RESUMO
Hammondia heydorni is a coccidian parasite believed to be nonpathogenic for naturally-infected animals, but it is biologically and genetically related to Neospora caninum, a worldwide cause of abortion in cattle. The major aim of the present work was to determine whether dogs shed H. heydorni oocysts after consuming in vitro generated tissue cysts of the parasite. In addition, we investigated cross-immunity between H. heydorni and N. caninum in mice. Two dogs were fed cultured cells containing tissue cysts of H. heydorni mixed with canned dog food, and a third dog (negative control) received only non-infected cells mixed with canned food. The two dogs that consumed in vitro produced tissue cysts shed high numbers of oocysts, which were induced to sporulate and tested positive for H. heydorni by a species-specific PCR. The third uninfected dog did not shed H. heydorni oocysts in the feces. Oocysts shed by the dogs induced the formation of encysted bradyzoites of H. heydorni on KH-R cells. Nineteen BALB/c mice were employed in the cross-immunity study. Nine mice were orally inoculated with 1×105 sporulated oocysts of H. heydorni and challenged with N. caninum tachyzoites 30days after infection with H. heydorni. Other ten mice, which did not receive H. heydorni oocysts, were infected with 2×105N. caninum tachyzoites. Thirty days after challenging with N. caninum, all mice were euthanized and N. caninum DNA in their tissues was quantified by real time PCR. No statistically significant difference in N. caninum DNA concentrations were observed between the two groups. We concluded that in vitro generated cysts of H. heydorni are biologically active, because they induced oocyst shedding in dogs. As no cross-protection occurred in mice inoculated with H. heydorni and challenged with N. caninum, it is suspected that these parasites do not express significant numbers of homologous proteins during infection, or the immune response of BALB/c mice after H. heydorni infection was not sufficient.
Assuntos
Coccidiose/veterinária , Doenças do Cão/imunologia , Neospora/imunologia , Sarcocystidae/genética , Sarcocystidae/imunologia , Animais , Linhagem Celular , Coccidiose/imunologia , Coccidiose/parasitologia , Proteção Cruzada , Doenças do Cão/parasitologia , Cães , Fezes/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neospora/genética , Oocistos/imunologiaRESUMO
The aim of this study was to examine the intra-uterine exposure to Sarcocystis spp. antigens, determining the number of foals with detectable concentrations of antibodies against these agents in the serum, before colostrum ingestion and collect data about exposure of horses to the parasite. Serum samples were collected from 195 thoroughbred mares and their newborns in two farms from southern Brazil. Parasite specific antibody responses to Sarcocystis antigens were detected using the indirect immunofluorescent antibody test (IFAT) and immunoblot analysis. In 84.1% (159/189) of the pregnant mares and in 7.4% (14/189) of foals we detected antibodies anti-Sarcocystis spp. by IFAT. All samples seropositive from foals were also positive in their respective mares. Serum samples of seropositive foals by IFAT, showed no reactivity on the immunoblot, having as antigens S. neurona merozoites. In conclusion, the intra-uterine exposure to Sarcocystis spp. antigens in horses was demonstrated, with occurrence not only in mares, but also in their foals, before colostrum ingestion these occurrences were reduced.
O objetivo deste estudo foi avaliar a exposição intrauterina ao Sarcocystis spp., para determinar o número de potros que possuem concentrações detectáveis de anticorpos contra esses agentes no soro, antes da ingestão do colostro, por meio da coleta de dados sobre a exposição a esses protozoários nos equinos. Amostras de soro foram coletadas de 195 éguas puro-sangue e seus respectivos potros recém-nascidos, em duas fazendas localizadas na região Sul do Brasil. Os testes utilizados na detecção de anticorpos específicos para o Sarcocystis foram a reação de imunofluorescência indireta (RIFI) e análise por meio de immunoblot. Pela RIFI, em 84,1% (159/189) das éguas e em 7,4% (14/189) dos potros foram detectados anticorpos anti-Sarcocystis spp. Todas as amostras soropositivas dos potros também foram positivas para suas respectivas mães. As amostras de soro dos potros soropositivos na RIFI, não apresentaram reatividade no immunoblot, tendo como antígenos merozoítos de S. neurona. Em conclusão, foi demonstrada a exposição intrauterina de Sarcocystis spp. em equinos, com ocorrência em éguas, porém, em seus respectivos potros, antes da ingestão de colostro a ocorrência foi reduzida.
