Diagnostic potential of a multi-antigen ELISA for feline leishmaniosis

Abstract Background Leishmania infantum is a sand fly-transmitted zoonotic protozoan, endemic in the Mediterranean basin and responsible for human, canine (CanL), and feline (FeL) leishmaniosis. While dogs are the primary reservoir host, a growing number of FeL cases have been reported in this region despite the absence of pathognomonic clinical signs and limited diagnostic tools. Herein, we evaluate the performance of seven serological tools for CanL in detecting antibodies to Leishmania in cats, aiming to improve FeL diagnosis. Methods Five ELISAs based on Leishmania -specific antigens (soluble promastigote Leishmania antigens, SPLA; recombinant Leishmania proteins K39 rK39, K28, and KDDR, and L. infantum cytosolic peroxiredoxin, LicTXNPx), indirect fluorescent antibody test (IFAT), and direct agglutination test (DAT) were compared for detecting anti- Leishmania antibodies in 274 cats. Blood samples from the same cats were molecularly tested. Statistical analysis was performed based on clustering of multivariate serological data. Reference serological profiles were first defined in a control group. Study group data were subsequently classified according to these profiles, with principal component analysis used for dimensionality reduction and graphical representation. Associations between seropositivity and clinicopathological alterations were determined using seropositivity thresholds. Results Cats exhibited attenuated and heterogeneous antibody responses to L. infantum serological tests. Agreement between individual tests was variable, with poor concordance when single markers were considered. Multivariate analysis, based on clustering of serological responses, showed that positivity to multiple antigens was associated with clinically affected cats. Positivity to multiple Leishmania -specific ELISA antigens was associated with diverse clinical presentations and prognostic laboratory alterations, including anaemia, thrombocytopenia, and hypergammaglobulinaemia. Conclusions Integrating multi-antigen ELISA, particularly rK39, SPLA, and LicTXNPx, into FeL diagnostic workflows, alongside molecular and clinical assessment, improves epidemiological surveillance, early detection, and disease management. These findings support the development of serological strategies tailored to feline hosts for enhanced surveillance and management. Graphical Abstract