Genetic diversity of human zoonotic leishmaniasis in Iberian Peninsula

Leishmaniasis caused by Leishmania (Leishmania) infantum is a zoonotic disease endemic in South Europe, from Portugal to the Middle East. The aim of the present study was to investigate the genetic diversity of L. infantum parasites in Iberian Peninsula. Twenty-four L. infantum strains isolated from immunocompetent patients with leishmaniasis from several localities of Portugal and Spain were studied. The use of kinetoplast DNA-PCR-restriction fragment length polymorphism as a molecular marker revealed intra-specific variation. No association was found between genotype and clinical form of the disease or patients age group. Two main clusters were identified with this marker: (i) zymodeme MON-1 strains and (ii) non-MON-1 strains. However, no association was found between strains variability and geographical distribution suggesting that parasite populations of different regions in the Iberian Peninsula are homogenous.     

Evaluation of 65% permethrin spot-on and deltamethrin-impregnated collars for canine Leishmania infantum infection prevention

During the 2004 and 2005 sand fly seasons, we evaluated the efficacy of a 65% spot-on solution of permethrin (Exspot, Schering & Plough) and deltamethrin-impregnated collar (Scalibor, Intervet) in reducing Leishmania infantum infection, in a canine leishmaniasis (CanL) endemic region (Liguria) in Italy. Immunofluorescent assay (IFA) revealed that three of 120 dogs (2.5%) treated with a 65% spot-on solution of permethrin, as three of 119 dogs (2.5%) treated with deltamethrin-impregnated collar have shown seroconversion after sand fly season. On the contrary, seroconversion was 15% in 188 untreated control dogs. Treatment reduced the risk of infection by 84%. The difference in treated dogs and control ones is highly significant (chi2 = 12.4; P = 0.0004). Our results show that treatment with 65% spot-on solution of permethrin and the deltamethrin-impregnated collar are effective in reducing the risk of acquiring L. infantum infection.     

Extranodal γδ-T-cell lymphoma in a dog with leishmaniasis

Abstract: An 8-year-old intact male mongrel dog with alopecia and weight loss was referred to the Veterinary Faculty of Naples. The dog had pale mucous membranes, enlarged prescapular lymph nodes, and splenomegaly. Laboratory abnormalities included anemia, thrombocytopenia, and hyperglobulinemia. Bone marrow aspirate smears contained numerous Leishmania amastigotes and an immunofluorescent antibody titer was strongly positive (1:1280) for leishmaniasis. The dog was treated with a combination of meglumine antimoniate and allopurinol for 60 days and showed clinical improvement. Two months after the end of treatment the dog was again referred because of relapse of leishmaniasis and the presence of a firm subcutaneous mass on the medial right thigh. Based on cytologic examination of fine needle aspirates of the mass, a diagnosis of large-cell lymphoma was made. Flow cytometry of tumor cells revealed γδ-T-cell lymphoma with a CD5+, CD3+, TCRγδ+, CD4−, CD8−, CD45RA+ immunophenotype. Using nested PCR, amastigotes were not detected in the neoplastic tissue. An association between leishmaniasis and hematopoietic tumors has been described rarely. γδ-T cells may be involved in the host response to this parasite, and prolonged antigenic stimulation and chronic immunosuppression (typical of leishmaniasis) play a crucial role in the etiopathogenesis of T-cell lymphoma.     

Periodic administration of allopurinol is not effective for the prevention of canine leishmaniosis (Leishmania infantum) in the endemic areas

A total of 95 clinically healthy and seronegative for Leishmania infantum dogs, residing an area highly endemic for canine leishmaniosis (CL) and living an outdoor life-style, were split into positive and negative groups, and then were randomly assigned to receive allopurinol (n = 51; 20 mg/kg once daily), or placebo (n = 44) for 1 week per month, from April to November. Forty per cent (38/95) of these dogs were not reexamined and retested at the end of the trial for reasons unrelated to CL. None of the remaining 57 dogs exhibited the symptomatic form of the disease at the end of the 1-year follow-up period. Of the 15 allopurinol-treated dogs that were non-infected (negative PCR and tissue smear microscopy) at the beginning of the trial, 6 (40% P = 0.03) became PCR-positive, of which 3 became also seropositive, at the end of the observation period. In contrast, only 1 of 7 (14.3%) placebo-treated non-infected dogs became PCR positive at the same time point. Of the 19 allopurinol-treated dogs that were infected (PCR-positive) at the beginning of the trial, 18 (94.7%) remained PCR-positive and one (5.3%) seroconverted, at the end of the observation period. Of the 16 initially infected and placebo-treated dogs, 14 (87.5%) remained PCR positive, of which one (6.7%) also turned positive by tissue smear microscopy. Therefore, it is concluded that the use of allopurinol, at the daily dose of 20 mg/kg, for 1 week per month, during the period of sandfly activity, does not prevent the infection of non-infected dogs by L. infantum, and, also, does not help in the elimination of the parasite from dogs with asymptomatic infections.     

