NcROP4 como um marcador de fase crônica no sorodiagnóstico da infecção por Neospora caninum

Abstract

Neospora caninum is an obligate intracellular parasite whick is able to infect a wide range of hosts. Since its first description, N. caninum has emerged as an important disease of cattle and dogs worldwide. The neosporosis diagnosis is classically performed by serological tests as indirect immunofluorescence (RIFI) and ELISA, being the first gold standard for serodiagnosis that aim to detect the presence of antibodies against the parasite. However, there are no commercial tests designed to assess the stage of infection, once the main clinical signs are reported during the acute phase or during infection reactivation. Several protein targets of this pathogen has been tested in order to check the phase of host infection with satisfactory results. However, new targets should be evaluated in order to develop an effective diagnostic for the stages of infection. Thus, the present study aimed to studied the potential of NcROP4 protein as a stage marker of N. caninum infection. For this purpose, we evaluated the expression and localization of NcROP4 during infection in HeLa cells to investigate the possible exposure of this protein to antibodies. Subsequently, bioinformatic analysis and phage display were developed aiming to predict and determine B cell epitopes of the NcROP4. Mice and cattle were experimentally infected to investigate the producing of IgG through indirect and reverse immunoenzyme assay against NcROP4 during infection. It was observed that the NcROP4 protein is secreted into the cytoplasm of HeLa cells during the invasion process, which allows its extravasation to the extracellular environment after cell lysis. Bioinformatics analysis showed that NcROP4 displays 23 regions that are potential B-cell epitopes and analysis of phage display have showed that mAb 20D2 binds to the region 360-400 amino acids of this protein. Also made sure that mice produce high avidity antibodies against recombinant NcROP4 (rNcROP4) after 30 days of infection (chronic phase) and that there is no difference between the IgG subclass (IgG1 and IgG2) in this recognition process. Moreover, experimentally infected cattle recognize rNcROP4 after 44 days of infection, featuring the recognizing of chronic phase. It was also observed that NcROP4 is recognized only during reinfection in reverse ELISA, in which there is the block an epitope of the protein. Thus, it is concluded that NcROP4 can be used as chronic phase marker during N. caninum infection, being an additional strategy in the serodiagnosis of neosporosis.