Seroprevalence and risk factors of Toxoplasma gondii in goats (Capra hircus) in the state of Espírito Santo, Southeast Brazil.

Toxoplasma gondii is described as a potential cause of abortion in goats and as a threat to public health. To estimate the prevalence of goats infected by T. gondii, in different cities in the Espírito Santo State, and to identify possible risk factors for infection a serological study was conducted. A total of 146 goat serum samples from the cities of Cariacica, Serra and Vila Velha were analyzed. The presence of IgG Class Immunoglobulins was serologically evaluated by Immunofluorescence antibody test (IFAT) and by Enzyme-linked Immunosorbent Assay (ELISA). The seroprevalence of anti-T. gondii was 46.6% (68/146) in both techniques and the same samples got the same results in both techniques. Among the analyzed sera, 70.6% (48/68) exhibited high-avidity IgG antibodies, and 29.4% (20/68) exhibited low-avidity IgG antibodies, suggesting that the infection was chronic in the infected animals. Female sex, age group over two years old, water from the public supply system, storage of food and supplies in an open and unprotected place, and the presence of a domestic cat on the property were identified as risk factors for T. gondii infection in goats. The state of Espirito Santo has a high frequency of infected goats, and this is the first research on caprine toxoplasmosis seroepidemiology in that region.

Tityus serrulatus (Scorpion): From the Crude Venom to the Construction of Synthetic Peptides and Their Possible Therapeutic Application Against Toxoplasma gondii Infection.

Toxoplasmosis, caused by Toxoplasma gondii, is a major public concern owing to its neurotropic nature and high morbidity and mortality rates in immunocompromised patients and newborns. Current treatment for this disease is inefficient and produces side effects. Inflammatory mediators produced during T. gondii infection (e.g., cytokines and nitric oxide) are crucial in controlling parasite replication. In this context, Tityus serrulatus venom (TsV) induces the production of inflammatory mediators by immune cells. Thus, this study aimed to isolate and identify the components of TsV with potential anti-T. gondii activity. TsV was extracted from scorpions and lyophilized or loaded onto a column to obtain its fractions. TsV subfractions were obtained using chromatography, and its amino acid sequence was identified and applied to peptide design using bioinformatics tools. The C57BL/6 mice and their harvested macrophages were used to test the anti-Toxoplasma activity of TsV components and peptides. TsV and its fraction F6 attenuated the replication of tachyzoites in macrophages and induced nitric oxide and cytokine (IL-12, TNF, and IL-6) production by infected cells, without host cell toxicity. Moreover, Su6-B toxin, a subfraction of F6, demonstrated anti-T. gondii activity. The partially elucidated and characterized amino acid sequence of Sub6-B demonstrated 93% similarity with T. serrulatus 2 toxin (Ts2). Ts2 mimetic peptides ("Pep1," "Pep2a," and "Pep2b") were designed and synthesized. Pep1 and Pep2a, but not Pep2b, reduced the replication of tachyzoites in macrophages. In vivo, treatment of T. gondii-infected mice with Pep1, Pep2a, or Pep2b decreased the number of cerebral cysts and did not induce hepatotoxicity in the animals. Taken together, our data show promising immunomodulatory and antiparasitic activity of TsV that could be explored and applied in future therapies for treating infectious parasitic diseases such as toxoplasmosis.

Sarcocystidae in wild birds of southeastern Brazil.

This study aimed to identify members of the Sarcocystidae family in naturally infected wild birds at a rescue center in the state of Minas Gerais, southeastern Brazil. The heart and brain of 44 wild birds were evaluated by bioassay in mice to detect T. gondii, and extracted DNA was used for nested PCR of the 18S ribosomal DNA gene to detect members of the Sarcocystidae family. The positive samples were sequenced, assembled, edited and compared with sequences deposited in GenBank. Toxoplasma gondii was isolated from six (13.6%) out of 44 birds. Toxoplasma gondii DNA was identified in 10/44 (22.7%) of the birds. The amplified sequences exhibited 100% similarity with the DNA of the ME49 strain of T. gondii. Sarcocystis DNA (99% similarity) was identified in 5/44 (11.4%) of the birds. T. gondii and Sarcocystis spp. are common in wild birds in Minas Gerais, Brazil.

Sarcocystidae in wild birds of southeastern Brazil / Sarcocystidae em aves silvestres do sudeste do Brasil

This study aimed to identify members of the Sarcocystidae family in naturally infected wild birds at a rescue center in the state of Minas Gerais, southeastern Brazil. The heart and brain of 44 wild birds were evaluated by bioassay in mice to detect T. gondii, and extracted DNA was used for nested PCR of the 18S ribosomal DNA gene to detect members of the Sarcocystidae family. The positive samples were sequenced, assembled, edited and compared with sequences deposited in GenBank. Toxoplasma gondii was isolated from six (13.6%) out of 44 birds. Toxoplasma gondii DNA was identified in 10/44 (22.7%) of the birds. The amplified sequences exhibited 100% similarity with the DNA of the ME49 strain of T. gondii. Sarcocystis DNA (99% similarity) was identified in 5/44 (11.4%) of the birds. T. gondii and Sarcocystis spp. are common in wild birds in Minas Gerais, Brazil.(AU)

