Ecological site distribution of sand fly species of Mokolo, an endemic focus of cutaneous leishmaniasis in northern Cameroon.

Leishmaniasis is a vector borne disease present in two major clinical forms (cutaneous and visceral) in the northern part of Cameroon. The disease is classified as a neglected tropical disease by the World Health Organization and thus, requires more attention. The aim of this study was to correlate the previously established composition and abundance of sand fly fauna with the putative vector status and the ecological behavior in the Mokolo cutaneous leishmaniasis focus to propose fighting strategies integrating vectors control. Over a 12-month period light traps were used for sand flies' collection in urban, peri-urban and sylvatic environment found in Mokolo, an endemic focus of leishmanisis in northern Cameroon, microscope and taxonomic keys were used for their identification. Nineteen (19) species were identified belonging to the genera Sergentomyia, and Phlebotomus. The influence of human population density on sand fly's species density and composition was assessed trough the evaluation of ecological distribution of sand flies in Mokolo. It came out that, Se. coronula and Se. thomsoni mandarai are strictly wild species and Ph. duboscqi, a domestic species. The other species are generalists.The number of Se. antennata and Se. adami decreases with the increase of the density of human population while Se. distincta, Se. vorax and Ph. duboscqi increase with the density of human population in the study site. Based on its previous reports in the Leishmania transmission in West Africa, Ph. duboscqi should still be considered as the main suspected vector in Mokolo. Ph. duboscqi, Se. distincta, Se. affinis ssp. vorax and Se. schwetzi are highly represented around human dwellings.

Montmorency Cherry Juice Consumption does not Improve Muscle Soreness or Inhibit Pro-inflammatory Monocyte Responses Following an Acute Bout of Whole-body Resistance Training.

Montmorency Cherry Juice (MCJ) may improve acute exercise recovery by attenuating inflammation and oxidative stress. However, the anti-inflammatory effects of MCJ on monocyte responses following resistance exercise have not been explored. Seven resistance-trained males (age: 22.9 ± 4.1 yrs; height: 1.8 ± 0.1 m; weight: 81.7 ± 13.2 kg) participated in this study. Participants completed a placebo-controlled crossover design, drinking either MCJ or placebo beverages, 7 days prior to completing an acute bout of unilateral resistance exercise. Statistical significance was assessed using a withinsubjects repeated measures ANOVA; alpha level p ≤ 0.05. Main effects for time were observed for changes in classical and intermediate monocytes (p ≤ 0.05), but no significant treatment effects were observed for monocyte subtypes p > 0.05. Classical monocytes (CD14+ CD16-) increased and peaked 24 hr post-exercise (placebo 1.14 ± 0.04 and MCJ 1.06 ± 0.06-fold). Intermediate monocytes peaked 48 hr post-exercise increasing 1.82 ± 0.41 and 2.01 ± 0.80- fold. Nonclassical monocytes peaked post-exercise (placebo 1.17 ± 0.31 and MCJ 1.02 ± 0.20-fold). Peak pain visual analog scale (VAS) occurred post-exercise for MCJ (3.63 ± 2.01-fold) and 72 hr post-exercise for placebo (4.26 ± 3.46- fold). IL-6 and pressure pain threshold (PPT) peaked 24 hr post-exercise (IL-6 placebo 3.83 ± 1.01- and MCJ 6.43 ± 3.43-fold) and (PPT placebo 86.37 ± 3.95% and MCJ 82.81 ± 2.90% of pressure needed at pre-exercise). Our data suggests MCJ consumption does not decrease muscle soreness, IL-6, or monocyte subset responses following a high-intensity resistance exercise protocol in resistance-trained males.

Comparison of percutaneous vs oral infection of hamsters with the hookworm Ancylostoma ceylanicum: Parasite development, pathology and primary immune response.

