Susceptibility of spiny rats (Proechimys semispinosus) to Leishmania (Viannia) panamensis and Leishmania (Leishmania) chagasi

The role of Proechimys semispinosus as reservoir of Leishmania (Viannia) panamensis on the Colombian Pacific coast was experimentally evaluated. The susceptibility to L. chagasi also was assessed to determine the utility of this rodent as a model for studying reservoir characteristics in the laboratory. Wild-caught animals were screened for natural trypanosomatid infections, and negative individuals were inoculated intradermally (ID) in the snout or feet with 10(7) promastigotes of L. panamensis. L. chagasi was inoculated intracardially (10(7) promastigotes) or ID in the ear (10(8) promastigotes). PCR-hybridization showed that 15% of 33 spiny rats were naturally infected with L. Viannia sp. Animals experimentally infected with L. panamensis developed non-ulcerated lesions that disappeared by the 7th week post-infection (p.i.) and became more resistant upon reinfection. Infectivity to sand flies was low (1/20-1/48 infected/fed flies) and transient, and both culture and PCR-hybridization showed that L. panamensis was cleared by the 13th week p.i. Animals inoculated with L. chagasi became subclinically infected and were non-infective to sand flies. Transient infectivity to vectors of spiny rats infected with L. panamensis, combined with population characteristics, e.g., abundance, exploitation of degraded habitats and high reproductive rates, could make them epidemiologically suitable reservoirs.

Study of the safety, immunogenicity and efficacy of attenuated and killed Leishmania (Leishmania) major vaccines in a rhesus monkey (Macaca mulatta) model of the human disease

We have compared the efficacy of two Leishmania (Leishmania) major vaccines, one genetically attenuated (DHFR-TS deficient organisms), the other inactivated [autoclaved promastigotes (ALM) with bacillus Calmete-Guérin (BCG)], in protecting rhesus macaques (Macaca mulatta) against infection with virulent L. (L.) major. Positive antigen-specific recall proliferative response was observed in vaccinees (79% in attenuated parasite-vaccinated monkeys, versus 75% in ALM-plus-BCG-vaccinated animals), although none of these animals exhibited either augmented in vitro gamma interferon (IFN-g) production or positive delayed-type hypersensitivity (DTH) response to the leishmanin skin test prior to the challenge. Following challenge, there were significant differences in blastogenic responses (p < 0.05) between attenuated-vaccinated monkeys and naïve controls. In both vaccinated groups very low levels of antibody were found before challenge, which increased after infective challenge. Protective immunity did not follow vaccination, in that monkeys exhibited skin lesion at the site of challenge in all the groups. The most striking result was the lack of pathogenicity of the attenuated parasite, which persisted in infected animals for up to three months, but were incapable of causing disease under the conditions employed. We concluded that both vaccine protocols used in this study are safe in primates, but require further improvement for vaccine application.

The 245 kb amplified chromosome of Leishmania (V.) braziliensis contains a biopterin transporter gene

Leishmania (V.) braziliensis M2903 presents a small linear and stable 245 kb chromosome originating from a genomic amplification. Similar amplifications present in other species of Leishmania contain a gene coding for a biopterin transporter. Since Leishmania is auxotrophic for this metabolite, this amplification could result from the need to better capture biotpterin from growth media under specific circumstances. In this paper we show that this gene is also present in L. (V.) braziliensis small chromosome, which shares sequences with other genomic amplifications already described.

Reverse transcription-polymerase chain reaction construction of plasmid-based, full-length cDNA libraries from Leishmania infantum for in vitro expression screening

We describe a streamlined reverse transcription-polymerase chain reaction methodology for constructing full-length cDNA libraries of trypanosomatids on the basis of conserved sequences located at the 5' and 3'ends of trans-spliced mRNAs. The amplified cDNA corresponded to full-length messengers and was amenable to in vitro expression. Fractionated libraries could be rapidly constructed in a plasmid vector by the TA cloning method (Invitrogen). We believe this is useful when there are concerns over the use of restriction enzymes and phage technology as well as in cases where expression of proteins in their native conformation is desired.

