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.

Galectin-3 is essential for reactive oxygen species production by peritoneal neutrophils from mice infected with a virulent strain of Toxoplasma gondii.

Toxoplasma gondii stimulates a potent pro-inflammatory response and neutrophils are involved in early infection. Galectin-3 (Gal-3) is an endogenous modulator of inflammatory processes and anti-infective agents, but its interaction with neutrophils in T. gondii infection is still unclear. Here, we evaluated the role of Gal-3 in peritoneal inflammation, reactive oxygen species (ROS) production by neutrophils and survival, after in vivo T. gondii infection with virulent RH strain, using Gal-3 deficient and wild type mice. Animals were inoculated with thioglycollate or tachyzoites, and peritoneal cells were harvested for analysis of the influx of leukocytes. Neutrophils were isolated from peritoneal exudates from infected mice and stimulated with phorbol myristate acetate (PMA) to evaluate ROS production by luminol-dependent chemiluminescence assay. Our results showed that: (1) Gal-3 upregulates peritoneal inflammation, with enhanced recruitment of neutrophils and lymphocytes after thioglycollate stimulation, but does not influence the enhanced neutrophil influx after early T. gondii infection; (2) Gal-3 upregulates ROS generation by inflammatory peritoneal neutrophils from infected mice, but downregulates its production in non-infected mice and (3) Gal-3 does not influence the survival of mice after infection with the virulent T. gondii strain. In conclusion, Gal-3 is essential for ROS generation by neutrophils in the initial acute phase of T. gondii infection and this phenomenon may constitute an attempt to control parasite growth during in vivo infection with the T. gondii virulent strain.

Antibody response and avidity of respiratory syncytial virus-specific total IgG, IgG1, and IgG3 in young children.

Respiratory syncytial virus (RSV) is a major cause of acute respiratory disease in infants and young children. Considering that several aspects of the humoral immune response to RSV infection remain unclear, this study aimed to investigate the occurrence, levels, and avidity of total IgG, IgG1, and IgG3 antibodies against RSV in serum samples from children ≤5 years old. In addition, a possible association between antibody avidity and severity of illness was examined. The occurrence and levels of RSV-specific IgG depended on age, with infants <3 months old displaying high levels of antibodies, which were probably acquired from the mother. Children ≥24 months old also showed frequent occurrence and high levels of IgG, which was produced actively during infection. In addition, the avidity assay showed that the avidity of RSV-specific total IgG and IgG1 was lower in infants <3 months old who had acute respiratory disease than in age-matched controls. The avidity of RSV-specific IgG detected in children ≥24 months old with lower respiratory infection was lower than that in children with upper respiratory infection. These results indicate that the presence of high avidity RSV-specific IgG antibodies may lead to better protection against RSV infection in children <3 months old, who may have a lower probability of developing disease of increased severity. In addition, children ≥24 months old with RSV-specific IgG antibodies of low avidity tended to develop more severe RSV illness. These findings may be helpful in establishing vaccination schedules when a vaccine becomes available.

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.

Evaluation of homologous, heterologous, and affinity conjugates for the serodiagnosis of Toxoplasma gondii and Neospora caninum in maned wolves (Chrysocyon brachyurus).

Use of serological tests in the diagnosis of infectious diseases in wild animals has several limitations, primarily the difficulty of obtaining species-specific reagents. Wild canids, such as maned wolves (Chrysocyon brachyurus), are highly predisposed to infection by Toxoplasma gondii and, to a lesser extent, to Neospora caninum. The aim of the present study was to evaluate homologous, heterologous, and affinity conjugates in enzyme-linked immunosorbent assays (ELISAs) and indirect fluorescent antibody tests (IFATs) for detecting immunoglobulin (Ig) G antibodies against T. gondii and N. caninum in maned wolves. Serum samples were obtained from 59 captive animals in Brazil and tested by ELISA for T. gondii serology and IFAT for N. caninum serology using 3 different enzymatic and fluorescent conjugates: homologous (guinea pig anti-maned wolf IgG-peroxidase and -fluorescein isothiocyanate [FITC]), heterologous (rabbit anti-dog IgG-peroxidase and -FITC), and affinity (protein A-peroxidase and -FITC). Seropositivity to T. gondii was comparable among the homologous (69.5%), heterologous (74.6%), and affinity (71.2%) enzymatic conjugates. A significant positive correlation was found between the antibody levels determined by the 3 enzymatic conjugates. The highest mean antibody levels (ELISA index = 4.5) were observed with the protein A-peroxidase conjugate. The same seropositivity to N. caninum (8.5%) was found with the homologous and heterologous fluorescent conjugates, but protein A-FITC was not able to detect or confirm any positive samples with homologous or heterologous conjugates. Our results demonstrate that homologous, heterologous, and affinity conjugates might be used in ELISA for serological assays of T. gondii in wild canids, whereas for N. caninum infection, only the homologous or heterologous fluorescent conjugates have been shown to be useful.

