Nested PCR for diagnosis of canine leishmaniosis in peripheral blood, lymph node and bone marrow aspirates

A nested polymerase chain reaction (PCR) assay was developed using primers selected from the genomic DNA of Leishmania infantum and applied to the diagnosis of leishmaniosis in peripheral blood in dogs. Blood of 39 dogs of different breeds, all sampled in Catalonia (Spain), were tested for leishmaniosis by enzyme linked immunosorbent assay (ELISA), western blotting (WB) and peripheral blood mononuclear cell (PBMC) culture and nested PCR. Twenty negative controls (healthy dogs less than 1-year-old that had not been exposed to a sandfly season) were also studied. Nineteen of the 39 dogs studied were positive by ELISA and/or WB, and 18 of these had a positive PBMC nested PCR. PBMC nested PCR was negative in all the remaining animals that were negative by serological examination, including the 20 negative controls. Parasitological examination and nested PCR of bone marrow and lymph node aspirate from the 19 dogs positive by serological examination, were also positive. These results indicate that PBMC nested PCR is a sensitive and specific tool to diagnose leishmaniosis in dogs. The use of PBMC has the advantage over bone marrow and lymph node aspirates in that it is a less invasive sample.     

Effects of allopurinol treatment on the progression of chronic nephritis in Canine leishmaniosis (Leishmania infantum)

Forty dogs with canine leishmaniosis (CL) participated in this study, which was designed to investigate the effect of allopurinol on the progression of the renal lesions associated with this disease. The animals were allocated into 5 groups. Group A dogs (n = 12) had neither proteinuria nor renal insufficiency, group B dogs (n= 10) had asymptomatic proteinuria, and group C dogs (n = 8) were proteinuric and azotemic. Two more groups, CA and CB, comprising 5 dogs each, served as controls for groups A and B, respectively. Group A, B, and C dogs received allopurinol PO (10 mg/kg q12h) for 6 months, whereas group CA and CB dogs were placebo-treated. Serum biochemistry profile, urinalysis, urine protein/creatinine ratio, and glomerular filtration rate (GFR) measurements were carried out at the beginning of the study, the 3rd month, and the 6th month, whereas renal biopsies were carried out only at the beginning and the end of the trial. Membranoproliferative glomerulonephritis was the most common cause of chronic renal failure. Mesangioproliferative and tubulointerstitial nephritis were detected even in group A and CA dogs. Allopurinol not only lowered proteinuria in group B dogs but also prevented the deterioration of GFR and improved the tubulointerstitial, but not the glomerular, lesions in both group A and group B dogs. Further, it resolved the azotemia in 5 of the 8 dogs admitted with 2nd stage chronic renal failure (group C). Consequently, treatment with allopurinol is advisable in CL cases with asymptomatic proteinuria or 1st-2nd stage chronic renal failure.     

Diagnosis of canine leishmaniasis by a polymerase chain reaction technique

A polymerase chain reaction (PCR) technique for the diagnosis of canine leishmaniasis on bone marrow samples was developed which amplified a 120 bp DNA fragment of the Leishmania kinetoplast DNA, common to all Leishmania species. Forty-five of 46 dogs in which leishmaniasis had been diagnosed were positive with the PCR technique, whereas none of 41 healthy dogs gave a positive result. Fifteen dogs with leishmaniasis that had been treated for six months with N-methylglucamine antimoniate and allopurinol were also investigated. Seven were positive, implying that they remained infected despite the resolution of their clinical signs.     

Real-time PCR assay in Leishmania-infected dogs treated with meglumine antimoniate and allopurinol

A real-time PCR assay was exploited for monitoring the Leishmania DNA load in different tissues from 18 naturally-infected dogs before and after treatment with a combination of meglumine antimoniate (100mg/kg/day, subcutaneously) and allopurinol (10mg/kg/day, orally) for 30 days. After the combined therapy, allopurinol was continued at the same dose until the end of the observation period. Whole blood samples, lymph node aspirates, and skin biopsies were collected at the time of diagnosis, 1 month after starting therapy, and every 3 months for 2 years. In six dogs parasite load assessments continued every 6 months for a further 3 years. At each assessment, the dogs were examined for signs of disease and a clinical score was recorded. At diagnosis, the highest Leishmania DNA load was detected in lymph node aspirates. From 1-6 months post-therapy a general improvement in clinical conditions was recorded in all dogs, which correlated with a decrease in the parasite DNA load in all tested tissues, even though it was less pronounced in lymph node aspirates. In the period from 9-24 months post-therapy, a re-increase in parasite load was observed in the tissues of some dogs, concomitant with a disease relapse. The results show that the combined therapy with meglumine antimoniate and allopurinol promoted a clinical improvement which was accompanied by a reduction in the parasitic load in the blood, skin and lymph nodes but, even after long period of allopurinol administration alone, Leishmania may persist in dog tissues.     

