DIFFERENCES IN THE ANTIGENIC PROFILE AND INFECTIVITY OF MURINE MACROPHAGES OF LEISHMANIA (VIANNIA) PARASITES

J. Parasitol., 96(3), 2010, pp. 509 515 F American Society of Parasitologists 2010 DIFFERENCES IN THE ANTIGENIC PROFILE AND INFECTIVITY OF MURINE MACROPHAGES OF LEISHMANIA (VIANNIA) PARASITES Nubia E. Matta*À, Lea Cysne-Finkelstein*, Gerzia Maria C. MachadoÀ, Alda Maria Da-Cruz*`, and Leonor LeonÀ Departamento de Biología, Universidad Nacional de Colombia, Bogotá, Colombia. e-mail: nemattac@unal.edu.co ABSTRACT: The antigenic profile and infectivity were compared between 3 recent Leishmania (Viannia) isolates from the Amazonian region (Instituto Nacional de Pesquisas da Amazonia [INPA] strains) and 3 World Health Organization (WHO) reference species (Leishmania guyanensis, Leishmania braziliensis, and Leishmania naiffi). Differences were observed in the peak and extent of promastigote growth. The WHO reference strains exhibited significantly higher exponential growth as promastigotes than INPA strains. In the immunoblot analyses, the INPA strains revealed several specific peptide fragments, as well as the greatest recognition frequencies by sera from Leishmania sp. infected ; among the latter, antigens derived from L. naiffi were the most frequently recognized. In vitro infection was carried out using mice peritoneal macrophages; all strains were able to enter the macrophages, but only L. amazonensis was able to reproduce. A striking observation was that L. naiffi exhibited the longest survival time inside the macrophages. Our data strongly suggest the application of recently isolated parasites as sources of antigen for diagnosis procedures. Moreover, L. naiffi species possesses several characteristics relevant for its use as a source of novel antigens to be explored in the design of diagnostic tools and vaccines. American cutaneous leishmaniasis (ACL) is a widespread parasitic disease in most Latin American countries. Cutaneous leishmaniasis (CL), with single, or multiple, ulcerated skin lesions represents the most frequent form of the disease. ACL is caused by protozoans from several Leishmania species belonging to the subgenera Leishmania and Viannia. Leishmania (Viannia) parasites represent a biologically diverse group of microorganisms having considerable inter- and intraspecies variability (Cupolillo et al., 1994, 1998). For example, recent studies on Leishmania (V.) guyanensis show that, depending on the geographic site of isolation, there are differences in its reactivity to the monoclonal antibody B19, which is considered species-specific (McMahon-Pratt et al., 1982). More specifically, antibody B19 reacted with French Guiana and eastern Amazonian Brazil isolates, but not with parasites from Colombia or Manaus (Amazonas, Brazil) (Romero et al., 2002; Saravia et al., 2002). Considering that the epitopes recognized by B19 are surface membrane molecules, it is possible that the immune system may be exerting some selective pressure, resulting in changes in the expression of these molecules in various localities (Saravia et al., 2002). Alternatively, it is conceivable that the samples of parasites used to generate the monoclonal antibody B19 had different antigenic composition in comparison with those isolated in other geographic regions (Saravia et al., 2002). However, metacyclogenesis is associated with molecular changes to the cell surface, i.e., promastigote surface antigen (GP46), major surface protease (GP63), and lipophosphoglycan (LPG). Thus, these molecules are lost with serial passage (Brittingham et al., 2001; Beetham et al., 2003). The number of passages in culture of Leishmania sp. parasites is inversely correlated with the number of metacyclic forms and the lesion size induced in mice or hamsters after Received 30 June 2009; revised 20 September 2009, 21 November 2009; accepted 5 December 2009. * Laboratório de Imunoparasitologia, Instituto Oswaldo Cruz, FIO- CRUZ, Av. Brasil 4365, Pav. Leônidas Deane, 4u andar, Manguinhos, Rio de Janeiro-RJ, CEP 21040-900, Brazil. { Laboratório