Molecular probes for the identification of avian Haemoproteus and Leucocytozoon parasites in tissue sections by chromogenic in situ hybridization

BackgroundAvian haemosporidian parasites can cause severe disease in their hosts due to excessive exo-erythrocytic merogony and anaemia caused by blood stages. Notably, the development of megalomeronts by species of Haemoproteus and Leucocytozoon has been associated with mortalities in birds. Diagnosis of lethal infections is currently accomplished by the detection of parasites' tissue stages in histological sections combined with PCR and sequencing. However, sequences frequently are not reliably obtained and the generic discrimination of exo-erythrocytic tissue stages based on morphological characters is challenging. Therefore, the present study aimed at developing specific molecular probes for the identification of Haemoproteus spp. and Leucocytozoon spp. in histological sections using chromogenic in situ hybridization.MethodsParasite subgenus-specific oligonucleotide probes were designed to target the 18S ribosomal RNA of Haemoproteus species (subgenus Parahaemoproteus) and Leucocytozoon spp. (subgenus Leucocytozoon) and were in situ hybridized to sections from formalin-fixed, paraffin-embedded tissue samples determined positive for these parasites by PCR and histopathology. To confirm the presence of parasites at sites of probe hybridization, consecutive sections were stained with haematoxylin-eosin and examined.ResultsParahaemoproteus- and Leucocytozoon-specific probes labelled erythrocytic and exo-erythrocytic stages of Haemoproteus spp. and Leucocytozoon spp., respectively. Binding of probes to parasites was consistent with detection of the same exo-erythrocytic meronts in consecutive haematoxylin-eosin-stained sections. Cross-reactivity of the probes was ruled out by negative chromogenic in situ hybridization when applied to samples positive for a parasite of a genus different from the probes' target.ConclusionsChromogenic in situ hybridization using 18S ribosomal RNA-specific oligonucleotide probes reliably identifies and discriminates Haemoproteus and Leucocytozoon parasites in tissue sections and enables unequivocal diagnosis of haemosporidioses.

• Avian malaria and related haemosporidioses in wild birds: molecular tools for improved post-mortem diagnosis of haemosporidial disease and an investigation of haemosporidian-associated mortalities in native thrushes.

Loro Parque Stiftung
Noahs Path
Nature Research Centre
Veterinärmedizinische Universität Wien
Loro Parque Fdn