Down-modulation of nitric oxide production in murine macrophages treated with crude plant extracts from the Brazilian Cerrado.

Several plant species from the Cerrado biome in Brazil are popularly used as herbal medicines for its reputed analgesic, anti-acid, anti-microbial, anti-inflammatory and anti-tumoral properties, among others. It has been reported that some plant extracts interfere in the production of nitric oxide (NO), an important inflammatory mediator. In the present study, we investigated the effect of hexanic and ethanolic extracts from three plant species on NO production by LPS/IFN-gamma-activated J774 macrophages based on traditional use. The cytotoxic effect of the crude extracts was determined by the thiazolyl blue test (MTT) to measure cell viability. Serjania lethalis stem extracts and Cupania vernalis leaf extracts significantly inhibited NO production, while extracts from Casearia sylvestris var. lingua were inactive or showed low activity on NO production, or were very cytotoxic. The ethanolic stem bark and leaf extracts of Serjania lethalis and Cupania vernalis, respectively, almost completely inhibited the production of NO by J774 macrophages. It can be concluded that the selected extracts are potential sources of active compounds that might be used as anti-inflammatory agents.

Trophoblast-macrophage crosstalk on human extravillous under Toxoplasma gondii infection.

INTRODUCTION: The interaction between human extravillous trophoblasts and macrophages has an important role in implantation and placentation. However, any dysfunction in this communication system is associated with pregnancy pitfalls, and a Toxoplasma gondii infection can be a potential problem in this crosstalk. Therefore, the aim of this study was to assess the influence of infected macrophages on cytokine production and the incidence of apoptosis in T. gondii-infected extravillous trophoblast cells. METHODS: HTR-8/SVneo cells were treated with supernatant from macrophages infected or not by T. gondii (conditioned medium) in order to analyze apoptosis and cytokine production in comparison to uninfected control conditions. RESULTS: The IL-6 secretion by HTR-8/SVneo cells increased synergistically by treatment with conditioned medium and T. gondii infection. The apoptosis index of HTR-8/SVneo cells was also upregulated by treatment with conditioned medium and infection. In addition, a low expression of Fas/CD95 and a high soluble FasL release were observed during infection, although no significant change was observed in the proliferation of T. gondii. DISCUSSION: The parasite modulates the high apoptosis index in HTR-8/SVneo cells in order to favor its establishment inside its host cells. On the other hand, the conditioned medium from uninfected macrophages restores the apoptosis rates, although the effect of the infection seems to be stronger. In conclusion, our results showed that T. gondii infection in human extravillous trophoblasts is able to modulate the trophoblast-macrophage crosstalk.

Immunoenzymatic assy (ELISA) in mucocutaneous leishmaniasis, kala-azar, and Chagas' disease: an epimastigote Trypanosoma cruzi antigen able to distinguish between anti-Trypanosoma and anti-Leishmania antibodies.

