Inhibition of nitric oxide production of activated mice peritoneal macrophages is independent of the Toxoplasma gondii strain

BACKGROUND Toxoplasma gondii causes toxoplasmosis and is controlled by activated macrophages. However, infection of macrophages by tachyzoites induces TGF-β signaling (TGF-s) inhibiting nitric oxide (NO) production. NO inhibition may be a general escape mechanism of distinct T. gondii strains. OBJECTIVES To evaluate in activated macrophages the capacity of T. gondii strains of different virulence and genetics (RH, type I; ME-49, type II; VEG, type III; P-Br, recombinant) to evade the NO microbicidal defense system and determine LC3 loading to the parasitophorous vacuole. METHODS Activated peritoneal macrophages were infected with the different T. gondii strains, NO-production was evaluated by the Griess reagent, and inducible nitric oxide synthase expression, TGF-s, and LC3 localisation assayed by immunofluorescence. FINDINGS Only RH persisted in macrophages, while VEG was more resistant than P-Br and ME-49. All strains induced TGF-s, degradation of inducible nitric oxide synthase, and NO-production inhibition from 2 to 24 h of infection, but only RH sustained these alterations for 48 h. By 24 h of infection, TGF-s lowered in macrophages infected by ME-49, and P-Br, and NO-production recovered, while VEG sustained TGF-s and NO-production inhibition longer. LC3 loading to parasitophorous vacuole was strain-dependent: higher for ME-49, P-Br and VEG, lower for RH. All strains inhibited NO-production, but only RH sustained this effect probably because it persisted in macrophages due to additional evasive mechanisms as lower LC3 loading to parasitophorous vacuole. MAIN CONCLUSIONS These results support that T. gondii can escape the NO microbicidal defense system at the initial phase of the infection, but only the virulent strain sustain this evasion mechanism.

Serological evidence of exposure to Toxoplasma gondii and Neospora caninum in free-ranging Orinoco goose (Neochen jubata) in Brazil / Evidência sorológica de exposição à Toxoplasma gondii e Neospora caninum em Ganso-do-Orinoco (Neochen jubata) de vida livre no Brasil

Abstract Toxoplasma gondii and Neospora caninum are Apicomplexan intracellular protozoan parasites that affect numerous animal species, thus leading to severe diseases and economic losses, depending on the vertebrate species involved. The role of the avian species in maintaining and transmission of these coccidia has been studied for several years as they tend to serve as a potential source of infection for mammals and humans. The present study aimed to assess the serological exposure of Orinoco goose (Neochen jubata) to T. gondii and N. caninum. Between 2010 and 2013, 41 free-ranging Orinoco geese were captured in the Araguaia River, Brazil. The presence and titration of IgY antibodies to both coccidia were assayed via indirect immunofluorescent antibody test (IFAT). While IgY antibodies for N. caninum were present in 5 animals, with titers of 20, the antibodies for T. gondii were found in 35 animals, with titers ranging from 20 to 640. Considering that the Orinoco goose's meat is consumed by the local population in the studied area, it may represent an important source of T. gondii infection for humans. Due to its migratory behavior, this goose may play a pivotal role in the natural dispersion of both parasites. Furthermore, molecular studies are required for genotyping the isolates of T. gondii that occurs in this avian species.

Resumo Toxoplasma gondii e Neospora caninum são parasitas protozoários intracelulares do philo Aplicomplexa que afetam uma vasta gama de espécies animais, causando sérias doenças e levando a perdas econômicas, dependendo da espécie envolvida. O papel das aves na manutenção e transmissão destes coccídios tem sido estudado por anos, já que eles são potenciais fontes de infecção para outros animais e humanos. O objetivo deste estudo foi avaliar a exposição do Ganso-do-Orinoco (Neochen jubata) a T. gondii e N. caninum por meio de técnicas sorológicas. Entre os anos de 2010 e 2013, 41 Gansos-do-Orinoco de vida livre foram capturados no Vale do Rio Araguaia, Brasil. A presença e titulação de anticorpos IgY para ambos os coccídios foi obtida utilizando-se a Reação de Imunofluorescência Indireta (RIFI). Enquanto a presença de anticorpos IgY para N. caninum foi detectada em 5 aves, com titulação 20, anticorpos para T. gondii foram encontrados em 35 aves, com títulos variando de 20 a 640. Considerando que a carne do Ganso-do-Orinoco é uma fonte de alimento para a população da área estudada, a ave pode representar uma importante fonte de infecção de T. gondii para humanos. Devido ao seu comportamento migratório, esta espécie assume grande importância na dispersão de ambos os parasitas. Estudos moleculares são necessários a fim de caracterizar genotipicamente os isolados de T. gondii que ocorrem nesta espécie de ave.