Detection of amastigote-like forms in the valve of Phlebotomus papatasi infected with Leishmania major

A massive and homogeneous amount of amastigote-like forms was detected in the stomodeal valve (SV) and the thoracic mid-gut (TMG) of Leishmania major-infected Phlebotomus papatasi, which received a second blood meal 13 to 21 days post-infection on healthy anaesthetized hamsters. After re-feeding, the infected sand flies were dissected out to examine the morphology of the parasite in SV, TMG and the abdominal mid-gut (AMG). Different promastigote forms were seen in the infected flies. Among these included typical promastigotes (nectomonads and haptomonads), paramastigotes, metacyclic promastigotes and, in some samples, the here-reported amastigote-like forms. The Leishmania amastigote-like forms were detected in the SV of sand flies with 14, 18 and 21 days of infection as well as in the TMG at 13 and 18 days post-infection. However, the amastigote-like forms were not detected in the AMG. Factors such as the acidic pH predominating the TMG and the SV, as well as the temperature of the ingested blood, among others, are suggested as contributing to the transformation of the typical promastigotes into the amastigote-like forms. The significance of this finding is discussed and the possible biological advantage for transmission of Leishmania is considered.

Epidemiological and clinical features of Leishmania (Viannia) braziliensis American cutaneous and mucocutaneous leishmaniasis in the State of Espírito Santo, Brazil

Between 1985 and 2000, epidemiological surveys of the American tegumentary leishmaniasis (ATL) were carried out in several rural and urban communities in Espírito Santo, Brazil. A total of 100 stocks of Leishmania (comprising isolates from both human and canine hosts with ATL) were identified by two methods of molecular characterization, using specific monoclonal antibodies and multilocus enzyme electrophoresis. Parasite isolates from 19 municipalities were found to belong to the same zymodeme and serodeme type as of the Leishmania (Viannia) braziliensis reference strain. In contrast, our genotyping studies have shown intra-specific variation among these parasites (comparisons of the variability of the internal transcribed spacers between the small and large subunits of the rRNA genes of the 22 stocks studiedrevealed at least 11 genotypes). Two main clusters of L. (V.) braziliensis genotypes were observed, representing parasites collected from different endemic regions in the state, where transmission reflects distinct eco-epidemiological features. Infection with this pathogen was associated with the characteristic disease forms, but neither the clinical outcome nor the response to treatment could be related to the genetic polymorphism of the isolates, as defined by using the proposed methodology.

Detection of pathogenic bacteria in skin lesions of patients with chiclero's ulcer: reluctant response to antimonial treatment

We investigated the bacterial flora present in skin lesions of patients with chiclero's ulcer from the Yucatan peninsula of Mexico using conventional culture methods (11 patients), and an immunocolorimetric detection of pathogenic Streptococcus pyogenes (15 patients). Prevalence of bacteria isolated by culture methods was 90.9% (10/11). We cultured, from chiclero's ulcers (60%), pathogenic bacterial such as Staphylococcus aureus (20%), S. pyogenes (1.6%), Pseudomonas aeruginosa (1.6%), Morganella morganii (1.6%), and opportunist pathogenic bacteria such as Klebsiella spp. (20.0%), Enterobacter spp. (20%), and Enterococcus spp. (20%). We also cultured coagulase-negative staphylococci in 40% (4/10) of the remaining patients. Micrococcus spp. and coagulase-negative staphylococci constituted the bacterial genuses more frequently isolated in the normal skin of patients with chiclero's ulcer and healthy individuals used as controls. We also undertook another study to find out the presence of S. pyogenes by an immunocolorimetric assay. This study indicated that 60% (9/15) of the ulcerated lesions, but not normal controls, were contaminated with S. pyogenes. Importantly, individuals with purulent secretion and holding concomitant infections with S. pyogenes, S. aureus, P. aeruginosa, M. morganii, and E. durans took longer to heal Leishmania (L.) mexicana infections treated with antimonial drugs. Our results suggest the need to eliminate bacterial purulent infections, by antibiotic treatment, before starting antimonial administration to patients with chiclero's ulcer.

