Abstract. We studied the reservoir competency of canines with distinct clinical presentations of Leishmania chagasi infection. The parasitologic status of asymptomatic and symptomatic dogs was determined by standard culture methods Infectivity was assessed by multiple xenodiagnoses with Lutzomyia longipalpis, over a period of 2-11 months. Asymptomatic dogs were non-infective (0 of 5) while 2 of 7 oligosymptomatic dogs infected L. longipalpis, transmitting the parasites at low rates (range 0.9-5.2% of engorged flies). Polysymptomatic dogs transmitted L. chagasi more frequently (4 of 8 dogs) and reached higher infection rates (range 5.0-22.5% of engorged flies). The skin of the ear tended to be more infective to sand flies than that of the abdomen. Polymerase chain reaction hybridization (PCR-H) was a sensitive method for detection of L. chagasi, yielding the highest positive rate in serum (16 of 17 dogs) with no distinction between clinical groups. No association between skin positivity by PCR-H and infectivity to sand flies was found. The infectivity of dogs from clinically comparable groups from Colombian and Mediterranean foci differed. This may be a reflection of varied nutritional conditions or vector competency of distinct sand fly species.

In regions where leishmaniasis is endemic, clinical disease is usually reported more frequently among males than females. This difference could be due to disparate risks of exposure of males and females, but genderrelated differences in the host response to infection may also play a role. Experimental studies of the influence of gender on Leishmania infection have not included parasites of the subgenus Viannia, which is the most common cause of cutaneous leishmaniasis in the Americas. Mice are not readily susceptible to infection by Leishmania (Viannia) spp., but cutaneous infection of hamsters with L. (V.) panamensis or L. (V.) guyanensis resulted in chronic lesions typical of the human disease caused by these parasites. Strikingly, infection of male hamsters resulted in significantly greater lesion size and severity, an increased rate of dissemination to distant cutaneous sites, and a greater parasite burden in the draining lymph node than infection in female animals. Two lines of evidence indicated this gender-related difference in disease evolution was determined at least in part by the sex hormone status of the animal. First, prepubertal male animals had smaller and/or less severe cutaneous lesions than adult male animals. Second, infection of testosterone-treated female animals resulted in significantly larger lesions than in untreated female animals. The increased severity of disease in male compared to female animals was associated with significantly greater intralesional expression of interleukin-4 (IL-4) (P ‫؍‬ 0.04), IL-10 (P ‫؍‬ 0.04), and transforming growth factor ␤ (TGF-␤) (P < 0.001), cytokines known to promote disease in experimental leishmaniasis. There was a direct correlation between the expression of TGF-␤ mRNA and lesion size (Spearman's correlation coefficient ‫؍‬ 0.873; P < 0.001). These findings demonstrate an inherent risk of increased disease severity in male animals, which is associated with a more permissive immune response.The outcome of Leishmania infection depends on several biological traits of both the host and the infecting parasite strain. A number of epidemiological studies indicate that leishmaniasis occurs more frequently among adult males than females (3,13,38). It is unclear whether this difference is due merely to dissimilar risks of exposure because of the distinct activities of males and females or whether gender-related differences in the host immune response play a role in resistance and susceptibility to infection.Epidemiological data for children Ͻ15 years of age, where both genders would seem to have similar risks of infection, indicate that boys are threefold more likely to develop visceral leishmaniasis than girls (35). Similarly, the disease rate for cutaneous leishmaniasis in an area of Brazil where the disease is endemic was shown to be 50% higher in males than females in all age groups, including children, who are expected to have comparable risks of exposure for both sexes (13). In studies of several different endemic foci in both the New and Old Worlds, re...

