Abstract

S. mansoni cysteine protease identified via Immunoproteomics as serodiagnostic antigen. • S. mansoni infection rate in stray cats was significantly higher than domestic cats in Shanghai. The zoonotic cestode Spirometra mansoni (Cestoda: Diphyllobothriidae), characterized by a complex life cycle involving plerocercoid larvae (spargana) in amphibians, reptiles, and mammals, as well as adult intestinal stages in carnivorous mammals, particularly felids and canids, presents significant risks to both veterinary and public health. To address diagnostic challenges in feline infections, potential serodiagnostic antigens were characterized using an integrated immunoproteomic and molecular approach. Four subcutaneous infections in nude mouse demonstrated plerocercoid larval migration from the dorsal to the cervical regions within 3 days post-infection (dpi). Total protein extracts from plerocercoid larvae underwent two-dimensional electrophoresis (2-DE) and immunoblotting with hyperimmune rabbit serum, revealing 229 immunoreactive spots among 588 resolved proteins. MALDI-TOF/TOF mass spectrometry combined with bioinformatics analysis identified S. mansoni cysteine protease (SmCP) as a key diagnostic antigen. The smcp gene was prokaryotically expressed, and the recombinant SmCP protein (rSmCP) exhibited strong immunoreactivity, as confirmed by Western blot with sera from infected mice and cats. Immunofluorescence assays (IFA) localized SmCP predominantly to the tegument of plerocercoid larvae. An rSmCP-based ELISA was developed and demonstrated 100% specificity against heterologous parasitic sera. Specific antibodies were detectable in infected mice as early as 6 dpi (OD 450 nm = 0.76 ± 0.18), with peak titers observed at 21 dpi (OD 450 nm = 1.82 ± 0.07). Field testing of cat sera collected in Shanghai revealed a significantly higher seroprevalence in stray cats (28.70%, 33/115) than in domestic cats (8.22%, 6/73; χ 2 =11.29, p  < 0.01). These findings not only