas vaginalis is a human infective parasite responsible for trichomoniasis-the most common, non-viral, sexually transmitted infection worldwide. T. vaginalis resides exclusively in the urogenital tract of both men and women. In women, T. vaginalis has been found colonizing the cervix and vaginal tract while in men it has been identified in the upper and lower urogenital tract and in secreted fluids such as semen, urethral discharge, urine, and prostatic fluid. Despite the over 270 million cases of trichomoniasis annually worldwide, T. vaginalis continues to be a highly neglected organism and thus poorly studied. Here we have developed a male mouse model for studying T. vaginalis pathogenesis in vivo by delivering parasites into the murine urogenital tract (MUT) via transurethral catheterization. Parasite burden was assessed ex-vivo using a nanoluciferase-based gene expression assay which allowed quantification of parasites pre- and post-inoculation. Using this model and read-out approach, we show that T. vaginalis can be found within MUT tissue up to 72 hrs post-inoculation. Furthermore, we also demonstrate that parasites that exhibit increased parasite adherence in vitro also have higher parasite burden in mice in vivo. These data provide evidence that parasite adherence to host cells aids in parasite persistence in vivo and molecular determinants found to correlate with host cell adherence in vitro are applicable to infection in vivo. Finally, we show that co-inoculation of T. vaginalis extracellular vesicles (TvEVs) and parasites results in higher parasite burden in vivo. These findings confirm our previous in vitro-based predictions that TvEVs assist the parasite in colonizing the host. The establishment of this pathogenesis model for T. vaginalis sets the stage for identifying and examining parasite factors that contribute to and influence infection outcomes., Competing Interests: The authors disclose no competing interests that could be perceived to bias this work., (Copyright: © 2023 Molgora et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In order to evaluate and compare the specific immune response of pregnant women (PW) chronically infected with Toxoplasma gondii , with and without gestational diabetes mellitus (GDM), and the humoral response of their respective newborns (NB), the study was carried out on 81 PW (34 GDM and 47 controls) from whose medical records the results of the oral glucose tolerance test (OGTT) were obtained, and blood samples were collected at the third trimester of pregnancy; also, on 45 NBs (20 GDM and 25 controls) from whom umbilical cord blood samples were obtained. Humoral immunity was analyzed by measuring anti- T. gondii total IgG, IgG subclasses and IgG avidity. To evaluate cellular immunity, peripheral blood mononuclear cells (PBMC) from 32 PW (16 GDM and 16 controls) were cultured, supernatant cytokines were determined, and flow cytometry was performed to analyze the expression at lymphocytes of surface molecules, cytokines and transcription factors. All PW and NBs were positive for total IgG, and the prevalent subclass was IgG1. There was a negative correlation between the OGTT glycemia of PW and the levels of total IgG, IgG1 and IgG avidity. The IgG avidity of the GDM group was significantly lower than the control group. Patients from the GDM group had a higher number of T lymphocytes expressing markers of cell activation and exhaustion (CD28 and PD-1). In the presence of T. gondii soluble antigen (STAg) the amount of CD4 + T cells producing IFN-γ, IL-10 and IL-17 was significantly lower in the GDM group, while there was no difference between groups in the number of CD4 + CD25 High FOXP 3 +LAP+ functional Treg cells. Additionally, under STAg stimulus, the secretion of IL-17, IL-4, TNF and IL-2 cytokines at PBMCs culture supernatant was lower in the GDM group. In conclusion, there was a correlation between the increase in blood glucose and the decrease in levels of anti- T. gondii antibodies, associated with the decreased IgG avidity in patients who develop GDM. Also, the GDM group had decreased immune responses in Th1, Th2 and Th17 profiles, suggesting an association between GDM and the negative modulation of the humoral and cellular immune responses against T. gondii ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest., (Copyright © 2022 Oliveira-Scussel, Ferreira, Resende, Ratkevicius-Andrade, Gomes, Paschoini, De Vito, Farnesi-de-Assunção, Silva, Mineo, Rodrigues and Rodrigues.)

