First Molecular Report of Haemaphysalis shimoga and Haemaphysalis bispinosa Infesting Captive Sambar Deer (Rusa unicolor) in Malaysia, with Detection of Anaplasma phagocytophilum and Theileria Co-infection.

PURPOSE: Haemaphysalis (subgenus Kaiseriana) ticks are important vectors of tick-borne pathogens in tropical Asia, yet data on tick species infesting captive wildlife in East Malaysia remain scarce. This study provides the first molecular characterization of Haemaphysalis ticks and detection of their associated pathogens from captive Sambar deer (Rusa unicolor) at a biopark in Bintulu, East Malaysia, and a slaughterhouse in Gombak, West Malaysia. METHODS: A total of 60 ticks were collected from five Sambar deer during 2021-2022 and morphologically identified. Subsequently, DNA was extracted from 49 specimens; however, due to specimen degradation, only seven samples retained sufficient DNA quality and quantity for molecular analysis. These seven ticks were molecularly identified by using the mitochondrial cox1 sequencing and screened for Anaplasma and Theileria species via 16S rDNA and 18S rDNA sequencing, with phylogenetic relationships inferred by maximum likelihood analysis. RESULTS: Two Haemaphysalis species were identified: Haemaphysalis shimoga (97.88-99.27% identity) and Haemaphysalis bispinosa (99.5-100% identity), each resolved in well-supported monophyletic clades. Five ticks (four H. shimoga and one H. bispinosa) were infected with Theileria capreoli (98.83-100%) or T. cervi (95.07-99.74%), and H. bispinosa was positive for Anaplasma phagocytophilum (99.53-100%). Moreover, H. bispinosa specimen harbored a concurrent triple infection with T. cervi, T. capreoli, and A. phagocytophilum. CONCLUSIONS: This study provides the first molecular evidence of H. shimoga and H. bispinosa infesting captive Samber deer, with detection of Anaplasma phagocytophilum and Theileria co-infection in Malaysia and highlights the occurrence of tick-associated pathogens in these specimens in Malaysia.