Integrative molecular and serum proteomic insights into subclinical Theileria infection in captive Fea's muntjac (Muntiacus feae) and red muntjac (Muntiacus vaginalis) in Thailand

The Fea's muntjac (Muntiacus feae) and the northern red muntjac (Muntiacus vaginalis) are Asian cervids maintained in captive conservation programmes in Thailand, yet information on blood-borne pathogens in these species remains limited. This study aimed to investigate Theileria spp. infection in 34 captive muntjacs from four facilities across Thailand using PCR, Sanger sequencing, phylogenetic analysis, and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based serum proteomics. All animals appeared clinically healthy, and no hemoparasites were observed on blood smears. Nevertheless, Theileria spp. DNA was detected by PCR in every individual, indicating subclinical infection. Babesia spp. were below the detection limit of the PCR assay, while members of the Anaplasmataceae were not detected. Phylogenetic analysis of 15 sequences positioned most isolates among T. capreoli-associated (9/15) or other cervid-associated Theileria lineages (5/15), while one isolate (1/15) was positioned separately; relationships involving the study isolates were generally weakly supported. Serum proteomics identified 414 Theileria-associated proteins consistently detected in both host species. Functional annotation revealed proteins involved in invasion (rhoptry-associated proteins), proteolysis (aspartyl proteases and calpain-like proteins), ubiquitin-mediated regulation, vesicular trafficking, and RNA processing, providing preliminary functional insight into Theileria-associated biological processes during subclinical infection. These findings suggest a potential role of captive muntjacs in parasite maintenance where competent vectors are present and demonstrate the value of integrating molecular detection with proteomic profiling to investigate host-parasite interactions in wildlife.