Resolution of an infection with Leishmania braziliensis confers complete protection to a subsequent challenge with Leishmania major in BALB/c mice

Both Leishmania major and L. braziliensis induce cutaneous leishmaniasis in BALB/c mice. Whereas BALB/c mice die of infection with L. major, they cure an infection with L. braziliensis. We report here that after curing an infection with L. braziliensis, BALB/c mice are resistant to challenge with L. major. When challenged with L. major, L. braziliensis pre-treated BALB/c mice mounted a delayed-type hypersensitivity response to L. major and produced high amounts of interferon-gamma (IFN-gamma) but low amounts of interleukin-4. The IFN-gamma produced by the L. braziliensis pre-infected mice was involved in the protection seen against L. major challenge since treating the mice with a neutralizing anti-IFN-gamma abrogated the protection. This suggests that cross-reactive antigen epitopes exist between L. braziliensis and L. major and that pre-infection with L. braziliensis primes BALB/c mice to epitopes on L. major that can elicit a protective Th1 response to the parasite.     

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.     

Distribution of sand flies (Diptera: Psychodidae) at different altitudes in an endemic region of American cutaneous leishmaniasis in the State of Espírito Santo, Brazil.

The involvement of different sand fly species in the transmission of American cutaneous leishmaniasis (ACL) at different altitudes was evaluated in the municipality of Afonso Cláudio in the State of Espírito Santo, Brazil, from November 1995 to February 1997. CDC light traps and Shannon traps baited with human volunteers were hung simultaneously at three altitudes of a river valley: (a) 650-750 m in an area of active ACL transmission; (b) 750-850 m in a transitional area; and (c) 850-950 m in an area where no ACL transmission occurred. A total of 13,363 specimens belonging to 28 species was collected. The five most abundant man-biting species were Lutzomyia intermedia, which constituted 24.3% of the total, Lu. migonei (22.3%), Lu. whitmani (15.4%), Lu. fischeri (14.9%) and Lu. monticola (5.8%). Analysis of the distribution of these species at the three altitudes provided evidence that Lu. fischeri and Lu. monticola were not involved in ACL transmission, whereas Lu. migonei and Lu. whitmani might act as secondary vectors of Leishmania (Viannia) braziliensis and Lu. intermedia was probably the principal vector of the parasite. These results reinforce those published in the existing literature, which indicate that Lu. intermedia is the main vector of Le. (V.) braziliensis in Southeast Brazil, while Lu. migonei and Lu. whitmani are of secondary importance.     

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.     

Dissociation between vasodilation and Leishmania infection-enhancing effects of sand fly saliva and maxadilan.

In this study, the ability of maxadilan and Lutzomyia longipalpis salivary gland lysate to enhance the infection of CBA mice by Leishmania major and of BALB/c mice by L. braziliensis was tested. No difference was observed between sizes of lesion in CBA mice infected with L. major and treated or not with salivary gland lysate or maxadilan, although they were injected in concentrations that induced cutaneous vasodilation. Although parasites were more frequently observed in foot pads and spleens of animals treated with maxadilan than in the animals treated with salivary gland lysate or saline, the differences were small and not statistically significant. The lesions in BALB/c mice infected with L. braziliensis and treated with maxadilan were slightly larger than in animals that received Leishmania alone. Such differences disappeared 14 weeks after infection, and were statistically significant only in one of two experiments.     

Experimental comparison of anthropophily between geographically dispersed populations of Lutzomyia whitmani (Diptera: Psychodidae)

Lutzomyia whitmani, a major vector of cutaneous leishmaniasis in Brazil, occupies diverse habitats from the Amazon forest canopy to suburban animal pens. Three mitochondrial lineages of Lu. whitmani ('Amazonian', 'North-South' and 'North-east') have parapatric distributions coinciding with different ecological zones. We assessed the host preferences of populations representing the three lineages in standardized field experiments, and found that Lu. whitmani in all sites were significantly more attracted to humans than to dogs or chickens. Females from a southerly population of the North-South lineage showed the greatest degree of anthropophily. Lu. whitmani from Amazonia were also strongly attracted to human baits, contradicting previously published accounts. Intraspecific comparisons in non-Amazonian sites suggest that Lu. whitmani is less anthropophilic than Lu. intermedia but more so than Lu. longipalpis. No significant difference was detected in anthropophily between Lu. whitmani in the Amazon and either Lu. dendrophyla or Lu. gomezi. Anthropophilic behaviour was demonstrated in the same site for Lu. complexa, Lu. flaviscutellata and Lu. brachyphalla, but not for Lu. infraspinosa.     