Comparison of parasitological, immunological and molecular methods for the diagnosis of leishmaniasis in dogs with different clinical signs

Aiming to improve the diagnosis of canine leishmaniasis (CanL) in an endemic area of the Northwest region of S?o Paulo State, Brazil, the efficacy of parasitological, immunological and molecular diagnostic methods were studied. Dogs with and without clinical signs of the disease and positive for Leishmania, by direct parasite identification on lymph node smears and/or specific antibody detection by ELISA, were selected for the study. According to the clinical signs, 89 dogs attending the Veterinary Hospital of UNESP in Ara?atuba (SP, Brazil) were divided into three groups: symptomatic (36%), oligosymptomatic (22%) and asymptomatic (22%). Twenty-six dogs from an area non-endemic for CanL were used as negative controls (20%). Fine-needle aspiration biopsies (FNA) of popliteal lymph nodes were collected and Diff-Quick-stained for optical microscopy. Direct immunofluorescence, immunocytochemistry and parasite DNA amplification by PCR were also performed. After euthanasia, fragments of popliteal lymph nodes, spleen, bone marrow and liver were collected and processed for HE and immunohistochemistry. Parasite detection by both HE and immunohistochemistry was specifically more effective in lymph nodes, when compared with the other organs. Immunolabeling provided higher sensitivity for parasite detection in the tissues. In the symptomatic group, assay sensitivity was 75.61% for direct parasite search on Diff-Quick-stained FNAs, 92.68% for direct immunofluorescence, 92.68% for immunocytochemistry and 100% for PCR; the corresponding values in the other clinical groups were: 32, 60, 76 and 96% (oligosymptomatic), and 39.13, 73.91, 100 and 95.65% (asymptomatic). Results of the control animals from the CanL non-endemic area were all negative, indicating that the methods used were 100% specific.     

Mixed Ehrlichia canis, Hepatozoon canis, and presumptive Anaplasma phagocytophilum infection in a dog

A 5-month-old, female, mongrel dog was admitted to the Clinic of Companion Animal Medicine, Aristotle University of Thessaloniki, Greece, with depression, anorexia, fever, peripheral lymphadenopathy, splenomegaly, oculonasal discharge, nonregenerative anemia, and mild thrombocytopenia. Cytology of Giemsa-stained buffy coat, bone marrow, and lymph node aspiration smears revealed numerous morulae in mononuclear leukocytes and in neutrophils, and Hepatozoon canis gamonts in neutrophils. The dog was seropositive to Ehrlichia canis (immunofluorescence assay [IFA]) and Hepatozoon canis (ELISA) but not to Anaplasma phagocytophilum (IFA). A nested polymerase chain reaction performed on bone marrow aspirates was positive for E canis. This method was not applied for the detection of A phagocytophilum. Treatment with doxycycline and imidocarb dipropionate resulted in both clinical and parasitologic cure. This is the first reported case of a mixed infection with E canis, H canis, and presumptive A phagocytophilum. The findings emphasize the value of cytology in offering a quick and inexpensive diagnosis in mixed tick-borne infections of dogs.     

Comparison of different tissue sampling for PCR-based diagnosis and follow-up of canine visceral leishmaniosis

