Assessment of PCR in the detection of Leishmania spp in experimentally infected individual phlebotomine sandflies (Diptera: Psychodidae: Phlebotominae)

DNA amplification by the polymerase chain reaction (PCR) was applied in the investigation of the presence of Leishmania (Kinetoplastida: Trypanosomatidae) parasites in single phlebotomine sandflies. Three phlebotomine/parasite pairs were used: Lutzomyia longipalpis/Leishmania chagasi, Lutzomyia migonei/Leishmania amazonensis and Lutzomyia migonei/Leishmania braziliensis, all of them incriminated in the transmission of visceral or cutaneous leishmaniasis. DNA extraction was performed with whole insects, with no need of previous digestive tract dissection or pooling specimens. The presence of either mouse blood in the digestive tract of the sandflies or the digestive tract itself did not interfere in the PCR. Infection by as few as 10 Leishmania sp. per individual were sufficient for DNA amplification with genus-specific primers. Using primers for L. braziliensis and L. mexicana complexes, respectively, it was possible to discriminate between L. braziliensis and L. amazonensis in experimentally infected vectors (L. migonei).     

Cytokine control of Leishmania infection in the BALB/c mouse: enhancement and inhibition of parasite growth by local administration of IL-2 or IL-4 is species and time dependent

The therapeutic potential of locally injected interleukin-2 (IL-2) or interleukin-4 (IL-4) was studied in the footpads of Leishmania mexicana or Leishmania major infected BALB/c mice. The disease state was measured both pathologically, by measuring lesion size, and parasitologically, by counting total parasite numbers from infected footpads. IL-2 (0.5 microgram/dose) or IL-4 (0.1 microgram/dose) was administered either early, 1 day and/or 15 days after infection, or late, after palpable lesions had developed. Results differed markedly depending on which Leishmania species was used and at what time during the course of disease that therapy commenced. Both L. major and L. mexicana infections, as measured by footpad thickness and parasite number, were exacerbated if IL-4 was injected into the infected footpads early, during the first two weeks of infection. Paradoxically, late intralesional injection (i.e. after measurable lesions had developed) of IL-4 markedly inhibited both lesion size and parasite growth in L. major, though not L. mexicana, infected mice. IL-2 had no measurable effect on the course of L. major infections no matter when or how often, the infected footpads of mice were treated. However, early administration of IL-2 did exacerbate L. mexicana lesion and parasite growth while late treatment had no effect. Generally, but not always, increases in footpad size correlated with increases in parasite number.     

Amyloidosis in experimental tegumentary leishmaniasis in mice

Renal and hepatosplenic amyloidosis was found in chronic cutaneous leishmaniasis in mice infected with 10(6) purified amastigotes from lesions produced by the H21 strain of Leishmania mexicana amazonensis. After 1 year a progressive lesion leading to metastasis was observed in most animals.     

Early infection with Leishmania major restrains pathogenic response to Leishmania amazonensis and parasite growth

Experimental models of infection with Leishmania spp. have provided knowledge of several immunological events involved in the resistance mechanism used by the host to restrain parasite growth. It is well accepted that concomitant immunity exists, and there is some evidence that it would play a major role in long-lasting acquired resistance to infection. In this paper, the resistance to Leishmania amazonensis infection in C57BL/6 mice infected with Leishmania major was investigated. C57BL/6 mice, which spontaneously heal lesions caused by infection with L. major, were infected with L. amazonensis at different times before and after L. major. We demonstrated that C57BL/6 mice previously infected with L. major restrain pathogenic responses induced by L. amazonensis infection and decrease parasite burdens by one order of magnitude. Co-infected mice showed production of IFN-gamma in lesions similar to mice infected solely with L. major, but higher TNF-alpha and nitric oxide synthase (iNOS) mRNA expression was observed. Surprisingly, the restrained pathogenic response was not related to IL-10 production, as evidenced by lower levels of both mRNA, protein expression in lesions from co-infected mice and in co-infections in IL-10(-/-) mice. Examination of the inflammatory infiltrate at the site of infection showed a reduced number of monocytes and lymphocytes in L. amazonensis lesions. Additionally, differential production of the CCL3/MIP-1 alpha and CCL5/RANTES was observed. We suggest that the control of lesion progression caused by L. amazonensis in C57BL/6 mice pre-infected with L. major is related to the induction of a down-regulatory environment at the site of infection with L. amazonensis.     

