Experimental canine mucocutaneous leishmaniasis (Leishmania braziliensis braziliensis)

Four mongrel dogs were intradermically inoculated with 3 x 10(6) Leishmania braziliensis braziliensis promastigotes. Three out of the four animals developed cutaneous lesions respectively 4, 7, and 8 months after. The fourth dog did not develop lesion at the inoculation site, but a mucosal ulcer was seen 16 months after the inoculum. Clinical, histopathological, and serological findings were similar to what is found in natural canine infection as well as in the human disease. These results suggest that dogs may be an useful model for L. b. braziliensis infection.     

Cutaneous leishmaniasis caused by members of Leishmania braziliensis complex in Nayarit, State of Mexico

An epidemiological study was carried out in the northern Mexican state, Nayarit. Fourteen patients with possible cutaneous leishmaniasis skin lesions gave positive Montenegro skin tests. Biopsies were taken from the skin ulcer and analyzed by polymerase chain reaction (PCR) with specific primers for the Leishmania mexicana complex; however all biopsies were not amplified. PCR carried out with specific primers for the L. braziliensis complex resulted in the amplification of all patient DNA. DNA from 12 out of 14 biopsies gave positive amplification with primers species specific for L. (Viannia) braziliensis and hybridized with a species specific L. (V.) braziliensis probe. These results demonstrate the presence in Nayarit of at least two members of the L. braziliensis complex. Most of the cutaneous lesions were caused by L. (V.) braziliensis and two by another species belonging to the L. braziliensis complex. As far as we are aware, this is the first report of L. (V.) braziliensis in Nayarit. The main risk factor associated with the contraction of this disease in Nayarit is attributed to working on coffee plantations.     

Zoonotic cutaneous leishmaniasis due to Leishmania (Viannia) braziliensis associated with domestic animals in Venezuela and Brazil

After outbreaks of cutaneous leishmaniasis in Solano State, Venezuela, 5% of the population had parasitized ulcers while after similar outbreaks in Mesquita, Rio de Janeiro State, Brazil, 9% had the disease. In these foci children, including some under six years of age, were affected. There was no significant difference in the occurrence of the disease according to sex or type of employment. In Solano, 3% of dogs and 28% of donkeys had parasitized lesions, while in Mesquita these indices were 19.8% and 30.8% respectively. The parasite from man, dogs and equines was identified as Leishmania (Viannia) braziliensis, by zymodeme and serodeme characterization. In these foci there is evidence suggesting that leishmaniasis is a zoonosis, possibly with equines and dogs as reservoirs, although both a wild enzootic cycle and the role of man as a source of infection can not be ruled out. Transmission is assumed to occur peridomestically by sandfly vectors such as Lutzomyia panamensis in Venezuela and Lutzomyia intermedia in Brazil. Information about the origin of these foci suggests that infected equines may be an important factor in the dissemination of the parasite in a peridomestic situation where these sandflies are abundant.     

Leishmania braziliensis spp. 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(4) amastigotes of Leishmania braziliensis 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 parasitizations 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 greater frequency between 60 and 120 days post-inoculation. The use of this model for the study of mucocutaneous parasitism by L. braziliensis 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. braziliensis.     

Responses of Leishmania (Viannia) braziliensis cutaneous infection to N-methylglucamine antimoniate in the rhesus monkey (Macaca mulatta) model

The antileishmanial efficacy of the reference drug N-methylglucamine antimoniate (Glucantime) was evaluated in groups of rhesus monkeys with acute and chronic Leishmania (Viannia) braziliensis cutaneous infection. The therapeutic responses in experimental animals to either a low dose (5 mg/kg body wt/day for 28 days) or a routine dose (20 mg/kg/day for 28 days) of pentavalent antimony were similar to those reported in the human disease. Primates were cured of their lesions after treatment, but with cryptic parasitism and/or relapse. The rhesus model of L. (V.) braziliensis cutaneous leishmaniasis therefore provides an additional resource for preclinical trials with newer drugs.     

Concurrent cutaneous,visceral and ocular leishmaniasis caused by Leishmania (Viannia) braziliensis in a kidney transplant patient

Although cases of leishmaniasis co-infection have been described in acquired immunodeficiency syndrome patients as well as those who have undergone organ transplants, to our knowledge, the present report is the first documented case of simultaneous cutaneous, visceral and ocular leishmaniasis due to Leishmania (Viannia) braziliensis in a transplant patient. The patient had been using immunosuppressive drugs since receiving a transplanted kidney. The first clinical signs of leishmaniasis included fever, thoracic pain, hepatosplenomegaly, leucopenia and anemia. The cutaneous disease was revealed by the presence of amastigotes in the skin biopsy. After three months, the patient presented fever with conjunctive hyperemia, intense ocular pain and low visual acuity. Parasites isolated from iliac crest, aqueous humor and vitreous body were examined using a range of molecular techniques. The same strain of L. (V.) braziliensis was responsible for the different clinical manifestations. The immunosuppressive drugs probably contributed to the dissemination of Leishmania.     

