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.

Pathogenicity and phenotypic sulfadiazine resistance of Toxoplasma gondii isolates obtained from livestock in northeastern Brazil

Toxoplasma gondii is the causative protozoan agent of toxoplasmosis, which is a common infection that is widely distributed worldwide. Studies revealed stronger clonal strains in North America and Europe and genetic diversity in South American strains. Our study aimed to differentiate the pathogenicity and sulfadiazine resistance of three T. gondii isolates obtained from livestock intended for human consumption. The cytopathic effects of the T. gondii isolates were evaluated. The pathogenicity was determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using a CS3 marker and in a rodent model in vivo. Phenotypic sulfadiazine resistance was measured using a kinetic curve of drug activity in Swiss mice. IgM and IgG were measured by ELISA, and the dihydropteroate synthase (DHPS) gene sequence was analysed. The cytopathic effects and the PCR-RFLP profiles from chickens indicated a different infection source. The Ck3 isolate displayed more cytopathic effects in vitro than the Ck2 and ME49 strains. Additionally, the Ck2 isolate induced a differential humoral immune response compared to ME49. The Ck3 and Pg1 isolates, but not the Ck2 isolate, showed sulfadiazine resistance in the sensitivity assay. We did not find any DHPS gene polymorphisms in the mouse samples. These atypical pathogenicity and sulfadiazine resistance profiles were not previously reported and served as a warning to local health authorities.

PREVALENCE, ISOLATION, AND GENETIC CHARACTERIZATION OF TOXOPLASMA GONDII IN CHICKEN

Toxoplasma gondii (T. gondii) is one of the most successful parasites in the world because of its capability of infecting all warm-blooded animals. It has been reported that up to one third of the world population is infected with this parasite. Chickens are recognized as good indicators of the environmental T. gondii oocysts contamination because they obtain food from the ground. Thus, the prevalence of T. gondii in chicken provides more insight related to public health concern from T. gondii. Previous studies have shown a high isolation rate from free-range chickens raised in the United States. The objectives of this study were to evaluate the microbial safety and infection of T. gondii in free-range chickens available at the grocery stores and farms for the consumers to purchase and genotype T. gondii isolates. Chicken hearts were obtained from the local markets and also from the farms raising free- range chickens. Heart juice was obtained from cavities of each heart. Modified agglutination test (MAT) for detection of IgG antibodies was conducted with those heart juice samples with titer of 1:5, 1:25, and 1: 100. Each seropositive heart was pepsin digested and bioassayed into a group of two mice. Six weeks post inoculation (p.i.) mice were bled and euthanized to examine the infection of T. gondii. In addition, multiplex multilocus nested PCR-RFLP was performed to genetically characterize T. gondii isolates with eleven PCR-RFLP markers including SAG1, SAG2, altSAT2, SAG3, BTUB, GRA6, c22-8, c29-a, L358, PK1, and Apico. One hundred fifty from a total of 997 samples (15.0%) were found seropositive for T. gondii. No viable T. gondii was isolated from chicken hearts that were sampled. A total of four genotypes were identified, including one new genotype and three previously identified genotypes. The results suggest that T. gondii oocysts could present in the environment and infect the food animals. T. gondii prevalence in chicken hearts could reflect the environmental contamination of T. gondii and prevalence information can be used to manage T. gondii infection risk.