CTL responses to Leishmania mexicana gp63-cDNA vaccine in a murine model

Immunity to Leishmania is believed to be strongly dependent upon the activation of Th1 immune responses, although the exact role of cytotoxic T lymphocytes (CTLs) has not yet been determined. The aims of this study were to establish a suitable cytotoxicity assay to measure CTL activity and to compare immunity induced by Leishmania mexicana gp63 cDNA via i.m. injection and gene gun immunization in the BALB/c mouse model. The CTL activity was evaluated by short-term ⁵¹Cr-release cytotoxicity assays against CT26 tumour cells transfected with L. mexicana gp63 cDNA and dendritic cells (DCs) loaded with soluble Leishmania antigen (SLA) as targets. The results clearly demonstrated that higher protection to L. mexicana infection was induced by gene gun DNA-immunization vs. i.m. injection. Cytotoxic T lymphocyte activity of splenocytes was observed in mice immunized either with L. mexicana gp63 cDNA or SLA and long-lived CTL activity was observed in immunized and/or re-challenged mice but not naïve mice infected with the parasite.     

Leishmania amazonensis infections in Oryzomys acritus and Oryzomys nitidus from Bolivia

Three of thirteen Oryzomys acritus, Emmons and Patton 2005 (Rodentia: Muridae: Sigmodontinae) and 3 of 17 Oryzomys nitidus, Thomas 1884, collected from No?l Kempff National Park, Bolivia, from 2002 to 2005, tested positive for Leishmania (Leishmania) amazonensis or L. (L.) mexicana and negative for Leishmania (Viannia) spp. using the polymerase chain reaction (PCR). Based on previous records of L. (L.) amazonensis in humans, rodents, and sand flies from Bolivia, and the geographic distributions of L. (L.) amazonensis and L. (L.) mexicana, it was concluded that the Oryzomys were infected with L. (L.) amazonensis. These results identify two additional species of Oryzomys as hosts of L. (L.) amazonensis, and identify an ecological region of Bolivia where L. (L.) amazonensis is enzootic.     

Differentiation and limited proliferation of isolated Leishmania mexicana amastigotes at 27 degrees C.

We have demonstrated here that while many amastigotes of both Leishmania mexicana amazonensis and Leishmania mexicana mexicana differentiate to promastigotes when placed in culture at 27 degrees C, many others remain as amastigotes in their proliferative cycle. We have used this system to examine the effects of the anti-leishmanial compounds amphotericin B, 4-pentenoate and sodium stibogluconate (Pentostam) on the proliferation and differentiation of isolated extracellular amastigotes. Amphotericin B and 4-pentenoate showed little preferential effect on amastigote proliferation over promastigote proliferation although Leishmania mexicana mexicana (strain M379) was generally more resistant to these compounds than Leishmania mexicana amazonensis (strain LV78). This relative resistance was also observed in axenic cultures of proliferating promastigotes. L.m. mexicana amastigote differentiation was inhibited by amphotericin B and 4-pentenoate. Pentostam displayed a greater effect on amastigote differentiation than proliferation in both sub-species although again, a higher concentration was required to produce the same effect in L.m. mexicana.     

Influence of vertebrate blood meals on the development of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis in the sand fly Lutzomyia migonei (Diptera: Psychodidae)

The effect of blood meals from humans and seven domestic, wild, or laboratory animals (dogs, horses, chickens, rats, opossums, mice, and hamsters) on the development of Leishmania braziliensis and L. amazonensis was studied in the sand fly Lutzomyia migonei. The development of L. braziliensis and L. amazonensis exhibited peripylarian and suprapylarian patterns of development, respectively, in the sand fly gut with all blood meals tested. The blood meal sources influenced the infection rate of the sand flies. In both the Leishmania species, the highest parasite density was obtained with blood from wild rats followed by skunk, human, and horse. The epidemiological significance of these observations may be related to the distribution of leishmaniasis and needs to be evaluated further.     

Leishmaniasis in Brazil: XII. Observations on cross-immunity in monkeys and man infected with Leishmania mexicana mexicana, L. m. amazonensis, L. braziliensis braziliensis, L. b. guyanensis and L. b. panamensis.

