Cell mediated immunity and specific IgG1 and IgG2 antibody response in natural and experimental canine leishmaniosis

In the present study, we have followed up Leishmania infantum infection in dogs: (1) naturally infected; (2) experimentally infected with amastigotes; and (3) experimentally infected with culture promastigotes. The main objective was to evaluate the differences of the humoral and cellular immune responses of each group. Sera from 12 beagle dogs were analysed for total anti-leishmanial antibodies and IgG1 and IgG2 subclasses by enzyme-linked immunosorbent assay (ELISA). Lymphoproliferation to L. infantum antigen was also performed. All naturally infected animals were symptomatic with a marked humoral response. Dogs inoculated with amastigotes were asymptomotic and presented lower antibody titres than naturally infected. Dogs inoculated with culture promastigotes were asymptomotic with no significant humoral response. Strong proliferative responses to Leishmania antigen was observed in dogs inoculated with promastigotes. In our experimental model, IgG1 antibody levels presented a similar pattern in all infected animals, and IgG2 reactivity was high in naturally infected dogs.     

Serological evaluation of experimentally infected dogs by LicTXNPx-ELISA and amastigote-flow cytometry

Canine visceral leishmaniasis (CVL) is characterized by a high incidence of asymptomatic infections. Because of the high prevalence of asymptomatic dogs in the endemic areas of visceral leishmaniasis (VL), a sensitive test is required for an accurate diagnosis. In this study, we evaluated the detection of symptomatic and asymptomatic Leishmania infantum infection in dogs using the secreted LicTXNPx antigen (Leishmania infantum cytosolic tryparedoxin peroxidase) in an ELISA format and compared it to soluble Leishmania antigens from promastigote or amastigote forms (SPLA and SALA) and two other unrelated secreted Leishmania proteins (LiTXN1 and TDR1). Moreover, we evaluated the diagnostic potential using the promastigote or amastigote-flow cytometric methodologies. The assays utilized sera collected from a cohort of L. infantum experimentally infected dogs, in which the intravenous or intradermal parasite injection mimics a symptomatic or asymptomatic pattern of infection, respectively. Our study indicated that anti-LicTXNPx antibodies were present in both symptomatic and asymptomatic experimental infections. Among the different Leishmania recombinant proteins tested, LicTXNPx showed a good predictive correlation with total soluble promastigote or amastigote Leishmania antigens, suggesting this antigen as a good candidate for a marker in either symptomatic or asymptomatic infection. The use of flow cytometry using both forms of live parasites was also tested with the same group of dogs. Amastigotes were shown to have more advantages than promastigotes for the serological diagnostic in both symptomatic and asymptomatic dogs, since higher continuous levels of anti-amastigote antibodies were detected during the course of experimental infection. Moreover, additional studies were done using sera from non-infected dogs and clinically asymptomatic and symptomatic dogs with confirmed naturally occurring L. infantum infections. The sensitivities of amastigote and promastigote flow cytometry were 96% vs. 89%, respectively, while the specificity for both was 93.2%. Therefore, our findings showed for the first time the potential of amastigote-flow cytometry regarding their applicability to detect both symptomatic and asymptomatic VL canine infections.     

Utility of diagnostic tests used in diagnosis of infection in dogs experimentally inoculated with a North American isolate of Leishmania infantum infantum

