RESUMO
This study aimed to isolate and genotype T. gondii from Brazilian wildlife. For this purpose, 226 samples were submitted to mice bioassay and screened by PCR based on 18S rRNA sequences. A total of 15 T. gondii isolates were obtained, including samples from four armadillos (three Dasypus novemcinctus, one Euphractus sexcinctus), three collared anteaters (Tamandua tetradactyla), three whited-lipped peccaries (Tayassu pecari), one spotted paca (Cuniculus paca), one oncilla (Leopardus tigrinus), one hoary fox (Pseudalopex vetulus), one lineated woodpecker (Dryocopus lineatus) and one maned wolf (Chrysocyon brachyurus). DNA from the isolates, originated from mice bioassay, and from the tissues of the wild animal, designated as "primary samples", were genotyped by PCR-restriction fragment length polymorphism (PCR/RFLP), using 12 genetic markers (SAG1, SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L258, PK1, CS3 and Apico). A total of 17 genotypes were identified, with 13 identified for the first time and four already reported in published literature. Results herein obtained corroborate previous studies in Brazil, confirming high diversity and revealing unique genotypes in this region. Given most of genotypes here identified are different from previous studies in domestic animals, future studies on T. gondii from wildlife is of interest to understand population genetics and structure of this parasite.
RESUMO
Brazil is one of the regions with the highest prevalences of Toxoplasma gondii in humans and animals. Because free-range chickens become infected by feeding from ground contaminated with oocysts, the prevalence of T. gondii in this host has been widely used as an indicator of the strains prevalent in the environment. The genetic variability among T. gondii isolates from different healthy and sick hosts all over the world has been recently studied. Three clonal genetic lineages (Types I, II and III) were initially recognised as predominant in Western Europe and the United States. T. gondii strains are genetically diverse in South America. In Brazil, recombination plays an important role in strain diversification. The objective of this study was to genetically characterise T. gondii isolates from free-range chickens from Espírito Santo state, Southeast region, Brazil, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). A total of 44 isolates among 47 previously described isolates (TgCkBr234-281) from free-range chickens were included in this study. Strain typing was performed using 12 PCR-RFLP markers: SAG1, SAG2, alt. SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico and CS3. Eleven genotypes were identified. Ten isolates (23%) were grouped into four novel genotypes. Four isolates, distributed in four counties, corresponded to the Type BrI lineage, the genotype found most frequently in Brazil. No clonal Types I, II or III lineages were found. Two novel genotypes were represented by single isolates. Unique alleles were identified for the markers SAG1, c22-8 and CS3, and for the first time, a unique allele was found for the marker SAG3. Although a large number of T. gondii genotypes have already been identified from a variety of animal hosts in Brazil, new genotypes are continuously identified from different animal species. This study confirmed the diversity of T. gondii in Brazil and demonstrates clonal Type I, II and III lineages are rare in this country.
Assuntos
Galinhas/parasitologia , Glicoproteínas de Membrana/genética , Reação em Cadeia da Polimerase/veterinária , Doenças das Aves Domésticas/parasitologia , Proteínas de Protozoários/genética , Toxoplasma/genética , Toxoplasmose Animal/parasitologia , Alelos , Animais , Sequência de Bases , Brasil/epidemiologia , Marcadores Genéticos , Variação Genética , Genótipo , Dados de Sequência Molecular , Filogenia , Polimorfismo de Fragmento de Restrição , Doenças das Aves Domésticas/epidemiologia , Alinhamento de Sequência , Análise de Sequência de DNA , Toxoplasma/classificação , Toxoplasma/isolamento & purificação , Toxoplasmose Animal/epidemiologiaRESUMO
Toxoplasma gondii isolates are highly diverse in domestic animals from Brazil. However, little is known about the genetics of this parasite from wild mammals in the same region. Reveal genetic similarity or difference of T. gondii among different animal populations is necessary for us to understand transmission of this parasite. Here we reported isolation and genetic characterisation of three T. gondii isolates from wild animals in Brazil. The parasite was isolated by bioassay in mice from tissues of a young male red handed howler monkey (Alouatta belzebul), an adult male jaguarundi (Puma yagouaroundi), and an adult female black-eared opossum (Didelphis aurita). The monkey and the jaguarundi had inhabited the Zoo of Parque Estadual Dois Irmãos, Pernambuco State, Northeastern Brazil, for 1 year and 8 years, respectively. The wild black-eared opossum was captured in São Paulo State, Southeastern Brazil, and euthanised for this study because it was seropositive for T. gondii (titre 1:100 by the modified agglutination test, MAT). Ten PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) markers, SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico, were used to genotype the isolates. T. gondii was isolated from the brain and heart homogenate of the monkey, the muscle homogenate of the jaguarundi, and the heart homogenate of the black-eared opossum. This was the first isolation of T. gondii from a neotropical felid from Brazil. The isolate from the monkey (TgRhHmBr1) was not virulent in mice, whereas the isolates from the jaguarundi (TgJagBr1) and the black-eared opossum (TgOpBr1) were virulent in mice. The genotype of the isolate from the monkey has been identified in isolates from a goat and ten chickens in the same region of Brazil, suggesting that it may be a common lineage circulating in this region. The genotypes of the isolates from the jaguarundi and the black-eared opossum have not been previously reported. Although there are already 88 genotypes identified from a variety of animal hosts in Brazil, new genotypes are continuously being identified from different animal species, indicating an extremely high diversity of T. gondii in the population.
