First report of the use of meglumine antimoniate for treatment of canine leishmaniasis in a pregnant dog

Canine leishmaniasis during pregnancy is rarely reported, even in countries where the infection in dogs is endemic. The authors report a case of a 4 yr old bitch with leishmaniasis treated with meglumine antimoniate during pregnancy. The pregnancy and delivery were normal and the bitch presented improvement of the infection during treatment. Three puppies died within 2 days of birth and tested negative via real-time PCR for L. infantum. The two surviving puppies were followed clinically, serologically, and by real-time PCR until 1 yr of age with no evidence of congenital leishmaniasis. L. infantum DNA was detected with real-time PCR analysis of uterine tissue from the bitch at the time of ovariohysterectomy. PCR analysis was performed after an ovariohysterectomy of the bitch that was performed two months after parturition. Meglumine antimoniate use in the pregnant bitch may have prevented vertical transmission of leishmaniasis.     

Canine visceral leishmaniasis: comparison of in vitro leishmanicidal activity of marbofloxacin, meglumine antimoniate and sodium stibogluconate

The control of canine leishmaniasis largely depends on the success of treatment. Drugs currently available to treat this disease are toxic and partially effective. The curative effect of marbofloxacin, a third-generation fluoroquinolone developed for veterinarian individual treatment, was evaluated in vitro in the presence of Leishmania infantum promastigotes and dog-monocyte-derived macrophages; meglumine antimoniate and sodium stibogluconate were used as comparative treatments. We observed that the killing of Leishmania promastigotes and intracellular amastigotes by marbofloxacin was dose-dependent. We demonstrated that successful treatment of canine infected macrophages for 48 h was possible with 500 microg/ml of marbofloxacin. Leishmanicidal activity acted through a TNF-alpha and nitric oxide pathway and correlated with the generation of nitric oxide (NO(2)) production by monocytes derived macrophages from infected (23+/-5 microM) or healthy (21+/-6 microM) dogs, in comparison with NO(2) concentration in infected/non-treated macrophages (< 3 microM, P<0.01). This significant induced parasiticidal effect correlated with extensive elimination of amastigotes by macrophages derived from infected (11+/-5) and healthy dogs (6+/-2), when compared to infected/non-treated macrophages (530+/-105 and 472+/-86 amastigotes, respectively, P< 0.01). Marbofloxacin was shown to be non-toxic at 500 microg/ml in vitro and no cell apoptosis was observed. The molecule was able to induce a parasitic process after significant elimination of amastigotes in leishmania-infected dog macrophages. We propose that marbofloxacin, compared to standard chemotherapeutic agents (meglumine antimoniate and sodium stibogluconate), could be an effective and pragmatic oral route alternative to treat canine leishmaniasis.     

Evaluation of renal and hepatic functions in dogs naturally infected by visceral leishmaniasis submitted to treatment with meglumine antimoniate

The present study aimed to evaluate the renal and hepatic responses in eight dogs with visceral leishmaniasis submitted to treatment with meglumine antimoniate and to verify the occurrence of possible side effects. Urinalysis, hepatic and renal function tests were carried out in all animals at up to seven moments. After the end of a six-month observation period, all dogs were euthanized. Before the beginning of the experiment urinary and biochemical alterations were observed in four dogs due to the changes caused by the parasite itself. These alterations included the presence of renal cells, cylindruria, proteinuria, azotemia, hyperproteinemia and hypoalbuminemia. One dog died on the third day after treatment because an aggravation of the clinical picture, probably due to the medication. During the course of the study, an increase in hepatic enzymes was verified in two animals. Sixty days after the beginning of the treatment four dogs showed remission of clinical signs. The other three were asymptomatic with persistent biochemical alterations. From these, two presented recurrence of clinical signs about 150 days after the beginning of the treatment while in the other, hyperproteinemia persisted. Meglumine antimoniate was not efficient to treat dogs with severe renal dysfunction and the side effects observed were pain at the site of injection and the probable transient hepatotoxicity, evidenced by biochemical examinations, but without the presence of 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.     

