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.

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.

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.

Experimental infection of pregnant queens with two major Brazilian clonal lineages of Toxoplasma gondii

Toxoplasma gondii isolates from Brazil are biologically and genetically different from European and North America isolates. Recently, four genotypes were considered the common clonal lineages in Brazil and were designated as types BrI, BrII, BrIII, and BrIV. The pathogenicity of two major Brazilian lineages was investigated after oral inoculation of queens in the middle third of their pregnancies with T. gondii cysts. Twelve pregnant queens without T. gondii antibodies were distributed in group A (infected with a type BrI isolate); group 2 (infected with type BrIII isolate), and group 3 (non-infected control). Infection with type BrI isolate caused toxoplasmosis manifestations and abortion from one litter. Toxoplasmosis manifestations besides premature stillbirth of one litter were observed in queens infected with type BrIII isolate. Indirect fluorescence antibody test showed T. gondii antibodies in all eight infected queens at 30 days after inoculation. In two 10-day-old kittens of the same litter (group 1), titers of 16 and 64 were detected. At the same time, titers of 16, 32, and 32 were detected in three kittens from the same litter (group 2). Experimental infection with tissue cysts from a type BrI and type BrIII isolates of T. gondii developed similar reproductive disturbance in primary infected pregnant queens.

Avaliação da reatividade de anticorpos anti-Toxoplasma gondii pela técnica de citometria de fluxo como indicadores de lesão ocular em soros de recém-nascidos com toxoplasmose congênita

A retinocoroidite é a manifestação mais comum causada pela infecção congênita por Toxoplasma gondii. Devido a gravidade das lesões oculares que podem até levar à perda completa da visão, a detecção precoce da toxoplasmose congênita e da lesão ocular são essenciais para o tratamento. Este trabalho possuiu o objetivo de avaliar a aplicabilidade da pesquisa de anticorpos IgG e das subclasses IgG1, IgG2, IgG3 e IgG4 anti-T. gondii por citometria de fluxo como marcador laboratorial das diferentes formas de lesões retinocoroidais na toxoplasmose congênita. Foram analisadas 88 amostras de soro de recém-nascidos com toxoplasmose congênita, sendo 25 sem lesão ocular (SL), 10 com lesão ocular ativa (RA), 26 com lesão ocular ativa e cicatricial (RAC) e 27 com lesão ocular cicatricial (RC). Foram também utilizadas 19 amostras de soro de recém-nascidos não infectados que apresentaram IgG positivo após o nascimento (NI). Essas amostras foram obtidas a partir de soros de recémnascidos participantes de um programa de triagem neonatal realizado em Minas Gerais realizado nos anos de 2006 e 2007. Os resultados demonstraram que os recém-nascidos com toxoplasmose congênita apresentam maior reatividade de anticorpos IgG total e subclasses IgG1, IgG2 e IgG3 do que indivíduos não infectados. No grupo não infectado, o único anticorpo com mais de 50% de indivíduos com alta reatividade de anticorpos foi IgG4. Ao comparar os grupos de indivíduos com toxoplasmose congênita, foi observado que o grupo RAC, seguido de RC, apresentou maior reatividade principalmente para os anticorpos IgG1 e IgG3 comparado aos recém-nascidos dos grupos RA e SL, enquanto que pacientes do grupo RA apresentaram maior reatividade para IgG4 do que indivíduos dos outros grupos. IgG1 foi a única subclasse capaz de diferenciar os grupos NI, SL dos grupos RAC e RC. Também foi avaliado o índice de avidez de IgG total, que não permitiu estabelecer nenhum critério de diferenciação das formas de lesão ocular causadas pelo T. gondii. Portanto, a citometria de fluxo demonstrou que pode ser um método laboratorial complementar para ser utilizado como indicador das diferentes lesões oculares causadas pela toxoplasmose congênita.