Therapy of Venezuelan patients with severe mucocutaneous or early lesions of diffuse cutaneous leishmaniasis with a vaccine containing pasteurized Leishmania promastigotes and bacillus Calmette-Guerin: preliminary report

Severe mucocutaneous (MCL) and diffuse (DCL) forms of American cutaneous leishmaniasis (ACL) are infrequent in Venezuela. Chemotherapy produces only transitory remission in DCL, and occasional treatment failures are observed in MCL. We have evaluated therapy with an experimental vaccine in patients with severe leishmaniasis. Four patients with MCL and 3 with early DCL were treated with monthly intradermal injections of a vaccine containing promastigotes of Leishmania (Viannia) braziliensis killed by pasteurization and viable Bacillus Calmette- Guerin. Clinical and immunological responses were evaluated. Integrity of protein constituents in extracts of pasteurized promastigotes was evaluated by gel electrophoresis. Complete remission of lesions occurred after 5-9 injections in patients with MCL or 7-10 injections in patients with early DCL. DCL patients developed positive skin reactions, average size 18.7 mm. All have been free of active lesions for at least 10 months. Adverse effects of the vaccine were limited to local reactivity to BCG at the injection sites and fever in 2 patients. Extracts of pasteurized and fresh promastigotes did not reveal differences in the integrity of protein components detectable by gel electrophoresis. Immunotherapy with this modified vaccine offers an effective, safe option for the treatment of patients who do not respond to immunotherapy with vaccine containing autoclaved parasites or to chemotherapy .

Comparison between splenic and lymph node aspirations as sampling methods for the parasitological detection of Leishmania chagasi infection in dogs

The sensitivities of spleen and lymph node cultures for the diagnosis of canine visceral leishmaniasis were compared in 64 anti-Leishmania antibody positive dogs from an endemic area in Brazil. The sensitivity of spleen cultures for Leishmania detection was 97.9%; in lymph node cultures it was 25%. Positive spleen culture was more frequent (p = 0.048, Fisher's exact probability test) in symptomatic (28 out of 33 animals) than in asymptomatic animals (19 out of 31 animals). These results support the use of spleen instead of lymph node aspiration as the choice method for the parasitological diagnosis of the infection.

Leishmania braziliensis: partial control of experimental infection by interleukin-12 p40 deficient mice

Resistance to infection by Leishmania major has been associated with the development of a Th1 type response that is dependent on the presence of interleukin 12 (IL-12). In this work the involvement of this cytokine in the response to infection by L. braziliensis, a less virulent species in the mouse model, was evaluated. Our results show that while interferon (IFN-g) deficient (-/-) mice inoculated L. braziliensis develop severe uncontrolled lesions, chronic lesions that remained under control up to 12 weeks of infection were observed in IL-12p40 -/- mice. IL 12p40 -/- mice had fewer parasites in their lesions than IFN-g-/- mice. Lymph node cells from IL-12p40 -/- were capable of producing low but consistent levels of IFN-g suggestive of its involvement in parasite control. Furthermore, as opposed to previous reports on L. major-infected animals, no switch to a Th2 response was observed in IL-12p40 -/- infected with L. braziliensis.

Anti-leishmanial activity of alkaloidal extract from Aspidosperma ramiflorum

Infections due to protozoa of the genus Leishmania are a major worldwide health problem, with high endemicity in developing countries. The drugs of choice for the treatment of leishmaniasis are the pentavalent antimonials (SbV), which present renal and cardiac toxicity. Besides, the precise chemical structure and mechanism of action of these drugs are unknown up to date. In order to find new drugs against leishmaniasis, we have been studying extracts of Brazilian trees. In the present study, we have evaluated the effectiveness of an alkaloid extract of Aspidosperma ramiflorum Muell. Arg. (Apocynaceae), against the extracellular forms promastigotes of L. (L.) amazonensis and L. (V.) braziliensis. The alkaloid extract of A. ramiflorum was much more effective against L. (L.) amazonensis (LD50 < 47 µg/ml) than L. (V.) braziliensis. Based on these in vitro results against L. (L.) amazonensis new studies should be made to find the compounds with anti-leishmanial activity.

