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.

Leishmanial infection: analysis of its first steps. A review

The first steps in leishmaniasis are critical in determining the evolution of the disease. Major advances have recently been done in understanding this crucial moment. Fundamental research in parasite-vector interaction, parasite biology, insect saliva, and vertebrate host response have shed new light and uncovered a most fascinating and complex moment in leishmaniasis. We review here some of these aspects and we try to connect them in a logical framework.

Seasonal transmission of Leishmania (Leishmania) mexicana in the state of Campeche, Yucatan Peninsula, Mexico

In the Yucatan Peninsula, Mexico, localized cutaneous leishmaniasis (LCL) caused by Leishmania (Leishmania) mexicana is a typical wild zoonosis restricted to the forest, and humans are only accidentally involved. The transmission of L. (L.) mexicana has been related to the patient's occupation: "chicleros"(gum collectors) and agricultural workers. The objective of this study was to document L. (L.) mexicana seasonally of transmission in endemic areas of LCL in the state of Campeche, Yucatan Peninsula, Mexico. The timing of incidence of LCL in humans during 1993-1994, as well as the rate and time of infection in rodents and sand flies between February 1993 and March 1995 were analyzed. Rodents and sand flies were found infected between November and March, when men carried out their field activities and are exposed. Based on results analyzed, it is concluded that L. (L.) mexicana in the endemic area of LCL in the state of Campeche, Yucatan Peninsula, Mexico, presents a seasonal transmission restricted to the months of November to March. The knowledge of the timing of the transmission cycle in an endemic area of leishmaniasis is very important because intervention measures on the high-risk focus and population might be restricted.

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.

Leishmania major-specific B cells are necessary for Th2 cell development and susceptibility to L. major LV39 in BALB/c mice.

B lymphocytes are considered to play a minimal role in host defense against Leishmania major. In this study, the contribution of B cells to susceptibility to infection with different strains of L. major was investigated in BALB/c mice lacking mature B cells due to the disruption of the IgM transmembrane domain (microMT). Whereas BALB/c microMT remained susceptible to infection with L. major IR173 and IR75, they were partially resistant to infection with L. major LV39. Adoptive transfer of naive B cells into BALB/c microMT mice before infection restored susceptibility to infection with L. major LV39, demonstrating a role for B cells in susceptibility to infection with this parasite. In contrast, adoptive transfer of B cells that express an IgM/IgD specific for hen egg lysozyme (HEL), an irrelevant Ag, did not restore disease progression in BALB/c microMT mice infected with L. major LV39. This finding was likely due to the inability of HEL Tg B cells to internalize and present Leishmania Ags to specific T cells. Furthermore, specific Ig did not contribute to disease progression as assessed by transfer of immune serum in BALB/c microMT mice. These data suggest that direct Ag presentation by specific B cells and not Ig effector functions is involved in susceptibility of BALB/c mice to infection with L. major LV39.

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.

Anti-leishmania effector functions of CD4+ Th1 cells and early events instructing Th2 cell development and susceptibility to Leishmania major in BALB/c mice.

Resistance and susceptibility to infection with the intracellular parasite, Leishmania major, are mediated by parasite-specific CD4+ Th1 and Th2 cells, respectively. It is well established that the protective effect of parasite-specific CD4+ Th1 cells is largely dependent upon the IFN-gamma produced. However, recent results indicate that the effect of Th1 cells on resolution of lesions induced by L. major in genetically resistant mice also requires a functional Fas-FasL pathway of cytotoxicity. In contrast to resistant mice, susceptible BALB/c mice develop aberrant Th2 responses following infection with L. major and consequently suffer progressive disease. These outcomes clearly depends upon the production of interleukin 4 (IL-4) early after infection. We have shown that a burst of IL-4 mRNA, peaking in draining lymph nodes of BALB/c mice 16 hrs after infection, occurs within CD4+ T cells that express V beta 4-V alpha 8 T cell receptors. In contrast to control and V beta 6-deficient mice, V beta 4-deficient BALB/c mice were resistant to infection, demonstrating the role of these cells in Th2 development. The early IL-4 response was absent in these mice, and Th1 responses occurred following infection. The LACK antigen of L. major induced comparable IL-4 production in V beta 4-V alpha 8 CD4+ T cells. Thus, the IL-4 required for Th2 development and susceptibility to L. major is produced by a restricted population of V beta 4-V alpha 8 CD4+ T cells after cognate interaction with a single antigen from this complex parasite. The IL-4 produced rapidly by these CD4+ T cells induces within 48 hours a state of unresponsiveness to IL-12 among parasite-specific CD4+ T cell precursors by downregulating the IL-12 receptor beta 2 chain expression.

