Cross-species genetic exchange between visceral and cutaneous strains of Leishmania in the sand fly vector

Genetic exchange between Leishmania major strains during their development in the sand fly vector has been experimentally shown. To investigate the possibility of genetic exchange between different Leishmania species, a cutaneous strain of L. major and a visceral strain of Leishmania infantum, each bearing a different drug-resistant marker, were used to coinfect Lutzomyia longipalpis sand flies. Eleven double–drug-resistant progeny clones, each the product of an independent mating event, were generated and submitted to genotype and phenotype analyses. The analysis of multiple allelic markers across the genome suggested that each progeny clone inherited at least one full set of chromosomes from each parent, with loss of heterozygosity at some loci, and uniparental retention of maxicircle kinetoplast DNA. Hybrids with DNA contents of approximately 2n, 3n, and 4n were observed. In vivo studies revealed clear differences in the ability of the hybrids to produce pathology in the skin or to disseminate to and grow in the viscera, suggesting polymorphisms and differential inheritance of the gene(s) controlling these traits. The studies, to our knowledge, represent the first experimental confirmation of cross-species mating in Leishmania, opening the way toward genetic linkage analysis of important traits and providing strong evidence that genetic exchange is responsible for the generation of the mixed-species genotypes observed in natural populations.Kinetoplastid protozoan parasites of the genus Leishmania are transmitted by a sand fly bite into the skin of the mammalian host. More than 20 different species of Leishmania are known to produce disease in humans, ranging from localized, self-limiting cutaneous lesions that develop at the site of inoculation to visceralizing infections that are fatal in the absence of treatment (1). These diverse clinical outcomes have generally clear parasite species and strain associations, although the ability of visceral strains to occasionally produce cutaneous disease, and vice versa, is also well described. The specific contribution of parasite genotype to disease outcome remains largely unknown. Studies by Zhang et al. demonstrated that the introduction of Leishmania donovani-specific genes into Leishmania major increased their ability to grow in the viscera of BALB/c mice, but in no case reached the level of infection produced by the visceral strains (2, 3), suggesting that these tissue tropisms are under multigenic control.The Leishmania genome is organized in 36 chromosomes for the Old World species, such as L. major, L. donovani, and Leishmania infantum, and in 35 and 34 for the New World species Leishmania braziliensis and Leishmania mexicana, respectively (4, 5). Although approximately a diploid organism, aneuploidy, including mosaic aneuploidy, is now known to be widespread (6–10); we refer to the nearly diploid state as “2n” here. A series of population genetic studies have suggested that clonal lineages have experienced at least occasional bouts of genetic exchange both within and between species (11–16), and the frequency of these recombination events underlies the continuing clonality vs. sexuality debate (17).By using drug-resistance markers, the first direct demonstration of genetic exchange between different L. major strains was reported (18) and possibly between L. donovani lines using fluorescent markers (19). In each case, the hybrids were generated between the extracellular promastigote stages in the sand fly vector. These and more recent studies (20, 21) critically inform the population genetics data by formally demonstrating that Leishmania are capable of intraspecies and even intraclonal mating. The present studies were designed to provide, to our knowledge, the first experimental demonstration of cross-species mating and to explore the heritability of the genes controlling tissue-specific tropisms. By using L. major and L. infantum parental lines bearing independent drug-resistance markers and that reproduce in mice their respective human cutaneous and visceral tropisms, hybrid progeny were recovered from coinfected Lutzomyia longipalpis sand flies. Detailed genotyping and phenotyping of 11 progeny clones are described.     

