Chemotherapy of canine leishmaniosis

Visceral leishmaniosis is a widespread and potentially fatal disease of dogs and humans common in the Mediterranean region, the Middle East, and South America. Canine leishmaniosis is most frequently treated with the drugs meglumine antimoniate, allopurinol, amphotericin B, or a combination of meglumine antimoniate and allopurinol. Therapy with the currently used drugs often achieves temporary clinical improvement and changes in immunologic parameters with restoration of the ability to mount parasite-specific cell mediated responses and decrease in anti-leishmanial antibody titers. However, treatment usually does not prevent relapse of disease or eliminate parasite carriage. Due to the current lack of an ultimate and effective therapy for canine leishmaniosis, new drugs, delivery systems and treatment strategies are necessary to achieve a consistent parasitological cure in infected dogs.     

Nodular lesions of the tongue in canine leishmaniosis

In this case report, an atypical clinical presentation of leishmaniosis in a dog with multiple nodular lesions of the tongue is described. Haematological and biochemical analysis, serological test for Leishmania infantum antibodies and biopsy samples from several nodules of the tongue for histopathological examination were made. The final diagnosis of leishmaniosis was based upon the observation of amastigotes in the bioptic samples. It is recommended to consider leishmaniosis among the list of differentials of mucosal nodular lesions, at least in endemic areas.     

Serum ferritin and paraoxonase-1 in canine leishmaniosis

Ferritin and paraoxonase-1 (PON-1) were measured in dogs experimentally infected by Leishmania infantum (during experimental infection and following treatment) and also in naturally-infected dogs which presented different degrees of proteinuria. Experimentally-infected dogs were monitored for 7 months post-infection, then treated for 3 months with allopurinol, and their response to therapy was followed for 11 additional months. Naturally-infected dogs were staged based on the urine protein/creatinine (UPC) ratio into three groups as follows: group 1 (non-proteinuric; UPC ratio: <0.2), group 2 (borderline proteinuric; UPC ratio: 0.2–0.5) and group 3 (proteinuric; UPC ratio >0.5). An increase in serum ferritin values and a decrease in PON-1 activity were observed 2 months after infection. Both analytes returned to preinfection values following treatment. Significantly higher concentrations of ferritin were observed in dogs classified as either borderline or proteinuric when compared with non-proteinuric dogs whereas serum PON-1 activity was decreased only in proteinuric dogs.     

Histopathological differences between canine idiopathic sebaceous adenitis and canine leishmaniosis with sebaceous adenitis

Sebaceous adenitis (SA) may be idiopathic (ISA) or associated with other disorders. The purpose of the present study was to compare the cutaneous histopathology of SA in cases in which Leishmania organisms were detected by immunohistochemistry (IHC) with that of cases diagnosed as ISA. Skin sections of 29 patients were evaluated histologically and divided into two groups, one characterized by several epidermal and subepidermal lesions, a granulomatous to pyogranulomatous nodular to diffuse dermatitis involving the sebaceous glands and a positive IHC for Leishmania spp. The other group was characterized by orthokeratotic hyperkeratosis, follicular keratosis with different degrees of pyogranulomatous to granulomatous SA, lack of nodular dermatitis and a negative IHC for Leishmania spp. Hidradenitis was present in both groups. From these results it can be concluded that SA in canine Leishmaniosis (CL) is usually present together with a nodular to diffuse dermal infiltrate and epidermal and subepidermal lesions, and that SA in the absence of dermal inflammation is probably not associated with or suggestive of CL, even in regions where the disease is endemic.     

Cutaneous immune mechanisms in canine leishmaniosis due to Leishmania infantum

Canine leishmaniosis (CanL) caused by the parasite Leishmania infantum is a systemic disease with variable clinical signs. The disease is endemic in the Mediterranean countries and dogs are the main domestic reservoir of the parasite. The quite complicated immune response against the parasite is crucial for the evolution of CanL infection with the skin playing a major role in its immunopathogenesis.After the inoculation of Leishmania promastigotes into the dermis by sand fly bites, complement factors, Langerhan's cells, neutrophils, fibroblasts and keratinocytes are involved in the activation of the innate arm of the skin immune system, with the macrophages and dendritic cells to play a major key role.The effective activation of cellular immunity is the cornerstone of dog's resistance against the parasite. Promastigotes reaching the dermis are engulfed, processed and transferred by APCs to draining lymph nodes to stimulate naïve T-cells for proliferation and differentiation into armed effector T-cells. Th1 cells activate the infected macrophages to kill Leishmania, whereas Th2 cells divert the immune response to humoral immunity and down regulation of cellular immunity with Th1 cell anergy. Inhibition of co-stimulatory molecules expression by infected macrophages contributes to T-cell anergy. In canine subclinical infections cutaneous lymphocytic infiltrate and parasites are absent, as opposed to dogs with clinical leishmaniosis. CD8+ cells constitute a significant population of cellular immunity in CanL since they outnumber CD4+ cells in the dermis, producing IFN-γ in sub clinically infected dogs and high levels of IL-4 in dogs with clinical leishmaniosis.Numerous B-lymphocytes have been shown to heavily infiltrate the dermis at least in exfoliative dermatitis in CanL. A mixed Th1/Th2 cytokine profile has been found in the dermis of naturally infected with L. infantum dogs. In the skin of dogs with clinical leishmaniosis, where plasma cells outnumber T lymphocytes in the dermal infiltrate, there is an overproduction of IL-4, IL-13 and TNF-α leading to Th2-biased humoral immune response. The issue of humoral immunity polarization in CanL remains controversial. Much still needs to be learned about other mechanisms underlying the complex interaction between the skin immune system and the parasite.     

