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1.
Int J Food Microbiol ; 314: 108380, 2020 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-31707174

RESUMO

The protozoan parasite Toxoplasma gondii can infect all warm-blooded animals and it causes the disease toxoplasmosis. Meat containing viable T. gondii tissue cysts is considered one of the main sources of human infection. The relative importance of the different types of meat depends, not only on the prevalence of T. gondii infection in the different livestock species, but also on consumed volumes and preparation habits. To take these factors into account and to estimate the relative contribution of different meat products to human infection, a quantitative risk assessment model for meat-borne T. gondii infection was previously developed. However, at the time, the effect of salting on parasite viability was estimated based on a single experiment. In recent years, data using salting methods that are more in line with processing of meat products have come available. Literature data on the effect of salting on T. gondii viability were collected and used to fit a predictive model. In addition to the new salting model, a lower concentration of bradyzoites in cattle, more specific heating profiles, and more recent consumption data were implemented in the QMRA model for meat-borne T. gondii infection in the Netherlands. Results show that beef remains the most important source, as it contributed 84% of the total number of predicted infections in the Dutch population, followed by pork (12%), mutton (3.7%), lamb (0.2%) pork/beef mixed products (0.1%), and veal (0.01%). The predicted number of T. gondii infections is reasonably in line with epidemiological data. At the product level, filet americain (a raw beef spread) alone contributed 80% of the total predicted infections in the base model, but scenario analyses demonstrate that its contribution is highly dependent on the salting parameters. A clear identification of the most risky meat products is important, as interventions focussing on these products could have a great impact on reducing T. gondii disease burden in the Netherlands. For that reason, it is important that the effects of salting and other processing methods are evaluated in line with industrial processing and incorporated in quantitative risk assessment models for meat-borne toxoplasmosis.


Assuntos
Manipulação de Alimentos/métodos , Microbiologia de Alimentos/métodos , Microbiologia de Alimentos/estatística & dados numéricos , Produtos da Carne/parasitologia , Toxoplasma/fisiologia , Toxoplasmose/epidemiologia , Animais , Bovinos , Humanos , Países Baixos/epidemiologia , Prevalência , Carne Vermelha/parasitologia , Medição de Risco , Carneiro Doméstico , Suínos , Toxoplasma/isolamento & purificação , Toxoplasmose/parasitologia , Toxoplasmose/prevenção & controle
2.
Invest Ophthalmol Vis Sci ; 60(14): 4652-4660, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31743940

RESUMO

Purpose: Retinal damage in ocular toxoplasmosis reflects Toxoplasma gondii-induced cell lysis and reactive inflammation. Human retinal histopathology demonstrates the presence of neutrophils, but activities of this leukocyte subset are unstudied. We conducted in vitro experiments to evaluate roles for neutrophils as retinal taxis for T. gondii and as contributors to the inflammation. Methods: Human neutrophils were isolated from peripheral blood. Migration to disease-relevant chemokines was evaluated in transwells, seeded with human retinal endothelial cells for some assays, using neutrophils infected with GT-1 strain T. gondii tachyzoites. Neutrophils were cocultured with T. gondii-infected ARPE-19 and primary human retinal pigment epithelial cells, and production of reactive oxygen species (ROS) was estimated by dihydroethidium reaction. Proteins produced by T. gondii-infected ARPE-19 cells were profiled by immunoarray, and candidate neutrophil-activating proteins were targeted with specific blocking antibody in coculture assays. Results: Infection with T. gondii arrested neutrophil migration across retinal endothelium regardless of the presence of CXCL8. Migration to CXCL1, CXCL2, and CXCL8 also was significantly inhibited in infected neutrophils. Neutrophils generated more ROS when cocultured with infected versus uninfected ARPE-19 cells and three of four primary retinal pigment epithelial cell isolates. Infected ARPE-19 cells augmented the synthesis of 12 neutrophil-activating proteins also expressed by primary retinal pigment epithelial cells. Antibody blockade of granulocyte-macrophage colony-stimulating factor, interleukin-6 (IL-6) and IL-18 significantly reduced ROS production by neutrophils cocultured with T. gondii-infected ARPE-19 cells. Conclusions: Our findings support involvement of neutrophils in retinal inflammation, but not parasite transport, in the setting of ocular toxoplasmosis.


