Single and concomitant experimental infectionsby Endotrypanum spp. and Leishmania (Viannia) guyanensis (Kinetoplastida: Trypanosomatidae) in the Neotropical sand fly Lutzomyia longipalpis (Diptera: Psychodidae)

Lutzomyia longipalpis females received single and mixed infections with Endotrypanum and Leishmania. Two biological parameters were analyzed: the percentage of infected females and the distribution of flagellates in the gut of the females. The principal comparisons were performed between (1) two strains of Endotrypanum, (2) cloned versus primary sample of one strain of Endotrypanum, (3) Endotrypanum versus Leishmania guyanensis, and (4) the pattern of flagellates behaviour by optical microscopy in females with single or mixed infection versus the identification of parasites isolated from digestive tracts by isoenzyme electrophoresis. Flagellates of Endotrypanum showed distinct patterns of infection suggesting that there is variation between and within strains. The distribution of Endotrypanum and L. guyanensis differed significantly in relation to the colonization of the stomodeal valve. In co-infection with L. guyanensis, a large number of flagellates were seen to be plentifully infecting the stomodeal valve in significantly more specimens than in females infected by Endotrypanum only. However, the electrophoretic profiles of isoenzymes of parasites recovered from all co-infected specimens corresponded to Endotrypanum. This suggests that the mere correlation sand fly infection-biochemical analysis of isolates may induce parasitological incorrect consideration.

Leishmania (Viannia) lainsoni: occurrence of intracellular promastigote forms in vivo and in vitro

Experimental chronic (45-day-old) skin lesion in hamster hind foot induced by Leishmania (Viannia) lainsoni infection showed the presence of promastigote forms in the tissue, inside parasitophorous vacuoles, as assessed by transmission electron microscopy. Experimental in vitro interaction (24 and 48 h) between Leishmania (V.)lainsoni and J774-G8 macrophage cells also demonstrated the same profile. This morphological aspect is unusual, since in this parasite genus only amastigote forms have been described as the resistant and obligate intracellular forms.

Activity of Brazilian and Bulgarian propolis against different species of Leishmania

Extracts of propolis samples collected in Brazil and Bulgaria were assayed against four Leishmania species - Leishmania amazonensis, L. braziliensis, L. chagasi from the New World, and L. major from the Old World - associated to different clinical forms of leishmaniasis. The composition of the extracts has been previously characterized by high temperature high resolution gas chromatography coupled to mass spectrometry. Considering the chemical differences among the extracts and the behavior of the parasites, it was observed significant differences in the leishmanicidal activities with IC50/1 day values in the range of 2.8 to 229.3 µg/ml . An overall analysis showed that for all the species evaluated, Bulgarian extracts were more active than the ethanol Brazilian extract. As the assayed propolis extracts have their chemical composition determined it merits further investigation the effect of individual components or their combinations on each Leishmania species.

Interferon-gamma is required for the late but not early control of Leishmania amazonensis infection in C57Bl/6 mice

The critical role of interferon-gamma (IFN-g) in the resistance of C57Bl/6 mice to Leishmania major is widely established but its role in the relative resistance of these animals to L. amazonensis infection is still not clear. In this work we use C57Bl/6 mice congenitally deficient in the IFN-g gene (IFN-g KO) to address this issue. We found that IFN-g KO mice were as resistant as their wild-type (WT) counterparts at least during the first two months of infection. Afterwards, whereas WT mice maintained lesion growth under control, IFN-g KO mice developed devastating lesions. At day 97 of infection, their lesions were 9-fold larger than WT controls, concomitant with an increased parasite burden. At this stage, lesion-draining cells from IFN-g KO mice had impaired capacity to produce interleukin-12 (IL-12) and tumour necrosis factor-a in response to parasite antigens whereas IL-4 was slightly increased in comparison to infected WT mice. Together, these results show that IFN-g is not critical for the initial control of L. amazonensis infection in C57Bl/6 mice, but is essencial for the developmente of a protective Th1 type immune response in the later stages.

Effects of Brazilian propolis on Leishmania amazonensis

Leishmaniasis, an endemic parasitosis that leads to chronic cutaneous, mucocutaneous or visceral lesions, is part of those diseases, which still requires improved control tools. Propolis has shown activities against different bacteria, fungi, and parasites. In this study we investigated the effect of four ethanolic extracts of typified propolis collected in different Brazilian states, on Leishmania amazonensis performing assays with promastigote forms, extracellular amastigotes, and on infected peritoneal macrophages. Ethanolic extracts of all propolis samples (BRG, BRPG, BRP-1, and BRV) were capable to reduce parasite load as monitored by the percentage of infected macrophages and the number of intracellular parasites. BRV sample called red propolis, collected in the state of Alagoas, and containing high concentration of prenylated and benzophenones compounds, was the most active extract against L. amazonensis. The anti-Leishmania effect of BRV sample was increased in a concentration and time dependent manner. BRV treatment proved to be non-toxic to macrophage cultures. Since BRV extract at the concentration of 25 µg/ml reduced the parasite load of macrophages while presented no direct toxic to promastigotes and extracellular amastigotes, it was suggested that constituents of propolis intensify the mechanism of macrophage activation leading to killing of L. amazonensis. Our results demonstrate, for the first time, that ethanolic extracts of Brazilian propolis reduce L. amazonensis infection in macrophages, and encourage further studies of this natural compound in animal models of leishmaniasis.