Eight female beagles were infected with 1 x 10(7) (low dose, LD) or 2 x 10(8) (high dose, HD) promastigotes of a North American isolate of Leishmania infantum infantum (LIVT-1 strain) isolated from naturally infected Virginia Foxhounds. Two female beagles served as negative controls and 2 male beagles chronically infected (> 3 years) with Leishmania infantum chagasi were positive controls. Bone marrow (BM) and lymph node (LN) aspirates were collected every 6-8 weeks for cytologic evaluation, parasite culture, and polymerase chain reaction (PCR). Serum samples were collected monthly for determination of serologic responses by indirect fluorescent antibody test (IFAT) and diagnostic rK39 antigen. Cultures of BM and LN aspirates and cytology evaluation were consistently positive in positive control dogs during the course of study. Negative control dogs were negative on BM and LN cultures and on cytologic evaluation of aspirates. Amastigotes were present on cytological examination of BM aspirates in 2 experimentally infected dogs. Cultures of LN aspirates were positive on 22 samples, whereas BM cultures were positive on 12 samples for all dogs. IFA titers ranged from 0 to 1 :400 in experimentally infected dogs during the course of the study. Recombinant K39 immunoassay tests were consistently positive in positive control dogs and in the HD dogs by approximately 8 weeks after infection. BM PCR products were identified more consistently in the HD dogs compared with the LD dogs. Kappa statistics indicated PCR correlated better with cultures and cytology than did IFAT or the rK39 immunoassay results in the experimentally infected dogs.     

Immune responses in vaccinated dogs with autoclaved Leishmania major promastigotes.

A comparative study was undertaken on the immunogen power of autoclaved Leishmania major promastigotes (ALM) vaccines given simultaneously with either BCG or saponin against canine leishmaniasis. The humoral immune response was assessed by ELISA and western blotting. The cellular immune response was evaluated by the lymphocyte transformation test. Dogs vaccinated simultaneously with ALM and saponin showed high antibody titres to crude L. infantum antigens after the first vaccine booster and reacted with several antigens, with molecular weights from 26 to 108 kDa by western blotting. However, the lymphocyte proliferation of these dogs to the crude L. infantum antigen was not significantly different from the control group. In contrast, in dogs vaccinated simultaneously with ALM and BCG, the antibody titres to crude antigen were low. Their sera reacted with the same proteins recognised by sera from dogs vaccinated simultaneously with ALM and saponin by western blotting. However, the 85-kDa protein was only identified by sera taken from dogs vaccinated simultaneously with ALM and BCG. These latter exhibited specific lymphocyte proliferation to the L. infantum antigen. This cell proliferation was observed for approximately 9 months after the first dose of the vaccine. This study indicates that a combination of ALM as the vaccine and BCG as the adjuvant, in the dog model, was successful in inducing a cell immune response, which is implicated in protection of dogs against a Leishmania infection.     

Lymphocytes of dogs immunised with purified excreted-secreted antigens of Leishmania infantum co-incubated with Leishmania infected macrophages produce IFN gamma resulting in nitric oxide-mediated amastigote apoptosis

The role of nitric oxide (NO) in the anti-leishmanial activity has been confirmed both in vitro and in vivo. Recently, we demonstrated that NO-mediated apoptosis-like amastigote death pathway is an important and highly regulated mechanism used for the clearance of Leishmania within infected murine macrophages stimulated to produce NO endogenously. To further characterize these important effector mechanisms in dog, a natural host-reservoir of L. infantum/L. chagasi, we have developed an ex vivo infection model of canine macrophages. Exposure of L. infantum-infected macrophages to autologous peripheral lymphocytes derived from dogs immunised with purified excreted-secreted antigens of L. infantum promastigotes (LiESAp) formulated with muramyl dipeptide (MDP) as adjuvant resulted in a significant leishmanicidal effect due to interferon (IFN)-gamma dependent macrophage activation. Concomitant accumulation of NO(3)(-)/NO(2)(-) in supernatants of co-cultured cells and in situ staining of parasites with terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) and YOPRO-1 showed that NO-mediated apoptosis of intracellular L. infantum amastigotes is occurring in canine macrophages as previously observed in mouse models. Monitoring these parameters in dogs after immunisation and before experimental challenge can represent a useful and easy way to rapidly evaluate vaccine candidates against canine visceral leishmaniasis.     