The effects of prednisone on haemostasis in leishmaniotic dogs treated with meglumine antimoniate and allopurinol

Thirty dogs naturally infected with Leishmania infantum were studied in order to determine the effects of treatment on haemostatic function. The animals were divided randomly into two treatment groups: Group 1 received meglumine antimoniate and allopurinol; Group 2 dogs were given the same treatment plus prednisone. Ten healthy animals were used as untreated controls. Clinical examination and determination of platelet aggregation, coagulation factors and biochemical parameters were undertaken before treatment and after 15, 30 and 60 days. A significant improvement in platelet aggregation was detected after 60 days in Group 1, but only after 15 days in Group 2. In both treated groups, platelet aggregation was lower than in the control group at the end of the study. The results suggest that prednisone may be a useful tool in the treatment of haemostatic disorders during canine leishmaniosis. The potential benefits and risks due to the use of corticosteroids in the treatment of leishmaniosis are discussed.     

Skin lesions in canine leishmaniasis

Forty-three dogs affected by canine leishmaniasis (CL) with skin lesions have been studied clinically and histopathologically. Identification of leishmaniads in tissues was achieved using indirect immunoperoxidase staining. According to both macroscopic and microscopic lesions, four different dermatological patterns have been observed. Twenty-six dogs showed alopecia and desquamation as the main skin lesions. Histologically a diffuse non-suppurative dermatitis, with numerous leishmaniads inside macrophages was present. Ten animals showed skin ulcerations on limbs, especially over articulations. Histologically, the number of leishmaniads was very reduced. Five animals presented a generalised nodular disease. Each nodule corresponded with a focal accumulation of macrophages, multinucleated giant cells and lymphocytes, with a high number of parasites. Finally, two dogs presented generalised skin pustules. The possible pathogenesis and the differential diagnosis of each form is discussed.     

Proteomic analysis in canine leishmaniasis

In horses, Recurrent Airway Obstruction (RAO) is an allergic disease that involves IgE mediated Type I Hypersensitivity responses. The development of this type of allergy involves a series of events that begins with reaginic antibodies, mainly IgE and some IgG subclasses. These reaginic antibodies bind with high affinity, via the Fc portion, to FcεRI receptors on the membrane of mast cells and basophils. Once bound, environmental allergens cross-link the antibodies, which results in mast cell degranulation leading to the production of histamine and other chemical mediators that act together to induce airway inflammation. RAO-affected horses present with coughing, respiratory distress, airway obstruction and poor performance. The aspect of the RAO has been extensively studied, yet the precise sequence of events is still not well understood. Therefore, this study proposes a bioassay for reaginic antibody detection from horse serum of RAO-affected individuals, in order to determine the etiology of disease, which mediate immediate type reactions. The technique involves measuring in vitro calcium mobilization in RBL-2H3 cells following incubation with horse serum from affected or unaffected horses and one of the RAO antigens (Aspergillus fumigatus). The results presented here demonstrate that 30% of RAO-affected horses react positively in this in vitro bioassay, whereas unaffected horses do not. This bioassay may facilitate further research on RAO and other allergic diseases in horses.     

Use of domperidone in the treatment of canine visceral leishmaniasis: a clinical trial

The aim of this study was to evaluate the effects of domperidone, a dopamine D2 receptor antagonist, in dogs naturally infected by Leishmania infantum. Ninety-eight dogs were treated with single-agent domperidone at 1mg/kg twice a day orally for 1 month. Clinical, serological, biochemical and immunological examinations were conducted for the following 12 months. Domperidone was effective in controlling and reducing clinical signs and antibody titre. Significant decreases in reciprocal serum antibodies were seen in 74.3% of the dogs with mild clinical signs and 40% of the dogs became seronegative. In dogs with several clinical signs and high antibody titres, clinical improvement occurred in 86% of animals and the reciprocal serum antibody titres decreased in 38% of these dogs. A significant increase was noted in the immune cellular status, as measured by the leishmanin skin test and a lymphocyte proliferation assay.     

