Role of TLR2/MyD88 in the production of specific IgM and IgG antibodies during the immunization of mice against Neospora caninum.

Neospora caninum is a parasite relevant to the veterinary field. Innate and adaptive responses against N. caninum induce effector mechanisms that limit parasite replication, but little is known about their role in humoral response. Our work aimed to verify whether key molecules in the TLR2/MyD88-mediated response would impact the production of specific IgM and IgG antibodies in mice during immunization with soluble antigens of N. caninum. We observed that lack of IFN-gamma did not negatively affect the production of specific antibodies. However, mice genetically deficient in Toll-like receptor 2, Myeloid differentiation factor 88, Interleukin 12 and inducible nitric oxide synthase presented significant decrease in antibody levels against N. caninum antigens, which also reflected in the diversity of the antigen recognized by their serum. In that sense, we show here that molecules within this innate recognition pathway may present a direct impact in the induction of an antibody response against N. caninum.

C57BL/6 mice immunized with synthetic peptides from Toxoplasma gondii surface and microneme immunodominant antigens are able to decrease parasite burden in the brain tissues.

Toxoplasmosis is a disease caused by Toxoplasma gondii, an intracellular protozoan able to infect a wide range of hosts. The infection is particularly severe in immunocompromised patients or during pregnancy, circumstances in which the parasite could find a more favorable microenvironment to replicate and invade host tissues. The current treatment consists in toxic drugs for the patients, being not appropriate for the fetuses and immunodeficient patients. So far, there is a lack of available vaccine to prevent the disease. The present study aimed to evaluate the immune response induced by peptides derived from parasite immunodominant proteins from key components, as surface, rhoptry, microneme and dense granule antigens. A panel of eleven peptides was selected considering the highest scores for B cell epitope prediction by in silico analyses. The peptides were divided in groups, according to the parasite organelle locations, and used to immunize C57BL/6 mice. The animals were submitted to three doses of immunization and infected by 10 cysts of T. gondii ME49 strain. Blood samples were collected and used to measure the production of antibodies and cytokines, while the brains were collected to determine the parasite burden by quantitative polymerase chain reaction (qPCR). It was found that synthetic peptides from all targets were able to induce IgG synthesis in immunized mice, as well as to modulate the Th1/Th2 cytokine production, particularly the MIC and SRS groups, which presented the IFN-γ/IL-10 and TNF-α/IL-10 ratios 30 and 10 times higher, respectively, when compared with non-immunized group. Interestingly, the animals from MIC and SRS groups had significantly lower levels of T. gondii DNA in their brains. In summary, it can be concluded that peptides mainly from SRS and MIC parasite components constitute relevant targets to design vaccine candidates against parasite burden observed during chronic toxoplasmosis.

Assessment of experimental infection for dogs using Gallus gallus chorioallantoic membranes inoculated with Neospora caninum

The aim of this study was to evaluate parasitism kinetics and tissue lesions in the first week of infection by Neospora caninum in dogs fed Gallus gallus chorioallantoic membranes (CMs) previously infected in ovo. Five two-month-old pups were used. Each dog was given five CMs that were previously infected with N. caninum via the oral route. Four animals were euthanized in the first week of infection. All four dogs had their stools examined one week prior to and up to the day they were euthanized. The stools of the uneuthanized dog were collected for 30 days. After euthanasia, organ sections were utilized for histopathology, immunohistochemistry, indirect immunofluorescent tissue reactions, PCR and real-time PCR to detect parasites. Necropsy revealed that the small and large intestines, spleen, and lungs were affected. No oocysts or N. caninum DNA were identified in the stool samples. Real-time PCR was the most sensitive technique used to detect the protozoa in tissues, which were identified in 41% of the analyzed samples. Our results indicate that an experimental model using previously infected CMs appears to be a useful model for the study of the host-parasite relationship during the infection's acute phase.

O objetivo deste estudo foi avaliar a cinética de parasitismo e lesões teciduais, na primeira semana de infecção por Neospora caninum, em cães alimentados com membranas corioalantóicas (MCs) de Gallus gallus, previamente infectadas in ovo. Foram utilizados cinco filhotes de dois meses de idade. Cada cão recebeu cinco MCs previamente infectadas com N. caninum, por via oral. Quatro animais foram eutanasiados na primeira semana de infecção. Todos os quatro cães tiveram suas fezes examinadas uma semana antes e até o dia em que foram eutanasiados. O cão que não foi eutanasiado teve suas fezes colhidas durante 30 dias. Depois da eutanasia fragmentos de órgãos foram processados para histopatologia, imuno-histoquímica, reação de imunofluorescência indireta em tecidos, PCR e PCR em tempo real para detecção do parasito. A necropsia revelou que os intestinos delgado e grosso, baço e pulmões foram os órgãos afectados. Oocistos de N. caninum não foram identificados nas amostras de fezes. A PCR em tempo real foi a técnica mais sensível para detectar o protozoário nos tecidos, sendo identificados em 41% das amostras analisadas. Os nossos resultados indicam que o modelo experimental utilizando MCs evidenciou ser um bom modelo para estudar a relação parasito-hospedeiro durante a fase aguda da infecção.