Leishmania braziliensis ssp. in the nasal mucosa of guinea pigs inoculated in the tarsi

Two lots of 20 young male guinea pigs were inoculated subcutaneously in the tarsi with 10 (elevated to fourth potency) amastigotes of Leishmania braziliensis or L. b. guyanensis to study the susceptibility of this Neotropical hystricomorph rodent the autochthonous parasites. Almost 50% of the animals showed lesions in the inoculation site and had parazitations that were infective to hamsters, as shown by inoculating homogenates of the dermal lesion, of the spleen, of the liver, and of the nasal mucosa into hamsters at 20, 40, 60 and 120 days after inoculation of the guinea pig. Smears of the above organs showed the presence of amastigotes. Parasites inoculated into the tarsi were detected early in the skin, spleen, and liver of the guinea pig host. Blood cultures made by cardiopuncture on sacrifice of the guinea pigs were uniformly negative. The nasal mucosa of nearly all animals positive in the skin or viscera was invaded early by the parasites, although with grater frequency between 60 and 120 days post-inoculation. The use of this model for the study of mucocutaneous parasitism by L. brasiliensis is discussed, together with the phenomena of parasitism at a distance from the inoculation site, the temperature of the body regions affected, and the possible genetic influence on susceptibility of the guinea pig to L. brasiliensis.

New records of phlebotomine sand flies (Diptera: Psychodidae) from Ecuador

The plebotomine sand fly fauna of Ecuador was surveyed in two 3-month collecting trips made in 1988 and 1990. A total of 12 provinces were visited, including three (Bolivar, Loja and Morona Santiago) from wich no previous records to phlebotomines existed. Forty-six species were collected, 13 of wich, together with 1 subspecies and 1 genus (Warileya) represented new records for the country. This survey increases the known number of species in Ecuador to 60. The distribuition of Ecuadorian sand flies is discussed in the light of these new findings.

Antigenic differences among Leishmania amazonensis isolates and their relationship with distinct clinical forms of the disease

Immunoblot analysis was used to investigate antigenic differences among clinical isolates of Leishmania amazonensis and their role in the etiology of the diseases. Western blots of promastigote homogenates were analyzed with either monoclonal antibodies (MAbs) specific for the L. mexicana complex (M-4, M-6, M-9 and M-11) or polyclonal sera from L. amazonensis infected patients with the various forms of clinical disease. In the case of the MAbs, no significant variation was observed among the strains of L. amazonensis, isolated from cases of cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), diffuse cutaneous leishmaniasis (DCL), visceral leishmaniasis (VL) or post kala-azar dermal leishmaniasis (PKDL), in either the relative morbility (Mr) or the quantitative amount (intensity) of the antigenic determinats. In the case of the sera of the infected patients, the patterns of antigenic reactivity of these strains revealed that, despite showing the presence of shared antigens, differences were observed between some of the antigenic components of the various isolates of L. amazonensis that were recognized by a single serum. Differences were also demonstrated between the antigenic determinants of a single isolate of L. amazonensis that were recognized by the different patient's sera. No apparent association was consistently found, however, between the Mr components identified in these isolates and clinical form of the disease or the geographical area of isolation. In addition, the spectrum of antigens recognized by the sera from patients with the same clinical form were not identical; although in some instances, similar Mr antigens were shared. These results indicate that isolates of L. amazonensis are not antigenically identical (homogeneous) and that the immune responses (antibodies) observed among infected patients are heterogeneous.

Development of Leishmania (Viannia) braziliensis Vianna, 1911 in Lutzomyia intermedia (Lutz & Neiva, 1912) (Diptera: Psychodidae: Phlebotominae) under experimental conditions

The development of Leishmania (Viannia) braziliensis in experimentally infected Lutzomyia intermedia, showed colonization of the hindgut from 48h after the infective blood-meal, and the migration flagellates to the foregut, with a massive infection of the cardia at the 5th day post infection. Up to 10 days following the infective blood-meal, very few parasites were seen in the pharynx and cibarium. The role of L. intermedia as a vector of cutaneous leishmaniasis is discussed according to the estabilished criteria.

Rapid diagnostic tests for non-malarial febrile illness in the tropics.

The recent roll-out of rapid diagnostic tests (RDTs) for malaria has highlighted the decreasing proportion of malaria-attributable illness in endemic areas. Unfortunately, once malaria is excluded, there are few accessible diagnostic tools to guide the management of severe febrile illnesses in low resource settings. This review summarizes the current state of RDT development for several key infections, including dengue fever, enteric fever, leptospirosis, brucellosis, visceral leishmaniasis and human African trypanosomiasis, and highlights many remaining gaps. Most RDTs for non-malarial tropical infections currently rely on the detection of host antibodies against a single infectious agent. The sensitivity and specificity of host-antibody detection tests are both inherently limited. Moreover, prolonged antibody responses to many infections preclude the use of most serological RDTs for monitoring response to treatment and/or for diagnosing relapse. Considering these limitations, there is a pressing need for sensitive pathogen-detection-based RDTs, as have been successfully developed for malaria and dengue. Ultimately, integration of RDTs into a validated syndromic approach to tropical fevers is urgently needed. Related research priorities are to define the evolving epidemiology of fever in the tropics, and to determine how combinations of RDTs could be best used to improve the management of severe and treatable infections requiring specific therapy.

