Mecanismos protetores induzidos pelo tratamento prévio com antígeno solúvel de taquizoítas (STAg) no intestino delgado de camundongos BALB/c e C57BL/6 infectados oralmente por Toxoplasma gondii

T. gondii can infect the gut mucosa by direct invasion of epithelial cells in the small intestine and these cells may respond directly to infection promoting a local immune response. C57BL/6 mice orally infected with a high parasitic load of T.gondii are highly susceptible, presenting a lethal ileitis. Recently, it was demonstrated that pretreatment with STAg protects C57BL/6 mice against intestinal pathology in oral T. gondii infection. To investigate the mechanisms induced by STAg in the small intestine in oral T.gondii infection, BALB/c and C57BL/6 mice were treated with STAg 48 hours before oral infection with 30 ME-49 cysts and sacrificed at 8 days of infection. Previous treatment with STAg were able of decrease parasitism and pathology in peripheral organs of BALB/c and C57BL/6 mice and induced a increase in amounts of goblet cells, IgA positive cells, Paneth cells and expression of cryptidin in the small intestine of both lineages of mice, moreover BALB/c mice presented higher amount of these cells comparing with C57BL/6 mice. The results suggests that STAg is able of promoting protective mechanisms in both lineages of mice, although these protection is more evidenced in BALB/c mice, and these mechanisms could be in part mediated by increase in goblet, Paneth and local secretion of IgA in the small intestine of mice orally infected with T.gondii.

Utilização de peptídeos sintéticos derivados de moléculas imunodominantes de Toxoplasma gondii para diagnóstico sorológico da toxoplasmose em ovinos e galinhas caipiras

Chapter I - The obligate intracellular parasite Toxoplasma gondii is the causative agent of toxoplasmosis, a disease that affects humans and generates economic losses in farm animals. When prevention fails disease refers to the diagnosis and subsequent treatment if the individual is diagnosed as positive. Therefore, the development of new accurate diagnostic tools for detecting T. gondii infection is a need in particular to determine the environmental source of infection which can result in more appropriate public health policies against different routes of infection and prevent potential damage that toxoplasmosis can cause when animals are infected. Chapter II - The domestic chicken (Gallus gallus domesticus) are considered epidemiological sentinels, still representing a major source of recombinant strains when predated by cats, it is common to find them with multiple infections. We evaluate the diagnostic potential of six synthetic peptides (SAG2Y, MIC1, M2AP, GRA10, ROP2 and ROP7) predicted in silico from tachyzoites immunodominant markers of T. gondii in samples from naturally infected chickens, comparing synthetic peptides with antigen total soluble (STAg). In general, our results demonstrated that reactivity rates and positivity for these peptides are similar to the STAg, and the ROP7 peptide and the combination of peptides MIC1+ROP2 have significant sensitivity, confirming them as potential diagnostic tools for the diagnosis of toxoplasmosis in chickens. Chapter III - Sheep (Ovis aries) are commonly infected with Toxoplasma gondii due to his eating habits. Infection in pregnant sheep can have serious consequences such as embryonic death, fetal resorption, mummification, and neonatal death. One concern regarding the infection in these animals is that the meat can be a source of contamination to humans and other carnivores. Therefore perform accurate diagnosis in these animals is of fundamental importance. In the present study we evaluated the potential of new synthetic peptides as a diagnostic tool. Synthetic peptides (SAG2Y, SRS52A, MIC14, GRA4, GRA10 and GRA15) were predicted in silico from immunodominant proteins of T. gondii. We determine the levels of IgG antibodies using sera obtained from two farms in the city of Uberlândia. Analyzing the results together, the peptide combination GRA10+GRA15 (Accuracy = 0,82) showed better characteristics compared with the other mixtures. This preparation could be better analyzed with an antigenic mixture potential use in the diagnosis of toxoplasmosis in sheep and other species.

Enrofloxacina e toltrazuril reduzem a infecção por Toxoplasma gondii em células trofoblásticas humanas e em explantes de vilos placentários humanos de terceiro trimestre