O objetivo deste estudo foi identificar membros da família Sarcocystidae em aves silvestres de vida livre naturalmente infectadas e resgatadas no estado de Minas Gerais, Brasil. Coração e cérebro de 44 aves silvestres foram avaliados por bioensaio em camundongos para detecção de T. gondii e extração de DNA para Nested-PCR do gene 18S do DNA ribossomal de membros da família Sarcocystidae. As amostras positivas foram sequenciadas, analisadas, editadas e comparadas com sequências depositadas no GenBank. Toxoplasma gondii foi isolado de seis (13,6%) das 44 aves. DNA de T. gondii foi identificado em 10/44 (22,7%) das 44 aves. As sequências amplificadas exibiram 100% de similaridade com o DNA da cepa ME49 de T. gondii. DNA de Sarcocystis (99% de similaridade) foi identificado em 5/44 (11,4%) das 44 aves. T. gondii e Sarcocystis spp. são encontrados, comumente, em aves silvestres no estado de Minas Gerais, Brasil.(AU)

Putative biomarkers for early diagnosis and prognosis of congenital ocular toxoplasmosis.

In the present study we have evaluated the performance of several immunological biomarkers for early diagnosis and prognosis of congenital toxoplasmosis. Our results showed that ex vivo serum levels of CXCL9, and the frequencies of circulating CD4+CD25+ T-cells and T. gondii-specific IFN-γ+CD4+ T-cells measured 30-45 days after birth presented high accuracy to distinguish T. gondii-infected infants from healthy age-matched controls (Global Accuracy/AUC = 0.9; 0.9 and 0.8, respectively). Of note was the enhanced performance (Accuracy = 96%) achieved by using a combined stepwise analysis of CD4+CD25+ T-cells and CXCL9. In addition, high global accuracy (AUC = 0.9) with elevated sensitivity (Se = 98%) was also reached by using the total frequency of in vitro IFN-γ-producing T. gondii-specific T-cells (∑ IFN-γ+ CD4+ & CD8+) as a biomarker of congenital toxoplasmosis. Furthermore, the analysis of in vitro T. gondii-specific IL5+CD4+ T-cells and IFN-γ+NK-cells displayed a high accuracy for early prognosis of ocular lesion in infant with congenital toxoplasmosis (Global Accuracy/AUC = 0.8 and 0.9, respectively). Together, these findings support the relevance of employing the elements of the cell-mediated immune response as biomarkers with potential to endorse early diagnosis and prognosis of congenital ocular toxoplasmosis to contribute for a precise clinical management and effective therapeutic intervention.

Toxoplasma gondii: Cytokine responses in mice reinfected with atypical strains.

This study aimed to elucidate the cellular immune response against Toxoplasma gondii in chronically infected mice reinfected with different strains of the parasite to elucidate the immunological basis for chronicity or virulence and to uncover the involvement of genes that encode virulence proteins and modulate the immune response. BALB/c mice were infected by oral gavage with non-virulent D8 strain and challenged 45 days post-infection with virulent EGS or CH3 strains. Cytokine measurement was performed 2 days post-challenge in cell extracts of the small intestine and 2, 7, and 14 days post-challenge in serum. Virulence gene allele type of these strains was analyzed. Challenged mice survived by avoiding exacerbated inflammation and inhibiting the overproduction of cytokines. Local and systemic cytokine response in challenged mice was similar to chronic controls and quite distinct in mice acutely infected with the EGS or CH3 strains. Allelic combinations of the virulence genes ROP5/ROP18 was predictive of virulence in mice when tested in these T. gondii strains. Other allelic combinations of rhoptries and dense granules genes showed discrepancies.

Seroprevalence of Brucella ovis-epididymitis, smooth-Brucella, leptospirosis, toxoplasmosis, and Maedi-Visna in sheep slaughtered in Minas Gerais State, Brazil / Soroprevalência de Brucella ovis, Brucella lisa, leptospirose, toxoplasmose e Maedi-Visna em ovinos abatidos em Minas Gerais, Brasil

The present study aimed to estimate the prevalence of antibodies against Brucella ovis-epididymitis, smooth-Brucella, leptospirosis, toxoplasmosis and Maedi-visna in sheep slaughtered in Minas Gerais, Brazil and to study their simultaneous occurrence, including caseous lymphadenitis, at sheep and flock levels. The study was conducted at a sheep slaughterhouse with Federal Inspection Service. Sera from 594 animals from 21 flocks were collected, in 2007. The agar gel immunodiffusion (AGID) was employed to detect anti-B. ovis and anti-Maedi Visna antibodies, whereas Rose Bengal (RB) and the 2-mercaptoethanol test (2ME) were used to test anti-smooth Brucella antibodies. For the detection of anti-Leptospiraantibodies, sera were examined by microscopic agglutination test (MAT), while for the detection of IgG antibodies to Toxoplasma gondii ELISA was used. Prevalence of antibodies against smooth Brucella, B. ovis-epididimitis, Leptospiraspp., toxoplasmosis and Maedi-Visna found in sheep from Minas Gerais was 0.00%, 24.04%, 25.96%, 10.46% and 3.08%, respectively; whereas the seroprevalence in flocks was 0.00%, 80.95%, 90.48%, 71.43% and 23.81%, respectively. Moreover, when data on antibodies anti-Corynebacterium pseudotuberculosis, previously obtained, were included, about 60% of the flocks showed animals that were exposed to four or more of the studied agents. However, only 25.47% of the sheep exhibited simultaneously antibodies against more than one pathogen. Thus, data from the present study on sheep slaughtered in Minas Gerais, Brazil, showed no antibodies to smooth-Brucella and a low frequency of antibodies anti-Maedi Visna lentivirus, and a high and widespread seroprevalence of B. ovis, Leptospira spp., and T. gondii among animals and flocks.(AU)

