Experimental pathogenicity of a presumed monoxenous trypanosomatid isolated from humans in a murine model

Two strains of a presumed lower trypanosomatid isolated from immunocompetent and HIV-infected humans in French West Indies were investigated in vitro and in vivo in a murine experimental model. The ability of parasites to grow in vitro in bone marrow-derived macrophages and their virulence in vivo were assessed. For in vivo infection, two groups of BALB/c mice were inoculated either by the subcutaneous or intravenous route with 10(7) promastigotes at day 0. Infection was monitored by measuring parasite load in liver, spleen, foot pad, popliteal, and mesenteric lymph nodes and brain from day 7 to day 150 post-infection using a microtitration technique. Parasites multiplied in mouse macrophages in vitro. In vivo, both strains proved infective to mice and capable of visceralization and dissemination in the popliteal and mesenteric lymph nodes, liver, spleen, and even brain. Both strains elicited a strong humoral response against trypanosomatid antigen in mice, which cross-reacted with Leishmania antigen. Contrasting with the straightforward dissemination of parasites, the infection was strikingly well tolerated by the murine host with no clinical signs and minimal tissue changes around parasitized macrophage infiltrates.     

Synthesis and in vitro evaluation of leishmanicidal and trypanocidal activities of N-quinolin-8-yl-arylsulfonamides

In the present paper 12 N-quinolin-8-yl-arylsulfonamides synthesized by coupling 8-aminoquinolines with various arylsulfonylchlorides were assayed in vitro against Leishmania amazonensis, Leishmania chagasi and Trypanosoma cruzi strains. This series of new compounds were found to be selective for Leishmania spp. promastigote and amastigote forms. The most active compound was the N-(8-quinolyl)-3,5-difluoro-benzenesulfonamide 10 with an IC₅₀ against L. amazonensis and L. chagasi of 2.12 and 0.45 μM, respectively. The less cytotoxic biphenyl derivative 7 was very effective against intracellular L. amazonensis with a reduction of macrophage cell infection of 82.1% at 25 μM. In addition, a copper complex 17 of an inactive ligand was readily synthesized and showed high leishmanicidal and trypanocidal activity against both extra and intracellular forms.     

Molecular evolution of the MLO gene family in Oryza sativa and their functional divergence

The Leishmania genome project has identified new genes at a rapid rate. The 32.8-megabase haploid genome of Leishmania major (Friedlin strain) is published and the comparative analysis of genome sequences of two other species, Leishmania infantum and Leishmanai braziliensis has been done. The haploid genome of Leishmania major (Friedlin strain) has around 8272 protein-coding genes, of which only 36% can be ascribed a putative function. Out of these open reading frames around 910 Leishmania major genes have no orthologs in the other two Tritryp genomes. These “Leishmania -restricted” genes hold a potential as novel drug targets and potential vaccine candidates. Open reading frame, ORFF, is a single copy gene located on the chromosome 35 as a part of the multigene LD1 locus. Indirect immunofluorescence study and creation of ORFF-GFP fusion showed that ORFF is localized in the DNA containing compartments of Leishmania donovani, the nucleus and the kinetoplast. In order to characterize ORFF gene of L. donovani, we have created ORFF over-expressors and single allele deletion mutants by homologous replacement strategy. ORFF is likely to be an important gene for the parasite growth since results from over-expression studies and characterization of ORFF heterozygous knockout mutants reveal marked alterations in the cell cycle phenotype compared to the wild-type parasites. Flowcytometry based cell cycle analysis showed selective increase in the DNA synthetic phase of the ORFF over-expressors and a subversion of the same in heterozygous knockouts of ORFF suggesting its potential role in cell cycle progression.     