de Bioquimica de Tripanossomatídeos, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365, Pav. Leônidas Deane, 4u andar, Manguinhos, Rio de Janeiro-RJ, CEP 21040-900, Brazil. { Laboratório Interdisciplinar de Pesquisas Medicas, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365, Pav. Leônidas Deane, 4u andar, Manguinhos, Rio de Janeiro-RJ, CEP 21040-900, Brazil. DOI: 10.1645/GE-2241.1 inoculation (Rey et al., 1990; Cysne-Finkelstein et al., 1998). Moreover, nitric oxide (NO) production is altered, reinforcing the association of NO with metacyclogenesis (Genestra et al., 2003). It has been also observed that noninfective parasites (with no metacyclic forms detected) showed a higher rate of growth than infective promastigotes (Genestra et al., 2003). Collectively, these observations may have profound implications when comparing studies that have used WHO Leishmania reference strains (which have undergone multiple culture passages) to those that use more recently isolated local Leishmania sp. parasites. Therefore, we decided to compare WHO Leishmania reference strains to recently isolated parasites, by focusing on their immunoreactivity to human sera from with different parasitic infections and their infectivity of murine macrophages in vitro. MATERIALS AND METHODS Leishmania strains Three WHO reference strains (Leishmania (V.) braziliensis, Leishmania (V.) guyanensis, and Leishmania (V.) naiffi) and 3 isolates of the same species from Amazonian stored at the Instituto Nacional de Pesquisas da Amazonia (INPA) were used. Parasites were maintained in Schneider s Insect medium, supplemented with 20% of heat-inactivated fetal calf serum (HI-FCS) and 2% human urine (Howard et al., 1991) at 26 C, ph 7.2. The INPA s strain infectivity was maintained through passages in hamsters prior to experiments. Parasites recovered from hamsters were seeded in NNN medium and then in Schneider s medium, and were used after no more than 8 serial passages in vitro. The WHO reference strains were maintained by subculturing in NNN medium and used without previous passage in animal models. As the control of the macrophage infection experiments, L. (Leishmania) amazonensis was used. Designations and sources of the Leishmania species employed in this study are presented in Table I. Growth curves Procedures for growing Leishmania sp. promastigotes in vitro have been reported previously (Grimaldi et al., 1987). Briefly, promastigotes (5 3 10 5 ) from 3-day-old cultures were seeded in sterile culture bottles (Corning, Lowell, Massachusetts) containing 5 ml of Schneider s medium, as described in previous paragraph, and incubated at 26 C. Every 24 hr, 0.05-ml aliquots were collected, and the number of parasites was counted using a Neubauer s chamber. Preparation of samples for immunoblot analysis From each strain, the total antigens were obtained as described previously (Leon et al., 1990), with some modifications. Briefly, 509

510 THE JOURNAL OF PARASITOLOGY, VOL. 96, NO. 3, JUNE 2010 TABLE I. Characteristics of Leishmania species used in this study: (1) Laboratorio de Pesquisa em Leishmaniose, Instituto Oswaldo Cruz (IOC); and (2) Instituto Nacional de Pesquisas da Amazonia (INPA). Species International code Source Donor Leishmania (Viannia) braziliensis.mhom/br/75/m2903.man 1 Leishmania (Viannia) guyanensis.mhom/br/75/m4147.man 1 Leishmania (Viannia) naiffi.mdas/br/79/m5533.armadillo 1 Leishmania (Viannia) braziliensis.mhom/br/95/im4102.man 2 Leishmania (Viannia) guyanensis.mhom/br/95/im4216.man 2 Leishmania (Viannia) naiffi.mhom/br/86/im2764.man 2. Leishmania (Leishmania) amazonensis.mhom/br/77ltb0016.man 1. promastigotes at the late log phase of growth in Schneider s medium were harvested by centrifugation (15,000 g for 10 min at 4 C) and washed twice in phosphate-buffered saline (PBS). Pellets were resuspended in an antiproteolytic buffer (40 mm NaCl, 10 mm sodium ethylenediaminotetraacetate [EDTA], 1 mm phenylmethylsulfonylfluoride [PMSF], 10 mm iodoacetamide, 5 mm 1,10-phenanthroline, and 10 mm Tris, ph 