Susceptibility to visceral leishmaniasis in the domestic dog is associated with MHC class II polymorphism

Zoonotic visceral leishmaniasis (VL) is a disease of dogs, humans and other animals caused by the intracellular macrophage parasite Leishmania infantum. We examined the relationship between DLA class II alleles ( DRB1, DQA1, DQB1) and the course of infection in a cohort of Brazilian mongrel dogs exposed to natural L. infantum infection. DLA alleles were typed by sequence-based typing. DLA-DRB1 genotype was significantly associated with levels of anti- Leishmania IgG and parasite status assessed by PCR. Dogs with DLA-DRB1*01502 had higher levels of specific IgG and an increased risk of being parasite positive compared with dogs without this allele, controlling for other alleles and significant variables. No significant associations were seen for DLA-DQA1 or DLA-DQB1 alleles. These results suggest that the DLA-DRB1 locus plays a role in determining susceptibility to canine VL. As the domestic dog is the main reservoir for human infection, the identification of genetic factors influencing canine resistance or susceptibility to VL may provide insights into the immunology and potential control through vaccination of VL.     

Canine cutaneous leishmaniasis caused by neotropical Leishmania infantum despite of systemic disease: A case report

Visceral leishmaniasis is an anthropozoonosis caused by a protozoan Leishmania infantum (syn. Leishmania chagasi). Here, we report a typical case of canine cutaneous leishmaniasis due to L. infantum infection without any other systemic symptom in one dog in the city of Rio de Janeiro, Brazil. A mongrel female dog was admitted in a veterinary clinic with reports of chronic wounds in the body. Physical examination revealed erosive lesions in the limbs, nasal ulcers, presence of ectoparasites and seborrheic dermatitis. Blood samples and fragments of healthy and injured skin were collected. The complete hemogram revealed aregenerative normocytic normochromic anemia and erythrocyte rouleaux, and biochemical analysis revealed normal renal and hepatic functions. Cytology of the muzzle and skin lesions suggested pyogranulomatous inflammatory process. The histopathology of a skin fragment was performed and revealed suspicion of protozoa accompanied by necrotizing dermatitis. The diagnosis of leishmaniasis was accomplished by positive serology, isolation of Leishmania from the skin lesion, and also by molecular test (PCR targeting the conserved region of Leishmania kDNA). Culture was positive for damaged skin samples. PCR targeting a fragment of Leishmania hsp70 gene was performed employing DNA extracted from damaged skin. RFLP of the amplified hsp70 fragment identified the parasite as L. infantum, instead of Leishmania braziliensis, the main agent of cutaneous leishmaniasis in Rio de Janeiro. Characterization of isolated promastigotes by five different enzymatic systems confirmed the species identification of the etiological agent. Serology was positive by ELISA and rapid test. This case warns to the suspicion of viscerotropic Leishmania in cases of chronic skin lesions and brings the discussion of the mechanisms involved in the parasite tissue tropism.     

Leishmaniosis due to Leishmania infantum in a FIV and FelV positive cat with a squamous cell carcinoma diagnosed with histological, serological and isoenzymatic methods

Leishmaniosis caused by Leishmania infantum is an endemic zoonosis present in the Mediterranean area. Canidae (dog and fox) constitute the main reservoir hosts for the parasite, whilst wild rodents or the cat can be carriers of the protozoan and are considered as secondary potential reservoirs. This paper describes a case of disseminated feline leishmaniosis with cutaneous (ulcerative), visceral (spleen and lymph nodes) and blood involvement in a FIV-FelV positive cat. The microscopic identification of the Leishmania infection was initially made on a skin biopsy of the temporal area, where a squamous cell carcinoma was diagnosed. The diagnosis of the disease was achieved by several serological techniques (ELISA, IFAT and Western-blot). The strain was obtained by blood culture, characterized by electrophoresis of isoenzymes and identified as Leishmania infantum zymodeme MON-1. Since the infection due to L. infantum is a zoonosis, the potential feline reservoir should be more investigated. Serological analysis by Western blot on domestic cats provides a useful tool. In veterinary practice, feline leishmaniosis should be systematically included in the differential diagnosis when compatible cutaneous lesions are present, especially in the endemic areas of canine leishmaniosis.     

