Phenotypic and genotypic characterization of two Toxoplasma gondii isolates in free-range chickens from Uberlândia, Brazil.

The aim of this study was to determine the seroprevalence of Toxoplasma gondii infection in free-range chickens from Uberlândia, Minas Gerais state, Brazil, and characterize the genotypic and phenotypic features of two isolates of this parasite, considering the importance of these hosts in the epidemiology of toxoplasmosis. Serum samples from 108 free-range chickens were obtained from ten different districts, and submitted to the modified agglutination test (MAT) for the presence of anti-T. gondii antibodies, and brain and heart tissue samples from infected chickens were processed for mouse bioassay. An overall seroprevalence of 71·3% was found and antibody titres ranged from 16 to 4096. After confirmation of seropositivity by mouse bioassay, the determination of the T. gondii genotypes of two isolates was performed by PCR-RFLP, using primers for the following markers: SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, new SAG2, Apico and CS3. These T. gondii isolates, designated TgChBrUD1and TgChBrUD2, were obtained from heart samples of free-range chickens. The TgChBrUD1 isolate belonged to ToxoDB PCR-RFLP genotype 11 and the TgChBrUD2 isolate belonged to ToxoDB PCR-RFLP genotype 6. Both isolates demonstrated high virulence in a rodent model, with the TgChBrUD1 isolate able to induce brain cysts, in accord with its pattern of multiplication rates in human fibroblast culture. Taken together, these results reveal high prevalence of T. gondii infection in free-range chickens throughout Uberlândia, indicating an important degree of oocyst environmental contamination and the existence of considerable risk for T. gondii transmission to humans by consumption of free-range chicken as a food source.

Susceptibility to Toxoplasma gondii proliferation in BeWo human trophoblast cells is dose-dependent of macrophage migration inhibitory factor (MIF), via ERK1/2 phosphorylation and prostaglandin E2 production.

INTRODUCTION: Macrophage migration inhibitory factor (MIF) participates in the immune response to Toxoplasma gondii, triggers ERK1/2 and prostaglandin E2 (PGE2) activation, but there is limited information on these mechanisms in human trophoblast. The present study aimed to verify the role of MIF in the ERK1/2 phosphorylation and PGE2 production, as well as its effect on the susceptibility to T. gondii in BeWo cells. METHODS: BeWo cells were treated with increasing concentrations of recombinant MIF (rMIF) and/or T. gondii-soluble tachyzoite antigen (STAg) and analyzed for ERK1/2 phosphorylation and PGE2 production by Western blotting and ELISA, respectively. Cells were also treated with increasing concentrations of rMIF, rPGE2, or ERK1/2 inhibitor and tested for T. gondii proliferation. The supernatants of cells treated with rPGE2 were assayed for cytokine production by ELISA or CBA. RESULTS: ERK1/2 phosphorylation and PGE2 production increased when the cells were treated with low MIF concentrations while the parasitism control occurred only at high MIF concentrations. STAg was unable to change ERK1/2 phosphorylation or PGE2 release. BeWo cells demonstrated increased T. gondii proliferation and reduced production of pro-inflammatory cytokines when treated with PGE2, while PD98059 diminished the parasite proliferation. DISCUSSION: The intracellular mechanisms triggered by MIF are dose-dependent in BeWo cells, and PGE2 is an important factor for the persistence of T. gondii at the maternal fetal interface. CONCLUSION: MIF was unable to control T. gondii infection in BeWo cells at low concentrations since ERK1/2 and PGE2 expression were activated, demonstrating a critical effect of these mediators favoring parasite proliferation.

Review: putative roles for the macrophage migratory inhibitory factor at the maternal fetal interface.

Complex and dynamic networks of molecules participate in the essential interactions between maternal organism, placenta and fetus in a healthy and successful pregnancy. Macrophage migratory inhibitory factor (MIF) is one of several molecules produced at implantation sites; MIF is mostly expressed by trophoblast cells. This has led to expectations of MIF's relevance as a partner in the maternal/fetal dialog. MIF is known by its biological interactions and functional roles as an activator of innate immunity, regulating subsequent adaptive responses, which include inhibition of migration of mononuclear cells in vitro, antagonism of glucocorticoids, and regulation of expression of Toll-like receptor 4. Beyond roles in the inflammatory response, MIF can interfere with proliferative activities in different cell types, as well as with cell death pathways. This intriguing factor found at the human, porcine, ovine, bovine and rodent maternal-fetal interfaces is present in a time- and spatially-dependent manner, indicating regulatory roles in the process of embryo implantation, placental development, maintenance of pregnancy and birth. Here, we will review MIF participation in placental physiology, including new evidence for a dialog with uterine cells, and a potential role in protection of uterine decidual cells.