BACKGROUND: Hundreds of millions of people in poor countries continue to suffer from disease caused by bloodfeeding hookworms. While mice and rats are not reliably permissive hosts for any human hookworm species, adult Golden Syrian hamsters are fully permissive for the human and animal pathogen Ancylostoma ceylanicum. Similar to humans, hamsters may be infected with A. ceylanicum third-stage larvae orally or percutaneously. Oral infection typically leads to consistent worm yields in hamsters but may not accurately reflect the clinical and immunological manifestations of human infection resulting from skin penetration. METHODOLOGY/PRINCIPAL FINDINGS: In this study we compared host responses following percutaneous infection to those utilizing an established oral infection protocol. Infected hamsters exhibited a dose-dependent pathology, with 1000 percutaneous larvae (L3) causing anemia and adult worm recovery comparable to that of 50 orally administered L3. A delayed arrival and maturity of worms in the intestine was observed, as was variation in measured cellular immune responses. A long-term study found that the decline in blood hemoglobin was more gradual and did not reach levels as low, with the nadir of disease coming later in percutaneously infected hamsters. Both groups exhibited moderate growth delay, an effect that was more persistent in the percutaneously infected group. Fecal egg output also peaked later and at lower levels in the percutaneously infected animals. In contrast to orally infected hamsters, antibody titers to larval antigens continued to increase throughout the course of the experiment in the percutaneous group. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that the route of infection with A. ceylanicum impacts disease pathogenesis, as well as humoral and cellular immune responses in an experimental setting. These data further validate the utility of the Golden Syrian hamster as a model of both oral and percutaneous infection with human hookworms.

Leishmaniasis in Cameroon and neighboring countries: An overview of current status and control challenges.

Leishmaniasis causes the ninth largest disease burden among infectious diseases but remains a very neglected tropical disease. Although the disease is endemic in Cameroon and some neighboring countries, data on its epidemiology are very scanty. The present review summarizes the available information on leishmaniasis in the central region of Africa. According to available records, Cameroon, Chad and Nigeria have been identified as endemic foci of both cutaneous (CL) and visceral leishmaniasis (VL). In addition, the phlebotomine vectors of leishmaniasis have been reported in these three countries and also in Congo and the Central African Republic. Although Gabon, Central African Republic, Equatorial Guinea and Congo are all situated next to the above leishmaniasis-endemic countries and are characterized by similar landscapes and vegetation, they lack published reports of autochthonous cases of leishmaniasis. Considering that many cases of the disease might remain unreported, it might not be an overstatement to recommend that research should be carried out in Gabon, Equatorial Guinea, Central African Republic and Congo to identify cases of leishmaniasis (CL and/or VL), the parasite and vector species, and the mammalian reservoir host. This review updates data on leishmaniasis and its insect vector in the geographical region of Central Africa. Such updates are basic requirement for the development of successful control programmes in individual countries and the whole region. In order to address the shortcomings identified in the present review, the authors recommend training of more scientists in leishmaniasis epidemiology in the region that should be accompanied by necessary funding. This training must be multidisciplinary and include development of laboratory and field skills for studies of the parasite, the vector, the reservoir, the vegetation and the soil in potential endemic foci. In addition, prospective studies involving geographers and other experts should develop a disease risk map of the Central Africa region.

Investigations on the effects of anti-Leishmania major serum on the progression of Leishmania infantum infection in vivo and in vitro - implications of heterologous exposure to Leishmania spp.

Leishmaniasis is a vector-borne parasitic disease caused by protozoa of the genus Leishmania. Twenty different species are known to cause disease in humans with varying degrees of pathology. These diseases are transmitted throughout the geographic range of phlebotomine sandflies, found between the latitudes 50°N and 40°S. This study explores antibody dependent enhancement (ADE) as the cause of disease exacerbation in heterologous exposure of L. major primed mice to L. infantum challenge. BALB/c mice received serum from L. major infected or naive mice. All mice were challenged with L. infantum and tissue parasite burdens were recorded. Animals that received anti-L. major serum exhibited significantly higher parasite burdens. Surprisingly, these parasite burdens were higher than those of mice infected with L. major and challenged with L. infantum. In vitro phagocytosis assays were carried out to measure parasite uptake in the presence of naive vs. anti-L. major serum. J774A.1 murine monocytes were cultured with either L. major or L. infantum in the presence of anti-L. major serum, naive serum, or no serum. Significantly higher rates of L. major uptake by J774A.1 cells occurred in the presence of anti-L. major serum, but no measurable increase of L. infantum phagocytosis was seen. Our results suggest that increased disease severity observed in vivo in mice previously exposed to L. major and challenged with L infantum is not a result of extrinsic ADE. We speculate that intrinsic ADE, due to biased memory T cell responses caused by Fcγ signaling, could account for disease exacerbation seen in the animal model.