O presente estudo teve como objetivo estimar a prevalência de anticorpos contra Brucella ovis (epididimite ovina), Brucellalisa, leptospirose, toxoplasmose e Maedi-visna em ovinos abatidos em Minas Gerais, Brasil, e estudar sua ocorrência simultânea, incluindo linfadenite caseosa, nos ovinos e nos rebanhos. O estudo foi realizado em um abatedouro de ovinos com Serviço de Inspeção Federal. Soros de 594 animais de 21 rebanhos foram coletados, em 2007. A imunodifusão em gel de ágar (IDGA) foi empregada para detectar anticorpos anti-B. ovis e anticorpos anti-Maedi Visna, enquanto o teste do antígeno acidificado tamponado (AAT) e o teste de 2-mercaptoetanol (2ME) foram utilizados para testar anticorpos anti-Brucella lisa. Para a detecção de anticorpos anti-Leptospira, os soros foram examinados pelo teste de aglutinação microscópica (MAT), enquanto que para a detecção de anticorpos IgG para Toxoplasma gondii, foi usado o ELISA. A prevalência de anticorpos anti-Brucella lisa, B. ovis, Leptospira spp., toxoplasmose e Maedi-Visna encontrados em ovinos de Minas Gerais foi de 0,00%, 24,04%, 25,96%, 10,46% e 3,08%, respectivamente; enquanto a soroprevalência em rebanhos foi de 0,00%, 80,95%, 90,48%, 71,43% e 23,81%, respectivamente. Além disso, quando dados de anticorpos anti-Corynebacterium pseudotuberculosis, previamente obtidos, foram incluídos, cerca de 60% dos rebanhos apresentaram animais expostos a quatro ou mais dos agentes estudados. No entanto, apenas 25,47% dos ovinos exibiram simultaneamente anticorpos contra mais de um patógeno. Assim, os dados do presente estudo sobre ovinos abatidos em Minas Gerais, Brasil, mostram que ausência de anticorpos anti-Brucella lisa e baixa frequência de anticorpos anti-Maedi Visna, e uma soroprevalência alta e generalizada de B. ovis, Leptospira spp. e T. gondii entre animais e rebanhos.(AU)

Seroprevalence of Brucella ovis-epididymitis, smooth-Brucella, leptospirosis, toxoplasmosis, and Maedi-Visna in sheep slaughtered in Minas Gerais State, Brazil / Soroprevalência de Brucella ovis, Brucella lisa, leptospirose, toxoplasmose e Maedi-Visna em ovinos abatidos em Minas Gerais, Brasil

The present study aimed to estimate the prevalence of antibodies against Brucella ovis-epididymitis, smooth-Brucella, leptospirosis, toxoplasmosis and Maedi-visna in sheep slaughtered in Minas Gerais, Brazil and to study their simultaneous occurrence, including caseous lymphadenitis, at sheep and flock levels. The study was conducted at a sheep slaughterhouse with Federal Inspection Service. Sera from 594 animals from 21 flocks were collected, in 2007. The agar gel immunodiffusion (AGID) was employed to detect anti-B. ovis and anti-Maedi Visna antibodies, whereas Rose Bengal (RB) and the 2-mercaptoethanol test (2ME) were used to test anti-smooth Brucella antibodies. For the detection of anti-Leptospiraantibodies, sera were examined by microscopic agglutination test (MAT), while for the detection of IgG antibodies to Toxoplasma gondii ELISA was used. Prevalence of antibodies against smooth Brucella, B. ovis-epididimitis, Leptospiraspp., toxoplasmosis and Maedi-Visna found in sheep from Minas Gerais was 0.00%, 24.04%, 25.96%, 10.46% and 3.08%, respectively; whereas the seroprevalence in flocks was 0.00%, 80.95%, 90.48%, 71.43% and 23.81%, respectively. Moreover, when data on antibodies anti-Corynebacterium pseudotuberculosis, previously obtained, were included, about 60% of the flocks showed animals that were exposed to four or more of the studied agents. However, only 25.47% of the sheep exhibited simultaneously antibodies against more than one pathogen. Thus, data from the present study on sheep slaughtered in Minas Gerais, Brazil, showed no antibodies to smooth-Brucella and a low frequency of antibodies anti-Maedi Visna lentivirus, and a high and widespread seroprevalence of B. ovis, Leptospira spp., and T. gondii among animals and flocks.(AU)