Serosurvey for Leishmania spp, Toxoplasma gondii, Trypanosoma cruzi and Neospora caninum in neighborhood dogs in Curitiba-Paraná, Brazil / Inquérito sorológico para Leishmania spp, Toxoplasma gondii, Trypanosoma cruzi e Neospora caninum em cães comunitários em Curitiba-Paraná, Brasil

Abstract Neighborhood dogs may act as reservoirs for several zoonotic protozoan infections, particularly in urban areas, thus constituting a potential public health threat. Accordingly, the aim of the present study was to evaluate the exposure of neighborhood dogs to four protozoan pathogens in public areas with high levels of human movement in Curitiba, southern Brazil. Blood samples from 26 neighborhood dogs were screened by means of the indirect immunofluorescent antibody test (IFAT) for Leishmania spp., Toxoplasma gondii, Trypanosoma cruzi and Neospora caninum, and a questionnaire was answered by the respective keeper. A total of 8/26 dogs (30.7%) seroreactive to T. gondii, 3/26 (11.5%) to N. caninum and 2/26 (7.7%) to both were identified. All the samples were seronegative for T. cruzi and Leishmania spp. Pathogen seroreactivity was not associated with the daily human movements or other epidemiological variables investigated (p > 0.05). In conclusion, the low seroprevalence for T. gondii and N. caninum indicated low environmental and food risk for animal infection and the seronegativity for Leishmania spp. and T. cruzi may reflect the absence of these pathogens in urban areas of Curitiba. Moreover, neighborhood dogs may be used as environmental sentinels for the presence of protozoan pathogens and their vectors.

Resumo Cães comunitários podem atuar como reservatórios para algumas zoonoses causadas por protozoários, principalmente em áreas urbanas, constituindo potencial ameaça à saúde pública. Portanto, o objetivo do presente estudo foi avaliar a exposição de cães comunitários a quatro protozoários em áreas públicas com alta circulação de pessoas, em Curitiba, Sul do Brasil. Amostras de sangue de 26 cães comunitários foram testadas pela reação de imunofluorescência indireta (RIFI) para Leishmania spp., Toxoplasma gondii, Trypanosoma cruzi e Neospora caninum, e um questionário foi respondido pelo respectivo mantenedor. Um total de 8/26 (30,7%) foram sororreagentes para T. gondii, 3/26 (11,5%) para N. caninum e 2/26 (7,7%) para ambos. Todas as amostras foram soronegativas para T. cruzi e Leishmania spp. Não houve associação entre sororreatividade para os patógenos pesquisados e o tráfego diário de pessoas e outras variáveis epidemiológicas analisadas (p > 0.05). Conclui-se a baixa soroprevalência para T. gondii e T. cruzi indica baixo risco ambiental e alimentar para a infecção dos animais, e a soronegatividade para Leishmania spp. e T. cruzi pode refletir a ausência desses patógenos em áreas urbanas de Curitiba. Além disso, os cães comunitários podem atuar como sentinelas ambientais quanto à presença de protozoários e seus vetores.

Anti-Toxoplasma gondii and anti-Neospora caninum antibodies in capybaras (Hydrochoerus hydrochaeris) from Itu Municipality, São Paulo / Anticorpos anti-Toxoplasma gondii e anti-Neospora caninum em capivaras (Hydrochoerus hydrochaeris) do Município de Itu, São Paulo

Abstract Capybaras (Hydrochoerus hydrochaeris) are the largest rodents found in South America. The aim of the present study was to investigate the occurrence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in 170 free-living capybaras in a residential park area in Itu Municipality, São Paulo State, Brazil. Serum samples were tested by indirect fluorescent antibody test (IFAT) for T. gondii (IFAT ≥ 1:16) and N. caninum (IFAT ≥ 1:50). Among the 170 samples analyzed, 10% (17/170) and 0% (0/170) were seropositive for T. gondii and N. caninum, respectively. This study confirms the widespread presence of T. gondii and reinforces the role of capybaras in the life cycle of this parasite. Capybaras may not be important as intermediate hosts of N. caninum in the studied environment.

Resumo As capivaras (Hydrochoerus hydrochaeris) são os maiores roedores encontrados na América do Sul. O objetivo do presente estudo foi investigar a ocorrência de anticorpos anti-Toxoplasma gondii e anti-Neospora caninum em 170 capivaras de vida livre, habitando em um condomínio fechado no Município de Itu, Estado de São Paulo, Brasil. Amostras de soro foram testadas por meio da Reação de Imunofluorescência Indireta (RIFI) para T. gondii (RIFI ≥ 1:16) e N. caninum (RIFI ≥ 1:50). Entre as 170 amostras analisadas, 10% (17/170) e 0% (0/170) foram positivas para T. gondii eN. caninum, respectivamente. O presente estudo confirma a ampla distribuição de T. gondii e reforça o papel das capivaras no ciclo biológico desse parasita. Provavelmente, as capivaras não têm um papel importante como hospedeiros intermediários de N. caninum no ambiente estudado.

Isolation and Genotyping of Toxoplasma gondii Strains in Ovine Aborted Fetuses in Khorasan Razavi Province, Iran

Toxoplasmosis is an important zoonotic disease that can cause abortion in humans and animals. The aim of this study was isolation and subsequent genotyping of Toxoplasma gondii isolates in ovine aborted fetuses. During 2012-2013, 39 ovine aborted fetuses were collected from sheep flocks in Khorasan Razavi Province, Iran. The brain samples were screened for detection of the parasite DNA by nested PCR. The positive brain samples were bioassayed in Webster Swiss mice. The serum samples of mice were examined for T. gondii antibodies by IFAT at 6 weeks post inoculation, and T. gondii cysts were searched in brain tissue samples of seropositive mice. The positive samples were genotyped by using a PCR-RLFP method. Subsequently, GRA6 sequences of isolates were analyzed using a phylogenetic method. The results revealed that T. gondii DNA was detected in 54% (20/37, 95% CI 38.4-69.0%) brain samples of ovine aborted fetuses. In bioassay of mice, only 2 samples were virulent and the mice were killed at 30 days post inoculation, while the others were non-virulent to mice. The size of cysts ranged 7-22 µm. Complete genotyping data for GRA6 locus were observed in 5 of the 20 samples. PCR-RLFP results and phylogenetic analysis revealed that all of the isolated samples were closely related to type I. For the first time, we could genotype and report T. gondii isolates from ovine aborted fetuses in Khorasan Razavi Province, Iran. The results indicate that the T. gondii isolates are genetically related to type I, although most of them were non-virulent for mice.