Areas where mucocutaneous leishmaniasis, kala-azar and Chagas' disease are prevalent often overlap in Latin America. Due to a variable degree of cross-reactivity between the three etiological agents and antibodies synthesized during the course of the disease, a test able to distinguish between them is greatly needed. Saline-extracted antigen from live Trypanosoma cruzi epimastigotes behaved towards Chagas' disease sera in immunoenzymatic assays (ELISA) with the same specificity and sensitivity as the antigen used routinely for such tests. In tests using mucocutaneous leishmaniasis or kala-azar sera, the live T. cruzi antigen showed a much lower geometric mean titer (GMT) than the standard T. cruzi antigen. In ELISA tests with L. braziliensis antigen a higher GMT was seen with kala-azar sera than with those from cases of mucocutaneous leishmaniasis. Also, sera from Vale do Ribeira leishmaniasis showed higher GMT with an L. donovani antigen than with a homologous one. Such discrepancies were not seen in immunofluorescence tests employing the same sera and antigens.

Evaluation of a synthetic tripeptide as antigen for detection of IgM and IgG antibodies to Trypanosoma cruzi in serum samples from patients with Chagas disease or viral diseases.

An enzyme-linked immunosorbent assay (ELISA) has been developed to detect IgG and IgM antibodies in human sera against a synthetic tripeptide derived from a hybrid peptide containing 3 specific epitopes from Trypanosoma cruzi. This assay was compared in Brazil with one using conventional antigen, the alkaline crude extract. Serum samples were divided into positive (40 samples) or negative (107 samples) for Chagas disease. Positive samples included 9 serum samples from patients with acute Chagas disease, while negative samples included 57 samples from patients suffering from viral diseases. The total percentages of IgG positive samples from patients with chronic Chagas disease for alkaline extract and synthetic tripeptide were 93.5% and 100%, respectively. All samples from patients with acute Chagas disease were confirmed positive for IgM antibodies by using both the tripeptide and the alkaline extract. However, the results for anti-T. cruzi IgM in the group of chronic Chagas disease patients demonstrated that 41.9% were positive for IgM with the alkaline extract, while the synthetic peptide showed a significantly lower number of positive samples (12.9%). The serum samples from healthy people showed similar results for both antigens. However, 40% of the serum samples from patients presenting with viral diseases were IgM positive for Chagas disease when assayed with conventional antigen; with the synthetic tripeptide as antigen, 100% of this group of samples were found to be negative. Thus, as the results of ELISA with synthetic tripeptide showed higher rates of sensitivity and specificity than ELISA with conventional antigen, the former should be included as a laboratory tool in the serodiagnosis of Chagas disease.

Total and specific anti-Trypanosoma cruzi immunoglobulin E in pericardial fluid samples from patients with chronic Chagas disease.

Levels of total and specific anti-Trypanosoma cruzi immunoglobulin E (IgE) were determined by immunoenzymatic assay among 101 samples of pericardial fluid from patients who had died in one trypanosomiasis endemic area in central Brazil. These samples were divided into 6 groups. Group I, 17 samples from patients with the cardiac form of Chagas disease; group II, 11 samples from patients with the digestive form of Chagas disease, presenting megaoesophagus and/or megacolon; group III, 41 samples from patients with the indeterminate form of Chagas disease; group IV, 4 samples from patients with both cardiac and digestive forms of Chagas disease; group V, 5 samples from patients who suddenly died and were seropositive for T. cruzi antibodies; group VI, 23 samples, used as a control group, which came from patients seronegative for T. cruzi antibodies. Significantly high levels of total IgE were observed in groups I, II, III, IV and V when compared with group VI (mean concentrations 708-1157 iu/mL compared with 394 iu/mL). In groups I-V, 32 samples (41%) had specific anti-T. cruzi IgE antibodies. The individual percentage positivity rates in these groups were 64.7% (group I), 45.4% (group II), 34.1% (group III), nil (group IV), and 40.0% (group V). A significant correlation between total IgE and specific anti-T. cruzi IgE was observed only in the samples from patients with the cardiac form of Chagas disease (group I).

Changes in the behavioral and immunological parameters of the mollusk Biomphalaria tenagophila induced by disruption of the circadian cycle as a consequence of continuous illumination.