Assuntos
Neutrófilos/fisiologia , Epitélio Pigmentado da Retina/metabolismo , Toxoplasmose Ocular/imunologia , Adulto , Linhagem Celular , Ensaios de Migração de Leucócitos , Movimento Celular/fisiologia , Quimiocinas/metabolismo , Técnicas de Cocultura , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Interleucina-18/metabolismo , Interleucina-6/metabolismo , Ativação de Neutrófilo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Epitélio Pigmentado da Retina/parasitologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Toxoplasma/fisiologia
3.
Exp Parasitol ; 207: 107775, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31628896

RESUMO

The ability to differentiate from the proliferative (tachyzoite) to the latent (bradyzoite) stage of isolates of Toxoplasma gondii recombinant genotypes (I/II/III and I/III) and reference strains from a clonal line (RH and ME49) was investigated in this study. Two isolates from chicken (#114 and #277; ToxoDB) and 3 from pigs (#114; ToxoDB) were the subjects for evaluation. The isolates were grown in cell culture under 2 different conditions: culture medium at pH 7.0 (neutral, without stress induction) or pH 8.0 (alkaline, stress inducing). After 4 days, the cultures were fixed and the events resulting from infection and induction were labeled. T. gondii cysts were labeled using Dolichos biflorus-FITC lectin (DBL-cysts) and free tachyzoites or vacuolar were labeled using an anti-T. gondii polyclonal antibody followed by an Alexa 594-conjugated secondary antibody (DBL-negative structures compatible with parasite structures - lysis plaques or vacuole). Differences in DBL-cysts formation in vitro in response to exogenous stress were observed between recombinant genotype isolates and the typical genotypes. The differences in conversion rates and the patterns of lysis plate production between genotype I/III isolates (#114) indicate that care should be taken when extrapolating the in vitro phenotypic characteristics of parasites from the same genotype.


Assuntos
Galinhas/parasitologia , Genótipo , Doenças das Aves Domésticas/parasitologia , Doenças dos Suínos/parasitologia , Toxoplasma/fisiologia , Toxoplasmose Animal/parasitologia , Análise de Variância , Animais , Brasil , Linhagem Celular , Meios de Cultura/química , Imunofluorescência , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Microscopia de Fluorescência , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Ovinos , Suínos , Toxoplasma/classificação , Toxoplasma/genética
4.
Exp Parasitol ; 207: 107781, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31626796

RESUMO

The paradigm that Toxoplasma gondii infection generates sterilizing protective immunity was broken by case studies in which reinfections were observed in immunocompetent pregnant women in the chronic phase of toxoplasmosis. Since then, several murine models have suggested that immunoprotection against a previous T. gondii infection may be violated after reinfection with strains of different genotypes. This study aimed to evaluate the dissemination of the parasite after reinfection with the virulent TgCTBr9 and EGS strains in BALB/c mice chronically infected with the avirulent TgCTBr5 strain. Three mice were euthanized at 2, 4, 8, 12, 24 and 48 h post challenge (p.c.) and at 7, 14 and 30 days p.c. Intestines, mesenteric lymph nodes, lungs and brains were collected for PCR-RFLP. Blood samples were collected to measure total IgG, IgG1 and IgG2a by ELISA. The reinfected animals survived and presented reduced morbidity after challenge with the virulent strains. Mice challenged with the TgCTBr9 strain showed a slight increase in anti-T. gondii IgG1. The spread of the TgCTBr5 strain was observed to occur earlier than the dissemination of the virulent TgCTBr9 or EGS strains. The TgCTBr9 strain was observed in the mesenteric lymph node at 7 days post challenge (d.p.c.); in the intestine and lungs at 14 d.p.c.; and in the brain at 30 d.p.c. EGS strain was demonstrated in the mesenteric lymph node and lung at 7 d.p.c and in the intestine and brain at a later time point. The immune response promoted by the primary infection with the avirulent strain (TgCTBr5) protected the animals from death after challenge with the virulent strains (TgCTBr9 or EGS).


Assuntos
Anticorpos Antiprotozoários/sangue , Toxoplasma/fisiologia , Toxoplasmose Congênita/parasitologia , Animais , Peso Corporal , Encéfalo/parasitologia , Brasil , Feminino , Genótipo , Humanos , Imunoglobulina G/sangue , Intestinos/parasitologia , Pulmão/parasitologia , Linfonodos/parasitologia , Mesentério , Camundongos , Camundongos Endogâmicos BALB C , Morbidade , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Recidiva , Toxoplasma/genética , Toxoplasma/imunologia , Toxoplasma/patogenicidade , Toxoplasmose Congênita/imunologia , Virulência
5.
Vet Res ; 50(1): 66, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31533826

RESUMO

Although it is known that gestation could influence the clinical course of ovine toxoplasmosis, the precise effect of the term of gestation when sheep are infected are yet mostly unknown. The aim of this study was to evaluate the peripheral and placental immune responses developed in pregnant sheep after experimental infection with Toxoplasma gondii at different times of gestation. Thirty-six pregnant sheep were allocated in different groups, orally inoculated with sporulated oocysts of T. gondii at early, mid and late gestation and culled within 30 days post-infection. The peripheral humoral and cytokine responses were evaluated, as well as the transcription of cytokines at the placenta. Serological analysis revealed that, regardless the term of gestation when infected, specific IgG against T. gondii were detected from day 8 post-infection and there was an early peripheral release of IFN-γ at the first week post-infection followed by a short peak of IL10 and TNF-α at the second week post-infection. There were no significant differences in this response between infected groups. At the placenta, a similar increase in transcription of IFN-γ, and TNF-α was found at the three terms of gestation, while IL-4 increased mainly at the first and second terms and IL-10 transcription was higher at the last term. While these findings show that both Th1 and Th2 cytokines play a key role in the pathogenesis of ovine toxoplasmosis and that placental and peripheral immune responses do not closely correlate, there seems to be no clear modulation of these responses along the gestation.


