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

Abstract

Background: Avian 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. Methods: Parasite 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. Results: Parahaemoproteus-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. Conclusions: Chromogenic 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.

Haemosporidioses in wild Eurasian blackbirds (Turdus merula) and song thrushes (T. philomelos): an in situ hybridization study with emphasis on exo-erythrocytic parasite burden

“…A small number of the samples was already analysed earlier by Dinhopl et al [25], however, they were screened using the primers Palu-F and Palu-R by Martínez et al [29], which do not allow detection of Leucocytozoon parasites. In order to re-evaluate these samples using a current standard PCR assay [30] and recently developed molecular probes [28], they were included in this study as well. Tissue samples were taken from several organs, including heart, lung, liver, spleen, kidney, brain, gizzard and intestine in most cases and in some also skeletal muscle and bursa of Fabricius.…”

“…For the detection of parasites in tissue sections, FFPE tissue blocks of each individual bird were cut into 1-2 µm thick sections, one of which was stained with haematoxylin and eosin (HE) and the remaining ones were subjected to CISH. Chromogenic in situ hybridization was performed following previously established protocols using genus-specific oligonucleotide probes for the detection of Plasmodium, Haemoproteus, and Leucocytozoon [14,28]. In cases of mixed infections, as determined by PCR and sequencing, several sections were separately incubated with all relevant probes.…”

“…Traditionally, exo-erythrocytic parasite stages are examined by histological techniques, but their detection is cumbersome because the stages are often small and easily confounded with cellular structures or debris. In order to facilitate the detection in tissue sections and identification of parasites belonging to the three major genera, molecular probes have been developed [14,28].…”

Penguins are competent hosts of Haemoproteus parasites: the first detection of gametocytes, with molecular characterization of Haemoproteus larae

“…Unless gametocytes are present in the bloodstream, arthropod vectors are not capable of transmitting the parasite to another host. Haemoproteus species have generally been considered relatively harmless in the past [24], but cases of lethal haemoproteosis have been reported in captive birds [22,23,25,26] and haemoproteosis may possibly be lethal to penguins as well.…”

Haemosporidioses in wild Eurasian blackbirds (Turdus merula) and song thrushes (T. philomelos): an in situ hybridization study with emphasis on exo-erythrocytic parasite burden

“…Traditionally, exo-erythrocytic parasite stages are examined by histological techniques, but their detection is cumbersome because the stages are usually small and easily confounded with cellular structures or debris. In order to facilitate the detection in tissue sections and identification of parasites belonging to the three major genera, molecular probes have been developed [14,28].…”