O presente estudo teve como objetivo estimar a prevalência de anticorpos contra Brucella ovis (epididimite ovina), Brucellalisa, leptospirose, toxoplasmose e Maedi-visna em ovinos abatidos em Minas Gerais, Brasil, e estudar sua ocorrência simultânea, incluindo linfadenite caseosa, nos ovinos e nos rebanhos. O estudo foi realizado em um abatedouro de ovinos com Serviço de Inspeção Federal. Soros de 594 animais de 21 rebanhos foram coletados, em 2007. A imunodifusão em gel de ágar (IDGA) foi empregada para detectar anticorpos anti-B. ovis e anticorpos anti-Maedi Visna, enquanto o teste do antígeno acidificado tamponado (AAT) e o teste de 2-mercaptoetanol (2ME) foram utilizados para testar anticorpos anti-Brucella lisa. Para a detecção de anticorpos anti-Leptospira, os soros foram examinados pelo teste de aglutinação microscópica (MAT), enquanto que para a detecção de anticorpos IgG para Toxoplasma gondii, foi usado o ELISA. A prevalência de anticorpos anti-Brucella lisa, B. ovis, Leptospira spp., toxoplasmose e Maedi-Visna encontrados em ovinos de Minas Gerais foi de 0,00%, 24,04%, 25,96%, 10,46% e 3,08%, respectivamente; enquanto a soroprevalência em rebanhos foi de 0,00%, 80,95%, 90,48%, 71,43% e 23,81%, respectivamente. Além disso, quando dados de anticorpos anti-Corynebacterium pseudotuberculosis, previamente obtidos, foram incluídos, cerca de 60% dos rebanhos apresentaram animais expostos a quatro ou mais dos agentes estudados. No entanto, apenas 25,47% dos ovinos exibiram simultaneamente anticorpos contra mais de um patógeno. Assim, os dados do presente estudo sobre ovinos abatidos em Minas Gerais, Brasil, mostram que ausência de anticorpos anti-Brucella lisa e baixa frequência de anticorpos anti-Maedi Visna, e uma soroprevalência alta e generalizada de B. ovis, Leptospira spp. e T. gondii entre animais e rebanhos.(AU)

Seroprevalence of Brucella ovis-epididymitis, smooth-Brucella, leptospirosis, toxoplasmosis, and Maedi-Visna in sheep slaughtered in Minas Gerais State, Brazil / Soroprevalência de Brucella ovis, Brucella lisa, leptospirose, toxoplasmose e Maedi-Visna em ovinos abatidos em Minas Gerais, Brasil

The present study aimed to estimate the prevalence of antibodies against Brucella ovis-epididymitis, smooth-Brucella, leptospirosis, toxoplasmosis and Maedi-visna in sheep slaughtered in Minas Gerais, Brazil and to study their simultaneous occurrence, including caseous lymphadenitis, at sheep and flock levels. The study was conducted at a sheep slaughterhouse with Federal Inspection Service. Sera from 594 animals from 21 flocks were collected, in 2007. The agar gel immunodiffusion (AGID) was employed to detect anti-B. ovis and anti-Maedi Visna antibodies, whereas Rose Bengal (RB) and the 2-mercaptoethanol test (2ME) were used to test anti-smooth Brucella antibodies. For the detection of anti-Leptospira antibodies, sera were examined by microscopic agglutination test (MAT), while for the detection of IgG antibodies to Toxoplasma gondii ELISA was used. Prevalence of antibodies against smooth Brucella, B. ovis-epididimitis, Leptospira spp., toxoplasmosis and Maedi-Visna found in sheep from Minas Gerais was 0.00%, 24.04%, 25.96%, 10.46% and 3.08%, respectively; whereas the seroprevalence in flocks was 0.00%, 80.95%, 90.48%, 71.43% and 23.81%, respectively. Moreover, when data on antibodies anti-Corynebacterium pseudotuberculosis, previously obtained, were included, about 60% of the flocks showed animals that were exposed to four or more of the studied agents. However, only 25.47% o

O presente estudo teve como objetivo estimar a prevalência de anticorpos contra Brucella ovis (epididimite ovina), Brucella lisa, leptospirose, toxoplasmose e Maedi-visna em ovinos abatidos em Minas Gerais, Brasil, e estudar sua ocorrência simultânea, incluindo linfadenite caseosa, nos ovinos e nos rebanhos. O estudo foi realizado em um abatedouro de ovinos com Serviço de Inspeção Federal. Soros de 594 animais de 21 rebanhos foram coletados, em 2007. A imunodifusão em gel de ágar (IDGA) foi empregada para detectar anticorpos anti-B. ovis e anticorpos anti-Maedi Visna, enquanto o teste do antígeno acidificado tamponado (AAT) e o teste de 2-mercaptoetanol (2ME) foram utilizados para testar anticorpos anti-Brucella lisa. Para a detecção de anticorpos anti-Leptospira, os soros foram examinados pelo teste de aglutinação microscópica (MAT), enquanto que para a detecção de anticorpos IgG para Toxoplasma gondii, foi usado o ELISA. A prevalência de anticorpos anti-Brucella lisa, B. ovis, Leptospira spp., toxoplasmose e Maedi-Visna encontrados em ovinos de Minas Gerais foi de 0,00%, 24,04%, 25,96%, 10,46% e 3,08%, respectivamente; enquanto a soroprevalência em rebanhos foi de 0,00%, 80,95%, 90,48%, 71,43% e 23,81%, respectivamente. Além disso, quando dados de anticorpos anti-Corynebacterium pseudotuberculosis, previamente obtidos, foram incluídos, cerca de 60% dos rebanhos apresentaram a

