PCR-based detection and genotyping of haematozoa (Protozoa) parasitizing eagle owls, Bubo bubo

Abstract

We genetically analysed haematozoa parasites (Protozoa) isolated from nestling eagle owls (Bubo bubo) in Toledo province, Central Spain. A total of 206 nestlings from 74 nests were screened for parasites of the genera Leucocytozoon, Plasmodium, and Haemoproteus using a very efficient polymerase chain reaction (PCR) approach that amplifies a partial segment of the mitochondrial cytochrome b gene of these parasites. PCR-based detection and sequence analyses revealed a unique lineage of Leucocytozoon (EO1) parasitizing nestling eagle owls. Ocular examination of blood smears identified these parasites as Leucocytozoon ziemanni, the only species deemed valid in owls based on morphology. Preliminary phylogenetic analyses using homologous published sequences isolated from other owl species suggest that L. ziemanni constitutes a monophyletic clade that may be composed by a complex of genetically differentiated cryptic species.

Factors associated with the geographic distribution of leucocytozoa parasitizing nestling eagle owls (Bubo bubo): a local spatial-scale analysis

“…Positive or negative infections were scored running 2 ll of the final PCR product on a 2% agarose gel stained with ethidium bromide and determining the presence/absence of a band of the expected size under UV light. In a previous study we found that all infections detected by ocular screening of blood smears gave positive PCR amplifications whereas only 83.17% of positive amplifications are also detected by ocular methods (Ortego and Cordero 2009). Thus, as also established in several previous studies (e.g.…”

“…Data on host specificity of the studied lineage is also an interesting issue to be addressed in the future. L. ziemanni, the only leucocytozoid species recognized in owls, probably constitutes a complex of cryptic species shared by a variable number of host species (Krone et al 2008;Ishak et al 2008;Ortego and Cordero 2009). Phylogenetic analyses and data on sequence divergence derived from recent studies suggest that the lineage infecting eagle owls could be shared with other coexisting owl species and horizontal transmissions may be relevant (Krone et al 2008;Ishak et al 2008;Ortego and Cordero 2009).…”

“…A second visit was conducted when nestlings were 25-40 days old to obtain blood samples and determine the prevalence and intensity of blood parasite infections. The only blood parasite infecting eagle owls in the study population is Leucocytozoon ziemanni (Ortego et al 2007a;Ortego and Cordero 2009) and all nestlings which eventually become infected by fledging had detectable gametocytes in the blood smears from age 25 days old (Ortego et al 2007a). Thus, absences of L. ziemanni after this age are not likely to be due to unreliable sampling before infections are patent (Ortego et al 2007a).…”

“…In recent years, DNA sequencing has greatly contributed to understanding several aspects of this hostparasite system, including relevant information on their ecology and evolution (Perkins and Schall 2002;Sehgal et al 2006;Hellgren et al 2007Hellgren et al , 2008; Krone et al 2008;Martinsen et al 2008;Ishak et al 2008;Ortego and Cordero 2009). Recent phylogenetic information is also of great relevance because several apparent cryptic species may show different transmission times or vectors, and previous studies disregarding this variability may confound the factors influencing parasite transmission (Sehgal et al 2006;Pérez-Tris et al 2007;Ishak et al 2008) or the consequences on host fitness (Marzal et al 2008;Ortego et al 2008).…”

“…Molecular techniques have now revealed that nestling eagle owls are parasitized by a unique lineage (EO1) of Leucocytozoon ziemanni (synonym to L. danilewskyi; Valkiūnas 2004) in our study population from Central Spain (Ortego and Cordero 2009). The prevalence of this lineage among individual nestlings is 52.7 and 68.9% of the nests had at least one infected nestling (Ortego and Cordero 2009). Leucocytozoids infecting nestling eagle owls are a good model system for exploring the factors influencing transmission of avian haematozoa for several reasons.…”