8.0). Parasites were then disrupted by 10 repeated cycles of freezing and thawing, followed by 15 min ultrasonication at 65 MHz (BRANSONICH ultrasonic cleaner, Wilmington, North Carolina). This preparation was centrifuged (14,000 rpm/10 min), and supernatant was considered to contain soluble antigens, which were used in immunoblot assays. Protein concentration was determined by spectrophotometry (Spectronic 21 Milton Roy Company, Ivyland, Pennsylvania) (Johnstone and Thorpe, 1982). Final protein concentration was adjusted at 1 mg/ml. Samples were stored in aliquots at 220 C until use. Study groups Blood samples were obtained from 4 groups of subjects: (1) 23 suffering from CL from the endemic area of L. guyanensis (CL-Lg); (2) 8 with CL due to L. braziliensis (CL-Lb); (3) 12 healthy donors living in endemic areas for L. guyanensis or L. braziliensis; (4) 4 with Chagas disease. The following criteria were used for diagnosis: (1) type of lesion and epidemiological data compatible with American tegumentary leishmaniasis (ATL); (2) positive Montenegro skin test (MST), a delayed-type hypersensitivity to leishmania antigens; (3) presence of immunoglobulin G and/or M Leishmania-specific serum antibodies; and (4) detection of Leishmania sp. parasites in lesion by microscopic examination of histological sections from biopsy samples or by culture in NNN modified medium. This study was approved by the Committee of Ethics of the Fundação Oswaldo Cruz, Ministério da Saúde, Rio de Janeiro, Brazil. Informed consent was obtained from all individuals who participated in the study. Western blot analysis Total protein fractions were resolved by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) using 10% slab gels in nonreducing conditions together with prestained SDS molecular weight markers (Sigma, St. Louis, Missouri). Electrophoresis was performed in a Hoefer Amersham SE 660 apparatus, at 40 ma for 4 hr. Polypeptides were transblotted onto nitrocellulose membrane (0.45 mm), according to the procedure described by Towbin et al. (1979). After transference, lanes containing molecular mass standards were cut and stored. The nitrocellulose sheets were blocked with PBS, 0.5% Tween-20 (PBS-T) (Bio-Rad, Hercules, California), and 10% nonfat milk for 90 min at room temperature, and then washed in PBS-T for 30 min under agitation. Membranes were incubated overnight with the antisera from infected with L.(V.) braziliensis or L.(V.) guyanensis (1:100 in PBS-T), and then washed 6 times with PBS-T and incubated for 1 hr with antihuman immunoglobulin G antibody coupled to peroxidase (1:1,000 in PBS-T; Sigma). Next, lanes were washed again 6 times with PBS-T for 15 min each. Antigen-antibody reactions were detected by the peroxidase activity on H 2 O 2 and in the presence of 3939-diaminobenzidine (Graham and Karnovsky, 1965). Molecular weights are expressed as kda. Murine macrophage culture Murine resident peritoneal macrophages obtained from 6- to 8-wk-old, male BALB/c mice were placed in cold, serum-free RPMI 1640 medium, supplemented with 1 mm of L-glutamine, 1 mm of HEPES (N-[2- hydroxyethyl] piperazine-n9-[2-ethanesulfonic acid]), 100 IU of penicillin, 100 mg/ml of streptomycin, and 1 mm of sodium pyruvate. Macrophages were seeded on tissue plate wells containing round cover slips (3 3 10 6 cells/well) and then incubated for 2 hr at 37 C in a 5% CO 2 atmosphere. Nonadherent cells were removed by washing with RPMI 1640 medium, supplemented with 10% HI-FCS. Cells were maintained under the same culture conditions for 24 hr before infection. Evaluation of the infection of the different Leishmania species and isolates Leishmania guyanensis, L. braziliensis, and L. naiffi promastigotes from INPA isolates and WHO reference strains were harvested in late log growth phase. Parasites were washed in culture medium and resuspended in a medium containing 1% HI-FCS and incubated overnight with peritoneal adherent cells (10 parasites/cell, 3 3 10 7 parasites/well), in a CO 2 incubator at 37 C. After this period, free parasites were removed by