Emergence of Zoonotic Canine Leishmaniasis in the United States: Isolation and Immunohistochemical Detection of Leishmania infantum from Foxhounds from Virginia.

ABSTRACT. Previously considered an exotic disease, canine leishmaniasis caused by Leishmania infantum has recently been detected within the foxhound population in the United States and parts of Canada. Leishmania infantum is the etiologic agent of visceral leishmaniasis in many areas of the world and dogs are considered a major reservoir host for human Leishmania infections. Human visceral leishmaniasis has recently emerged as an opportunistic infection among individuals co-infected with HIV/AIDS and in persons taking immunosuppressive drugs. We report the isolation of L. infantum from 3 naturally infected foxhounds from Virginia by culture of popliteal lymph node and bone marrow, and the development of an immunohistochemical test to detect the parasite in tissues.     

The human cytokine response to Leishmania major early after exposure to the parasite in vitro

Leishmaniasis is caused by the protozoan parasite Leishmania spp. In murine leishmaniasis, a T helper cell type-I (Th1) response, characterized by the secretion of interferon (IFN)-gamma is necessary for clearing the infection. whereas a Th2 response, accompanied by the production of interleukin (IL)-5, can exacerbate the disease. Moreover, the early cytokine milieu is thought to play an important role in determining the outcome of infection. In human leishmaniasis little is known about this early cytokine response. Because of this, we cocultured human peripheral blood mononuclear cells (PBMC) with Leishmania major in vitro and measured the production of IFN-gamma, IL-5, and IL-10. We also treated PBMC cultures with various cytokines and neutralizing anticytokines. We found that the principal cytokine produced was IFN-gamma and that its production was regulated by IL-10 and IL-12. In contrast, only low levels of Th2 cytokines such as IL-5 were produced. Therefore, the Th1-Th2 dichotomy that exists in inbred strains of mice does not appear to apply to the response of humans to L. major. Rather, Th2 cytokines may play a role in regulating IFN-gamma production.     

Interleukin-12 augments a Th1-type immune response manifested as lymphocyte proliferation and interferon gamma production in Leishmania infantum-infected dogs

The dog is the major reservoir for human visceral leishmaniasis caused by Leishmania infantum. Interleukin-12 is considered to have an essential role in the development of both innate and adaptive immunity to Leishmania spp. and other intracellular pathogens. This study focused on the influence of IL-12 in experimental and natural canine visceral leishmaniasis. Responses of peripheral blood mononuclear cells to IL-12, interleukin-10 and Leishmania soluble antigen were evaluated in L. infantum experimentally infected oligosymptomatic beagles, uninfected beagles, naturally infected polysymptomatic dogs, and their matched uninfected controls. Leishmania soluble antigen induced strong peripheral blood mononuclear cells proliferation both in experimentally infected dogs (median stimulation index [SI]=15.01), and in naturally infected dogs (SI=8.86), but not by cells from the control groups. IL-12 addition further enhanced cell proliferation in naturally (SI=14.95), but not in experimentally infected animals. Peripheral blood mononuclear cells from experimentally infected dogs were able to produce significant amounts of IFN-gamma (3.39 ng/ml) upon LSA stimulation, but no such production was detected in cells from naturally infected or control animals. Interestingly, addition of IL-12 reversed the inhibitory effect of LSA on IFN-gamma production by cells from polysymptomatic naturally infected dogs and the uninfected beagles (4.84 and 7.45 ng/ml, respectively), and further increased IFN-gamma production by peripheral blood mononuclear cells from experimentally infected oligosymptomatic dogs (29.28 ng/ml). IFN-gamma mRNA expression correlated well with IFN-gamma production. Addition of IL-10 to Leishmania soluble antigen stimulated peripheral blood mononuclear cells inhibited proliferation and IFN-gamma production in experimentally infected dogs. Thus, the ability of IL-12 to augment IFN-gamma production by peripheral blood mononuclear cells from dogs with experimental or natural symptomatic canine visceral leishmaniasis makes it a good candidate for cytokine therapy in dogs that are refractory to current therapy.     