Skin Lesions in Canine Leishmaniasis (Kala-Azar): A Clinical and Histopathological Study on 22 Spontaneous Cases in Greece

Abstract— Skin lesions occurring in 22 dogs with leishmaniasis (Kala-azar) were studied clinically and histopathologically, so as to better characterize the range of abnormalities encountered in this disease. The main clinical dermatological patterns observed were exfoliative dermatitis (90.9 per cent of dogs), ulcerations (63.6 per cent), onychogryposis (54.5 per cent), sterile pustular dermatitis (13.6 per cent), and paronychia (13.6 per cent). Most of the dogs (86.4 per cent) had more than one type of skin lesion. The most commonly seen histopathological patterns were granulomatous perifolliculitis and sebaceous adenitis (68.2 per cent), superficial and deep perivascular dermatitis (54.5 per cent), and interstitial dermatitis (50.0 per cent). Parasites, mostly within macrophages, were detected in the skin biopsies from 50.0 per cent of the dogs. Résumé— Les lesions cutanés de 22 chiens atteints de leishmaniose ont étéétudiées cliniquement et histopathologiquement, afin de mieux caractériser les anomalies observées lors de cette maladie. La principale manifestation dermatologique observée était une dermatitie exfoliative (90,9% des chiens), des ulcérations (63,6%), une onychogriphose (54,5%) une dermite pustuleuse stérile (13,6%) et une paronychie (13,6%). La plupart des chiens (84,6%) avaient plus d'un type de lésion cutanée. Les lesions histopathologiques les plus communes étaient des périfolliculites granulomateuses et des adénites sébacées (68,2%), une dermite superficielle et profonde périvasculire (54,5%), une dermite intersticielle (50%). Les parasites, généraiement dans les macrophages, ont été isolés dans une biopsie sur deux. Zusammenfassung— Hautveränderungen, die bei 22 Hunden mit Leishmaniose (Kala-azar) auftraten, wurden klinisch und histopathologisch untersucht, um die Spannweite der Veränderungen, die bei dieser Krankheit auftreten, besser charakterisieren zu können. Als hauptsächliche klinische dermatologische Symptome wurden exfoliative Dermatitis (90,9% der Hunde), Ulzerationen (63,6%), Onychogrypose (54,5%)), sterile pustulöse Dermatitis (13,6%) und Paronychie (13,6%) beobachtet. Die meisten Hunde (86,4%) wiesen mehr als einen Typ Hautveränderung auf. Die häufigsten histopathologischen Befunde bestanden in granulomatöser Perifollikulitis und Talgdrüsenadenitis (68,2%), oberflächlicher und tiefer perivaskulärer Dermatitis (54,5%) und interstitieller Dermatitis (50,0%). Die Parasiten, die meistens in den Makrophagen liegen, wurden in den Hautbiopsien bei 50,0% der Hunde entdeckt. Resumen Se estudiarion clínica e histopatológicamente las lesiones cutáneas padecidas por 22 perros con leismaniasis (Kala-azar), con el propósito de caracterizar el rango de abnormalidades producidas por la enfermedad. La sprincipales características patológicias observadas fueron: dermatitis exfoliativa (90.9% de los perros), ulceraciones (63.6%), onicogriposis (54.5%), dermatitis postular esteril (13.6%), y paronquia (13.6%). La mayoría de los perros, (86.4%), presentaban más de un tipo de lesion dérmica. Los modelos histopatológicos reconocidos más frequentemente fueron: perifoliculitis granulomatosa y adenitis sebácea (68.2%)), dermatitis perivascular superficial y profunda (54.5%), y dermatitis intersticial (50%). Las biopsias cutáneas detectaron parásitos, la mayoría en los macrófagos, en 50% de los perros.     

Use of PCR-RFLP to identify Leishmania species in naturally-infected dogs

Tissue imprints on Giemsa stained slides from dogs were used to investigate the presence of Leishmania amastigotes by either optical microscopy (OM) or Polymerase chain reaction (PCR) detection of DNA. Samples from skin, spleen, lymph node, liver and bone marrow from a Leishmaniasis endemic area dogs where Leishmania (Leishmania) chagasi and Leishmania (Viannia) braziliensis are sympatric were studied. Dogs were initially diagnosed by Indirect Immunofluorescence (IIF), as which 39 were IIF positive (≥1:40) and 16 negative. The IIF positive dogs were clinically grouped as symptomatic (n = 15), oligosymptomatic (n = 12) and asymptomatic (n = 12). Although PCR positivity was higher in symptomatic dogs, specially their skin samples, there was no significant difference among clinical groups or organs examined. Ten (62.5%) out of 16 IIF and OM negative animals were positive for PCR in at least one organ. Forty-eight positive PCR amplicons were further submitted to RFLP for Leishmania identification. All dogs were infected with L. (L.) chagasi except one, infected with L. (V.) braziliensis. PCR was more efficient than IIF and OM to diagnose canine visceral Leishmaniasis (CVL), regardless of the organ examined and the clinical form present. The use of PCR together with serology helps determining the extension of sub clinical infection in CVL endemic areas and provides a better estimate of the number of dogs to be targeted for control measures. In conclusion, our data reinforce the need for a specific diagnosis of canine infection in areas where diverse Leishmania species are sympatric and demonstrate that PCR–RFLP can be used to identify Leishmania species in dog tissue imprint stained slides.