Serological and parasitological follow-up in dogs experimentally infected with Leishmania infantum and treated with meglumine antimoniate.

Six healthy beagle dogs were infected with Leishmania infantum (MCAN/ES/92/BCN-83/MON-1) by intravenous inoculation of 5 x 10(7) promastigotes and two others were used as controls. When animals showed clinical signs of disease at 29, 37, 41 and 45 weeks post-infection (p.i.), they were treated with meglumine antimoniate (20.4 mg Sb/kg/12 h) subcutaneously for two periods of 10 days each. Sera were tested periodically for Leishmania antibodies by Dot-ELISA, ELISA and Western blot (WB). Aspirates of popliteal lymph node (PLN), peripheral blood sample (PB) and healthy skin were cultured in NNN and Schneider's medium. PLNs were positive between 8 and 20 weeks p.i. and in one animal PB was positive 6 weeks p.i. Samples of healthy skin, obtained before treatment, were also positive. Dot-ELISA and ELISA detected specific antibodies at an early stage between 4 and 12 weeks p.i and surpassed the cut-off between 16-24 weeks p.i., while the WB was positive between 10-19 weeks p.i. The pattern of bands revealed during the first stages of infection was variable and only in two cases did the positivity start with bands of low molecular weight (12-14 kD); the number of bands increased until 15-24 weeks p.i., after which sera revealed a complete pattern of bands, from 12 to 85 kD, in the antigen of Leishmania. After treatment the clinical improvement of the animals was accompanied by a decrease in antibody titers (Dot-ELISA and ELISA) although the parasites remained in the PLN. This was reflected in the WB by a decrease in the intensity of bands, especially those in the region of 12-30 kD. A new increase in the antibody levels between 3 and 5 months after terminating the therapy was detected in the WB by a restoration of the initial complete pattern of bands.     

Evaluation of secondary haemostasis in canine leishmaniasis

Secondary haemostasis was evaluated in 26 dogs with leishmaniasis and 10 normal dogs by measurements of modified one-stage prothrombin time (m-OSPT), activated partial thromboplastin time (APTT), thrombin time, fibrinogen concentration and fibrin degradation products. There were no significant differences between the groups in the m-OSPT, fibrinogen concentration, or levels of fibrin degradation products. The APTT was significantly (P = 0.006) longer in the infected dogs than in the control group, and in infected dogs with alanine aminotransferase (ALT) activities > 50 U/litre. There was a significant linear regression between ALT and APTT. Thrombin time was significantly (P = 0.003) longer in the infected dogs than in the normal dogs. There were no significant differences between the thrombin times of sick dogs with different levels of creatinine or activities of ALT, or between male and female dogs, whether diseased or normal.     

Acute phase protein response in experimental canine leishmaniasis

Acute phase proteins (APPs) have been proposed as useful markers for the diagnosis and monitoring of treatment of dogs infected by Leishmania infantum. However, the kinetics and behavior of these proteins in canine leishmaniasis is still unknown. The aim of this study was to monitor the kinetics of APPs in dogs experimentally infected with L. infantum, before, during and after therapy against canine leishmaniasis. Levels of serum haptoglobin, serum amyloid A and C-reactive protein from 6 infected beagles, positive by both PCR and parasite culture, were monitored for 7 months post-infection. The dogs were then treated for 3 months with allopurinol (20 mg mg/kg/day PO), and their response to therapy was followed for 11 additional months. Levels of Immunoglobulins G and M were recorded during these 21 months and compared. Experimental infection with L. infantum amastigotes induced an increase in all APPs studied which was statistically significant 2 months after infection for all proteins. Clinical recovery was accompanied by a significant decrease of all APPs 1 month after the beginning of treatment. However, differences were found between the APPs in both magnitude and duration of serum level elevations. The increase in total IgG and IgM was delayed in comparison to APPs and contrarily to the APPs, these immunoglobulins did not significantly decrease with treatment. In conclusion, the results of this study suggest that APPs could be used as early markers for disease as well as for monitoring the response to treatment in canine leishmaniasis.     