Differential apoptosis in BeWo cells after infection with highly (RH) or moderately (ME49) virulent strains of Toxoplasma gondii is related to the cytokine profile secreted, the death receptor Fas expression and phosphorylated ERK1/2 expression.

INTRODUCTION: Alterations of apoptosis are commonly associated with pregnancy complications and abortion. Modulation of apoptosis is a relevant feature of Toxoplasma gondii infection and it is related to parasite strain types. The aim of the present study was to evaluate the possible factors that are involved in the differential apoptosis of BeWo cells infected with distinct T. gondii strain types. METHODS: Human trophoblastic cells (BeWo cell line) were infected with RH or ME49 strains, the cytokine production was measured and the phosphorylation of anti-apoptotic ERK1/2 protein was analyzed. Also, cells were treated with different cytokines, infected with RH or ME49 strain, and analyzed for apoptosis index and Fas/CD95 death receptor expression. RESULTS: ME49-infected BeWo cells exhibited a predominantly pro-inflammatory cytokine profile, whereas cells infected with RH strain had a higher production of anti-inflammatory cytokines. Also, the incidence of apoptosis was higher in ME49-infected cells, which have been treated with pro-inflammatory cytokines compared to cells infected with RH and treated with anti-inflammatory cytokines. Moreover, Fas/CD95 expression was higher in cells infected with either ME49 or RH strain and treated with pro-inflammatory cytokines compared to anti-inflammatory cytokine treatment. The phosphorylation of ERK1/2 protein increased after 24 h of infection only with the RH strain. CONCLUSION: These results suggest that opposing mechanisms of interference in apoptosis of BeWo cells after infection with RH or ME49 strains of T. gondii can be associated with the differential cytokine profile secreted, the Fas/CD95 expression and the phosphorylated ERK1/2 expression.

Trophoblast cells are able to regulate monocyte activity to control Toxoplasma gondii infection.

INTRODUCTION: Toxoplasma gondii is an intracellular parasite that causes severe disease when the infection occurs during pregnancy. Trophoblast cells constitute an important maternal-fetal barrier, with monocytes concentrating around them. Thus, interactions between trophoblasts and monocytes are important for maintaining a successful pregnancy, especially in cases of infection. This study aimed to evaluate the role of trophoblast cells (BeWo line) on monocyte (THP-1 line) activity in the presence or absence of T. gondii infection. METHODS: THP-1 cells were stimulated with supernatants of BeWo cells, previously infected or not with T. gondii, and then infected with parasites. The supernatant of both cells were collected and analyzed for cytokine production and T. gondii proliferation in THP-1 cells was determined. RESULTS: The results showed that after infection, the pattern of cytokines secreted by THP-1 and BeWo cells was characterized as a pro-inflammatory profile. Furthermore, supernatant of BeWo cells infected or not, was able to change the cytokine profile secreted by infected THP-1 cells, and this supernatant became THP-1 cells more able to control T. gondii proliferation than those that had not been stimulated. DISCUSSION: This effect was associated with secretion of interleukin (IL)-6 by the THP-1 cells and soluble factors secreted by BeWo cells, such as IL-6 and MIF. CONCLUSION: Together, these results suggest that trophoblast cells are able to modulate monocyte activity, resulting in the control of T. gondii infection and subsequent maintenance of pregnancy.

Azithromycin and spiramycin induce anti-inflammatory response in human trophoblastic (BeWo) cells infected by Toxoplasma gondii but are able to control infection.