Application of different techniques to detect Toxoplasma gondii in slaughtered sheep for human consumption / Utilização de diferentes técnicas para detecção de Toxoplasma gondii em ovinos abatidos para consumo humano

Abstract The aim of this study was to investigate occurrence of Toxoplasma gondii in sheep slaughtered in the state of Alagoas, Brazil, by means of different diagnosis techniques. Serum samples and tissues from 100 slaughtered sheep were used. To detect antibodies, the indirect immunofluorescence antibody test (IFAT) was used, and tissues from seropositive animals (cut-off ≥1:64) were submitted to Polymerase Chain Reaction (PCR) and immunohistochemistry (IHC). To assess the concordance between the direct techniques, the kappa test was used. In the IFAT, it was observed that 14% (14/100) of the ovine samples were serum-positive. In the PCR, 21.43% (3/14) of the animals were positive and in IHC, it was observed that 7.14% (1/14) were positively stained for T. gondii in cerebral tissue. Histopathologically, the predominant finding was the presence of mononuclear cell infiltrate in the heart and a perivascular cuff in the cerebrum and cerebellum. The concordance between the direct diagnosis techniques was moderate (k=0.44). Thus, it is important to use different direct techniques in diagnosing toxoplasmosis in naturally infected sheep.

Resumo O objetivo deste estudo foi pesquisar a ocorrência de Toxoplasma gondii em ovinos abatidos no Estado de Alagoas, Brasil por meio de diferentes técnicas de diagnóstico. Foram utilizadas amostras de soros e tecidos de 100 ovinos abatidos. Para a pesquisa de anticorpos foi utilizada a Reação de Imunofluorescência Indireta (RIFI), e os tecidos dos animais soropositivos (ponto de corte ≥1:64) foram submetidos às técnicas de Reação de Cadeia da Polimerase (PCR) e Imunohistoquímica (IHQ). Para o estudo da concordância entre as técnicas diretas foi empregado o teste Kappa. Na RIFI, 14% (14/100) das amostras foram soro-positivas. Na PCR, 21,43% (3/14) dos animais foram positivos e, na IHC, 7,14% (1/14) apresentaram marcação positiva para T. gondii no tecido cerebral. Na histopatologia, o achado predominante foi o infiltrado celular mononuclear no coração e manguito perivascular no cérebro e cerebelo. A concordância entre as técnicas diretas de diagnóstico foi moderada (K= 0,44). Desse modo, é importante utilizar diferentes técnicas diretas no diagnóstico da toxoplasmose em ovinos naturalmente infectados.

An atypical Toxoplasma gondii genotype in a rural Brazilian dog co-infected with Leishmania (Viannia) braziliensis

Toxoplasmosis and leishmaniasis are two worldwide zoonoses caused by the protozoan parasites Toxoplasma gondii and Leishmania spp., respectively. This report describes the clinical and laboratorial findings of a co-infection with both parasites in a 4-year-old female dog suspected of ehrlichiosis that presented anemia, thrombocytopenia, hypoalbuminemia, hyperglobulinemia, tachyzoite-like structures to the lung imprints, and polymerase chain reaction (PCR) results positive for T. gondii (kidney, lung, and liver) and Leishmania spp. Co-infection with Toxoplasma gondii and Leishmania braziliensis was confirmed by sequencing; restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) confirmed an atypical T. gondii genotype circulating in dogs that has been reported to cause human congenital toxoplasmosis.

Toxoplasmosis in a Pet Peach-Faced Lovebird (Agapornis roseicollis)

Toxoplasma gondii atypical type II genotype was diagnosed in a pet peach-faced lovebird (Agapornis roseicollis) based on histopathology, immunohistochemistry, and multilocus DNA typing. The bird presented with severe neurological signs, and hematology was suggestive of chronic granulomatous disease. Gross post-mortem examination revealed cerebral hemorrhage, splenomegaly, hepatitis, and thickening of the right ventricular free wall. Histologic sections of the most significant lesions in the brain revealed intralesional protozoan organisms associated with malacia, spongiform changes, and a mild histiocytic response, indicative of diffuse, non-suppurative encephalitis. Immunohistochemistry confirmed the causative organisms to be T. gondii. DNA isolated from the brain was used to confirm the presence of T. gondii DNA. Multilocus genotyping based on SAG1, altSAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico markers demonstrated the presence of ToxoDB PCR-RFLP genotype #3 and B1 gene as atypical T. gondii type II. The atypical type II strain has been previously documented in Australian wildlife, indicating an environmental transmission route.