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

Avaliação da ocorrência de anticorpos anti-Toxoplasma gondii, em soros de caprinos do estado de São Paulo, e associação com variáveis epidemiológicas, problemas reprodutivos e riscos à saúde pública

Objetivando avaliar a ocorrência de anticorpos anti-Toxoplasma gondii, em caprinos leiteiros do estado de São Paulo, e verificar possíveis associações com idade, sexo, presença de gatos, problemas reprodutivos e potenciais riscos à saúde pública, foram considerados soros de 923 caprinos, de ambos os sexos e idade acima de três meses, provenientes de 17 propriedades de diferentes municípios. Para o diagnóstico, utilizou-se a reação de imunofluorescência indireta (RIFIe"16) e, também, um inquérito sobre saúde, a fim de se coletarem informações epidemiológicas e de esfera reprodutiva de todos os capris. Os resultados foram discutidos no nível de 5% de significância. Do total das 17 propriedades, foram diagnosticados 15 focos de T. gondii, com positividade entre 2,70% e 81,25%. Não foram verificadas associações entre freqüência de soropositividade e sexo dos animais nem ocorrência de falhas reprodutivas, nos capris. Constatou-se influência positiva na taxa de anticorpos anti-T. gondii pelo aumento da idade dos caprinos e presença de gatos, nos capris. Além de a enfermidade encontrar-se amplamente difundida no estado de São Paulo, o risco eminente de transmissão de T. gondii à saúde pública também deve ser considerado, uma vez que se encontraram focos onde se comercializavam produtos "in natura", como leite e carne.

Myeloid differentiation factor 88 is required for resistance to Neospora caninum infection

Neospora caninum is an intracellular parasite that causes major economic impact on cattle raising farms, and infects a wide range of warm-blooded hosts worldwide. Innate immune mechanisms that lead to protection against this parasite are still unknown. In order to investigate whether myeloid differentiation factor 88 (MyD88) is required for resistance against N. caninum, genetically deficient mice (MyD88(-/-)) and wild type littermates were infected with live tachyzoites and the resistance to infection was evaluated. We found that sub-lethal tachyzoite doses induced acute mortality of MyD88(-/-) mice, which succumbed to infection due to uncontrolled parasite replication. Higher parasitism in MyD88(-/-) mice was associated with the lack of IL-12 production by dendritic cells, delayed IFN-gamma responses by NKT, CD4(+) and CD8(+) T lymphocytes, and production of high levels of IL-10. MyD88(-/-) mice replenished with IL-12 and IFN-gamma abolished susceptibility as the animals survived throughout the experimental period. We conclude that protective IFN-gamma-mediated immunity to N. caninum is dependent on initial MyD88 signaling, in a mechanism triggered by production of IL-12 by dendritic cells. Further knowledge on Toll-like receptor recognition of N. caninum antigens is encouraged, since it could generate new prophylactic and therapeutic tools to control parasite burden.

Toxoplasma gondii Antibodies in Wild White-Lipped Peccary (Tayassu pecari) From Peru

In the Peruvian Amazon, the white-lipped peccary (Tayassu pecan) is a desirable game species and is important for the local rural economy. Blood samples from 101 white-lipped peccaries from Peru were collected from 3 different conservation areas located in the municipalities of Manu and Tambopata, southeastern region of the Peruvian Amazon. Antibodies were assayed using the modified agglutination test (MAT, cut of value of 25). Antibodies to Toxoplasma gondii were found in 89.1% (90 of 101) of animals, with titers of 1:25 in 9, 1:50 in 25, 1:100 in 20, 1:200 in 14, 1:400 in 12, 1:800 in 9, and 1:3,200 in 1; 87.7% and 89.2% of males and females, respectively, tested positively, and no association (P >= 0.05) with gender and occurrence of antibodies was observed.

Transplacental transmission of Toxoplasma gondii in reinfected pregnant female canines