Toxoplasma gondii is an important pathogen which may cause fetal infection if primary infection. Our previous studies have used human choriocarcinoma trophoblastic cells (BeWo cell line) as experimental model of T. gondii infection involving placental microenvironment. This study aimed to examine the effects of azithromycin and spiramycin against T. gondii infection in BeWo cells. Cells were treated with different concentrations of the macrolide antibiotics and analyzed first for cell viability using thiazolyl blue tetrazole (MTT) assay. As cell viability was significantly decreased with drug concentrations higher than 400 µg/mL, the concentration range used in further experiments was from 50 to 400 µg/mL. The number of infected cells and intracellular replication of T. gondii decreased after treatment with each drug. The infection induced up-regulation of the macrophage migration inhibitory factor (MIF), which was also enhanced in infected cells after treatment with azithromycin, but not with spiramycin. Analysis of the cytokine profile showed increase TNF-α, IL-10 and IL-4 production, but decreased IFN-γ levels, were detected in infected cells and treated with each drug. In conclusion, treatment of human trophoblastic BeWo cells with with azithromycin or spiramycin is able to control the infection and replication of T. gondii. In addition, treatment with these macrolides, especially with azityromycin induces an anti-inflammatory response and high MIF production, which can be important for the establishment and maintenance of a viable pregnancy during T. gondii infection.

Evaluation of vertical transmission of Toxoplasma gondii in Calomys callosus model after reinfection with heterologous and virulent strain.

Toxoplasma gondii is an obligate intracellular protozoan parasite that causes a variety of clinical syndromes, but the infection is severe in immunocompromised individuals and during pregnancy due to the possibility of transplacental transmission of the parasite causing congenital toxoplasmosis. Vertical transmission of the parasite usually occurs when females are primarily infected during pregnancy. Calomys callosus is resistant to T. gondii ME49 strain, which presents a moderate virulence and congenital disease occurs only during the acute phase of infection. The aim of this study was to determine whether vertical transmission occurs when females of C. callosus chronically infected with ME49 strain of T. gondii are reinfected with a highly virulent strain (RH, type I). Females were infected with cysts of the ME49 strain. On the 1st day of pregnancy, animals were reinfected with tachyzoites of the RH strain. In the 19th day of pregnancy, placentas and embryos were processed for morphological analysis, immunohistochemistry and for detection of the parasite by PCR and mouse bioassay. Morphological and immunohistochemical analyses revealed the presence of parasites only in placental tissues. Mouse bioassay results showed seroconversion only in mice that were inoculated with placental tissues. Also, T. gondii DNA was detected only in placental samples. Congenital toxoplasmosis does not occur in C. callosus females chronically infected with the moderately virulent ME49 strain of T. gondii and reinfected with the highly virulent RH strain, thus indicating that primary T. gondii infection before pregnancy leads to an effective long-term immunity preventing transplacental transmission to the fetus.

Action of neuwiedase, a metalloproteinase isolated from bothrops neuwiedi venom, on skeletal muscle: an ultrastructural and immunocytochemistry study

The damaging effects of neuwiedase, a non-hemorrhagic snake venom metalloproteinase from P-I class, on gastrocnemius muscle are studied herein. Following neuwiedase injection, ultrastructural alterations were detected early showing disarrangement of skeletal muscle fibers (characterized by discontinuity of Z lines), mitochondrial swelling, and disruption of plasma membrane and basal lamina. Degradation of skeletal muscle and the appearance of an amorphous substance, primarily composed of cellular debris, were noted after 24 hours. The presence of neuwiedase at the injection site (detected by immunocytochemistry) revealed highly specific labeling of myofibril components of damaged myocytes. In addition, proteolysis of muscle proteins assayed through myofibrils extracted from gastrocnemius muscle indicated that neuwiedase provoked degradation of myofibrils, especially myosin. These results suggest that skeletal muscle damage, induced by neuwiedase, is probably due to its proteolytic action on myofibrils, which are responsible for the maintenance of the cellular architecture.

Apoptosis and S phase of the cell cycle in BeWo trophoblastic and HeLa cells are differentially modulated by Toxoplasma gondii strain types.

