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.     

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.     

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).     

Factors associated with seroprevalence to Mycobacterium paratuberculosis in small-ruminant farms in the Madrid region (Spain)

A cross-sectional study was conducted in a population of small ruminants in the Madrid region (Spain) to determine the Mycobacterium paratuberculosis seroprevalence and to identify farm factors possibly associated with paratuberculosis (PTB). Farming-management information and sera were collected from 60 sheep or goat flocks. The relationship between seropositivity and the variables in the questionnaire was assessed by unconditional logistic regression, followed by random-effects logistic regression analysis to adjust for overdispersion between herds. The seroprevalence to M. paratuberculosis was 11.7% (64 out of 546) using agar–gel immunodiffusion assay (AGID). According to the sensitivity and specificity of the AGID test the true prevalence could be as high as 44%. A herd size of between 200–400 head and the presence of foreign breeds and their crosses were significantly associated with seropositivity (OR=4.05 and OR=4.32, respectively). A higher replacement rate was also associated with seroprevalence to M. paratuberculosis (24.2% in positive herds vs. 18.1% in negative). All these three factors were related to more intensive management in the surveyed area. In contrast, membership of a professional livestock association appeared to be a protective factor against PTB (OR=0.28). No variables related to veterinary assistance were associated with seroprevalence, probably reflecting the current lack of interest in PTB on the part of the animal-health administration and veterinary services in Spain.     

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.     

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.     

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.     

A randomised, blinded, placebo-controlled clinical trial with allopurinol in canine leishmaniosis.

A total of 45 non-uremic dogs, with clinical signs indicating leishmaniosis, entered the study. Diagnosis was confirmed by indirect immunofluorescence assay (IFA) on serum and polymerase chain reaction (PCR) on bone marrow samples. The dogs were randomly allocated into Group A (n=37) that received allopurinol (10mg/kg B.W., per os, twice daily) for 4 consecutive months, and Group B (n=8) that were placebo-treated. Clinical signs were scored just before and at monthly intervals throughout the study period, in a blinded and independent fashion. Complete blood count, serum biochemistry profile, urinalysis, lymph node and bone marrow parasitology, IFA and enzyme-linked immunosorbent assay (ELISA) serology and bone marrow PCR were carried out at the beginning and at the end of the trial. A total of three Group A and one Group B dogs died of end stage kidney disease that developed during the trial. In Group A animals that endured the trial there was a significant improvement in the general body condition, conjunctivitis, peripheral lymphadenopathy, splenomegaly, masticatory muscle atrophy, ulcerative stomatitis, epistaxis, exfoliative dermatitis, cutaneous ulcerations, blepharitis and nasodigital hyperkeratosis. The same observation was made for anemia, lymphopenia, hyperproteinemia, hyperglobulinemia, hyperphosphatemia, increased alkaline phosphatase activity and the low albumin/globulin ratio. By contrast, no improvement of any kind was seen in Group B dogs. Lymph node and bone marrow parasite numbers were significantly decreased in Group A animals. In Group B, that occurred only in the lymph nodes. Apart from remission of clinical signs and restoration to normal of clinicopathological abnormalities, allopurinol did not eliminate Leishmania organisms, as the PCR result on bone marrow was still positive in all the dogs that finished the trial.     

Two cases of feline visceral and cutaneous leishmaniosis in Spain

This paper describes clinical signs and lesions in two cases of leishmaniosis--one visceral and one cutaneous in the cat (Felis catus domesticus). The diagnosis was achieved by a combination of serology, light and electron microscopic studies. The vague nature of the clinical signs observed in both cases was particularly striking, and clinical features were similar to many other diseases commonly found in cats. Therefore, the use of various investigations to detect leishmaniosis (serum chemistry, serology and histopathology) is highly recommended in cases where clinical signs do not respond to conventional treatment.     

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.     

A survey on canine leishmaniasis and phlebotomine sand flies in central Italy

Zoonotic visceral leishmaniasis (ZVL) is a vector-transmitted zoonosis caused by the parasitic protozoan Leishmania infantum. Bloodsucking sand flies of the subfamily Phlebotominae are the obligatory insect hosts, and the dog is the only domestic reservoir.This study reports data from a survey of canine infection and sand fly phlebotomine monitoring in the province of Perugia in central Italy. The overall seroprevalence in a total of 100 dogs tested was 8% (95% confidence interval: 3.8-15.6%). Data analysis revealed that serological positivity was statistically associated with age (p-value = 0.03) and the area where dogs lived. Standard blacklight traps employed for sampling Culicoides midges in bluetongue disease surveillance were used in phlebotomine monitoring. A total of 5698 sand flies were collected and the two species, Leishmania competent vectors, were identified, Phlebotomus perfiliewi (50%) and Phlebotomus perniciosus (30%).     

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

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

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.     

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.     

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.