Immunoglobulin G and E responses in various stages of canine leishmaniosis

Pathogenesis in visceral leishmaniosis is associated with depressed cellular immunity and a significant rise of antileishmanial antibodies. We assessed the relative levels of immunoglobulin E anti-Leishmania infantum, together with those of IgG, IgG1 and IgG2, using the enzyme-linked immunosorbent assay (ELISA) test, in non-infected and infected dogs with or without symptoms, and their association with symptoms to differentiate the stages of the infection. The expression of all immunoglobulins (IgG, IgG1, IgG2 and IgE) was higher in symptomatic dogs than in all other categories. IgG and IgG2 expression was higher in the infected asymptomatic group than in the non-infected group, whereas IgG1 and IgE expression was only higher in symptomatic animals. This correlation between the expression of IgG1 and IgE and the pathology of leishmaniosis points to their potential role as markers of the active disease.     

Epidemiological aspects of canine visceral leishmaniosis in the Islamic Republic of Iran

An epidemiological study to examine the sero-prevalence of zoonotic visceral leishmaniosis (ZVL) among domestic and wild canines in endemic foci of Iran was carried out during 1999-2003 to assess the distribution of the disease and the possible association between infection in dogs, wild canines and people. Anti-leishmanial antibodies were detected by the direct agglutination test (DAT). Parasitological study was performed for all captured wild canines and were detected in some of the seropositive dogs with specific clinical signs (n=107). Serum samples (n=1568) were collected from domestic dogs in villages that are known endemic foci of human visceral leishmaniosis (HVL). Wild canine sera were collected from jackals (Canis aureus, n=10), foxes (Vulpes vulpes, n=10) and wolves (Canis lupus, n=10). Of the 1568 serum sampled collected from domestic dogs, 222 (14.2%) were positive by DAT (1:320 and above). No statistically significant difference was found between male (15.2%) and female (11.8%) sero-prevalence (P=0.083). Dogs of 8 years and above showed the highest sero-prevalence (40.6%). Only 23.9% of the seropositive domestic dogs had clinical signs. Parasitology and serology tests that were performed in 30 wild canines showed 10% these animals were infected by Leishmania infantum. Ten out of 11 Leishmania spp. isolated from the dogs and wild canines were identified as L. infantum and one other as L. tropica by molecular and biochemical techniques. For the first time in Iran, L. infantum and L. tropica were isolated from viscera of both a wolf and a domestic dog.     

Nodular granulomatous glossitis as the sole clinical sign in canine leishmaniosis

A 5.5‐year‐old, intact male Rottweiler dog was admitted with a history of multifocal nodular tongue lesions which progressively deteriorated during the previous year. Physical examination revealed several reddish nodules with central depression on the surface of the tongue in an otherwise healthy dog. Clinicopathologic abnormalities included eosinophilia and hyperproteinemia. Lingual nodule cytopathology, histopathology, and immunohistochemistry revealed Leishmania spp. amastigotes and a severe granulomatous glossitis. The dog was also seroreactive to L infantum antigens by an indirect immunofluorescence assay. Clinical reevaluation 3 months after the institution of treatment with allopurinol and miltefosine indicated that the nodular lesions had completely regressed. In endemic areas, lingual nodular lesions may rarely be the sole clinical sign of canine leishmaniosis. Standard medical treatment may provide an excellent prognosis.     