In this study, different types of tissue sampling for PCR-based diagnosis and follow-up of canine visceral leishmaniosis were compared. Skin, whole blood and lymph node samples were collected from 95 naturally infected dogs living in South Italy, where the disease is endemic. Twenty-nine of these 95 dogs, treated with meglumine administered concurrently with allopurinol for 30 days, and then with allopurinol alone, were monitored during a period of 2 years. The DNA extracted from the clinical specimens was amplified by PCR using as target DNA a 116-bp fragment in the constant region of the kinetoplast DNA minicircle. PCR analysis was more sensitive than indirect immunofluorescence antibody test in detecting Leishmania infection in symptomatic dogs: 99% of lymph node samples resulted positive, whereas 94% of blood samples and 95% of skin samples gave a positive result. PCR analysis of samples from dogs followed up 2 years showed that: (1) all subjects resulted positive in at least one of the three types of samples; (2) all time the dogs had a relapse, PCR resulted positive in all three types of samples; (3) when dogs were apparently healthy, PCR analysis was positive on skin and lymph node samples, but not always on blood samples. Since lymph node sampling is invasive and sometimes difficult in healthy asymptomatic dogs, our results suggest that, independently from the presence or not of cutaneous lesions, skin biopsy represents a good substratum for PCR-based diagnosis and follow-up of canine visceral leishmaniosis.     

Clinical and serological follow-up in dogs with visceral leishmaniosis treated with allopurinol and sodium stibogluconate

Seven dogs with parasitologically proven clinical visceral leishmaniosis (Leishmania infantum infection) were treated with a combination of allopurinol and sodium stibogluconate. The dogs received first orally 15 mg/kg of allopurinol every 12 h until the clinical signs improved, in the following 1 month period allopurinol at same dose and subcutaneously 30 mg/kg of sodium stibogluconate combination were given daily and at the end of the combined treatment, allopurinol was continued alone at the same dose till the end of 8 months. During the treatment period, dogs were supported by additional proteins, vitamins, and minerals. A long acting insecticide (collar or drop) was also used in order to prevent further parasite transmission. Follow-up was maintained by clinical, clinicopathological evaluation, and parasitological examination of lymph node, serology using the indirect immunofluorescent antibody test (IFAT). Before treatment commenced, the most important clinical signs were exfoliative dermatitis, ulcerations, peripheral lymhadenopathy, pale mucous membranes, weight loss, and ocular lesions. Clinicopathological findings included commonly anaemia, hyperproteinaemia, hyperglobulinaemia and hypoalbuminaemia. Before the treatment, amastigotes were seen in six of the seven dogs by examination of lymph node aspiration, and IFAT-titers were positive in all dogs. At the end of 8 months treatment, remission of clinical signs, restoration to normal of clinicopathological abnormalities were noticed. Lymph node aspiration was performed on three out of the seven dogs at the end of the treatment because of the very small sizes of the lymph nodes, and no amastigotes were observed. Although the mean IFAT-titer of the dogs were significantly (P < 0.001) lower compared with pretreatment, IFAT-titers of dogs were still positive. No relapses occurred during treatment period and a 6-24-month duration after the end of therapy. Based on the above results, long-term use of allopurinol combined with sodium stibogluconate together with support treatment concluded to have enough therapeutic efficacies in the treatment of dogs with visceral leishmaniosis. Observations of the cases for possible relapses were still going on and insecticide application was carefully carrying on in order preventing a possible re-infection.     

Detection of Ehrlichia canis by PCR in different tissues obtained during necropsy from dogs surveyed for naturally occurring canine monocytic ehrlichiosis

A molecular study for the detection of Ehrlichia canis was carried out on tissues obtained at necropsy from randomly selected dogs with the intention of investigating naturally-occurring canine ehrlichiosis. The tissues evaluated for the presence of E. canis included lymph nodes, spleen, liver, bone marrow, and blood. Eight of the 18 dogs included were found to be positive for E. canis by polymerase chain reaction (PCR) and sequencing of the 16S rRNA gene. Two dogs were positive for Anaplasma platys of which one dog was co-infected with E. canis and A. platys. Blood (5/8) and lymph nodes (5/8) were the tissues found to yield the highest number of positive E. canis PCR results with 7/8 dogs positive in the blood or lymph node. E. canis and A. platys DNA could be amplified by PCR when tissue samples were obtained 72h after the time of death.     