Leishmania (Leishmania) amazonensis-induced cutaneous leishmaniasis in the primate Cebus apella: a model for vaccine trials

A primate model of leishmaniasis was developed with the objective of future vaccine testing. Lesion development and immunological parameters were studied upon primary and secondary infections. Seven Cebus apella were injected subcutaneously with 2×10⁶ Leishmania (Leishmania) amazonensis promastigotes. Erythematous nodules appeared 19–29 days p.i., which disappeared 100 days p.i. Four months later, six of the monkeys were challenged with the same inoculum; three of them developed erythematous nodules after 7 days p.i., with ulcer formation in two of these subjects. The lesions were short-lived and all were cured 40 days post challenge. Anti-Leishmania IgG antibodies were detected and they increased after the challenge infection. Leishmania antigen-induced lymphoproliferation was found 1 month post-primary infection, which coincided with IFN-γ production and lesion development. It decreased to control levels afterwards, but at the time of the challenge dose, it was significantly above the initial level. After the challenge infection, it first increased then decreased sharply at 40 days post-challenge, coinciding with the healing of the lesion. It increased again to a higher level at 60 days post-challenge. Leishmania (Leishmania) amazonensis-infection in C. apella did not induce complete protection against a secondary infection with a homologous parasite although specific antibody production and lymphoproliferation with IFN-γ production were observed. This fact indicates that vaccine has to be better than infection in the induction of protective immunity, and raises a question on in vitro parameters that should be considered as a counterpart of expected protection induced by vaccine candidate. In addition, we conclude that this is a useful primate model for the evaluation of candidate vaccines.     

Rapid identification of Leishmania species from formalin-fixed biopsy samples by polymorphism-specific polymerase chain reaction

The precise identification and classification of Leishmania species is important for public health surveillance since different species cause different clinical features of the disease. A highly specific polymerase chain reaction (PCR) panel was developed to enable the identification of the five major Leishmania species that cause New World cutaneous leishmaniases. The primers used for this panel were designed to distinguish the polymorphism in sequences of commonly amplified DNA bands of the parasites produced by arbitrarily primed PCR. These polymorphism-specific PCR diagnoses were performed with formalin-fixed biopsy specimens of the leishmanial lesions from four patients in Ecuador and one hamster skin lesion, and these lesions were determined to be caused by Leishmania (Viannia) panamensis, L. (Leishmania) mexicana, and L. (L.) amazonensis. The PCR panel may offer an important and practical approach to the standardized identification of Leishmania species in field examinations.     

Expression of heat shock protein in host macrophages correlates with a protective potential against infection with Leishmania major in mice

We studied the relationship between the expression of 65-kDa heat shock protein (HSP65) and resistance of mice to infection with Leishmania major (L. major), an obligate intracellular protozoan. C57BL/6 (B6) mice, a strain genetically resistant to L. major infection, expressed high level of HSP65 in their peritoneal and draining lymph node macrophages after infection, whereas susceptible BALB/c mice expressed only slightly at the early stage of infection. This protein was not expressed in the parasite itself and macrophages of non-infected mice. We examined the role of T cells in the expression of HSP65 by using SCID mice grafted with the fetal thymus from B6 or BALB/c mice (B6-TG or BALB-TG mice, respectively). Either BALB-TG or B6-TG mice were reconstituted with T cells derived from each grafted fetal thymus. B6-TG mice were markedly resistant against infection with L. major, compared with BALB-TG mice. Furthermore, the HSP65 expression in macrophages of thymus-grafted mice was similar to that of the thymus-donor type. That is, B6-TG mice expressed a high level of this protein, whereas BALB-TG mice did in lower level than B6-TG mice. These results show that T cells are necessary to express HSP65 and the expression correlates with a protective potential of T cells against infection with L. major.     