In the present investigation we studied some behavioral and immunological parameters of adult gastropod mollusk, Biomphalaria tenagophila, which have been reproducing for several generations under laboratory conditions. One group of gastropods was kept on a 14-h light/10-h dark cycle, corresponding to a regular circadian cycle, and another group was exposed to continuous light for 48 h. Animals were studied along (behavioral groups) or immediately after (immunological groups) 48 h of regular circadian cycle or continuous light conditions. Stopping/floating, dragging and sliding were the behavioral aspects considered (N = 20 for regular cycle; N = 20 for continuous illumination) and number of hemocytes/microl hemolymph was the immunological parameter studied (N = 15 for regular cycle, N = 14 for continuous illumination). Animals under continuous illumination were more active (sliding = 33 episodes, dragging = 48 episodes) and displayed a lower number of hemocytes (78.0 +/- 24. 27/microl) when compared with mollusks kept on a regular circadian cycle (sliding = 18 episodes, dragging = 27 episodes; hemocytes = 157.6 +/- 53.27/microl). The data are discussed in terms of neural circuits and neuroimmunological relations with the possible stressful effect of continuous illumination.

Immune response to respiratory syncytial virus in young Brazilian children.

We have evaluated the cellular and humoral immune response to primary respiratory syncytial virus (RSV) infection in young infants. Serum specimens from 65 patients <=12 months of age (39 males and 26 females, 28 cases <3 months and 37 cases > or = 3 months; median 3 3.9 months) were tested for anti-RSV IgG and IgG subclass antibodies by EIA. Flow cytometry was used to characterize cell surface markers expressed on peripheral blood mononuclear cells (PBMC) from 29 RSV-infected children. There was a low rate of seroconversion in children <3 months of age, whose acute-phase PBMC were mostly T lymphocytes (63.0 +/- 9.0%). In contrast, a higher rate of seroconversion was observed in children >3 months of age, with predominance of B lymphocytes (71.0 +/- 17.7%). Stimulation of PBMC with RSV (2 x 10(5) TCID50) for 48 h did not induce a detectable increase in intracellular cytokines and only a few showed a detectable increase in RSV-specific secreted cytokines. These data suggest that age is an important factor affecting the infants' ability to develop an immune response to RSV.

Seroprevalence of Toxoplasma gondii and Neospora caninum in captive maned wolves (Chrysocyon brachyurus) from southeastern and midwestern regions of Brazil.

The main purpose of the present study was to investigate the occurrence of antibodies against T. gondii and N. caninum in captive maned wolves from Brazil, considering that little information is available at the literature about infections by these parasites in this wild animal. Serum samples were obtained from 59 maned wolves originated from six zoos and from one ecological reserve of the southeastern and midwestern regions of Brazil. To detect IgG antibodies against T. gondii, an ELISA protocol was used and the results were expressed as ELISA reactivity indexes (EI). Serology for N. caninum was carried out by indirect fluorescent antibody test (IFAT) and cut-off titers were established at 1:25 dilution. From the total of the analyzed samples, 44 (74.6%) were seropositive for T. gondii and only 5 (8.5%) for N. caninum. Seropositivity for T. gondii ranged from 0 to 100% in the seven different origin locals, with rates over 50% among the six zoos, whereas no positivity was found in the samples from ecological reserve. For N. caninum, seroprevalence varied from 0 to 50% in the different locals, with the highest rates also detected in zoos. Seroprevalence for T. gondii was strongly related with age, with rates significantly higher among adult wolves (91.7%) when compared to newborn or young animals. Seropositive samples for N. caninum were found predominantly in adult wolves. For both parasites, seroprevalence did not show a significant distinction in relation to gender. Although seroprevalence for T. gondii was significantly higher when compared to N. caninum in the Brazilian captive maned wolves tested, these findings reflect the great exposure of this species to T. gondii and, in lower extension, to N. caninum. Also, the present study demonstrated for the first time the presence of antibodies to N. caninum in wild life from South America.

Detection of IgG antibodies to Neospora caninum and Toxoplasma gondii in dogs examined in a veterinary hospital from Brazil.