Sequence Diversity in MIC6 Gene among Toxoplasma gondii Isolates from Different Hosts and Geographical Locations

Toxoplasma gondii is an opportunistic protozoan parasite that can infect almost all warm-blooded animals including humans with a worldwide distribution. Micronemes play an important role in invasion process of T. gondii, associated with the attachment, motility, and host cell recognition. In this research, sequence diversity in microneme protein 6 (MIC6) gene among 16 T. gondii isolates from different hosts and geographical regions and 1 reference strain was examined. The results showed that the sequence of all the examined T. gondii strains was 1,050 bp in length, and their A + T content was between 45.7% and 46.1%. Sequence analysis presented 33 nucleotide mutation positions (0-1.1%), resulting in 23 amino acid substitutions (0-2.3%) aligned with T. gondii RH strain. Moreover, T. gondii strains representing the 3 classical genotypes (Type I, II, and III) were separated into different clusters based on the locus of MIC6 using phylogenetic analyses by Bayesian inference (BI), maximum parsimony (MP), and maximum likelihood (ML), but T. gondii strains belonging to ToxoDB #9 were separated into different clusters. Our results suggested that MIC6 gene is not a suitable marker for T. gondii population genetic studies.

Genetic Diversity of Toxoplasma gondii Strains from Different Hosts and Geographical Regions by Sequence Analysis of GRA20 Gene

Toxoplasma gondii is a eukaryotic parasite of the phylum Apicomplexa, which infects all warm-blood animals, including humans. In the present study, we examined sequence variation in dense granule 20 (GRA20) genes among T. gondii isolates collected from different hosts and geographical regions worldwide. The complete GRA20 genes were amplified from 16 T. gondii isolates using PCR, sequence were analyzed, and phylogenetic reconstruction was analyzed by maximum parsimony (MP) and maximum likelihood (ML) methods. The results showed that the complete GRA20 gene sequence was 1,586 bp in length among all the isolates used in this study, and the sequence variations in nucleotides were 0-7.9% among all strains. However, removing the type III strains (CTG, VEG), the sequence variations became very low, only 0-0.7%. These results indicated that the GRA20 sequence in type III was more divergence. Phylogenetic analysis of GRA20 sequences using MP and ML methods can differentiate 2 major clonal lineage types (type I and type III) into their respective clusters, indicating the GRA20 gene may represent a novel genetic marker for intraspecific phylogenetic analyses of T. gondii.

Sequence Variation in Superoxide Dismutase Gene of Toxoplasma gondii among Various Isolates from Different Hosts and Geographical Regions

Toxoplasma gondii, an obligate intracellular protozoan parasite of the phylum Apicomplexa, can infect all warm-blooded vertebrates, including humans, livestock, and marine mammals. The aim of this study was to investigate whether superoxide dismutase (SOD) of T. gondii can be used as a new marker for genetic study or a potential vaccine candidate. The partial genome region of the SOD gene was amplified and sequenced from 10 different T. gondii isolates from different parts of the world, and all the sequences were examined by PCR-RFLP, sequence analysis, and phylogenetic reconstruction. The results showed that partial SOD gene sequences ranged from 1,702 bp to 1,712 bp and A + T contents varied from 50.1% to 51.1% among all examined isolates. Sequence alignment analysis identified total 43 variable nucleotide positions, and these results showed that 97.5% sequence similarity of SOD gene among all examined isolates. Phylogenetic analysis revealed that these SOD sequences were not an effective molecular marker for differential identification of T. gondii strains. The research demonstrated existence of low sequence variation in the SOD gene among T. gondii strains of different genotypes from different hosts and geographical regions.

Polimorfismos en los genes de dihidrofolato-reductasa ( dhfr ) y dihidropteroato-sintasa ( dhps ) y modelado estructural del gen dhps en aislamientos colombianos de Toxoplasma gondii / Gene polymorphisms in the dihydrofolate reductase ( dhfr ) and dihydropteroate synthase ( dhps ) genes and structural modelling of the dhps gene in Colombian isolates of Toxoplasma gondii

Introducción. No existen reportes sobre las variaciones en la secuencia de los genes blanco de los medicamentos anti- Toxoplasma en aislamientos provenientes de Suramérica. Objetivo. Clonar y secuenciar los genes de la dihidrofolato-reductasa ( dhfr ) y la dihidropteroato-sintetasa ( dhps ) de la cepa de referencia RH y de dos aislamientos colombianos de Toxoplasma gondii. Materiales y métodos. Se obtuvieron dos aislamientos de T. gondii en líquido céfalorraquídeo de pacientes colombianos positivos para HIV con toxoplasmosis cerebral. Se extrajo el ADN de los genes dhfr y dhps y se amplificaron mediante reacción en cadena de la polimerasa (PCR). Los productos fueron clonados en el vector pGEM-T y secuenciados. Resultados. Se encontró un cambio de adenina por guanina (A « G) en la posición 235 del exón 2 del gen dhps , dos cambios de guanina por citocina (G « C) en las posiciones 259 y 260 y un cambio de timina por guanina (T « G) en la posición 371 del exón 4 del gen dhps. Por análisis bioinformático, en este último exón se identificó un polimorfismo no sinónimo en la región codificante, que podría llevar al cambio de una Glu (CAA o CAG) por una His (codificada por los codones AAU o AAC). Se calculó el modelo estructural de la enzima dihidropteroato-sintetasa (DHPS) de T. gondii y se identificaron las modificaciones en la estructura secundaria ocasionadas por las mutaciones. Conclusiones. La metodología estandarizada puede servir como base para la búsqueda de polimorfismos en muestras de pacientes con diferentes manifestaciones clínicas de toxoplasmosis y para establecer su posible relación con los cambios en la sensibilidad a los antifolatos y la reacción al tratamiento.