Experimental American leishmaniasis and Chagas'' disease in the Brazilian squirrel monkey: cross immunity and electrocardiographic studies of monkeys infected with Leishmania braziliensis and Trypanosoma cruzi

, and 1988. Experimental American leishmaniasis and Chagas' disease in the Brazilian squirrel monkey: cross immunity and electrocardiographic studies of monkeys infected with Leishmania braziliensis and Trypanosoma cruzi. International Journal for Parasitology 18: 1053–1059. Adult, laboratory-bred squirrel monkeys (Saimiri sciureus) previously infected with either Leishmania braziliensis braziliensis or L. b. panamensis were challenge infected with blood-form trypomastigotes of Trypanosoma cruzi (Brazil strain). Monkeys previously infected with T. cruzi were challenged with stationary phase promastigote forms of L. b. braziliensis. Monkeys were examined during the course of challenge for evidence of infection, electrocardiographic alterations and parasite-specific antibody responses. T. cruzi epimastigotes were cultured from the blood of monkeys up to 3 months after challenge with this parasite. Unulcerated cutaneous lesions appeared and persisted in monkeys challenged with L. b. braziliensis. The formation of satellite lesions was observed in one monkey. Increased QRS intervals were not observed in T. cruzi challenged monkeys without prior cardiac irregularities and QRS left axis shifts were observed in only two of these monkeys. Elevated titers of parasite binding IgM and IgG specific for both T. cruzi and L. braziliensis were observed in all monkeys following challenge. These results indicate that prior infection with T. cruzi or L. braziliensis does not protect against heterologous challenge infection with these organisms. However, prior infection with Leishmania parasites may provide some protection against chagasic cardiopathies.     

Canine experimental infection: intradermal inoculation of Leishmania infantum promastigotes

Five mixed breed dogs were inoculated intradermally (ID) with cultured virulent stationary phase promastigotes of Leishmania infantum Nicole, 1908 stocks recently isolated. Parasite transformations in the skin of ID infected dogs were monitored from the moment of inoculation and for 48 h, by skin biopsies. Anti-Leishmania antibody levels were measured by indirect immunofluorescence assay, counterimmunoelectrophoresis and direct agglutination test, and clinical conditions were examined. Thirty minutes after ID inoculation the first amastigotes were visualised and 3 to 4 h after inoculation the promastigotes were phagocytized by neutrophils and by a few macrophages. These cells parasitised by amastigotes progressively disappeared from the skin and 24 h after inoculation parasites were no longer observed. Local granulomes were not observed, however, serological conversion for antibodies anti-Leishmania was achieved in all dogs. Direct agglutination test was the only technique positive in all inoculated dogs. Amastigotes were found in the popliteal lymph node in one dog three months after inoculation. This work demonstrates that, with this inoculum, the promastigotes were transformed into amastigotes and were up taken by neutrophils and macrophages. The surviving parasites may have been disseminated in the canine organism, eliciting a humoral response in all cases.     

LFR1 ferric iron reductase of Leishmania amazonensis is essential for the generation of infective parasite forms

The protozoan parasite Leishmania is the causative agent of serious human infections worldwide. The parasites alternate between insect and vertebrate hosts and cause disease by invading macrophages, where they replicate. Parasites lacking the ferrous iron transporter LIT1 cannot grow intracellularly, indicating that a plasma membrane-associated mechanism for iron uptake is essential for the establishment of infections. Here, we identify and functionally characterize a second member of the Leishmania iron acquisition pathway, the ferric iron reductase LFR1. The LFR1 gene is up-regulated under iron deprivation and accounts for all the detectable ferric reductase activity exposed on the surface of Leishmania amazonensis. LFR1 null mutants grow normally as promastigote insect stages but are defective in differentiation into the vertebrate infective forms, metacyclic promastigotes and amastigotes. LFR1 overexpression partially restores the abnormal morphology of infective stages but markedly reduces parasite viability, precluding its ability to rescue LFR1 null replication in macrophages. However, LFR1 overexpression is not toxic for amastigotes lacking the ferrous iron transporter LIT1 and rescues their growth defect. In addition, the intracellular growth of both LFR1 and LIT1 null parasites is rescued in macrophages loaded with exogenous iron. This indicates that the Fe(3+) reductase LFR1 functions upstream of LIT1 and suggests that LFR1 overexpression results in excessive Fe(2+) production, which impairs parasite viability after intracellular transport by LIT1.     