washing the monolayers with serum-free medium. Infected macrophages were maintained in RPMI 1640 with 10% HI-FCS for 24 or 48 hr. Cover slips were fixed in methanol and stained with Giemsa. Intracellular numbers of parasites were assessed microscopically (Carl Zeiss, Axioskop, Göttingen, Germany). The percentage of infected macrophages and intracellular parasite numbers per 100 macrophages were determined by counting 200 cells per cover slip. Data analysis Results were analyzed by 1-way ANOVA test using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, California), and a P-value,0.05 was deemed significant. Growth kinetics RESULTS The species compared here showed differences with regard to the peak of growth, culture density, and infective ability. WHO reference strains exhibited higher levels of growth than recent isolates. The peak of growth for L. braziliensis and L. guyanensis was reached at day 5 by both the WHO and INPA isolates, whereas L. naiffi strains, from both sources, reached its growth peak at day 6 (Fig. 1). Previous data have shown that noninfective parasites, which do not have metacyclic forms detected, usually show a high rate of growth as compared to the infective promastigotes (Genestra et al., 2003). Our results corroborate these data. Percentages of macrophage infection To investigate infectivity differences between WHO and INPA strains, the percentage of infected macrophages and the number

MATTA ET AL. DIFFERENCES OF LEISHMANIA (V.) PARASITES 511 FIGURE 1. The curves show kinetics of Leishmania growth maintained in Schneider medium supplemented with 20% fetal calf serum plus 2% human urine at 26 C. (A) WHO reference strains. (B) INPA strains. Individual points represent mean ± SEM. of amastigotes per 100 macrophages were determined. Leishmania amazonensis LTB 0016, used as a positive control for infection, consistently showed 100% success; indeed, it was the only species that exhibited an increase in the number of intracellular amastigotes (Fig. 2a c). All the L. (Viannia) species analyzed were internalized by macrophages within the first 24 hr (Fig. 2a c). Whereas the initial parasite recruitment was high, a marked reduction of intracellular parasites was always observed, indicating that they were not able to replicate inside the macrophage. Since the macrophages are capable of killing promastigotes after uptake (Pearson et al., 1981), an estimation of the number of amastigotes found 72 hr subsequent to initial infection should give a reliable picture of the infective potential of a parasite population, as suggested by Sacks and Perkins (1984). Leishmania naiffi survived the for the longest period and maintained the highest number of intracellular parasites 48 hr after macrophage exposure, infecting 3 times more macrophages than the other species (P, 0.05) (Fig. 2c). These data clearly indicate that the clearance of L. braziliensis or L. guyanensis species was faster than that observed for L. naiffi (Fig. 2a, b). Differences in the antigenic profile The antibody response to total antigenic fractions from L. guyanensis, L. braziliensis, and L. naiffi from both sources was evaluated using immunoblot analysis. Overall, 35 to 50 protein bands from the leishmanial preparations could be determined by silver staining, independent of the source of parasites. Relatively few peptides were observed in the upper molecular weight range (molecular mass.80 kda). However, numerous bands in the middle and lower molecular weight ranges were observed. The pattern of antibody recognition to the 6 leishmanial antigen preparations was determined with sera from the 2 CL patient groups (infected with either L. braziliensis or L. guyanensis), from a healthy control group, and sera from Chagas disease. both CL groups of showed numerous antigens in the molecular weight ranging from 26 to 116 kda. Differentially recognized bands in the Western blot assay in sera from L. guyanensis (CL-Lg) or L. braziliensis (CL-Lb) infected, comparing WHO reference strains and INPA strains, are shown in Table II.

