Himmel et al. Parasites Vectors (2019) 12:282 https://doi.org/10.1186/s13071-019-3536-2 Parasites & Vectors RESEARCH Open Access Molecular probes for the identifcation of avian Haemoproteus and Leucocytozoon parasites in tissue sections by chromogenic in situ hybridization Tanja Himmel1, Josef Harl1, Anna Kübber‑Heiss2, Cornelia Konicek3, Nuhacet Fernández4, Carles Juan‑Sallés5, Mikas Ilgūnas6, Gediminas Valkiūnas6 and Herbert Weissenböck1* Abstract Background: Avian haemosporidian parasites can cause severe disease in their hosts due to excessive exo‑eryth‑ rocytic 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‑eryth‑ rocytic tissue stages based on morphological characters is challenging. Therefore, the present study aimed at devel‑ oping specifc molecular probes for the identifcation of Haemoproteus spp. and Leucocytozoon spp. in histological sections using chromogenic in situ hybridization. Methods: Parasite subgenus‑specifc 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‑fxed, parafn‑embedded tissue samples determined positive for these parasites by PCR and histopathology. To confrm the presence of parasites at sites of probe hybridization, consecutive sections were stained with haematoxylin–eosin and examined. Results: Parahaemoproteus‑ and Leucocytozoon‑specifc 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 diferent from the probes’ target. Conclusions: Chromogenic in situ hybridization using 18S ribosomal RNA‑specifc oligonucleotide probes reliably identifes and discriminates Haemoproteus and Leucocytozoon parasites in tissue sections and enables unequivocal diagnosis of haemosporidioses. Keywords: In situ hybridization, Haemoproteus, Leucocytozoon, Birds, Pathology, Exo‑erythrocytic meronts, 18S ribosomal RNA, Haemosporidians *Correspondence: [email protected] 1 Institute of Pathology, Department of Pathobiology, University of Veterinary Medicine Vienna, Vienna, Austria Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Himmel et al. Parasites Vectors (2019) 12:282 Page 2 of 10 Background parasites have been identifed to cause morbidity and Avian haemosporidian parasites (Haemosporida) are mortality in several bird species. Severe haemosporidi- widespread almost all over the world and infect birds of oses caused by these parasites have been repeatedly diverse families and orders. Te majority of described reported in both captive [12–15, 17–25] and wild birds parasites belong to the genera Plasmodium, Haemo- [9, 10, 16, 18, 26] and were commonly associated with the proteus and Leucocytozoon, comprising more than 200 development of megalomeronts in diverse organs of their morphologically distinct species [1]. Te life-cycle of hosts. haemosporidians in the avian host includes development Unequivocal determination of parasitic tissue stages both in tissues (exo-erythrocytic merogony) and blood in haemosporidian infections is difcult. Diagnosis of cells. Haemosporidians always undergo exo-erythrocytic haemosporidian infections in lethal cases is primar- merogony producing meronts in tissue cells before they ily achieved by necropsy followed by histopathological infect blood cells and form gametocytes, the parasite examination and PCR-based testing. Parasites can be stage infective for vectors [2]. detected in routinely stained histological sections; how- Te pathogenic impact of haemosporidians on the host ever, microscopic examination is time-consuming and can be variable at diferent developmental stages, and parasites can be overlooked or confounded with nuclear severity of infection in avian hosts depends on many fac- fragments in necrotic tissue or cell debris. Although mor- tors [3]. Generally, haemosporidian infection is subclini- phological features can indicate their generic identity, cal in birds; however, it is well established that parasites the correct assignment of parasites is often challenging, not only cause haematological disorders but can also especially in cases of aberrant infections due to unusual elicit severe damage to organs due to marked merogony morphology and location in atypical hosts [2]. Tere are in tissues [2]. For example, Plasmodium infections can several reports presenting severe cases of haemosporidi- result in excessive multiplication of parasites in various oses, in which conclusive determination of the parasites organs including the brain, where meronts cause block- based on tissue stages was not achieved, particularly in age of capillaries, ultimately leading to death of the host cases of Haemoproteus and Leucocytozoon infections [10, [4]. Such lethal infections were frequently reported in 26–28]. In many cases, identifcation of involved parasites immunologically naive hosts, which are highly suscepti- is accomplished by histological analysis in combination ble to infection, e.g. penguins [5–7]. However, there are with PCR and sequencing. However, even sensitive PCR also cases, albeit less numerous, which demonstrate mor- does not always reliably detect parasites, especially in talities linked to malaria in wild birds inhabiting regions co-infections, which are common in birds [29] hamper- with haemosporidian transmission [8–10]. Exo-eryth- ing correct attribution of detected genotypes to morpho- rocytic merogony of Haemoproteus and Leucocytozoon, logical stages observed in histology. Tis is a particularly the most diverse avian haemosporidian genera besides sensitive issue in distinguishing growing (young) exo- Plasmodium, has been investigated only fragmentarily erythrocytic meronts of Plasmodium and Haemoproteus because of methodological difculties to access these life- parasites, which usually are morphologically identical at cycle stages, particularly in wild animals. Data on com- this stage of development [1]. plete tissue merogony are available only for a few of these Te aim of the present study was to develop molecular blood parasite species [2]. Experimental research might probes for the specifc detection of Haemoproteus (sub- provide convincing data [1], but is challenging to design genus Parahaemoproteus) and Leucocytozoon (subgenus with Haemoproteus and Leucocytozoon parasites, mainly Leucocytozoon) parasites in formalin-fxed, parafn wax- because of difculties to obtain sporozoites, the only embedded (FFPE) tissue samples by using chromogenic infective stage that can initiate exo-erythrocytic devel- in situ hybridization (CISH). Tis method has already opment of these haemosporidians in vertebrate hosts. been established for the detection of Plasmodium spe- Cultivation and experimental infection of vectors (spe- cies by using probes which target the 18S ribosomal cies of Ceratopogonidae, Hippoboscidae and Simuliidae) RNA (rRNA) of parasites [7]. Based on these convinc- are needed for such experiments. With rare exceptions, ing results, molecular probes targeting the 18S rRNA Haemoproteus and Leucocytozoon parasites were usually of Haemoproteus species and Leucocytozoon spp. were considered to be relatively benign in avian hosts, par- designed. ticularly in wildlife [11]. Tat hampered research in this feld of parasitology and veterinary medicine for better Methods understanding virulence of haemosporidian infections. Probe design Numerous veterinary observations and feld case reports Oligonucleotide probes were designed to target the contradicted this opinion, but they did not receive due 18S rRNA of parasites from the avian haemosporidian attention [12–16]. Haemoproteus and Leucocytozoon genera Haemoproteus (subgenus Parahaemoproteus) Himmel et al. Parasites Vectors (2019) 12:282 Page 3 of 10 and Leucocytozoon (subgenus Leucocytozoon). Hae- Tissue samples mosporidian 18S ribosomal DNA (rDNA) sequences Formalin-fxed, parafn wax-embedded (FFPE) tissue were isolated from genomes published in NCBI Gen- samples including various organs from infected wild Bank [30] (accession numbers X13706, AY625607, birds were obtained from the archive of the Institute of PRJEB9073, PRJEB9074) and aligned with sequences Pathology and the Research