Early histopathology of experimental infection with Leishmania donovani in hamsters

The extracellular promastigote stage of Leishmania donovani is inoculated by a phlebotomine sandfly into the skin of a susceptible host, after which visceral dissemination and clinical disease may ensue. Using a hamster model we examined the histopathology of early infection with L. donovani after intradermal inoculation of cultured promastigotes. The initial response was a mixed polymorphonuclear (PMN)-mononuclear phagocyte infiltrate, noted between 1 and 24 hr after inoculation, which became primarily mononuclear by 48 hr. Parasites were initially found intracellularly in both PMN's and mononuclear phagocytes, but by 48 hr they had assumed amastigote-like morphology and were found exclusively in macrophages. The number of parasites per infected macrophage increased during the first week after inoculation, suggesting that intracellular replication of the organism was taking place. This was followed by the formation of granulomas between 4 and 6 wk. By 8 wk intracellular parasites were largely gone. The histologic response was consistent with early destruction of parasites in PMN's, and survival and replication of L. donovani in macrophages. Cutaneous infection with the parasite was eventually controlled locally, coincident with granuloma formation. Despite these local responses, the organism was able to disseminate and eventually produce typical visceral leishmaniasis.     

Bioluminescent Leishmania expressing luciferase for rapid and high throughput screening of drugs acting on amastigote-harbouring macrophages and for quantitative real-time monitoring of parasitism features in living mice

In this study, we have established conditions for generating Leishmania amazonensis recombinants stably expressing the firefly luciferase gene. These parasites produced significant bioluminescent signals for both in vitro studies and the development of an in vivo model, allowing the course of the parasitism to be readily monitored in real time in the living animals such as laboratory mice. First, a model was established, using parasite-infected mouse macrophages for rapidly determining the activity of drugs against intracellular amastigotes. Results indicated that recombinant Leishmania can be reliably and confidently used to monitor compounds acting on intracellular amastigote-harbouring macrophages. Secondly, temporal analyses were performed following inoculation of metacyclic promastigotes into the ear dermis of BALB/c mice and the bioluminescent light transmitted through the tissue was imaged externally using a charge coupled device (CCD) camera. Bioluminescent signals, measured at the inoculation site and in the draining lymph node of mice containing these parasites correlated well with the more classical quantification of parasites. These assays prove that the real-time bioluminescent assay is not only sensitive but also more rapid than culture-base techniques allowing to monitor parasite-load before any clinical signs of leishmaniasis are detectable. In short, this luciferase imaging study is useful to monitor the efficacy of anti-leishmanial drugs on live cell culture and to trace leishmanial infection in animal models.     

Leishmania braziliensis spp. in the nasal mucosa of guinea pigs inoculated in the tarsi.

Two lots of 20 young male guinea pigs were inoculated subcutaneously in the tarsi with 10(4) amastigotes of Leishmania braziliensis braziliensis or L. b. guyanensis to study the susceptibility of this Neotropical hystricomorph rodent the autochthonous parasites. Almost 50% of the animals showed lesions in the inoculation site and had parasitizations that were infective to hamsters, as shown by inoculating homogenates of the dermal lesion, of the spleen, of the liver, and of the nasal mucosa into hamsters at 20, 40, 60, and 120 days after inoculation of the guinea pig. Smears of the above organs showed the presence of amastigotes. Parasites inoculated into the tarsi were detected early in the skin, spleen, and liver of the guinea pig host. Blood cultures made by cardiopuncture on sacrifice of the guinea pigs were uniformly negative. The nasal mucosa of nearly all animals positive in the skin or viscera was invaded early by the parasites, although with greater frequency between 60 and 120 days post-inoculation. The use of this model for the study of mucocutaneous parasitism by L. braziliensis is discussed, together with the phenomena of parasitism at a distance from the inoculation site, the temperature of the body regions affected, and the possible genetic influence on susceptibility of the guinea pig to L. braziliensis.     