Introduction: There are no reports describing polymorphisms in target genes of anti- Toxoplasma drugs in South American isolates. Objective: This study sought to perform cloning and sequencing of the dihydrofolate reductase ( dhfr ) and dihydropteroate-synthase ( dhps ) genes of the reference Rh strain and two Colombian isolates of Toxoplasma gondii . Materials and methods: Two isolates were obtained from the cerebrospinal fluid of HIV-infected patients with cerebral toxoplasmosis. A DNA extraction technique and PCR assay for the dhfr and dhps genes were standardized, and the products of amplification were cloned into Escherichia coli and sequenced. Results: One polymorphism (A « G) was found at position 235 of exon 2 in the dhps gene. In addition, two polymorphisms (G « C) at positions 259 and 260 and one polymorphism (T « G) at position 371 within exon 4 of the dhps gene were detected. In this last exon, a bioinformatic analysis revealed a non-synonymous polymorphism in the coding region that could lead to the substitution of Glu (CAA or CAG) for His (encoded by codons AAU or AAC). A structural model of the T. gondii DHPS protein was calculated, and the results revealed modifications in secondary structure due to mutations. Conclusions: The methods described in this study can be used as a tool to search for polymorphisms in samples from patients with different clinical manifestations of toxoplasmosis and to examine their relationship with the therapeutic response.

Efecto inmunosupresor de la infección por Trypanosoma musculi (Mastigophora: Trypanosomatidae) en la toxoplasmosis experimental / Immunosuppressor effect of Trypanosoma musculi (Mastigophora: Trypanosomatidae) on experimental toxoplasmosis

The immunosuppression caused by species of the gender Trypanosoma has been widely documented. The influence over experimental infections with Toxoplasma gondii is evident when using Trypanosoma lewisi, a natural parasite of white rats. We decided to test the effect of Trypanosoma musculi from mice, an organism with very similar biological characteristics to T. lewisi, to see if this trypanosomatid could induce a similar effect. Four groups of Swiss mice were inoculated with T. musculi previously to infection with T. gondii, and we determined the survival of the animals, as well as the number of cysts developed in the brain of survivors. We isolated and tested different strains of T. gondii from different sources. In a first experiment, the animals were previously inoculated with T. musculi at different times prior to the infection with Toxoplasma; this allowed us to determine that the immunosuppression process resulted more evident when T. musculi inoculation was made four days before. In a second experiment, we used different inoculi dose and found that it did not influenced the process. Furthermore, the results were negative when evaluating if the amount of the inoculated trypomastigote influenced the process. In order to demonstrate if there were differences in the immnosuppressive effect, related to Toxoplasma strains, groups of mice were inoculated with brain cysts of TFC, TLP, TLW and TBT strains. Excluding the TLP strain, that resulted to be very pathogenic regardless the previous inoculation with T. lewisi, the other strains kept the same pattern of immunosuppression in mice, whose survival time was shorter as the presence of cysts in the brain was higher. These observations were in agreement with an experi- mental immunosuppression model, associated with immunosuppressive diseases, specially cancer and AIDS.

La prevalencia de infecciones por Toxoplasma gondii en el ser humano es de 5-90% según la zona geográfica; en Costa Rica por ejemplo, la seroprevalencia es de un 58%, por lo que es importante comprender algunos procesos inmunológicos, propios en estas afectaciones parasitarias. Con el objeto de determinar si el Trypanosoma musculi ejerce procesos de inmunosupresión sobre Toxoplasma gondii se realizó un experimento en el que se inocularon ratones Swiss con T. musculi cuatro, cinco, seis y siete días previos a la infección con T. gondii, ocurriendo la inmunosupresión cuando la inoculación con T. musculi fue hecha cuatro días antes. Además, la cantidad de tripomastigotos inoculados no influyó en el proceso. Se probaron tres cepas de T. gondii aisladas de las heces de un gato casero (TFC), de un Leopardus pardalis (TLP), de un Leopardus wiedii y de la carne de un Bos taurus (TBT). La cepa TLP resultó ser muy patógena, matando a los animales en un tiempo corto, independientemente de la inoculación con T. musculi; para las otras cepas se mantuvo el patrón de inmunosupresión en los ratones. Se reporta entonces un modelo experimental de inmunosupresión, aspecto muy en boga en este momento, por su relación con enfermedades que inducen esta condición en el ser humano, especialmente a enfermedades como el cáncer y el SIDA. Este modelo es más fácil de aplicar experimentalmente que el correspondiente con T. lewisi previamente descrito, el cual usa ratas blancas de más difícil manejo que los ratones usados en este estudio.

Detection and molecular analysis of Toxoplasma gondii and Neospora caninum from dogs with neurological disorders / Detecção e análise molecular de Toxoplasma gondii e Neospora caninum em cães com distúrbios neurológicos

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.