Serologic evidence of mixed infection involving the zoonoses leishmaniasis and leptospirosis in Greek dogs

Leishmaniasis, an important zoonosis, was serologically found to coexist with leptospirosis, another important zoonosis. The proportion of dogs positive to leishmaniasis was approximately 36%. Significant differences were observed between dogs located in greater Athens and those from rural Greece. Although dogs from either of the groups had a similar chance to be infected, rural dogs had significantly (p<0.01) higher titers (1/1,600) than dogs from greater Athens. Thirty two of the 344 dog serum samples examined had positive antibody titers against Leptospira spp., but only 13 of them had titers of 1/400 or over to both of the infectious agents. Whilst noted differences in the antibody titers of samples with evidence of leishmaniasis and leptospirosis were not observed their coexistence may complicate the clinical outcome of cases of mixed infection.     

Leishmania donovani: immunostimulatory cellular responses of membrane and soluble protein fractions of splenic amastigotes in cured patient and hamsters

Visceral leishmaniasis (VL), caused by the intracellular parasite Leishmania donovani, L. chagasi and L. infantum is characterized by defective cell-mediated immunity (CMI) and is usually fatal if not treated properly. An estimated 350 million people worldwide are at risk of acquiring infection with Leishmania parasites with approximately 500,000 cases of VL being reported each year. In the absence of an efficient and cost-effective antileishmanial drug, development of an appropriate long-lasting vaccine against VL is the need of the day. In VL, the development of a CMI, capable of mounting Th1-type of immune responses, play an important role as it correlate with recovery from and resistance to disease. Resolution of infection results in lifelong immunity against the disease which indicates towards the feasibility of a vaccine against the disease. Most of the vaccination studies in Leishmaniasis have been focused on promastigote--an infective stage of parasite with less exploration of pathogenic amastigote form, due to the cumbersome process of its purified isolation. In the present study, we have isolated and purified splenic amastigotes of L. donovani, following the traditional protocol with slight modification. These were fractionated into five membranous and soluble subfractions each i.e MAF1-5 and SAF1-5 and were subjected for evaluation of their ability to induce cellular responses. Out of five sub-fractions from each of membrane and soluble, only four viz. MAF2, MAF3, SAF2 and SAF3 were observed to stimulate remarkable lymphoproliferative, IFN-γ, IL-12 responses and Nitric Oxide production, in Leishmania-infected cured/exposed patients and hamsters. Results suggest the presence of Th-1 type immunostimulatory molecules in these sub-fractions which may further be exploited for developing a successful subunit vaccine from the less explored pathogenic stage against VL.     

IL-10 and TGF-beta control the establishment of persistent and transmissible infections produced by Leishmania tropica in C57BL/6 mice

Leishmania tropica is the causative agent of Old World anthroponotic cutaneous leishmaniasis, which is characterized by lesions that take an extended period of time to heal, often resulting in disfiguring scars, and are more refractory to treatment than leishmaniasis caused by Leishmania major. Immunologic studies involving experimental animal models of L. tropica infection are virtually nonexistent. In the current study, infectious-stage L. tropica were used to establish dermal infections in C57BL/6 and BALB/c mice. In both strains, the lesions were slow to develop and showed minimal pathology. They nonetheless contained a stable number of between 10(4) and 10(5) parasites for over 1 year, which were efficiently picked up by a natural sand fly vector, Phlebotomus sergenti. Control of parasite growth depended on the development of a Th1 response, as C57BL/6 mice genetically deficient in Th1 cytokines and BALB/c mice treated with Abs to IFN-gamma harbored significantly more parasites. By contrast, IL-10-deficient mice harbored significantly fewer parasites throughout the infection. To further study the immunologic mechanisms that may prevent efficient clearance of the parasites, IL-10 and TGF-beta signaling were abrogated during the chronic phase of infection in wild-type C57BL/6 mice. Distinct from chronic L. major infection, IL-10 blockade alone had no effect on L. tropica, but required simultaneous treatment with anti-TGF-beta Abs to promote efficient parasite clearance from the infection site. Thus, chronic infection with L. tropica appears to be established via multiple suppressive factors, which together maintain the host as a long-term reservoir of infection for vector sand flies.     