Assuntos
Imunidade Humoral/imunologia , Placenta/imunologia , Doenças dos Ovinos/imunologia , Toxoplasma/fisiologia , Toxoplasmose Animal/imunologia , Animais , Anticorpos Antiprotozoários , Feminino , Idade Gestacional , Oocistos/fisiologia , Gravidez , Ovinos , Doenças dos Ovinos/parasitologia , Fatores de Tempo , Toxoplasmose Animal/parasitologia
6.
BMC Genomics ; 20(1): 705, 2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31506064

RESUMO

BACKGROUND: Infection with Toxoplasma gondii is thought to damage the brain and be a risk factor for neurological and psychotic disorders. The immune response-participating chemokine system has recently been considered vital for brain cell signaling and neural functioning. Here, we investigated the effect of the deficiency of C-C chemokine receptor 5 (CCR5), which is previously reported to be associated with T. gondii infection, on gene expression in the brain during T. gondii infection and the relationship between CCR5 and the inflammatory response against T. gondii infection in the brain. RESULTS: We performed a genome-wide comprehensive analysis of brain cells from wild-type and CCR5-deficient mice. Mouse primary brain cells infected with T. gondii were subjected to RNA sequencing. The expression levels of some genes, especially in astrocytes and microglia, were altered by CCR5-deficiency during T. gondii infection, and the gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis revealed an enhanced immune response in the brain cells. The expression levels of genes which were highly differentially expressed in vitro were also investigated in the mouse brains during the T. gondii infections. Among the genes tested, only Saa3 (serum amyloid A3) showed partly CCR5-dependent upregulation during the acute infection phase. However, analysis of the subacute phase showed that in addition to Saa3, Hmox1 may also contribute to the protection and/or pathology partly via the CCR5 pathway. CONCLUSIONS: Our results indicate that CCR5 is involved in T. gondii infection in the brain where it contributes to inflammatory responses and parasite elimination. We suggest that the inflammatory response by glial cells through CCR5 might be associated with neurological injury during T. gondii infection to some extent.


Assuntos
Encéfalo/citologia , Encéfalo/parasitologia , Perfilação da Expressão Gênica , Receptores CCR5/deficiência , Toxoplasma/fisiologia , Animais , Astrócitos/metabolismo , Astrócitos/parasitologia , Encéfalo/metabolismo , Técnicas de Inativação de Genes , Camundongos , Microglia/metabolismo , Microglia/parasitologia , Receptores CCR5/genética
7.
Microbiol Res ; 227: 126293, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31421715

RESUMO

T. gondii is a major opportunistic pathogen chronically infecting nearly one third of the world's population. Due to the high infection and mortality rates in immunocompromised patients and newborns, the extent or magnitude of T. gondii pathogenesis is determined mainly by host-pathogen interactions. T. gondii utilizes specialized secretory proteins to modify host cellular factors and facilitate invasion and replication. This review provides update on the recent progress in this field of research with particular emphasis on the T. gondii secretory proteins and their role in invasion and pathogenesis.


Assuntos
Transporte Proteico/fisiologia , Proteínas de Protozoários/metabolismo , Toxoplasma/fisiologia , Toxoplasma/patogenicidade , Animais , Interações Hospedeiro-Parasita , Humanos , Estágios do Ciclo de Vida , Toxoplasmose/parasitologia
8.
Res Vet Sci ; 125: 382-389, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31404885

RESUMO

Toxoplasma gondii is an intracellular opportunistic, parasitic protozoan. Microglia have been classified into two main types: M1 (classically activated macrophages) and M2 (alternatively activated macrophages). BV2 cells were used in this study, together with lipopolysaccharide (LPS) and interferon (IFN)-γ or interleukin (IL)-4, which were used to induce resting microglia. Expression levels of M1/M2 markers were determined at both mRNA and protein levels, using PCR, western blot, and flow cytometry. Furthermore, cells were infected with T. gondii PLK strain, and the dynamic changes in M1/M2 marker expression levels were determined. An in vitro polarization model was successfully established. Expression of Nos2 and M1-associated markers was significantly upregulated at 12 h post-infection in BV2 cells. Further, the JAK/STAT1 and NF-κB signaling pathways were also activated following T. gondii infection. This demonstrated that T. gondii infection induces M1-type microglial polarization in vitro. The present study demonstrated that T. gondii infection affects microglial activation in vitro and elucidated the effects of activated microglia on T. gondii proliferation. This data may serve as a useful reference for more detailed elucidation of interactions between T. gondii and the innate immune system.