T-cell responses associated with resistance to Leishmania infection in individuals from endemic areas for Leishmania (Viannia) braziliensis

Subclinical or asymptomatic infection is documented in individuals living in endemic areas for leishmaniasis suggesting that the development of an appropriate immune response can control parasite replication and maintain tissue integrity. A low morbidity indicates that intrinsic factors could favor resistance to Leishmania infection. Herein, leishmanial T-cell responses induced in subjects with low susceptibility to leishmaniasis as asymptomatic subjects were compared to those observed in cured cutaneous leishmaniasis (CCL) patients, who controlled the disease after antimonial therapy. All of them have shown maintenance of specific long-term immune responses characterized by expansion of higher proportions of CD4+ as compared to CD8+ Leishmania reactive T-lymphocytes. Asymptomatic subjects had lower indexes of in vitro Leishmania induced lymphoproliferative responses and interferon-gamma (IFN-gamma) production in comparison to CCL patients. On the other hand, interleukin (IL-10) production was much higher in asymptomatics than in CCL, while no differences in IL-5 levels were found. In conclusion, long lived T-cell responses achieved by asymptomatic individuals differed from those who had developed symptomatic leishmaniasis in terms of intensity of lymphocyte activation (proliferation or IFN-gamma) and regulatory mechanisms (IL-10). The absence of the disease in asymptomatics could be explained by their intrinsic ability to create a balance between immunoregulatory (IL-10) and effector cytokines (IFN-gamma), leading to parasite destruction without producing skin tissue damage. The establishment of profiles of cell-mediated immune responses associated with resistance against Leishmania infection is likely to make new inroads into understanding the long-lived immune protection against the disease.

Isolation and molecular identification of Leishmania ( Viannia ) peruviana from naturally infected Lutzomyia peruensis (Diptera: Psychodidae) in the Peruvian Andes

Leishmania (Viannia) peruviana was isolated from 1/75 Lutzomyia peruensis captured during May 2006 in an endemic cutaneous leishmaniasis region of the Peruvian Andes (Chaute, Huarochiri, Lima, Peru). Sand fly gut with promastigotes was inoculated into a hamster and the remaining body was fixed in ethanol. L. (Viannia) sp. was determined by polymerase chain reaction (PCR), and Leishmania species through molecular genotyping by PCR-restriction fragment length polymorphism analyses targeting the genes cpb and hsp70, resulting L. (V.) peruviana. The infected sand fly appeared 15 days after the rains finished, time expected and useful real time data for interventions when transmission is occurring.

Analysis and chromosomal mapping of Leishmania (Leishmania) amazonensis amastigote expressed sequence tags

The characterization of expressed sequence tags (ESTs) generated from a cDNA library of Leishmania (Leishmania) amazonensis amastigotes is described. The sequencing of 93 clones generated new L. (L.) amazonensis ESTs from which 32% are not related to any other sequences in database and 68% presented significant similarities to known genes. The chromosome localization of some L. (L.) amazonensis ESTs was also determined in L. (L.) amazonensis and L. (L.) major. The characterization of these ESTs is suitable for the genome physical mapping, as well as for the identification of genes encoding cysteine proteinases implicated with protective immune responses in leishmaniasis.

Triatoma mexicana (Hemiptera: Reduviidae) in Guanajuato, Mexico: house infestation and seasonal variation

Triatoma mexicana was described by Herrich-Schaeffer in 1848. In 1940, a male specimen was found in Hidalgo. In 1970, this species was recorded in the state of Queretaro. Later, it was registered in Guanajuato and San Luis Potosi. In the present paper we performed an investigation in 545 dwellings from three counties in the state of Guanajuato, Mexico, from March 2003 to May 2004. The search and capture of triatomines were seasonally performed indoors and outdoors. Entomological indexes were calculated. The risk and no risk relations between triatomine presence and housing construction materials were analyzed. Fourteen triatomines were collected indoors and 151 outdoors. The vectors were collected in houses built with either risky and non-risky materials. Adults go indoors but do not settle there, hence, no relationship was found between the building materials and infestation of houses. Conventional interventions like house improvement or insecticide spraying are not efficient for the control of T. mexicana, because its developmental cycle is accomplished outdoors in the area surrounding the houses.