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.     

Experimental infection of Lutzomyia whitmani in dogs infected with Leishmania braziliensis braziliensis

Lutzomyia (N.) whitmani was infected on leishmaniotic lesions of three out of nine dogs infected with Leishmania braziliensis braziliensis. The infectivity rates in these sandflies were 8.3% (1/12), 7.1% (1/14) and 1.8% (3/160), respectively. In addition, 180 Lu. whitmani fed on non-ulcerated regions of one of the infected dogs and none became infected. We emphasize the vector potentiality of Lu. whitmani for L.b. braziliensis in the endemic region of Três Bra?os, Bahia, Brazil.     

Preliminary results in favor of the existence of a major surface antigen, specific for Leishmania braziliensis braziliensis

The comparative surface antigen study of 10 bolivian strains and 1 brazilian reference strain from L. b. braziliensis sub-species shows an important homogeneity within this group. All the strains tested so far present similar antigenic patterns with a major antigen at 72 kD. On the contrary, the comparative analysis of surface antigens of L. b. braziliensis with those of L. b. guyanensis, L. b. panamensis, L. mexicana amazonensis and L. donovani chagasi shows a large antigenic heterogeneity between the Leishmania sub-species and species we studied. The 72 kD antigen was only detected on L. b. braziliensis surface.     

Experimental infection with Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis in the marmoset, Callithrix penicillata (Primates:Callithricidae)

Fourteen marmosets (Callithrix penicillata) were inoculated intradermally with promastigotes and/or amastigotes of Leishmania (Viannia) braziliensis (L. (V) b.) strains MHOM/BR/83/LTB-300 MHOM/BR/85/LTB-12 MHOM/BR/81/LTB-179 and MHOM/BR/82/LTB-250. The evolution of subsequent lesions was studied for 15 to 75 weeks post-inoculation (PI). All but 3 of the L. (V) b. injected marmosets developed a cutaneous lesion at the point of inoculation after 3 to 9 weeks, characterized by the appearance of subcutaneous nodules containing parasites. Parasites were isolated by culture (Difco Blood Agar) from all 11 positive animals. The maximum size of the lesions was variable and ranged between 37 mm2 to 107 mm2. Ulceration of primary nodules became evident after 3 to 12 weeks in all infected marmosets, but was faster and larger in 5 of the 11 animals. The active lesions persisted in 9 out of 11 Callithrix until the end of the observation period, which varied from 15-75 weeks. In 3 animals spontaneous healing of their lesions (13 to 25 weeks, PI) was observed but with cryptic parasitism. In another 2 infected animals there was regression followed by reactivation of the cutaneous lesions. The appearance of smaller satellite lesions adjacent to primary ones, as well as metastatic lesions to the ear lobes, were documented in 2 animals. Promastigotes of L. (Leishmania) amazonensis (L. (L) a.) MHOM/BR/77/LTB-16 were inoculated in 1 marmoset. This animal remained chronically infected for 6 months and the lesion developed in a similar manner to L. (V) b. infected marmosets. No significant differences in clinical and parasitological behaviour were observed between promastigote or amastigote derived infections of the 2 species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative zymographic analysis of metallopeptidase of Leishmania (Viannia) peruviana and Leishmania (Viannia) braziliensis isolates from Peru

American tegumentary leishmaniasis (ATL) in Peru is mainly associated with Leishmania (Viannia) peruviana and L. (V.) braziliensis. These parasites are genetically related, and their characterization as distinct species is controversial. Despite their genetic similarity, each species is associated with different clinical manifestations of ATL; L. (V.) peruviana causes only cutaneous leishmaniasis, whereas L. (V.) braziliensis can cause both cutaneous and mucocutaneous leishmaniasis. Because the primary cutaneous lesions caused by infection with these species are indistinguishable, it is necessary to develop a suitable method to differentiate them in order to prevent possible metastasis to oropharyngeal mucosa. In the present study, we investigated the proteolytic profile of L. (V.) peruviana and L. (V.) braziliensis isolates from Peru by zymographic analysis in SDS-PAGE copolymerized with gelatin. Enzymes were characterized according to their pH range of activity and sensitivity to distinct peptidase inhibitors. We observed that L. (V.) peruviana isolates displayed three proteolytic bands with molecular masses ranging from 55 to 80kDa, whereas L. (V.) braziliensis isolates showed six proteolytic activities between 55 and 130kDa. Using specific inhibitors, we determined that these proteolytic activities are due to metallopeptidases and present optimal activity between the pH range 5.5 and 10.0. Our results suggest that the expression of metallopeptidases in L. (V.) peruviana and L. (V.) braziliensis isolates from Peru is species-specific.     