BackgroundNew drugs are needed to treat visceral leishmaniasis (VL) because the current therapies are toxic, expensive, and parasite resistance may weaken drug efficacy. We established a novel ex vivo splenic explant culture system from hamsters infected with luciferase-transfected Leishmania donovani to screen chemical compounds for anti-leishmanial activity.Methodology/Principal FindingsThis model has advantages over in vitro systems in that it: 1) includes the whole cellular population involved in the host-parasite interaction; 2) is initiated at a stage of infection when the immunosuppressive mechanisms that lead to progressive VL are evident; 3) involves the intracellular form of Leishmania; 4) supports parasite replication that can be easily quantified by detection of parasite-expressed luciferase; 5) is adaptable to a high-throughput screening format; and 6) can be used to identify compounds that have both direct and indirect anti-parasitic activity. The assay showed excellent discrimination between positive (amphotericin B) and negative (vehicle) controls with a Z' Factor >0.8. A duplicate screen of 4 chemical libraries containing 4,035 compounds identified 202 hits (5.0%) with a Z score of <–1.96 (p<0.05). Eighty-four (2.1%) of the hits were classified as lead compounds based on the in vitro therapeutic index (ratio of the compound concentration causing 50% cytotoxicity in the HepG2 cell line to the concentration that caused 50% reduction in the parasite load). Sixty-nine (82%) of the lead compounds were previously unknown to have anti-leishmanial activity. The most frequently identified lead compounds were classified as quinoline-containing compounds (14%), alkaloids (10%), aromatics (11%), terpenes (8%), phenothiazines (7%) and furans (5%).Conclusions/SignificanceThe ex vivo splenic explant model provides a powerful approach to identify new compounds active against L. donovani within the pathophysiologic environment of the infected spleen. Further in vivo evaluation and chemical optimization of these lead compounds may generate new candidates for preclinical studies of treatment for VL.

The P-8 proteoglycolipid complex , an amastigote antigen of L. pifanoi, has been demonstrated to induce protection in mouse models, as well as to induce Tc1/Th1-like cellular responses in American cutaneous leishmaniasis patients. Because the immunization with P-8 PGLC in the murine model does not appear to be genetically restricted, we have studied the reactivity of the P-8 PGLC in L. infantum infected dogs. In this study, it is shown that PBMC from experimentally infected dogs (asymptomatic, oligosymptomatic) significantly proliferated in response to soluble leishmanial antigen (SLA) or the P-8 PGLC. Further, quantification of the gene expression induced by the stimulation with P-8 in asymptomatically infected dogs showed an up-regulation of IFN-γ and TNF-α, which were three to four-fold higher than that induced by soluble Leishmania antigen (SLA). While no measurable induction of IL-10 was observed, low levels of IL-4 mRNA were observed in response to both P-8 and SLA antigens. Thus, our studies establish that P-8 is recognized by infected canines and elicits a potentially curative/protective Th1-like immune response. The identification of Leishmania antigens that elicit appropriate immune responses across different host species (humans, canine) and disease manifestations (cutaneous or visceral) could be an advantage in generating a general vaccine for leishmaniasis.

We determined that the site of inoculation (foot or snout) influences the clinical evolution and immune responses of hamsters infected with Leishmania (Viannia) panamensis. Hamsters infected in the snout showed (i) a more rapid and severe lesion evolution at multiple time points (P < 0.05), (ii) a more extensive inflammatory infiltrate and tissue necrosis, (iii) a higher tissue parasite burden, (iv) a higher antibody titre (P < 0.01), but lower antigen-specific spleen cell proliferative response (P = 0.02), and (v) a slower response to anti-leishmanial drug treatment (P < 0.002). In both inoculation groups there was co-expression of type 1 (IFN-gamma and IL-12) and some type 2 (IL-10 and TGF-beta, but not IL-4) cytokines in the cutaneous lesions and spleen. Early in the course of infection, hamsters infected in the snout showed higher expression of splenic IL-10 (P = 0.04) and intra-lesional IFN-gamma (P = 0.02) than foot infections. No expression of IL-12p40 or IL-4 was detected. During the chronic phase, snout lesions expressed more IFN-gamma (P = 0.001), IL-12p40 (P = 0.01), IL-10 (P = 0.009) and TGF-beta (P = 0.001), and the level of expression of each of these cytokines correlated with lesion size (P < or = 0.01). These results suggest that the site of infection influences the clinical outcome in experimental cutaneous leishmaniasis, and that the expression of macrophage-deactivating type 2 cytokines and/or an exaggerated type 1 proinflammatory cytokine response may contribute to lesion severity.