Transplacental transmission of Toxoplasma gondii causes congenital toxoplasmosis, one of the most severe forms of infection. The ability of the parasite to survive intracellularly largely depends on the blocking of different proapoptotic signaling cascades of the host cells. During pregnancy, however, alterations in the incidence of apoptosis are associated with abnormal placental morphology and function. The aim of this study was to evaluate the incidence of apoptosis and cell proliferation in trophoblastic (BeWo cell line) and uterine cervical (HeLa cell line) cells infected with a highly virulent RH strain or a moderately virulent ME49 strain of T. gondii. BeWo and HeLa cells were infected with RH or ME49 tachyzoites (2:1 and 5:1; parasite:cell) or medium alone (control). After 2 h, 6 h and 12 h of incubation, cells were fixed in 10% formalin and analyzed by immunohistochemistry to determine the apoptosis (expression of cytokeratin 18 neo-epitope--clone M30) and cell in S phase (expression of proliferating cell nuclear antigen--PCNA) indices. RH strain-infected BeWo and HeLa cells showed a lower apoptosis index than non-infected controls, whereas a higher apoptosis index was found in ME49 strain-infected cells compared to controls. In addition, RH-infected cells displayed lower apoptosis index than ME49-infected cells, even though active caspase-3 was detected in both cell types infected with either RH or ME49 strains as well in non-infected cells in all analyzed times of infection. Also, the cell S phase indices were higher in ME49 strain-infected BeWo and HeLa cells as compared to non-infected controls and RH strain-infected cells. These results indicate that RH and ME49 strains of T. gondii possess opposing mechanism of interference in apoptosis and cell cycle S phase of both BeWo and HeLa cells and these differences can be associated to evasion strategies of the parasite to survive inside the host cells.

Azithromycin inhibits vertical transmission of Toxoplasma gondii in Calomys callosus (Rodentia: Cricetidae).

Toxoplasma gondii infection during pregnancy may cause severe consequences to the embryo. Current toxoplasmosis treatment for pregnant women is based on the administration of spiramycin or a drug combination as sulphadiazine-pyrimethamine-folinic acid (SPFA) in cases of confirmed fetal infection. However, these drugs are few tolerated and present many disadvantages due to their toxic effects to the host. The aim of this study was to evaluate the effectiveness of different treatments on the vertical transmission of T. gondii, including azithromycin, Artemisia annua infusion, spiramycin and SPFA in Calomys callosus as model of congenital toxoplasmosis. C. callosus females were perorally infected with 20 cysts of T. gondii ME49 strain at the day that a vaginal plug was observed (1st day of pregnancy - dop). Treatment with azithromycin, A. annua infusion, and spiramycin started at the 4th dop, while the treatment with SPFA started at the 14th dop. Placenta and embryonic tissues were collected for morphological and immunohistochemical analyses, mouse bioassay and PCR from the 15th to 20th dop. No morphological changes were seen in the placenta and embryonic tissues from females treated with azithromycin, spiramycin and SPFA, but embryonic atrophy was observed in animals treated with A. annua infusion. Parasites were found in the placenta and fetal (brain and liver) tissues of animals treated with SPFA, A. annua infusion and spiramycin, although the number of parasites was lower than in non-treated animals. Parasites were also observed in the placenta of animals treated with azithromycin, but not in their embryos. Bioassay and PCR results confirmed the immunohistochemical data. Also, bradyzoite immunostaining was observed only in placental and fetal tissues of animals treated with SPFA. In conclusion, the treatment with azithromycin showed to be more effective, since it was capable to inhibit the vertical transmission of T. gondii in this model of congenital toxoplasmosis.

BeWo trophoblast cell susceptibility to Toxoplasma gondii is increased by interferon-gamma, interleukin-10 and transforming growth factor-beta1.

The present study aimed to investigate BeWo trophoblast cell susceptibility to Toxoplasma gondii infection under stimulation with anti-inflammatory cytokines in comparison with HeLa cells. Both cell types were submitted to different treatments with recombinant cytokines [interleukin (IL)-10 and transforming growth factor (TGF)-beta1] or the respective antibodies (anti-IL-10 and anti-TGF-beta) before and after T. gondii infection. The effect of interferon (IFN)-gamma was also assessed alone or in combination with anti-inflammatory cytokines or the respective antibodies after the parasite infection. Cells were fixed, stained and parasites quantified under light microscopy to evaluate intracellular replication (mean number of parasites per cell in 100 infected cells) and infection index (percentage of infected cells per 100 examined cells). In contrast with HeLa cells, treatments with IL-10 or TGF-beta1 induced a considerable augmentation in both T. gondii intracellular replication and invasion into BeWo cells. In addition, treatment with IFN-gamma alone or associated with IL-10 or TGF-beta1 increased the same parameters in BeWo cells, whereas the opposite effect was observed in HeLa cells. When endogenous IL-10 or TGF-beta was blocked, both BeWo and HeLa cells were able to control the parasite infection only in the presence of IFN-gamma. Together, these results indicate that the higher susceptibility of BeWo cells to T. gondii may be due to immunomodulation mechanisms, suggesting that the role of trophoblast cells in maintaining a placental microenvironment favourable to pregnancy may facilitate the infection into the placental tissues.