Canine leishmaniasis chemotherapy: dog's clinical condition and risk of Leishmania transmission

The aim of this study was to investigate whether treatment against canine leishmaniasis reduced the presence of Leishmania in the healthy skin of dogs, affecting the capacity of parasite transmission. A total of 37 dogs from an endemic region of leishmaniasis were studied. Thirteen symptomatic animals revealed parasites in the bone marrow and eight had also in the skin. Five of the 22 dogs that had been treated with meglumine antimoniate alone, meglumine antimoniate or trifluralin followed by allopurinol or just with allopurinol had the parasite in bone marrow but none showed Leishmania in the skin. One dog that was treated only with aminosidine was polisymptomatic and had parasites in bone marrow and skin. The different treatments used in this study did not completely eliminate the parasite allowing relapses to occur when the treatment is discontinued, but the use of meglumine antimoniate or allopurinol, alone or combined may improve dogs clinical condition and reduce or eliminate the parasite from the skin decreasing the probability of Leishmania transmission.     

Determination of the number of mast cells in lymph node, bone marrow, and buffy coat cytologic specimens from dogs.

Cytologic samples of popliteal lymph node, proximal femoral bone marrow, and the buffy coat fraction of blood were obtained from 56 dogs. The number of mast cells on 1 slide of each sample was determined by microscopic examination. Eleven of 46 slides of lymph node aspirate contained mast cells (range, 1 to 16; mean, 6.4; median, 5 mast cells/slide). Fifty-one bone marrow aspirate slides were evaluated. Two of these contained a single mast cell. None of the 53 buffy coat smear slides examined contained any mast cells. These results indicated that in clinically normal dogs, a few to several mast cells may be encountered in smears of lymph node aspirate, mast cells are rare in smears of bone marrow aspirate, and mast cells are absent from smears of buffy coat.     

Leishmania (Leishmania) chagasi is not vertically transmitted in dogs.

The most frequent and most important mode of human or canine visceral leishmaniasis (CVL) transmission is through the bite of infected sand flies. This study investigates Leishmania (Leishmania) chagasi vertical transmission in offspring of naturally infected dogs. Thus 63 puppies from 18 female dogs with CVL were used. Parasite presence was evaluated through parasitologic and histopathologic examination of lymphatic organs, as well as polymerase chain reaction (PCR) on samples from adults (milk, uterus, placenta, spleen, liver and bone marrow) and offspring (spleen, liver, lymph nodes and bone marrow). PCR sensitivity and specificity were calculated using a microscope as the gold standard on samples of bone marrow, spleen and liver. Specificity was 100% for all organs and sensitivity was 100% for bone marrow, 71.4% for spleen and 66.6% for liver. Bone marrow smears (n = 63), histopathology and imprint of spleen (n = 25), liver (n = 25) and lymph nodes (n = 25) were performed to evaluate congenital transmission in the 63 offspring. PCR was done on 92 samples collected from 56 of the offspring. No test performed on the offspring was positive. It was not possible to confirm vertical transmission of CVL (95% confidence interval for the observed prevalence), despite positive PCR in the placenta of seropositive adults.     

Effect of Peganum harmala (wild rue) extract on experimental ovine malignant theileriosis: pathological and parasitological findings

Malignant theileriosis of sheep is a highly fatal, acute or subacute disease is caused by the tick-borne protozoan parasite, Theileria hirci. In this investigation ten healthy male lambs aged 5-6 months were randomly divided into two groups, A and B and were kept in isolated tick-proof pens. They were treated for internal and external parasite before commencement of the experiment. The lambs were experimentally infected with T. hirci by placing ticks Hyalomma anatolicum anatolicum infected with T. hirci on them. The ticks used in this survey had originally been isolated from sheep and colonies of them were established in an insectarium. Before and after infection rectal temperatures and clinical signs of the lambs were recorded, blood and prescapular lymph node smears were prepared and examined to determine the extent of the parasitaemia, and blood samples were analyzed to evaluate their haemoglobin (Hb) and packed cell volume (PCV) rates. Three days after the commencement of a febrile reaction and appearance of the schizonts in the lymph node smears, treatment of the lambs in Group A with an extract containing the alkaloids of Peganum harmala (wild rue) was commenced. Group B lambs were kept untreated controls. Before treatment there were no significant differences in the rectal temperature, parasitaemia rate, and the Hb and PCV values between animals in the two groups but after treatment significant differences in these values was detected (P < 0.05). After treatment, the clinical signs and parasites in the lymph node smears of the animals in Group A disappeared and they all animals recovered. These parameters in the animals of Group B progressed until their death. Pathological studies showed the characteristic lesions of theileriosis in lambs in Group B, but not in Group A. The results indicate a therapeutic effect of the alkaloids of P. harmala for treatment of ovine malignant theileriosis.     