Assuntos
Macrófagos/parasitologia , Microglia/parasitologia , Toxoplasma/fisiologia , Animais , Linhagem Celular , Proliferação de Células , Regulação da Expressão Gênica , Interferon gama/farmacologia , Interleucina-4/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/classificação , NF-kappa B/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais
9.
PLoS Pathog ; 15(8): e1007923, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31449558

RESUMO

IL-1ß is a potent pro-inflammatory cytokine that promotes immunity and host defense, and its dysregulation is associated with immune pathology. Toxoplasma gondii infection of myeloid cells triggers the production and release of IL-1ß; however, the mechanisms regulating this pathway, particularly in human immune cells, are incompletely understood. We have identified a novel pathway of T. gondii induction of IL-1ß via a Syk-CARD9-NF-κB signaling axis in primary human peripheral blood monocytes. Syk was rapidly phosphorylated during T. gondii infection of primary monocytes, and inhibiting Syk with the pharmacological inhibitors R406 or entospletinib, or genetic ablation of Syk in THP-1 cells, reduced IL-1ß release. Inhibition of Syk in primary cells or deletion of Syk in THP-1 cells decreased parasite-induced IL-1ß transcripts and the production of pro-IL-1ß. Furthermore, inhibition of PKCδ, CARD9/MALT-1 and IKK reduced p65 phosphorylation and pro-IL-1ß production in T. gondii-infected primary monocytes, and genetic knockout of PKCδ or CARD9 in THP-1 cells also reduced pro-IL-1ß protein levels and IL-1ß release during T. gondii infection, indicating that Syk functions upstream of this NF-κB-dependent signaling pathway for IL-1ß transcriptional activation. IL-1ß release from T. gondii-infected primary human monocytes required the NLRP3-caspase-1 inflammasome, but interestingly, was independent of gasdermin D (GSDMD) cleavage and pyroptosis. Moreover, GSDMD knockout THP-1 cells released comparable amounts of IL-1ß to wild-type THP-1 cells after T. gondii infection. Taken together, our data indicate that T. gondii induces a Syk-CARD9/MALT-1-NF-κB signaling pathway and activation of the NLRP3 inflammasome for the release of IL-1ß in a cell death- and GSDMD-independent manner. This research expands our understanding of the molecular basis for human innate immune regulation of inflammation and host defense during parasite infection.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Monócitos/metabolismo , NF-kappa B/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Quinase Syk/metabolismo , Toxoplasmose/metabolismo , Proteínas Adaptadoras de Sinalização CARD/genética , Células Cultivadas , Humanos , Inflamassomos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Monócitos/imunologia , Monócitos/microbiologia , NF-kappa B/genética , Proteínas de Ligação a Fosfato/genética , Transdução de Sinais , Quinase Syk/genética , Toxoplasma/fisiologia , Toxoplasmose/imunologia , Toxoplasmose/microbiologia
10.
Acta Parasitol ; 64(3): 612-616, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31286354

RESUMO

PURPOSE: Toxoplasma gondii is a protozoan from phylum Apicomplexa, which causes the toxoplasmosis infection; this one exhibits an apicoplast organelle which assists in the metabolism of isoprenoids and other pivotal mediators for the parasite survival. Statins are drugs that inhibit cholesterol synthesis, blocking the conversion of the substrate HMG-CoA to mevalonate, thus preventing the initial processes of the biosynthesis of these precursors, both in humans and parasite. Our goal was to verify whether the Toxoplasma gondii (RH strain) tachyzoites form pretreated with pravastatin and simvastatin in association with pyrimethamine and sulfadiazine at low concentrations could affect the infection processes, suggesting direct action on protozoa intracellular proliferation through the inhibition of isoprenoids in the parasite's apicoplast. METHODS: To have the adhesion, infection, and parasite proliferation during experimental infection investigated, HeLa cells (105) were subjected to a 24-hour infection by T. gondii tachyzoites forms of RH strain (5 × 105) pretreated for 30 min with pravastatin and/or simvastatin combined or not with pyrimethamine and sulfadiazine. RESULTS: Combined with conventional drugs at low concentrations pravastatin and simvastatin inhibit the adhesion, invasion, and intracellular proliferation of T. gondii in HeLa cells which are similar to the positive control. CONCLUSION: Pravastatin and simvastatin in association with pyrimethamine and sulfadiazine at low concentrations can be regarded as a promising, effective alternative to toxoplasmosis treatment with reduced side effects.