Assuntos
Alouatta/parasitologia , Didelphis/parasitologia , Doenças dos Macacos/parasitologia , Puma/parasitologia , Toxoplasma/isolamento & purificação , Toxoplasmose Animal/parasitologia , Animais , Animais Selvagens , Animais de Zoológico , Anticorpos Antiprotozoários , Bioensaio/veterinária , Brasil , DNA de Protozoário/genética , Feminino , Genótipo , Masculino , Camundongos , Reação em Cadeia da Polimerase/veterinária , Toxoplasma/classificação , Toxoplasma/genética , Toxoplasma/patogenicidade , VirulênciaRESUMO
Toxoplasma gondii affects mainly warm-blooded animals, including birds. Even though previous experimental data indicate that raptors are resistant to clinical infection, there is no information regarding the susceptibility of Brazilian birds of prey to T. gondii. The present study aimed to observe how the crested caracara, a common raptor in Brazil, interacts with T. gondii using an experimental model. Seven crested caracaras, seronegative for T. gondii, were separated into infected (n=5) and control groups (n=2). Birds from the infected group were fed T. gondii-infected Calomys callosus, a rodent present in Brazilian savanna and described as highly susceptible to infection by the parasite, for three consecutive days, while control animals were fed non-infected rodents. All infected birds produced T. gondii-specific IgG antibodies that were firstly detected at day 7 post-infection, with peak production detected between 15 and 30dpi. No significant alterations in clinical and hematological parameters were observed throughout the experimental period, and parasites were sparsely found in muscular tissues after the birds were euthanized. In conclusion, our results demonstrated that crested caracaras are resistant to oral infection with T. gondii, suggesting that the host-parasite relationship between both species has reached a remarkable equilibrium.
Assuntos
Doenças das Aves/parasitologia , Toxoplasma/imunologia , Toxoplasmose Animal/parasitologia , Animais , Anticorpos Antiprotozoários/sangue , Bioensaio , Doenças das Aves/imunologia , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Camundongos , Aves Predatórias , Toxoplasmose Animal/imunologiaRESUMO
The intraocular pressure (IOP) and its correlations with arterial carbon dioxide partial pressure (PaCO2) and arterial pH were studied in five crested caracaras (Caracara plancus) anesthetized with isoflurane (ISO) and sevoflurane (SEV). Baseline IOP values were measured in both eyes (M0). Brachial artery was previously catheterized to obtain blood gas and cardiorespiratory analysis. Anesthesia was induced with 5 percent ISO and maintained with 2.5 percent for 40 minutes. IOP measurements and blood samples were evaluated in different moments until the end of the procedure. After recovering, a second anesthesia was induced with 6 percent SEV and maintained with 3.5 percent. Parameters were evaluated at the same time points of the previous procedure. IOP reduced significantly (P= 0.012) from M0 at all time points and no significative changes were observed between ISO and SEV anesthesias. Correlation between IOP and PaCO2 and between PIO and blood pH were found only for SEV. IOP and blood pH decreased in parallel with IOP, whereas values of PaCO2 increased in caracaras anesthetized with isoflurane and sevoflurane.(AU)
Avaliou-se a pressão intra-ocular (PIO) e estimaram-se as correlações entre PIO e pressão de dióxido de carbono (PaCO2) e pH arterial de cinco caracarás (Caracara plancus), anestesiados com isofluorano (ISO) ou sevofluorano (SEV). Valores basais da PIO foram aferidos em ambos os olhos (M0). Cateterizou-se previamente a artéria braquial para obtenção de parâmetros hemogasométricos e cardiorrespiratórios. Anestesia foi induzida com ISO a 5V por cento e mantida por 40 minutos com 2,5V por cento. PIO e amostras de sangue foram avaliadas em diferentes momentos até o final do procedimento. Após recuperação, uma segunda anestesia foi realizada com SEV a 6 por cento e mantida com 3,5 por cento. Os parâmetros foram aferidos nos mesmos momentos estabelecidos previamente. A PIO decresceu significativamente (P=0,012) de M0 em todos os momentos e não houve diferença estatística entre ISO e SEV. Correlações significativas entre PIO e PaCO2 e entre PIO e pH sangüíneo foram observadas apenas para a anestesia com SEV. O pH sangüíneo decresceu paralelamente a PIO, enquanto a PaCO2 aumentou, em carcarás anestesiados com isofluorano e sevofluorano.(AU)
Assuntos
Animais , Pressão Intraocular , Gasometria/métodos , Gasometria/veterinária , Anestésicos Inalatórios/administração & dosagem , Falcões/sangueRESUMO
The intraocular pressure (IOP) and its correlations with arterial carbon dioxide partial pressure (PaCO2) and arterial pH were studied in five crested caracaras (Caracara plancus) anesthetized with isoflurane (ISO) and sevoflurane (SEV). Baseline IOP values were measured in both eyes (M0). Brachial artery was previously catheterized to obtain blood gas and cardiorespiratory analysis. Anesthesia was induced with 5 percent ISO and maintained with 2.5 percent for 40 minutes. IOP measurements and blood samples were evaluated in different moments until the end of the procedure. After recovering, a second anesthesia was induced with 6 percent SEV and maintained with 3.5 percent. Parameters were evaluated at the same time points of the previous procedure. IOP reduced significantly (P= 0.012) from M0 at all time points and no significative changes were observed between ISO and SEV anesthesias. Correlation between IOP and PaCO2 and between PIO and blood pH were found only for SEV. IOP and blood pH decreased in parallel with IOP, whereas values of PaCO2 increased in caracaras anesthetized with isoflurane and sevoflurane.