Leishmania braziliensis isolates differing at the genome level display distinctive features in BALB/c mice

Leishmania braziliensis is the species responsible for the majority of cases of human cutaneous leishmaniasis in Brazil. In the present study, L. braziliensis isolates from two different geographic areas in Brazil were studied by RAPD, using arbitrary primers. We also evaluated other biological features of these two isolates. We compared (a) the clinical features they initiate or not once delivered subcutaneously as stationary-phase promastigotes in the footpad of BALB/c mice; (b) the parasite load in both the footpad and the draining lymph node; (c) the cytokines present in the supernatant of cultures of the cell suspensions from the draining lymph nodes; and (d) the cell types present at the site of parasite delivery. The results show that the L. braziliensis strain from Ceará (H3227) is genotypically different from the L. braziliensis strain from Bahia (BA788). H3227-parasitized mice developed detectable lesions, whereas BA788-parasitized mice did not. Fifteen days post parasite inoculation there was an increase in the numbers of macrophages and lymphocytes in the footpads, whatever the parasite inoculum. Parasite load at the inoculation site--namely the footpad--did not differ significantly; in draining lymph nodes, however, it increased over the period under study. Early after parasite inoculation, the cells recovered from the draining lymph nodes of BA788-parasitized mice produced higher levels of IFN-gamma, a feature coupled to a higher number of NK cells. Later, after the parasite inoculation, there was an increased content of IL-12p70 and IL-10 in the supernatant of cells recovered from the lymph nodes of H3227-parasitized mice. This comparative analysis points out that L. braziliensis isolates differing in their genomic profiles do establish different parasitic processes in BALB/c mice.     

Evolutionary implications of Leishmania amastigotes in circulating blood cells of lizards

Amastigotes of 2 Leishmania species are reported from the Pakistani lizards Teratoscincus scincus (Gekkonidae) and Agama agilis (Agamidae) collected in western Baluchistan and north-central Sind, respectively. Parasites were seen only in blood cells primarily within thrombocytes, and were detected on smears of peripheral blood. Slides made at 3-day intervals for 38 days from an infected hatchling T. scincus demonstrated an increase with time in the mean number of amastigotes/infected thrombocyte. No evidence of an infection focus in fixed cells of the viscera was found. It is suggested, in view of reports of amastigotes in circulating blood cells of hosts belonging to 5 genera, collected in 5 countries from India to France, that saurian Leishmania may behave simply as parasites of circulating blood cells, thus illustrating an early stage in the adaptation of leishmanias to the vertebrate host.     

Intradermal inoculations of low doses of Leishmania major and Leishmania amazonensis metacyclic promastigotes induce different immunoparasitic processes and status of protection in BALB/c mice

In order to simulate the natural long term parasitisms which may occur in mammals infected with Leishmania, cutaneous leishmaniases due to Leishmania major or Leishmania amazonensis were induced using a model based on the inoculation of 10-1000 metacyclic promastigotes into the ear dermis of BALB/c mice. The final outcome of these parasitisms was dependent upon the number of inoculated parasites. Only some of the mice inoculated with ten parasites displayed cutaneous lesions, whereas most mice infected with 100 metacyclics and all mice infected with 1000 metacyclics developed progressive lesions. We found, using the latter experimental conditions, that the onset of the pathology was associated with: (a) parasite multiplication in the inoculation site and the draining lymph node correlating with an increase of the lymph node cell number, especially in L. major-infected mice; and (b) the detection of lymph node cells, at least in part CD4(+) T lymphocytes, able to produce high levels of interferon-gamma, interleukin (IL)-4, IL-10 and IL-13. Thereafter, mice infected by L. major harboured few parasites in the ear and had a 100-fold reduction in lymph node parasite load between 23 and 40 weeks post-inoculation. In contrast, the parasite loads of L. amazonensis-infected mice remained stable in the ear and increased in nodes during the same period of time. Only L. major-infected mice that exhibited cutaneous lesions in the primary site were resistant to the re-inoculation of 1000 metacyclic promastigotes, whereas all L. amazonensis-primary infected mice remained susceptible to a second homologous challenge. These results are the first to document that a status of resistance to re-infection, referred to concomitant immunity, is coupled to the development of primary progressive lesions in L. major-infected BALB/c mice. Such a protective status is absent in L. amazonensis-infected BALB/c mice.     