TGF-beta-associated enhanced susceptibility to leishmaniasis following intramuscular vaccination of mice with Leishmania amazonensis antigens

Leishmania amazonensis and Leishmania braziliensis are the main causal agents of anergic diffuse cutaneous leishmaniasis and hyperergic mucosal leishmaniasis in man, respectively. In this work we demonstrate that intramuscular vaccination of BALB/c mice with whole antigens of L. amazonensis (LaAg) but not L. braziliensis (LbAg) results in increased susceptibility to cutaneous leishmaniasis. LaAg vaccination resulted in an increased capacity of the draining lymph nodes to produce IL-10 and TGF-beta during antigen recall responses. In vitro cultivation with LaAg but not LbAg induced increased apoptosis of CD8+ T cells. Following infection with L. amazonensis, LaAg-vaccinated mice produced significantly more TGF-beta and a higher serum IgG1/IgG2a antibody ratio compared with LbAg-vaccinated and non-vaccinated animals. The association of TGF-beta with enhanced susceptibility to infection was confirmed in mice co-vaccinated with LaAg and neutralizing anti-TGF-beta antibodies. Upon parasite challenge, these animals developed much smaller lesion sizes and parasite burdens, comparable with non-vaccinated controls. The disease-promoting effect of LaAg vaccination is not a general event, as in contrast to BALB/c, the disease outcome in C57Bl/6 mice was unaltered. Together, these findings indicate that species-specific components of L. amazonensis activate overt TGF-beta production that predisposes more susceptible individuals to aggravated disease following vaccination.     

Lutzomyia (Nyssomyia) whitmani s.l . (Antunes & Coutinho, 1939)(Diptera: Psychodidae: Phlebotominae): geographical distribution and the epidemiology of American cutaneous leishmaniasis in Brazil--mini-review

The aim of the present study, in view of the widespread geographical distribution of Lutzomyia (Nyssomyia) whitmani s.l. in Brazil, in close association with the regions of transmission of Leishmania (Viannia) braziliensis and L. (V.) shawi, is to investigate the distribution of this sand fly species and American cutaneous leishmaniasis (ACL) in relationship to vegetation and landscape ecology throughout Brazil. Thematic maps were elaborated by the MapInfo programme, giving information on the spatial distribution of L. whitmani s.l., in accordance with types of vegetation and foci of ACL. With regards to the known areas of transmission of ACL in Brazil, it is notable that L. (N.) whitmani s.l. occurs in most of them, where it has been implicated as a possible vector of L. (V.) braziliensis. The presence of L. whitmani s.l. has been registered in 26 states, the one exception being Santa Catarina; in some states such as Roraima, Acre, Tocantins, and Mato Grosso do Sul this sand fly species has been recorded in a large number of municipalities. L. whitmani s.l. has been found in association with a variety of vegetation types, including the Amazonian forest, savanna ("campos cerrados"), and northeastern savanna ("caatingas nordestinas" or "savana estépica").     

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.     

Vector competence of some Neotropical sandflies for the Leishmania (Viannia) braziliensis complex