Lymphoma (malignant lymphoma, lymphosarcoma) in the dog

This report describes seven cases of canine lymphoma involving three German shepherd dogs, two boxers and two mongrels with a male to female ratio of 1–3 to 1. Their mean age and body weight was four years (range 1–5 to 8) and 31 kg (range 21 to 40), respectively. Six of the animals were euthanased at their owner's request and one died. Excessive lymph node enlargement, splenomegaly, hepatomegaly, blood lymphocytosis and proteinuria were the most striking clinical and clinicopathological findings. All the cases except one presented systemic signs and three developed secondary leukaemia because of bone marrow involvement. The anatomical types were classified according to the World Health Organization's criteria as thymic (one dog) and generalised (six dogs) and the clinical stage as IV (four dogs) and V (three dogs). Extranodal lymphomas involving the kidneys, meninges and the alimentary tract were seen in two cases. Histologically, the lymphomas were classified as lymphoblastic in four dogs, and in the remaining animals as lymphoblastic with plasmacytoid differentiation, lymphocytic with plasmacytoid differentiation, and poorly differentiated (stem cell) with one type appearing in each dog.     

Study of efficacy of miltefosine and allopurinol in dogs with leishmaniosis

Visceral leishmaniosis is a life-threatening disease of medical, social and economic importance in endemic areas. It is an opportunistic infection in immunocompromised patients, including human immunodeficiency virus-positive subjects. Dogs are the main reservoir of Leishmania infantum. The aim of this study was to evaluate the efficacy of miltefosine and allopurinol for the control of human leishmaniosis using the dog as a model. The study included 28 sick dogs treated with miltefosine (2 mg/kg/day PO) administered concurrently with allopurinol (10 mg/kg/day, PO) for 30 days, and then with allopurinol alone, at the same dosage, for 1 year. Eight dogs (four of which relapsed) received a second cycle of miltefosine within 6 months of the first cycle. Efficacy was measured by real-time polymerase chain reaction assay on whole blood samples and lymph node aspirates, collected at baseline and every 3 months for 12 months. Of the total number of animals (28), two showed renal insufficiency and died after the start of therapy with miltefosine. Two other dogs presented some side effects to treatment, such as nausea, vomiting and reduction in white and red blood cell counts, and these animals were excluded from the follow-up. The results showed that the first cycle of therapy with miltefosine and allopurinol induced a drastic and progressive reduction of L. infantum load in lymph node aspirates but the second cycle did not eliminate the parasite.     

Immunophenotypic comparison of blood and lymph node from dogs with lymphoma

Peripheral blood and lymph node tissue from 12 dogs with lymphoma was immunophenotyped. Additionally, the bone marrow was immunophenotyped in 6 dogs. The lymphomas were characterized as B-cell in 11 dogs and T-cell in 1 dog. Immunophenotypic patterns in the peripheral blood and bone marrow were variable. The trend in dogs with B-cell lymphoma was normal to increased percentage of IgG-positive cells, decreased percentage of pan-T-positive cells, decreased percentage of CD4-positive cells, and decreased CD4/CD8 ratio. Simultaneous immunophenotyping of lymph node, blood and bone marrow cannot be recommended routinely without further studies to document its value as an independent prognostic indicator. However, it is potentially useful for tumor staging and monitoring remission, especially in lymphoma patients with a leukemic phase.     

Combination Allopurinol and Antimony Treatment versus Antimony Alone and Allopurinol Alone in the Treatment of Canine Leishmaniasis (96 Cases)