Investigation of Dalea parryi (Fabaceae) metabolites for anthelmintic activity against the human pathogenic hookworm Ancylostoma ceylanicum.

The US Southwest plant Dalea parryi (Fabaceae) was investigated as part of an ongoing study of the potential of plant compounds for anthelmintic activity to the human pathogenic hookworm Ancylostoma ceylanicum. This has resulted in the isolation of three previously undescribed isoflavonoid metabolites, denoted parryans A-C, a chalcone, six pterocarpans, and three known compounds from the roots of D. parryi. Parryans A and B express a rarely-seen O-prenyl substituent. Structures of the previously undescribed compounds were established using 1D and 2D NMR spectroscopy and mass spectrometry. The relative and absolute configurations of the undescribed stereoisomers were assigned using chemical shift and coupling constant data and comparisons of specific rotations to published data. The most active of the isolated compounds only expressed a 17% reduction in survival of A. ceylanicum adult hookworm in an ex vivo assay at 50 µg/mL after 5 days exposure. Toxicity, ranging from 47 to 93% reduction in survival of mammalian splenocytes was expressed by four of the compounds.

Inventory and taxonomy of phlebotomine sand flies of the Mokolo leishmaniasis focus, northern Cameroon, with description of new Sergentomyia taxa (Diptera: Psychodidae).

Leishmaniasis is endemic in northern Cameroon. However, the sand fly vectors have not been incriminated. A sand fly species inventory was generated by integrating a number of techniques. Miniature light traps were used for collecting sand flies in a variety of ecotopes found across the area, and a morphological and molecular identification approach for taxonomic confirmation was undertaken. In a pilot survey conducted in September 2012, we captured 687 sand flies, 259 of which were morphologically identified to species level. They represent 14 species of the genera Sergentomyia and Grassomyia. No Phlebotomus spp. were found. A second series of collections was carried out during 2013 in five different environmental setups: two urban, two peri-urban/rural and one sylvatic; 14,036 sand flies (6665 males and 7371 females) were collected. A total of 5926 females and 98 males were morphologically identified to species level, representing 19 species of the genera Sergentomyia, Grassomyia and Phlebotomus, including Ph. duboscqi, a known vector of cutaneous leishmaniasis in the region. Two new taxa were found and are described: Sergentomyia (Sintonius) thomsoni mandarai ssp. nov. and Se. coronula sp. nov. Our study is the first to report the following species in Cameroon: Se. (Sin.) thomsoni (as ssp. nov. mandarai), Se. (Ser.) cincta, Se. (Sin.) affinis ssp. vorax, Se. (Sin.) adami, Se. (Sin.) herollandi, and Se. (Sin.) christophersi. In addition, some morphologically atypical Sergentomyia specimens (combination of Ser. x Sin. traits) were recorded. A checklist of 32 species reports from Cameroon is presented.

First detection of Leishmania donovani in sand flies from Cameroon and its epidemiological implications.

OBJECTIVES: A sound knowledge of the vector-host-parasite transmission dynamics is a prerequisite for adequate control measures of vector-borne diseases. To achieve this, an entomological investigation was conducted in the cutaneous leishmaniasis (CL) endemic focus of Mokolo District, northern Cameroon to identify the insect vector(s) of the disease. METHODS: Phlebotomine sand flies were collected in and around Mokolo using New Standard CDC Miniature Light Traps. Individual sand flies were used for morphological species identification, and the remainder of the body for DNA analysis. Sand flies were demonstrated to harbour Leishmania spp. parasites using ITS1 PCR. Mitochondrial vertebrate-specific Cytochrome b -PCR was used to identify blood meals ingested by female sand flies. PCR amplicons were sequenced for Leishmania and blood sources discrimination. RESULTS: This study revealed the presence of Leishmania donovani complex DNA (n = 1) in Phlebotomus duboscqi and of lizard-borne Leishmania tarentolae-like DNA (n = 3) in Sergentomyia spp. in 79 sand fly specimens from Mokolo district. CONCLUSIONS: The causative agent of CL could not be detected in potential vectors. Instead, we found evidence for visceral leishmaniasis (VL) parasites in Phlebotomus duboscqi as well as enzootic reptile parasites in the Mokolo area. We recommend that an epidemiological survey be carried out in the area to evaluate the prevalence and eventually describe the clinical manifestations of VL in the human population. Political instability in neighbouring countries and the resulting refugee migration are likely explanations for the emergence of VL in Mokolo.