Abstract. To evaluate the vector competence of some Lutzomyia spp. (Diptera: Psychodidae) for Leishmania (Viannia) spp. (Kinetoplastida: Trypanosoma-tidae), experimental infections of anthropophilic sandflies from the Colombian Pacific coast were performed, through membrane feeding and xenodiagnosis on hamsters infected with Le.(V.)braziliensis or Le.(V.)panamensis. Wild-caught or F, generation females of Lutzomyia gomezi, Lu.hartmanni, Lu.panamensis and Lu. trapidoi were allowed to feed on hamster lesions and then maintained at 26^oC and >80% r.h. on a sugar-water diet until dissection on the fifth day post-infection (p.i.).
Despite similar infection rates (range 37–44%) in both Lu.gomezi and Lu.trapidoi , infections were heavier (>100 parasites) in the latter species. Infections of Lu.trapidoi with Le.braziliensis ( n = 21) and Le.panamensis (n = 27) showed parasite migration toward the foregut, with promastigote colonization of the stomodeal valve and appearance of infective forms. In contrast, infections of Lu.gomezi with Le. braziliensis (n = 10) and Le.panamensis (n = 5) were light (<50 parasites) and usually restricted to the pylorus. In Lu.hartmanni , only a few promastigotes were found in the pylorus and midgut of 3/8 specimens infected with Le.braziliensis , and no Le.panamensis developed (n = 19). By day 5 p.i., promastigote colonization of the hind- and midgut by Le.panamensis was observed in 2/4 Lu.panamensis but not Le.braziliensis (n = 3).
It was concluded that Lu.trapidoi is a more efficient vector than Lu.gomezi for both Le.braziliensis and Le.panamensis , and that Lu.hartmanni and Lu.panamensis are of minor importance for Leishmania transmission in this endemic area.     

Leishmania tropica infection, in comparison to Leishmania major, induces lower delayed type hypersensitivity in BALB/c mice

Leishmania tropica and L. major are etiologic agents of human cutaneous leishmaniasis. Delayed type hypersensitivity (DTH) is an immunologic response that has been frequently used as a correlate for protection against or sensitization to leishmania antigen. In BALB/c mice, L. tropica infection results in non-ulcerating disease, whereas L. major infection results in destructive lesions. In order to clarify the immunologic mechanisms of these 2 different outcomes, we compared the ability of these 2 leishmania species in induction of DTH response in this murine model. BALB/c mice were infected with L. major or L. tropica, and disease evolution and DTH responses were determined. The results show that the primary L. major infection can exacerbate the secondary L. major infection and is associated with DTH response. Higher doses of the primary L. major infection result in more disease exacerbation of the secondary L. major infection as well as higher DTH response. L. tropica infection induces lower DTH responses than L. major. We have previously reported that the primary L. tropica infection induces partial protection against the secondary L. major infection in BALB/c mice. Induction of lower DTH response by L. tropica suggests that the protection induced against L. major by prior L. tropica infection may be due to suppression of DTH response.     

Cutaneous inflammatory disorder in integrin alphaE (CD103)-deficient mice

The integrin alpha(E)beta(7) is thought to play an important role in the localization of mucosal, but not of cutaneous T lymphocytes. Thus, it was surprising that 89% of adult alpha(E)(-/-) mice on the 129/Sv x BALB/c background developed inflammatory skin lesions without an apparent infectious etiology. Skin inflammation correlated with alpha(E) deficiency in mice with a mixed 129/Sv x BALB/c background, but not in mice further backcrossed to BALB/c and housed in a second animal facility. These studies suggested that alpha(E) deficiency, in combination with other genetic and/or environmental factors, is involved in lesion development. The lesions were infiltrated by CD4(+) T cells and neutrophils, and associated with increased expression of inflammatory cytokines. Furthermore, skin inflammation resulted from transfer of unfractionated alpha(E)(-/-) splenocytes into scid/scid mice, but not from transfer of wild-type splenocytes, suggesting that the lesions resulted from immune dysregulation. We also studied the role of alpha(E)beta(7) in a murine model of hyperproliferative inflammatory skin disorders that is induced by transfer of minor histocompatibility-mismatched CD4(+)/CD45RB(high) T cells into scid/scid mice under specific environmental conditions. Under housing conditions that were permissive for lesion development, transfer of alpha(E)-deficient CD4(+)/CD45RB(high) T cells significantly exacerbated the cutaneous lesions as compared with lesions observed in mice reconstituted with wild-type donor cells. These experiments suggested that alpha(E)-expressing cells play an important role during the course of cutaneous inflammation. In addition, they suggest that alpha(E)beta(7) deficiency, in combination with other genetic or environmental factors, is a risk factor for inflammatory skin disease.     