Background: Toxoplasma gondii is an obligate intracellular parasite that can infect several species, including humans, and can cause severe damage to the fetus when the infection occurs during pregnancy. The environment and/or food contamination are critical to spreading the infection. Human milk is rich in nutrients and bioactive elements that provide growth and development of the immune system of the newborn. All isotypes of immunoglobulins are present in human colostrum and they are produced from systemic or local sources. Breastfeeding protects the infant against various pathogens, but there is no conclusive study to detect IgG subclasses in colostrum against T. gondii . Therefore, the aim of this study was to detect and evaluate the presence of antibody isotypes against T. gondii in paired samples of serum and colostrum., Methods: The study included 283 puerperal patients. ELISA (Enzyme-Linked Immunosorbent Assay) for detection of anti- T. gondii -specific IgM, IgA, and IgG isotypes and IgG1, IgG3, and IgG4 subclasses were conducted on paired samples of serum and colostrum., Results: It was found that 45.9%, 6.0%, and 2.1% of serum samples and 45.2%, 7.1%, and 2.1% of colostrum samples were positive for IgG, IgM, and IgA, respectively. Specific IgG1, IgG3, and IgG4 were positive, respectively, in 98.5%, 54.6%, and 44.6% of serum samples, in contrast with 56.9%, 78.5%, and 34.6% of colostrum samples. Thus, the predominant reactivity of IgG subclasses against T. gondii was IgG1 in serum and IgG3 in colostrum. The higher percentage of positive samples and higher levels of anti- T. gondii IgG3 antibodies were observed in colostrum, when compared to serum samples, suggesting a local production of this subclass. IgG3 and IgG1 subclasses presented different percentages of positivity in serum and colostrum. Only the IgG1 subclass showed a significant correlation between the levels of anti- T. gondii in serum and colostrum, suggesting that IgG1 in breast milk comes from a systemic source. IgG4 showed a similar percentage of positivity in both sample types, but no significant correlation was observed between their levels., Conclusion: Colostrum presents representative levels of IgM, IgA, IgG1, IgG3, and IgG4 antibodies specific to T. gondii . The detection of these antibodies presents the potential for diagnostic application of colostrum samples to better identify the diagnostic status of T. gondii infection, especially during the acute phase. In addition, breastfeeding can also be a possible source of protective antibodies for the newborn against toxoplasmosis, an anthropozoonosis maintained by environmental infection, which interferes in the public health of many countries.

The development of molecular genetics has greatly enhanced the study of the biology and pathology associated with parasites of the phylum Apicomplexa. While the molecular tools are highly developed for the apicomplexan Toxoplasma gondii, the closely related parasite Neospora caninum lacks efficient tools for genetic manipulation. To enable efficient homologous recombination in N. caninum, we targeted the Ku heterodimer DNA repair mechanism in the genomic reference strain, Nc-Liverpool (NcLiv), and show that deletion of Ku80 results in a destabilization and loss of its partner Ku70. Disruption of Ku80 generated parasites in which genes are efficiently epitope tagged and only short homology regions are required for gene knockouts. We used this improved strain to target novel nonessential genes encoding dense granule proteins that are unique to N. caninum or conserved in T. gondii. To expand the utility of this strain for essential genes, we developed the auxin-inducible degron system for N. caninum using parasite-specific promoters. As a proof of concept, we knocked down a novel nuclear factor in both N. caninum and T. gondii and showed that it is essential for survival of both parasites. Together, these efficient knockout and knockdown technologies will enable the field to unravel specific gene functions in N. caninum, which is likely to aid in the identification of targets responsible for the phenotypic differences observed between these two closely related apicomplexan parasites. IMPORTANCE Neospora caninum is a parasite with veterinary relevance, inducing severe disease in dogs and reproductive disorders in ruminants, especially cattle, leading to major losses. The close phylogenetic relationship to Toxoplasma gondii and the lack of pathogenicity in humans drives an interest of the scientific community toward using N. caninum as a model to study the pathogenicity of T. gondii. To enable this comparison, it is important to develop efficient molecular tools for N. caninum, to gain accuracy and save time in genetic manipulation protocols. Here, we have developed base strains and protocols using the genomic reference strain of N. caninum to enable efficient knockout and knockdown assays in this model. We demonstrate that these tools are effective in targeting known and previously unexplored genes. Thus, these tools will greatly improve the study of this protozoan, as well as enhance its ability to serve as a model to understand other apicomplexan parasites.

