Prevalence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in sheep from Mossoro, Rio Grande do Norte, Brazil

Toxoplasma gondii is an obligate intracellular parasite with a variety of hosts, responsible for reproductive problems and economic losses in sheep flocks. Neospora caninum was recently identified and its clinical presentation in sheep is similar to that of toxoplasmosis, which can cause repeated abortions, though less frequently in this species. In order to confirm the prevalence of these agents in the city of Mossoro, Rio Grande do Norte, Brazil, 409 serum samples from adult sheep (364 females and 45 males) were tested by the indirect immunofluorescence antibody test, using cut-off point at a dilution of 1:64 and 1:50 for T. gondii and N. caninum, respectively. From the 35 properties examined, 23 (65.7%)had at least one seropositive animal for T gondii and six (17.1%) for N. caninum. The prevalence of seropositive animals for T. gondii was 20.7% and for N. caninum 1.8%. There was no association between the presence of the agent`s antibody and gender, reports of reproductive problems and presence of dogs and/or cats in the properties. T. gondii is well distributed and N. caninum has low prevalence in sheep and in the properties of the studied region. (C) 2008 Elsevier B.V. All rights reserved.

Evaluation of IFA, MAT, ELISAs and immunoblotting for the detection of anti-Toxoplasma gondii antibodies in paired serum and aqueous humour samples from experimentally infected pigs

The study evaluated the efficiency of diagnostic laboratory methods to detect anti-Toxoplasma gondii antibodies in paired serum and aqueous humour samples from experimentally infected pigs. 18-mixed breed pigs were used during the experiment; these were divided into two groups, G I (infected group, n = 10) and G2 (uninfected group, n = 8). Infection was performed with 4 x 10(4) VEG strain oocysts at day 0 by the oral route in G1 animals. All pigs were euthanized at day 60, when retina, aqueous humour, and blood samples were collected. Anti-T gondii antibody levels were assessed in serum (s) and aqueous humour (ah) by indirect immunofluorescence assay (IFA), modified agglutination test (MAT), m-ELISA (using crude membranes from T gondii tachyzoites as antigen) and r-ELISA (using rhoptries from T gondii tachyzoites as antigen). Polymerase chain reactions (PCR) of samples from the retina were performed by using Tox4 and Tox5 primers. Antibody titers of G1 animals ranged from 128 to 1024 and from 16 to 256 in serum and aqueous humour, respectively. There were differences in the correlation coefficients between IFA(s) x IFA (ah) (r = 0.62, P = 0.05), MAT(s) x MAT (ah) (r = 0.97, P < 0.0001); however, there was no significant difference between r-ELISA(s) x r-ELISA (ah) (r = 0. 14, P = 0.7). Antibodies present in serum and aqueous humour recognized similar antigens. Samples of retina were positive by PCR in 30% (3/10) of infected pigs. G2 animals remained without antibody levels and were PCR negative throughout the experiment. These results suggest that the use of a combination of tests and immunoblotting for paired aqueous humour and serum samples could improve the sensitivity and specificity for the diagnosis of ocular toxoplasmosis. (c) 2007 Elsevier Ltd. All rights reserved.

Genetic diversity among Toxoplasma gondii isolates of small ruminants from Brazil: Novel genotypes revealed

Recent studies indicated that Toxoplasma gondii isolates of many domestic animal hosts from Brazil are genetically and biologically different from those in USA and Europe. Despite of high pathogenicity of this parasite to small ruminants, the epidemiology and genetic diversity of T. gondii in these animals are not well understood in Brazil. In this study, a total of 28 T. gondii samples (16 isolates from sheep in Sao Paulo state, and 12 isolates from goats in the states of Sao Paulo and Rio Grande do Norte) were genotyped using genetic markers SAG1, SAG2, SAG3, BTUB, CRAG, c22-8, c29-2, L358, PK1, Apico and CS3. Eleven genotypes were identified from these T. gondii isolates. Eight isolates (4 from sheep and 4 from goats) were grouped into the common clonal type Brl lineage. One sheep isolate was grouped to the type BrIII lineage. Five isolates grouped to three previously identified genotypes in Brazil, and 13 isolates grouped to six novel genotypes. Mixed genotype was found in one isolate from goat in Sao Paulo. No classical clonal Type I. II or III isolates were found, confirming previous reports that these clonal lineages are rare in Brazil. The allele types at the CS3 locus are strongly linked to mouse virulence of the parasite. The results of this study indicate that even though a large number of T. gondii genotypes have been identified from a variety of animal hosts in Brazil, high percentage of new genotypes are continuously identified from different animal species, suggesting extremely high diversity of T. gondii in the population. (C) 2010 Elsevier B.V. All rights reserved.

Analysis of marine bivalve shellfish from the fish market in Santos city, Sao Paulo state, Brazil, for Toxoplasma gondii