Flow cytometric analysis of cellular immune responses in dogs experimentally infected with a North American isolate of Leishmania infantum

Canine leishmaniasis caused by Leishmania infantum is endemic in the foxhound population in North America. Studies of canine leishmaniasis in the Mediterranean basin indicate a role for both CD4+ and CD8+ lymphocytes with clinical illness and in asymptomatic dogs. Limited information is available on the strain of L. infantum infecting foxhounds in North America. The present study investigated changes in cellular immune responses in dogs experimentally infected with 1x10(7) (low dose, LD; N=4) or 2x10(8) (high dose, HD; N=4) promastigotes of a United States isolate of L. infantum and control dogs (N=2) for 72 weeks. Density gradient separation was used to enrich for peripheral blood lymphocytes from canine blood. Lymphocyte subsets (CD4+ and CD8+) were quantified by flow cytometric analysis. Lymphocyte population expression levels over the course of the present study were compared to clinical status of the dog and antibody responses in infected and control dogs. No significant differences (P>0.05) were observed in either CD4+ or CD8+ lymphocyte expression in of the groups over the experimental period. This study suggests that the cellular immune responses to North American L. infantum in experimentally infected dogs may differ from other strains of L. infantum.     

Immune and clinical parameters associated with Leishmania infantum infection in the golden hamster model

For experimental infections with viscerotropic strains of Leishmania, a suitable animal model is not yet defined. In the present work, we have reappraised the use of golden hamster (Mesocricetus auratus) as an experimental model for infection with Leishmania infantum. Groups of hamsters were challenged by the intracardial route with doses ranging from 10(3) to 10(5) infectious promastigotes and the animals were monitored for 1-year follow-up period. The outcome of the infection was assessed by clinical symptoms of leishmaniasis, parasite loads in both liver and spleen, humoral response to Leishmania antigens and antibody levels in kidneys. The humoral response was analysed using either crude antigens (by ELISA and Western blotting) or several recombinant Leishmania antigens (Hsp70, Hsp83, LiP2a, LiP2b, H2A, H3 and KMP-11). From the analysis of all these parameters, we established the existence of three groups of animals: symptomatic or susceptible, oligosymptomatic, and resistant. Given the parallelism existing between the outcomes of Leishmania-infection in hamsters, dogs and humans, we believe that our data illustrate that the hamster is an excellent experimental model to study visceral leishmaniasis and for the design of vaccine development.     

Peripheral blood mononuclear cell supernatants from asymptomatic dogs immunized and experimentally challenged with Leishmania chagasi can stimulate canine macrophages to reduce infection in vitro

Leishmania chagasi is the causative agent of visceral leishmaniasis in both humans and dogs in the New World. The dog is the main domestic reservoir and its infection displays different clinical presentations, from asymptomatic to severe disease. Macrophages play an important role in the control of Leishmania infection. Although it is not an area of intense study, some data suggest a role for canine macrophages in parasite killing by a NO-dependent mechanism. It has been proposed that control of human disease could be possible with the development of an effective vaccine against canine visceral leishmaniasis. Development of a rapid in vitro test to predict animal responses to Leishmania infection or vaccination should be helpful. In this study, an in vitro model was established to test whether peripheral blood mononuclear cell (PBMC) supernatants from dogs immunized with promastigote lysates and infected with L. chagasi promastigotes could stimulate macrophages from healthy dogs in order to control parasite infection. PBMC from a majority of the immunized and experimentally infected dogs expressed IFN-gamma mRNA and secreted IFN-gamma when stimulated with soluble L. chagasi antigen (SLA) in vitro. Additionally, the supernatants from stimulated PBMC were able to reduce the percentage of infected donor macrophages. The results also indicate that parasite killing in this system is dependent on NO, since aminoguanidine (AMG) reversed this effect. This in vitro test appears to be useful for screening animal responses to parasite inoculation as well as studying the lymphocyte effector mechanisms involved in pathogen killing by canine macrophages.     