Avaliou-se a pressão intra-ocular (PIO) e estimaram-se as correlações entre PIO e pressão de dióxido de carbono (PaCO2) e pH arterial de cinco caracarás (Caracara plancus), anestesiados com isofluorano (ISO) ou sevofluorano (SEV). Valores basais da PIO foram aferidos em ambos os olhos (M0). Cateterizou-se previamente a artéria braquial para obtenção de parâmetros hemogasométricos e cardiorrespiratórios. Anestesia foi induzida com ISO a 5V por cento e mantida por 40 minutos com 2,5V por cento. PIO e amostras de sangue foram avaliadas em diferentes momentos até o final do procedimento. Após recuperação, uma segunda anestesia foi realizada com SEV a 6 por cento e mantida com 3,5 por cento. Os parâmetros foram aferidos nos mesmos momentos estabelecidos previamente. A PIO decresceu significativamente (P=0,012) de M0 em todos os momentos e não houve diferença estatística entre ISO e SEV. Correlações significativas entre PIO e PaCO2 e entre PIO e pH sangüíneo foram observadas apenas para a anestesia com SEV. O pH sangüíneo decresceu paralelamente a PIO, enquanto a PaCO2 aumentou, em carcarás anestesiados com isofluorano e sevofluorano.
Assuntos
Animais , Anestésicos Inalatórios/administração & dosagem , Falcões/sangue , Gasometria/métodos , Gasometria/veterinária , Pressão IntraocularRESUMO
Use of serological tests in the diagnosis of infectious diseases in wild animals has several limitations, primarily the difficulty of obtaining species-specific reagents. Wild canids, such as maned wolves (Chrysocyon brachyurus), are highly predisposed to infection by Toxoplasma gondii and, to a lesser extent, to Neospora caninum. The aim of the present study was to evaluate homologous, heterologous, and affinity conjugates in enzyme-linked immunosorbent assays (ELISAs) and indirect fluorescent antibody tests (IFATs) for detecting immunoglobulin (Ig) G antibodies against T. gondii and N. caninum in maned wolves. Serum samples were obtained from 59 captive animals in Brazil and tested by ELISA for T. gondii serology and IFAT for N. caninum serology using 3 different enzymatic and fluorescent conjugates: homologous (guinea pig anti-maned wolf IgG-peroxidase and -fluorescein isothiocyanate [FITC]), heterologous (rabbit anti-dog IgG-peroxidase and -FITC), and affinity (protein A-peroxidase and -FITC). Seropositivity to T. gondii was comparable among the homologous (69.5%), heterologous (74.6%), and affinity (71.2%) enzymatic conjugates. A significant positive correlation was found between the antibody levels determined by the 3 enzymatic conjugates. The highest mean antibody levels (ELISA index = 4.5) were observed with the protein A-peroxidase conjugate. The same seropositivity to N. caninum (8.5%) was found with the homologous and heterologous fluorescent conjugates, but protein A-FITC was not able to detect or confirm any positive samples with homologous or heterologous conjugates. Our results demonstrate that homologous, heterologous, and affinity conjugates might be used in ELISA for serological assays of T. gondii in wild canids, whereas for N. caninum infection, only the homologous or heterologous fluorescent conjugates have been shown to be useful.
Assuntos
Anticorpos Antiprotozoários/sangue , Canidae/parasitologia , Coccidiose/veterinária , Neospora/imunologia , Toxoplasma/imunologia , Toxoplasmose Animal/diagnóstico , Marcadores de Afinidade/normas , Animais , Animais de Zoológico , Brasil/epidemiologia , Coccidiose/diagnóstico , Coccidiose/epidemiologia , Coccidiose/imunologia , Ensaio de Imunoadsorção Enzimática/veterinária , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Imunoglobulina G/sangue , Toxoplasmose Animal/epidemiologia , Toxoplasmose Animal/imunologiaRESUMO
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 (EI). 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.