Sensitivity of a vacuum aspiratory culture technique for diagnosis of localized cutaneous leishmaniasis in an endemic area of Leishmania (Viannia) braziliensis transmission.

Sixty eight patients with localized cutaneous leishmaniasis from an area with Leishmania (Viannia) braziliensis transmission had cultures performed with a modified Marzochís vacuum aspiratory puncture technique to establish sensitivity and contamination rate with this new method. Overall sensitivity of three aspirates was 47.1%; (CI95% 39.4; 59.4) significantly greater than the sensitivity of a single one aspirate. Fungal contamination was observed in 6/204 (2.9%) inoculated culture tubes. We recommend that this useful technique should be adopted as routine for primary isolation of L. (V.) braziliensis from localized cutaneous ulcers.     

Epidemiological characteristics of American cutaneous leishmaniasis in an endemic region of the State of Bahia. III. Phlebotomine fauna

The phlebotomine fauna is highly varied in Três Bra?os, an endemic area of american cutaneous leishmaniasis, situated in the cacao growing region in the southeast of Bahia State, Brazil. Thirty spécies of the Lutzomyia genus were identified in 13,535 specimens collected between 1976 and 1984. Lutzomyia whitmani was the dominant species accounting for 99% of flies in the peridomicile and 97.5% of those caught in homes. In the forest the predominant species were Lu. ayrozai and Lu. yuilli. Lu. whitmani accounted for only 1.0% of the specimens examined. Lu. flaviscutellata, the proven vector of Leishmania mexicana amazonensis, was also collected in small numbers. Lu. wellcomei, a known vector of L. braziliensis braziliensis in the Serra dos Carajás, Pará, Brazil was not encountered in the Três Bra?os region where the parasite causing human infections is usually L.b. braziliensis. Although we have not encountered a natural infection with leishmanial promastigotes in 1,832 females of the various species examined, we discuss the probability that Lu. whitmani is the vector of L.b. braziliensis in the region maintaining transmission in dogs and man.     

Leishmaniasis in Bolivia. II. The involvement of Psychodopygus yucumensis and Psychodopygus llanosmartinsi in the selvatic transmission cycle of Leishmania braziliensis braziliensis in a lowland subandean region

An epidemiological survey of the vectors of cutaneous leishmaniasis ("espúndia" type) was carried out in the Alto Beni region of Bolivia, an area of Andean foothills at the Eastern limit of the Amazonian lowlands. The climate is typical wet tropical (15 degrees S latitude). Anthropophilic phlebotomine sandfly species were sampled at 20 sites, all forested. The importance of species from the Psychodopygus group, already suspected as a vector in the transmission of Leishmania from the braziliensis complex, was confirmed by: the aggressiveness and diversity of the species encountered (83% of catches, nine species), the discovery of a new anthropophilic species, P. yucumensis and the isolation of a strain of Leishmania braziliensis braziliensis indistinguishable from human strains from the same area, from two species, P. llanosmartinsi and P. yucumensis.     

Leishmania braziliensis isolates differing at the genome level display distinctive features in BALB/c mice

Leishmania braziliensis is the species responsible for the majority of cases of human cutaneous leishmaniasis in Brazil. In the present study, L. braziliensis isolates from two different geographic areas in Brazil were studied by RAPD, using arbitrary primers. We also evaluated other biological features of these two isolates. We compared (a) the clinical features they initiate or not once delivered subcutaneously as stationary-phase promastigotes in the footpad of BALB/c mice; (b) the parasite load in both the footpad and the draining lymph node; (c) the cytokines present in the supernatant of cultures of the cell suspensions from the draining lymph nodes; and (d) the cell types present at the site of parasite delivery. The results show that the L. braziliensis strain from Ceará (H3227) is genotypically different from the L. braziliensis strain from Bahia (BA788). H3227-parasitized mice developed detectable lesions, whereas BA788-parasitized mice did not. Fifteen days post parasite inoculation there was an increase in the numbers of macrophages and lymphocytes in the footpads, whatever the parasite inoculum. Parasite load at the inoculation site--namely the footpad--did not differ significantly; in draining lymph nodes, however, it increased over the period under study. Early after parasite inoculation, the cells recovered from the draining lymph nodes of BA788-parasitized mice produced higher levels of IFN-gamma, a feature coupled to a higher number of NK cells. Later, after the parasite inoculation, there was an increased content of IL-12p70 and IL-10 in the supernatant of cells recovered from the lymph nodes of H3227-parasitized mice. This comparative analysis points out that L. braziliensis isolates differing in their genomic profiles do establish different parasitic processes in BALB/c mice.     