Advantages of real-time PCR assay for diagnosis and monitoring of canine leishmaniosis

The aim of the present study is to highlight the advantages of real-time quantitative PCR intended to aid in the diagnosis and monitoring of canine leishmaniosis. Diagnosis of canine leishmaniosis is extremely challenging, especially in endemic areas, due to the diverse and non-specific clinical manifestations, and due to the high seroprevalence rate in sub-clinical dogs. Veterinarian clinicians are usually confronted with cases that are compatible with the disease, and with several diagnostic tests, sometimes with contradictory results. We have developed a new TaqMan assay, targeting the kinetoplast, applied to 44 samples of bone marrow aspirate or peripheral blood. The dynamic range of detection of Leishmania DNA was established in 7 logs and the limit of detection is 0.001 parasites in the PCR reaction. At the time of diagnosis parasitemia ranges from less than 1 to 10(7)parasites/ml. The ability to quantify the parasite burden allowed: (i) to elucidate the status of positive dogs by conventional PCR, although larger studies are necessary to clarify the dividing line between infection and disease, (ii) to estimate the kinetics of the parasite load and the different response to the treatment in a follow-up and (iii) to validate blood as less invasive sample for qPCR. The continuous data provided by real-time qPCR could solve the dilemma for the clinician managing cases of canine leishmaniosis by differentiating between Leishmania-infected dogs or dogs with active disease of leishmaniosis.     

Cellular immunophenotyping of exfoliative dermatitis in canine leishmaniosis (Leishmania infantum)

Lymphocyte subsets, major histocompatibility complex (MHC)-II expressing cells and number of amastigotes in the epidermis and dermis were investigated immunohistochemically in 48 dogs with patent leishmaniosis, with or without exfoliative dermatitis (ED) to study the immunopathogenesis of this common cutaneous form of the disease. Skin biopsies were obtained and compared for ED sites (group A, n = 26), normal-appearing skin from the same animals (group B, n = 24), and leishmanial dogs not exhibiting ED (group C, n = 22), and normal controls (group D, n = 22). The CD3+, CD45RA+, CD4+, CD8+ (CD8a+), CD21+, and MHC-II+ cells and leishmania amastigotes were identified immunohistochemically and counted with the aid of an image analysis system. Pyogranulomatous to granulomatous dermatitis, expressed in various histopathological patterns, was noticed in all groups A and B and in half of group C dogs. In the epidermis, the low number of T-cells and their subsets did not differ significantly between groups A and B, but CD8+ outnumbered CD4+ lymphocytes in both groups. MHC-II+ expression on epidermal keratinocytes was intense in the skin with and without lesions from dogs with ED but not in group C dogs. CD3+, CD8+ and MHC-II+ cells were fewer in group C compared to group A and B dogs. In the dermis, CD3+ cells in group A animals were mainly represented by the CD8+. CD45RA+ and CD21+ cells were also seen in high numbers. MHC-II expression, potentially in lymphocytes, fibroblasts, dendritic cells, and macrophages was intense. The numbers of all cellular subpopulations in the dermis were significantly different between the groups, being highest in group A and lowest in group D. In sebaceous adenitis sites, CD4+ outnumbered CD8+ cells in contrast to the neighbouring dermis and the epidermis. The number of CD21+ and CD45RA+ cells was much lower in the inflamed sebaceous glands compared to the dermis. Finally, the number of amastigotes in the normal-appearing skin was significantly higher in the ED dogs (group B) than in those not exhibiting this cutaneous form of the disease (group C).     

Regional parasite density in the skin of dogs with symptomatic canine leishmaniosis

In canine leishmaniosis, the parasitic density of the skin may be important for the infection of sandflies, and increased accumulation of inflammatory cells infected with Leishmania is believed to occur in dermal areas subjected to mechanical trauma. Parasite density and inflammatory responses in the upper and lower dermis of three body sites: flank (control site), dorsal muzzle (sandfly feeding site), and footpads (mechanical stress sites) were thus investigated in 15 dogs with symptomatic leishmaniosis. Parasite density did not differ between the control and tested sites or between the upper and lower dermis, apart from the footpads where it was higher in the upper dermis, and there was no correlation with severity of the macroscopic lesions or inflammatory infiltrate, except for the lower footpad dermis. No selective accumulation of the parasite in the muzzle that would favour its transmission to sandflies occurred, and the mechanical stress imposed on the footpads was not associated with increased parasitic density, or with inflammatory infiltrate.     

Identification of two highly performing Leishmania infantum antigens for serodiagnosis of canine leishmaniosis

In the aim of improving serodiagnosis of canine leishmaniosis, we analysed the humoral immune response of dog against Leishmania infantum parasite. The antigenic reaction of L. infantum polypeptides with sera from 31 dogs with parasitologically confirmed leishmaniosis was studied by using the immunoblot technique. Electrophoretic profile of the parasite extract showed more than 50 polypeptides, with molecular weights ranging from 12 to 170 kDa. Among these polypeptides, 37 antigen components, ranging from 14 to 91 kDa, were recognised by antibodies of L. infantum infected dogs. Three polypeptides (14, 16 and 76 kDa) reacted with all of the 31 serum samples. The other most frequently recognised antigens were those of 29.5, 32, 46, 59 and 66 kDa with a sensitivity of 87.1%, 93.6%, 96.8%, 87.1% and 80.6%, respectively. The 14 and 16 kDa bands were the most intense and remained detectable until a serum dilution of 1:6400. No reaction of these two major antigens was observed with sera collected from 50 Leishmania-free dogs, living in the leishmaniosis-free region of Rabat in Morocco, whereas the crude antigen used in IFAT or ELISA lead to three false positive results. Four antigen components of 29, 41, 55, and 70 kDa were recognised by some sera samples from negative controls. These results demonstrated the potential interest of the fractions of 14 and 16 kDa in immunodiagnosis of canine leishmaniosis.     