A total of 163 dogs with neuromuscular, respiratory and/or gastrointestinal disorders, was admitted at the Veterinary Hospital, Federal University of Uberlândia, Brazil, and submitted to serology for Toxoplasma gondii and Neospora caninum. Assays for T. gondii included indirect haemagglutination (IHA), indirect fluorescent antibody (IFAT-Tg), immunoenzymatic (ELISA), and immunoblotting (IB-Tg). Assays for N. caninum included IFAT-Nc and immunoprecipitation (IP-Nc). Based on concordant results by three serological tests (IHA, IFAT-Tg and ELISA) for T. gondii, and divergent results further confirmed by IB-Tg for reactivity to TgSAG1, the 163 sera were divided into two groups: 59 (36%) Tg-seropositive samples and 104 (64%) Tg-seronegative samples. Antibodies to Neospora were detected in 11 (6.7%) out of 163 analyzed dog sera, with 5 (3.1%) samples reactive to both parasites (Tg+/Nc+), and 6 (3.7%) reactive only to Neospora (Tg-/Nc+). Antibodies only to T. gondii were found in 54 (33%) samples. Among the 11 Neospora-positive sera analyzed by IB-Tg, the five sera Tg+/Nc+ showed strong reactivity to Toxoplasma antigens, especially to TgSAG1 (p30). No reactivity was observed to TgSAG1 in the six samples Tg-/Nc+. By IP-Nc, two highly immunodominant antigens (29 and 35kDa proteins) were recognized by all 11 IFAT-Nc positive sera. Our results suggest that the infection by N. caninum can be concomitantly present in dogs from this area, although less common, and therefore should be considered in the differential clinical diagnosis with T. gondii in dogs presenting neuromuscular, respiratory and/or gastrointestinal disorders.

Lectin used in the purification process of Toxoplasma gondii tachyzoites.

Toxoplasma gondii tachyzoites were purified from mouse peritoneal exudates using lectins to remove the host cells. Best results were obtained with phytohemagglutinin P at a concentration of 0.01% (W/V), which provided a 99.7% pure Toxoplasma suspension. The process was reproducible and easy to perform. Toxoplasmas so obtained were infective and served as sources of high quality antigens for immunofluorescence, hemagglutination, and complement fixation tests.

Detection of Toxoplasma gondii soluble antigen, SAG-1(p30), antibody and immune complex in the cerebrospinal fluid of HIV positive or negative individuals.

Active infection by T. gondii was evaluated by immunoassay for soluble SAG-1 (p30), the major surface antigen from T. gondii, specific antibodies and immune complexes in human cerebrospinal fluid (CSF) samples. A total of 263 samples of CSF were collected from hospitalized patients presenting neurological disorders and analyzed for antibodies to HIV. Patients were divided into two groups: HIV positive (n = 96) or HIV negative (n =167). The results of the assays showed that 45% of all samples were positive for soluble SAG-1. Toxoplasma Ag/Ab immune complexes were detected in 19% of the CSF samples and 62% were positive for T. gondii- specific IgG. A combination of these assays in the presence of clinical findings consistent with active Toxoplasma infection may predict the presence of toxoplasmic encephalitis. Moreover, detection of soluble SAG-1 in the CSF of these individuals appears consistent with active infection.

Toxoplasmosis in naturally infected deer from Brazil.

Serum samples from 107 cervids were examined for Toxoplasma gondii antibodies using indirect hemagglutination (IHA), indirect immunofluorescence (IFA), enzyme linked immunosorbent assay (ELISA) and Dot-ELISA. Samples were obtained from 66 marsh deer (Blastocerus dichotomus) in the State of São Paulo (Brazil) and from 41 pampas deer (Ozotocerus bezoarticus) in the State of Goiás (Brazil). Antibodies to T. gondii were found in 23 (22%) of the deer, with 18 and 5 positive samples, respectively, for B. dichotomus and O. bezoarticus. The highest prevalence of T. gondii antibodies were young adults (32%), following by adults (27%) and fawns (13%). Only one serum sample (8%) from a newborn fawn was positive in the serological tests. The convenience of the Dot-ELISA test is obvious when compared with other serological tests for both laboratory or field surveys, mainly due to its features of practicability and reagent stability.

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.