Nonhealing infection despite Th1 polarization produced by a strain of Leishmania major in C57BL/6 mice

Experimental Leishmania major infection in mice has been of immense interest because it was among the first models to demonstrate the importance of the Th1/Th2 balance to infection outcome in vivo. However, the Th2 polarization that promotes the development of nonhealing cutaneous lesions in BALB/c mice has failed to adequately explain the mechanisms underlying nonhealing forms of leishmaniasis in humans. We have studied a L. major strain from a patient with nonhealing lesions that also produces nonhealing lesions with ulcerations and high parasite burden in conventionally resistant C57BL/6 mice. Surprisingly, these mice develop a strong, polarized, and sustained Th1 response, as evidenced by high levels of IFN-gamma produced by Leishmania-specific cells in the draining lymph node and in the ear lesion, and an absence of IL-4 or IL-13. The parasites fail to be effectively cleared despite high level induction of inducible NO synthase in the lesion, and despite their sensitivity to killing by IFN-gamma-activated macrophages in vitro. Infection of IL-10(-/-) mice, blockade of the IL-10R, or depletion of CD25(+) cells during the chronic phase promotes parasite killing, indicating that IL-10 and regulatory T cells play a role in rendering the Th1 responses ineffective at controlling infection in the skin. Mice with nonhealing primary lesions are nonetheless resistant to reinfection in the other ear. We suggest that nonhealing infections in animal models that are explained not by aberrant Th2 development, but by overactivation of homeostatic pathways designed to control inflammation, provide better models to understand nonhealing or reactivation forms of leishmaniasis in humans.     

Canine leishmaniasis in the Rif mountains (Moroccan Mediterranean coast): a seroepidemiological survey

A sero-epidemiological survey has been conducted in several localities of the province of Nador to investigate canine leishmaniasis in the North-Eastern slope of the Rif mountains (Mediterranean coast of Morocco). Serum samples collected from 257 dogs were analysed using indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA) to detect anti-Leishmania infantum antibodies. Forty eight (18.7%) of the screened dogs were IFAT positive and 54 (21.0%) were ELISA positive; the concordance of the two methods was 96.1%. The prevalence of infection is significantly higher in dogs more than four years of age whereas no significant difference in prevalence of infection was seen between males and females. The frequent symptoms observed in seropositive dogs were the enlargement of lymph nodes (57.4%), emaciation (51.9%) and skin involvement (25.9%). However, 38.9% of those dogs showed no one of the major symptoms of visceral leishmaniasis. Leishmania isolated from three of the examined dogs was identified as L. infantum MON-1. These results show that the North-Eastern slope of the Rif mountains is one of the most active Mediterranean areas of visceral leishmaniasis and confirm that the dog is the main reservoir of L. infantum.     

Transplacental transmission of a North American isolate of Leishmania infantum in an experimentally infected beagle

Leishmania infantum, an etiologic agent of zoonotic visceral leishmaniasis, is widespread among foxhounds in the United States. Although sand flies are widely distributed throughout the United States, epidemiological data do not support a major role for sand flies in the transmission of L. infantum in foxhounds in this country. Congenital transmission of human visceral leishmaniasis is reported in humans and might also occur in dogs. We have previously isolated L. infantum from Virginia foxhounds and used this isolate (LIVT-1) to experimentally infect beagles. Four female beagles, chronically infected with LIVT-1, were bred to a male beagle chronically infected with L. infantum chagasi. One beagle was able to maintain her pregnancy, and 4 puppies were delivered by cesarean section. One puppy was malformed and autolytic at delivery, and tissues were not collected or analyzed. The remaining puppies were killed at the time of cesarean section, and selected tissues were collected for parasite culture and PCR. Promastigotes were not cultured from tissues in any of the puppies. Leishmania sp. DNA was detectable by PCR in liver, bone marrow, and heart from all 3 puppies and in the spleen, lymph node, kidney, and placenta in 2 puppies. Placental tissue from the dam was PCR negative. This is the first report of maternal transmission of a North American isolate of L. infantum from an experimentally infected dog.     

