Partial protection against tissue cysts formation in pigs vaccinated with crude rhoptry proteins of Toxoplasma gondii

A vaccine containing crude Toxoplasma gondii rhoptry proteins incorporated in the immunostimulating complexes (ISCOM) adjuvant was tested in pigs for protecting against tissue cyst formation. For this, 38 mixed breed pigs were divided into four groups, G1 (vaccinated challenged, n = 10) received two doses (100 mu g/dose) of the rhoptry vaccine at days 0 and 21, G2 (vaccinated challenged, n = 10) received viable tachyzoites (7 x 10(7)) of the RH strain at day 0, G3 (unvaccinated challenged, n = 10) and G4 (unvaccinated unchallenged, n = 8). Pigs were challenged with 4 x 10(4) VEG strain oocysts 57 days later. The G1 pigs produced high IgG antibody levels in the indirect enzyme-linked immunosorbent assay (ELISA) after the second dose of rhoptry vaccine, but were not clinically protected against a high dose oocyst challenge. Partial protection was observed in G1 at the chronic phase of infection, when compared with G3. Pigs in group 2 developed high antibody levels and were protected against clinic signs. T gondii was not detected in two (G1) and three (G2) pigs by mouse bioassay. The results indicate partial protection in pigs vaccinated with a rhoptry vaccine. (c) 2005 Elsevier B.V. All rights reserved.

Use of near-infrared Raman spectroscopy to detect IgG and IgM antibodies against Toxoplasma gondii in serum samples of domestic cats

Near-infrared Raman spectroscopy (NIRS) is a particularly promising technique that is being used in recent years for many biomedical applications. Optical spectroscopy has gained increasing prominence as a tool for quantitative analysis of biological samples, clinical diagnostic, concentration measurements of blood metabolites and therapeutic drugs, and analysis of the chemical composition of human tissues. Toxoplasmosis is an important zoonosis in public health, and domestic cats are the most important transmitters of the disease. This disease can be detected by several serological tests, which usually have a high cost and require a long time. The goal of this work was to investigate a new method to diagnosis Toxoplasma gondii infections using NIRS. In order to confirm antibody detection, 24 cat blood scrum samples were analyzed by the Raman spectra, from which 23 presented positive serology to toxoplasmosis and one was a reference negative serum. Characteristic Raman peaks allowed differentiation between negative and positive sera, confirming the possibility of antibody detection by Raman spectroscopy. These results give the first evidence that this technique can be useful to quantify antibodies in cat sera.

Histopathological analysis of the reproductive system of male dogs experimentally infected with Toxoplasma gondii

O presente trabalho teve como objetivo descrever eventuais alterações histopatológicas no sistema reprodutor (testículo e epidídimo) de cães machos experimentalmente infectados com Toxoplasma gondii. Para tal, 10 animais sorologicamente negativos para T. gondii foram selecionados e distribuídos em três grupos experimentais: GI - três cães inoculados com 2,0 x 10(5) oocistos da cepa P, GII - três cães infectados com 1,0 x 10(6) taquizoítos da cepa RH e GIII - quatro cães mantidos como controle. Pesquisa de anticorpos (IFI) contra T. gondii foi realizada. A infecção por T. gondii confirmou-se pela soroconversão de todos os machos infectados a partir do 7° e do 14° dia pós-inoculação (DPI) para cães que receberam taquizoítos e oocistos respectivamente. Decorridos 70DPI, realizou-se, em todos os cães, orquiectomia, e amostras (testículo e epidídimo) foram coletadas e processadas histologicamente para leitura em microscópio óptico. As seguintes alterações foram diagnosticadas: infiltrado inflamatório mononuclear leve e moderado em epidídimo, edema celular moderado, degeneração hidrópica e fibrose intersticial moderada em túbulos seminíferos. Os resultados histopatológicos do presente trabalho, aliados ao isolamento do T. gondii em fragmentos de testículo e epidídimo pela imunoistoquímica, juntamente com os resultados encontrados na literatura por outros autores em diferentes tecidos, permitem inferir que as alterações encontradas nos cães infectados com o respectivo protozoário são sugestivas de infecção toxoplásmica.