RNA polymerase I promoter and splice acceptor site recognition affect gene expression in non-pathogenic Leishmania species

Leishmania (Sauroleishmania) tarentolae has biotechnological potential for use as live vaccine against visceral leishmaniasis and as a system for the over expression of eukaryotic proteins that possess accurate post-translational modifications. For both purposes, new systems for protein expression in this non-pathogenic protozoan are necessary. The ribosomal RNA promoter proved to be a stronger transcription driver since its use yielded increased levels of recombinant protein in organisms of both genera Trypanosoma or Leishmania. We have evaluated heterologous expression systems using vectors with two different polypyrimidine tracts in the splice acceptor site by measuring a reporter gene transcribed from L. tarentolae RNA polymerase I promoter. Our data indicate that the efficiency of chloramphenicol acetyl transferase expression changed drastically with homologous or heterologous sequences, depending on the polypyrimidine tract used in the construct and differences in size and/or distance from the AG dinucleotide. In relation to the promoter sequence the reporter expression was higher in heterologous lizard-infecting species than in the homologous L. tarentolae or in the mammalian-infecting L. (Leishmania) amazonensis.

First encounter of subclinical human Leishmania (Viannia) infection in State of Rio Grande do Sul, Brazil

The objective of the present study was to evaluate the specificity of the Montenegro skin test (MST) in an area in Brazil, state of Grande do Sul State (RS), which was considered to be non-endemic for leishmaniasis. Sixty subjects presented a positive MST and were reevaluated by clinical examination, serology and polymerase chain reaction (PCR) of peripheral blood for the detection of subclinical Leishmania infection. None of the subjects presented clinical signs or symptoms of current leishmaniasis or a history of the disease.Leishmania (Viannia) DNA was detected in blood by PCR and hybridization in one subject. The PCR skin test-positive individual remained asymptomatic throughout the study. Clinical examination showed no scars suggestive of past cutaneous leishmaniasis. Human subclinical infection with Leishmania (Viannia) in RS was confirmed by PCR. This is the first report of subclinical infection with this parasite in the human population of this area.

In vitro and in vivo activity of meglumine antimoniate produced at Farmanguinhos-Fiocruz, Brazil, against Leishmania (Leishmania) amazonensis, L (L.) chagasi and L (Viannia) braziliensis

The leishmanicidal activity of four batches of meglumine antimoniate, produced in Farmanguinhos-Fiocruz, Brazil (TAMs), was assessed and compared to Glucantime®-Aventis Pharma Ltda. Using the amastigote-like in vitro model, the active concentrations of Sb v varied from 10µg/ml to 300 µg/ml for L. (L.) chagasi and from 50µg/ml to 300µg/ml for L. (L.) amazonensis, with no statistically significant differences among the four batches of TAMs and Glucantime®. The inhibitory concentrations (IC50) determined by the amastigote-infected macrophage model for TAM01/03 and Glucantime® were, respectively: 26.3µg/ml and 127.6µg/ml for L. chagasi, 15.4µg /ml and 22.9µg/ml for L. amazonensis, and 12.1µg/ml and 24.2µg/ml for L. (V.) braziliensis. The activities of the four batches of TAMs were confirmed in an in vivo model by assessing, during eight weeks skin lesions caused by L. braziliensis in hamster that were treated with 20mg Sb v/Kg/day for 30 consecutive days. The meglumine antimoniate produced by Farmanguinhos was as effective as the reference drug, Glucantime®-Aventis, against three species of Leishmania that are of medical importance in Brazil.

Isolation and molecular identification of Leishmania chagasi from a bat (Carollia perspicillata) in northeastern Venezuela

This report describes the isolation of a Leishmania chagasi strain from a bat (Carollia perspicillata), and its identification using biological methods and molecular characterization. The parasites were isolated in an artificial culture medium from a blood sample extracted from a bat heart. The isolate was then inoculated into the footpads of Balb/c mice, which subsequently developed a typical nodular leishmanial lesion; the parasites were confirmed as Leishmania by smear and histopathology. Molecular characterization of the parasites was performed by polymerase chain reaction with species-specific primers, kDNA restriction pattern following Hae III endonuclease digestion and dot blot hybridization using a kDNA probe. This report demonstrates that bats can be hosts for L. chagasi species and suggests the need for studies to determine whether they may be involved in foci of visceral leishmaniasis.

Experimental infection parameters in Galea spixii (Rodentia: Caviidae) with Leishmania infantum chagasi

In order to better understand the epidemiological transmission network of leishmaniasis, an endemic disease in Northeast Brazil, we investigated the susceptibility of Spix yellow-toothed cavies (Galea spixii) to the Leishmania infantum chagasi parasite. Nine cavies were experimentally infected, separated into three groups and monitored at 30, 90 and 180 days, respectively. Amastigotes were identified in the spleen slides of two cavies killed 180 days after infection. Antibodies against the L. i. chagasi were identified in one of the cavies. This demonstrates that G. spixii is in fact capable of maintaining a stable infection by L. i. chagasi without alterations in biochemical and hematological parameters of the host and without perceivable micro and macroscopic lesions.