Analysis of the humoral immune response against total and recombinant antigens of Leishmania infantum: correlation with disease progression in canine experimental leishmaniasis

Leishmaniasis by Leishmania infantum in the Mediterranean Basin constitutes an important problem in both human and veterinary medicine. Based in both the importance of canids as reservoirs for the human disease and the fact that the canine disease may be an excellent model for the human condition, the present work has been conducted to analyze clinical and immune mechanisms associated with canine experimental leishmaniasis. Six-month-old mixed-breed dogs were intravenously infected with L. infantum promastigotes and the infection course was monitored along a 343 days-period. On day 75 post-infection (p.i.), amastigotes were observed in the lymph nodes of all dogs. The analysis of the humoral response against total L. infantum antigens by both ELISA and Western blotting evidenced a correlation between the levels of IgG isotypes (IgG1 and IgG2) and disease progression. It was observed that in those animals showing either a regressive or an oligosymptomatic form of the disease, the anti-Leishmania IgG1 antibodies were undetectable whereas those animals developing active disease showed high levels of anti-Leishmania IgG1 antibodies. Additionally, the time-course of antibody production against L. infantum recombinant antigens in the experimentally infected dogs has been analyzed. The present data suggest that reactivity against the heat-shock protein 70 (HSP70) may be used as diagnostic marker of early steps of infection, and that the appearance of anti-histone antibodies is associated with progression of infection to disease status.     

Canine visceral leishmaniasis: dog infectivity to sand flies from non-endemic areas

Canine visceral leishmaniasis (VL), caused by Leishmania infantum (Leishmania chagasi in the New World), is a zoonotic, endemic disease in Western Europe and Latin America. The potential spreading to new regions was suggested by the appearance of canine VL among foxhounds in the US. Although the sand fly vectors in the major foci of transmission have been described, no information exists on other sand flies that could propagate the infection outside endemic areas. We evaluated the capacity of Lutzomyia shannoni (Dyar) and Lutomyia youngi (Feliciangeli & Murillo), which are widely distributed in the New World, to acquire L chagasi (Cunha and Chagas) infections. A high proportion of L youngi were infected after feeding on an oligosymptomatic dog (51 per cent) or a polysymptomatic individual (95 per cent), but the intensity of infection was low (< 200 promastigotes/fly). L shannoni became infected only by feeding on the polysymptomatic dog, and the infection rate was lower (9 per cent) than in Lutzomyia longipalpis (36 per cent), and Lutzomyia evansi (Nunez-Tovar) (Lutz and Neiva) (38 per cent), but the intensity of infection (200 to > 500 promastigotes/fly) was comparable (L longipalpis) or higher (L evansi) than in the New World vectors. It is hypothesised that the presence of infected dogs in areas where L shannoni or L youngi occur could initiate new endemic cycles of VL in both South and North America.     

A Leishmania infantum multi-component antigenic protein mixed with live BCG confers protection to dogs experimentally infected with L. infantum

The capacity of a quimeric protein, formed by the genetic fusion of five antigenic determinants from four Leishmania proteins, formulated with BCG, to protect dogs against Leishmania infantum infection is described. The data showed that after i.v. administration of 500,000 parasites of the L. infantum M/CAN/ES/96/BCN150 strain, zymodeme MON-1, the animals became infected as suggested by the humoral response against the parasite antigens. All control unvaccinated dogs had parasites in the lymph nodes at day 150 post-infection. One of these unvaccinated infected dog was parasite negative at day 634 behaving, thus, as resistant. In contrast, only 50% of the immunized dogs had parasites in the lymph nodes at day 150 post-infection. Four of these dogs became parasite negative by day 634 post-infection. The control animals developed at various times during the follow-up period clinical symptoms associated with Leishmaniasis. The control diseased dogs developed also in the liver and spleen some of the abnormal histological features associated with natural visceral Leishmaniasis. The immunized dogs, however, were not only normal at the clinical but also at the anatomo-pathological level. A positive delayed type hypersensitivity (DTH) response was observed in nine of the immunized protected dogs. The data indicated that Q+BCG confers 90% protection against infection and at least 90% protection at the clinical level.     