Toxoplasma gondii: Evaluation of an intranasal vaccine using recombinant proteins against brain cyst formation in BALB/c mice

The purpose of this work was to evaluate protective activity against brain cyst formation in BALB/c mice intranasally vaccinated with recombinant proteins from Toxoplasma gondii. The recombinant proteins rROP2, rGRA5 and rGRA7 were used in vaccine preparation. Thirty-three female mice were divided into three groups, these animals received two doses by intranasal route at days 0 and 21 as follows; group 1 (G1, n = 11) received 12.5 mu g of each recombinant protein plus 0.5 mu g of cholera toxin, group 2 (G2, n = 11) received phosphate buffer saline (PBS) plus 0.5 mu g of cholera toxin, and group 3 (G3, n = 11) received PBS only. At challenge day (day 33) three animals from each group were euthanatized for IgA measure from intestine. Mice were infected orally with 50 cysts from the VEG strain at day 33. At challenge day the G1 animals had high immunoglobulin A levels, however, they only showed IgG antibody titers against rROP2 and rGRAT Animals from G1 also exhibited strong resistance to cyst formation compared with the control group (G3, P < 0.05). However, we did not observe differences in protection against brain cyst formation between G1 and G2 (P > 0.1). These results indicate that intranasal immunization in BALB/c mice with recombinant proteins rROP2, rGRA5 and rGRA7 associated with cholera toxin induced partial protection, when compared with G3, against tissue cyst formation after oral infection with tissue cysts from T gondii. (c) 2007 Elsevier B.V. All rights reserved.

Detection of Toxoplasma gondii oocysts in environmental samples from public schools

The number of Toxoplasma gondii oocysts that can be found in random environmental samples is probably low; in addition, these cysts may be confused with Hammondia spp. and Neospora spp. oocysts. The aim of the present work was to evaluate the presence of T. gondii oocysts in the soil of public elementary schools in the northwest area of the state of São Paulo, Brazil using mouse bioassays. A comparison was made between the different available bioassay techniques, such as squash, histopathology, immunohistochemistry and indirect fluorescent antibody test (IFAT). T. gondii was isolated by bioassay in mice (squash brain samples) from 22.58%(7/31) of the school playgrounds. Immunohistochemistry and IFAT showed positive results in 32.26% (10/31) and 25.80% (8/31) of samples, respectively. The sensitivity and specificity of the immunohistochemistry method were 85.71% and 83.33%, respectively. The IFAT results showed 100% sensitivity and 95.83% specificity. The presence of T. gondii was not detected in histopathological examinations. The results of the present study strongly suggest that T. gondii oocysts are widely distributed in elementary public schools in the region that was evaluated, likely constituting the main contamination source for these children. Educational programs directed at reducing environmental contamination with T. gondii would eventually lower the cost of treating humans for clinical toxoplasmosis. It is also possible to conclude that the use of IFAT in mouse bioassays can be recommended without the need for brain cysts research, which is extremely difficult and laborious. (C) 2010 Elsevier B.V. All rights reserved.

Occurrence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in cats with outdoor access in São Luís, Maranhão, Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The protective effects of Baypamun®HK in mice experimentally infected with Toxoplasma gondii

O presente trabalho teve como objetivo verificar os efeitos do Baypamun®HK sobre a sobrevida de camundongos albinos, experimentalmente infectados com Toxoplasma gondii amostra RH, submetidos ou não ao tratamento com sulfadiazina-pirimetamina, bem como sobre a formação e número de cistos cerebrais. Quatro grupos de 20 camundongos foram inoculados com 10(5) taquizoítos, via subcutânea, e submetidos a diferentes tratamentos. Os testes sorológicos foram realizados pela técnica de imunofluorescência indireta, e, dos animais que sucumbiram, amostras de cérebro, pulmão e fígado foram retiradas para exame citológico e de cérebros para a pesquisa de cistos. Ao final de 60 dias todos os sobreviventes foram sacrificados. Isoladamente o Baypamun®HK não apresentou uma ação protetora contra a infecção pelo T. gondii, mas sua associação com o tratamento específico pela sulfadiazina-pirimetamina promoveu maior sobrevida dos animais e uma resposta de anticorpos mais intensa e duradoura.