Semi-quantitative analysis of cytokine expression in asymptomatic canine leishmaniasis

The dog is the main reservoir of Leishmania infantum, the parasite responsible for visceral leishmaniasis in Mediterranean countries. The infection in dogs shows different clinical presentations, from subclinical/asymptomatic to a fully developed disease, depending on the host's immune responses. The Th1/Th2 dichotomy is not clear in the different forms of canine leishmaniasis, since the data available from studies of immunity response in canine leishmaniasis are scarce and fragmented. The present work describes the cytokine expression in peripheral blood mononuclear cells (PBMC) obtained from asymptomatic dogs experimentally infected with L. infantum that present a cellular protective immune response. The results obtained from freshly isolated PBMC showed expressions of TNF-alpha, IL-2, IFN-gamma, IL-10 and IL-18 mRNA, similar to those from non-infected dogs. However, there was almost no expression of IL-4 mRNA detected in the asymptomatic infected dogs compared to the control dogs. Unspecific stimulation with ConA promoted the expression in a greater or lower degree of all the cytokines studied. In vitro stimulation of PBMC with soluble leishmanial antigen (SLA) promoted the expression of IL-2, IFN-gamma, TNF-alpha, IL-18, IL-4, IL-6 and IL-10 mRNA, with the two first being specifically induced. Although both Th1 and Th2 cytokines are produced, cell mediated immunity observed in these L. infantum-infected asymptomatic dogs depended on the preferential expression of Th1 cytokines.     

Diagnosis of canine visceral leishmaniasis: biotechnological advances

Human visceral leishmaniasis (HVL) is endemic in the tropical and sub-tropical regions of Africa, Asia, the Mediterranean, Southern Europe and South and Central America, with approximately 500,000 new cases reported annually. As dogs are considered to be the major reservoirs for HVL, the accurate diagnosis of disease in these animals is important. Diagnosis of canine visceral leishmaniasis (CVL) is performed mainly by direct parasitological methods that can yield false-negative results, either because of the very low number of Leishmania spp. organisms in clinical samples (bone marrow and lymph nodes) or because morphological identification is difficult. In addition, these methods are invasive. Conventional serological techniques are limited by cross-reactivity with other parasitic diseases and because several technical procedures have not been standardised. The development of polymerase chain reaction based approaches and immunoassays based on the use of recombinant antigens aimed at improving the sensitivity and specificity of CVL diagnosis is discussed.     

Clinical and parasitological evaluation of dogs naturally infected by Leishmania (Leishmania) chagasi submitted to treatment with meglumine antimoniate

Aiming to evaluate the efficacy of the treatment of canine visceral leishmaniasis, to verify the occurrence of a possible disease relapse, and to search for the presence of the parasites after the end of the treatment, seven dogs naturally infected by Leishmania (Leishmania) chagasi were used. The dogs were subjected to a treatment with 75 mg/kg meglumine antimoniate subcutaneously every 12 h for 21 days, and followed-up for a period of 6 months. During the whole experimental period the animals wore deltamethrin collars and were kept in a screened kennel to avoid reinfection. Lymph node and bone marrow aspiration biopsy was carried out to search for the parasite at seven moments: before the treatment, 30, 60, 90, 120, 150 and 180 days after the start of the treatment. After the end of the experiment all dogs were humanely euthanized. Then, spleen and liver "imprints" and in vitro cultures were carried out to search for amastigote forms of the parasite. During the treatment all animals presented remission of symptoms. However, two dogs were observed to present new symptoms in the course of the experiment. At the end of the experiment, the presence of amastigote forms of the parasite was evidenced in five of the seven dogs. This enabled us to conclude that the treatment promoted clinical cure but did not eliminate the parasites completely.     

