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Leishmaniasis is a parasitic disease of worldwide spread. It is caused by protozoa of the genus Leishmania and is transmitted to animals and humans through the bite of sand flies. In Brazil, leishmaniasis is one of the zoonoses of major importance and expansion. The objective of this work is to describe the clinical, pathological, immunohistochemical and molecular findings of cutaneous leishmaniasis by Leishmania enriettii in guinea pig (Cavia porcellus). Three animals had nodular and alopecia lesions on the muzzle, ears and ulcerated lesions on the distal extremities of the pelvic limbs. The males (2) also had diffuse thickening of the scrotal skin. Samples of the ulcerated cutaneous lesions were evaluated by cytology which were observed as amastigote forms of Leishmania. One of the animals was euthanized and necropsied. Histopathology showed abundant dermal infiltrate of macrophages, plasma cells, lymphocytes and multinucleated giant cells. Numerous macrophages contained parasitoid vacuoles with amastigote forms, evidenced by immunohistochemical examination. The molecular characterization based on the SSUrDNA gene identified the species as L. enrietti. The diagnosis of cutaneous leishmaniasis in these cases was based on pathological findings and confirmed by immunohistochemistry, PCR and sequencing.(AU)

A leishmaniose é uma doença parasitária de distribuição mundial. É causada por protozoários do gênero Leishmania e é transmitida para animais e seres humanos por meio da picada de flebotomíneos. No Brasil, a leishmaniose é uma das zoonoses de maior importância e expansão. O objetivo deste trabalho é descrever os achados clínicos, patológicos, imuno-histoquímicos e moleculares de leishmaniose cutânea por Leishmania enriettii em cobaia (Cavia porcellus). Três animais apresentavam lesões nodulares e alopécicas no focinho e orelhas, além de lesões ulceradas nas extremidades distais dos membros pélvicos. Nos machos (2), foi observado espessamento difuso da pele escrotal. Amostras das lesões cutâneas ulceradas foram avaliadas por citologia, nas quais foram observadas formas amastigotas de Leishmania. Um dos animais foi submetido à eutanásia e necropsiado. Na histopatologia, foi observado infiltrado dérmico abundante de macrófagos, plasmócitos, linfócitos e com células gigantes multinucleadas. Numerosos macrófagos continham vacúolos parasitóforos com formas amastigotas, evidenciados por meio do exame de imuno-histoquímica. A caracterização molecular baseada no gene de SSUrDNA identificou a espécie como L. enrietti. O diagnóstico de leishmaniose cutânea nesses casos foi baseado nos achados patológicos e confirmado pelas técnicas de imuno-histoquímica, PCR e sequenciamento.(AU)

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BACKGROUND: Despite their importance in animal and human health, the epidemiology of species of the Leishmania enriettii complex remains poorly understood, including the identity of their biological vectors. Biting midges of the genus Forcipomyia (Lasiohelea) have been implicated in the transmission of a member of the L. enriettii complex in Australia, but the far larger and more widespread genus Culicoides has not been investigated for the potential to include vectors to date. METHODOLOGY/PRINCIPAL FINDINGS: Females from colonies of the midges Culicoides nubeculosus Meigen and C. sonorensis Wirth & Jones and the sand fly Lutzomyia longipalpis Lutz & Nevia (Diptera: Psychodidae) were experimentally infected with two different species of Leishmania, originating from Australia (Leishmania sp. AM-2004) and Brazil (Leishmania enriettii). In addition, the infectivity of L. enriettii infections generated in guinea pigs and golden hamsters for Lu. longipalpis and C. sonorensis was tested by xenodiagnosis. Development of L. enriettii in Lu. longipalpis was relatively poor compared to other Leishmania species in this permissive vector. Culicoides nubeculosus was not susceptible to infection by parasites from the L. enriettii complex. In contrast, C. sonorensis developed late stage infections with colonization of the thoracic midgut and the stomodeal valve. In hamsters, experimental infection with L. enriettii led only to mild symptoms, while in guinea pigs L. enriettii grew aggressively, producing large, ulcerated, tumour-like lesions. A high proportion of C. sonorensis (up to 80%) feeding on the ears and nose of these guinea pigs became infected. CONCLUSIONS/SIGNIFICANCE: We demonstrate that L. enriettii can develop late stage infections in the biting midge Culicoides sonorensis. This midge was found to be susceptible to L. enriettii to a similar degree as Lutzomyia longipalpis, the vector of Leishmania infantum in South America. Our results support the hypothesis that some biting midges could be natural vectors of the L. enriettii complex because of their vector competence, although not Culicoides sonorensis itself, which is not sympatric, and midges should be assessed in the field while searching for vectors of related Leishmania species including L. martiniquensis and "L. siamensis".