INTRODUÇÃO: Toxoplasma gondii e Neospora caninum são parasitas Apicomplexa responsáveis por doenças sistêmicas em muitas espécies de animais, incluindo cães, o que representa grande importância em animais de estimação. MÉTODOS: Este estudo teve como objetivo determinar a prevalência da infecção de T. gondii e N. caninum em 50 cães com sinais neurológicos internados no Hospital Veterinário da Universidade Estadual Paulista (UNESP) na Cidade de Botucatu, Brasil. Todos os animais foram examinados para detecção de anticorpos por IFAT para ambos os parasitas. Tecidos de animais positivos foram analisados por bioensaio em camundongos (T. gondii) e gerbilos (N. caninum) e o DNA foi pesquisado por PCR. Amostras positivas para T. gondii por PCR foram analisadas por meio de análise de restrição de fragmentos polimórficos (restriction fragment length polymorphism-polymerase chain reaction - RFLP-PCR), utilizando-se 11 marcadores: SAG1, SAG2 (5'-3'SAG2 e, alt.SAG2), SAG3, Btub, GRA6, L358, c22 -8, C29-6, PK1 e Apico e o marcador CS3 para análise de virulência. RESULTADOS: Os anticorpos específicos foram detectados em 11/50 animais (22%; IC95% 12,8-35,3%) para T. gondii, e 7/50 (14%; IC95% 7,0-26,3%) para N. caninum. No bioensaio e PCR, 7/11 (63,6%; IC95% 34,9-84,8%) das amostras foram positivas para T. gondii, e 3/7 (42,9%; IC95% 15,7-75,5%) para N. caninum. Três diferentes genótipos foram identificados. Apenas um foi único. CONCLUSÕES: Estes dados confirmam a presença de T. gondii e N. caninum em cães do Brasil, e demonstra a importância do T. gondii como sentinela para a infecção e a variação genotípica deste parasita no Brasil.

Efeitos da infecção crônica por Toxoplasma gondii sobre a parede intestinal de gatos domésticos / The effects of the infection caused by Toxoplasma gondii on the cat duodenal wall

O objetivo deste trabalho foi analisar os efeitos da infecção causada pelo Toxoplasma gondii sobre a parede do duodeno de gatos. Foram utilizados seis gatos (Felis catus), com cerca de três meses de vida, distribuídos aleatoriamente em Grupo controle (G1; n = 3) e Grupo infectado (G2; n = 3). Os animais do G2 receberam, por via oral, 200 cistos teciduais da cepa ME49 (tipo II) do T. gondii. Após 40 dias, os animais foram submetidos à eutanásia, laparotomia e retirada do duodeno, que foi fixado em solução de Bouin e submetido à rotina histológica para obtenção de cortes transversais de 3 µm. Os cortes foram corados com Hematoxilina-Eosina (HE), Azan, Ácido Periódico de Schiff (PAS), Alcian-Blue e Tricrômio de Mallory. Realizou-se uma avaliação qualitativa da parede intestinal e medidas comparativas entre os dois grupos, com relação: à espessura da túnica mucosa, túnica muscular, parede total, altura dos vilos, profundidade das criptas e altura dos enterócitos e seus núcleos. As células caliciformes, os linfócitos intraepiteliais e as células de Paneth foram quantificados. Os resultados mostraram que a infecção levou à atrofia da túnica mucosa, túnica muscular e parede intestinal do duodeno de gatos do G2 (p < 0,05). A altura dos enterócitos apresentou um aumento significativo (p < 0,05) nos animais do G2. Na avaliação qualitativa, as fibras colágenas ocupavam visivelmente uma maior área dos estratos da parede intestinal, o que sugere que estejam aumentadas. Observou-se a redução da secreção de sulfomucinas e o aumento das células de Paneth nesses mesmos animais (p < 0,05).

This paper analyzes the effects of the infection caused by Toxoplasma gondii on the cat duodenal wall. Six cats (Felis catus) with 3-month-old were randomly divided into Control Group (G1; n = 3) and Infected Group (G2; n = 3). The animals from G2 received orarilly 200 T. gondii tissye cysts of ME49-strain (type II). After 40 days, the animals were submitted to euthanasia, laparotomy and had their duodenum removed, fixed in Bouin solution and submitted to histological routine obtaining 3 µm transverse cuts. The cuts were stained with Hematoxylin-Eosin (HE), Azan, Periodic acid - Schiff (PAS), Alcian-Blue, and Mallory trichrome. Qualitative assessment of the intestine wall as well as comparative measurements with respect to the thickness of mucosa, muscle tunic, total wall, the height of the villous, the depth of the crypts, and the height of the enterocytes and their nuclei were carried out. Calciform cells, the intraepithelial lymphocytes, and the Paneth cells were quantified. The results showed that the infection led to the atrophy of the mucosa, muscle tunic, and the intestinal wall of the duodenum of G2 cats (p < 0.05). The enterocytes height presented significant (p < 0.05) increase for G2 animals. According to the qualitative analysis, the collagen fibers were visibly taken a broader area on the intestinal wall layers, what suggests they have increased in size. Decrease in the sulphomucins secretion and the increase of Paneth cells were observed for these animals (p < 0.05).