Histopathologic patterns of nephropathy in naturally acquired canine visceral leishmaniasis

Although the nephropathy of visceral leishmaniasis (VL) is known both in humans and dogs, histopathologic alterations have not been thoroughly studied. We examined renal alterations in 55 dogs with naturally acquired VL compared with five noninfected dogs from an endemic area in northeastern Brazil. Glomerulonephritis was found in 55 dogs, interstitial alterations in 53 dogs, and tubular changes in 43 dogs with VL. The glomerular alterations found were minor glomerular abnormalities (n = 8, 14.5%), focal segmental glomerulosclerosis (n = 10, 18.2%), mesangial proliferative glomerulonephritis (n = 17, 32.7%), membranoproliferative glomerulonephritis, (n = 18, 30.9%), crescentic glomerulonephritis (n = 1, 1.8%), and chronic glomerulonephritis (n = 1, 1.8%). Morphometric and ultrastructural studies complemented the analysis. The five control animals exhibited no glomerular alterations. The glomerular lesions were related to functional alterations. Considering that the alterations of canine and human nephropathy in VL are very similar, the data obtained in this study constitute an important contribution to the understanding of canine and human VL nephropathy.     

Laboratory tests for diagnosing and monitoring canine leishmaniasis

Although several reviews on canine leishmaniasis have been published, none thoroughly described clinicopathologic abnormalities and their clinical usefulness. The aim of this review was to provide information concerning current diagnostic tests relevant for clinical pathologists and from a practical perspective. Specifically, in canine leishmaniasis, nonregenerative normocytic normochromic anemia, thrombocytopenia, or leukogram changes may be present. Clinical chemistry and urinalysis may indicate renal dysfunction (azotemia, decreased urine specific gravity, proteinuria) and an inflammatory/immune response (increased acute phase proteins [APP] or α₂‐ and/or γ‐globulins). Although a potential gammopathy is usually polyclonal, it may also appear oligo‐ or monoclonal, especially in dogs coinfected by other vector‐borne pathogens. When lesions are accessible to fine‐needle aspiration (lymphoadenomegaly, nodular lesions, joint swelling), cytology is strongly advised, as the presence of Leishmania amastigotes in a pattern of pyogranulomatous inflammation or lymphoplasmacytic hyperplasia is diagnostic. If the cytologic pattern is inconclusive, the parasite should be identified by histology/immunohistochemistry or PCR on surgical biopsies. Alternatively, cytology and PCR may be performed on bone marrow samples where amastigotes, along with erythroid hypoplasia, myeloid hyperplasia, plasmacytosis, or secondary dysmyelopoiesis can be observed. Dogs with overt leishmaniasis generally have high antibody titers, while low titers predominate in immunologically resistant infected dogs or in exposed dogs with no parasite confirmation. Quantitative serology is recommended in clinically suspect dogs as high‐titer antibodies titers may confirm the clinical diagnosis. In confirmed and treated dogs, renal function and inflammatory/immune response variables should be periodically monitored.     

Incidences of canine leishmaniasis in an endemic area of southern Italy

Canine leishmaniasis (CanL), caused by Leishmania infantum, is widely distributed in many Mediterranean countries and is considered endemic in southern and central Italy with prevalence reaching up to 48.4%. Determination of the incidence would be useful as a measure of the risk of infection, then to evaluate the usefulness of control measures and to estimate whether a new focus is autochthonous or imported. This study was performed on two sites in the Apulia region of southern Italy, namely sites A and B. A total of 262 dogs were included in the evaluation of incidence, 94 farm dogs from site A and 168 dogs (92 farm and 76 kennel dogs) from site B. The incidence of infection was determined by using two different approaches: in site A by means of incidence density rate (IDR); in site B by the yearly seroconversion rate. In site A, the IDR was calculated at 4.25% dog-years; in site B the yearly incidence rate was of 9.52% (6.5% and 13.1% in farm and kennel dogs, respectively). The strength and weakness of the two different approaches (i.e. annual monitoring or monthly interval monitoring) for calculating the incidence of CanL in an endemic area have been discussed.