Leishmania (Viannia) braziliensis: epidemiology of canine cutaneous leishmaniasis in the State of Paraná (Brazil)

The present study examines the role that dogs play in the maintenance of the Leishmania cycle in the State of Paraná, Southern Brazil. Dogs were examined in three regions where cutaneous leishmaniasis is endemic or epidemic (R1-Vale da Ribeira; R2-Central region of Paraná State and R3-Northern region). To determine serum prevalence rates ELISA was used. In regions endemic for Trypanosoma cruzi (R1 and R3), serum from dogs seroreactive towards Leishmania antigen was subjected to T. cruzi adsorption in order to eliminate cross-reaction with common antigen epitopes. Concomitantly, dogs with cutaneous lesions were biopsied to isolate and identify parasites using RAPD. Leishmania were classified by the phenetic method using the Jaccard coefficient of similarity, and grouped by Unweighted Pair-Group Method using an Arithmetic Average (UPGMA). A total of 410 dogs were studied. In R1 (Vale da Ribeira) 159 dogs were evaluated of which 10 had anti-Leishmania antibody. In R2 (Central Paraná), 39 animals were examined of which 8 were seropositive. In R3 (the North) 212 dogs were evaluated of which 39 animals were seropositive. Thirteen dogs had cutaneous lesions and the parasites were isolated from a dog with mucocutaneous lesion in R1, two animals with simple skin lesions in R2 and 10 dogs with multiple lesions in R3. The identification of the parasite by molecular methods showed it to be L. (Viannia) braziliensis. Based on this information, the role of domestic dogs in Leishmania infection of cutaneous leishmaniasis in Paraná is discussed.     

Canine leishmaniosis--new concepts and insights on an expanding zoonosis: part two

Canine leishmaniosis is a widely spread zoonosis that is potentially fatal to humans and dogs. Infection with Leishmania infantum is considerably more prevalent than clinical disease, and infected dogs with no signs of disease might, potentially, transmit infection. Diagnosis of asymptomatic infection by serology is insufficient and PCR markedly increases its sensitivity. A new therapy exclusively for canine leishmaniosis is needed because current drugs do not reliably eliminate infection and might provoke resistance. Protection against sand-fly bites by topical insecticides is effective in reducing infection, and recent development of vaccines has indicated that prevention by vaccination is feasible. Integrated prevention with topical insecticides reducing the feeding of vectors and vaccination blocking early infection would be the basis of successful control programs.     

A single intradermal administration of soluble leishmanial antigen and plasmid expressing interleukin-12 protects BALB/c mice from Leishmania major infection.

In murine leishmaniasis, the induction of the T-helper type 1 (Th1) response contributes to infection resistance, whereas the establishment of the Th2 response makes the mice susceptible to infection. Interleukin-12 (IL-12) plays a pivotal role in the diversification of immune responses to the Th1 type. In this study, we tested whether the co-administration of IL-12 expression plasmid which compose p35 and p40 subunits and soluble leishmanial antigen (SLA) will skew the susceptible BALB/c mice to Th1 response and protect from leishmaniasis. When the mice were intradermally injected with the combination of IL-12 plasmid and SLA 7 days prior to the challenge with 1x10(6) promastigotes of Leishmania major, the local lesions completely healed and the parasite burden in the local lymph nodes significantly decreased. The cured mice attained long-term immunity, and were resistant to any subsequent rechallenge of the lethal dose of the parasite. The protective effect was associated with the development of a Th1 response, as demonstrated by the enhanced level of antigen-specific interferon-gamma (IFN-gamma) and dominant production of IgG2a in the serum. In contrast, the administration of empty plasmid plus SLA or IL-12 plasmid alone failed to protect the disease and shape the Th1 response. Furthermore, the protective efficiency induced by the vaccination was clearly prevented by the injection of either neutralizing anti-IL-12 mAb or anti-IFN-gamma mAb. The IL-12 expression plasmid is thus an effective adjuvant for the elicitation of a protective Th1 response against leishmaniasis and is therefore, considered to be appropriate for vaccinations that require the induction of Th1 type immunity.     