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Since the World Health Organization declared the global COVID-19 state of emergency in early 2020, several vaccine candidates have emerged to control SARS-CoV-2, and some of them have been approved and implemented in vaccination campaigns worldwide. Although clinical trials for these vaccines have been carried out using highly controlled methods with accurate immunological tests, clinical questionnaires did not include questions concerning the physical activity profile among volunteers. It has been well established that physical activity plays a pivotal role in the immune response after vaccination, led by the activation of cytokines, antibodies, and cells. This concept should have been considered when evaluating the efficacy of COVID-19 vaccine candidates, particularly in elderly and obese people. Here, we discuss data from the literature providing strong evidence regarding the importance of analyzing physical activity parameters to improve the accuracy of clinical trials on assessing the efficacy of vaccine candidates.

Neospora caninum is a protozoan associated with abortions in ruminants and neuromuscular disease in dogs. Classically, the immune response against apicomplexan parasites is characterized by the production of proinflammatory cytokines, such as IL-12, IFN-γ and TNF. TNF is mainly produced during the acute phases of the infections and binds to TNF receptor 1 (CD120a, p55, TNFR1) activating a variety of cells, hence playing an important role in the induction of the inflammatory process against diverse pathogens. Thus, in this study, we aimed to evaluate the role of TNF in cellular and humoral immune responses during N. caninum infection. For this purpose, we used a mouse model of infection based on wildtype (WT) and genetically deficient C57BL/6 mice in TNFR1 ( Tnfr1 -/- ). We observed that Tnfr1 -/- mice presented higher mortality associated with inflammatory lesions and increased parasite burden in the brain after the infection with N. caninum tachyzoites. Moreover, Tnfr1 -/- mice showed a reduction in nitric oxide (NO) levels in vivo . We also observed that Tnfr1 -/- mice showed enhanced serum concentration of antigen-specific IgG2 subclass, while IgG1 production was significantly reduced compared to WT mice, suggesting that TNFR1 is required for regular IgG subclass production and antigen recognition. Based on our results, we conclude that the TNF-TNFR1 complex is crucial for mediating host resistance during the infection by N. caninum ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ferreira França, Silva, Silva, Ramos, Miranda, Mota, Santiago, Mineo and Mineo.)