Antileishmanial activity of lapachol analogues

The antileishmanial activity of lapachol, isolapachol, and dihydrolapachol, along with soluble derivatives (potassium salt) and acetate was obtained. All the compounds were assayed against metacyclic promastigotes of two different species of Leishmania associated to tegumentar leishmaniasis: L. amazonensis and L. braziliensis. All compounds presented significant activity, being isolapachol acetate the most active against promastigotes, with IC50/24h = 1.6 ± 0.0 µg/ml and 3.4 ± 0.5 µg/ml for, respectively, L. amazonensis and L. braziliensis. This compound was also assayed in vivo against L. amazonensis and showed to be active. Its toxicity in vitro was also established, and at concentration similar to the IC50, no toxicity was evidenced. In all experiments, pentamidine isethionate was used as a reference drug. The present results reinforce the potential use of substituted hydroxyquinones and derivatives as promising antileishmanial drugs and suggest a continuing study within this class of compounds.

Experimental infection of Leishmania (L.) chagasi in a cell line derived from Lutzomyia longipalpis (Diptera:Psychodidae)

The present work describes the in vitro infection of a cell line Lulo, derived from Lutzomyia longipalpis embryonic tissue, by Leishmania chagasi promastigotes. This infection process is compared with a parallel one developed using the J774 cell line. The L. chagasi MH/CO/84/CI-044B strain was used for experimental infection in two cell lines. The cells were seeded on glass coverslips in 24-well plates to reach a final number of 2 x 10(5) cells/well. Parasites were added to the adhered Lulo and J774 cells in a 10:1 ratio and were incubated at 28 and 37ºC respectively. After 2, 4, 6, 8, and 10 days post-infection, the cells were extensively washed with PBS, fixed with methanol, and stained with Giemsa. The number of internalized parasites was determined by counting at least 400 cultured cells on each coverslip. The results showed continuous interaction between L. chagasi promastigotes with the cell lines. Some ultrastructural characteristics of the amastigote forms were observed using transmission electron microscopy. The highest percentage of infection in Lulo cells was registered on day 6 post-infection (29.6%) and on day 4 in the J774 cells (51%). This work shows similarities and differences in the L. chagasi experimental infection process in the two cell lines. However, Lulo cells emerge as a new model to study the life-cycle of this parasite.

Leishmania (Viannia) lainsoni (Kinetoplastida: Trypanosomatidae), a divergent Leishmania of the Viannia subgenus: a mini review

Leishmania (Viannia) lainsoni is the Leishmania species that presents the most distinct biological (morphology, growth in axenic culture medium), biochemical (enzymatic electrophoresis profile), and molecular biology characteristics, when compared to other species of the Viannia subgenus. Development of promastigote forms of this parasite attached to the wall of the pyloric and hind gut regions of sand fly vectors is a solid characteristic that allows its positioning in the Viannia subgenus. However, taxonomic data from biochemical and molecular techniques on this Leishmania species are still not conclusive. It is evident the difficulty in taxonomically positioning this borderline Leishmania species. In this review we present the data accumulated since L. (Viannia) lainsoni has been described and we discuss its position in the Viannia subgenus.

Isolation and isoenzyme characterization of Leishmania (Viannia) braziliensis from a case of human cutaneous leishmaniasis in northeast centre of the state of São Paulo

The diagnosis of human cutaneous leishmaniasis in small towns is sometimes made without the species identification of the Leishmania, even in areas without previous epidemiological surveys. Here we report the isolation of a Leishmania strain from a patient of Rincão, state of São Paulo, that was identified by isoenzyme characterization as L. (Viannia) braziliensis. Sand fly collections were made in the area where the patient live in order to investigate the likely vector species.