Seroprevalence of Brucella ovis-epididymitis, smooth-Brucella, leptospirosis, toxoplasmosis, and Maedi-Visna in sheep slaughtered in Minas Gerais State, Brazil / Soroprevalência de Brucella ovis, Brucella lisa, leptospirose, toxoplasmose e Maedi-Visna em ovinos abatidos em Minas Gerais, Brasil

The present study aimed to estimate the prevalence of antibodies against Brucella ovis-epididymitis, smooth-Brucella, leptospirosis, toxoplasmosis and Maedi-visna in sheep slaughtered in Minas Gerais, Brazil and to study their simultaneous occurrence, including caseous lymphadenitis, at sheep and flock levels. The study was conducted at a sheep slaughterhouse with Federal Inspection Service. Sera from 594 animals from 21 flocks were collected, in 2007. The agar gel immunodiffusion (AGID) was employed to detect anti-B. ovis and anti-Maedi Visna antibodies, whereas Rose Bengal (RB) and the 2-mercaptoethanol test (2ME) were used to test anti-smooth Brucella antibodies. For the detection of anti-Leptospira antibodies, sera were examined by microscopic agglutination test (MAT), while for the detection of IgG antibodies to Toxoplasma gondii ELISA was used. Prevalence of antibodies against smooth Brucella, B. ovis-epididimitis, Leptospira spp., toxoplasmosis and Maedi-Visna found in sheep from Minas Gerais was 0.00%, 24.04%, 25.96%, 10.46% and 3.08%, respectively; whereas the seroprevalence in flocks was 0.00%, 80.95%, 90.48%, 71.43% and 23.81%, respectively. Moreover, when data on antibodies anti-Corynebacterium pseudotuberculosis, previously obtained, were included, about 60% of the flocks showed animals that were exposed to four or more of the studied agents. However, only 25.47% o

O presente estudo teve como objetivo estimar a prevalência de anticorpos contra Brucella ovis (epididimite ovina), Brucella lisa, leptospirose, toxoplasmose e Maedi-visna em ovinos abatidos em Minas Gerais, Brasil, e estudar sua ocorrência simultânea, incluindo linfadenite caseosa, nos ovinos e nos rebanhos. O estudo foi realizado em um abatedouro de ovinos com Serviço de Inspeção Federal. Soros de 594 animais de 21 rebanhos foram coletados, em 2007. A imunodifusão em gel de ágar (IDGA) foi empregada para detectar anticorpos anti-B. ovis e anticorpos anti-Maedi Visna, enquanto o teste do antígeno acidificado tamponado (AAT) e o teste de 2-mercaptoetanol (2ME) foram utilizados para testar anticorpos anti-Brucella lisa. Para a detecção de anticorpos anti-Leptospira, os soros foram examinados pelo teste de aglutinação microscópica (MAT), enquanto que para a detecção de anticorpos IgG para Toxoplasma gondii, foi usado o ELISA. A prevalência de anticorpos anti-Brucella lisa, B. ovis, Leptospira spp., toxoplasmose e Maedi-Visna encontrados em ovinos de Minas Gerais foi de 0,00%, 24,04%, 25,96%, 10,46% e 3,08%, respectivamente; enquanto a soroprevalência em rebanhos foi de 0,00%, 80,95%, 90,48%, 71,43% e 23,81%, respectivamente. Além disso, quando dados de anticorpos anti-Corynebacterium pseudotuberculosis, previamente obtidos, foram incluídos, cerca de 60% dos rebanhos apresentaram a

Tissue dissemination and humoral response after experimental reinfection with atypical Toxoplasma gondii strains obtained from congenital human toxoplasmosis in Brazil.

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).

Toxoplasma gondii: isolation, biological and molecular characterisation of samples from free-range Gallus gallus domesticus from countryside Southeast Brazil / Toxoplasma gondii: isolamento, caracterização biológica e molecular de amostras de Gallus gallus domesticus no interior do Sudeste brasileiro

Toxoplasma gondii presents a high prevalence worldwide, infecting several animals. Felines are considered the definitive hosts and among the intermediate hosts we highlight mammals and birds. The man can become infected by ingesting tissue cysts present in birds and mammals. Biological and molecular aspects of T. gondii allows a better understanding of the epidemiology of toxoplasmosis. This work is a serologic screening of 58 chickens grown (Gallus gallus domesticus) for human consumption in Espírito Santo State, by means of indirect haemagglutination assay (IHA). Thirteen chickens tested positive for anti-T. gondii antibodies. The heart and brain of five positive chickens were harvested, treated with pepsin and inoculated separately, in two Swiss mice, intraperitoneally. Tachyzoites were observed in the peritoneum of all the animals, between seven and 10 days after the inoculum. Ten isolates were obtained and biologically characterised in BALB/c mice inoculated with 101 to 104 tachyzoites. All isolates were classified as virulent or intermediately virulent. Isolates were genotyped by means of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, revealing three different genotypes. None of the isolates exhibited the clonal type I, II or III genotype. No genotypic differences were observed between the isolates from the brain or heart from the same bird.(AU)