Cross-immunity trials in monkeys (Cebus apella apella) and observations on experimental and natural infections in man confirm the separate identity of L. mexicana mexicana, L. m. amazonensis, L. b. braziliensis, L. b. guyanensis and L. b. panamensis. Neither L. m. mexicana nor L. m. amazonensis infections gave protection against subsequent challenge with parasites of the L. braziliensis complex; but recovery from infection with subspecies of L. braziliensis in most cases gave firm resistance to infection with the mexicana parasites. The failure of certain New World leishmanias to immunize against each other has an important bearing on taxonomy, future attempts to prepare vaccines against Leishmania, epidemiology and diagnosis.     

Different patterns of disease in two inbred mouse strains infected with a clone of Leishmania mexicana amazonensis

We have infected BALB/c and C57BL/6 mice with a cloned Leishmania mexicana amazonensis population, obtained from the "Maria" strain. Progression of infection and histopathological examination confirmed the extreme susceptibility of BALB/c mice and the resistant pattern of the C57 BL/6. Anti-Leishmania antibody titers were higher in BALB/c than in C57BL/6 mice through the period of infection. Tests of delayed type hypersensitivity reaction with Leishmania antigens were positive in both strains in the beginning of the infection, but were negative later on in BALB/c mice. Our results are similar to those obtained with mixed parasite populations, and rule out the possibility of selection among different parasite subpopulations as responsible for the divergent course of the disease exhibited by these two strains of mice.     

Preliminary study towards a novel experimental model to study localized cutaneous leishmaniasis caused by Leishmania (Leishmania) mexicana

There is not an experimental model of localized cutaneous leishmaniasis (LCL) caused by Leishmania (Leishmania) mexicana. The aim of the present study was to characterize the clinical and histological features of Peromyscus yucatanicus experimentally infected with L. (L.) mexicana. A total of 54 P. yucatanicus (groups of 18) were inoculated with 1x10(6) promastigotes of L. (L.) mexicana in the base of the tail. They were euthanized at three and six months post experimental infection. The control group was inoculated with RPMI-1640. The predominant clinical sign observed was a single ulcerated lesion in 27.77% (5/18) and in 11.11% (2/18) P. yucatanicus at three and six months respectively. The histological pattern described as chronic granulomatous inflammation with or without necrosis was found in 7/7 (100%) biopsies of euthanized P. yucatanicus at three (n = 5) and six (n = 2) months, respectively. These results resembled clinical and histological features caused by L. (L.) mexicana in humans, and support the possibility to employ P. yucatanicus as a novel experimental model to study LCL caused by this parasite.     

Herbicides to curb human parasitic infections: in vitro and in vivo effects of trifluralin on the trypanosomatid protozoans.

Leishmaniasis is a major tropical disease for which current chemotherapies, pentavalent antimonials, are inadequate and cause severe side effects. It has been reported that trifluralin, a microtubule-disrupting herbicide, is inhibitory to Leishmania amazonensis. In this study, the in vitro effect of trifluralin on different species of trypanosomatid protozoans was determined. In addition to L. amazonensis, trifluralin is effective against Leishmania major and Leishmania tropica, which cause cutaneous infections, Leishmania donovani, which causes visceral disease, Leishmania panamensis, which may cause mucocutaneous infection, and Trypanosoma brucei, an important human and veterinary pathogen. Moreover, most encouragingly, trifluralin is effective in vivo as a topical ointment against L. major and Leishmania mexicana murine cutaneous leishmaniasis. Thus, trifluralin is a promising lead drug for several related, prevalent tropical diseases: leishmaniasis, trypanosomiasis of animals, and, possibly, African trypanosomiasis in humans.     

Pathophysiology of experimental leishmaniasis: the role of parasite physiology in the development of metastatic disease

This paper addresses the issue of how physiological properties of Leishmania determine the pattern of development of disseminated leishmaniasis in the mammalian host. It presents direct experimental evidence from in vivo studies that species of Leishmania differ in their capacity to multiply in cutaneous and visceral sites which results in differences in the pattern and rate of development of leishmaniasis. It was found that Leishmania mexicana amazonensis begins to multiply in the cutaneous site of inoculation within 7 days. Parasites, detected in the liver and spleen at 4 weeks, increased 100-fold during the next 4 months. However, the slow multiplication of L. mexicana amazonensis in the liver and spleen was more apparent than real. Parasites implanted in those organs of athymic nude mice by an intravenous injection were rapidly eliminated with a half-time of 16 hr. Thus, the parasites found in small numbers in the liver during the development of disseminated cutaneous disease in mice are most likely those which have been recently removed from the blood. Those few parasites that are not removed from the blood can establish metastatic foci in distant cutaneous sites, and replicate progressively once there.     