The classical treatment for congenital toxoplasmosis is based on combination of sulfadiazine and pyrimethamine plus folinic acid. Due to teratogenic effects and bone marrow suppression caused by pyrimethamine, the establishment of new therapeutic targets is indispensable to minimize the undesirable effects and improve the control of the infection. Previous studies demonstrated that enrofloxacin and toltrazuril were able to control the infection triggered by Neospora caninum and Toxoplasma gondii. Therefore, the aim of this present study was evaluate the efficacy of enrofloxacin and toltrazuril in the control of T. gondii proliferation in human trophoblast cells (BeWo lineage) and in human villous explants from third trimester. BeWo cells and villous were treated with several concentrations of enrofloxacin, toltrazuril, sulfadiazine, pyrimethamine or association (sulfadiazine + pyrimethamine) in other to verify their viability by MTT or LDH assay, respectively. Next, BeWo cells were infected with T. gondii RH (2F1 clone) or ME49 strain, whereas villous were infected only with RH strain (2F1 clone), after, both cells and villous were treated or not with the same antibiotics and analyzed to T. gondii intracellular proliferation by beta-galactosidase assay (for RH strain) or blue toluidine staining (for ME49 strain). ELISA was performed in the supernatant to evaluate the cytokine profile. Enrofloxacin and toltrazuril did not change strongly the viability in cells and villous. Furthermore, the drugs decreased the parasite intracellular proliferation regardless T. gondii strain in BeWo cells and villous explants when compared to untreated and infected conditions. In BeWo cells infected by RH, enrofloxacin induced high levels of IL-6 low levels of MIF, while both cytokines were upregulated by enrofloxacin and toltrazuril in BeWo cells infected by ME49 strain. Additionally, in villous explantes, enrofloxacin induced high MIF production. Thus, enrofloxacin and toltrazuril were able to control the parasitism in BeWo cells and villous explants, and probably it occurs by modulation of immune response in these cells or tissues and direct action on parasite, but future experiments are necessary to verify this hypothesis.

Toxoplasma gondii seropositive rates of animals living on Svalbard

Samples (blood or tissue fluid) from 594 arctic foxes (Alopex lagopus), 390 Svalbard reindeer (Rangifer tarandus platyrhynchus), 361 sibling voles (Microtus rossiaemeridionalis), 17 walruses (Odobenus rosmarus), 149 barnacle geese (Branta leucopsis), 58 kittiwakes (Rissa tridactyla), and 27 glaucous gulls (Larus hyperboreus) from Svalbard and nearby waters were assayed for antibodies against Toxoplasma gondii using a direct agglutination test. The proportion of seropositive animals was 43% in arctic foxes, 7% in barnacle geese, and 6% (1 of 17) in walruses. There were no seropositive Svalbard reindeer, sibling voles, glaucous gulls, or kittiwakes. The prevalence in the arctic fox was relatively high compared to previous reports from canid populations. There are no wild felids in Svalbard and domestic cats are prohibited, and the absence of antibodies against T gondii among the herbivorous Svalbard reindeer and voles indicates that transmission of the parasite by oocysts is not likely to be an important mechanism in the Svalbard ecosystem. Our results suggest that migratory birds, such as the barnacle goose, may be the most important vectors bringing the parasite to Svalbard. In addition to transmission through infected prey and carrion, the age-seroprevalence profile in the fox population suggests that their infection levels are enhanced by vertical transmission.

Ocorrência da infecção por Toxoplasma gondii e avaliação da imunização em caprinos do sertão do cabugi, Rio Grande do Norte

Toxoplasmosis is one zoonosis caused by Toxoplasma gondii protozoan. Goats, amongst the production animals, are one of the species most susceptible to this parasite, being one them main involved agents in ovine and goat abortions, determining great economic losses and implications for public health, since the presence it parasite in the products of goat origin, consist in one of the main sources of infection for the man. In this study 244 blood samples in 8 farms situated in 4 cities from the Sertão do Cabugi region, Rio Grande do Norte State, northeast of Brazil and, tested by ELISA assay. The results had shown a prevalence of 47.13% for anti- T. gondii antibodies and a significant association between positivity and variable evaluated as age, locality and property. The IgG avidity assay evaluated in 115 positive samples was carried to discriminate acute and chronic infection. Twelve samples (10.4%) had presented antibodies of low avidity while 103 (89.6%) presented high avidity antibodies; indicating that most of the animals was precocious exposure to the parasite. Significant difference was verified only for the variable sex. We also evaluate the capacity of recombinant adenoviruses codifying SAG1, SAG2, SAG3 and CMV in inducing activation of specific immune response in goat. These 109 animals received 109 pfu of the AdSAG1, AdSAG2, AdSAG3, AdCMV or PBS in vaccine protocol with 3 immunizations. Serum samples of the each animal, before and after mmunization, had been submitted to the ELISA. The results demonstrate that the immunizations had induced the production of IgG antibodies specific against T. gondii proteins

Serological evidence of Toxoplasma gondii in hunted wild boar from Portugal

Toxoplasmosis is an important parasitic zoonosis with a worldwide distribution, being the parasitic disease with the highest occurrence in Europe. Wild boar has an important role in the epidemiological cycle of Toxoplasma gondii as an intermediate host, that can potentially infect humans when the meat is consumed raw or undercooked. The purpose of this work was to determine the presence of antibodies to T. gondii in serum of hunted wild boar. During the hunting season 2011/2012, sera samples were collected from 97 wild boar and tested for IgG antibodies to T. gondii, using the modified agglutination test. Twenty out of the 97 wild boar (20.6%) were seropositive for T. gondii IgG antibodies. Multivariate logistic regression analysis showed that males and older animals were associated with T. gondii seropositivity. These results show that T. gondii has an important presence in wild boar population from Portugal, suggesting a potential zoonotic risk for humans when wild boar meat or meat products are consumed raw or undercooked.

