Abstract
BACKGROUND AND AIM: Zoonotic malaria poses an emerging challenge in Southeast Asia, where rapid urbanization and deforestation increase human-wildlife interactions. Macaques are important natural reservoirs for Plasmodium species due to their evolutionary proximity to humans. Understanding host-specific immune responses to malaria in free-living macaques may aid biomarker discovery and guide surveillance strategies. This study aimed to characterize serum proteomic profiles in three wild macaque species, Macaca fascicularis (Mf), Macaca leonina (Ml), and Macaca arctoides (Ma), naturally infected with zoonotic Plasmodium spp., and to identify conserved proteins with biomarker potential. MATERIALS AND METHODS: Blood samples from 61 macaques (25 Ml, 32 Ma, 4 Mf) were screened for Plasmodium species using nested polymerase chain reaction (PCR). Serum proteomes were analyzed using liquid chromatography-tandem mass spectrometry, followed by bioinformatics-based differential protein expression analysis, pathway enrichment, gene ontology classification, and construction of protein-protein interaction (PPI) networks. Comparative analyses were performed across species to identify conserved expression signatures. RESULTS: Nested PCR confirmed Plasmodium infections in 20.00% of Ml, 50.00% of Ma (34.78% zoonotic), and 100% of Mf samples. Proteomic profiling identified 9,525 serum proteins, of which 698 were differentially expressed across species. Thirty-six proteins formed robust PPI subnetworks linked to immune defense, apoptosis, DNA repair, calcium signaling, and cytoskeletal remodeling. Ml exhibited predominant protein upregulation, whereas Mf and Ma showed downregulation trends, indicating species-specific immune adaptations. Notably, six of nine previously reported malaria-associated proteins (including CARD domain-containing protein, interleukin 1 receptor-associated kinase 1, and non-specific serine/threonine protein kinase) were consistently expressed across all species, supporting their potential as conserved biomarkers. CONCLUSION: Free-living macaques demonstrate distinct proteomic responses to Plasmodium infection, with Ml mounting a stronger immune response relative to Ma and Mf. The identification of conserved immune-related proteins highlights their translational potential as biomarkers for zoonotic malaria in humans. These findings advance the understanding of host-parasite interactions in natural macaque populations and provide a foundation for selecting optimal primate models, improving surveillance, and developing targeted interventions against zoonotic malaria.