七种实验动物对沙鼠利什曼原虫感受性的研究

本文报道3株近交系小鼠、沙鼠和仓鼠对沙鼠利什曼原虫的感受性。结果表明,BALB/c株小鼠,长爪沙鼠和黑线仓鼠对沙鼠利什曼有较好的感受性。CFW株小鼠感染后不久即可自愈,而C57 BL株鼠则抗拒感染。除了黑线仓鼠的睾丸在感染原虫后偶可产生转移性皮肤损害外,其余实验动物的感染均仅局限在接种部位的皮肤,进一步表明沙鼠利什曼是一种亲皮肤性的原虫。     

Requirement of SIRPα for protective immunity against Leishmania major

Signal regulatory protein α (SIRPα) is a transmembrane protein that binds the protein tyrosine phosphatases SHP-1 and SHP-2 through its cytoplasmic region and is abundantly expressed on dendritic cells and macrophages. Wild-type (WT) C57BL/6 mice are known to be resistant to Leishmania major infection. We here found that C57BL/6 mice that express a mutant version of SIRPα lacking most of the cytoplasmic region manifested increased susceptibility to L. major infection, characterized by the marked infiltration of inflammatory cells in the infected lesions. The numbers of the parasites in footpads, draining lymph nodes and spleens were also markedly increased in the infected SIRPα mutant mice, compared with those for the infected WT mice. In addition, soluble leishmanial antigen-induced production of IFN-γ by splenocytes of the infected SIRPα mutant mice was markedly reduced. By contrast, the ability of macrophages of SIRPα mutant mice to produce nitric oxide in response to IFN-γ was almost equivalent to that of macrophages from WT mice. These results suggest that SIRPα is indispensable for protective immunity against L. major by the induction of Th1 response.     

Parasites may exit immunocompromised northern pig-tailed macaques(Macaca leonina) infected with SIVmac239

Parasites can increase infection rates and pathogenicity in immunocompromised human immunodeficiency virus(HIV) patients. However, in vitro studies and epidemiological investigations also suggest that parasites might escape immunocompromised hosts during HIV infection.Due to the lack of direct evidence from animal experiments, the effects of parasitic infections on immunocompromised hosts remain unclear. Here,we detected 14 different parasites in six northern pig-tailed macaques(NPMs) before or at the 50 th week of simian immunodeficiency virus(SIV) infection by ELISA. The NPMs all carried parasites before viral injection. At the 50 th week after viral injection, the individuals with negative results in parasitic detection(i.e., 08247 and 08287) were characterized as the Parasites Exit(PE) group, with the other individuals(i.e., 09203, 09211, 10205, and 10225) characterized as the Parasites Remain(PR) group. Compared with the PR group, the NPMs in the PE group showed higher viral loads, lower CD4~+T cells counts, and lower CD4/CD8 rates. Additionally, the PE group had higher immune activation and immune exhaustion of both CD4~+and CD8~+T cells. Pathological observation showed greater injury to the liver, cecum, colon,spleen, and mesenteric lymph nodes in the PE group.This study showed more seriously compromised immunity in the PE group, strongly indicating that parasites might exit an immunocompromised host.