512 THE JOURNAL OF PARASITOLOGY, VOL. 96, NO. 3, JUNE 2010 FIGURE 2. Peritoneal macrophages obtained from BALB/c mice infected with promastigotes from late log phase of growth, from the INPA Leishmania strains and WHO reference strains at a 10:1 ratio (Leishmania: macrophage): L. (V.) braziliensis; L. (V.) guyanensis; L. (V.) naiffi. L. (L.) amazonensis was used as control. The data were obtained at 0, 24, and 48 hr postinfection. At the indicated times, cover slips with cells were fixed stained with Giemsa and examined by light microscopy for the percentage of infected macrophages, and the number of intracellular parasites. Two-hundred macrophages were counted each time. Results are shown as the mean ± SEM from 3 samples in each group. (A) Percentage of infected macrophages. (B) Number of parasites/100 cells. In the immunoblots, the serum antibodies from the documented bands of 62, 55, 50.6, and 30.6 kda more often in INPA leishmanial extracts than in WHO extracts. Another protein band (36.6 kda) showed cross-reactivity with sera from Chagas disease. Finally, a band of 48.5 kda was found in all 3 types of sera, i.e., sera from CL, Chagas disease, and healthy donors. When comparing WHO or INPA Leishmania antigens, differences were observed in the number of bands and the frequency of recognition. The highest frequencies were detected in INPA antigenic preparations, which were the only ones that produced the 62, 50.6, 42, and 30.6 kda bands (Table II). There was not a specific band recognized in the WHO extracts; however, the peptide of 72.5 kda presented the highest frequencies in these preparations. In the L. naiffi extract, 2 protein fractions of 30.6 and 42 kda, which were not found in the other antigenic preparations, were detected. In addition, the highest frequencies of detection of 36.6, 48.5, and 55 kda bands were also seen in the L. naiffi extract. In summary, antigens from a heterologous species (L. naiffi) are well recognizable in the sera of L. guyanensis and L. braziliensis infected.

MATTA ET AL. DIFFERENCES OF LEISHMANIA (V.) PARASITES 513 TABLE II. Differential recognized bands in the Western blot assay by L. guyanensis (CL-Lg) and L. braziliensis (CL-Lb) infected, comparing WHO reference strains and INPA strains (kda: kilodaltons). Origin Molecular weight of bands (kda) WHO antigens CL-Lg CL-Lb DISCUSSION INPA antigens CL-Lg CL-Lb Strains. % Recognition L. guyanensis 62 0 0 39 71 50.6 0 0 22 43 L. braziliensis 62 0 0 35 43 50.6 0 0 22 57 L. naiffi 62 0 0 52 43 50.6 0 0 22 14 42 0 0 52 57 30.6 0 0 17 29 It has been shown that the number of passages in culture of Leishmania spp. parasites is negatively correlated with the number of metacyclic forms in cultures and the lesion size induced in mice or hamsters after parasite inoculation (Rey et al., 1990; Cysne- Finkelstein et al., 1998). In addition, membrane molecules such as GP63 are selectively lost after serial passage. When cultured in vitro for relatively longer periods of time, it appears that clones that develop are better adapted to experimental conditions. Our data show that these clones maintain their growth profiles, but WHO strains had higher rates of replication when compared with INPA isolates. This observation was previously made by Genestra et al. (2003). The mechanism by which serial passage causes attenuation may involve changes in the level and stability of mrna (Beetham, 2003). A striking observation in this study was that all species were found inside macrophages in the infection assays, independent of their number of subcultures, and that no significant differences in the infection index were detected. This may reflect active phagocytic function, or it may be mediated by ligand-receptor interactions, or both. If the second mechanism is operational, it could indicate that the molecules necessary for the invasion of host cells did not change significantly in the WHO species. On the one hand, it has been postulated that Leishmania sp. parasites have redundancy in molecules involved in virulence mechanisms, such as LPG or GP63 (Brittingham et al., 1995; Yao et al., 2003), which could operate in a complementary manner, depending on the relative abundance of these molecules. On the other hand, it is possible that these parasites have differences in their replication ability inside of macrophages, but this likelihood was not evaluated in the present study. Recently, it was demonstrated that both virulent