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 phagocyted 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.

Cell populations in lesions of cutaneous leishmaniasis of leishmania (L.) amazonensis- infected rhesus macaques, Macaca mulatta

The cellular nature of the infiltrate in cutaneous lesion of rhesus monkeys experimentally infected with Leishmania (L.) amazonensis was characterized by immunohistochemistry. Skin biopsies from infected animals with active or healing lesions were compared to non-infected controls (three of each type) to quantitate inflammatory cell types. Inflammatory cells (composed of a mixture of T lymphocyte subpopulations, macrophages and a small number of natural killer cells and granulocytes) were more numerous in active lesions than in healing ones. T-cells accounted for 44.7 ± 13.1% of the infiltrate in active lesions (versus CD2+= 40.3 ± 5.7% in healing lesions) and T-cell ratios favor CD8+ cells in both lesion types. The percentage of cells expressing class II antigen (HLA-DR+) in active lesions (95 ± 7.1%) was significantly higher (P < 0.005) from the healing lesions (42.7 ± 12.7%). Moreover, the expression of the activation molecules CD25 (@ 16%), the receptor for interleukin-2, suggests that many T cells are primed and proliferating in active lesions. Distinct histopathological patterns were observed in lesions at biopsy, but healing lesions contained more organized epithelioid granulomas and activated macrophages, followed by fibrotic substitution. The progression and resolution of skin lesions appears to be very similar to that observed in humans, confirming the potential for this to be used as a viable model to study the immune response in human cutaneous leishmaniasis.

Action of pentoxifylline on experimental cutaneous leishmaniasis due to Leishmania (Leishmania) amazonensis

In the animal model of leishmaniasis caused by Leishmania (Leishmania) amazonensis there is a complex mechanism of the host-parasite interaction. The present study was performed to interfere with the inflammatory reaction to the parasites, through immune modulation. Female C5BL/6 isogenic mice were used, some of which were inoculated on the right ear and others on the right footpad with 3.10(6) stationary phase promastigotes of the MHOM/BR/PH8 strain of L. (L.) amazonensis, and were allocated in three groups: the first received pentoxifylline 8mg/kg every 12 h, since the first day; the second one received the same dose since the 40th day of infection and a control group that did not receive any treatment. All the ears excised were analyzed to determine the variation in weight between both ears and for histopathological analyses. A quantification of the parasites was done using the limiting dilution assay. A significant reduction of the number of parasites, was observed among the animals treated which had an accordingly significant reduction on the weight of the ears. Pentoxifylline reduced the macrophages propensity to vacuolation and induced a more effective destruction of the parasites by these cells. Moreover, the group that began the treatment later did not show the same effectiveness.

Evidence for a neotropical origin of Leishmania

Contradictory biogeographic hypotheses for either a Neotropical or a Palaearctic origin of the genus Leishmania have been proposed. Hypotheses constructed on the basis of biogeographic data must be tested against an independent dataset and cannot be supported by biogeographic data alone. In the absence of a fossil record for the Leishmania these two hypotheses were tested against a combined dataset of sequences from the DNA polymerase A catalytic subunit and the RNA polymerase II largest subunit. The phylogeny obtained provided considerable support for a Neotropical origin of the genus Leishmania and leads us to reject the hypothesis for a Palaearctic origin.

Speculations on the origin and evolution of the genus Leishmania

Recently two hypotheses have been proposed for the evolution of Leishmania involving respectively a Neotropical or Paleartic origin for the species. Here an alternative proposal on the phylogeny of Leishmania based on the major divisions within the genus is presented. In this hypothesis a Neotropic origin is retained for L. (Viannia) and Paraleishmania, a recently desribed section within the genus Leishmania, while an African origin is proposed for L. (Leishmania) and possibly Sauroleishmania. The current distribution of Leishmania in the Neotropics is explained as the product of multiple introductions of Leishmania parasites into the New World. Problems with organismal identity in Sauroleishmania and the use of molecular sequence data in inferring phylogenies are also discussed.

Sand fly evolution and its relationship to Leishmania transmission

The evolutionary relationships of sand flies and Leishmania are discussed in this report, which draws distinctions between co-association, co-evolution and co-speciation (or co-cladogenesis). Examples focus on Phlebotomus vectors of Le. infantum and Le. major in the Mediterranean subregion.

