Molecular and Morphological Analyses of Leucocytozoon Parasites (Haemosporida: Leucocytozoidae) in Raptors From Thailand.

INTRODUCTION: Leucocytozoon spp. causes a vector-borne disease that is nonpathogenic in domestic and wild birds. To date, there was no report of leucocytozoonosis in raptors from Thailand. METHODS: This study was carried out to perform morphological and molecular analyses of Leucocytozoon in 400 raptors at a rehabilitation center at Kasetsart University, Thailand during a 7-year period. The nested PCR was used to amplify the cytochrome b gene of Leucocytozoon with primers HaemNF1 and HaemNR3 as the primary reaction. RESULTS: The light microscopic examination revealed Leucocytozoon gametocytes in five raptors; three diurnal raptors [two Crested Goshawks (CGs, Accipiter trivirgatus) and one Eastern Imperial Eagle (EIE, Aquila heliaca)], and two nocturnal raptors (one Oriental Scops-Owl (OSO, Otus sunia,) and one Short-eared Owl, Asio flammeus) and two species were identified: Leucocytozoon danilewskyi in both owl species and L. californicus in two CGs. The PCR method revealed more infection rate (2.0%, 8/400) than the light microscopic method including one Barred Eagle-Owl (BEO, Bubo sumatranus), one Brown Hawk Owl (BHO, Ninox scutulata) and one OSO. A phylogeny revealed that sequences from one SEO and one OSO were clustered with L. danilewskyi and the three Leucocytozoon sequences from diurnal raptors were clustered with L. californicus. The other three sequences from a BHO, a BEO and an OSO were ambiguous. CONCLUSION: This study combined morphological, morphometric and molecular phylogenetic analyses to identify L. danilewskyi in two species of owls, L. californicus in three diurnal raptors, and unknown species in three other owls, representing the first records of leucocytozoon infection in raptors from Thailand.

Morphological and molecular characterization of avian trypanosomes in raptors from Thailand.

From September 2012 to May 2018, blood samples from 364 raptors (mostly adults) were collected and screened for trypanosomes and haemosporidians by microscopic examination and nested polymerase chain reactions (PCR). Trypanosoma spp. were identified in 15 birds from eight different species. Light microscopy revealed 14 cases of infection with Trypanosoma cf. corvi, including one each in black-shouldered kite (Elanus caeruleus, n = 49), Brahminy kite (Haliastur indus, n = 50), and spotted owlet (SO, Athene brama, n = 27); two mountain hawk-eagles (Spizaetus nipalensis, n = 3); and three each in Asian barred owlets (ABO, Glaucidium cuculoides, n = 27), barn owls (BO, Tyto alba, n = 65) and collared scops owls (CSO, Otus lettia, n = 41). In addition, one case of infection with T. avium was identified in an oriental scops owl (OSO, Otus sunia, n = 2). All infected raptors showed very low parasitemia levels. The PCR detected more three positives in one CSO, one Japanese sparrowhawk (Accipiter gularis), and one OSO. The sensitivity and specificity of the PCR method were 93.3% and 99.1%, respectively. The overall infection rate was very low (4.9%). The highest infection rate was recorded in cold-dry season (9.9%). Coinfection of Plasmodium with trypanosomes was found in all three ABOs. Coinfection with Haemoproteus spp. was found in one BO, three CSOs, and one SO. Coinfection with Haemoproteus spp. and Leucocytozoon danilewskyi was found in the OSO. Microfilarias were detected in one ABO and one CSO. The ultrastructure of trypomastigotes of T. cf. corvi in an ABO revealed fine structures. All small subunit ribosomal RNA (SSU rRNA) sequences belong to two clades: T. avium and T. corvi-culicavium complex/group. SSU rRNA gene amplification was not successful in one BO. The raptors with trypanosome infections showed normal hematological values and healthy appearance. Furthermore, this is the first report of T. avium in a nocturnal raptor from Thailand.