Administration of monoclonal anti-IFN-gamma antibodies in vivo abrogates natural resistance of C3H/HeN mice to infection with Leishmania major

C3H/HeN mice that are naturally resistant to cutaneous disease and systemic infections with the protozoan parasite, Leishmania major, were treated at the time of infection, and weekly thereafter, with mouse anti-rat IFN-gamma mAb or an irrelevant antibody of similar isotype. Anti-IFN-gamma-treated mice developed cutaneous lesions; parasites spread to the regional lymph nodes and then metastasized to spleens and livers. The course of disease in these animals was similar to that of genetically susceptible BALB/c mice. Two exceptions in the pathology of L. major infections were noted between BALB/c and anti-IFN-gamma-treated C3H/HeN mice: 1) BALB/c mice died of systemic complications, whereas C3H/HeN mice did not, and 2) multinucleated giant cells were observed in lymph nodes and spleens of infected BALB/c mice, whereas these cells were not observed in infected C3H/HeN mice. Control mice, those treated with either saline or irrelevant antibody of the same isotype as the anti-IFN-gamma monoclonal, showed no evidence of cutaneous disease (development of footpad lesions) or systemic infection (by histopathology). Total abrogation of the natural resistance of C3H/HeN mice could be achieved by treatment with as little as 0.5 mg/mouse/wk of anti-IFN-gamma antibody, or by a single dose of 1 mg/mouse anti-IFN-gamma antibody administered at the time of parasite inoculation. If antibody treatment was delayed for as little as 1 wk after parasite inoculation, the infections in treated animals resembled that of untreated or control antibody-treated mice: no cutaneous lesions (by footpad swelling or viable counts of leishmania in footpad tissue) or systemic disease (by microscopic analysis of touch preparations of internal organs, and histopathology of same). The production of IFN-gamma during the initial interaction of the parasite and host cells appears to be a major component of genetic control of natural resistance to infection with L. major in C3H/HeN mice.     

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.     

The role of interleukin-10 in susceptibility of BALB/c mice to infection with Leishmania mexicana and Leishmania amazonensis

Recent studies have demonstrated the critical role of IL-10 in susceptibility to cutaneous and visceral leishmaniasis caused by Leishmania major and Leishmania donovani, respectively. To determine whether IL-10 also plays a similar role in the susceptibility and pathogenesis of cutaneous leishmaniasis caused by the New World species, L. mexicana and L. amazonensis, we analyzed their course of infection in IL-10-deficient BALB/c mice and their wild-type counterparts. Although IL-10-deficient mice infected with either L. mexicana or L. amazonensis failed to control the lesion progression, we did observe consistently lower levels of infection in IL-10(-/-) mice compared with wild-type BALB/c mice. We also observed increased IFN-gamma and NO production and higher levels for IL-12p40 and IL-12Rbeta(2) mRNA in cells from IL-10(-/-) mice compared with cells from BALB/c mice. The mRNA levels for IL-4, which increased significantly in both IL-10(-/-) and BALB/c mice, were comparable. When treated with anti-IL-4 mAb, IL-10(-/-) mice resolved the infection more effectively and had significantly fewer parasites in their lesions compared with similarly treated BALB/c mice. These findings suggest that IL-10, although not the dominant mediator of susceptibility of BALB/c mice to infection with L. mexicana and L. amazonensis, does play a significant role in regulating the development of a protective Th1-type response. However, effective resolution of infection with these New World parasites requires neutralization of both IL-4 and IL-10.     

Cutaneous leishmaniasis in anti-IgM-treated mice: enhanced resistance due to functional depletion of a B cell-dependent T cell involved in the suppressor pathway