Ethnopharmacological Relevance: Sulfadiazine and pyrimethamine are the two drugs used as part of the standard therapy for toxoplasmosis, however; they may cause adverse side effects and fail to prevent relapse in many patients, rendering infected individuals at risk of reactivation upon becoming immunocompromised. Extracts from various parts of Annona muricata have been widely used medicinally for the management, control and/or treatment of several human diseases, acting against parasites that cause diseases in humans., Aim of the Study: This study was performed to investigate the action of the ethanolic extract of A. muricata (EtOHAm) and its fractions in the control of the apicomplexan parasite Toxoplasma gondii in vitro and in vivo, and the effect of EtOHAm on the inflammatory response and lipid profile alteration induced by in vivo T. gondii infection., Materials and Methods: The cytotoxicity of EtOHAm and its fractions ethyl acetate (EtOAcAm), n-butanol (BuOHAm), aqueous (H 2 OAm), hexane (HexAm) and dichloromethane (CH 2 Cl 2 Am) was evaluated in NIH/3T3 fibroblasts using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The cells were infected with T. gondii, treated with the extracts, and parasite proliferation was analyzed. For the in vivo experiments, C57BL/6 mice were orally infected with T. gondii and, treated with different concentrations of extract fractions that were effective in vitro (EtOHAm, EtOAcAm, HexAm and CH 2 Cl 2 Am). Tissue parasitism, histological alterations, systemic cytokine and lipid profile were investigated., Results: EtOHAm, EtOAcAm, BuOHAm, H 2 OAm presented low cytotoxicity until doses of 200 μg/mL, while HexAm and CH 2 Cl 2 Am presented toxicity from doses of 100μg/mL. EtOHAm, HexAm and CH 2 Cl 2 Am decreased the parasitism in vitro, presenting a therapeutic index of 2.62, 2.44, and 2.96, respectively. In vivo, EtOHAm, HexAm and CH 2 Cl 2 Am improved the survival rate of infected animals, however, only EtOHAm was able to decrease the parasitism in the small intestine and lung. Additionally, EtOHAm decreased the systemic interferon (IFN)-γ and tumor necrosis factor (TNF) systemically in infected mice, and was able to maintain the triglycerides and very-low-density lipoprotein (VLDL) lipid fractions at similar levels to uninfected animals. Although treatment with EtOHAm could not control the inflammation induced by oral infection in the tissues analyzed, it was able to preserve the number of goblet cells in the small intestine., Conclusions: Ethanolic A. muricata leaf extract could be considered as a good candidate for the development of a complementary/alternative therapy against toxoplasmosis, and also as an anti-inflammatory alternative for decreasing TNF and IFN-γ concentrations and lipid fractions in specific diseases., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Heme oxygenase-1 (HO-1) enzyme exerts beneficial effects at the maternal-fetal interface, especially in trophoblasts, being involved in survival and maturation of these cell phenotypes. Trophoblast cells play essential roles throughout pregnancy, being the gateway for pathogens vertically transmitted, such as Toxoplasma gondii . It was previously shown that HO-1 activity was involved in the control of T. gondii infection in vivo ; however, its contribution in trophoblast cells during T. gondii infection, remain undefined. Thus, this study aimed to investigate the influence of HO-1 in T. gondii -infected BeWo and HTR-8/SVneo human trophoblast cells. For this purpose, trophoblast cells were infected and the HO-1 expression was evaluated. T. gondii -infected BeWo cells were treated with hemin or CoPPIX, as inducers of HO-1, or with bilirubin, an end-product of HO-1, and the parasitism was quantified. The involvement of p38 MAPK, a regulator of HO-1, and the cytokine production, were also evaluated. It was found that T. gondii decreased the HO-1 expression in BeWo but not in HTR-8/SVneo cells. When treated with the HO-1 inducers or bilirubin, BeWo cells reduced the parasite proliferation. T. gondii also decreased the p38 MAPK phosphorylation in BeWo cells; on the other hand, HO-1 induction sustained its activation. Finally, the IL-6 production was upregulated by HO-1 induction in T. gondii -infected cells, which was associated with the control of infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Almeida, Mota, Mineo, Ferro, Barbosa and Silva.)

Neospora caninum poses as a considerable threat to animal health and generates significant economic impact in livestock production worldwide. Here, we have investigated the mechanism that underlies the participation of the inflammasome complex and Reactive Oxygen Species (ROS) in the regulation of immune responses during N. caninum infection. For that purpose, we used in vitro (bone marrow derived macrophages) and in vivo mouse models of infection. Our results show that NLRP3 and NLRC4 receptors, alongside with ASC and Caspase-1, are required for proper activation of the inflammasome during N. caninum infection. As expected, the engagement of these pathways is crucial for IL-1α, IL-1β, and IL-18 production, as well as the induction of pyroptosis. Our results also show that N. caninum induces ROS production dependent of the inflammasome assembly, which in its turn also depends on MyD88/NF-κB-induced ROS to maintain its activation and, ultimately, lead to restriction of parasite replication., (Copyright © 2020 Mota, Lima-Junior, Ferreira França, Aguillón Torres, Barros, Santiago, Silva, Mineo, Zamboni and Mineo.)