Assuntos
Antiprotozoários/farmacologia , Estágios do Ciclo de Vida/efeitos dos fármacos , Pravastatina/farmacologia , Pirimetamina/farmacologia , Sinvastatina/farmacologia , Sulfadiazina/farmacologia , Toxoplasma/efeitos dos fármacos , Toxoplasmose/parasitologia , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Células HeLa , Humanos , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/fisiologia , Toxoplasmose/tratamento farmacológico
11.
Exp Parasitol ; 204: 107721, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31288023

RESUMO

BACKGROUND: Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan able to infect humans and it is common in pregnant women. During pregnancy and lactation, there are changes in the concentration of 17ß-estradiol (E2), progesterone (Prg), and prolactin (PRL). It is known that a proinflamatory response reduces the susceptibility to be infected, and this response may change according to hormonal impairment. Monocytes and macrophages are the main barrier against many intracellular microorganisms, due to their ability to produce cytokines. The aim of this work was to determine the effect of E2, progesterone, and PRL on the infective capacity of T. gondii, proinflamatory immune response modulation and the expression of hormonal receptors on THP-1 cell stimulated with T. gondii. METHODS: The THP-1 cells were infected with 1500 T. gondii tachyzoites, of RH strain. Stimuli were conducted with recombinant PRL (200 ng/mL), E2 (40 nM) y Prg (40 nM). MTT assays were performed to evaluate cellular viability. Western blot assays were carried out to evaluate the expression of the hormonal receptors (PRLR, ERα, and ERß). Cytokines produced were measured with a magnetic bead kit directed to 17 cytokines. RESULTS: Stimuli with E2 and Prg increased T. gondii infection in monocytes after 48 h; however, no differences in infection were observed in PRL stimulus. The E2 decreased the secretion of IL-12 and IL-1ß and PRL did not modify the production of these cytokines in THP-1 cells stimulated with T. gondii; however, both hormones increased the production of IL-10. Besides, PRL augmented the production of IL-4 and IL-13. In contrast, Prg reduced these cytokines. Our results show that T. gondii induces the expression of ERα and ERß and lowers PRLR. The hormones modify the expression of the receptors of other hormones: Prg decreases PRLR, ERß and increases ERα; E2 diminishes PRLR; and PRL decreases ERα and ERß expression. CONCLUSION: The hormones can increase T. gondii infection and could be mediating an anti-inflammatory response in THP-1 cells. T. gondii induces changes in the expression of hormonal receptors.


Assuntos
Citocinas/metabolismo , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Receptores da Prolactina/metabolismo , Células THP-1/metabolismo , Toxoplasma/fisiologia , Animais , Corantes , Estradiol/metabolismo , Feminino , Humanos , Camundongos , Progesterona/metabolismo , Prolactina/metabolismo , Isoformas de Proteínas/metabolismo , Células THP-1/imunologia , Células THP-1/parasitologia , Sais de Tetrazólio , Tiazóis , Toxoplasma/crescimento & desenvolvimento
12.
Acta Parasitol ; 64(3): 514-519, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31187391

RESUMO

INTRODUCTION: Toxoplasma gondii is a widely distributed parasite and of great importance to human and animal health. METHODS: The objective of this study was to assess the prevalence of T. gondii antibodies and risk factors associated with the infection in sheep in the Northwest region of the State of Rio Grande do Sul, Brazil; this region has a very high rate of human ocular toxoplasmosis. Ovine sera were tested by the modified agglutination test (cut-off 1:25). RESULTS: T. gondii antibodies were detected in 70.2% (224 of 319). According to the logistic regression, the most significant factors associated were age and cat access to food stock facility. CONCLUSION: Preventive measures are discussed to reduce the risk of transmission of this zoonosis.


Assuntos
Doenças dos Ovinos/epidemiologia , Toxoplasmose Animal/epidemiologia , Toxoplasmose Ocular/veterinária , Testes de Aglutinação , Animais , Anticorpos Antiprotozoários/sangue , Brasil/epidemiologia , Doenças Endêmicas/economia , Doenças Endêmicas/estatística & dados numéricos , Doenças Endêmicas/veterinária , Feminino , Masculino , Ovinos , Doenças dos Ovinos/sangue , Doenças dos Ovinos/parasitologia , Toxoplasma/imunologia , Toxoplasma/fisiologia , Toxoplasmose Animal/sangue , Toxoplasmose Animal/diagnóstico , Toxoplasmose Animal/parasitologia , Toxoplasmose Ocular/sangue , Toxoplasmose Ocular/diagnóstico , Toxoplasmose Ocular/parasitologia
13.
Acta Vet Hung ; 67(2): 204-211, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31238734

RESUMO

Consumption of undercooked or raw pork is considered a significant risk factor for human infection with Toxoplasma gondii. In this study, we investigated the genetic structure of 18 T. gondii strains obtained from slaughter pigs from Northern Serbia (mainly Vojvodina). The examined samples originated from eight pigs from large commercial farms, six backyard pigs and four free-range Mangalica pigs, all found to be positive for either viable T. gondii or T. gondii DNA. Genotyping was attempted from both pig tissues and mouse brains from the bio-assays using a multiplex multilocus nested polymerase chain reaction-restriction fragment length polymorphism (Mn-PCR-RFLP) method with seven markers (GRA6, alt. SAG2, PK-1, BTUB, C22-8, CS3 and Apico). Identification was achieved for nine T. gondii isolates. Seven isolates were classified as type II and two as type III. These results are consistent with previous studies on animal isolates from Serbia as well as with previous reports that type III is more frequently found in samples from Southern Europe than in those from other parts of the continent.


