Toxoplasma gondii: cloning, sequencing, expression, and antigenic characterization of ROP2, GRA5 and GRA7

Toxoplasma gondii is an intracellular obligate protozoan, which infects humans and warm-blooded animals. The aim of the present study was to clone the rop2, gra5 and gra7 genes from T. gondii RH strain and to produce recombinant proteins. The rop2, gra5 and gra7 gene fragments produced by polymerase chain reaction were cloned into the pET102/D-TOPO(R) vector which contains thioredoxin and polyhistidine tags at the C-and N-ends, respectively, and is expressed in Escherichia coli BL21(DE-3). The expression fusion proteins were found almost entirely in the insoluble form in the cell lysate. These recombinant proteins were purified with an Ni-NTA column. Concentrations of the recombinant antigens produced in the E. coli BL21-star ranged from 300 to 500 mu g/mL growth media, which was used to immunize rabbits. We observed an identity ranging from 96 to 97% when nucleotide sequences were compared to GenBank database sequences. Immunocharacterization of proteins was made by indirect immunofluorescence assay. These proteins will be used for serodiagnosis and vaccination.

Genotyping of Toxoplasma gondii strains isolated from dogs with neurological signs

From 111 dogs with neurological signs admitted in this research in a 22-month period, brain samples of 34 animals were inoculated in mice in order to isolate Toxoplasma gondii. From these 34 dogs, 9 strains of T. gondii were isolated and the genetic characterization performed by restriction analysis (RFLP) of the SAG-2 gene. RFLP analysis showed that four of them were classified as Type I, and five as Type Ill. The present report is the first description of genotyping of T. gondii isolated from brain samples of naturally infected dogs, in Brazil. (C) 2004 Elsevier B.V. All rights reserved.

Leptospira spp. and Toxoplasma gondii antibodies in vampire bats (Desmodus rotundus) in Botucatu region, SP, Brazil

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

Prevalence of antibodies against Toxoplasma gondii among Brazilian white-eared opossums (Didelphis albiventris)

Considering that Toxoplasma gondii is a parasite of global importance which affects several animal species including humans, the current study aimed to investigate the prevalence of antibodies against T. gondii among 72 white-eared opossums (Didelphis albiventris) from Botucatu Municipality (22 degrees 53'S 48 degrees 26'W), São Paulo State, Brazil. The investigation was carried out from January 2008 to December 2009, when the animals had their blood samples collected and subjected to the modified agglutination test (MAT); 12 specimens had brain samples bioassayed in mice. Seroprevalence was 5.5% (n = 4) and bioassays were negative. Older animals had higher prevalence of antibodies against T. gondii. Opossums in closer contact with the urban environment are likely more exposed to T. gondii than animals from the sylvatic environment. (C) 2011 Elsevier B.V. All rights reserved.

Anticorpos contra Toxoplasma gondii em equinos com histórico de ataxia

Stelmann U.J.P., Silva R.C., Langoni H., Borges A.S. & Amorim R.M. [Antibodies against Toxoplasma gondii in horses with history of ataxia]. Anticorpos contra Toxoplasma gondii em equinos corn historic de ataxia. Revista Brasileira de Medicina Veterinaria, 33(4):200-202, 2011. Programa de Pos-Graduacao em Medicina Veterinaria, Departamento de Clinica Veterinaria, Faculdade de Medicina Veterinaria e Zootecnia, Universidade Estadual Paulista Julio de Mesquita Filho, Campus de Botucatu, Distrito de Rubiao Junior, s/n, Botucatu, SP 18618-970, Brasil. E-mail: stelmann.ppgctia@gmail.comThe frequency of antibodies in blood serum and cerebrospinal fluid against Toxoplasma gondii in horses with a history of ataxia from the state of São Paulo, Brazil was carried out. Modified agglutination test was used to determine antibodies against T gondii, considering as positive samples with titers >= 2. of the blood samples tested, only 8 of 23 (34.78%) were positive, while CSF samples were negative when used the same technique. According to the negative results obtained for CSF, we concluded that T gondii was not the etiological agent of the myeloencephalitis in studied horses with neurological manifestation of ataxia, while the blood serological results indicated a previously exposure to T gondii.

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)

Toxoplasma gondii: host-parasite interaction and behavior manipulation

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

Isolation and multilocus genotyping of Toxoplasma gondii in seronegative rodents in Brazil

Synanthropic rodents, mainly rats and mice, become ecologically associated with men due to changes in their ecosystems caused by human activities. These animals may take part in the epidemiological cycles of several diseases, including toxoplasmosis. The presence of serum antibodies to Toxoplasma gondii in 43 rodents captured in the urban area of Umuarama, PR, Brazil, was verified by modified agglutination test (MAT). Brain and heart samples were also collected and bioassayed in mice for the isolation of the parasite. Isolated samples were analyzed by 12 multilocus genotyping. Although all rodents were seronegative, the parasite was isolated in one mouse (Mus musculus) and one rat (Rattus rattus). Genotyping showed that these samples were similar to those previously isolated from cats in the state of Parana, Brazil. (C) 2010 Elsevier B.V. All rights reserved.