Toxoplasma gondii apresenta alta prevalência mundial, capaz de infectar diversos animais. Felinos são considerados os hospedeiros definitivos e entre os hospedeiros intermediários destacamos os mamíferos e as aves. O homem pode se infectar ingerindo cistos teciduais presentes na carne das aves e mamíferos. O conhecimento dos aspectos biológicos e moleculares do parasito possibilitam melhor entendimento da epidemiologia da toxoplasmose. Neste trabalho foi realizada triagem sorológica por hemaglutinação indireta (HI) em 58 galinhas caipiras (Gallus gallus domesticus) utilizadas para consumo humano, provenientes do estado do Espírito Santo, Brasil. Treze galinhas apresentaram sorologia positiva para T. gondii. O coração e o cérebro de cinco galinhas positivas foram colhidos, tratados com pepsina e inoculados separadamente, em dois camundongos Swiss, por via intraperitoneal. Observou-se taquizoítos no peritônio de todos os camundongos, entre sete e 10 dias após o inóculo. Foram obtidos 10 novos isolados de T. gondii os quais foram estudados em camundongos BALB/C inoculados com 101 a 104 taquizoítos por animal. Todos os isolados foram considerados virulentos ou de virulência intermediária. A caracterização molecular dos isolados, realizada por PCR-RFLP, demonstrou a ocorrência de três genótipos distintos. Nenhum isolado apresentou genótipo clonal ou linhagem clonal do Brasil. Não foi observada diferença molecular (PCR-RFLP) entre os isolados obtidos a partir do cérebro ou do coração da mesma ave. Dois isolados já haviam sido relatados na literatura como causadores de doenças em humanos.(AU)

Toxoplasma gondii: isolation, biological and molecular characterisation of samples from free-range Gallus gallus domesticus from countryside Southeast Brazil / Toxoplasma gondii: isolamento, caracterização biológica e molecular de amostras de Gallus gallus domesticus no interior do Sudeste brasileiro

Abstract Toxoplasma gondii presents a high prevalence worldwide, infecting several animals. Felines are considered the definitive hosts and among the intermediate hosts we highlight mammals and birds. The man can become infected by ingesting tissue cysts present in birds and mammals. Biological and molecular aspects of T. gondii allows a better understanding of the epidemiology of toxoplasmosis. This work is a serologic screening of 58 chickens grown (Gallus gallus domesticus) for human consumption in Espírito Santo State, by means of indirect haemagglutination assay (IHA). Thirteen chickens tested positive for anti-T. gondii antibodies. The heart and brain of five positive chickens were harvested, treated with pepsin and inoculated separately, in two Swiss mice, intraperitoneally. Tachyzoites were observed in the peritoneum of all the animals, between seven and 10 days after the inoculum. Ten isolates were obtained and biologically characterised in BALB/c mice inoculated with 101 to 104 tachyzoites. All isolates were classified as virulent or intermediately virulent. Isolates were genotyped by means of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, revealing three different genotypes. None of the isolates exhibited the clonal type I, II or III genotype. No genotypic differences were observed between the isolates from the brain or heart from the same bird.

Resumo Toxoplasma gondii apresenta alta prevalência mundial, capaz de infectar diversos animais. Felinos são considerados os hospedeiros definitivos e entre os hospedeiros intermediários destacamos os mamíferos e as aves. O homem pode se infectar ingerindo cistos teciduais presentes na carne das aves e mamíferos. O conhecimento dos aspectos biológicos e moleculares do parasito possibilitam melhor entendimento da epidemiologia da toxoplasmose. Neste trabalho foi realizada triagem sorológica por hemaglutinação indireta (HI) em 58 galinhas caipiras (Gallus gallus domesticus) utilizadas para consumo humano, provenientes do estado do Espírito Santo, Brasil. Treze galinhas apresentaram sorologia positiva para T. gondii. O coração e o cérebro de cinco galinhas positivas foram colhidos, tratados com pepsina e inoculados separadamente, em dois camundongos Swiss, por via intraperitoneal. Observou-se taquizoítos no peritônio de todos os camundongos, entre sete e 10 dias após o inóculo. Foram obtidos 10 novos isolados de T. gondii os quais foram estudados em camundongos BALB/C inoculados com 101 a 104 taquizoítos por animal. Todos os isolados foram considerados virulentos ou de virulência intermediária. A caracterização molecular dos isolados, realizada por PCR-RFLP, demonstrou a ocorrência de três genótipos distintos. Nenhum isolado apresentou genótipo clonal ou linhagem clonal do Brasil. Não foi observada diferença molecular (PCR-RFLP) entre os isolados obtidos a partir do cérebro ou do coração da mesma ave. Dois isolados já haviam sido relatados na literatura como causadores de doenças em humanos.

Toxoplasma gondii: isolation, biological and molecular characterisation of samples from free-range Gallus gallus domesticus from countryside Southeast Brazil.