Assuntos
Genótipo , Doenças dos Suínos/parasitologia , Toxoplasma/genética , Toxoplasmose Animal/parasitologia , Animais , Sérvia , Sus scrofa , Suínos , Toxoplasma/fisiologia
14.
PLoS One ; 14(6): e0217989, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31188858

RESUMO

Toxoplasma gondii is a single-celled intracellular apicomplexan parasite that causes toxoplasmosis. It is capable of infecting humans and nearly all warm-blooded animals including pigs, but cats are the only known definitive host. This ubiquitous zoonotic pathogen can cause abortion, stillbirth and fetal abnormalities, and has been associated with mental and behavioral changes in humans. Acute infection is potentially fatal in immunocompromised individuals. The present study aimed to assess the Toxoplasma seroprevalence in pigs, humans and cats after its initial reported detection in pigs about three decades ago in Cebu, Philippines. A total of 924 humans, 104 cats and 514 slaughter pigs were tested for antibodies against T. gondii using a commercial latex agglutination test. The results revealed positive detection rates of 26.3% (244/924) for humans, 42.3% (44/104) for cats and 13.4% (69/514) for slaughter pigs. Statistical analyses revealed that the area (P = 0.004), cat ownership (P = 0.020), the frequency of contact with cats (P < 0.0001) and consumption of street foods (P = 0.043) were significantly associated with seropositivity for T. gondii in humans. Meanwhile, the use of litter trays (P = 0.001) and contact with other animals (P = 0.007) were significantly associated with seropositivity in cats. The odds ratio for selected significant factors revealed that living in suburban areas (OR 1.66, 95% CI: 1.20-2.31), owning a cat (OR 1.482, 95% CI: 1.07-2.07) and eating street foods (OR 1.585, 95% CI: 1.01-2.48) were associated with an increased risk of T. gondii exposure in humans. In cats, the use of a litter tray (OR 4.5, 95% CI: 1.73-11.71) was associated with an increased risk of exposure. None of the profile parameters were found to be significantly associated with seropositivity in slaughter pigs (P > 0.05). This study is the first report of the serological detection of T. gondii in humans and cats in Cebu, Philippines, and the first assessment of the prevalence of the parasite in pigs in the area since its initial detection in 1982. This is also the first report documenting the seropositivity of T. gondii in pregnant women in the country. The confirmed seropositivity of T. gondii in Cebu, Philippines, in the present study implies the endemicity of toxoplasmosis in this area and highlights the need for routine testing and increased public awareness.


Assuntos
Doenças do Gato/epidemiologia , Doenças Endêmicas , Doenças dos Suínos/epidemiologia , Toxoplasma/patogenicidade , Toxoplasmose Animal/epidemiologia , Toxoplasmose Cerebral/epidemiologia , Matadouros , Adolescente , Adulto , Idoso , Animais , Anticorpos Antiprotozoários/sangue , Doenças do Gato/parasitologia , Doenças do Gato/transmissão , Gatos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Filipinas/epidemiologia , Gravidez , Fatores de Risco , Estudos Soroepidemiológicos , Suínos , Doenças dos Suínos/parasitologia , Doenças dos Suínos/transmissão , Toxoplasma/fisiologia , Toxoplasmose Animal/parasitologia , Toxoplasmose Animal/transmissão , Toxoplasmose Cerebral/parasitologia , Toxoplasmose Cerebral/transmissão
15.
mSphere ; 4(3)2019 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-31167946

RESUMO

Toxoplasma gondii is a ubiquitous obligate intracellular parasite that infects the nucleated cells of warm-blooded animals. From within the parasitophorous vacuole in which they reside, Toxoplasma tachyzoites secrete an arsenal of effector proteins that can reprogram host gene expression to facilitate parasite survival and replication. Gaining a better understanding of how host gene expression is altered upon infection is central for understanding parasite strategies for host invasion and for developing new parasite therapies. Here, we applied ribosome profiling coupled with mRNA measurements to concurrently study gene expression in the parasite and in host human foreskin fibroblasts. By examining the parasite transcriptome and translatome, we identified potential upstream open reading frames that may permit the stress-induced preferential translation of parasite mRNAs. We also determined that tachyzoites reduce host death-associated pathways and increase survival, proliferation, and motility in both quiescent and proliferative host cell models of infection. Additionally, proliferative cells alter their gene expression in ways that are consistent with massive transcriptional rewiring, while quiescent cells were best characterized by reentry into the cell cycle. We also identified a translational control regimen consistent with mechanistic target of rapamycin (mTOR) activation in quiescent cells and, to a lesser degree, in proliferative cells. This study illustrates the utility of the method for dissection of gene expression programs simultaneously in the parasite and host.IMPORTANCE Toxoplasma gondii is a single-celled parasite that has infected up to one-third of the world's population. Significant overhauls in gene expression in both the parasite and the host cell accompany parasite invasion, and a better understanding of these changes may lead to the development of new therapeutic agents. In this study, we employed ribosome profiling to determine the changes that occur at the levels of transcription and translation in both the parasite and the infected host cell at the same time. We discovered features of Toxoplasma mRNAs that suggest a means for controlling parasite gene expression under stressful conditions. We also show that differences in host gene expression occur depending on whether they are confluent or not. Our findings demonstrate the feasibility of using ribosomal profiling to interrogate the host-parasite dynamic under a variety of conditions.