Phlebotomine sand fly population dynamics in a leishmaniasis endemic peri-urban area in southern Italy

A 2-year survey was carried out from May to November 2008 and 2009 to study the sand fly species composition, its seasonal phenology and density in Apulia region (southern, Italy). The study was conducted in a dog shelter located in a new residential urban district where Leishmania infantum is endemic. Sand flies were collected using sticky traps from May to November, at about 7-day intervals. Temperature and relative humidity were recorded daily. In December 2008, general environmental improvements (e.g., the ground was covered with gravel and the vegetation present inside the cages was removed to facilitate cleaning) were made in the study area. The most diffused species during the whole study period were Phlebotomus perniciosus (2008, n = 248, 49.4%; 2009, n = 254, 50.6%) followed by Phlebotomus neglectus (2008, n = 76, 39.8%; 2009, n = 115, 60.2%) and Phlebotomus papatasi (2008, n = 5, 50.0%; 2009, n = 5, 50.0%). Four specimens of Phlebotomus perfiliewi were collected only in the first year. The number of Sergentomyia minuta specimens collected increased considerably in the second (n = 548, 86.2%) in comparison to the first year (n = 88, 13.8%). The highest number of phlebotomine sand flies was collected in July and August when a mean temperature from 27.09 to 28.02 °C and mean relative humidity from 47.28 to 56.36% were recorded. The variations in phlebotomine sand fly species diversity and abundance recorded in this study were related to climatic and environmental factors. Data here presented confirm that sand flies easily adapt to the urban environments and that the may represent a public health concern for L. infantum and other pathogen transmission also in similar urban environment of southern Europe.     

DNA vaccination with KMP11 and Lutzomyia longipalpis salivary protein protects hamsters against visceral leishmaniasis

It was recently shown that immunization of hamsters with DNA plasmids coding LJM19, a sand fly salivary protein, partially protected against a challenge with Leishmania chagasi, whereas immunization with KMP11 DNA plasmid, a Leishmania antigen, induced protection against L. donovani infection. In the present study, we evaluated the protective effect of immunization with both LJM19 and KMP11 DNA plasmid together. Concerning the protection against an infection by L. chagasi, immunization with DNA plasmids coding LJM19 or KMP11, as well as with both plasmids combined, induced IFN-γ production in draining lymph nodes at 7, 14 and 21 days post-immunization. Immunized hamsters challenged with L. chagasi plus Salivary Gland Sonicate (SGS) from Lutzomyia longipalpis showed an enhancement of IFN-γ/IL-10 and IFN-γ/TGF-β in draining lymph nodes after 7 and 14 days of infection. Two and five months after challenge, immunized animals showed reduced parasite load in the liver and spleen, as well as increased IFN-γ/IL-10 and IFN-γ/TGF-β ratios in the spleen. Furthermore, immunized animals remained with a normal hematological profile even five months after the challenge, whereas L. chagasi in unimmunized hamsters lead to a significant anemia. The protection observed with LJM19 or KMP11 DNA plasmids used alone was very similar to the protection obtained by the combination of both plasmids.     

Antigenic diversity in maxadilan, a salivary protein from the sand fly vector of American visceral leishmaniasis

The salivary protein maxadilan (MAX) is a vasodilator and immunomodulator from the sand fly vector of the protozoan parasite Leishmania chagasi. Vaccinating BALB/c mice with sand fly salivary gland extracts or with MAX protects the host against L. major infection. Because of the potential use of MAX in an anti-Leishmania vaccine, we characterized the vertebrate host IgG response to MAX in the present study. Our immunochemical analysis indicated that antibodies to MAX were detected in BALB/c mice, as well as in pigs and humans, from a area in Nicaragua endemic for Lutzomyia longipalpis. Previous studies demonstrate that the MAX protein is polymorphic on the amino acid level. Our findings suggested that naturally occurring MAX variants were recognized specifically by the host immune system and antigenicity appeared to be associated with amino-acid sequence variability. Thus, antigenic diversity of MAX and possibly of other arthropod salivary proteins may dictate the development of vector-based vaccines(s).     

Man-biting sand fly species and natural infection with the Leishmania promastigote in leishmaniasis-endemic areas of Ecuador

