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.     

Cutaneous leishmaniosis in a horse in southern Germany caused by Leishmania infantum

This report describes a case of cutaneous leishmaniosis in a horse in southern Germany. Diagnosis is based on histopathology, immunohistochemistry and electron microscopy. The protozoan was identified as Leishmania infantum via PCR and restriction fragment length polymorphism. The horse did not show specific Leishmania antibodies. The lesions healed completely within 6 months without any specific treatment. Since neither the infected horse nor its dam had ever left their rural area, autochthonous infection in Germany cannot be excluded. Factors possibly influencing the epidemiological situation are discussed.     

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).     

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.     

Claw histopathology and parasitic load in natural cases of canine leishmaniosis associated with Leishmania infantum

Histological lesions and the presence of Leishmania spp. amastigotes in claw tissues were investigated in 40 dogs with leishmaniosis, with (16/40--group A) or without (24/40--group B) generalized onychogryphosis. Following euthanasia, the entire third phalanx with intact claw was amputated, formalin fixed, decalcified in a formic acid solution, embedded in paraffin, sectioned longitudinally and stained with haematoxylin and eosin, and acid orcein-Giemsa. Nested polymerase chain reaction (PCR) was used for the detection of Leishmania amastigotes. Lichenoid mononuclear infiltration (all dogs in group A, 21 of 24 dogs in group B), basal keratinocyte vacuolation (nine of 16 dogs in group A, 15 of 24 dogs in group B) and dermoepidermal clefting (13 of 16 dogs in group A, 18 of 24 dogs in group B) were the most prominent histopathological findings. There was no difference in the frequency and severity of these lesions between the two groups. Leishmania amastigotes could not be visualized in the dermis of any of the H&E sections, but their presence was demonstrated by nested PCR in three of 16 dogs in group A and two of 24 dogs in group B. However, the frequency of positive nested PCRs was not significantly different between the two groups. In conclusion, claw histopathology in symptomatic dogs with leishmaniosis, either with or without onychogryphosis is mainly characterized by mononuclear lichenoid dermatitis with or without interface dermatitis and dermoepidermal clefting, and is not accompanied by substantial local parasitism.     

Leishmania infantum in wild animals in endemic areas of southern Italy

Leishmania infantum infection in wildlife is increasingly reported in Europe, but scant data are available in Italy so far. This study aimed to investigate the circulation of L. infantum among sylvatic hosts in Sicily (southern Italy), a highly endemic area for canine leishmaniosis, through serological and molecular tools. Target tissues (skin, spleen, lymph nodes) collected from 71 European rabbits, 2 European hares, 7 red foxes, 11 European wildcats and 1 pine marten, were qPCR analysed for the detection of L. infantum DNA. Additionally, 40 rabbits, older than one year, were serologically screened for specific anti-Leishmania antibodies. Leishmania infantum was molecularly diagnosed in 5.4% (n = 5) of the examined animals (3/71 European rabbits, 2/7 red foxes). In many of the qPCR positive animals (4/5), the parasite DNA was more prevalent in visceral than cutaneous tissues. None of the positive animal showed signs of disease and/or macroscopic alterations of organs; low parasitic burden in all positive tissue samples was also recorded. Only one rabbit serum (i.e., 2.5%) tested positive for anti-Leishmania antibodies. The seropositive rabbit was in good health status and no amastigotes were observed in lymph-node aspirate and blood smears.This study provides first evidence of L. infantum infection in wild animals from Sicily (southern Italy). Despite the low prevalence of infection here reported, the circulation of the Leishmania in wild reservoirs in Sicily remains worthy of future investigations for a better understanding of their role in the epidemiology of the disease as well as to fine-tune control strategies in the area.     

Disseminated Leishmania infantum infection in two sibling foxhounds due to possible vertical transmission

Two sibling foxhounds born to a Leishmania seropositive bitch were presented after testing seropositive for Leishmania. Leishmania infantum infection was detected via histopathology, culture, and quantitative polymerase chain reaction (q-PCR). This is the first report of natural infection with Leishmania infantum with the possibility for vertical transmission in North America.     

Detection of Leishmania infantum DNA by fret-based real-time PCR in urine from dogs with natural clinical leishmaniosis

The aim of this study was to detect Leishmania infantum DNA by real-time PCR in urine from different groups of dogs with clinical leishmaniosis. Urine from 10 clinically healthy dogs and 43 dogs with clinical leishmaniosis diagnosed by positive serology and/or bone marrow PCR were studied. The group of 43 dogs with clinical leishmaniosis was divided into three subgroups: 13 dogs with renal insufficiency and proteinuria (urine protein-creatinine ratio greater than one), 13 dogs with only proteinuria, and 17 dogs with neither renal insufficiency nor proteinuria. The detection of Leishmania DNA was performed by light cycler real-time PCR using hybridization probes in each urine sample. Leishmania positive PCR was found in 47% (20/43) of the urine from leishmaniotic dogs, while all urine from clinically healthy dogs were negative. The percentages of positive Leishmania PCR were 85% (11/13) in dogs with renal insufficiency and proteinuria, 23% (3/13) in dogs with proteinuria and 35% (6/17) in dogs with neither renal insufficiency nor proteinuria. Dogs with renal insufficiency and proteinuria presented a statistical significant greater percentage of positive Leishmania PCR in urine when compared with the other subgroups (P<0.02). This study demonstrates the presence of Leishmania DNA in urine of dogs with leishmaniosis. Those dogs with severe renal damage present a greater number of Leishmania parasites in urine.     