Depression of virus-specific cytotoxic T-cell responses during murine malaria

Mice with self-limiting P. yoelii or fatal P. berghei infections exhibited a markedly impaired ability to mount specific splenic cytotoxic T-lymphocyte responses to immunization with infectious ectromelia (EV), vaccinia (VAC), or lymphocytic choriomeningitis viruses (LCMV). Lymph node responsiveness, however, was not impaired. Primary CTL responses were depressed in mice immunized 7 days after P. berghei infection, while in P. yoelii-infected mice, depressed responses were detected only during the period corresponding with maximal parasitemia (days 9-12). Secondary VAC-specific CTL responses in vitro by spleen cells of mice previously immunized during P. yoelii infection were also depressed if UV-inactivated rather than infectious VAC was used for immunization. In addition, spleen cells of mice already immune to VAC failed to yield normal secondary CTL responses in vitro during the period of maximal P. yoelii parasitaemia. Collectively, these findings indicate that, during patent malaria infections, priming for and expression of virus-specific CTL responses may be inhibited.     

New administration model of trans-chalcone biodegradable polymers for the treatment of experimental leishmaniasis

The present study was designed to investigate a new administration model and the antileishmanial activity of a semi-synthetic chalcone, benzylideneacetophenone (trans-chalcone). The antileishmanial activity of this product was first tested in vitro against promastigotes of L. braziliensis, L. tropica, L. infantum and L. amazonensis. An in vivo experiment was carried out using subcutaneous administration of trans-chalcone and implants of synthetic biodegradable polymers, polylactic acid (PLA) and polylactic/glycolic acid (PLGA). This compound showed potent inhibitory effects on the growth of all Leishmania strains examinated. Subcutaneous administration of trans-chalcone at a single dose of 4 mg/kg of body weight reduced lesion development in mice infected with L. amazonensis. A similar inhibition of the lesion growth in mice treated with trans-chalcone and pentamidine was observed. PLA and PGLA implants of trans-chalcone at 4 mg/kg were administered to mice infected with L. amazonensis. PLGA implants induced a highest reduction in the lesion size (31.25%) than PLA implants (10.75%). Treatment in vitro with trans-chalcone at IC50, completely inhibited the pathogenicity of this parasite in vivo. The development of this model provides a new practical technique for delivering drugs and can be useful for experimental leishmaniasis treatment.     

Isolation of Leishmania mexicana amazonensis from the bone marrow in a case of American visceral leishmaniasis

The first documented human case of visceral leishmaniasis caused by L. mexicana amazonensis is reported. Leishmania were isolated from bone marrow aspirate material from a typical visceral leishmaniasis patient. Further characterization by isoenzyme electrophoresis and by a panel of species- and subspecies-specific monoclonal antibodies established its classification as L. m. amazonensis.     

Surface antigenic change during differentiation of a parasitic protozoan, Leishmania mexicana: Identification by monoclonal antibodies.

The fusion of SP2/0 myeloma cells with spleen cells from mice immunized with Leishmania mexicana amazonensis promastigotes produced hybridoma clones. Indirect immunofluorescent antibody assay with live leishmanias showed that the monoclonal antibody 6H12 recognized only the antigens bound to the surface of L. mexicana amazonensis promastigotes. It also showed that the antibody bound to neither amastigotes of this species nor to other Leishmania species--i.e., L. braziliensis braziliensis, L. tropica, and L. donovani. Monoclonal antibodies from three other clones (4D11, 4H9, and 6A11) were found to compete with 6H12 for binding to L. mexicana promastigotes. With lysates of [35S]methionine-labeled promastigotes, all four monoclonal antibodies precipitated the same triplet set of protein bands at the approximately equal to 68,000-dalton region, whereas another monoclonal antibody (6G5) precipitated a different band at approximately equal to 90,000 daltons. During differentiation of L. mexicana amazonensis from amastigotes to promastigotes, there was a 4- to 8-fold increase above the initial level in the binding of 6H12 monoclonal antibody to leishmanias, as detected by enzyme-linked immunosorbent assay and quantitative fluorometric assay, respectively. Thus, we have demonstrated the use of monoclonal antibodies as probes for antigens that change during leishmanial differentiation.     