Phenolic Metabolites of Dalea ornata Affect Both Survival and Motility of the Human Pathogenic Hookworm Ancylostoma ceylanicum.

Hookworms are ubiquitous human parasites, infecting nearly one billion people worldwide, and are the leading cause of anemia and malnutrition in resource-limited countries. Current drug treatments rely on the benzimidazole derivatives albendazole and mebendazole, but there is emerging resistance to these drugs. As part of a larger screening effort, using a hamster-based ex vivo assay, anthelmintic activity toward Ancylostoma ceylanicum was observed in the crude extract of aerial parts of Dalea ornata. These studies have led to the isolation and characterization of phenolic metabolites 1-10. The structures were determined by 1D and 2D NMR spectroscopy, and the absolute configuration of 1 was assigned using electronic circular dichroism data. The new compound, (2S)-8-(3-methylbut-2-en-1-yl)-6,7,4'-trihydroxyflavanone (1), was weakly active at 7.3 µM, with 17% reduction in survival of the hookworms after 5 days. The rotenoids deguelin (9) and tephrosin (10), predictably perhaps, were the most active, with complete worm mortality observed by day 4 (or earlier) at 6.3 and 6.0 µM, respectively. The effects of 1-10 on hookworm motility and on toxicity to hamster splenocytes were also explored as important measures of treatment potential.

Impaired T-cell-dependent protection against Leishmania major infection in HIV-positive patients is associated with worsened disease outcome.

Cutaneous leishmaniasis (CL) patients coinfected with HIV are known to show a more severe, prolonged course of disease; the immunological basis is not known. We now assessed clinical features, sera and skin biopsies of HIV(+) and HIV(-) patients with CL to identify drivers of increased susceptibility to Leishmania. CL lesion numbers, surface, and healing duration were significantly increased in HIV(+) as compared to HIV(-) patients (2.5, 14 and >4-fold, respectively). Patients with HIV infection exhibited lower serum Leishmania-specific IgG levels and decreased IL-6 and IL-8. Most importantly, dramatically decreased numbers of CD4(+) T cells (approximately eightfold), but not CD8(+) cells, together with fewer CXCR3(+) Th1 cells, fewer Foxp3(+) effector/regulatory T cells, and reduced levels of IFN-γ expression were found in lesional skin. Our findings suggest that compromised CD4(+) T-cell responses may be responsible for worsened disease outcome leading to defects in parasite elimination in the absence of sufficient numbers of IFN-γ-producing Th1 cells.

Previous exposure to a low infectious dose of Leishmania major exacerbates infection with Leishmania infantum in the susceptible BALB/c mouse.

The geographic distribution of Leishmania major overlaps with several other species of Leishmania. This study seeks to examine what effect previous exposure to L. major has on the outcome of infection with Leishmania infantum, the agent of virulent visceral leishmaniasis. The L. major immune response is well characterized by a strong Th1 response leading to resolution and protection against subsequent re-infection. A contrasting Th2 immune response leads to disseminated disease, while the role Th17 cytokines may play in Leishmania infection is still being explored. The cytokine profile, antibody titer, and parasite burden were evaluated in the susceptible BALB/c mouse after L. infantum infection in either naïve mice or those previously infected with a low/self-healing dose of L. major. Only IL-4 expression in mice previously exposed to L. major was found to be significantly increased over controls, a cytokine with an ambiguous role in L. infantum infection. However, disease exacerbation, with a notably higher parasite burden, was observed in the L. major exposed mice compared to the L. infantum only. Cross-reactive antibodies were seen in both groups of infected mice regardless of their immune history. Studies have shown a role for opsonizing antibodies leading to increased disease in visceral leishmaniasis. We speculate that cross-reactive antibodies may be playing a role in augmenting visceral disease in mice with immunological memory to L. major.

Clinical features and epidemiology of cutaneous leishmaniasis and Leishmania major/HIV co-infection in Cameroon: results of a large cross-sectional study.