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.     

Feline leishmaniasis due to Leishmania (Leishmania) amazonensis in Mato Grosso do Sul State, Brazil

A case of leishmaniasis in a domestic cat (Felis domesticus) is described. The animal showed a single, nodular lesion on the nose and many nodules of different size on the ears and digital regions of all the paws. Diagnosis was made by microscopic detection of amastigotes in Giemsa-stained smears from the lesions. By monoclonal antibodies the aetiological agent was identified as Leishmania (Leishmania) amazonensis, one of the seven species implicated in human leishmaniasis in Brazil. The clinical signs in feline leishmaniasis are unspecific and similar to those observed in other diseases such as cryptococcosis and in sporotrichosis, commonly found in cats. Leishmaniasis should therefore, be added to the differential diagnosis by feline veterinary practitioners and adequate investigations should carried out for dermal leishmaniasis in the area where the feline infection is detected.     

Leishmania infantum and Neospora caninum simultaneous skin infection in a young dog in Italy.

Leishmania infantum, the agent of canine leishmaniasis in Mediterranean countries, and Neospora caninum, a recently recognized protozoal pathogen in dogs, were diagnosed in a 9-month-old Argentine Dogo dog. Both skin lesions and neurological signs were present. Histopathology of cutaneous lesions revealed a suppurative, diffuse dermatitis with numerous intracellular protozoa. Serology was positive for both L. infantum (1:640) and N. caninum (1:800). Double-label immunohistochemical staining of skin samples with hyperimmune serum from L. infantum-infected dogs was positive for protozoa within macrophages, while the polyclonal antibody specific for N. caninum showed positive reactions for protozoa in endothelial cells and fibroblasts. Transmission electron microscopy (TEM) confirmed the infection with both protozoa. This is, to the authors' knowledge, the first case of simultaneous infection with L. infantum and N. caninum in a dog. It is possible that the immunosuppressive effects of Leishmania infection or long-term steroid therapy may have been a contributing factor to the development of N. caninum in this dog.     

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.     

Immune response to Leishmania infantum in healthy horses in Spain

Leishmania infantum infection has recently been described in horses in Europe. We report the results of a study on the immune response to L. infantum in horses living in an area endemic for leishmaniosis in NE Spain. Two ELISAs using protein A and anti-horse IgG conjugates were adapted to measure specific antibodies to L. infantum in horse sera. A lymphocyte proliferation assay (LPA) of peripheral blood mononuclear cells to L. infantum antigen was also performed to detect specific cellular immune response to Leishmania. Anti-L. infantum antibodies were detected in the serum of 16 of the horses studied (n=112) using the protein A assay but not in the assay using the anti-horse IgG conjugate. Specific lymphocyte proliferation was observed in 20 out of 55 horses. This study shows that horses in the area studied mount specific immune responses to L. infantum, and must therefore be considered among the species exposed to the parasite in this region. The infrequency of leishmaniosis in horses suggests that the immune response in this species is effective in controlling the infection.     

Epidemiological evaluation of Leishmania infantum zoonotic transmission risk in the recently established endemic area of Northwestern Italy

Leishmania infantum infection had been expanding into new areas due to changes in vector and host biology. Zoonotic visceral leishmaniasis has become endemic in previously unsuitable areas as vectors find favourable climatic conditions and an increasing number of reservoir dogs are moved between traditionally and new endemic areas. Monitoring vector and disease expansion in areas of recent colonization is needed to understand transmission mechanisms and patterns of disease establishment. Here, we studied the infection status of 815 human blood donors and of 803 sympatric dogs from five, newly endemic, areas in Northwestern Italy. In autochthonous dogs, the seroprevalence of anti‐L. infantum antibodies, recorded by Western blot, reached 42.22%, while in humans, the seroprevalence was of 16.81%. No significant correlation between the infection status of dogs and that of their human owners was found, but L. infantum infection was recorded in the different study areas with significant levels of diversity. Restriction fragment length polymorphism showed a high genetic variability of the circulating strains and gave useful insights on patterns of disease establishment into a naïve area.     

Immunity to Leishmania infantum in the Dog: Resistance and Disease

Veterinary Research Communications -