Studies on the topical treatment of experimental cutaneous leishmaniasis: the therapeutic effect of methyl benzethonium chloride and the aminoglycosides, gentamicin and paromomycin

BALB/c mice infected with either Leishmania major or Leishmania mexicana were treated twice a day for 10 days with an ointment containing 15% gentamicin or paromomycin, with or without 12% methylbenzethonium chloride (MBCl). It was found that topical application of either paromomycin or MBCl cured the parasite lesion, and that combined treatment with the two compounds had an additive effect. However, after four days' therapy there was a severe inflammatory response at the treatment site, and in most experiments mice relapsed and renewed lesion growth was observed. It is suggested that a non-specific inflammatory reaction may be an important component of the therapeutic response. In further experiments, L. major infected mice treated with paromomycin and MBCl which had cured but not relapsed 58 days after treatment were challenged with a similar dose of the homologous parasite. Lesions developed 16 days post-infection, and the number of parasites recovered from these lesions was similar to that recovered from lesions in control mice. Therefore no protective immunity had been induced by chemotherapy.     

Immunomodulatory effect of Canova medication on experimental Leishmania amazonensis infection

This study investigates the action of Canova medication (CM) on experimental infection by Leishmania (Leishmania) amazonensis, utilizing in vitro and in vivo assays. For the in vitro tests, Balb/c mouse peritoneal macrophages (5x10(5) cells in 500 microl of culture medium, supplemented with 10% fetal calf serum, penicillin (100 U/ml) and streptomycin (0.1 mg/ml) (were distributed in 24-well plates and CM was added at concentrations of 20 or 40%. Twenty-four hours later, the macrophages were infected with Leishmania amastigotes in culture medium. The effect of CM on macrophages leishmanicidal activity in 24 and 48 h cultures was evaluated by determining infection index and measuring nitric oxide (NO) production. The in vivo tests were performed in mice infected with 10(7)L. (L.) amazonensis promastigotes injected in to the right hind footpad (25 microl in phosphate buffered saline). The progression of the lesions was examined over a 9-week period by measuring footpad swelling, and the parasite load in regional lymph nodes and spleen. The in vitro results showed that at 40% CM reduced the infection index, and induced NO production in the elicited macrophages, which suggests that the inhibitory effect on infection index may be mediated by NO. In the in vivo infection, when administered, orally or subcutaneously in mice, CM reduced infection by L. (L.) amazonensis in the paws, resulting in smaller lesions. CM treatment also decreased parasite load in the regional popliteal lymph nodes and in the spleen. These results suggest that CM modulates experimental infection by L. (L.) amazonensis, controlling infection progression and limiting dissemination.     

Inefficacy of metronidazole in experimental infections of Leishmania donovani, L. mexicana, and Trypanosoma brucei brucei

Metronidazole has been claimed in several earlier reports to be active in human cases of leishmaniasis and trypanosomiasis. Its efficacy against the protozoa causing these diseases was tested in hamsters infected with Leishmania mexicana or L. donovani, and in mice infected with Trypanosoma brucei brucei. In separate experiments, hamsters were either inoculated intradermally into the nose with 5 million amastigotes of L. mexicana or intracardially with 10-30 million amastigotes of L. donovani, and mice were infected intraperitoneally with 30 million T. b. brucei. Metronidazole was administered in four oral doses on alternate days for a total of 375 mg/kg to hamsters and 500 mg/kg to mice. Sodium stibogluconate (Pentostam) served as a positive control. In hamsters the extent of infection was assessed by the appearance of flagellates in blood agar cultures of nose and spleen, by counting amastigotes in nose and liver impression smears, and by measuring the size of nose lesions. Ultrastructure of nose lesions before and after treatment with metronidazole or Pentostam was also evaluated. Infection in mice was assessed by the extent of parasitemia and/or survival to 30 days. In no case did metronidazole-treated animals differ from untreated controls. Metronidazole shows no activity against experimental infections of leishmaniasis or trypanosomiasis in these animal models.