Twelve pregnant female canines, naturally infected with Toxoplasma gondii, were reinfected with T. gondii: three (GI) received tachyzoites subcutaneously (1.0 x 107), three (GII) were orally inoculated with oocysts (1.5 x 104), and six (GIII) were kept as a nonreinfected control group. All the reinfected female canines (GI and GII) miscarried or presented fetal death, while only one GIII female presented a stillborn in a litter of four pups (P < 0.01). Fever, lymphoadenopathy, miscarriage, and fetal death were the main clinical alterations observed. The highest serological titers detected through the indirect fluorescence antibody test (IFAT) were 1,024 (GI) and 4,096 (GII). In group III, the titers ranged between 64 and 256. By bioassays in mice, T. gondii was isolated in 17 organs of the reinfected adult canines, in 11 of the control group, and in 20 of the neonates. Positive immunostaining of cysts and/or tachyzoites were observed in 26 canine tissues (14 from GI and GII and ten from GIII). The agent was detected by immunohistochemistry in the encephalon of a neonate and in the spinal cord of a stillborn, thus, confirming that T. gondii infected canine fetuses, provoking miscarriages, even in bitches that presented primoinfection.

Population structure and mouse-virulence of Toxoplasma gondii in Brazil

Recent studies found that isolates of Toxoplasma gondii from Brazil were biologically and genetically different from those in North America and Europe. However, to date only a small number of isolates have been analysed from different animal hosts in Brazil. In the present study DNA samples of 46 T. gondii isolates from cats in 11 counties in Sao Paulo state, Brazil were genetically characterised using 10 PCR restriction fragment length polymorphism markers including SAG1, SAG2, SAG3, STUB, GRA6, c22-8, c29-2, L358, PKI and Apico. An additional marker, CS3, that locates on chromosome VIIa and has previously been shown to be linked to acute virulence of T. gondii was also used to determine its association to virulence in mice. Genotyping of these 46 isolates revealed a high genetic diversity with 20 genotypes but no clonal Type I, II or III lineage was found. Two of the 46 isolates showed mixed infections. Combining genotyping data in this study with recent reported results from chickens, dogs and cats in Brazil (total 125 isolates) identified 48 genotypes and 26 of these genotypes had single isolates. Four of the 48 genotypes with multiple isolates identified from different hosts and locations are considered the common clonal lineages in Brazil. These lineages are designated as Types BrI, BrII, BrIII and BrIV. These results indicate that the T. gondii population in Brazil is highly diverse with a few successful clonal lineages expanded into wide geographical areas. In contrast to North America and Europe, where the Type II clonal lineage is overwhelmingly predominant, no Type II strain was identified from the 125 Brazil isolates. Analysis of mortality rates in infected mice indicates that Type BrI is highly virulent, Type BrIII is non-virulent, whilst Type BrII and BrIV lineages are intermediately virulent. In addition, allele types at the CS3 locus are strongly linked to mouse-virulence of the parasite. Thus, T. gondii has an epidemic population structure in Brazil and the major lineages have different biological traits. (C) 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Genotyping of Toxoplasma gondii isolates from free range chickens in the Pantanal area of Brazil

The aim of this paper was to genetically characterize Toxoplasma gondii isolates from free range chickens in regions of Brazilian territory in the state of Mato Grosso do Sul (MS) where T. gondii strains have never been studied. In total, T. gondii isolates from 22 free range chickens were included in this study. Fifty chickens from Eldorado, thirty from Rio Verde and ten from Aquidauana were sampled between January and April 2007. In relation to the genetic diversity of T. gondii isolates from chickens in MS, the magnitude of the diversity in the isolates sampled in this study was comparable to the overall diversity in a composite data set. These 22 isolates in MS revealed 11 genotypes, whereas the 321 isolates ever genotyped in Brazil have revealed 95 genotypes. The values of Simpson`s Diversity Index for the whole population of T. gondii isolates in Brazil, the whole population of T. gondii isolates from chickens in Brazil and the population surveyed in this study were 0.97, 0.95 and 0.90, respectively. Seven of the 11 genotypes revealed from chicken isolates from MS are newly described genotypes and six of them each have a single isolate. In conclusion, the results obtained from isolates in MS corroborate previous studies on T. gondii isolates in Brazil, thus confirming their diversity and atypicality. Nonetheless, the applicability of PCR-RFLP markers for epidemiological inferences remains controversial. (c) 2011 Elsevier B.V. All rights reserved.