Fatores de risco associados à infecção por Toxoplasma gondii em ovinos e caprinos no estado de Pernambuco

Objetivou-se com este estudo investigar a participação de Toxoplasma gondii em falhas reprodutivas em pequenos ruminantes de criatórios situados na Zona da Mata e no Agreste do Estado de Pernambuco e que apresentavam histórico de distúrbios reprodutivos. Foram selecionadas 12 propriedades das quais se coletaram amostras de 262 animais, sendo 167 caprinos e 95 ovinos. Realizou-se a pesquisa de anticorpos anti-Toxoplasma gondii, utilizando-se a técnica da Imunofluorescência Indireta (RIFI). Foram aplicados questionários investigativos nas propriedades visitadas para identificar os fatores de risco associados à infecção. em 100% das propriedades foram encontrados animais soropositivos. Para T. gondii, das 167 amostras de soro caprino analisadas, 31,7% foram positivas, enquanto que na espécie ovina, das 95 amostras, 16,9% foram positivas. Para a espécie ovina, não foram observadas associações significativas. Para os caprinos, houve associação significativa (p<0,05) para as variáveis: manejo intensivo (OR=2,40), exploração leiteira (OR=2,10), animais procedentes de outros estados (OR=7,89) e monta natural (OR=5,69). Conclui-se que a infecção pelo T. gondii encontra-se disseminada nos rebanhos de caprinos e ovinos estudados e que medidas sanitárias devem ser adotadas para controlar os fatores de risco identificados neste estudo.

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 (RIFIe16) 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.

Detection and molecular analysis of Toxoplasma gondii and Neospora caninum from dogs with neurological disorders

Introduction: Toxoplasma gondii and Neospora caninum are related Apicomplexa parasites responsible for systemic diseases in many species of animals, including dogs. Methods: This study aimed to determine the occurrence of T. gondii and N. caninum infections in 50 dogs with neurological signs that were admitted to the Veterinary Hospital of Universidade Estadual Paulista, City of Botucatu, Brazil. All animals were screened for antibodies using an immunofluorescent antibody test for both parasites. Tissues of positive animals were bioassayed in mice (T. gondii) and gerbils (N. caninum), and DNA was analyzed using the polymerase chain reaction (PCR). Positive samples for T. gondii by PCR were typed using restriction fragment length polymorphism-PCR for 11 markers: SAG1, SAG2 (5'-3'-SAG2 and alt.SAG2), SAG3, Btub, GRA6, L358, c22-8, c29-6, PK1 and Apico, and CS3 marker for virulence analysis. Results: Specific antibodies were detected in 11/50 (22%; 95% confidence interval (CI95%), 12.8-35.3%) animals for T. gondii and 7/50 (14%; CI95%, 7.02-26.3%) for N. caninum. In the bioassay and PCR, 7/11 (63.6%; CI95%, 34.9-84.8%) samples were positive for T. gondii and 3/7 (42.9%; CI95% I, 15.7-75.5%) samples were positive for N. caninum. Three different genotypes were identified, but only 1 was unique. Conclusions: These data confirm the presence of T. gondii and N. caninum in dogs from Brazil, indicating the importance of this host as a sentinel of T. gondii for human beings, and the genotypic variation of this parasite in Brazil.

Detection of Toxoplasma gondii in the milk of experimentally infected Wistar female rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Genotypic characterization of Toxoplasma gondii in sheep from Brazilian slaughterhouses: New atypical genotypes and the clonal type II strain identified

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Chlamydophila psittaci and Toxoplasma gondii infection in pigeons (Columba livia) from São Paulo State, Brazil