Assuntos
Animais , Anticorpos Antiprotozoários/análise , Cavalos/parasitologia , Diagnóstico Pré-Natal/veterinária , Sarcocystis/patogenicidade , Encefalomielite/veterinária , Immunoblotting , Immunoblotting/veterinária , Troca Materno-Fetal , Estudos SoroepidemiológicosRESUMO
The aim of this study was to examine the intra-uterine exposure to Sarcocystis spp. antigens, determining the number of foals with detectable concentrations of antibodies against these agents in the serum, before colostrum ingestion and collect data about exposure of horses to the parasite. Serum samples were collected from 195 thoroughbred mares and their newborns in two farms from southern Brazil. Parasite specific antibody responses to Sarcocystis antigens were detected using the indirect immunofluorescent antibody test (IFAT) and immunoblot analysis. In 84.1% (159/189) of the pregnant mares and in 7.4% (14/189) of foals we detected antibodies anti-Sarcocystis spp. by IFAT. All samples seropositive from foals were also positive in their respective mares. Serum samples of seropositive foals by IFAT, showed no reactivity on the immunoblot, having as antigens S. neurona merozoites. In conclusion, the intra-uterine exposure to Sarcocystis spp. antigens in horses was demonstrated, with occurrence not only in mares, but also in their foals, before colostrum ingestion these occurrences were reduced.(AU)
O objetivo deste estudo foi avaliar a exposição intrauterina ao Sarcocystis spp., para determinar o número de potros que possuem concentrações detectáveis de anticorpos contra esses agentes no soro, antes da ingestão do colostro, por meio da coleta de dados sobre a exposição a esses protozoários nos equinos. Amostras de soro foram coletadas de 195 éguas puro-sangue e seus respectivos potros recém-nascidos, em duas fazendas localizadas na região Sul do Brasil. Os testes utilizados na detecção de anticorpos específicos para o Sarcocystis foram a reação de imunofluorescência indireta (RIFI) e análise por meio de immunoblot. Pela RIFI, em 84,1% (159/189) das éguas e em 7,4% (14/189) dos potros foram detectados anticorpos anti-Sarcocystis spp. Todas as amostras soropositivas dos potros também foram positivas para suas respectivas mães. As amostras de soro dos potros soropositivos na RIFI, não apresentaram reatividade no immunoblot, tendo como antígenos merozoítos de S. neurona. Em conclusão, foi demonstrada a exposição intrauterina de Sarcocystis spp. em equinos, com ocorrência em éguas, porém, em seus respectivos potros, antes da ingestão de colostro a ocorrência foi reduzida.(AU)
Assuntos
Animais , Cavalos/parasitologia , Sarcocystis/patogenicidade , Diagnóstico Pré-Natal/veterinária , Anticorpos Antiprotozoários/análise , Encefalomielite/veterinária , Troca Materno-Fetal , Estudos Soroepidemiológicos , Immunoblotting , Immunoblotting/veterináriaRESUMO
Besnoitia besnoiti is a cyst-forming parasite that has been associated with economic losses in Africa and Europe. Besnoitiosis is considered as a re-emergent disease in the European continent. It is unknown whether cattle are exposed to B. besnoiti in the Americas, thus the aim of this study was to serologically investigate antibodies against B. besnoiti in a total of 2014 cattle serum samples from two states from Brazil. All samples were evaluated by IFAT and part of the positive sera was tested by Western blot (WB) using tachyzoites extracts under non-reducing condition. A total of 3.48% (70/2014) of the tested sera reacted positively by IFAT with titers of 200 (85.7%), 400 (10%) and 800 (4.3%). When 47 positive samples were assessed by WB a range of antigens from 7 to 206 kDa was recognized by the IFAT-positive sera. The results are suggestive of exposure of Brazilian cattle to B. besnoiti due to the titers (≥ 200) observed for some sera using IFAT. However, the antigens recognized by the IFAT-positive animals did not completely match with the WB patterns previously described by other working groups. It is possible that Brazilian cattle are exposed to B. besnoiti strains with different antigenic composition of those described in the European and African continent. Further studies are needed to confirm the presence of B. besnoiti or other Besnoitia species in Brazilian cattle.