Infections in immunocompetent and immune-deficient mice with promastigotes of a North American isolate of Leishmania infantum

Leishmania infantum, an etiologic agent of zoonotic visceral leishmaniasis, is widespread among foxhounds in the United States. Experimental infections with a North American isolate of L. infantum were evaluated using two inoculation routes in immunocompetent and immunosuppressed mouse strains. Groups of 2-5 interferon gamma gene knockout (IFN-gamma-KO) (BALB/c-Ifng), inducible nitric oxide synthase (NOS) gene knockout (iNOS-KO) (C57BL/6), B-cell-deficient (microMT) (C57BL/6), and BALB/c mice were intravenously (i.v.) or subcutaneously (s.c.) inoculated with various doses of promastigotes of the LIVT-1 strain of L. infantum. None of the mice developed clinical signs of leishmaniasis during the 8-9 weeks of the study. Promastigotes were cultured from spleens of all i.v.-infected mice by 3 days post culture. Spleens from s.c.-infected mice inoculated with greater than 1 x 10(6) parasites became culture positive 3-24 days post culture, but promastigotes were not cultured from mice infected with 1 x 10(5) or 5 x 10(5) LIVT-1 promastigotes. Histological lesions were prominent in the livers of i.v.-infected mice but were mild to nonexistent in s.c. infection. Serological responses were low and transient determined by indirect fluorescent antibody testing in all groups. These results indicate that the i.v. route of infection is superior to the s.c. route in a mouse model of North American leishmaniasis and that mice lacking INF-gamma, iNOS or mice that are B-cell-deficient are not more susceptible to acute infection.     

Nitric oxide production by macrophages of dogs vaccinated with killed Leishmania infantum promastigotes.

Human visceral leishmaniosis is endemic in Southern Italy, where the dog is the main reservoir of viscerotropic strains of Leishmania infantum. The release of nitric oxide (NO) by interferon (IFN)-gamma-activated macrophages is an important leishmanicidal mechanism in several animal species. In this work NO production, phagocytosis and killing capacity of monocyte-derived dog macrophages were evaluated in vitro before and after administration of a vaccine composed of killed Leishmania infantum promastigotes. Moreover, IFN-gamma content was measured in concanavalin A-activated dog peripheral blood mononuclear cell (PBMC) supernatants employed for macrophage stimulation. Phagocytosis, killing capacity and NO production by canine macrophages increased significantly 1 month after vaccine administration, and the increase also persisted 5 months later. In addition, the amount of IFN-gamma in PBMC supernatants was significantly higher after vaccination. Overall, our results suggest the usefulness of evaluating the in vivo protective role of this promastigote preparation in dogs.     

Validation of a Leishmania infantum ELISA rapid test for serological diagnosis of Leishmania chagasi in dogs

Canine visceral leishmaniasis (CVL) is caused by Leishmania donovani complex parasites including L. donovani, Leishmania infantum and Leishmania chagasi. As some studies suggest that L. chagasi and L. infantum may be very similar or even the same species, the aim of the present study was to evaluate a commercial rapid ELISA test, originally designed for L. infantum, in the diagnosis of CVL in dogs naturally infected by L. chagasi. A total of 400 serum canine samples, including 283 positive dogs for CVL from an endemic area, 86 clinically healthy dogs from a non-endemic area and 31 dogs seropositive for confounding infectious agents (Trypanosoma cruzi, Toxoplasma gondii, Neospora caninum, Babesia canis and Ehrlichia canis) were used for test validation. An overall sensitivity of 94.7% (95% CI=91.41-97.01%) and specificity of 90.6% (95% CI=83.80-95.21%) was found, with a high degree of agreement (k=0.8445) to the indirect ELISA. When confounding infectious diseases were excluded, specificity increased to 100% (95% CI=95.8-100%), with a higher degree of agreement (k=0.8928). In conclusion, the commercial kit designed for L. infantum was a highly sensitive and specific device for detection of L. chagasi infection in dogs, which indicates high immunoreactivity similarities between L. infantum and L. chagasi.     