and avirulent Leishmania major strains were able to invade macrophages, but only the first replicated once inside the cell (Brodskyn et al., 2000). It has been suggested that to sustain active infections within macrophages, parasites should preserve a detectable expression of trypanothione reductase (TR) (Castro-Pinto et al., 2007) and a high level of nitric oxide (NO) production (Genestra et al., 2006). TR and NO help parasites to maintain infection by neutralizing toxic free radicals (nitrogenated and oxygenated) produced by host cells. Several studies from our group have already shown that noninfective parasites have a low expression of TR, as well as low production of NO. These data could explain why parasites with no detectable metacyclic forms were unable to sustain the in vitro and in vivo infections (Genestra et al., 2006; Castro-Pinto et al., 2007). Interestingly, in the parasite-macrophage interaction assays, L. (V.) naiffi species exhibited the slowest clearance, and this difference was significant when compared with L. guyanensis or L. braziliensis species at 24 hr. Campos et al. (2008) correlated the low infectivity of L. (V.) naiffi with high NO production from macrophages, which results in a quick clearance of this parasite. Our results suggest that the low infectivity of L. (V.) naiffi may be related to an induced and well-balanced cytokine microenvironment. Recently, our group demonstrated that although L. naiffi antigens were able to expand in vitro a large quantity of T- cell lymphocytes from suffering from cutaneous leishmaniasis, the IFN-c levels produced were quite well balanced (Matta, 2005), which contrasted with the response detected from mucosal leishmaniasis stimulated with L. braziliensis antigens, where there is a high lymphoproliferative response, accompanied by high IFN-c levels. It has also been proposed that some organisms maximize their infectivity over pathology or mortality of their hosts. This means that less virulent organisms may replace virulent microorganisms as a consequence of a more efficient way of exploiting host clusters (Gandon et al., 2001). The association of Leishmania spp. with macrophages at the cellular level has been characterized as akin to symbiosis (Chang, 1983), apparently due primarily to their ability to preserve the structural, if not functional, integrity of infected cells (Chang et al., 2003). This notion could be extended to the in vivo observations of the natural infections due to L. naiffi species. Taken together, these results suggest a direct relationship between the lesion s size in the animal and the infectivity of macrophages in vitro. However, the present results with L. naiffi did not follow such a trend, since we found a high percentage of parasitized macrophages in vitro, while lesions are not produced by this species in hamsters, and CL infection in humans is self-limiting (Lainson, 1997). In the present study, we compared antigens derived from 3 different Leishmania species, i.e., L. (V.) braziliensis, L. (V.) guyanensis, and L. (V.) naiffi, with sera from infected by L. (V.) braziliensis or L. (V.) guyanensis. The Leishmania species analyzed shared bands of 26.6, 48.5, 50.6, 55, 62, and 72.5 kda, supporting the close relationship between species of the subgenus Viannia, and indicating the presence of common epitopes, consistent with previous findings (Chiller et al., 1990; Cuba Cuba et al., 2001; Gonçalves et al., 2002). When comparing the frequencies of recognition between WHO and INPA leishmania antigens, notable differences were found in the number of detected bands and the frequencies with which they appear when using the antisera from Leishmania spp. infected. A protein fraction was apparently present in all antigen preparations, i.e., 48.5 kda, but its frequency of recognition was broadly variable among the groups compared. There are several possible interpretations for this observation. First, there might be polypeptides that have very similar molecular weight but that have differing amino acid composition, and, therefore, different interactions between these proteins and the variable regions of the antibodies from the patient sera may occur. Second,