Assuntos
Perfilação da Expressão Gênica , Interações Hospedeiro-Parasita/genética , Ribossomos/genética , Toxoplasma/genética , Vacúolos/parasitologia , Células Cultivadas , Fibroblastos/parasitologia , Humanos , Fases de Leitura Aberta , Proteínas de Protozoários/genética , RNA Mensageiro , Toxoplasma/fisiologia , Transcriptoma
16.
PLoS Biol ; 17(6): e3000060, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31233488

RESUMO

Apicomplexan parasites invade host cells in an active process involving their ability to move by gliding motility. While the acto-myosin system of the parasite plays a crucial role in the formation and release of attachment sites during this process, there are still open questions regarding the involvement of other mechanisms in parasite motility. In many eukaryotes, a secretory-endocytic cycle leads to the recycling of receptors (integrins), necessary to form attachment sites, regulation of surface area during motility, and generation of retrograde membrane flow. Here, we demonstrate that endocytosis operates during gliding motility in Toxoplasma gondii and appears to be crucial for the establishment of retrograde membrane flow, because inhibition of endocytosis blocks retrograde flow and motility. We demonstrate that extracellular parasites can efficiently incorporate exogenous material, such as labelled phospholipids, nanogold particles (NGPs), antibodies, and Concanavalin A (ConA). Using labelled phospholipids, we observed that the endocytic and secretory pathways of the parasite converge, and endocytosed lipids are subsequently secreted, demonstrating the operation of an endocytic-secretory cycle. Together our data consolidate previous findings, and we propose an additional model, working in parallel to the acto-myosin motor, that reconciles parasite motility with observations in other eukaryotes: an apicomplexan fountain-flow-model for parasite motility.


Assuntos
Movimento Celular/fisiologia , Endocitose/fisiologia , Toxoplasma/metabolismo , Actinas/metabolismo , Animais , Adesão Celular/fisiologia , Extensões da Superfície Celular/fisiologia , Proteínas de Membrana/metabolismo , Miosinas/metabolismo , Parasitos , Proteínas de Protozoários/metabolismo , Via Secretória/fisiologia , Toxoplasma/fisiologia
17.
Artigo em Inglês | MEDLINE | ID: mdl-31245299

RESUMO

Toxoplasma gondii is a successful protozoan parasite that cycles between definitive felid hosts and a broad range of intermediate hosts, including rodents and humans. Within intermediate hosts, this obligate intracellular parasite invades the small intestine, inducing an inflammatory response. Toxoplasma infects infiltrating immune cells, using them to spread systemically and reach tissues amenable to chronic infection. An intact immune system is necessary to control life-long chronic infection. Chronic infection is characterized by formation of parasite cysts, which are necessary for survival through the gastrointestinal tract of the next host. Thus, Toxoplasma must evade sterilizing immunity, but still rely on the host's immune response for survival and transmission. To do this, Toxoplasma exploits a central cost-benefit tradeoff in immunity: the need to escalate inflammation for pathogen clearance vs. the need to limit inflammation-induced bystander damage. What are the consequences of sustained inflammation on host biology? Many studies have focused on aspects of the immune response that directly target Toxoplasma growth and survival, commonly referred to as "resistance mechanisms." However, it is becoming clear that a parallel arm of the immune response has evolved to mitigate damage caused by the parasite directly (for example, egress-induced cell death) or bystander damage due to the inflammatory response (for example, reactive nitrogen species, degranulation). These so-called "disease tolerance" mechanisms promote tissue function and host survival without directly targeting the pathogen. Here we review changes to host metabolism, tissue structure, and immune function that point to disease tolerance mechanisms during Toxoplasma infection. We explore the impact tolerance programs have on the health of the host and parasite biology.


