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.     

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.     

Atypical forms of canine leishmaniosis

Canine leishmaniosis is a common disease in the Mediterranean area, but sporadic cases in dogs having travelled through endemic regions are also reported. The disease's evolution is usually chronic and symptoms are either non-specific (fever, weight loss, lethargy, enlarged lymph nodes), dermatological, renal or ocular. The purpose of this article is to review the literature and to describe our own experience of certain atypical forms of canine leishmaniosis. These include specific skin lesions, monoclonal gammopathy, renal failure (without any other signs), chronic colitis, haemostatic problems and disorders of the cardiovascular, respiratory and musculo-skeletal systems.     

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.     

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.     

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.     

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.     

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.     

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).     

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.     

The first report of autochthonous non-vector-borne transmission of canine leishmaniosis in the Nordic countries

Background

Leishmania spp. are zoonotic protozoans that infect humans and other mammals such as dogs. The most significant causative species in dogs is L. infantum. In dogs, leishmaniosis is a potentially progressive, chronic disease with varying clinical outcomes. Autochthonous cases of canine leishmaniosis have not previously been reported in the Nordic countries.

Results

In this report we describe the first diagnosed autochthonous cases of canine leishmaniosis in Finland, in which transmission via a suitable arthropod vector was absent. Two Finnish boxers that had never been in endemic areas of Leishmania spp., had never received blood transfusions, nor were infested by ectoparasites were diagnosed with leishmaniosis. Another dog was found with elevated Leishmania antibodies. A fourth boxer dog that had been in Spain was considered to be the source of these infections. Transmission occurred through biting wounds and semen, however, transplacental infection in one of the dogs could not be ruled out.Two of the infected dogs developed a serious disease and were euthanized and sent for necropsy. The first one suffered from membranoproliferative glomerulonephritis and the second one had a chronic systemic disease. Leishmania sp. was detected from tissues by PCR and/or IHC in both dogs. The third infected dog was serologically positive for Leishmania sp. but remained free of clinical signs.

Conclusions

This case report shows that imported Leishmania-infected dogs may pose a risk for domestic dogs, even without suitable local arthropod vectors.     

Epidemiology of canine leishmaniosis in Catalonia (Spain) the example of the Priorat focus.

An epidemiological survey of canine leishmaniosis was conducted in the Priorat, a rural region in the Northeast of Spain, for 10 years (1985-1994). Seroprevalence throughout the region, determined by dot-ELISA and IFI, was 10.2% (8-12%). Forty percent of the dogs studied had a low level of anti-Leishmania antibodies, whereas only 50% were seronegative. Only one-third of the seropositive dogs had evident symptoms of the disease. Annual incidence of the disease was 5.7% and the level of endemicity was stable during the study. Four Leishmania zymodemes (MON-1, MON-29, MON-77, MON-105) were present in the focus, and their distribution in the different hosts is discussed. Apart from dogs and foxes, no other reservoir host has been found in the region.     

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.     

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.     

The importance of canine leishmaniosis in non-endemic areas, with special emphasis on the situation in Germany

This review article summarizes the situation of canine leishmaniosis in Germany. Published case studies on infections with Leishmania (L.) infantum in either humans or dogs are analyzed. Diagnosed cases of infections by Leishmania spp. in humans and animals are not a notifiable disease in Germany or other European countries. Taking this into consideration one may assume that there might be a significant gap between the analyzed and reported cases and the infectious status within the country. The reported case studies and results from surveys indicate that the majority of all L. infantum infections are acquired during travelling in endemic regions, predominantly the Mediterranean region. However there are cases reported from human infections and growing number of cases in dogs, where the case history may indicate an autochthonous infection within Germany, a country within a non-endemic region. The current data from entomological field studies proved the presence of two phlebotomine sand fly species. Phlebotomus (P.) mascittii, an anthropophilic sand fly species and P. perniciosus a proven vector of L. infantum. The impact from a growing leishmania-positive dog population within Germany, the distribution of at least two sand fly species, one with vector potential in the light of climate change and other non-vectorial transmissions are summarized.