Detection of Leishmania infantum in naturally infected Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae) and Canis familiaris in Misiones, Argentina: the first report of a PCR-RFLP and sequencing-based confirmation assay

In this study, a genotypification of Leishmania was performed using polimerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing techniques to identify species of Leishmania parasites in phlebotomine sand flies and dogs naturally infected. Between January-February of 2009, CDC light traps were used to collect insect samples from 13 capture sites in the municipality of Posadas, which is located in the province of Misiones of Argentina. Sand flies identified as Lutzomyia longipalpis were grouped into 28 separate pools for molecular biological analysis. Canine samples were taken from lymph node aspirates of two symptomatic stray animals that had been positively diagnosed with canine visceral leishmaniasis. One vector pool of 10 sand flies (1 out of the 28 pools tested) and both of the canine samples tested positively for Leishmania infantum by PCR and RFLP analysis. PCR products were confirmed by sequencing and showed a maximum identity with L. infantum. Given that infection was detected in one out of the 28 pools and that at least one infected insect was infected, it was possible to infer an infection rate at least of 0.47% for Lu. longipalpis among the analyzed samples. These results contribute to incriminate Lu. longipalpis as the vector of L. infantum in the municipality of Posadas, where cases of the disease in humans and dogs have been reported since 2005.     

A new stable focus of canine leishmaniasis in northern Italy

A new stable focus of canine leishmaniasis (CanL) was identified in a coastal Adriatic area of the Emilia-Romagna Region in Northern Italy. Following the first clinical cases observed starting from 1998, a seroepidemiological survey was carried out on owned dogs from two communes and on animals housed in dog pounds of the Rimini province. Sixteen out of 612 dogs (2.6%) resulted positive to the IFA test. The 16 positive dogs all came from the two communes, with seroprevalences of 3 and 6%, respectively. The autochthonous origin of the infection was confirmed in all the cases. The parasitological investigation led to the isolation and identification of the parasite as Leishmania infantum Zymodeme MON 1. An entomological survey showed that Phlebotomus perniciosus and P. perfiliewi are present in this area and that P. perfiliewi was very abundant in one collection site. The risk of the establishment of a permanent transmission of the infection in the area, previously considered CanL-free, must be analysed in view of further investigations to be extended also to neighbouring areas.     

Leishmania infantum: mixed T-helper-1/T-helper-2 immune response in experimentally infected BALB/c mice

The main goal of the present study was to characterise the course of infection and immunological responses developed by Leishmania infantum infected BALB/c mice. Parasite load was determined by Real-time TaqMan PCR while cytokine and Immunoglobulin G (IgG) production were assessed by ELISA. Leishmania DNA was detected in spleen and liver as soon as day 1 post-inoculation (pi) and the parasitism was sustained until the end of the experiment. The cytokine kinetics in spleen and liver was generally associated with the oscillations of parasite load. Overall, it was not observed a distinct Th1 or Th2 pattern of cytokine production during the time of experiment. The infected mice developed a mixed immune response, with concomitant production of IFN-gamma, IL-4 and IL-10, both in spleen and liver, and both IgG isotypes. However, our results suggest that, compared to liver, the spleen is more susceptible to L. infantum infection.     

IL-17 Mediates Immunopathology in the Absence of IL-10 Following Leishmania major Infection

Leishmaniasis, resulting from infection with the protozoan parasite Leishmania, consists of a wide spectrum of clinical manifestations, from healing cutaneous lesions to fatal visceral infections. A particularly severe form of cutaneous leishmaniasis, termed mucosal leishmaniasis, exhibits decreased IL-10 levels and an exaggerated inflammatory response that perpetuates the disease. Using a mouse model of leishmaniasis, we investigated what cytokines contribute to increased pathology when IL-10-mediated regulation is absent. Leishmania major infected C57BL/6 mice lacking IL-10 regulation developed larger lesions than controls, but fewer parasites. Both IFN-γ and IL-17 levels were substantially elevated in mice lacking the capacity to respond to IL-10. IFN-γ promoted an increased infiltration of monocytes, while IL-17 contributed to an increase in neutrophils. Surprisingly, however, we found that IFN-γ did not contribute to increased pathology, but instead regulated the IL-17 response. Thus, blocking IFN-γ led to a significant increase in IL-17, neutrophils and disease. Similarly, the production of IL-17 by cells from leishmaniasis patients was also regulated by IL-10 and IFN-γ. Additional studies found that the IL-1 receptor was required for both the IL-17 response and increased pathology. Therefore, we propose that regulating IL-17, possibly by downregulating IL-1β, may be a useful approach for controlling immunopathology in leishmaniasis.