Assuntos
Resistência à Doença/imunologia , Interações Hospedeiro-Parasita/imunologia , Toxoplasma/imunologia , Toxoplasmose/imunologia , Animais , Interações Hospedeiro-Parasita/fisiologia , Humanos , Tolerância Imunológica , Imunidade Inata/imunologia , Inflamação , Toxoplasma/patogenicidade , Toxoplasma/fisiologia , Toxoplasmose/parasitologia
18.
mBio ; 10(2)2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31040242

RESUMO

Toxoplasma gondii is an obligate intracellular parasite that establishes a favorable environment in the host cells in which it replicates. We have previously reported that it uses MYR-dependent translocation of dense granule proteins to elicit a key set of host responses related to the cell cycle, specifically, E2F transcription factor targets, including cyclin E. We report here the identification of a novel Toxoplasma effector protein that is exported from the parasitophorous vacuole in a MYR1-dependent manner and localizes to the host's nucleus. Parasites lacking this inducer of host cyclin E (HCE1) are unable to modulate E2F transcription factor target genes and exhibit a substantial growth defect. Immunoprecipitation of HCE1 from infected host cells showed that HCE1 efficiently binds elements of the cyclin E regulatory complex, namely, DP1 and its partners E2F3 and E2F4. Expression of HCE1 in Neospora caninum, or in uninfected human foreskin fibroblasts (HFFs), showed localization of the expressed protein to the host nuclei and strong cyclin E upregulation. Thus, HCE1 is a novel effector protein that is necessary and sufficient to impact the E2F axis of transcription, resulting in co-opting of host functions to the advantage of Toxoplasma IMPORTANCE Like most Apicomplexan parasites, Toxoplasma gondii has the remarkable ability to invade and establish a replicative niche within another eukaryotic cell, in this case, any of a large number of cell types in almost any warm-blooded animals. Part of the process of establishing this niche is the export of effector proteins to co-opt host cell functions in favor of the parasite. Here we identify a novel effector protein, HCE1, that the parasites export into the nucleus of human cells, where it modulates the expression of multiple genes, including the gene encoding cyclin E, one of the most crucial proteins involved in controlling when and whether a human cell divides. We show that HCE1 works through binding to specific transcription factors, namely, E2F3, E2F4, and DP1, that normally carefully regulate these all-important pathways. This represents a new way in which these consummately efficient infectious agents co-opt the human cells that they so efficiently grow within.


Assuntos
Ciclina E/biossíntese , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Miosina Tipo I/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/fisiologia , Fatores de Virulência/metabolismo , Células Cultivadas , Fatores de Transcrição E2F/metabolismo , Fibroblastos/parasitologia , Humanos , Ligação Proteica , Transporte Proteico
19.
PLoS Pathog ; 15(5): e1007670, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31121005

RESUMO

To elicit effective invasion and egress from infected cells, obligate intracellular parasites of the phylum Apicomplexa rely on the timely and spatially controlled exocytosis of specialized secretory organelles termed the micronemes. The effector molecules and signaling events underpinning this process are intricate; however, recent advances within the field of Toxoplasma gondii research have facilitated a broader understanding as well as a more integrated view of this complex cascade of events and have unraveled the importance of phosphatidic acid (PA) as a lipid mediator at multiple steps in this process.


Assuntos
Cálcio/metabolismo , GMP Cíclico/metabolismo , Exocitose/fisiologia , Organelas/metabolismo , Ácidos Fosfatídicos/metabolismo , Toxoplasma/fisiologia , Toxoplasmose/parasitologia , Animais , Interações Hospedeiro-Parasita , Humanos , Organelas/parasitologia , Transporte Proteico , Proteínas de Protozoários/metabolismo , Transdução de Sinais
20.
Nat Microbiol ; 4(7): 1208-1220, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31036909

RESUMO

The protozoan parasite Toxoplasma gondii has co-evolved with its homeothermic hosts (humans included) strategies that drive its quasi-asymptomatic persistence in hosts, hence optimizing the chance of transmission to new hosts. Persistence, which starts with a small subset of parasites that escape host immune killing and colonize the so-called immune privileged tissues where they differentiate into a low replicating stage, is driven by the interleukin 12 (IL-12)-interferon-γ (IFN-γ) axis. Recent characterization of a family of Toxoplasma effectors that are delivered into the host cell, in which they rewire the host cell gene expression, has allowed the identification of regulators of the IL-12-IFN-γ axis, including repressors. We now report on the dense granule-resident effector, called TEEGR (Toxoplasma E2F4-associated EZH2-inducing gene regulator) that counteracts the nuclear factor-κB (NF-κB) signalling pathway. Once exported into the host cell, TEEGR ends up in the nucleus where it not only complexes with the E2F3 and E2F4 host transcription factors to induce gene expression, but also promotes shaping of a non-permissive chromatin through its capacity to switch on EZH2. Remarkably, EZH2 fosters the epigenetic silencing of a subset of NF-κB-regulated cytokines, thereby strongly contributing to the host immune equilibrium that influences the host immune response and promotes parasite persistence in mice.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , NF-kappa B/metabolismo , Proteínas de Protozoários/metabolismo , Transdução de Sinais/genética , Toxoplasma/fisiologia , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Citocinas/metabolismo , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Expressão Gênica , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Carga Parasitária , Regiões Promotoras Genéticas , Multimerização Proteica , Proteínas de Protozoários/genética , Toxoplasma/genética , Toxoplasma/imunologia , Toxoplasmose/metabolismo , Toxoplasmose/parasitologia
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