Toxoplasma gondii Infection in Hunted Wild Boars (Sus scrofa): Heart Meat Juice as an Alternative Sample to Serum for the Detection of Antibodies

Toxoplasmosis is a global zoonosis caused by the protozoan parasite Toxoplasma gondii. Detection of antibodies to T. gondii in serum samples from hunted animals may represent a key step for public health protection. It is also important to assess the circulation of this parasite in wild boar population. However, in hunted animals, collection of blood is not feasible and meat juice may represent an alternative sample. The purpose of the present study was to evaluate heart meat juice of hunted wild boars as an alternative sample for post-mortem detection of antibodies to T. gondii by modified agglutination test (MAT). The agreement beyond chance between results from meat juice assessed with Cohen’s kappa coefficient revealed that the 1:20 meat juice dilution provided the highest agreement. McNemars’s test further revealed 1:10 as the most suitable meat juice dilution, as the proportion of positive paired samples (serum and meat juice from the same animal) did not differ at this dilution. All together, these results suggest a reasonable accuracy of heart meat juice to detect antibodies to T. gondii by MAT and support it as an alternative sample in post-mortem analysis in hunted wild boars.

Epidemiological studies on toxoplasma gondii infection in a one health perspective

The objective of the present Ph.D. thesis was to investigate with a One Health approach the epidemiological patterns of T. gondii infection in Italy, to better understand the transmission dynamics of the parasite, following different research lines. The results of a retrospective analysis in animals and human showed the widespread distribution of T. gondii in the study area, with specific antibodies found in various animal species and human populations, indicating its constant presence across diverse environments. The environment plays a significant role in T. gondii's epidemiology. Migratory aquatic birds, rodents, wolves, and wild boars were investigated as sentinels of their spread, highlighting the potential transmission across geographic areas and infection risks for wildlife in natural settings. The study also provided insights into seroprevalence in wolves. Dogs, subjected to serological investigations exhibited risk factors for T. gondii infection, such as cohabitation with cats, coprophagy behaviours, and continuous outdoor. Correlation between serological evidence of exposure to T. gondii and pathological anxiety in large-size dogs was observed, and the consumption of raw meat was associated with a higher risk of infection in these animals. Results of the investigations conducted in this thesis, demonstrate the dynamic nature of T. gondii infection in cattle, characterized by new infections and declining antibody levels over the production cycle. The study also describes a co-infection between T. gondii and Sarcocystis hominis in bovine eosinophilic myositis. In the final part of the Thesis, a comprehensive genotyping of T. gondii in Italy reveals the predominance of Type II strains, particularly in cases of ovine abortion and fatal toxoplasmosis among captive Lemur catta. This approach enhances our understanding of the parasite's genetic diversity and transmission patterns, vital for effective management of its impact on human and animal health in Italy.

Utilização, em politransfundidos, da pesquisa de anticorpos igm anti-trypanosoma cruzi e anti-toxoplasma gondii para detectar infecções pós-transfusionais recentes

Consideram os Autores que a pesquisa de anticorpos IgM no soro é tática capaz de revelar recentes infecções pós-transfusionais. Por isso, decidiram usar esse tipo de mensuração relativamente a grupo constituído por 101 politrans-fundidos, tendo abordado especificamente as aquisições de doença de Chagas e toxoplasmose. Através da investigação que realizaram, só em duas oportunidades encontraram anticorpos IgM anti-Trypanosoma cruzi ou anti-Toxoplasma gondii e, portanto, não evidenciaram expressivo panorama tradutor de processos há pouco tempo contraídos, como ainda, por meio de anticorpos IgG não identificaram números expressivos de pessoas com essas protozooses. No entanto, detectaram a expressiva taxa de 4,9% de casos de doença de Chagas muito provavelmente decorrentes da hemoterapia. A despeito da relevância não acentuada dos resultados que obtiveram, julgaram os Autores ser válido estimular a efetivação de outros estudos congêneres e correlatos, aptos a contribuir para aqui-latamento de riscos pertinentes à prática hemoterápica.