Toxoplasma gondii in experimentally infected Bos taurus and Bos indicus semen and tissues

Dezoito bovinos foram inoculados com Toxoplasma gondii e distribuídos aleatoriamente em três grupos de seis bovinos cada: GI (2,5x10(5) oocistos da cepa P), GII (5,0x10(6) taquizoítos da cepa RH) e GIII (controle). Exames clínicos, sorológicos e parasitêmicos foram realizados. Pesquisas do parasito, por meio da bioprova e pela técnica de Reação em Cadeia pela Polimerase (PCR), foram realizadas no sêmen e em fragmentos de musculatura esquelética, linfonodos, cérebro, retina, baço, fígado, pulmão, testículo, epidídimo e vesícula seminal. Amostras de sangue e sêmen foram colhidas nos dias -2, -1, 1, 3, 5, 7, 14 e, semanalmente, até o 84º dia pós-infecção (DPI). Os bovinos inoculados (GI e GII) apresentaram hipertermia do 3º ao 16º DPI. Anticorpos contra T. gondii foram detectados (IFI) no 5º DPI (1:16), em ambos grupos inoculados (oocistos e taquizoítos), atingindo picos de 1:4096 no 7º DPI. Surtos parasitêmicos ocorreram em todos os bovinos infectados, principalmente do 7º ao 28º DPI, independente da cepa e inóculo utilizados. O bioensaio revelou a presença do parasito em amostras seminais dos bovinos infectados com oocistos (GI) e taquizoítos (GII), em diversas datas experimentais, entre o 7º e 84º DPI. Parasitismo tissular por T. gondii foi diagnosticado por meio da bioprova e pela técnica da PCR, em vários fragmentos de tecidos e/ou órgãos. Os achados sugerem a possibilidade da ocorrência da transmissão sexual do T. gondii na espécie bovina.

Anticorpos em bovinos (Bos indicus e Bos taurus) e bubalinos (Bubalus bubalis) inoculados com oocistos de Toxoplasma gondii. Estudo comparativo

Três animais de cada espécie (Bos indicus, Bos taurus e Bubalus bubalis) foram inoculados, via oral, com 2×10(5) oocistos de Toxoplasma gondii. Seis outros animais, dois de cada espécie, foram mantidos como testemunhas. A resposta de anticorpos avaliada por meio da reação de imunofluorescência indireta iniciou-se a partir do quinto dia pós-inoculação (DPI) nos zebuínos e bubalinos, e no sétimo DPI nos taurinos. Os títulos sorológicos nos taurinos permaneceram elevados até o final do experimento (70º DPI), alcançando níveis máximos (1:16.384) entre o 42º e 49º DPI. Nos zebuínos e bubalinos o maior título de anticorpos anti-Toxoplasma foi de 1:256. A resposta de anticorpos mais ou menos acentuada não está necessariamente relacionada à sensibilidade ao T. gondii.

Toxoplasma gondii and Neospora caninum serological status of different canine populations from Uberlândia, Minas Gerais

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Anticorpos contra Toxoplasma gondii em estudantes de medicina veterinária de Campo Grande, MS, Brasil

Amostras de soro obtidas de estudantes do curso de Medicina Veteriná ria da Universidade para o Desenvolvimento do Estado e da Região do Pantanal, Campo Grande, MS, Brasil, foram examinadas para a presença de anticorpos contra Toxoplasma gondii. Dos 145 soros testados, 44 (30,34%) foram positivos na hemaglutinação, com título igual ou superior a 1:16. Não foram observadas associações entre as caracterí sticas epidemiológicas examinadas, tais como hábitos alimentares (ingestão de carne bovina crua ou malpassada, vegetais crus/não lavados, produtos lácteos não pasteurizados) ou contato constante com cães e a presença de anticorpos contra T. gondii, exceto pelo percentual significativamente maior de estudantes soropositivos que relataram ter contato freqüente com gatos (P=0,03).

Seroprevalence of Toxoplasma gondii and Neospora caninum in captive maned wolves (Chrysocyon brachyurus) from southeastern and midwestern regions of Brazil

The main purpose of the present study was to investigate the occurrence of antibodies against T gondii and N. caninum in captive maned wolves from Brazil, considering that little information is available at the literature about infections by these parasites in this wild animal. Serum samples were obtained from 59 maned wolves originated from six zoos and from one ecological reserve of the southeastern and midwestern regions of Brazil. To detect IgG antibodies against T gondii, an ELISA protocol was used and the results were expressed as ELISA reactivity indexes (131). Serology for N. caninum was carried out by indirect fluorescent antibody test (IFAT) and cut-off titers were established at 1:25 dilution. From the total of the analyzed samples, 44 (74.6%) were seropositive for T gondii and only 5 (8.5%) for N. caninum. Seropositivity for T gondii ranged from 0 to 100% in the seven different origin locals, with rates over 50% among the six zoos, whereas no positivity was found in the samples from ecological reserve. For N. caninum, seroprevalence varied from 0 to 50% in the different locals, with the highest rates also detected in zoos. Seroprevalence for T gondii was strongly related with age, with rates significantly higher among adult wolves (91.7%) when compared to newborn or young animals. Seropositive samples for N. caninum were found predominantly in adult wolves. For both parasites, seroprevalence did not show a significant distinction in relation to gender. Although seroprevalence for T gondii was significantly higher when compared to N. caninum in the Brazilian captive maned wolves tested, these findings reflect the great exposure of this species to T gondii and, in lower extension, to N. caninum. Also, the present study demonstrated for the first time the presence of antibodies to N. caninum in wild life from South America. (C) 2004 Elsevier B.V. All rights reserved.