Neospora caninum infection is an important cause of neuromuscular disease in dogs and abortion in cattle, leading to significant economic losses in beef and dairy industries. The protective immunity against apicomplexan parasites, specifically Toxoplasma gondii and N. caninum, is typically achieved by inducing an IL-12-driven Th1 immune response. IL-12 stimulates IFN-γ production, which activates Inducible Nitric Oxide Synthase (iNOS) and promotes consequent Nitric Oxide (NO) synthesis, classically described as one of the main effector mechanisms for parasite elimination. Here, we aimed to evaluate the role played by iNOS during N. caninum infection. Our results show that N. caninum infection in C57BL/6 wild type (WT) mice induce NO production in vivo and in vitro. In agreement, iNOS deficient mice, as well as WT mice treated with iNOS inhibitor aminoguanidine, succumbed during acute infection with a dose lethal to 50 % of the WT mice, and presented significant increase in parasite load when submitted to sub-lethal infection protocols. Interestingly, the lack of control of parasite proliferation observed in iNOS -/- mice was associated with notable CNS inflammation and increased production of the main systemic proinflammatory cytokines (IL-12, IFN-γ, IL-6, TNF and IL-17A). Taken together, our findings show that iNOS plays an important role in restricting N. caninum replication, while also modulates the inflammatory process induced by the infection., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Toxoplasmosis is a disease caused by Toxoplasma gondii, an intracellular protozoan able to infect a wide range of hosts. The infection is particularly severe in immunocompromised patients or during pregnancy, circumstances in which the parasite could find a more favorable microenvironment to replicate and invade host tissues. The current treatment consists in toxic drugs for the patients, being not appropriate for the fetuses and immunodeficient patients. So far, there is a lack of available vaccine to prevent the disease. The present study aimed to evaluate the immune response induced by peptides derived from parasite immunodominant proteins from key components, as surface, rhoptry, microneme and dense granule antigens. A panel of eleven peptides was selected considering the highest scores for B cell epitope prediction by in silico analyses. The peptides were divided in groups, according to the parasite organelle locations, and used to immunize C57BL/6 mice. The animals were submitted to three doses of immunization and infected by 10 cysts of T. gondii ME49 strain. Blood samples were collected and used to measure the production of antibodies and cytokines, while the brains were collected to determine the parasite burden by quantitative polymerase chain reaction (qPCR). It was found that synthetic peptides from all targets were able to induce IgG synthesis in immunized mice, as well as to modulate the Th1/Th2 cytokine production, particularly the MIC and SRS groups, which presented the IFN-γ/IL-10 and TNF-α/IL-10 ratios 30 and 10 times higher, respectively, when compared with non-immunized group. Interestingly, the animals from MIC and SRS groups had significantly lower levels of T. gondii DNA in their brains. In summary, it can be concluded that peptides mainly from SRS and MIC parasite components constitute relevant targets to design vaccine candidates against parasite burden observed during chronic toxoplasmosis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

It has been described that the metalloprotease BmooMP-alpha-I purified from Bothrops moojeni snake venom is able to hydrolyze the TNF molecule. However, this observation has been based mainly on in vitro investigation, in addition to molecular modeling and docking approaches. Considering that there is no in vivo study to demonstrate the biological effects of this enzyme, the major aim to the present work was to investigate whether the BmooMP-alpha-I has any anti-inflammatory efficacy by setting up a murine experimental design of colitis induced by dextran sulfate sodium (DSS). For this purpose, C57BL/6 mice were divided into six groups, as follows: (i) animals without intestinal inflammation, (ii) animals without intestinal inflammation treated with BmooMP-alpha-I (50 μ g/animal/day), and (iii) animals with intestinal inflammation induced by 3% of DSS, (iv) mice with intestinal inflammation induced by DSS and treated with BmooMP-alpha-I enzyme at the 50, 25, or 12.5 μ g/animal/day dosages by intraperitoneal route. Clinical signs of colitis were observed daily for calculating the morbidity scores, cytokine measurements, and histological features. We observed that the animals treated with different doses of the enzyme presented a remarkable improvement of colitis signs, as confirmed by a significant increase of the intestine length in comparison to the DSS group. Also, no difference was observed between the groups treated with the enzyme or vehicle, as the colon length of these animals was slightly lower than that of the group of healthy animals, without induction of intestinal inflammation. The cytokine quantification in supernatants of intestinal tissue homogenates showed a significant reduction of 38% in IFN-gamma levels, when the animals were treated with 50 μ g of the BmooMP-alpha-I compared to the animals receiving DSS only. A significant reduction of 39% in TNF levels was also observed in all doses of treatment with BmooMP-alpha-I, in addition to a significant reduction of 35% in the amount of IL-12p40. Histological examinations revealed that the BmooMP-alpha-I 50 μ g treated group preserved colon architecture and goblet cells and reduced the ulcer area, when compared with DSS mice, which showed typical inflammatory changes in tissue architecture, such as ulceration, crypt dilation, loss of tissue architecture, and goblet cell depletion, accompanied by a significant cell infiltration. In conclusion, our results suggest that the improvement of clinical scores and histological findings related to BmooMP-alpha-I treatment in this experimental model could be attributed to the metalloprotease ability to modulate cytokine production locally at the inflamed intestine. These findings highlight the potential anti-inflammatory role and effectiveness of this enzyme as a therapeutic alternative in this type of immunopathological condition., Competing Interests: The authors declare that there is no conflict of interests regarding the publication of this paper.