IL-4 rapidly produced by V beta 4 V alpha 8 CD4+ T cells instructs Th2 development and susceptibility to Leishmania major in BALB/c mice.

BALB/c mice develop aberrant T helper 2 (Th2) responses and suffer progressive disease after infection with Leishmania major. These outcomes depend on the production of interleukin-4 (IL-4) early after infection. Here we demonstrate that the burst of IL-4 mRNA, peaking in draining lymph nodes of BALB/c mice 16 hr after infection, occurs within CD4+ T cells that express V beta 4 V alpha 8 T cell receptors. In contrast to control and V beta 6-deficient BALB/c mice, V beta 4-deficient BALB/c mice were resistant to infection, demonstrating the role of these cells in Th2 development. The early IL-4 response was absent in these mice, and T helper 1 responses occurred following infection. Recombinant LACK antigen from L. major induced comparable IL-4 production in V beta 4 V alpha 8 CD4+ cells. Thus, the IL-4 required for Th2 development and susceptibility to L. major is produced by a restricted population of V beta 4 V alpha 8 CD4+ T cells after cognate interaction with a single antigen from this complex organism.

The utility of rhesus monkey (Macaca mulatta) and other non-human primate models for preclinical testing of Leishmania candidate vaccines

Leishmaniasis causes significant morbidity and mortality, constituting an important global health problem for which there are few effective drugs. Given the urgent need to identify a safe and effective Leishmania vaccine to help prevent the two million new cases of human leishmaniasis worldwide each year, all reasonable efforts to achieve this goal should be made. This includes the use of animal models that are as close to leishmanial infection in humans as is practical and feasible. Old world monkey species (macaques, baboons, mandrills etc.) have the closest evolutionary relatedness to humans among the approachable animal models. The Asian rhesus macaques (Macaca mulatta) are quite susceptible to leishmanial infection, develop a human-like disease, exhibit antibodies to Leishmania and parasite-specific T-cell mediated immune responses both in vivo and in vitro, and can be protected effectively by vaccination. Results from macaque vaccine studies could also prove useful in guiding the design of human vaccine trials. This review summarizes our current knowledge on this topic and proposes potential approaches that may result in the more effective use of the macaque model to maximize its potential to help the development of an effective vaccine for human leishmaniasis.

Detection of Leishmania infantum in naturally infected Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae) and Canis familiaris in Misiones, Argentina: the first report of a PCR-RFLP and sequencing-based confirmation assay

In this study, a genotypification of Leishmaniawas performed using polimerase chain reaction-restriction fragment length polymorfism (PCR-RFLP) and sequencing techniques to identify species of Leishmaniaparasites in phlebotomine sand flies and dogs naturally infected. Between January-February of 2009, CDC light traps were used to collect insect samples from 13 capture sites in the municipality of Posadas, which is located in the province of Misiones of Argentina. Sand flies identified as Lutzomyia longipalpiswere grouped into 28 separate pools for molecular biological analysis. Canine samples were taken from lymph node aspirates of two symptomatic stray animals that had been positively diagnosed with canine visceral leishmaniasis. One vector pool of 10 sand flies (1 out of the 28 pools tested) and both of the canine samples tested positively for Leishmania infantumby PCR and RFLP analysis. PCR products were confirmed by sequencing and showed a maximum identity with L. infantum. Given that infection was detected in one out of the 28 pools and that at least one infected insect was infected, it was possible to infer an infection rate at least of 0.47% for Lu. longipalpisamong the analyzed samples. These results contribute to incriminate Lu. longipalpis as the vector of L. infantumin the municipality of Posadas, where cases of the disease in humans and dogs have been reported since 2005.

