title: In situ hybridisation for the detection of Leishmania species in paraffin wax-embedded canine tissues using a digoxigenin-labelled oligonucleotide probe.
abstract: The diagnosis of canine leishmaniosis (CanL) is currently predominantly achieved by cytological or histological identification of amastigotes in biopsy samples, demonstration of specific anti-Leishmania antibodies and PCR-based approaches. All these methods have the advantage of being sensitive and more or less specific; nevertheless, most of them also have disadvantages. A chromogenic in situ hybridisation (ISH) procedure with a digoxigenin-labelled probe, targeting a fragment of the 5.8S rRNA was developed for the detection of all species of Leishmania parasites in routinely paraffin wax-embedded canine tissues. This method was validated in comparison with traditional techniques (histology, PCR), on various tissues from three dogs with histological changes consistent with a florid leishmaniosis. Amastigote forms of Leishmania gave clear signals and were easily identified using ISH. Various tissues from 10 additional dogs with clinical suspicion or/and a positive serological test but without histological presence of amastigotes did not show any ISH signals. Potential cross-reactivity of the probe was ruled out by negative outcome of the ISH against selected protozoa (including the related Trypanosoma cruzi) and fungi. Thus, ISH proved to be a powerful tool for unambiguous detection of Leishmania parasites in paraffin wax-embedded tissues.
authors.affiliation: Institute of Pathology and Forensic Veterinary Medicine, Department of Pathobiology, University of Veterinary Medicine, Veterinärplatz 1, 1210 Vienna, Austria.
authors.forename: N
authors.initials: N
authors.lastname: Dinhopl
authors.forename: M M
authors.initials: MM
authors.lastname: Mostegl
authors.forename: B
authors.initials: B
authors.lastname: Richter
authors.forename: N
authors.initials: N
authors.lastname: Nedorost
authors.forename: A
authors.initials: A
authors.lastname: Maderner
authors.forename: K
authors.initials: K
authors.lastname: Fragner
authors.forename: H
authors.initials: H
authors.lastname: Weissenböck
mesh_terms: D000818:Animals; D004076:Digoxigenin; D004283:Dog Diseases / Q000175:diagnosis*; D004285:Dogs; D017403:In Situ Hybridization / Q000379:methods / Q000662:veterinary*; D007891:Leishmania / Q000302:isolation & purification*; D007896:Leishmaniasis / Q000175:diagnosis / Q000662:veterinary*; D016612:Paraffin Embedding / Q000662:veterinary; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012341:RNA, Ribosomal, 5S / Q000032:analysis
chemical_list: D012341:RNA, Ribosomal, 5S; D004076:Digoxigenin