Cutaneous leishmaniasis (CL) is endemic in Central Africa, including Cameroon. However, data on its prevalence and co-infection with HIV are scarce. Here we present the results of a large cross-sectional study reporting the prevalence, clinical features and species identification of CL and HIV co-infection in northern Cameroon. A total of 32 466 subjects were clinically screened for CL during a door-to-door survey, followed by parasitological diagnosis in the field laboratory. Amongst the subjects surveyed, 146 (0.4%) were diagnosed with active CL. Seven (4.8%) of these 146 CL patients tested positive for HIV-1 and/or HIV-2. The number of lesions per CL patient ranged from 1 to 20. Three of the five subjects with >10 active lesions were co-infected with HIV. In both CL and HIV co-infected subjects, three successful parasite isolates were identified as Leishmania major by PCR. This first report of L. major/HIV co-infection in Cameroon and Central Africa confirms the endemicity of CL in the region and highlights a worsened CL pathology in HIV co-infected individuals. These findings provide important data necessary for the development and implementation of successful control programmes against CL and HIV in this geographical area.

Murine visceral leishmaniasis: IgM and polyclonal B-cell activation lead to disease exacerbation.

In visceral leishmaniasis, the draining LN (DLN) is the initial site for colonization and establishment of infection after intradermal transmission by the sand fly vector; however, little is known about the developing immune response within this site. Using an intradermal infection model, which allows for parasite visceralization, we have examined the ongoing immune responses in the DLN of BALB/c mice infected with Leishmania infantum. Although not unexpected, at early times post-infection there is a marked B-cell expansion in the DLN, which persists throughout infection. However, the characteristics of this response were of interest; as early as day 7 post-infection, polyclonal antibodies (TNP, OVA, chromatin) were observed and the levels appeared comparable to the specific anti-leishmania response. Although B-cell-deficient JhD BALB/c mice are relatively resistant to infection, neither B-cell-derived IL-10 nor B-cell antigen presentation appear to be primarily responsible for the elevated parasitemia. However, passive transfer and reconstitution of JhD BALB/c with secretory immunoglobulins, (IgM or IgG; specific or non-specific immune complexes) results in increased susceptibility to L. infantum infection. Further, JhD BALB/c mice transgenetically reconstituted to secrete IgM demonstrated exacerbated disease in comparison to WT BALB/c mice as early as 2 days post-infection. Evidence suggests that complement activation (generation of C5a) and signaling via the C5a receptor (CD88) is related to the disease exacerbation caused by IgM rather than cytokine levels (IL-10 or IFN-gamma). Overall these studies indicate that polyclonal B-cell activation, which is known to be associated with human visceral leishmaniasis, is an early and intrinsic characteristic of disease and may represent a target for therapeutic intervention.

What determines the success or failure of intracellular cutaneous parasites? Lessons learned from leishmaniasis.

Most parasitic skin infections are averted by very efficient strategies of preventing pathogen invasion. Innate immune cells such as mast cells, macrophages and dendritic cells are responsible for detecting parasites and for recruiting proinflammatory cells that help to contain and control the pathogen at sites of infection. This induces efficient adaptive immunity, which is crucially important for parasite control. Using the example of cutaneous leishmaniasis, we highlight how the skin utilizes different strategies to prevent skin infection and how containment of the infection to the skin site may reduce the harm that otherwise may result for the entire organism.

Role for nitric oxide in hookworm-associated immune suppression.

Hookworm infection is a major cause of anemia and malnutrition in resource-poor countries. Human and animal studies suggest that infection with these intestinal nematodes is associated with impaired cellular immunity, characterized by reduced lymphocyte proliferation in response to both parasite and heterologous antigens. We report here data from studies aimed at defining mechanisms through which hookworms modulate the host cellular immune response. Splenocytes and mesenteric lymph node (MLN) cells from hamsters infected with Ancylostoma ceylanicum showed minimal proliferation in response to mitogen at days 20 and 30 postinfection (p.i.), with partial recovery noted at day 70 p.i. The proliferative capacity of enriched splenocyte T-cell preparations from infected animals following stimulation with hookworm antigens was partially restored in the presence of antigen-presenting cells from uninfected hamsters. Analysis by fluorescence-activated cell sorting revealed that hookworm infection is associated with reduced percentages of both CD4(+) and surface immunoglobulin G-positive lymphocytes in the spleen and MLN cells. Splenocytes from infected hamsters also secreted more nitric oxide (NO) in culture than did those from naïve animals. Inhibition of NO secretion was associated with partial restoration of the proliferative capacity of splenocytes from infected animals in response to concanavalin A, suggesting a role for NO in mediating this effect. Together, these data demonstrate that hookworm infection is associated with impaired function of antigen-presenting cells and depletion of important lymphocyte subpopulations and also suggests a role for NO in parasite-induced immunosuppression.