Assuntos
Anticorpos Antiprotozoários/sangue , Doenças dos Bovinos/epidemiologia , Coccidiose/veterinária , Sarcocystidae/imunologia , Animais , Western Blotting , Brasil/epidemiologia , Bovinos , Doenças dos Bovinos/parasitologia , Coccidiose/epidemiologia , Técnica Indireta de Fluorescência para Anticorpo , Estudos SoroepidemiológicosRESUMO
Histological analysis is commonly used for a conclusive diagnosis of neosporosis. Immunohistochemistry (IHC) using monoclonal (mAb) and polyclonal (pAb) antibodies can improve diagnosis; however, the use of pAb may induce cross-reactivity with other related parasites. The aims of this study were to compare the performance of mAbs and their combinations with that of pAb in IHC and evaluate the usefulness of mAb to identify Neospora caninum infection in aborted bovine fetal tissues. For this purpose, mAbs targeting NcSRS2 (4.15.15) or NcGRA7 (4.11.5 and 1/24-12) and one pAb collected from a rabbit inoculated with N. caninum tachyzoites were tested by IHC. Artificial standardized tissue sections were prepared as positive controls using homogenized bovine brain spiked with cultured tachyzoites of N. caninum. The numbers of labeled parasites were counted in each positive control section. In addition, four equal proportional combinations of the mAbs were also analyzed in the IHC. Finally, the pAb and the best combination of mAbs obtained in the positive control experiments were tested with tissue sections of naturally-infected cattle. To confirm analytical specificity, mAbs and a pAb were tested with Toxoplasma gondii and Besnoitia besnoiti positive control slides and tissues sections from naturally infected cattle containing Sarcocystis spp. and B. besnoiti antigens. The mAb 4.15.15 detected 57% of the total parasites in sections while 4.11.5 and 1/24-12 were able to detect 49% and 41%, respectively. For the mAb combinations (I: 1/24-12+4.11.5, II: 1/24-12+4.15.15, III: 4.15.15+4.11.5, IV: 1/24-12+4.11.5+4.15.15), the detection capacity was 32.4%, 79.4%, 66.6% and 60.7% for each combination, respectively. The best mAb combination (1/24-12 and 4.15.15) and the pAb serum detected 100% (18/18) of naturally-infected animals. Sarcocystis spp. or B. besnoiti were not detected by mAb combinations in IHC, however the pAb cross-reacted with Sarcocystis spp. cysts. These results confirm the usefulness of mAb application in IHC to N. caninum.
Assuntos
Anticorpos Monoclonais/imunologia , Doenças dos Bovinos/parasitologia , Coccidiose/veterinária , Imuno-Histoquímica/veterinária , Neospora/imunologia , Feto Abortado/parasitologia , Aborto Animal/parasitologia , Animais , Bovinos , Doenças dos Bovinos/diagnóstico , Coccidiose/diagnóstico , Coccidiose/parasitologia , CoelhosRESUMO
The Toxoplasmatinae parasites Toxoplasma gondii, Neospora caninum and Hammondia spp. have carnivores as definitive hosts that shed the parasite oocysts in their feces. Birds that feed directly from the soil, such as chickens, are exposed to infection and may serve as indicators of the presence of the parasite in the environment and as a source of infection for other animals. The aims of this study were to determine the frequency of infection by these parasites in free ranging chickens, to test whether chickens are intermediate hosts of Hammondia spp., and to isolate N. caninum from chickens. One hundred chickens, which were raised in contact to cattle and dogs, were bought in five towns located in Bahia, Brazil. Blood and tissues (brain and heart) were used for serology, molecular tests and bioassay in mice for parasite isolation. T. gondii DNA was detected in 29 chickens, and N. caninum DNA was observed in six animals. Hammondia spp. DNA was not detected in tissues from any chicken. Tissues from eight N. caninum seropositive chickens were bioassayed in interferon-gamma gene knockout mice, but the mice did not become infected; T. gondii was isolated from six of 14 seropositive chickens after bioassay in outbreed Swiss mice. The authors concluded that: chickens seem to be better hosts for T. gondii when compared to N. caninum, based on the molecular and bioassay results; Hammondia spp. probably does not infect chickens or is rarely found in this animal species.
Assuntos
Anticorpos Antiprotozoários/sangue , Galinhas/parasitologia , Coccidiose/veterinária , Doenças das Aves Domésticas/parasitologia , Sarcocystidae/isolamento & purificação , Animais , Encéfalo/parasitologia , Brasil , Coccidiose/imunologia , Coccidiose/parasitologia , DNA de Protozoário/genética , Fezes/parasitologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Genótipo , Coração/parasitologia , Especificidade de Hospedeiro , Interferon gama/genética , Camundongos , Camundongos Knockout , Neospora/genética , Neospora/imunologia , Neospora/isolamento & purificação , Oocistos , Reação em Cadeia da Polimerase , Doenças das Aves Domésticas/imunologia , Sarcocystidae/genética , Sarcocystidae/imunologia , Toxoplasma/genética , Toxoplasma/imunologia , Toxoplasma/isolamento & purificação , Toxoplasmose Animal/imunologia , Toxoplasmose Animal/parasitologiaRESUMO
Neospora caninum naturally infects many mammal species, but has not previously been demonstrated in birds. We examined sera for N. caninum antibodies from 200 outdoor chickens and from 200 chickens confined indoors in the state of Bahia, Brazil. Seroprevalence was greater in outdoor chickens (23.5% versus 1.5%, P<0.001). PCR testing for N. caninum was positive in six of 10 seropositive chickens. Amplicons from two of these were sequenced and had 97-98% nucleotide identity with N. caninum. This finding extends the list of intermediate hosts of N. caninum to include birds and may have important epidemiological consequences.