Humoral immune responses among mucosal and cutaneous leishmaniasis patients caused by Leishmania braziliensis

Mucosal leishmaniasis is arguably the most morbid sequelae of cutaneous leishmaniasis. The importance of early diagnosis for effective therapy, coupled with the difficulty of diagnosing the disease parasitologically, prompted this investigation of humoral immune markers of mucosal disease. Promastigote soluble antigens of Leishmania braziliensis, isolated from cutaneous and mucosal lesions, were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis; antigens were identified by immunoblotting with parasite-specific IgG antibody-positive sera of patients with mucosal disease (n = 18) and cutaneous disease (n = 23). For antigens of the cutaneous parasite WR 2095, mucosal sera generally reacted intensely to antigens of 75, 66, and 45 kDa and weakly to 48-50-kDa antigens, whereas cutaneous sera generally detected weakly the first 3 antigens and intensely the latter doublet. The data suggest that the transition from the cutaneous antigenic profile to a mucosal antigenic profile could be used to predict mucosal disease in approximately half of mucosal patients. An additional finding was that antibodies present in the sera of patients with mucosal disease labeled a 66-kDa peptide of normal human lip mucosa more intensely than did cutaneous sera. Autoimmune processes stimulated by the reaction of IgG, originally directed against the 66-kDa of L. braziliensis, to the 66-kDa antigen of mucosal tissue may contribute to the clinical presentation of mucosal leishmaniasis.     

Detection of Leishmania braziliensis in human paraffin-embedded tissues from Tucumán, Argentina by polymerase chain reaction

American cutaneous leishmaniasis (ACL) is an endemic disease in Northern Argentina. We applied the polymerase chain reaction (PCR) followed by a hybridization labelled probe to 21 paraffin embedded human skin biopsies, already analyzed histologically, from leishmaniasis endemic areas in the province of Tucumán, Argentina. We used primers previously designed to detect a Leishmania-specific 120-base-pair fragment of kinetoplast DNA minicircle, other two primer pairs that amplify kDNA minicircles belonging to the L. braziliensis and L. mexicana complexes respectively, and specific oligonucleotide primers to detect L. (V.) braziliensis which amplify the sequence of the ribosomal protein L-14 of this species. The PCR-hybridization showed a sensitivity of 90.5% when compared to the histopathology test which was 61.9%. Five of the total samples analyzed were positive for the L. braziliensis complex whilst none was positive for the L. mexicana complex. The specific primers for L. (V.) braziliensis detected the parasite in four samples. These results are consistent with those reported for close endemic areas and demonstrate that the causative agent of human leishmaniasis in the analyzed cases was L. (V.) braziliensis. PCR should be used as a diagnostic tool for tegumentary leishmaniasis, especially in the mucosal form, and as a valuable technique for the identification of the Leishmania species that causes the disease in certain areas.     

Cutaneous leishmaniasis is frequent in equines from an endemic area in Rio de Janeiro, Brazil

In an endemic area of cutaneous leishmaniasis in Rio de Janeiro State where a mule had been found infected, a systematic search among equines was performed, resulting in the detection of Leishmania parasites in skin lesions of 30.8% of the animals, which included horses and mules. The eventual role of equines in the epidemiology of the human disease is being investigated.     

Recovery of Leishmania (Viannia) braziliensis from inoculated hamsters

Leishmania (Viannia) braziliensis has never been isolated from wild animals although it is apparently capable of inducing infections in man, dogs, and donkeys. An analysis of the standard hamster culture system for analyzing infectivity of Leishmania sp. was undertaken. Results indicate that for L. (V.) braziliensis, routine cultivation of aspirates taken from the inoculation sites of 1-mo-infected hamsters should be undertaken. Moreover, in at least 1 of the 3 strains examined, isolation of the parasite was only achieved after 84 days of cultivation.