Altered ligands reveal limited plasticity in the T cell response to a pathogenic epitope.

Experimental leishmaniasis offers a well characterized model of T helper type 1 cell (Th1)-mediated control of infection by an intracellular organism. Susceptible BALB/c mice aberrantly develop Th2 cells in response to infection and are unable to control parasite dissemination. The early CD4(+) T cell response in these mice is oligoclonal and reflects the expansion of Vbeta4/ Valpha8-bearing T cells in response to a single epitope from the parasite Leishmania homologue of mammalian RACK1 (LACK) antigen. Interleukin 4 (IL-4) generated by these cells is believed to direct the subsequent Th2 response. We used T cells from T cell receptor-transgenic mice expressing such a Vbeta4/Valpha8 receptor to characterize altered peptide ligands with similar affinity for I-Ad. Such altered ligands failed to activate IL-4 production from transgenic LACK-specific T cells or following injection into BALB/c mice. Pretreatment of susceptible mice with altered peptide ligands substantially altered the course of subsequent infection. The ability to confer a healer phenotype on otherwise susceptible mice using altered peptides that differed by a single amino acid suggests limited diversity in the endogenous T cell repertoire recognizing this antigen.

Enhancement of Leishmania amazonensis infection in BCG non-responder mice by BCG-antigen specific vaccine

Different patterns of cutaneous leishmaniasis can be induced when a challenge of alike dose of Leishmania amazonensis amastigotes in various inbred strains was applied. Two strains of mice, the Balb/c and C57 BL/10J, showed exceptional suscepbility, and 10(elevado a sexta potência) amastigotes infective dose lead, to ulcerative progressive lesions with cutaneous metastasis and loss by necrosis of leg on wich the footpad primary lesion occured. Lesions were also progressive but in a lower degree when C3H/HeN and C57BL/6 were infected. Lesions progress slowly in DBA/2 mice presenting lesions wich reach a discreet peack after 12 weeks, do not heal but do not uncerate. DBA/2 mice is, therefore, a good model for immunomodualtion. In attempt to determine the influence of BCG in vaccination schedule using microsomal fraction, DBA/2 became an excellent model, since it is also a non-responder to BCG. Vaccination of DBA/2 mice, receiving the same 10(elevado a sexta potência) BCG viable dose and 10 *g or 50 *g of protein content of microsomal fraction, lead to a progressive disease with time course similar to those observed in susceptible non-vaccinated C57BL/10J mice after 6 months of observation. An enhancement of infection in BCG non-responder mice suggests that use of BCG as immunostimulant in humans could be critical for both vaccination and immunoprophylactic strategies.

Immunogold labeling and cerium cytochemistry of the enzyme ecto-5'-nucleotidase in promastigote forms of Leishmania species

We have applied both enzyme cytochemistry and immunological labeling techniques to characterize the enzyme 5'-nucleotidase (5'-Nase), at the ultrastructural level, in promastigote forms of four Leishmania species: Leishmania amazonensis, Leishmania mexicana, Leishmania donovani and Leishmania chagasi. The cerium phosphate staining was localized at the surface of the cell body, the flagellum and the flagellar pocket membranes of all the parasites studied. The immunogold labelling technique confirmed these results. In this report we localized 5'-Nase in L. chagasi and L. amazonensis which have been implicated respectively in visceral and cutaneous forms of leishmaniasis. In addition, we confirmed the localization of this phosphomonoesterase in the other two species studied. The superior quality of the images, obtained with both methodologies, confirms that these parasites possess mechanisms capable of hydrolyzing nucleotide monophosphates, and that the expression of 5'-Nase is associated with the outer surface of the plasma membrane.

Annotated list of the Phlebotominae (Diptera) of Suriname

Phlebotomine sandflies were collected between 1952 and 1984 at 30 localities in the tropical rainforest and savanna regions of Suriname. Thirty-nine species were identified in the collections (2 Brumptomyia, 37 lutzomyia), including two known vectors of cutaneous leishmaniasis, Lutzomyia flaviscutellata and L. umbratilis. Nineteen of the species are new records for Suriname. In the rainforest region, the commonest phlebotomines were L. squatniventris maripaensis (79.8%), L. umbratilis (8.4%) and L. flaviscutellata (6.3%) in human bait catches, L. umbratilis (26.2%), L. infraspinosa (23.9%) and L. trichopyga (8.3%) in CDC light traps and L. umbratilis (84.3%), L. whitmani (6.8%) and L. shannoni (4.3%) in collections from tree trunks. The mean incidence of cutaneous leishmaniasis from 1979-1985 was 4.9 per 1000 inhabitants for the rainforest region and 0.66 per 1000 for Surinameas a whole.

