Prevalence of haemosporidia in Asian Glossy Starling with discovery of misbinding of Haemoproteus-specific primer to Plasmodium genera in Sarawak, Malaysian Borneo

Abstract

Background: Plasmodium, Haemoproteus and Leucocytozoon are three mainly studied blood parasites known to cause malarial and pseudomalarial infections in avian worldwide. Although Sarawak is a biodiversity hotspot, molecular data on blood parasite diversity in birds are absent. The objective of the study is to determine the prevalence of blood parasite in Asian Glossy Starlings (AGS), an urban bird with high population density in Sarawak and to elucidate the phylogenetic relationship with other blood parasite. Methods: Twenty-nine carcasses of juvenile AGS that were succumbed to death due to window collision were collected around the vicinity of Universiti Malaysia Sarawak. Nested-multiplex and nested PCR targeting the Cytochrome B gene were used to detect Plasmodium and Haemoproteus, and Leucocytozoon respectively. Two primer sets were used for Haemoproteus detection to increase detection sensitivity, with one being a genus-specific primer. Results: Fourteen samples (prevalence rate: 48.28%) were found positive for avian Plasmodium. Phylogenetic analysis divided our sequences into five lineages, pFANTAIL01, pCOLL4, pACCBAD01, pALPSIS01 and pALPSIS02, with two lineages being novel. No Haemoproteus and Leucocytozoon was found in this study. However, Haemoproteus-specific primer used amplified our Plasmodium samples, making the primer non-specific to Haemoproteus only. Conclusion: This is the first blood parasite detection study on AGS using carcasses and blood clot as sample source in Sarawak. Due to the scarcity of longer sequences from regions with high genetic plasticity, usage of genus-specific primers should be validated with sequencing to ensure correct prevalence interpretation.