Toxoplasma gondii presents a high prevalence worldwide, infecting several animals. Felines are considered the definitive hosts and among the intermediate hosts we highlight mammals and birds. The man can become infected by ingesting tissue cysts present in birds and mammals. Biological and molecular aspects of T. gondii allows a better understanding of the epidemiology of toxoplasmosis. This work is a serologic screening of 58 chickens grown (Gallus gallus domesticus) for human consumption in Espírito Santo State, by means of indirect haemagglutination assay (IHA). Thirteen chickens tested positive for anti-T. gondii antibodies. The heart and brain of five positive chickens were harvested, treated with pepsin and inoculated separately, in two Swiss mice, intraperitoneally. Tachyzoites were observed in the peritoneum of all the animals, between seven and 10 days after the inoculum. Ten isolates were obtained and biologically characterised in BALB/c mice inoculated with 101 to 104 tachyzoites. All isolates were classified as virulent or intermediately virulent. Isolates were genotyped by means of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, revealing three different genotypes. None of the isolates exhibited the clonal type I, II or III genotype. No genotypic differences were observed between the isolates from the brain or heart from the same bird.

Kinetics of parasite distribution after reinfection with genetically distinct strains of Toxoplasma gondii.

Recent data shows that prior infection by Toxoplasma gondii does not protect the host from subsequent reinfection even after the development of immunological memory. Although animal models for T. gondii reinfection were proposed after cases of natural human reinfection were described, little is known about the events that occur immediately after challenge. To further understand these events, BALB/c mice were chronically infected with D8 non-virulent strain (genotype ToxoDB#8 BrIII) and challenged with two different virulent strains: EGS (genotype ToxoDB #229) or CH3 strain (genotype ToxoDB #19). Primary infection protected animals from lethal challenge and morbidity was reduced. Reinfection was confirmed by PCR-RFLP, showing differences in the way the parasites spread in challenged animals. Parasites reached the lungs during early infection and a parasitism delay in the intestine was observed in D8+CH3 group. Parasites from challenge strains were not detected in the brain of D8+CH3 and in the intestine and brain of D8+EGS group. Previous infection with D8 strain of T. gondii protected against lethal challenges, but it did not prevent parasite spread to some organs.

Seroprevalence and risk factors for human toxoplasmosis in northeastern Brazil

Toxoplasmosis is one of the most prevalent parasitic infections in humans, causing severe clinical complications in immunocompromised patients or in foetuses. Estimates on the toxoplasmosis seroprevalence in human populations vary widely, even within the same region. This study aimed to estimate the prevalence of Toxoplasma gondii infection in the municipality of Santa Cruz, State of Rio Grande do Norte, Brazil, and identify the risk factors for toxoplasmosis. T. gondii IgG antibodies were measured in 66.2% of the studied population. The factors associated with infection were as follows: age above 45 years (OR = 7.4; 95% CI = 3.7-14.8); illiteracy (OR = 2.8; 95% CI = 1.6-5.0); the presence of more than three cats in the home (OR 2.0; 95% CI = 1.2-3.5); no water tank (OR = 2.0; 95% CI = 1.4-3.1); and consumption of raw or unpasteurized milk (OR = 2.0; 95% CI = 1.3-3.0). The risk factors identified were predominantly related to the ingestion of oocysts. This study may contribute to public health knowledge of toxoplasmosis and the definition of control programs, particularly for pregnant women from regions similar to the area studied.

Establishing tools for early diagnosis of congenital toxoplasmosis: Flow cytometric IgG avidity assay as a confirmatory test for neonatal screening.

The aim of this study was to evaluate the performance of conventional serology (Q-Preven™ and ELFAVIDAS™) and flow cytometry-based serologic tools for early serologic diagnosis of congenital toxoplasmosis. The study groups included prospectively confirmed cases of congenital toxoplasmosis (TOXO=88) and age-matching non-infected controls (NI=15).The results demonstrated that all samples tested positive/indeterminate for anti-T. gondii IgM screening at birth using air-dried whole blood samples. Serum samples collected at 30-45days after birth tested positive for ELFAVIDAS™ IgG in both groups. While all NI tested negative for ELFAVIDAS™ IgM and IgA, only 78% and 36% of TOXO tested positive for IgM and IgA, respectively. Flow cytometry-based anti-T. gondii IgM, IgA and IgG reactivity displayed moderate performance with low sensitivity (47.6%, 72.6% and 75.0%, respectively). Regardless the remarkable specificity of IgG1, IgG2 and IgG3 subclasses for early diagnosis, weak or moderate specificity was observed (Se=73.9%, 60.2% and 83.0%, respectively). The analysis of IgG avidity indices (AI) demonstrated the highest performance among the flow cytometry-based methods (Se=96.6%; Sp=93.3%), underscoring the low avidity index (AI<60%) within TOXO (97.0%) in contrast with the high avidity index (AI>60%) in NI (93%). Analysis of anti-T. gondii IgG and IgG3 reactivity for mother:infant paired samples may represent a relevant complementary tests for early diagnosis. In conclusion, a feasible high-standard algorithm (Accuracy=97.1%) was proposed consisting of Q-Preven™ IgM screening at birth, followed by ELFAVIDAS™ IgM and flow cytometric IgG avidity analysis at 30-45days after birth as a high performance tool for early serological diagnosis of congenital toxoplasmosis.