The aim of the present study was to evaluate the long-term clinical outcome for dogs with leishmaniasis that were treated with 3 different protocols: combined treatment with antimony and allopurinol, antimony alone, or allopurinol alone. Ninety-six dogs included in this study were determined to have leishmaniasis on the basis of (1) clinical features, (2) identification of the parasite in smears of lymph node, bone marrow aspirates, or skin biopsies, and (3) specific immunofluorescent assay. Three groups of dogs were defined: 45 dogs (group 1) were treated with antimony (100 mg/kg s.c. q24h) given concurrently for 1 month with allopurinol (15 mg/kg p.o. q12h), and then allopurinol alone for 8 months at the same dosage; 40 dogs (group 2) were treated with antimony alone according to the manufacturer's instructions (200 mg/kg s.c. q24h at 2-day intervals for 3-6 months); and 11 dogs (group 3) were treated with allopurinol alone (15 mg/kg p.o. q12h for 1-20 months). Information concerning signalment, history, physical examination findings, serologic testing and number of dogs becoming seronegative, outcome for each treated dog (clinical cure versus failure), and long-term survival were recorded. The numbers of the clinical cures versus failures were significantly different among the 3 groups (chi2 = 17.77, P < .001), between groups 1 and 2 (chi2 = 8.02, P < .01), between groups 2 and 3 (chi2 = 11.00, P < .01), and between groups 1 and 3 (chi2 = 16.52, P < .001). No significant difference between groups 1 and 2 was noted in the type of failure (relapse or death), serologic test results, and number of survival years (chi2 = 2.79, P > .05). The results of the present study indicate that antimony in combination with allopurinol produces better results than antimony alone or allopurinol alone for the treatment of the canine leishmaniasis. With combination treatment, duration of treatment with antimony is shorter and long-term administration of allopurinol is well tolerated.     

Bone marrow mastocytosis in dogs with myelosuppressive monocytic ehrlichiosis (Ehrlichia canis): a retrospective study

BACKGROUND: Bone marrow mastocytosis has been reported rarely in naturally occurring canine monocytic ehrlichiosis (CME). OBJECTIVES: The aims of the present study were to estimate the prevalence and magnitude of bone marrow mastocytosis in a case series of dogs with natural CME and to assess the association, if any, between mastocytosis and the clinical severity of the disease. METHODS: Seventy-six dogs with confirmed CME (Ehrlichia canis) were included in the study. Affected dogs were allocated into group A (n = 51) without bone marrow hypoplasia and group B (n = 25) with bone marrow hypoplasia. Twenty clinically healthy Beagles not previously exposed to E canis served as controls (group C). The main inclusion criteria for group A were documentation of normocellular to hypercellular bone marrow and complete clinical cure following a 4-week treatment with doxycycline, while those for group B were bone marrow hypoplasia and lack of response to doxycycline. Bone marrow aspirate smears from all 96 dogs were Giemsa-stained and examined for the presence of mast cells, which were calculated as a percentage of 1,000 nucleated cells (NCs). The prevalence of mastocytosis was compared among the 3 groups by the Pearson's chi-square test. RESULTS: Bone marrow mastocytosis (>0.1% of NCs) was found in 5 (20%) dogs in group B (range, 0.5-2.5% of NCs; median, 1% of NCs). One dog in each of groups A and C had 0.1% mast cells in the marrow. The prevalence of bone marrow mastocytosis in dogs in group B was significantly higher (P = .004) than in groups A and C. CONCLUSION: Bone marrow mastocytosis can be seen in a substantial number of dogs with E canis-induced myelosuppression.     

Clinical, Serologic, and Parasitologic Follow-Up after Long-Term Allopurinol Therapy of Dogs Naturally Infected with Leishmania infantum

Canine leishmaniasis usually is treated with antimony compounds, but frequent relapses, adverse effects, high costs, and development of resistance to long-term antimonial therapy emphasize the importance of searching for alternative antileishmanial drugs. Allopurinol was used at a dosage of 10 mg/kg/day PO to treat 10 dogs naturally infected with Leishmania infantum for a period of 2-24 months. Nine dogs recovered within 2-6 months of chemotherapy, and no relapses were observed during the treatment of up to 20 months. However, 3 of 4 dogs relapsed after treatment was discontinued. These dogs again recovered clinically when therapy was resumed. Parasite-specific immunoglobulin concentrations (IgG2) were high in all dogs before therapy and remained high even in clinically cured dogs during or after therapy. On the other hand, specific IgG1 reactions, which have been shown to be detectable in symptomatic animals, persisted in 7 dogs for long periods after clinical recovery. Three of these dogs relapsed within 2-4 weeks after interrupting therapy. However, 1 dog with no detectable specific IgG1 reaction at the end of therapy did not relapse in the following 4 months. Parasites could be detected in 8 of 9 dogs after clinical improvement by in vitro cultivation or polymerase chain reaction (PCR) testing of lymph node aspirates. In 4 of these dogs, parasites also were detected in blood samples by PCR. Hence, these clinically cured dogs must be regarded as reservoirs of Leishmania and allopurinol cannot be recommended in endemic areas.