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An active case detection approach with PCR diagnosis was used in the Ho District of the Volta Region, Ghana that identified individuals with active cutaneous leishmaniasis. Three isolates were successfully cultured and DNA sequences from these were analysed (ribosomal RNA internal transcribed spacer 1; ribosomal protein L23a intergenic spacer; RNA polymerase II large subunit), showing them to be Leishmania, identical to each other but different from all other known Leishmania spp. Phylogenetic analysis showed the parasites to be new members of the Leishmania enriettii complex, which is emerging as a possible new subgenus of Leishmania parasites containing human pathogens.

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Since 1996, there have been several case reports of autochthonous visceral leishmaniasis in Thailand. Here we report a case in a 52-year-old Thai male from northern Thailand, who presented with subacute fever, huge splenomegaly and pancytopenia. Bone marrow aspiration revealed numerous amastigotes within macrophages. Isolation of Leishmania LSCM1 into culture and DNA sequence analysis (ribosomal RNA ITS-1 and large subunit of RNA polymerase II) revealed the parasites to be members of the Leishmania enriettii complex, and apparently identical to L. martiniquensis previously reported from the Caribbean island of Martinique. This is the first report of visceral leishmaniasis caused by L. martiniquensis from the region. Moreover, the majority of parasites previously identified as "L. siamensis" also appear to be L. martiniquensis.

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Even for routine purposes, standard staining of Pneumocystis- or Leishmania-containing materials, e.g., with Giemsa or Diff-Quik, is often unsatisfactory due to poor contrast and to staining of irrelevant structures. In comparison, the bisbenzimide dye Hoechst 33258, a DNA-binding fluorochrome, allows a more precise analysis of such materials. Bisbenzimide stained all stages of these fungal or protozoal organisms with brilliant contrast against a uniformly dark background. The level of background luminescence and staining of detritus or non-DNA structures was very low. Organisms were stained both outside of and within phagocytic cells with equal intensity. Counting of individual microorganisms, e.g., in macrophages heavily parasitized with Leishmania or in Pneumocystis-infected bronchoalveolar lavage, was simplified and more precise. Air-dried cell suspensions, cytocentrifuge preparations, impression smears, or cryocut micrographs showed the advantages of bisbenzimide staining over Diff-Quik. Staining with bisbenzimide could be a valuable auxiliary technique for the analysis of material infected with a variety of microorganisms.

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Three cases of cutaneous leishmaniasis in guinea-pigs from a rural area near Curitiba (Paraná State, Brazil) are reported. The three parasite isolates were characterized by isoenzyme electrophoresis as Leishmania enriettii, of which two distinct zymodemes were observed.

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Two domestic guinea-pigs (Cavia porcellus), bought in Pinheros, São Paulo State, Brazil, were taken by their owners to a farm in the rural district of Capão Bonito, close to the Atlantic Forest, São Paulo, where they both developed tumour-like and ulcerating lesions on the ears. The causative agent was identified as Leishmania (L.) enriettii, based on biological characters and isoenzyme profiles. Sources of the parasite in wild mammals, and the possible sandfly vector species are discussed.

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