Detecção sorológica da infecção por Toxoplasma gondii em gatos errantes e domiciliados e sua avaliação hematológica / Serologic detection of toxoplasma gondii infection in stray and household cats and its hematologic evaluation

Aims: This study focused on the serologic detection of Toxoplasma gondii infection in two groups of cats:stray and household groups. In addition, hematologic assessment of seropositive and seronegative cats was done. Methods: Sixty cats were serologically tested for anti-Toxoplasma gondii antibodies using the latex agglutination test. Six collection sites for each group of cats were identified in the urban communities of Sta Rosa and San Pedro, Laguna, Philippines. The 60 cats collected were divided into 30 stray and 30 household cats. Results: Results revealed that 28 (46.67%) of the 60 cats were seropositive. There were more household cats (28.33%) which showed seropositivity compared to stray cats (18.33%), however the difference was statistically insignificant (p>0.05) . Hematologic tests through complete blood count showed significantly (p<0.05) higher number of seropositive cats with abnormalities on hemoglobin level, red blood cell count, segmenter (neutrophil) and monocyte counts compared to the control. Other parameters such as percent packed cell volume, white blood cell count, eosinophil and lymphocyte counts showed insignificant (p>0.05) results across seropositive cats and the control. Blood chemistry analysis showed significantly higher (p<0.05) potassium level irregularities in seropositive cats relative to the seronegative cats. Other parameters such as amylase, blood sugar, blood uric acid, creatinine and blood urea nitrogen were statistically insignificant (p>0.05). Conclusions: Although Toxoplasma gondii infection suggests possible cause of hematologic abnormalities, it is recommended that further studies on this aspect be done to provide more basic and clinical research information that would improve cat health management.

Atrofia neuronal mientérica no íleo de ratos infectados cronicamente por uma cepa genótipo i de Toxoplasma gondii / Myenteric neuronal atrop hy induced by chronic infection caused by a genotype i Toxoplasma gondii strain in the rat ileum / Atrofia neuronal mientérica en el íleon de ratas infectadas cronicamente por una cepa genotipo i de Toxoplasma gondii

A toxoplasmose é uma zoonose que desperta grande preocupação na saúde pública em nível mundial. No Brasil, os índices de soropositividade para Toxoplasma gondii variam entre 54% e 75% da população. Neste estudo objetivou-se avaliar os efeitos da infecção crônica, causada por uma cepa genótipo I de T. gondii, sobre o número e a morfometria de neurônios mientéricos do íleo terminal de ratos. Foram utilizados oito ratos (Rattus norvegicus) Wistar machos. O grupo experimental foi inoculado oralmente com 105 taquizoítos de uma cepa genótipo I de T. gondii. Após 30 dias de infecção, os animais foram submetidos à eutanásia, e por meio de laparotomia, o jejuno-íleo foi retirado, mensurado em seu comprimento e largura para cálculo de área. O íleo terminal de cada animal foi dissecado para confecção de preparados totais, os quais foram corados pela técnica de Giemsa. Não foram observadas alterações no peso corporal, comprimento ou área intestinal, apenas a largura do jejuno-íleo sofreu aumento. Não houve alteração significativa em relação à análise quantitativa dos neurônios. Na análise morfométrica, observou-se redução da área do pericário, do núcleo e do citoplasma dos neurônios mientéricos do grupo experimental. No que se refere à frequência de neurônios nas diferentes classes de intervalo da área do pericário, não houve alterações significativas. Entretanto, houve um aumento do número de neurônios cujos núcleos representavam de 21 a 30% de seu pericário e uma redução no número daqueles em que o núcleo representava 51-60% e mais que 71%.

Toxoplasmosis is a zoonosis which causes a great worldwide public health concern. In Brazil, T.gondii seropositivity indexes range from 54% to 75% of the population. This study assesses the effects of chronic infection caused by a genotype I Toxoplasma gondii strain over the amount and morphometrics of myenteric neurons of rat terminal ileum. Eight male Wistar rats (Rattus norvegicus) were used. The experimental group was orally inoculated with 105 tachyzoites from a genotype I strain of Toxoplasma gondii. The animals were submitted to euthanasia 30 days after the infection. Blood sampling was carried out by punctioning the retro-orbital plexus in order to detect anti-T.gondii seric antibodies. The ileum-jejunum was removed, measured with respect to its total length and width for the calculation of its area through laparotomy. The ileum from each animal was collected, dissected and stained with Giemsa. Alterations concerning body weight, length or intestinal area were not observed, however, the width of the ileum-jejunum had increased. There were no significant alterations with respect to the quantitative analysis. Reduction of the area of the perikarium, nucleus, and cytoplasm of the myenteric neurons from the experimental group were noticed through morphometric analysis. There were no significant alterations with respect to the incidence of neurons within the different interval classes of the perikarium area. However, there was an increase of the amount of neurons whose nuclei presented 21-30% of their perikarium as well as the reduction of those in which the nucleus presented 51-60%, and higher than 71%.

