Identification of suitable adjuvant for vaccine formulation with the Neospora caninum antigen NcSRS2.

The parasite Neospora caninum is the main cause of abortion in cattle in many countries around the world, so a vaccine is a rational approach method for the control of the disease. An effective vaccine should be able to prevent both, the horizontal and vertical transmission of N. caninum. In this study, the immune vaccinal response of the recombinant protein rNcSRS2 of N. caninum expressed in Pichia pastoris and formulated with water-in-oil emulsion, xanthan gum, and alum hydroxide was assessed in an experimental murine model. Groups of 10 Balb/c mice were subcutaneously inoculated with two doses of prNcSRS2 twenty-one days apart. After the second immunization, four mice from each group were euthanized, and splenocytes were stimulated ex vivo with recombinant protein. The IgG dynamics were evaluated by indirect ELISA, and the splenocytes cytokines transcription by qPCR. All groups elicited specific antibodies against prNcSRS2, with the water-in-oil group showing significantly (p ≤ .05) elevated titers compared to the other groups. The prNcSRS2 protein alone did not induce a significant ex vivo splenic transcription level of IFN-γ, TNF-α, IL-4, IL-10, and IL-12 cytokines, except for IL-17A, and the adjuvant associations with the prNcSRS2 protein induced different cytokine transcription profiles. The water-in-oil emulsion modulated the expression of TNF-α; the xanthan gum modulated IL-4, IL-10, and IL-12; and alum hydroxide modulated IFN-γ, TNF-α, IL-4, IL-10, and IL-12. In conclusion, it was found that the association of the recombinant prNcSRS2 protein with different adjuvants induced different levels of specific antibody, and a distinct splenic cytokine profile in an adjuvant-dependent manner. The mechanisms of adjuvancity activity is complex, so adjuvant formulation may help in the design of efficient vaccine to control Neosporosis.

Use of ELISA based on NcSRS2 of Neospora caninum expressed in Pichia pastoris for diagnosing neosporosis in sheep and dogs.

Neosporosis is a disease caused by the protozoon Neospora caninum that leads to significant economic losses in many countries. In the present study, we report on use of the recombinant protein NcSRS2 of N. caninum expressed in Pichia pastoris in an indirect immunoenzymatic assay (ELISA) for diagnosing neosporosis infection in sheep and dogs. We observed that the ELISA test yielded specificity of 94.5% and sensitivity of 100% for sheep and specificity of 93.3% and sensitivity of 100% for dogs. We observed that the sensitivity was higher than shown by the indirect fluorescent antibody test, and this was confirmed by means of Western blot. The results from this study suggest that the recombinant protein expressed in P. pastoris is a suitable antigen for use in immunodiagnosis to detect N. caninum in two important species exposed to this parasitosis.

Use of ELISA based on NcSRS2 of Neospora caninum expressed in Pichia pastoris for diagnosing neosporosis in sheep and dogs / Utilização de um ELISA baseado em NcSRS2 de Neospora caninum expressa em Pichia pastoris para diagnóstico de neosporose em ovinos e cães

Neosporosis is a disease caused by the protozoon Neospora caninum that leads to significant economic losses in many countries. In the present study, we report on use of the recombinant protein NcSRS2 of N. caninum expressed in Pichia pastoris in an indirect immunoenzymatic assay (ELISA) for diagnosing neosporosis infection in sheep and dogs. We observed that the ELISA test yielded specificity of 94.5% and sensitivity of 100% for sheep and specificity of 93.3% and sensitivity of 100% for dogs. We observed that the sensitivity was higher than shown by the indirect fluorescent antibody test, and this was confirmed by means of Western blot. The results from this study suggest that the recombinant protein expressed in P. pastoris is a suitable antigen for use in immunodiagnosis to detect N. caninum in two important species exposed to this parasitosis.

A neosporose é uma doença causada pelo protozoário Neospora caninum que leva a perdas econômicas importantes em muitos países. No presente estudo, é descrita a utilização da proteína recombinante NcSRS2 de N. caninum expressa em Pichia pastoris em um ensaio imunoenzimático indireto (ELISA) para o diagnóstico de infecção por Neospora em ovelhas e cães. Observou-se, que utilizando-se um ELISA, o teste produziu uma especificidade de 94,5% e uma sensibilidade de 100% para ovinos; e uma especificidade de 93,3% e sensibilidade de 100% para cães. Uma maior sensibilidade foi observada em relação à IFI que foi confirmada por Western blot. Os resultados deste estudo sugerem que a proteína recombinante expressa em P. pastoris é bom antígeno para ser utilizado no diagnóstico imunológico para detectar N. caninum em duas espécies importantes expostas a esta parasitose.

Expression of Neospora caninum NcSRS2 surface protein in Pichia pastoris and its application for serodiagnosis of Neospora infection.

Neospora caninum is considerd a major cause of abortion in cattle worldwide. The antigenic domain of NcSRS2 in N. caninum is an important surface antigen present in the membrane of this parasite. In the present study, the Pichia pastoris expression system proved to be a useful tool for the production of recombinant protein. The truncated NcSRS2 gene (by removal of the N-terminal hydrophobic sequence), was cloned in the vector pPICZalphaB, and integrated on the genome of the methylotrophic yeast P. pastoris. Subsequently, the NcSRS2 protein was expressed, purified, and characterized using naturally infected cattle sera and Mab 6xhistag. The recombinant protein NcSRS2 was present in the supernatant of the culture, where later it was concentrated and purified using ammonium sulfate (∼100 mg/ml). An indirect immunoenzymatic assay (ELISA) was performed using cattle sera from endemic N. caninum area.