Detection of Leishmania infection in paraffin-embedded skin biopsies of dogs using polymerase chain reaction.

Abstract

Canine leishmaniosis (CL) is a severe systemic infectious disease of the dog caused by protozoan parasites of the genus Leishmania. The disease is endemic in Mediterranean countries, parts of east Africa, India, and Central and South America. Classic CL is as a chronic wasting disease with anemia, intermittent pyrexia, arthritis, and generalized lymphadenopathy. The skin is one of the main organs affected by CL,17 and cutaneous lesions are highly variable. The most common cutaneous lesion is generalized scaling leading to large areas of alopecia.5,6 Clinical signs of CL are extremely variable, and diagnosis is often difficult. The diagnosis is usually made by direct observation of the parasite in bone marrow smears or by testing for specific antibodies in serum. In CL-endemic areas, however, pathologists often receive skin biopsies of dogs suspected of suffering from CL. Histopathologic pictures are not specific for CL and range from diffuse granulomatous dermatitis to lichenoid or pustular dermatitis.5,6 Under these conditions, a definitive diagnosis can only be established by detection of amastigotes of Leishmania. Often, however, very few amastigotes are present, and they may be overlooked in routine tissue examination.6 Immunohistochemistry has substantially improved the histopathologic diagnosis of CL,3,4 although doubtful cases still emerge when the presence of Leishmania amastigotes cannot be conclusively demonstrated. Recently, oligonucleotide primers with various specificities for Leishmania detection have been described,8,9,14 and the polymerase chain reaction (PCR) has been used to detect Leishmania DNA in bone marrow samples or fresh biopsies and in paraffin-embedded human skin biopsies.7 The aim of this study was to assess the ability of PCR to detect Leishmania DNA in paraffin-embedded canine skin biopsy specimens and to compare the results with those found by immunohistochemistry. Samples from 35 dogs were studied. Ten had confirmed CL diagnosed by clinical profile and course of disease, presence of anti-Leishmania antibodies, and observation of parasites in bone marrow. In addition, skin biopsies were taken from all 10 dogs, fixed in 4% formaldehyde, and embedded in paraffin, and sections were stained with hematoxylin and eosin. Immunohistochemical detection of Leishmania was accomplished using a standard immunoperoxidase protocol.4 A polyclonal anti-Leishmania antibody obtained from a rabbit was used as the first antiserum. In skin biopsies from these 10 dogs, lesions consistent with CL (an accumulation of macrophages in the superficial and deep dermis with some plasma cells, lymphocytes, and polynuclear neutrophils scattered between the macrophages) were observed, and im-