A countrywide surveillance of sand flies was performed to obtain information on their geographical distribution and natural infection by Leishmania protozoa in Ecuador. A total of 18,119 sand flies were collected by human landing collections during 32 years from 1982 to 2014, and 29 species were recognized. The most prevalent 10 species were Lutzomyia gomezi, Lu. robusta, Lu. hartmanni, Lu. shannoni, Lu. trapidoi, Lu. panamensis, Lu. maranonensis, Lu. ayacuchensis, Lu. tortura and Lu. yuilli yuilli, and their topographical and vertical distributions were identified. Among all the sand flies, only 197 (1.09%) flies of four Lutzomyia species, Lu. gomezi, Lu. trapidoi, Lu. tortura and Lu. ayacuchensis, were positive for Leishmania. Endotrypanum, a flagellate parasite not pathogenic to humans, were detected in five Lutzomyia species, Lu. robusta, Lu. hartmanni, Lu. trapidoi, Lu. panamensis and Lu. yuilli yuilli, suggesting wide vector-ranges of Endotrypanum species. These data on the genus Lutzomyia and their natural infections with Leishmania and Endotrypanum will be useful for transmission studies and surveillance of leishmaniasis.     

Immunomodulatory effects of Maxadilan and Phlebotomus papatasi sand fly salivary gland lysates on human primary in vitro immune responses

Leishmaniasis is a parasitic disease transmitted by the bite of Leishmania-infected sand flies. Here we show for the first time the ability of Maxadilan (Max), a vasodilatory peptide isolated from the sand fly Lutzomyia longipalpis, and salivary gland lysate (SGL) from Phlebotomus papatasi to decrease the secretion of Type 1 cytokines and to enhance the production of the Type 2 cytokine interleukin (IL)-6 by human peripheral blood mononuclear cells (PBMC) and monocytes. We found Max decreased the secretion of interferon (IFN)-gamma and IL-12p40 by PBMC and TNF-alpha by monocytes. SGL reduced the production of IFN-gamma by PBMC. In contrast, production of the Type 2 cytokine IL-6 was increased in Max or SGL-exposed cells. Finally, we determined that Max interacts with human cells through at least the pituitary adenylate cyclase activating polypeptide receptor. These results show that sand fly salivary gland components have an immunomodulatory effect on human cells, and this has important implications for the development of vaccines against leishmaniasis for humans.     

Effect of night time-intervals,height of traps and lunar phases on sand fly collection in a highly endemic area for canine leishmaniasis

The activity of phlebotomine sand flies was monitored in a sub-urban area of Sicily in order to acquire data on seasonality and to elucidate the effect of the night time-intervals, height of traps from ground and lunar phases on the abundance of the capture. The study was conducted in the farm of the University of Messina (Italy). Light traps were placed as in the following: biweekly, from dusk to dawn, and from May to November; for three consecutive nights from 18:00 to 6:00, with the net bag being changed every 2 h; for 30 days, at different heights from 18:00 to 6:00. A total of five species (i.e., Phlebotomus perniciosus, Phlebotomus neglectus, Phlebotomus sergenti, Phlebotomus perfiliewi, and Sergentomyia minuta), three of which are proven vectors of Leishmania infantum, were captured. The most abundant species was P. perniciosus (73.3%) followed by S. minuta (23.3%). The highest number of phlebotomine sand flies was collected in August and September with a peak of collection recorded in the evening (i.e., from 20:01 to 22.00). The number of phlebotomine sand flies collected at 50 cm above the ground was significantly higher (P = 0.041) than that captured at 150 cm. Results of this study shed light on the ecology of main phlebotomine species in the Mediterranean area, and on the influence of some factors, such as time and height of traps, on the light trap capture efficiency.     

Species co-occurrence and feeding behavior in sand fly transmission of American cutaneous leishmaniasis in western Venezuela

The structure of the Phlebotomine sand fly community from the Venezuelan Andes was studied using null-model tests. The analyses, at the living zones and altitudes scales, revealed C-scores larger than those expected by random, independently of the collection technique (P < 0.05). These results imply that sand fly species are non-aggregated at both scales. Random results for the variance of C-score and for the favored states hypothesis suggest that sand fly species belong to an unique guild. The latter is reinforced by the fact that anthropophilic and zoophilic species use in the same way a common resource (blood). Finally, we suggest additional approaches to study the role of the sand fly community structure on the genesis and dynamics of transmission of American cutaneous leishmaniasis.     

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.     