Pigeons (Columba livia) cohabit with humans in urban and rural areas, representing a public health problem since microorganisms are transmitted through the inhalation of dust from their dry feces (chlamydiosis) and through ingestion of their undercooked or poorly refrigerated meat (toxoplasmosis). This study aimed to evaluate the presence of Chlamydophila psittaci and Toxoplasma gondii in pigeons from four cities in São Paulo State, Brazil. C psittaci was evaluated through hemi-nested polymerase chain reaction (hnPCR) using cloacal and tracheal swabs, whereas T. gondii specific antibodies were assessed by means of modified agglutination test (MAT), mouse brain and muscle bioassay, and polymerase chain reaction (PCR). To confirm the infection in mice, T. gondii antibodies were assayed by using indirect fluorescent antibody test (IFAT). Considering C. psittaci, 40/238 (16.8%; 95%CI 12.6-22.1%) samples were positive according to hnPCR, especially for the cities of São Paulo (42.5%) and Bauru (35%). As regards T. gondii, 12/238 (5%; 95%CI 2.9-8.6%) serum samples were positive according to MAT. of these, five samples had titer equal to 1:8; six samples, 1:16; and one sample, 1:32. Bioassay, IFAT and PCR were negative for mouse toxoplasmosis. The absence of T. gondii antibodies suggests that pigeons may be infected with a low concentration of the agent, not detected by the antigen test. Thus, C. psittaci represents an actual problem concerning bird health. (C) 2010 Elsevier B.V. All rights reserved.

Serological profile of Toxoplasma gondii and Neospora caninum infection in commercial sheep from São Paulo State, Brazil

Neosporosis and toxoplasmosis are two important infections in young and adult sheep, leading to low production and abortion. This study aimed to determine the frequency of antibodies to Toxoplasma gondii and Neospora caninum in sheep from the eastern region of São Paulo State, Brazil. Serum samples (382) were collected from the sheep and assayed for T. gondii through modified agglutination test (MAT) and indirect fluorescent antibody test (IFAT), and for N. caninum antibodies, through IFAT, with cut-off titers equal to 16 (T. gondii) and 25 (N. caninum). All frozen samples were sent to the Center for Zoonoses Research (NUPEZO), Department of Veterinary Hygiene and Public Health (DHSVP), FMVZ, UNESP, for serological tests. A total of 71/382(18.6%) samples reacted to T. gondii, especially at titers 16 (28; 39.4%), 64(15; 21.1%), 256 (21; 29.6%) and 1024 (6; 8.5%) by MAT, and 16(34: 47.9%), 64 (18; 25.4%), 256 (14; 19.7%) and 1024 (5; 7%) by IFAT. As regards N. caninum, 49/382 (12.8%) samples reacted at titers 25 (17; 34.7%), 50 (11; 22.5%), 100 (11; 22.5%), and >= 200 (10; 20.4%). These animals presented infection but no clinical signs. Six and ten animals had high titers for toxoplasmosis and neosporosis. No significant association was observed between antibodies for both parasites (P = 0.535) according to Fisher's exact test, and no correlation was found between T. gondii (MAT) and N. caninum antibody titers (r = -0.0068; P=0.895), T. gondii (IFAT) and N. caninum antibody titers (r = -0.0025; P = 0.961). Thus, T. gondii and N. caninum infections were observed in farms located in São Paulo State, where sheep play an important economical role for the national and regional business. (C) 2010 Elsevier By. All rights reserved.

Toxoplasma gondii and Leishmania spp. infection in captive crab-eating foxes, Cerdocyon thous (Carnivora, Canidae) from Brazil

The zoonoses toxoplasmosis and leishmaniasis are important worldwide and also affect wild animals. Thus, the present study aimed to assess the prevalence of Leishmania spp. and Toxoplasma gondii antibodies in 52 serum samples from captive crab-eating foxes (Cerdocyon thous) kept in 17 zoos in São Paulo State, Brazil. Modified agglutination test (MAT, for toxoplasmosis) and indirect fluorescent antibody test (IFAT, for toxoplasmosis and leishmaniasis) were employed with heterologous anti-dog immunoglobulin. Antibodies to T. gondii were found in 19.2% animals, with an almost perfect concordance (kappa = 0.86; standard error = 9.31%; CI95% = 68.25-104.76%; P < 0.0001) and a strong correlation coefficient (rs = 0.87; P < 0.0001), which allows the use of heterologous anti-dog immunoglobulin to perform IFAT for toxoplasmosis in crab-eating foxes. No sample was positive for Leishmania spp. Toxoplasmosis infection occurs in wild animals from the studied Brazilian zoos, which indicates a probable environmental contamination, highlighting the importance of appropriate zoo management and the action of the parasite as a sentinel to human infection. (C) 2009 Elsevier B.V. All rights reserved.