The diagnosis of canine leishmaniosis (CanL) is currently predominantly achieved by cytological or histological identification of amastigotes in biopsy samples, demonstration of specific antiLeishmania 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 waxembedded tissues.OF the approximately 20 virulent species of Leishmania, a considerable number has been (Dantas-Torres 2007). Clinical disease in dogs, referred to as canine leishmaniosis (CanL) seems to be caused by only one species, L infantum (syn L chagasi) in many regions worldwide. These protozoal parasites of great medical and veterinary significance are transmitted by the bite of female blood-sucking phlebotomine sand fly vectors of the genera Phlebotomus in the Old World (Desjeux 1996) and Lutzomyia in the New World (Grimaldi and Tesh 1993).Based on serological surveys, it has been estimated that at least 2.5 million dogs are infected in the south-western European countries (Moreno and Alvar 2002). Although most of these dogs are clinically asymptomatic, they constitute a very significant part of the reservoir of L infantum, the aetiological agent of zoonotic visceral leishmaniosis (Dantas-Torres 2007 Frequently, skin biopsies and samples from lymphatic tissue are used for diagnosing CanL. Apart from the microscopic identification of amastigotes, IHC procedures are available. However, commercially available monoclonal antibodies failed to produce specific staining in paraffin wax-embedded tissue and alternatively used canine hyperimmune sera are not readily available for all laboratories and may be cross-reactive with related protozoa (Bourdoiseau and others 1997, Tafuri and others 2004). Thus the major aim of the present study was to fill this diagnostic gap and to establish an in situ hybridisation (ISH) procedure for Leishmania species which facilitates detection of parasites directly within the tissue and which should be specific and sensitive.
Material and methods
Tissue sam...