Lutzomyia migonei as putative vector of visceral leishmaniasis in La Banda, Argentina

Four autochthonous cases of human visceral leishmaniasis (VL) were reported in La Banda, Santiago del Estero from June 2007 to May 2008. In the vicinity of these cases there were 3/47 rK39 sero-positive dogs, and another 4 dogs with VL were reported by passive surveillance. The sero-positive dogs and infected humans lived within a 3.1 km radius. Phebotomine sand fly captures were performed twice during November/December 2007 and April 2008. In 20 of the 59 sampled sites in the areas of the human and canine cases (220 night/traps) 151 phlebotomine sand flies were collected and consisted of: Lutzomyia migonei 93%, Lutzomyia cortelezzii 5.6% and Lutzomyia neivai 1.4%. We propose that there was an enzootic cycle of VL with accidental human transmission due to L. migonei and suggest that there be a surveillance of human isolated cases of VL within the L. migonei dispersion area.     

Distribution of Lutzomyia ayacuchensis, the vector of Andean-type cutaneous leishmaniasis,at different altitudes on the Andean slope of Ecuador

Distribution of the vector species is a major risk factor for the endemicity of leishmaniasis. In the present study, the vertical distribution of Lutzomyia (Lu.) ayacuchensis, the vector of Leishmania (Leishmania) mexicana in the Ecuadorian Andes, was surveyed at different altitudes (300–2500 m above sea level) of the Andean slope. The vector species Lu. ayacuchensis was identified at an altitude of 650 m and a higher areas, and higher distribution ratio of the species was observed at higher altitudes. In addition, high ratios of L. (L.) mexicana infection were detected in higher areas, but none in lower populations of sand flies. Since an association between sand fly populations and vector competence is suggested in Lu. ayacuchensis, haplotype analysis was performed on the species from different altitudes of the study areas; however, no apparent difference was observed among populations. These results suggested that Lu. ayacuchensis in Andean slope areas of Ecuador has the potential to transmit L. (L.) mexicana and spread leishmaniasis in these areas.     

Leishmania infantum DNA detection in Phlebotomus tobbi in a new northern focus of visceral leishmaniasis in Iran

Objective

To identify the vector(s), the parasite and the species composition of sand flies in the district during May-October 2012.

Methods

For reaching our objectives we used polymerase chain reaction of kDNA, ITS1-rDNA, followed by restriction fragment length polymorphism.

Results

Two species of Phlebotomus sergenti and Phlebotomus tobbi were the most prevalent among 8 species identified comprising 51.1% and 32.9% respectively. Among the 160 specimens of female sand flies tested by polymerase chain reaction of kDNA, ITS1-rDNA, followed by restriction fragment length polymorphisms, only 1 out of 80 Phlebotomus tobbi (1.25%) were positive to Leishmania infantum parasites.

Conclusions

Our finding showed that Phlebotomus tobbi may play as a vector to circulate the parasite of Leishmania infantum among reservoir(s) and human.     

Development of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for Leishmania infection

Entomological monitoring of Leishmania infection in leishmaniasis endemic areas offers epidemiologic advantages for predicting the risk and expansion of the disease, as well as evaluation of the effectiveness of control programs. In this study, we developed a highly sensitive loop-mediated isothermal amplification (LAMP) method for the mass screening of sand flies for Leishmania infection based on the 18S rRNA gene. The LAMP technique could detect 0.01 parasites, which was more sensitive than classical PCR. The method was robust and could amplify the target DNA within 1 h from a crude sand fly template without DNA purification. Amplicon detection could be accomplished by the newly developed colorimetric malachite green (MG)—mediated naked eye visualization. Pre-addition of MG to the LAMP reaction solution did not inhibit amplification efficiency. The field applicability of the colorimetric MG-based LAMP assay was demonstrated with 397 field-caught samples from the endemic areas of Ecuador and eight positive sand flies were detected. The robustness, superior sensitivity, and ability to produce better visual discriminatory reaction products than existing LAMP fluorescence and turbidity assays indicated the field potential usefulness of this new method for surveillance and epidemiological studies of leishmaniasis in developing countries.     