Canine visceral leishmaniasis: relationships between clinical status, humoral immune response, haematology and Lutzomyia (Lutzomyia) longipalpis infectivity

The main source of Leishmania infantum infection in humans is a naturally infected dog. This study reports on the infectivity to phlebotomine sandflies (Lutzomyia longipalpis) of serologically positive mongrel dogs that differed in clinical status, haematology and humoral responses to immunoglobulin (Ig) G(T) (total anti-Leishmania IgG), IgG(1) and IgG(2) subclasses of antibody to crude antigen of L. infantum. Forty-five female L. longipalpis were allowed to feed directly on the ears of dogs classified as asymptomatic, oligosymptomatic or symptomatic before being dissected five days later. Promastigotes were detected in 88% of the dissected sandflies. The highest rate of infectivity to sandflies was found in symptomatic dogs, followed by oligosymptomatic and asymptomatic animals. The results suggest that dogs naturally infected with L. infantum with higher total IgG and IgG(2) concentrations and lower haematocrit levels were able to infect the highest proportion of L. longipalpis. No correlation was observed between anaemia and the intensity of clinical signs. Symptomatic dogs presented the highest infection rate and intensity of infection.     

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.     

The protective immune response produced in dogs after primary vaccination with the LiESP/QA-21 vaccine (CaniLeish?) remains effective against an experimental challenge one year later

Control of canine leishmaniasis is an important objective for the benefit of dogs living in or visiting endemic areas and for public health because of the zoonotic nature of this disease. Resistance or susceptibility to developing canine leishmaniasis after exposure to Leishmania infantum is primarily determined by the ability of the immune system to develop an appropriate Th1-dominated specific response to the parasite. For this reason there is a need for effective canine vaccines that can decrease the number of dogs developing progressive infections. In this study, we followed the impact of the LiESP/QA-21 canine vaccine (composed of excreted-secreted proteins of L. infantum and the QA-21 saponin adjuvant), recently launched commercially in Europe, on selected humoral and cellular immune parameters following an infectious intravenous challenge with L. infantum promastigotes administered one year after the primary vaccine course. We also followed parasitological parameters to determine the parasitological status of the challenged dogs. In contrast to controls, vaccinated dogs retained significantly stronger cell-mediated immune responses against the parasite despite a virulent challenge and had significantly lower mean parasite burdens at the end of the study, associated with a lower probability of developing active infections. These results confirm that the immune responses generated by vaccination with LiESP/QA-21 are still effective against an intravenous challenge one year after the primary vaccine course.     

Canine infection and the possible role of dogs in the transmission of American tegumentary leishmaniosis in Salta,Argentina