Seroprevalence of Toxoplasma gondii IgG antibody in HIV/AIDS-infected individuals in Maputo, Mozambique

OBJECTIVE To analyze the prevalence of IgG antibodies to Toxoplasma gondii in patients infected with HIV/AIDS and the association of demographic and social variables. METHODS Descriptive cross-sectional study that included the analysis of sociodemographic data and laboratory findings of 200 patients infected with HIV/AIDS treated in a laboratory unit in Maputo, Mozambique, in 2010. Individual data for all participants were collected with a self-administered questionnaire. Plasma samples were tested for IgG testing of anti- T. gondii using hemagglutination for the analysis of antibodies. RESULTS The seroprevalence of IgG anti- T. gondii was 46.0% (95%CI 39.2;52.9), 39.3% (95%CI 29.5;50.0) in men and 50.9% (95%CI 41.9;59.8) in women, with no difference between sex (OR 1.30; 95%CI 0.95;1.77; p = 0.12). Ages ranged from 10 to 60 years, with a higher prevalence of infection in older age groups, but with no significant difference between them. Regularly consuming cattle meat (OR 1.74; 95%CI 1.04;2.89, p = 0.05), breeding cats/dogs (OR 6.18; 95%CI 3.60;10.62, p < 0.000) and having regular contact with soil (OR 3.38; 95%CI 2.19;5.21; p < 0.000) were significantly associated with risk of latent infection. CONCLUSIONS Toxoplasmosis is an infection with high prevalence in Mozambique. Cultural and behavioral aspects increase the risk. Toxoplasmosis can be responsible in our environment by the great burden of morbidity and mortality associated with meningoencephalic injuries in patients with HIV/AIDS.

Usefulness of the detection of Toxoplasma gondii antigens in AIDS patients

Toxoplasmic encephalitis (TE) is a mayor cause of central nervous system infection in patients with acquired immunodeficiency syndrome (AIDS). Toxoplasma antibodies were detected in 56 of 79 patients with AIDS (71%), in the present study. Fourteen out of 57 seropositive patients developed TF (25%) and had Toxoplasma gondii antigen detected in their urine. For this, most of them received an effective therapy, with the subsequent disappearance of the symptoms and discontinuity of excretion of the T. gondii antigens. Our results suggest that the monitoring of T. gondii antigen in the urine of AIDS patients may be useful to decide on the proper time for therapy, as well as to avoid the beginning of neurologic signs in these patients.

Toxoplasma gondii infection in Brazilian domestic outpatient cats

The occurrence of Toxoplasma antibodies in domestic outpatient cats in the city of São Paulo was evaluated using the indirect immunofluorescence assay. A total of 248 blood samples obtained from male and female cats seen at the Veterinary Teaching Hospital at the University of São Paulo between February 1996 and January 1997 were tested. Of these, 17.7% were positive, with a 64 titer being detected in most animals. The frequence of Toxoplasma antibodies was significantly higher in older cats, those fed raw meat and those with free access to the outdoor environment. There was no significant difference in reactivity between males and females. We conclude that diet and free access to the outdoor environment were equally important as predisposing factors to the risk of Toxoplasma infection.

Prevalence of IgG antibodies specific to Toxoplasma gondii among blood donors in Recife, Northeast Brazil

A serological survey of Toxoplasma gondii infection in blood donors was carried out in order to identify seroprevalence in Recife, Brazil. Sera from 160 individuals (119 male and 41 female) were evaluated by using a Toxoplasma IgG-antibody enzyme immunoassay (Denka Seiken Co., LTD., Tokyo, Japan). The seropositive percentual for males (79.0%) showed to be higher (p < 0.05) than for females (63.4%). This percentage increases with age, ranging from 18.2% to 92.6% for individuals aging under 20 and 40-50 years old, respectively. For women of childbearing age (18-40 years) it was found a prevalence of 51.6%.

Seroprevalence of Toxoplasma gondii infection among indian people living in Iauareté, São Gabriel da Cachoeira, Amazonas, Brazil

The goal of this survey was to estimate the seroprevalence of Toxoplasma gondii infection in Iauareté, a multiethnic Indian community in the upper Rio Negro basin. We carried out a cross-sectional survey (n = 260), in order to obtain serum samples and demographic data. The sample was randomly selected, by family conglomerate analysis. Serodiagnosis was performed by an enzyme-linked immunosorbent assay and indirect immunofluorescence. Prevalence of reactivity was 73.5% (191/260), being higher in the older-age groups, reaching 95.7% (44/46) in the group aged 50 years or more. The majority of seropositive subjects had titers equal to or less than 1:64. Seroprevalence was greater in Indians belonging to the Hupda ethnic group (p = 0.03). According to the present survey, Indian people living in Iauareté have a high prevalence of antibodies to T. gondii. Demographic concentration and urbanization within low sanitation and poor hygiene backgrounds, as well as unfiltered water consumption, may be related to the high frequency of T. gondii seroprevalence observed in the studied area.