Assuntos
Anticorpos Antiprotozoários/sangue , Galinhas/parasitologia , Coccidiose/transmissão , Neospora/fisiologia , Criação de Animais Domésticos/métodos , Animais , Sequência de Bases , Doenças das Aves/parasitologia , Aves/parasitologia , Brasil , Galinhas/imunologia , DNA de Protozoário/análise , Reservatórios de Doenças/parasitologia , Técnica Indireta de Fluorescência para Anticorpo , Dados de Sequência Molecular , Neospora/genética , Neospora/imunologia , Estudos Soroepidemiológicos , Toxoplasma/imunologia , Toxoplasma/fisiologiaRESUMO
The frequency of antibodies to Neospora caninum (Protozoa: Apicomplexa) in dog sera was investigated. Blood samples from 100 mongrel dogs, captured in the streets of São Luís, State of Maranhão, were analized using imunofluorescent antibody test. Forty five percent of the dogs were positive, and the titers ranged from 1:50 to 1:800. No sex difference was observed for frequency of N. caninum (60% in males and 40% in females). This is the first report of antibodies to N. caninum in dogs from Maranhão.(AU)
Assuntos
Neospora/isolamento & purificação , Estudos Soroepidemiológicos , Anticorpos/análise , Anticorpos/isolamento & purificação , Técnica Indireta de Fluorescência para Anticorpo/métodos , CãesRESUMO
The frequency of antibodies to Neospora caninum (Protozoa: Apicomplexa) in dog sera was investigated. Blood samples from 100 mongrel dogs, captured in the streets of São Luís, State of Maranhão, were analized using imunofluorescent antibody test. Forty five percent of the dogs were positive, and the titers ranged from 1:50 to 1:800. No sex difference was observed for frequency of N. caninum (60 percent in males and 40 percent in females). This is the first report of antibodies to N. caninum in dogs from Maranhão.
Assuntos
Anticorpos/análise , Anticorpos/isolamento & purificação , Cães , Neospora/isolamento & purificação , Estudos Soroepidemiológicos , Técnica Indireta de Fluorescência para Anticorpo/métodosRESUMO
Three-hundred and eighty-five serum samples were taken from dairy cows on 90 farms in 12 counties from the northern region of the Paraná State, Brazil. The samples were analyzed by IFAT for the detection of anti-Neospora caninum and anti-Toxoplasma gondii IgG antibodies. Forty-five (12%) samples were seropositive to N. caninum, while 102 (26%) samples were seropositive to T. gondii. Only four animals were seropositive to both coccidia. No significant difference was observed between the N. caninum serology and any of the variables studied, such as dairy cattle management, milk production, reproductive problems, feeding, and presence of dogs, cats and rodents. These data suggest that neosporosis is present among dairy cattle in the studied geographic region and the simultaneous detection of serum positive animals to both types of coccidian parasite demonstrates the independent occurrence of these coccidia in dairy cows.(AU)
Foram analisadas, por meio da imunofluorescência indireta, 385 amostras de soros de vacas, pertencentes a 90 propriedades leiteiras de 12 municípios da região Norte do estado do Paraná. Foram observados 45 (12%) sororeagentes ao Neospora caninum e 102 (26%) ao Toxoplasma gondii. Apenas quatro animais apresentaram títulos de anticorpos para ambos os coccídios. Não foi observada diferença significativa na associação entre a sorologia do N. caninum e as variáveis relacionadas ao manejo, produção de leite, problemas reprodutivos, alimentação, presença de cães, gatos e roedores. Os resultados sugerem que neosporose e toxoplasmose estão disseminadas nos rebanhos leiteiros da região Norte do estado do Paraná, e a freqüência simultânea de anticorpos anti-N. caninum e anti-T. gondii, demonstra sua ocorrência independente em vacas leiteiras.(AU)