Susceptibility to vertical transmission of Toxoplasma gondii is temporally dependent on the preconceptional infection in Calomys callosus.

Toxoplasma gondii is an obligate intracellular parasite that causes a variety of clinical syndromes, but the infection is more severe in immunocompromised individuals and in cases of congenital toxoplasmosis. This study aimed to verify if the susceptibility to vertical transmission of Toxoplasma gondii is temporally dependent on the preconceptional infection in Calomys callosus. Twelve C. callosus females were infected with 20 cysts of T. gondii ME49 strain and divided into three groups of four animals that were mated after approximately 10 days (group 1), 30 days (group 2), and 50 days (group 3) of infection. The animals were sacrificed from the 17th to 20th day of pregnancy, when placentas and embryos were collected for morphological and immunohistochemical studies, mouse bioassay for evaluating seroconversion and PCR for detecting parasite DNA. Serum samples from C. callosus females and mice used in bioassay were analysed for the detection of IgG antibodies to T. gondii by ELISA. Detection of T. gondii was observed by mouse bioassay and PCR in placentas and embryos from C. callosus females infected around 10 days pre-conception. However, only placentas, but not embryos, from females infected around 30 and 50 days pre-conception showed positivity for parasite DNA and seroconversion by mouse bioassay. In conclusion, this study model shows that vertical transmission of T. gondii may take place when maternal infection occurs within one month before conception, thus demonstrating the time of preconceptional seroconversion that rule out a risk of congenital toxoplasmosis.

BeWo trophoblasts are unable to control replication of Toxoplasma gondii, even in the presence of exogenous IFN-gamma.

The ability of RH strain of Toxoplasma gondii to invade and grow into BeWo cells was investigated in the present study using IFN-gamma, l-tryptophan, or alpha-methyl-tryptophan treatments. HeLa cells were used in the same conditions for comparison purposes. It was demonstrated that BeWo cells are more permissive to T. gondii infection, making them more susceptible to this pathogen when compared to HeLa cells. Infection rates of BeWo cells do not show any significant alteration in different protocols using IFN-gamma. In addition, BeWo treated with l-tryptophan was unable to significantly increase parasite growth. In contrast, HeLa cells treated with IFN-gamma or IFN-gamma plus l-tryptophan are able to impair or increase, respectively, parasite replication, providing evidence that this indoleamine-2,3-dioxygenase-dependent phenomenon is operant in these cells, whereas it is inactive in BeWo. Therefore, our data support the hypothesis that the immunological mechanisms controlling infection at the maternal-fetal interface are different from those occurring in the periphery. At the same time that operating regulatory mechanisms work inside and outside the cells located at that microenvironment to prevent maternal rejection of the concept, these events might facilitate the progression of infection caused by intracellular pathogens, as T. gondii.

Effect of Toxoplasma gondii infection kinetics on trophoblast cell population in Calomys callosus, a model of congenital toxoplasmosis.

This work evaluated the kinetics of events that occur in the placenta of Calomys callosus after Toxoplasma gondii infection. Animals on the first day of pregnancy (dop) and virgin nonpregnant females were perorally infected with 20 cysts of T. gondii strain ME49. After 100 days of infection, the virgin animals were mated and received an additional 20 cysts on the first dop. The placentas and the embryos from both acutely and chronically infected animals were analyzed up to day 20 of pregnancy by morphological and immunocytochemical assays. Noninfected and infected animals exhibited placenta with normal morphology. From the seventh dop and infection onwards, liver and spleen cells of the infected animals contained several parasitophorous vacuoles. On the 13th day, the maternal blood present at the placental blood spaces contained T. gondii-infected leukocytes. Infected placental cells were only seen on the 15th dop, being the trophoblast giant cells, the first cell type to contain signs of the parasite internalization, followed by labyrinth zone cells 24 h later and spongiotrophoblast cells only after the 19th dop. Fetal liver and brain were infected by T. gondii concomitantly to the labyrinth cell infection. No signals of infection were observed on placentas and embryos from chronically infected animals. Therefore, considering the sequence of events leading to the infection of the various organs, it could be hypothesized that the placenta is infected later on during pregnancy, which may be related to the defense roles played by this structure. However, trophoblast giant cells are unable to completely stop the progression of T. gondii infection towards the fetal tissues. C. callosus was demonstrated to be a suitable experimental model to study the dynamics of congenital toxoplasmosis.