Reinfecção pelo Toxoplasma gondii Nicolle & Manceaux, 1909 em camundongos e gatos: estudo experimental

A reinfecção pelo Toxoplasma gondii foi estudada, experimentalmente, em 27 camundongos albinos e 25 gatos domésticos. A infecção e a reinfecção foram comprovadas por sinais clínicos, anticorpos sangüíneos à reação de Sabin & Feldman > 1/4, recuperação do parasita e, nos gatos, também pela eliminação de oocistos. Nos camundongos a mortalidade foi de 56%. A primo-infecção por cepa de baixa virulência ou a infecção por cepa mais virulenta, atenuada pela sulfamonometoxina, deram proteção à reinfecção por cepa mais virulenta, mortal em poucos dias. Esta proteção foi demonstrada pela sobrevivência de camundongos e menor mortalidade, 12,5'%. Nos gatos a mortalidade foi de 28%, A resposta sorológica à primo-infecção ocorreu em todos. Após a reinfecção houve elevação de títulos em 64% dos gatos, permanência em 32% e queda em 4%. A eliminação de oocistos foi observada em 60% dos gatos e só após inoculação de cistos, por via digestiva. A reeliminação após reinfecção, ocorreu em 25% dos gatos. Não foi observada eliminação após a 3.ª inoculação. Em 88,2% dos gatos houve recuperação do parasita em camundongo, mais freqüente nos linfonodos mesentéricos e no intestino delgado, persistindo neste cerca de 14 meses. Foi constatada certa relação entre o bom estado físico dos gatos e a resposta sorológica, a eliminação e a reeliminação de oocistos. Apesar da persistência de anticorpos sangüíneos a reinfecção experimental dos gatos foi observada, porém a reeliminação de oocistos foi pouco freqüente e em pequena quantidade.

Pesquisa de anticorpos IgM anti-Toxoplasma gondii por meio de técnica imunoenzimática reversa

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

Toxoplasmose aguda: avaliação da técnica de imunoensaio em camada delgada para a detecção de anticorpos IgM, anti-Toxoplasma gondii

Um método de reação antígeno-anticorpo denominado IMUNOENSAIO EM CAMADA DELGADA (ICD) foi padronizado e avaliado para o imunodiagnóstico da toxoplasmose aguda, através da detecção de anticorpos IgM, anti-Toxoplasma gondii (ICD-IgM). Um total de 300 amostras de soros, tendo ou não o perfil sorológico da toxoplasmose aguda, foi estudado pelo ICD-IgM e os resultados analisados quanto aos aspectos qualitativos e quantitativos em relação aos testes convencionais de imunofluorecência indireta para a detecção de IgM, (IFI-IgM) e de hemaglutinação pré e após tratamento com 2-mercaptoetanol (HA-2ME). Foram calculados os coeficientes de correlação entre os resultados fornecidos pelo ICD-IgM e as técnicas convencionais, de acordo com as concentrações do antígeno, assim como, foi verificada a influência dessas concentrações antigênicas nos índices relativos de sensibilidade e de especificidade. O estudo estatístico mostrou reprodutibilidades significativas dos resultados inter e intra testes, assim como no reaproveitamento do antígeno por 10 vezes. Os resultados de ICD-IgM mostraram que as concentrações antigênicas ideais foram de 70 a 100µg/ml, para o ensaio em questão, para se obter máximas sensibilidade e especificidade. Portanto, ICD-IgM, por fornecer resultados tão sensíveis e específicos quanto aqueles dos testes convencionais referidos, poderá ser empregado para fins diagnósticos.

Avidez de anticorpos IgG específicos como marcadores de infecção primária recente pelo Toxoplasma gondii

A caracterização de infecção primária recente pelo Toxoplasma gondii se apoia principalmente na presença, no soro, de anticorpos específicos IgM. Para fins diagnósticos de toxoplasmose aguda, ou de contágio recente, a possibilidade de outros marcadores é altamente desejável. Um marcador de infecção recente atualmente referido é a baixa afinidade ou avidez de anticorpos específicos IgG. Para avaliação do novo marcador, titularam-se os soros contra poliantígenos do T. gondii pelo teste imunoenzimático (ELISA), antes e após tratamento dos complexos antígeno-anticorpo formados, com solução de ureia 6 M como agente dissociante. O deslocamento de anticorpos de baixa avidez foi indicado por uma queda de títulos, calculada em porcentagem em relação aos títulos iniciais. Foram estudados 69 soros, 23 de cada um dos 3 perfis sorológicos sucessivos, observados na infecção, e que a caracterizam respectivamente como recente, em fase de transição e crônica. Os perfis foram determinados segundo os resultados de uma bateria de testes, incluindo os de imunofluorescência IgG e IgM, de captura de anticorpos IgM e de hemaglutinação. Para os soros de infecção crônica a queda observada foi de 3% ± 3%, de 34% ± 12% para toxoplasmose recente e de 12% ± 9% para a fase de transição. Conclue-se que a determinação da avidez de anticorpos IgG pode ser utilizada como marcador de infecção primária recente pelo T. gondii.