Acidic ribosomal proteins and histone H3 from Leishmania present a high rate of divergence

Another additional peculiarity in Leishmania will be discussed about of the amino acid divergence rate of three structural proteins: acidic ribosomal P1 and P2b proteins, and histone H3 by using multiple sequence alignment and dendrograms. These structural proteins present a high rate of divergence regarding to their homologous protein in Trypanosoma cruzi. At this regard, L. (V.) peruviana P1 and T. cruzi P1 showed 57.4% of divergence rate. Likewise, L. (V.) braziliensis histone H3 and acidic ribosomal P2 protein exhibited 31.8% and 41.7% respectively of rate of divergence in comparison with their homologous in T. cruzi.

Effect of Lutzomyia whitmani (Diptera: Psychodidae) salivary gland lysates on Leishmania (Viannia) braziliensis infection in BALB/c mice

Previous reports showed that Lutzomyia longipalpis saliva exacerbate Leishmania braziliensis infection in mice. The sand fly Lu. whitmani is one of the vectors of L. (Viannia) braziliensis (LVb), a causative agent of cutaneous leishmaniasis in the State of Ceará, Brazil. To determine whether saliva of Lu. whitmani could increase the infectivity of LVb in mice, we inoculated groups of BALB/c mice with LVb promastigotes in the presence or absence of the salivary glands lysate from Lu. whitmani. We found that coinjection with Lu. whitmani saliva increased size but not longevity of cutaneous LVb lesions in BALB/c mice, since the formed lesions gradually resolved. The mechanism(s) by which Lu. whitmani saliva might exacerbate LVb infection in BALB/c mice is speculated.

Skin reactions to thimerosal and Leishmania in dogs from a leishmaniasis endemic area: it is better to keep them apart

Positive Montenegro's skin test is a delayed type hypersensitivity reaction widely used as indicative of previous infection with Leishmania in both humans and dogs. Montenegro's antigen consists of a crude Leishmania antigen solution, usually containing thimerosal as preserving agent. In this work it is shown that a large proportion of dogs (11 out of 56) examined in an endemic area of leishmaniasis presented induration at the site of injection of a diluent containing thimerosal alone. This clearly demonstrates that thimerosal leads to a high number of false positive skin reactions in dogs and that its use in Montenegro's skin test antigenic preparations should be avoided.

Leishmania (Leishmania) major-infected rhesus macaques (Macaca mulatta) develop varying levels of resistance against homologous re-infections

Seven rhesus macaques were infected intradermally with 10(7) promastigotes of Leishmania (Leishmania) major. All monkeys developed a localized, ulcerative, self-healing nodular skin lesion at the site of inoculation of the parasite. Non-specific chronic inflammation and/or tuberculoid-type granulomatous reaction were the main histopathological manifestations of the disease. Serum Leishmania-specific antibodies (IgG and IgG1) were detected by ELISA in all infected animals; immunoblot analyses indicated that numerous antigens were recognized. A very high degree of variability was observed in the parasite-specific cell-mediated immune responses [as detected by measuring delayed-type hypersensitivity (DTH) reaction, in vitro lymphocyte proliferation, and gamma interferon (IFN-gamma) production] for individuals over time post challenge. From all the recovered monkeys (which showed resolution of the lesions after 11 weeks of infection), 57.2% (4/7) and 28.6% (2/7) animals remained susceptible to secondary and tertiary infections, respectively, but the disease severity was altered (i.e. lesion size was smaller and healed faster than in the primary infection). The remaining monkeys exhibited complete resistance (i.e. no lesion) to each rechallenge. Despite the inability to consistently detect correlates of cell-mediated immunity to Leishmania or correlation between resistance to challenge and DTH, lymphocyte transformation or IFN-gamma production, partial or complete acquired resistance was conferred by experimental infection. This primate model should be useful for measuring vaccine effectiveness against the human disease.

Identification of a differentially expressed mRNA in axenic Leishmania panamensis amastigotes

Differential display technique was applied in order to identify transcripts which are present in axenic amastigotes but not in promastigotes of the Leishmania panamensis parasites. One of them was cloned and the sequence reveals an open reading frame of 364 amino acids (aprox. 40 kDa). The deduced protein is homologous to the serine/threonine protein kinases and specially to the mitogen activates protein kinases from eukaryotic species. Southern blot analysis suggest that this transcript, named lpmkh, is present in the genome of the parasite as a single copy gene. These results could imply that lpmkh could be involved in the differentiation process or the preservation of amastigotes in axenic conditions.