Intradermal NKT cell activation during DNA priming in heterologous prime-boost vaccination enhances T cell responses and protection against Leishmania.

Heterologous prime-boost vaccination employing DNA-vaccinia virus (VACV) modality using the Leishmania homologue of receptors for activated C kinase (LACK) (p36) antigen has been shown to elicit protective immunity against both murine cutaneous and visceral leishmaniasis. However, DNA priming is known to have limited efficacy; therefore in the current study the effect of NKT cell activation using alpha-galactosyl-ceramide (alphaGalCer) during intradermal DNAp36 priming was examined. Vaccinated mice receiving alphaGalCer + DNAp36 followed by a boost with VVp36 appeared to be resolving their lesions and had at ten- to 20-fold higher reductions in parasite burdens. NKT cell activation during alphaGalCer + DNAp36 priming resulted in higher numbers of antigen-reactive effector CD4(+) and CD8(+) T cells producing granzyme and IFN-gamma, with lower levels of IL-10. Although immunodepletion studies indicate that both CD4 and CD8 T cells provide protection in the vaccinated mice, the contribution of CD4(+) T cells was significantly increased in mice primed with DNAp36 together with alphaGalCer. Notably 5 months after boosting, mice vaccinated with DNAp36 + alphaGalCer continued to show sustained and heightened T cell immune responses. Thus, heterologous prime-boost vaccination using alphaGalCer during priming is highly protective against murine cutaneous leishmaniasis, resulting in the heightened activation and development of CD4 and CD8 T cells (effector and memory T cells).

Assessment of laboratory and field assays of sunlight-induced killing of mosquito larvae by photosensitizers.

The effectiveness of light-induced killing of mosquito larvae in the presence of photosensitizers was studied with larvae of Aedes aegypti (L.), Anopheles stephensi (Liston), and Culex quinquefasciatus Say grown in the laboratory and of Cx. quinquefasciatus grown under field conditions. Tested photosensitizers included xanthene, chlorin, and porphyrin derivatives. All the larvae were treated at the fourth instar. Preliminary laboratory experiments showed a light-induced lethal effect of Rose Bengal (RB) on three species of mosquito larvae. Compared with other photosensitizers, RB seemed to be more efficient at even lower concentration than chlorin (e6) and chlorophyllin on Ae. aegypti larvae. Among the four porphyrin derivatives, i.e., chloroquinoline tetraphenyl propioamidoporphine, tetraphenyl porphine tetrasulfonate, hematoporphyrin (HP), and tetraphenylporphinepropionic acid porphine, HP was the only effective photosensitizer on Ae. aegypti larvae. The best conditions for field tests using RB were conducted on Cx. quinquefasciatus in Bobo-Dioulasso, Burkina Faso. The mortality induced by RB varied from 80 to 96% obtained with unfiltered cesspit water to 0.4 to 6.7% in cesspits with a heavy load of organic materials, thus providing the basis for further developments of this technique under field conditions.

Heterologous prime-boost vaccination with the LACK antigen protects against murine visceral leishmaniasis.

This study reports the efficacy of a heterologous prime-boost vaccination using DNA and vaccinia viruses (Western Reserve [WR] virus and modified [attenuated] vaccinia virus Ankara [MVA]) expressing the LACK antigen (Leishmania homologue of receptors for activated C kinase) and an intradermal murine infection model employing Leishmania infantum. At 1 month postinfection, vaccinated mice showed high levels of protection in the draining lymph node (240-fold reduction in parasite burden) coupled with significant levels of gamma interferon (20 to 200 ng/ml) and tumor necrosis factor alpha/lymphotoxin (8 to 134 pg/ml). Significant but lower levels of protection (6- to 30-fold) were observed in the spleen and liver. Comparable levels of protection were found for mice boosted with either LACK-WR or LACK-MVA, supporting the use of an attenuated vaccinia virus-based vaccine against human visceral leishmaniasis.