Neospora caninum is a protozoan parasite closely related to Toxoplasma gondii and has been studied for causing neuromuscular disease in dogs and abortions in cattle. It is recognized as one of the main transmissible causes of reproductive failure in cattle and consequent economic losses to the sector. In that sense, this study aimed to evaluate the role of Toll-like receptor 3 (TLR3)-TRIF-dependent resistance against N. caninum infection in mice. We observed that TLR3 -/- and TRIF -/- mice presented higher parasite burdens, increased inflammatory lesions, and reduced production of interleukin 12p40 (IL-12p40), tumor necrosis factor (TNF), gamma interferon (IFN-γ), and nitric oxide (NO). Unlike those of T. gondii , N. caninum tachyzoites and RNA recruited TLR3 to the parasitophorous vacuole (PV) and translocated interferon response factor 3 (IRF3) to the nucleus. We also observed that N. caninum upregulated the expression of TRIF in murine macrophages, which in turn upregulated IFN-α and IFN-β in the presence of the parasite. Furthermore, TRIF -/- infected macrophages produced lower levels of IL-12p40, while exogenous IFN-α replacement was able to completely restore the production of this key cytokine. Our results show that the TLR3-TRIF signaling pathway enhances resistance against N. caninum infection in mice, since it improves Th1 immune responses that result in controlled parasitism and reduced tissue inflammation, which are hallmarks of the disease., (Copyright © 2019 American Society for Microbiology.)

Toxoplasma gondii infection can cause abortions or congenital infection for a vast number of domestic animals and humans, leading to economic loss in veterinary sciences, as well as severe consequences for immunocompromised patients. Bidens pilosa Linné has been used in ethnopharmacology for treatment of diseases, as malaria, diabetes and hepatitis, in addition to its use as antioxidant, antiallergic, anti-inflammatory, and antiviral. The components of this plant have never been studied before for treatment of toxoplasmosis, and the conventional drugs currently used to treat this disease have high degree of toxicity. Thus, the aim of this study was to evaluate the effect of B. pilosa against T. gondii , by analyzing a total extract of this plant in parallel with a fraction obtained by precipitation in acetone. Also, it was assessed if the acetonic fraction could present lectinic activity, followed by its identification by mass spectrometry. It was observed with the experimental models designed that both total extract and acetonic fraction of B. pilosa were able to control T. gondii infection by in vitro and in vivo experiments, in addition to their low toxicity to host cells. Both total extract and acetonic fraction of this plant display capacity to impair replication of T. gondii tachyzoites. Interesting, the B. pilosa acetonic fraction treatment for 10 days after infection decreases significantly the number of T. gondii brain cyst in comparison with controls. The protein isolated from B. pilosa acetonic fraction was characterized as a novel lectin identified as maturase K. Taken together, these findings open new perspectives to treat patients infected by T. gondii . Future studies will be necessary to investigate the precise mechanism underlying the control of T. gondii infection to impair the replication of this parasite in the host cells after treatment with B. pilosa maturase K.