The aim of this study was to determine if Toxoplasma gondii are present in oysters (Crassostrea rhizophorae) and mussels (Mytella guyanensis) under natural conditions using a bioassay in mice and molecular detection methods. We first compared two standard protocols for DNA extraction, phenol-chloroform (PC) and guanidine-thiocyanate (GT), for both molluscs. A total of 300 oysters and 300 mussels were then acquired from the fish market in Santos city, Sao Paulo state, Brazil, between March and August of 2008 and divided into 60 groups of 5 oysters and 20 groups of 15 mussels. To isolate the parasite, five mice were orally inoculated with sieved tissue homogenates from each group of oysters or mussels. For molecular detection of T. gondii, DNA from mussels was extracted using the PC method and DNA from oysters was extracted using the GT method. A nested-PCR (Polymerase Chain Reaction) based on the amplification of a 155 bp fragment from the B1 gene of T. gondii was then performed. Eleven PCR-RFLP (Restriction Fragment Length Polymorphism) markers, SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, CS3 and Apico, were used to genotype positive samples. There was no isolation of the parasite by bioassay in mice. T. gondii was not detected in any of the groups of mussels by nested-PCR. DNA of T. gondii was apparently detected by nested-PCR in 2 groups of oysters (3.3%). Genotyping of these two positive samples was not successful. The results suggest that oysters of the species C. rhizophorae, the most common species from the coast of Sao Paulo, can filter and retain T. gondii oocysts from the marine environment. Ingestion of raw oysters as a potential transmission source of T. gondii to humans and marine mammals should be further investigated. (C) 2010 Elsevier B.V. All rights reserved.

Macrophage migration inhibitory factor is up-regulated in human first-trimester placenta stimulated by soluble antigen of Toxoplasma gondii, resulting in increased monocyte adhesion on villous explants

Considering the potential role of macrophage migration inhibitory factor (MIF) in the inflammation process in placenta when infected by pathogens, we investigated the production of this cytokine in chorionic villous explants obtained from human first-trimester placentas stimulated with soluble antigen from Toxoplasma gondii (STAg). Parallel cultures were performed with villous explants stimulated with STAB, interferon-gamma (IFN-gamma), or STAB plus IFN-gamma. To assess the role of placental MIF on monocyte adhesiveness to human trophoblast, explants were co-cultured with human myelomonocytic THP-1 cells in the presence or absence of supernatant from cultures treated with STAB (SPN), SPN plus anti-MIF antibodies, or recombinant MIF. A significantly higher concentration of MIF was produced and secreted by villous explants treated with STAB or STAB plus IFN-gamma after 24-hour culture. Addition of SPN or recombinant MIF was able to increase THP-1 adhesion, which was inhibited after treatment with anti-MIF antibodies. This phenomenon was associated with intercellular adhesion molecule expression by villous explants. Considering that the processes leading to vertical dissemination of T. gondii remain widely unknown, our results demonstrate that MIF production by human first-trimester placenta is up-regulated by parasite antigen and may play an essential role as an autocrine/paracrine mediator in placental infection by T. gondii.

Toxoplasma gondii in the peripheral blood of patients with acute and chronic toxoplasmosis

Background and aims Toxoplasmic retinochoroiditis may recur months or years after the primary infection. Rupture of dormant cysts in the retina is the accepted hypothesis to explain recurrence. Here, the authors present evidence supporting the presence of Toxoplasma gondii in the peripheral blood of immunocompetent patients. Methods Direct observation by light microscopy and by immunofluorescence assay was performed, and results were confirmed by PCR amplification of parasite DNA. Results The authors studied 20 patients from Erechim, Brazil, including acute infected patients, patients with recurrent active toxoplasmic retinochoroiditis, patients with old toxoplasmic retinal scars, and patients with circulating IgG antibodies against T gondii and absence of ocular lesions. Blood samples were analysed, and T gondii was found in the blood of acutely and chronically infected patients regardless of toxoplasmic retinochoroiditis. Conclusions The results indicate that the parasite may circulate in the blood of immunocompetent individuals and that parasitaemia could be associated with the reactivation of the ocular disease.

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.

Isolation and genetic characterisation of Toxoplasma gondii from a red-handed howler monkey (Alouatta belzebul), a jaguarundi (Puma yagouaroundi), and a black-eared opossum (Didelphis aurita) from Brazil

Toxoplasma gondii isolates are highly diverse in domestic animals from Brazil. However, little is known about the genetics of this parasite from wild mammals in the same region. Reveal genetic similarity or difference of T. gondii among different animal populations is necessary for us to understand transmission of this parasite. Here we reported isolation and genetic characterisation of three T. gondii isolates from wild animals in Brazil. The parasite was isolated by bioassay in mice from tissues of a young male red handed howler monkey (Alouatta belzebul), an adult male jaguarundi (Puma yagouaroundi), and an adult female black-eared opossum (Didelphis aurita). The monkey and the jaguarundi had inhabited the Zoo of Parque Estadual Dois Irmaos, Pernambuco State, Northeastern Brazil, for 1 year and 8 years, respectively. The wild black-eared opossum was captured in Sao Paulo State, Southeastern Brazil, and euthanised for this study because it was seropositive for T. gondii (titre 1:100 by the modified agglutination test, MAT). Ten PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) markers, SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico, were used to genotype the isolates. T. gondii was isolated from the brain and heart homogenate of the monkey, the muscle homogenate of the jaguarundi, and the heart homogenate of the black-eared opossum. This was the first isolation of T. gondii from a neotropical fetid from Brazil. The isolate from the monkey (TgRhHmBr1) was not virulent in mice, whereas the isolates from the jaguarundi (TgJagBr1) and the black-eared opossum (TgOpBr1) were virulent in mice. The genotype of the isolate from the monkey has been identified in isolates from a goat and ten chickens in the same region of Brazil, suggesting that it may be a common lineage circulating in this region. The genotypes of the isolates from the jaguarundi and the black-eared opossum have not been previously reported. Although there are already 88 genotypes identified from a variety of animal hosts in Brazil, new genotypes are continuously being identified from different animal species, indicating an extremely high diversity of T. gondii in the population. (C) 2010 Elsevier B.V. All rights reserved.