Host preferences of phlebotomine sand flies at a hypoendemic focus of canine leishmaniasis in central Italy

A survey was carried out on phlebotomine sand flies and their feeding habits at a hypoendemic focus of Leishmania infantum in Macerata province, central Italy. During two consecutive years (2000-2001), 1465 sand fly specimens (42.5% of which were males) were collected from a variety of diurnal resting sites in the municipality of Camerino. The most prevalent species was Phlebotomus perniciosus (76.6%), followed by P. papatasi (10.4%), Sergentomyia minuta (9.1%), Phlebotomus perfiliewi (3.3%) and P. mascittii (0.5%). Among the 842 females collected, 578 (68.6%) were blood-fed. Based on the results of blood meal analyses, P. perniciosus fed on man, dogs, equines, sheep and birds; P. perfiliewi on dogs, equines, sheep and birds; P. papatasi on dogs, equines and birds. Two specimens of P. mascittii fed on equines. Forage ratios (FRs) and host selectivity indices gave different results for the large domestic animals. More than 95% of the specimens collected inside a stable, dog kennel, sheep pen and chicken house were found to have fed on the animals housed in the respective shelters. In addition, at one collecting site where almost all the hosts mentioned above were present simultaneously, both P. perniciosus and P. perfiliewi were found to have fed on all five species, indicating that host choice was probably related to its availability (i.e. number and size) rather than specific attractiveness. The feeding habits of the two Leishmania vectors may have implications for the epidemiology of leishmaniasis in urban and peri-urban areas, where sand fly females deprived of other vertebrate hosts (particularly the larger species) may begin to bite humans and dogs more frequently.     

Drosophila melanogaster as a model host to dissect the immunopathogenesis of zygomycosis

Zygomycosis is an emerging frequently fatal opportunistic mycosis whose immunopathogenesis is poorly understood. We developed a zygomycosis model by injecting Drosophila melanogaster flies with a standardized amount of fungal spores from clinical Zygomycetes isolates to study virulence and host defense mechanisms. We found that, as opposed to most other fungi, which are nonpathogenic in D. melanogaster (e.g., Aspergillus fumigatus), Zygomycetes rapidly infect and kill wild-type flies. Toll-deficient flies exhibited increased susceptibility to Zygomycetes, whereas constitutive overexpression of the antifungal peptide Drosomycin in transgenic flies partially restored resistance to zygomycosis. D. melanogaster Schneider 2 phagocytic cells displayed decreased phagocytosis and caused less hyphal damage to Zygomycetes compared with that to A. fumigatus. Furthermore, phagocytosis-defective eater mutant flies displayed increased susceptibility to Zygomycetes infection. Classic enhancers of Zygomycetes virulence in humans, such as corticosteroids, increased iron supply, and iron availability through treatment with deferoxamine dramatically increased Zygomycetes pathogenicity in our model. In contrast, iron starvation induced by treatment with the iron chelator deferasirox significantly protected flies infected with Zygomycetes. Whole-genome expression profiling in wild-type flies after infection with Zygomycetes vs. A. fumigatus identified genes selectively down-regulated by Zygomycetes, which act in pathogen recognition, immune defense, stress response, detoxification, steroid metabolism, or tissue repair or have unknown functions. Our results provide insights into the factors that mediate host-pathogen interactions in zygomycosis and establish D. melanogaster as a promising model to study this important mycosis.     

Identification of the molecular attributes required for aminoglycoside activity against Leishmania

Leishmaniasis, a parasitic disease caused by protozoa of the genus Leishmania, affects millions of people worldwide. Aminoglycosides are mostly known as highly potent, broad-spectrum antibiotics that exert their antibacterial activity by selectively targeting the decoding A site of the bacterial ribosome, leading to aberrant protein synthesis. Recently, some aminoglycosides have been clinically approved and are currently used worldwide for the treatment of leishmaniasis; however the molecular details by which aminoglycosides induce their deleterious effect on Leishmaina is still rather obscure. Based on high conservation of the decoding site among all kingdoms, it is assumed that the putative binding site of these agents in Leishmania is the ribosomal A site. However, although recent X-ray crystal structures of the bacterial ribosome in complex with aminoglycosides shed light on the mechanism of aminoglycosides action as antibiotics, no such data are presently available regarding their binding site in Leishmania. We present crystal structures of two different aminoglycoside molecules bound to a model of the Leishmania ribosomal A site: Geneticin (G418), a potent aminoglycoside for the treatment of leishmaniasis at a 2.65-Å resolution, and Apramycin, shown to be a strong binder to the leishmanial ribosome lacking an antileishmanial activity at 1.4-Å resolution. The structural data, coupled with in vitro inhibition measurements on two strains of Leishmania, provide insight as to the source of the difference in inhibitory activity of different Aminoglycosides. The combined structural and physiological data sets the ground for rational design of new, and more specific, aminoglycoside derivatives as potential therapeutic agents against leishmaniasis.     