This paper has been retracted at the request of the authors.

The development of molecular genetics has greatly enhanced the study of the biology and pathology associated with parasites of the phylum Apicomplexa. We have established a system specifically designed for Neospora caninum, and used this system as a heterologous platform for the expression of foreign genes. Plasmid constructs containing fluorescent proteins or targeted genes of Toxoplasma gondii, driven by N. caninum promoters, have yielded robust expression and correct trafficking of target gene products as assessed by immunofluorescence assays and Western blot analyses. Using this approach, we here demonstrated that N. caninum expressing T. gondii's GRA15 and ROP16 kinase are biologically active and induced immunological phenotypes consistent with T. gondii strains. N. caninum expressing TgGRA15 differentially disturbed the NF-κB pathway, inducing an increased IL-12 production. On the other hand, N. caninum expressing TgROP16 induced host STAT3 phosphorylation and consequent reduction of IL-12 synthesis. These results indicate that heterologous gene expression in N. caninum is a useful tool for the study of specific gene functions and may allow the identification of antigenic targets responsible for the phenotypic differences observed between these two closely related apicomplexan parasites. Additionally, these observations may prove to be useful for the development of vaccine protocols to control toxoplasmosis and/or neosporosis.

Neospora caninum is an intracellular protozoan parasite that has drawn increasing interest due to its association with worldwide repetitive bovine abortions, which cause billionaire losses to the meat and dairy industries annually. Innate immunity plays an important role in infection control, and N. caninum activates the production of inflammatory mediators through toll-like receptors, NOD-like receptors, and mitogen-activated protein kinase signaling pathways. Advances in the knowledge of initial host-parasite interactions are desirable for the design of control measures against the infection, obliterating its pathogenesis. In that sense, we here aimed to describe the role of the innate C-type lectin receptor Dectin-1 during the infection by N. caninum . With that intent, we observed that the absence of Dectin-1, observed in genetically depleted (Dectin-1 -/- ) mice or competitively inhibited by an inert agonist [laminarin (LAM)], rescued 50% of the mice infected with lethal doses of N. caninum . Dectin-1 -/- and LAM-treated mice also presented a reduction in the parasite load during acute and chronic phases, associated with decreased inflammatory scores in the central nervous system. Among all the cell phenotypes that migrated to the initial site of infection, dendritic cells and macrophages gained subpopulations with high Dectin-1 surface expression. The impairment of the receptor in these cells led to a decreased parasite burden, as well as augmented production of IL-12p40. We also found that Dectin-1 + cells produced less reactive oxygen species (ROS) at the initial site of the infection, while mice deficient in NADPH oxidase isoform 2 (NOX2 -/- ) were not able to control parasite replication and produce IL-12p40, even upon LAM treatment. Interestingly, the absence of functional Dectin-1 did not alter the susceptibility of mice against closely related Toxoplasma gondii . In conclusion, the gathered data suggest that Dectin-1 is involved in the parasite-induced downmodulation of ROS, and other key molecules triggered for the control of N. caninum infection and are a promising target for future development of protocols intended for intervention against neosporosis.

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.

Neospora caninum is an apicomplexan parasite responsible for major economic losses due to abortions in cattle. Innate immune responses are crucial for host resistance against the infection, however the molecules involved in parasite recognition are still poorly understood. Nod2 is a cytosolic receptor that recognizes several pathogens and its role during N. caninum infection has not yet been described. In that sense, we evaluated the role of Nod2 in host response against this parasite. We found that infection of macrophages induced increased expression of Nod2, which colocalized with the parasites' vacuoles. Nod2-deficient macrophages showed an impaired induction of pro-inflammatory cytokines, increased production of modulatory molecules, and failure to restrict parasite replication. In vivo, Nod2-knockout mice showed a reduction of MAPK phosphorylation and proinflammatory cytokines, followed by decreased inflammation in target organs and increment in parasite burden. Surprisingly, these mice were partially resistant to lethal doses of tachyzoites. In addition, these phenomena were not observed in Rip2-/- mice. In conclusion, our study indicates that Nod2-dependent responses account for N. caninum elimination. On the other hand, the inflammatory milieu induced by this innate receptor provoked pathogenesis and death in severe experimental neosporosis.

