Application of in-situ hybridization for the detection and identification of avian malaria parasites in paraffin wax-embedded tissues from captive penguins

Abstract

In captive penguins, avian malaria due to Plasmodium parasites is a well-recognized disease problem as these protozoa may cause severe losses among valuable collections of zoo birds. In blood films from naturally infected birds, identification and differentiation of malaria parasites based on morphological criteria are difficult because parasitaemia is frequently light and blood stages, which are necessary for identification of parasites, are often absent. Post-mortem diagnosis by histological examination of tissue samples is sometimes inconclusive due to the difficulties in differentiating protozoal tissue stages from fragmented nuclei in necrotic tissue. The diagnosis of avian malaria would be facilitated by a technique with the ability to specifically identify developmental stages of Plasmodium in tissue samples. Thus, a chromogenic in-situ hybridization (ISH) procedure with a digoxigenin-labelled probe, targeting a fragment of the 18S rRNA, was developed for the detection of Plasmodium parasites in paraffin wax-embedded tissues. This method was validated in comparison with traditional techniques (histology, polymerase chain reaction), on various tissues from 48 captive penguins that died at the zoological garden Schönbrunn, Vienna, Austria. Meronts of Plasmodium gave clear signals and were easily identified using ISH. Potential cross-reactivity of the probe was ruled out by the negative outcome of the ISH against a number of protozoa and fungi. Thus, ISH proved to be a powerful, specific and sensitive tool for unambiguous detection of Plasmodium parasites in paraffin wax-embedded tissue samples.

In situ hybridization and sequence analysis reveal an association of Plasmodium spp. with mortalities in wild passerine birds in Austria

“…In contrast, non-indigenous birds, mainly penguins and puffins that are kept in zoos of temperate areas, succumb each year to avian malaria (Fleischmann et al 1968;Fix et al 1988;Loupal and Kutzer 1995;Sturrock and Tompkins 2007;Dinhopl et al 2011), because these birds are not adapted evolutionary and physiologically to haemosporidians. The causative species of avian malaria present in these birds are referred to as Plasmodium relictum (Fix et al 1988;Cranfield et al 1990), Plasmodium elongatum (Fleischmann et al 1968;Cranfield et al 1990;Graczyk et al 1994;Dinhopl et al 2011) and Plasmodium juxtanucleare (Grim et al 2003).…”

“…To resolve this problem, an ISH procedure was developed to specifically identify tissue stages of Plasmodium spp. (Dinhopl et al 2011). …”

“…The obtained nucleotide sequences were identified by BLAST search against the databases GenBank and MalAvi (Bensch et al 2009). In addition, these sequences were compared to previously obtained sequences from 13 captive penguins (Dinhopl et al 2011) infected with avian malaria parasites.…”

“…Chromogenic ISH was performed in all 233 cases according to a previously published protocol (Dinhopl et al 2011). In brief, 3-μm paraffin wax-embedded tissue sections were dewaxed and rehydrated.…”

“…The causative species of avian malaria present in these birds are referred to as Plasmodium relictum (Fix et al 1988;Cranfield et al 1990), Plasmodium elongatum (Fleischmann et al 1968;Cranfield et al 1990;Graczyk et al 1994;Dinhopl et al 2011) and Plasmodium juxtanucleare (Grim et al 2003). In a previous retrospective study, various tissue samples from 48 captive penguins that had died between 2000 and 2008 at the zoological garden Schönbrunn, Vienna, were examined by the use of in situ hybridization (ISH), specifically detecting Plasmodium parasites.…”