Antigenuria in visceral leishmaniasis: detection and partial characterisation of a carbohydrate antigen

The detection of antigen in the urine is increasingly being used for diagnosis of parasitic infections. A urinary antigen has recently been demonstrated in visceral leishmaniasis (VL), using a latex agglutination test. The results of our study show that the detected antigen is: heat-stable, precipitates with acetone and ethanol but not TCA, is sensitive to periodate and acid hydrolysis but not to pronase E, lipase, or neuraminidase. The antigen is a low molecular weight glycoconjugate that can be extracted by phenol-water, partitions into the aqueous phase when extracted with Triton X-114 or chloroform/methanol, and can be labelled by biotin hydrazide. Since this urinary antigen cannot be characterised by conventional SDS-PAGE and Western blotting, we used an affinity transfer blotting system in which antigens were captured onto nitro-cellulose paper previously coated with a specific antibody. Using this system a low molecular weight antigen (LMWA) spanning an area of the nitro-cellulose membrane corresponding to molecular weight of 5-20 kDa was detected in the urine of VL patients (from Nepal, Sudan, Brazil, Yemen and Spain) and of experimentally infected animals. No LMWA was detected in the urine of patients with malaria, schistosomiasis, or other nonparasitic diseases including typhoid and brucellosis. Immunoprecipitation, using antibody-coated latex, followed by immunoblotting showed that the LMWA is the target antigen in the previously described latex agglutination test ('KATEX'). The antigen is detectable in both the promastigote and amastigote stages of the parasite. Monoclonal antibodies (mAbs) against Leishmania glycoconjugates strongly react with this molecule. These results suggest that the detected antigen is highly specific and diagnostic for VL.     

Entomological investigation following the resurgence of human visceral leishmaniasis in southern Algeria

Visceral and cutaneous leishmaniasis are the main endemic vector born diseases in Algeria. In the Hoggar region (extreme south of the country) human visceral leishmaniasis (HVL) is known to be sporadic but during the last decade the number of cases has increased significantly. In 2010, a peak of HVL cases was registered mostly among children. Therefore an entomological survey and a retrospective study on HVL cases were carried out in order to explore the transmission of the disease. Among the sand fly caught Phlebotomus bergeroti was the most frequent species (68%) followed by Sergentomyia schwetzi (22%). In this work we describe the presence of Phlebotomus (Paraphlebotomus) kazeruni for the first time in the Hoggar region.     

Epidemiological studies of an outbreak of cutaneous leishmaniasis in the Rio Jequitinhonha Valley, Minas Gerais, Brazil.

We detected an outbreak of American cutaneous leishmaniasis in the Jequitinhonha River Valley, Minas Gerais, Brazil. Clinical and epidemiological aspects were studied for a period of two years. Data include results of physical examinations, Montenegro skin test and serology. In total 72 of the 299 individuals evaluated presented active lesions. Only one case out of these 72 patients showed the mucosal form of the disease. The precarious sanitary conditions, low educational level and low income found in the population studied demonstrated that, as with the other parasitic diseases, cutaneous leishmaniasis occurs with greater frequency in needy populations. A canine serological survey detected 20.3% (30/148) of dogs reactive to the Leishmania antigen. Lutzomyia intermedia was the predominant phlebotomine species and the majority of the specimens (84.9%) were captured in the peridomicile. Four samples from human and three from canine cases were isolated and characterised by PCR and isoenzymes as being Leishmania (Viannia) braziliensis. The peridomiciliary nature of the disease is discussed.