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.

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.

Background: Toxoplasmosis is a zoonosis caused by Toxoplasma gondii, an intracellular protozoan parasite able to infect a wide range of hosts, including humans. Congenital infection can cause severe damage to the fetus. Thus, it is important to detect antibodies against the parasite to confirm clinical manifestations. Considering that all immunoglobulin isotypes may be present in biological samples from newborns and their mothers, this study aimed to evaluate the ability to diagnose recent toxoplasmosis by using colostrum, as an alternative noninvasive way to obtain biological samples, as well as to determine correlation rates between antibodies from serum samples to detect IgG, IgM and IgA isotypes against T. gondii., Methods: A total of 289 puerperal women from Clinical Hospital of Federal University of Uberlândia (mean age: 24.8 years, range: 14 - 43 years) took part in this study. Serum and colostrum samples from these patients were analyzed using ELISA and immunoblotting assays for soluble antigens from T. gondii., Results: ELISA immunoassays with serum samples showed reactivity in 47.0, 6.9 and 2.8 % of samples to anti-T. gondii IgG, IgM and IgA, respectively, in comparison with colostrum samples, which showed reactivity in 46.0, 7.9 and 2.8 % of samples to the same isotypes. Also, significant correlation rates of anti-T. gondii antibody levels between serum and colostrum samples were observed. Interestingly, reactivity to IgM and/or IgA in colostrum and/or serum confirmed clinical manifestations of congenital toxoplasmosis in three newborns. Immunoblotting assays showed that it is possible to detect IgG, IgM and IgA antibodies against various antigens of T. gondii in serum and colostrum samples. IgG antibodies in serum and colostrum samples recognized more antigenic fractions than IgM and IgA antibodies. Serum IgG detected more antigenic fractions than IgG antibodies present in the colostrum of the same patient. In contrast, specific IgA present in colostrum recognized a higher number of antigens than IgA present in serum samples of the same patient., Conclusions: Overall, the results show that it is important to investigate the occurrence of congenital toxoplasmosis, even at puerperal period. Furthermore, this study demonstrates that T. gondii-specific IgG, IgM and IgA antibodies in serum and colostrum samples from puerperal women may be detected with a significant correlation, suggesting that colostrum may also be used as an alternative biological sample to efficiently diagnose recent human toxoplasmosis.

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Toxoplasma gondii is an intracellular parasite that causes relevant clinical disease in humans and animals. Several studies have been performed in order to understand the interactions between proteins of the parasite and host cells. SAG2A is a 22 kDa protein that is mainly found in the surface of tachyzoites. In the present work, our aim was to correlate the predicted three-dimensional structure of this protein with the immune system of infected hosts., Methods: To accomplish our goals, we performed in silico analysis of the amino acid sequence of SAG2A, correlating the predictions with in vitro stimulation of antigen presenting cells and serological assays., Results: Structure modeling predicts that SAG2A protein possesses an unfolded C-terminal end, which varies its conformation within distinct strain types of T. gondii. This structure within the protein shelters a known B-cell immunodominant epitope, which presents low identity with its closest phyllogenetically related protein, an orthologue predicted in Neospora caninum. In agreement with the in silico observations, sera of known T. gondii infected mice and goats recognized recombinant SAG2A, whereas no serological cross-reactivity was observed with samples from N. caninum animals. Additionally, the C-terminal end of the protein was able to down-modulate pro-inflammatory responses of activated macrophages and dendritic cells., Conclusions: Altogether, we demonstrate herein that recombinant SAG2A protein from T. gondii is immunologically relevant in the host-parasite interface and may be targeted in therapeutic and diagnostic procedures designed against the infection.