Quantitative toxoplasma gondii oocyst detection by a modified Kato Katz test using Kinyoun staining (KKK) in ME49 strain experimentally infected cats

We detected Toxoplasma gondii oocysts in feces of experimentally infected cats, using a Kato Katz approach with subsequent Kinyoun staining. Animals serologically negative to T. gondii were infected orally with 5x10² mice brain cysts of ME49 strain. Feces were collected daily from the 3rd to the 30th day after challenge. Oocysts were detected by qualitative sugar flotation and the quantitative modified Kato Katz stained by Kinyoun (KKK). In the experimentally infected cats, oocysts were detected from the 7th to 15th day through sugar flotation technique, but oocysts were found in KKK from the 6th to 16th day, being sensitive for a larger period, with permanent documentation. The peak of oocysts excretion occurred between the 8th to 11th days after challenge, before any serological positive result. KKK could be used in the screening and quantification of oocysts excretion in feces of suspected animals, with reduced handling of infective material, decreasing the possibility of environmental and operator contamination.

Antiretroviral activity of protease inhibitors against Toxoplasma gondii

The introduction of highly active antiretroviral therapy (HAART) has caused a marked reduction in the occurrence and severity of parasitic infections, including the toxoplasmic encephalitis (TE). These changes have been attributed to the restoration of cell-mediated immunity. This study was developed to examine the activity of six antiretroviral protease inhibitors (API) on Toxoplasma gondii tachyzoites. The six API showed anti-Toxoplasma activity, with IC50 value between 1.4 and 6.6 µg/mL. Further studies at the molecular level should be performed to clarify if the use of API could be beneficial or not for AIDS patients with TE.

IgG AVIDITY WESTERN BLOT USING Toxoplasma gondii rGRA-7 CLONED FROM NUCLEOTIDES 39-711 FOR SERODIAGNOSIS OF ACUTE TOXOPLASMOSIS

Toxoplasmosis is an important cause of congenital infection. The present study was performed to evaluate the usefulness of recombinant (r) GRA-7 cloned from nucleotides (n) 39-711 in discriminating between acute and chronic toxoplasmosis. First, commercial IgM, IgG and IgG avidity ELISAs were used to determine the serological profile of the sera. Serum samples were from 20 symptomatic patients with acute infection (low IgG avidity, IgM positive), 10 with chronic infection (high IgG avidity, IgM negative) and 10 with indeterminate IgG avidity (IgM positive) which were tested for IgG avidity status with an in-house developed IgG avidity Western blot using the rGRA-7 recombinant antigen. All 20 sera from cases of probable acute infection showed bands which either faded out completely or reduced significantly in intensity after treatment with 8 M urea, whereas the band intensities of the 10 serum samples from chronic cases remained the same. Of the 10 sera with indeterminate IgG avidity status, after treatment with 8 M urea the band intensities with six sera remained the same, two sera had completely faded bands and another two sera had significantly reduced band intensities. Discrimination between acute and chronic toxoplasmosis was successfully performed by the in-house IgG avidity Western blot.

CD19 LYMPHOCYTE PROLIFERATION INDUCED BY Bifidobacterium animalis subsp. lactis IN C57BL/6 MICE EXPERIMENTALLY INFECTED WITH Toxoplasma gondii

Toxoplasmosis is frequently acquired through the oral route by the ingestion of cysts or oocysts of Toxoplasma gondii. Once ingested, the parasites penetrate the intestinal epithelial cells and rapidly disseminate to all organs in the host. During T. gondii infection, the intestinal microbiota plays an important role in stimulating a protective immune response against the parasite. In this sense the use of probiotics is worthy of note since they are live microorganisms that have beneficial effects on the host through stimulation of the immune response that can be important in the control of T. gondii proliferation and dissemination in the host. In the present study, the action of the probiotic Bifidobacterium animalis subsp. lactis was investigated in C57BL/6 mice infected with oocysts of ME49 strain of T. gondii. The probiotic had an immunomodulatory action, inducing CD19 lymphocyte proliferation and consequently increasing anti-T. gondii antibody level.Bifidobacterium animalis subsp. lactisprovided protection in supplemented mice, compared to the control group. In addition, supplemented animals had milder inflammatory process in the small intestine, indicating that the probiotic protects the intestinal mucosa during infection with T. gondii. It was concluded that the probioticB. animalis subsp. lactis induces humoral immune response capable of providing protection against T. gondii infection.