A seroepidemiologic survey of canine visceral leishmaniosis among apparently healthy dogs in Croatia

Cross-sectional investigation was done on seroprevalence of Leishmania sp. infection among apparently healthy dogs in an area where canine leishmaniosis is endemic. Survey included 68 dogs living in the coastal city of Split, and 238 dogs living in 12 villages scattered in the hinterland. Each dog was clinically examined for the presence of some discrete signs compatible with leishmaniosis and by dot-ELISA modification determined the presence of anti-Leishmania antibodies. The titre 1:600 and higher was regarded as positive in the study. The seroprevalence ranged from 0 to 42.85%, depending on the location. 54.34% of the seropositive dogs had moderately enlarged lymph nodes and/or some discrete changes on the skin. In our parasitological study, Leishmania sp. was isolated from several seropositive animals that had some clinical signs and from a few which did not have any. Data analysis revealed that serological positivity to Leishmania sp. was not associated with a dog's outdoor lifestyle and utility, but was associated with the gender and age.     

One-year clinical and parasitological follow-up of dogs treated with marbofloxacin for canine leishmaniosis

The purpose of this international, multicentric, and non-comparative field trial was to obtain complementary data on long-term clinical and parasitological follow-up of dogs treated with marbofloxacin for canine leishmaniosis (CanL). Seventy-four dogs with clinical signs of CanL and without severe renal failure were recruited in France, Spain and Italy, and 61 of them were part of the analysis. Each dog was treated with palatable tablets of marbofloxacin at 2 mg/kg once a day for 28 days. A clinical and parasitological follow-up was performed regularly up to 12 months. Efficacy was demonstrated in 42 dogs (68.9%), within 51 days (mean value), 10 of them (23.8%) being clinically cured after 3 months. A decrease of 61% in the sum of clinical scores was observed after 3 months. Haemato-biochemical parameters improved in general, supporting the observed clinical efficacy. Relapse was observed in 20/38 dogs (52.6%) approximately 5.5 months after treatment completion. The blood parasite load generally developed in conformity with the clinical outcome, even if exceptions were not rare. Lymph nodes remained positive by culture or PCR for a long time, even in dogs for which a good clinical response was observed. Despite the incomplete parasite clearance, as is also the case with other anti-leishmanial drugs, these results nevertheless confirm the relevance of marbofloxacin as a CanL treatment.     

Atypical findings in 16 cases of canine ehrlichiosis

Sixteen cases of clinically and serologically diagnosed canine ehrlichiosis were studied retrospectively. Findings not previously reported included diffuse, pulmonary interstitial radiopacities; weak-positive results for blastomycosis, using agar gel immunodiffusion; normal platelet counts; hemorrhage despite platelet counts greater than 100,000/mm3, and positive results for platelet factor 3 and antinuclear antibody assays. These atypical findings showed that the differential diagnosis of canine ehrlichiosis, blastomycosis, immune-mediated thrombocytopenia, and primary nasal lesions may be confusing, warranting careful assessment. The increased release of platelet factor 3 and the finding of antinuclear antibodies supported an immune mechanism in the pathogenesis of canine ehrlichiosis, as suggested by previous experimental studies.     

Long term improvement in the treatment of canine leishmaniosis using an antimony liposomal formulation

Pharmacokinetic and clinical effectiveness of liposome-encapsulated N-methylglucamine antimoniate (LMA) was performed in dogs suffering from experimental leishmaniosis. LMA was compared with N-methylglucamine antimoniate (MGA), the same drug in its free form. Sb plasma concentrations for LMA were always higher than those for MGA. Mean residence time (MRT), half-life time (t(1/2)) and clearance (Cl) showed that Sb was eliminated slower after liposome administration. The high volume of distribution (Vd) obtained with LMA suggests that Sb could achieve therapeutic concentrations in parasite-infected tissues. Average plasma concentration at steady state (Css(ave)) shows that Sb body concentrations after LMA treatment (9.8 mg/kg Sb, each 24h) would be effective in Leishmania infantum canine infection. Comparing LMA with MGA in a 1-year follow-up we observed no relapses for LMA and total protein and gammaglobulin concentrations were within normal range, while for MGA both began to rise 3 months after treatment. Use of antimonial liposomal formulations may restore effectiveness to an existing drug and reduce toxicity.