Early serum biomarker networks in infants with distinct retinochoroidal lesion status of congenital toxoplasmosis.

The present study characterized the early changes in the serum chemokines/cytokine signatures and networks in infants with congenital-toxoplasmosis/(TOXO) as compared to non-infected-controls/(NI). TOXO were subgrouped according to the retinochoroidal lesion status as no-lesion/(NL), active-lesion/(ARL), active/cicatricial-lesion/(ACRL) and cicatricial-lesion/(CRL). The results showed that TOXO display prominent chemokine production mediated by IL-8/CXCL8, MIG/CXCL9, IP-10/CXCL10 and RANTES/CCL5. Additionally, TOXO is accompanied by mixed proinflammatory/regulatory cytokine pattern mediated by IL-6, IFN-γ, IL-4, IL-5 and IL-10. While TNF appears as a putative biomarker for NL and IFN-γ/IL-5 as immunological features for ARL, IL-10 emerges as a relevant mediator in ACRL/CRL. IL-8/CXCL8 and IP-10/CXCL10 are broad-spectrum indicators of ocular disease, whereas TNF is a NL biomarker, IFN-γ and MIG/CXCL9 point out to ARL; and IL-10 is highlighted as a genuine serum biomarker of ACRL/CRL. The network analysis demonstrated a broad chemokine/cytokine crosstalk with divergences in the molecular signatures in patients with different ocular lesions during congenital toxoplasmosis.

Genetic Polymorphisms and Phenotypic Profiles of Sulfadiazine-Resistant and Sensitive Toxoplasma gondii Isolates Obtained from Newborns with Congenital Toxoplasmosis in Minas Gerais, Brazil.

BACKGROUND: Previous Toxoplasma gondii studies revealed that mutations in the dhps (dihydropteroate synthase) gene are associated with resistance to sulfonamides. Although Brazilian strains are genotypically different, very limited data are available regarding the susceptibility of strains obtained from human to sulfonamides. The aim of this study was to evaluate the efficacy of sulfadiazine (SDZ) against Brazilian isolates of T. gondii and verify whether isolates present polymorphisms in the dhps gene. We also investigated whether the virulence-phenotype and/or genotype were associated with the profile of susceptibility to SDZ. METHODS: Five T. gondii isolates obtained from newborns with congenital toxoplasmosis were used to verify susceptibility. Mice were infected with 104 tachyzoites and orally treated with different doses of SDZ. The mortality curve was evaluated by the Log-rank test. The presence of polymorphisms in the dhps gene was verified using sequencing. A descriptive analysis for 11 Brazilian isolates was used to assess the association between susceptibility, genotype, and virulence-phenotype. RESULTS: Statistical analysis showed that TgCTBr03, 07, 08, and 16 isolates were susceptible to SDZ, whereas TgCTBr11 isolate presented a profile of resistance to SDZ. Nineteen polymorphisms were identified in dhps exons. Seven polymorphisms corresponded to non-synonymous mutations, with four being new mutations, described for the first time in this study. No association was found between the profile of susceptibility and the virulence-phenotype or genotype of the parasite. CONCLUSIONS: There is a high variability in the susceptibilities of Brazilian T. gondii strains to SDZ, with evidence of drug resistance. Despite the large number of polymorphisms identified, the profile of susceptibility to SDZ was not associated with any of the dhps variants identified in this study. Other genetic factors, not yet determined, may be associated with the resistance to SDZ; thus, further studies are needed as a basis for a more adequate toxoplasmosis treatment.

Cytokine Signatures Associated With Early Onset, Active Lesions and Late Cicatricial Events of Retinochoroidal Commitment in Infants With Congenital Toxoplasmosis.

BACKGROUND: Ocular toxoplasmosis is a prominent and severe condition of high incidence in Brazil. The current study provides new insights into the immunological events that can be associated with retinochoroiditis in the setting of congenital toxoplasmosis in human infants. METHODS: Flow cytometry of intracytoplasmic cytokines in leukocyte subsets following in vitro short-term antigenic recall in infants with congenital T. gondii infection. RESULTS: Our data demonstrates that whereas neutrophils and monocytes from T. gondii-infected infants display a combination of proinflammatory and regulatory cytokine profiles, natural killer cells showed a predominantly proinflammatory profile upon in vitro T. gondii stimulation. The proinflammatory response of CD4(+) and CD8(+) T cells, characterized by the production of interferon γ (IFN-γ) and interleukin 17 in patients with an active retinochoroidal lesion, revealed the presence of IFN-γ and tumor necrosis factor α during early and late immunological events. This specific proinflammatory pattern is associated with early events and active retinochoroidal lesion, whereas a robust monocyte-derived interleukin 10-mediated profile is observed in children with cicatricial ocular lesions. CONCLUSIONS: These findings support the existence of a progressive immunological environment concomitant with the initial, apical, and cicatricial phases in the process of retinochoroidal lesion formation in infants with congenital toxoplasmosis that may be relevant in the establishment of stage-specific clinical management.