Application two-dimensional gel electrophoresis coupled with LC-MS/MS to identify candidate serodiagnostic antigens for early detection Psoroptes ovis var. cuniculi infection

Currently, the 'gold standard' for diagnosis of Psoroptes ovis infections is detecting Psoroptes mites or eggs in skin scrapings under microscopy, but it is prone to be mis-diagnosed for detecting early infection of P. ovis. Hence, seeking a reliable diagnostic technique for detecting early-stage mite infections is extremely desirable. Enzyme linked immunosorbent assay (ELISA) has proven to be useful for the diagnosis of early-stage P. ovis infection. Thus, the purpose of this study was to screen serodiagnostic candidate antigens that can detect early P. ovis infection. Psoroptes ovis var. cuniculi wash proteins (PsoWA), which contained an enriched source of secretory and excretory antigens, were separated by two-dimensional gel electrophoresis (2-DE) and screened by immunoblot using sera from rabbits with early-stage Psoroptes infection (1 week and 3 weeks). Immunogenic proteins were submitted for sequencing by liquid chromatography tandem-mass spectrometry (LC-MS/MS) analyses. Three potential diagnostic antigens were identified (PsoSP3, Pso14-3-3(1) and Pso14-3-3(2)) in this study. These were further expressed in E. coli expression system to evaluate the serodiagnostic potential of these recombinant proteins for detecting early-stage P. ovis infection using an indirect ELISA (iELISA). Western blotting showed that 34 protein spots were recognized by rabbit sera of 1 week post-infection (wpi) and 3 wpi. The 2-DE results showed that a total of 199 proteins were detected with molecular weights varying from 20 to 100 kDa and isoelectric point (pI) from 4.1 to 9.3. Among these, 90 proteins were detected both at 1 wpi sera and 3 wpi sera, and the numbers of the specific identified proteins were 27 for 1 wpi sera and 82 for 3wpi sera. Moreover, rPsoSP3 showed better diagnostic efficacy than rPso14-3-3(1) and rPso14-3-3(2) in detecting early-stage P. ovis infection for its higher values of sensitivity, specificity and area under the receiver operating characteristic curve. Our study describes the first immunoproteomic analysis to identify early diagnostic candidate antigens of P. ovis, and the identified antigens of Psoroptes in our study have significant implications for the development of early-stage diagnostic tests. PsoSP3 is a promising early diagnostic antigen for detecting P. ovis var. cuniculi infection.