Some Leishmania species affect humans in two principal forms: visceral and cutaneous leishmaniosis (CL). Several studies have identified dogs as the main reservoirs of the visceral leishmaniosis (VL) caused by Leishmania infantum. The purpose of this work was to carry out a survey of the canine population associated with human cases of American tegumentary leishmaniosis (ATL), in order to establish the clinical, parasitological, serological and immunological characteristics of the canine disease, in an endemic region for both ATL and Chagas' disease in the province of Salta, in northwestern Argentina. Two hundred and eight dogs from the endemic area were examined and 41 (19.7%) of them presented lesions compatible with leishmaniosis. In order to investigate the presence of antibodies against Leishmania spp. and Trypanosoma cruzi, sera were screened by ELISA using two complex antigens from these parasites and, because of cross-reactions between them, a specific antigen for diagnosis of T. cruzi infection. Sixty-two (29.8%) of 208 dogs were positive for the complex antigen F45 from Leishmania and 50 (24%) were positive for the complex antigen F105 from T. cruzi. Nine dogs (4.3%) were positive for the specific Ag163B6-cruzipain suggesting that these dogs were truly infected with T. cruzi. Furthermore, three of these nine dogs presented Leishmania sp. in their skin lesions and therefore were considered as infected by both, T. cruzi and Leishmania parasites. The prevalence of Leishmania infection detected by lesions and/or positive serology was 27.4% (57/208). On the basis of previous observations regarding the clustered appearance of human ATL, the dog population was divided into two groups: zone A, dogs living within a 100 m radius from houses with human cases, and zone B, dogs living beyond this limit. The prevalence of ATL in dogs was significantly higher in zone A (34.6%) than in zone B (7.3%), suggesting a strong correlation between canine and human cases. The average time required for a parasitological diagnosis by microscopy was six times longer for dog samples than human ones, and the average number of parasites per 100 microscopic fields was 14-fold lower in canine samples. The high prevalence of Leishmania infection and the close association with human cases, demonstrated that dogs are a very susceptible host for Leishmania infection, but the scarcity of parasites in their lesions suggests that they may not be the main reservoir of the parasite in this endemic area.     

Infection of sandflies by a cat naturally infected with Leishmania infantum

Despite the recent reports of feline leishmaniosis from Southern Europe, cats are still regarded as unusual Leishmania hosts. A cat found chronically infected with Leishmania was submitted to xenodiagnosis. After being sedated, the animal was exposed to the bite of 100 laboratory-reared Phlebotomus perniciosus in a fine net cage for 90 min. Four out of 19 blood-fed sandflies (21%) showed motile promastigotes at the dissection. Parasites cultured from cat's lymph node and an infected fly were identical at PCR-RFLP genotyping and identified as Leishmania infantum MON-1, the main zymodeme responsible for human and canine leishmaniosis in Southern Europe. This is the first evidence of transmissibility of feline parasites to a proven vector, suggesting that cats may represent an additional domestic reservoir for L. infantum.     

Canine visceral leishmaniasis: comparison of in vitro leishmanicidal activity of marbofloxacin, meglumine antimoniate and sodium stibogluconate

The control of canine leishmaniasis largely depends on the success of treatment. Drugs currently available to treat this disease are toxic and partially effective. The curative effect of marbofloxacin, a third-generation fluoroquinolone developed for veterinarian individual treatment, was evaluated in vitro in the presence of Leishmania infantum promastigotes and dog-monocyte-derived macrophages; meglumine antimoniate and sodium stibogluconate were used as comparative treatments. We observed that the killing of Leishmania promastigotes and intracellular amastigotes by marbofloxacin was dose-dependent. We demonstrated that successful treatment of canine infected macrophages for 48 h was possible with 500 microg/ml of marbofloxacin. Leishmanicidal activity acted through a TNF-alpha and nitric oxide pathway and correlated with the generation of nitric oxide (NO(2)) production by monocytes derived macrophages from infected (23+/-5 microM) or healthy (21+/-6 microM) dogs, in comparison with NO(2) concentration in infected/non-treated macrophages (< 3 microM, P<0.01). This significant induced parasiticidal effect correlated with extensive elimination of amastigotes by macrophages derived from infected (11+/-5) and healthy dogs (6+/-2), when compared to infected/non-treated macrophages (530+/-105 and 472+/-86 amastigotes, respectively, P< 0.01). Marbofloxacin was shown to be non-toxic at 500 microg/ml in vitro and no cell apoptosis was observed. The molecule was able to induce a parasitic process after significant elimination of amastigotes in leishmania-infected dog macrophages. We propose that marbofloxacin, compared to standard chemotherapeutic agents (meglumine antimoniate and sodium stibogluconate), could be an effective and pragmatic oral route alternative to treat canine leishmaniasis.