Isolation of Leishmania infantum, zymodeme MON-1 from canine and human visceral leishmaniasis on Margarita Island, Venezuela

An increase in the incidence of human visceral leishmaniasis (HVL) has been detected in recent years on Margarita Island, located off the NE coast of Venezuela. Recent studies have revealed reactivity to rK39 antigen (Leishmania chagasi) in 20% of 541 sera from domestic dogs in endemic communities; PCR reactions were positive using primers for the L. donovani complex. Here we report that isolates from human and canine infection, identified by isoenzyme analysis, correspond to L. infantum, zymodeme MON-1. This appears to be the first isolation and identification of an isolate from HVL on Margarita Island and demonstrates the presence of this zymodeme in the canine population.

Dissociation between vasodilation and Leishmania infection-enhancing effects of sand fly saliva and maxadilan

In this study, the ability of maxadilan and Lutzomyia longipalpis salivary gland lysate to enhance the infection of CBA mice by Leishmania major and of BALB/c mice by L. braziliensis was tested. No difference was observed between sizes of lesion in CBA mice infected with L. major and treated or not with salivary gland lysate or maxadilan, although they were injected in concentrations that induced cutaneous vasodilation. Although parasites were more frequently observed in foot pads and spleens of animals treated with maxadilan than in the animals treated with salivary gland lysate or saline, the differences were small and not statistically significant. The lesions in BALB/c mice infected with L. braziliensis and treated with maxadilan were slightly larger than in animals that received Leishmania alone. Such differences disappeared 14 weeks after infection, and were statistically significant only in one of two experiments.

Frequency of Infection of Lutzomyia Phlebotomines with Leishmania braziliensis in a Brazilian Endemic Area as Assessed by Pinpoint Capture and Polymerase Chain Reaction

Leishmania infected of Lutzomyia spp. are rare in endemic areas. We tested the hypothesis that there is clustering of infected vectors by combining pinpoint capture with sensitive L. braziliensis kDNA minicircle specific PCR/dot blot in an endemic area in the State of Bahia. Thirty out of 335 samples (10 to 20 sand flies/sample; total of 4,027 female sand flies) were positive by PCR analysis and dot blot leading to a underestimated overall rate of 0.4% positive phlebotomines. However, 83.3% of the positive samples were contributed by a single sector out of four sectors of the whole studied area. This resulted in a rate of 1.5% Leishmania positive phlebotomines for this sector, far above rates of other sectors. Incidence of American cutaneous leishmaniasis cases for this sector was about twice that for other sectors. Our results show that there is a non-homogeneous distribution of Leishmania-infected vectors. Such a clustering may have implications in control strategies against leishmaniasis, and reinforces the necessity of understanding the ecological and geographical factors involved in leishmanial transmission.

Molecular Modeling Approaches for Determining Gene Function: application to a Putative Poly-A Binding Protein from Leishmania amazonensis (LaPABP)

The great expansion in the number of genome sequencing projects has revealed the importance of computational methods to speed up the characterization of unknown genes. These studies have been improved by the use of three dimensional information from the predicted proteins generated by molecular modeling techniques. In this work, we disclose the structure-function relationship of a gene product from Leishmania amazonensis by applying molecular modeling and bioinformatics techniques. The analyzed sequence encodes a 159 aminoacids polypeptide (estimated 18 kDa) and was denoted LaPABP for its high homology with poly-A binding proteins from trypanosomatids. The domain structure, clustering analysis and a three dimensional model of LaPABP, basically obtained by homology modeling on the structure of the human poly-A binding protein, are described. Based on the analysis of the electrostatic potential mapped on the model's surface and conservation of intramolecular contacts responsible for folding stabilization we hypothesize that this protein may have less avidity to RNA than it's L. major counterpart but still account for a significant functional activity in the parasite. The model obtained will help in the design of mutagenesis experiments aimed to elucidate the mechanism of gene expression in trypanosomatids and serve as a starting point for its exploration as a potential source of targets for a rational chemotherapy.