The molecular detection of different Leishmania species within sand flies from a cutaneous and visceral leishmaniasis sympatric area in Southeastern Brazil

Over the last 20 years, there has been an increase in the number of leishmaniasis cases in Brazil. Belo Horizonte (BH) is one of the most highly populated Brazilian cities that is affected by visceral leishmaniasis (VL). The health services in BH are coordinated by a central nucleus that is subdivided into nine sanitary districts. Historically, the highest level of human VL cases was found in the northeast sanitary district (NSD). The objective of our study was to detect Leishmania infection in the phlebotomine sand flies collected in the NSD by dissection and molecular approaches. Following the occurrence of human VL cases in 2005, entomological captures were performed from July 2006-June 2007. Out of the 245 sand flies dissected, only three Lutzomyia longipalpis spp contained flagellates. The female sand flies were grouped into 120 pools according to date, collection site and species, with approximately 10 individual sand flies in each pool. Subsquently, the DNA was extracted and Leishmania spp and other parasites were detected and identified by polymerase chain reaction (PCR) and PCR-restriction fragment length polymorfism. Leishmania infantum was present in at least 19% of the Lu. longipalpis collected, in 3.8% of the Nyssomiya whitmani collected, in 33.3% of the Evandromiya termitophila collected and in 14.3% of the Nyssomiya intermedia collected. When the females of the cortelezzii complex were compared with each other, 3.2% of the females were infected with Leishmania braziliensis, whereas 3.2% of the females were infected with trypanosomatids.

An experimental protocol for the establishment of dogs with long-term cellular immune reactions to Leishmania antigens

Domestic dogs are considered to be the main reservoirs of zoonotic visceral leishmaniasis. In this work, we evaluated a protocol to induce Leishmania infantum/Leishmania chagasi-specific cellular and humoral immune responses in dogs, which consisted of two injections of Leishmania promastigote lysate followed by a subcutaneous inoculation of viable promastigotes. The primary objective was to establish a canine experimental model to provide positive controls for testing immune responses to Leishmania in laboratory conditions. After inoculation of viable promastigotes, specific proliferative responses of peripheral blood mononuclear cells (PBMCs) to either Leishmania lysate or recombinant proteins, the in vitro production of interferon-γ by antigen-stimulated PBMCs and a significant increase in circulating levels of anti-Leishmania antibodies were observed. The immunized dogs also displayed positive delayed-type hypersensitivity reactions to Leishmania crude antigens and to purified recombinant proteins. An important finding that supports the suitability of the dogs as positive controls is that they remained healthy for the entire observation period, i.e., more than seven years after infection. Following the Leishmania antigen lysate injections, the infection of dogs by the subcutaneous route appears to induce a sustained cellular immune response, leading to an asymptomatic infection. This provides a useful model for both the selection of immunogenic Leishmania antigens and for immunobiological studies on their possible immunoprotective activities.

In vitro activity of amphotericin B cochleates against Leishmania chagasi

Cochleate delivery vehicles are a novel lipid-based system with potential for delivery of amphotericin B (AmB). In this study, the efficacy of cochleates was evaluated by examining the in vitro activity of AmB cochleates (CAMB) against Leishmania chagasi in a macrophage model of infection. We demonstrate that CAMB is nontoxic to macrophages at concentrations as high as 2.5 μg/mL, whereas the conventional formulation, AmB deoxycholate, showed high toxicity at this concentration. The in vitro activity of CAMB against L. chagasi was found to be similar to that of the reference drug AmB deoxycholate, with ED50s of 0.017 μg/mL and 0.021 μg/mL, respectively. Considering that L. chagasi affects organs amenable to cochleate-mediated delivery of AmB, we hypothesize that CAMB will be an effective lipid system for the treatment of visceral leishmaniasis.