Sand fly vectorial ecology in the state of São Paulo

Ecological aspect of sand fly distribution in the state of São Paulo, Brazil are described. The main man-biting species are Lutzomyia whitmani, Lu.pessoai, Lu.intermedia, Lu.migonei and Lu.fischeri. Their primary habitat is the forest but latter three of the above species are also encountered in domiciliary environment. Sylvatic species such as Lu.flaviscutellata bite man only rarely and Psychodopygus ayrozai seems to be more anthropophilic. The survival of sand flies in the residual forest and in cultivated areas where man has nearly destroyed the forest almost completely is analyzed. Over the last ten years the incidence of human American cutaneous leishmaniasis (ACL) has been increasing: human cases occurring within several municipalities in which there is overlapping with the distributon of domiciliary Lu.intermedia. New ACL microfoci are appearing in the state of São Paulo and these call for further study.

The development of species of Leishmania Ross, 1903 in Lutzomyia longipalpis (Lutz & Neiva, 1912)

The development of four isolates of Leishmania from foci of American cutaneous leishmaniasis was studied in Lutzomyia longipalpis. The suggestion that the differences in the development of the Leishmania in the invertebrate host are of great taxonomic significance was confirmed. The pattern of development of three strains was typical of parasites of the subgenus Leishmania, the other was similar to Leishmania of the subgenus Viannia. The identification of the strains using other criteria is in agreement with biological characterization. The results show that the morphological and morphometric study of promastigotes do not clearly define the taxonomic position of the parasites but other studies are needed to confirm this.

Interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) in pregnant C57BL/6 mice infected with L. major at different gestational periods.

BACKGROUND AND OBJECTIVE: To assess if gestational factors affect the resistance of C57BL/6 mice to L major infection, this study determined the levels of IL-4 and IFN-gamma in popliteal lymph node cells of pregnant C57BL/6 mice infected with L. major at 16 hours, 5 days-, 10 days- and 15 days- post plug by PCR, ELISA and BIOASSAY. DESIGN/SETTING: Experimental. RESULTS: Infected pregnant C57BL/6 mice developed larger cutaneous footpad lesions compared with non-pregnant C57BL/6 mice (that showed signs of resolution 7-10 weeks after infection). But, the lesions in infected pregnant C57BL/6 mice and infected non-pregnant C57BL/6 mice were not as large as in susceptible BALB/c mice. The mean litter weight was also reduced in pregnant infected C57BL/6 mice particularly in the groups infected at later stages of pregnancy (day 10- and day 15-post plug). The levels of both IL-4 and IFN-gamma increased with gestation in pregnant infected C57BL/6 mice compared with pregnant non-infected group, while only IL-4 was raised in pregnant infected mice compared with infected non pregnant mice. CONCLUSIONS: It may be concluded that increased IL-4 in pregnant infected C57BL/6 mice caused the transient susceptibility to L major infection while reduced litter weight was associated with increased IFN-gamma. These effects were pronounced in C57BI/6 mice infected with L major in late pregnancy.

Variation between geographical populations of Lutzomyia (Nyssomyia) whitmani (Antunes & Coutinho, 1939) sensu lato (Diptera:Psychodidae:Phlebotominae) in Brazil

Phylogenetic analysis of morphometric and biological characters indicated that there are two distinct forms of Lutzomyia whitmani in Brazil: one is present both north and south of the River Amazonas in the State of Pará while the other occurs in northeast Brazil, in the State of Ceará, and further south, including the type locality in State of Bahia. The Amazonian form is reportedly neither strongly anthropophilic nor synanthropic, and it is the vector of Leishmania shawi; whereas the southern form is often collected peridomestically, while biting man, and has been found infected with Le.(V.) braziliensis. The ratio of the length of the genital filaments to that the genital pump was found to be consistently smaller in males of the Amazonian populations. A middle repetitive DNA element was isolated by differentially screening a genomic library made using Amazonian material, and the sequence was diagnostic for this form of Lu. whitmani (being absent or occurring in low copy number in the southern form). The total evidence suggests there are at least two, geographically-isolated forms of Lu. whitmani, which may represent different cryptic species.

Persistent Infections by Leishmania (Viannia) braziliensis

Here we review the phenomenon of persistency in Leishmania (Viannia) braziliensis infections. In other Leishmania species where appropriate animal models exist, considerable advances in the understanding of basic immunologic mechanisms of persistency have been made; for a review see Aebisher (1994). On the contrary, the evidences of persistence in infections with L. braziliensis rest on studies of human clinical cases many of which we summarized and discussed in this work