The contribution of B cells and antibodies to either the resistance or susceptibility to cutaneous leishmaniasis has been investigated in mouse strains rendered B cell-deficient by treatment with anti-mouse IgM antisera from birth (mu-suppressed). These studies confirm that immunity to cutaneous disease in a normally resistant mouse strain (C3H/HeJ) is independent of antibody, but that B cells and/or antibodies are required for the evolution of suppressed DTH and the consequent disease susceptibility of BALB/c mice. Anti-IgM-treated BALB/c mice, which lacked detectable anti-leishmanial antibodies during the course of infection, displayed a sustained DTH response to leishmanial antigen and were able to control their cutaneous lesions. The enhanced resistance of mu-suppressed mice could be completely abrogated by transfer of suppressor T cells from infected control animals into mu-suppressed mice before their infection. Thus the suppressor T cells, which are generated during leishmanial infection in BALB/c mice, can effect suppression in the absence of antibody. Evidence that B cells or antibodies are required for the generation of suppressor T cells was demonstrated by using BALB/c mice in which suppressor T cells fail to be generated during infection as a result of prior sublethal irradiation. Splenic T cells from normal mice could overcome the resistance conferred by sublethal irradiation, whereas splenic T cells from mu-suppressed mice could not. Thus the enhanced resistance of mu-suppressed BALB/c mice appears to be a consequence of their lack of functional expression of a B cell-dependent T cell critical to the suppressor pathway.     

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.     

Suppressive effect of cyclosporin A on the development of Leishmania tropica-induced lesions in genetically susceptible BALB/c mice

The effect of cyclosporin A (CyA) application on the development of cutaneous lesions was analyzed in genetically susceptible BALB/c mice infected s.c. with Leishmania tropica promastigotes. Daily i.p. injections of CyA, beginning 2 days before or at the day of the infection, dose dependently inhibited the development of parasite-induced lesions; no effect on the lesions was observed, however, if CyA application was started 14 days after the infection. Cessation of CyA administration after having successfully suppressed the cutaneous lesions for a period of 42 days, resulted in the development of lesions within 3 days. CyA had no inhibitory effect on lesions developing in L. tropica infected hypothymic BALB/c nu/nu mice. CyA or CyA-containing mouse serum did not directly affect the viability and the growth rate of L. tropica promastigotes, suggesting that the effect of the agent was imposed on the cells participating in the formation of the cutaneous lesions. Quantitative analysis of the cell distribution in the spleens of infected mice revealed that CyA markedly suppressed the infection-associated numerical increase of splenocytes. Within the Thy-1+ lymphocyte compartment, CyA had its most pronounced effect on the Lyt-1+ T lymphocyte subset. Early in the disease, the frequency of splenic cells proliferating in response to L. tropica antigen in vitro was clearly inhibited by CyA; in the later stages of the infection, however, this effect could not be observed, indicating the presence of L. tropica-inducible T cells being relatively resistant to CyA. Taken together, our findings indicate that CyA reversibly inhibits or delays the parasite-induced expansion of Lyt-1+ splenic T lymphocytes, and thus suppresses the biological function of those T cells that are instrumental for the formation of cutaneous lesions in L. tropica-infected BALB/c mice.     

Isolation of Leishmania (Viannia) braziliensis from Lutzomyia spinicrassa (species group Verrucarum) Morales Osorno Mesa, Osorno and Hoyos 1969, in the Venezuelan Andean region

Natural infection with Leishmania spp. in phlebotomine sandflies was searched for during a longitudinal study carried out from July 1997 to July 1998 in the village Catarnica, Municipality Independencia, Táchira State. This hamlet is an old endemic focus of cutaneous leishmaniasis in the Venezuelan Andean region, which lies close to the Colombian border at 1,300 m a.s.l., in an agricultural area mainly used for cultivating coffee. Phlebotomine sandflies were collected using Shannon traps placed in the peridomestic habitat from 19:00 to 21:00 hs. Males were stored in alcohol 70 % while females were kept in Nunc vials with 10 % DMSO and cryopreserved in liquid nitrogen for subsequent dissection and identification. The most abundant anthropophilic species was Lutzomyia spinicrassa with 3,032 males and 4,290 females (85.4%). Among 1,633 (38%) females of Lu. spinicrassa dissected, 26 11.6%) were infected with promastigotes, while no natural infection was found in 209 females of other species. The flagellates were identified as Leishmania braziliensis braziliensis using PCR with species specific primers derived from nuclear DNA and hybridization using species specific probe labelled with digoxigenin. This parasite had been previously isolated from patients with cutaneous leishmaniasis from the same area. These results show Lu. spinicrassa as a new proven vector of Leishmania braziliensis in the Andean region of Venezuela.