Abstract
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.
Highlights
Avian haemosporidian parasites can cause severe disease in their hosts due to excessive exo-eryth‐ rocytic merogony and anaemia caused by blood stages
Haemosporidian 18S ribosomal DNA sequences were isolated from genomes published in NCBI GenBank [30] and aligned with sequences obtained from an analysis of nuclear 18S rDNA of a selection of Haemoproteus spp., Leucocytozoon spp. and Plasmodium spp. (JH, unpublished data) using MAFFT v. 7 [31]
This study demonstrates that Haemoproteus and Leucocytozoon parasite tissue stages can be readily detected and discriminated by chromogenic in situ hybridization (CISH) using 18S ribosomal RNA (rRNA)-specific probes
Summary
Avian haemosporidian parasites can cause severe disease in their hosts due to excessive exo-eryth‐ rocytic merogony and anaemia caused by blood stages. Plasmodium infections can result in excessive multiplication of parasites in various organs including the brain, where meronts cause blockage of capillaries, leading to death of the host [4]. Such lethal infections were frequently reported in immunologically naive hosts, which are highly susceptible to infection, e.g. penguins [5,6,7]. Experimental research might provide convincing data [1], but is challenging to design with Haemoproteus and Leucocytozoon parasites, mainly because of difficulties to obtain sporozoites, the only infective stage that can initiate exo-erythrocytic development of these haemosporidians in vertebrate hosts. Severe haemosporidioses caused by these parasites have been repeatedly reported in both captive [12,13,14,15, 17,18,19,20,21,22,23,24,25] and wild birds [9, 10, 16, 18, 26] and were commonly associated with the development of megalomeronts in diverse organs of their hosts
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