La toxoplasmosis es una zoonosis que despierta mucha preocupación en la salud pública a nivel mundial. En Brasil, los índices de soropositividad para Toxoplasma gondii varían entre 54% y 75% de la población. El objetivo de este estudio fue evaluar los efectos de la infección crónica, causada por una cepa genotipo I de T. gondii, sobre el número y la morfometría de neuronas mientéricas del íleon terminal de ratas. Fueron utilizadas 8 (ocho) ratas (Rattus norvegicus) Wistar machos. El grupo experimental fue inoculado oralmente con 105 taquizoítos de una cepa genotipo I de T. gondii. Después de 30 días de infección, los animales fueron sometidos a eutanasia, y a través de la laparotomía fue retirado el yeyuno-íleon, y así su anchura y longitud fueron medidas para cálculo del área. El íleon terminal de cada animal fue disecado para confección de preparados totales, los cuales fueron corados por la técnica de Giemsa. No fueron observadas alteraciones en el peso corporal, longitud o área intestinal, apenas en la anchura del yeyuno-íleon el cual sufrió aumento. No hubo alteraciones significativas con relación al análisis cuantitativo de las neuronas. En el análisis morfométrico, se observó reducción del área del pericario, del núcleo y del citoplasma de neuronas mientéricas del grupo experimental. Con relación a la incidencia de neuronas en las diferentes clases de intervalo del área del pericario, no hubo alteraciones significativas. Sin embargo, hubo un aumento en el número de neuronas cuyos núcleos representaban de 21 a 30% de su pericario y una reducción en el número de aquellos en que el núcleo representaba 51-60% y más que 71%.

Experimental reinfection of BALB/c mice with different recombinant type I/III strains of Toxoplasma gondii: involvement of IFN-³ and IL-10

To assess reinfection of BALB/c mice with different Toxoplasma gondii strains, the animals were prime infected with the non-virulent D8 strain and challenged with virulent recombinant strains. Thirty days after challenge, brain cysts were obtained from surviving BALB/c mice and inoculated in Swiss mice to obtain tachyzoites for DNA extraction and PCR-RFLP analysis to distinguish the different T. gondii strains present in possible co-infections. Anti-Toxoplasma immune responses were evaluated in D8-primed BALB/c mice by detecting IFN-³ and IL-10 produced by T cells and measuring immunoglobulin levels in serum samples. PCR-RFLP demonstrated that BALB/c mice were reinfected with the EGS strain at 45 days post prime infection (dpi) and with the EGS and CH3 strains at 180 dpi. High levels of IFN-³ were detected after D8 infection, with no significant difference between 45 and 180-day intervals. However, higher IL-10 levels and higher plasmatic IgG1 and IgA were detected from samples obtained 180 days after infection. BALB/c mice were susceptible to reinfection with different recombinant T. gondii strains and this susceptibility correlated with enhancement of IL-10 production.

Genotype of Toxoplasma gondii from Blood of Stray Cats in Gyeonggi-do, Korea

Genotyping of Toxoplasma gondii has been performed in 23 PCR positive blood samples from stray cats in Korea. We used 2 separate PCR-restriction fragment length polymorphism (RFLP) patterns of SAG2 gene, amplifying the 5'and 3'ends of the locus. The results revealed that all samples belonged to the type I clonal lineage. Although T. gondii organisms were not isolated from the samples, the results of the present study represent that stray cats with T. gondii infection should be seriously concerned in our environment. Adequate and continuous control programs of stray cats are needed to reduce the risk of transmission of T. gondii as a zoonotic infection threatening the public health.

Alterations of the myenteric plexus of the ileum and the descending colon caused by Toxoplasma gondii (genotype III) / Alterações do plexo mientérico do íleo e cólon descendente causadas por Toxoplasma gondii (genótipo III)

Alterations caused by a genotype III strain of Toxoplasma gondii were assessed with respect to the number and the morphometry of the myenteric neurons in the terminal ileum and the descending colon. Eighteen rats were divided into four groups: Acute Control Group (ACG, n=4); Acute Experimental Group (AEG, n=4); Chronic Control Group (CCG, n=5) and Chronic Experimental Group (CEG, n=5). NaCl solution was administered through gavage to the animals in the ACG and CCG. Toxoplasma gondii tachyzoites (10(4)) from a genotype III strain were orally administered to the AEG and CEG. Acute Groups were died after 24 hours, and the Chronic Groups after 30 days. Neuronal loss was not observed in both organs. The neurons atrophied in the terminal ileum as the opposite occurred with the neurons at the descending colon during the chronic phase of infection. In the terminal ileum, the neurons atrophied during the chronic phase of the infection as no alteration was found during the acute phase. For the descending colon, the neurons became hypertrophic during the chronic infection in opposition to the atrophy found during the acute phase.

Objetivou-se avaliar as alterações causadas por uma cepa genótipo III de Toxoplasma gondii, sobre o número e a morfometria de neurônios mientéricos, do íleo terminal e do cólon descendente. Dividiu-se dezoitos ratos em quatro grupos: controle agudo (GCA, n=4), experimental agudo (GEA, n=4), controle crônico (GCC, n=5) e experimental crônico (GEC, n=5). Os animais do GCA e GCC receberam solução de NaCl por gavagem, e os animais do GEA e GEC 10(4) taquizoítos de uma cepa genótipo III de T. gondii por via oral. Os grupos agudos após 24 horas foram mortos e os crônicos após 30 dias. Observou-se que não houve perda neuronal em ambos os órgãos. No íleo terminal, os neurônios atrofiaram-se na fase crônica da infecção, enquanto nenhuma alteração ocorreu na fase aguda. Já no cólon descendente, os neurônios tornaram-se hipertróficos na fase crônica da infecção, em oposição à atrofia observada na fase aguda.