The infection rate with Leishmania chagasi and the population dynamics of small mammals were studied in an undisturbed forest reserve (Colosó) and an area of highly degraded forest (San Andrés de Sotavento [SAS]) in northern Colombia, both endemic for visceral leishmaniasis. Live trapping of mammals was done every month, and species, age, sex and reproductive status determined. L. chagasi was detected in samples of skin or spleen by the polymerase chain reaction, after extraction of deoxyribonucleic acid using specific primers (DB8/AJS3), and dot blood hybridization. Didelphis marsupialis was found to be infected in Colosó (3/21, 14.3%) and SAS (13/137, 9.5%); its relative abundance was higher in SAS (93/113, 82% of the captures). Although Proechimys canicollis was also found to be infected in Colosó (3/34, 8.8%) and SAS (2/4), its relative abundance was much lower (4%) in SAS than in Colosó (56% of 77 animals captured). Sciurus granatensis, Marmosa robinsoni, Heteromys anomalus, Zygodontomys brevicauda and Metachirus nudicaudatus were less common, and no L. chagasi infection was detected in them.

BackgroundThe Syrian hamster, Mesocricetus auratus, has distinct immunological features and is uniquely susceptible to intracellular pathogens. Studies in hamsters are limited by the relative unavailability of tools to conduct immunological studies. To address this limitation we developed duplex real-time reverse transcriptase (RT) PCR assays for the relative quantification of the mRNAs of hamster cytokines, chemokines, and related immune response molecules.ResultsReal-time RT-PCR primers and probes were synthesized for analysis of interleukin (IL)-4, IFN-γ, TNF-α, IL-10, IL-12p40, TGF-β, IL-13, IL-21, chemokine ligand (CCL) 22, CCL17, Chemokine (C-C motif) receptor 4 and FoxP3 expression. Standard curves and validation experiments were performed for each real-time RT-PCR assay, allowing us to use the comparative Ct (2-ΔΔCt) method to calculate changes in gene expression. Application of the real-time RT PCR assays to a biological model was demonstrated by comparing mRNA expression in skin and lymph node tissues between uninfected and Leishmania panamensis infected hamsters.ConclusionsThe duplex real-time RT PCR assays provide a powerful approach for the quantification of cytokine transcription in hamsters, and their application to a model of cutaneous leishmaniasis suggests that a balanced type 1 and type 2 cytokine response contributes to the chronic, nonprogressive course of disease. These new molecular tools will further facilitate investigation into the mechanisms of disease in the hamster, not only for models of leishmaniasis, but also for other viral, bacterial, fungal, and parasitic infections.

e Leishmaniasis is a vector-borne zoonotic infection affecting people in tropical and subtropical regions of the world. Current treatments for cutaneous leishmaniasis are difficult to administer, toxic, expensive, and limited in effectiveness and availability. Here we describe the development and application of a medium-throughput screening approach to identify new drug candidates for cutaneous leishmaniasis using an ex vivo lymph node explant culture (ELEC) derived from the draining lymph nodes of Leishmania major-infected mice. The ELEC supported intracellular amastigote proliferation and contained lymph node cell populations (and their secreted products) that enabled the testing of compounds within a system that mimicked the immunopathological environment of the infected host, which is known to profoundly influence parasite replication, killing, and drug efficacy. The activity of known antileishmanial drugs in the ELEC system was similar to the activity measured in peritoneal macrophages infected in vitro with L. major. Using the ELEC system, we screened a collection of 334 compounds, some of which we had demonstrated previously to be active against L. donovani, and identified 119 hits, 85% of which were confirmed to be active by determination of the 50% effective concentration (EC 50 ). We found 24 compounds (7%) that had an in vitro therapeutic index (IVTI; 50% cytotoxic/effective concentration [CC 50 ]/EC 50 ) > 100; 19 of the compounds had an EC 50 below 1 M. According to PubChem searchs, 17 of those compounds had not previously been reported to be active against Leishmania. We expect that this novel method will help to accelerate discovery of new drug candidates for treatment of cutaneous leishmaniasis.