514 THE JOURNAL OF PARASITOLOGY, VOL. 96, NO. 3, JUNE 2010 although the same total protein quantity was deposited in each well, slight differences in the relative concentration of the different proteins can occur as a result of sample manipulation. Third, quantitative differences in the antigen-antibody complexes detected in the immunoblot assays are associated with the antibody concentrations present in the patient s sera. This concentration may be variable, i.e., due to difference in the duration of the infection, the infecting Leishmania species, and the immunological status of the. Changes in the antigenic composition and parasite phenotypes could determine the presence or absence of band(s) in the immunoblot assays. These antigenic changes may be relevant in the infection and in the intracellular survival of these parasites in macrophages. It is tempting to suggest that the 62 kda protein found specifically in INPA extracts may represent glycopeptide 63 (GP63, prominent in metacyclic forms) or leishmaniolysin, but further studies are required to corroborate this possibility. Overall, published studies suggest that this membrane protease, with molecular mass ranging from 63 to 68 kda, is an important ligand in cell to cell interactions and cell infectivity (Russell and Wilhelm, 1986; Kweider et al., 1987). Bands of 50.6 and 55 kda were also found with high frequencies in INPA extracts. Polypeptides with similar molecular weight have been found with variable reactivity among Leishmania spp. infected (Rodriguez et al., 1983; Nagakura et al., 1986; Jaffe et al., 1990; Gonçalves et al., 2002). It has been suggested that this fraction could also be the promastigote surface protein (gp63) with a relative molecular weight of 50 kda (determined under nonreducing conditions). Detailed analyses of the bands with high frequency of recognition in the INPA extract would be a key in the identification of important molecules implied in Leishmania spp. infection, as well as in the design, or improvement, of immunodiagnostic tests. Our results suggest that the observed antigenic differences in immunoblot assays among the parasites have no relevance in the macrophage infection process, at least using the present in vitro assay. Data showing that INPA isolates have the highest frequency of recognition and specific bands reinforce the importance of the use of recent isolates for diagnostic or immunoprophylactic purposes. Moreover, L. naiffi is presented as a good source of leishmanial antigens for the same purposes. ACKNOWLEDGMENTS This study was supported by grant 190025/01-8 from Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil, (CNPq). N.E.M. was sponsored by CNPq and Universidad Nacional de Colombia, Sede Bogotá. A.M.C. and L.L. are CNPq research fellows. IOC/FIOCRUZ internal funds RENOR/CAPES. The authors thank Dr. Elisa Cupolillo at the Coleção de Leishmania do Instituto Oswaldo Cruz (CLIOC WFCC WDCM731), and to INPA for providing Leishmania strains. LITERATURE CITED BEETHAM, J. K., J. E. DONELSON, AND R. R. DAHLIN. 2003. Surface glycoprotein PSA (GP46) expression during short and long-term culture of Leishmania chagasi. Molecular and Biochemical Parasitology 131: 109 117. BRITTINGHAM, A., M. A. MILLER, J. E. DONELSON, AND M. E. WILSON. 2001. Regulation of GP63 mrna stability in promastigotes of virulent and attenuated Leishmania chagasi. Molecular and Biochemical Parasitology 112: 51 59., C. J. MORRISON,W.R.MCMASTER,B.S.MCGWIRE,K.P.CHANG, AND D. M. MOSSER.1995. Role of the Leishmania surface protease GP63 in complement fixation, cell adhesion, and resistance to complement-mediated lysis. Journal of Immunology 155: 3102 3111. BRODSKYN, C. I., S. M. BEVERLEY, AND R. G. TITUS. 2000. Virulent or avirulent (dhfr-ts-) Leishmania major elicit predominantly a type-1 cytokine response by human cells in vitro. Clinical and Experimental Immunology 119: 299 304. CAMPOS, M. B., C. M. GOMES, A. A. DE SOUZA, R. LAINSON, C. E. CORBETT, AND F. T. SILVEIRA. 2008. In vitro infectivity of species of Leishmania (Viannia) responsible for American cutaneous leishmaniasis. Parasitology Research 103: 771 776. CASTRO-PINTO, D. B., E. SILVA, A.S.CUNHA, M.GENESTRA, R.M.LEO, F. P. MONTEIRO, AND L. L. LEON. 2007. Leishmania amazonensis trypanothione reductase: Evaluation of the effect of glutathione analogs on parasite growth, infectivity and enzyme activity. Journal of Enzyme Inhibition Medicinal Chemistry 22: 71 75. CHANG, K. P. 1983. Cellular and molecular mechanisms of intracellular symbiosis in leishmaniasis. International Review of Cytology 14(Suppl.): 267 305., S. G. REED, S. BRADFORD, B. S. MCGWIRE, AND L. SOONG. 2003. Leishmania model for microbial virulence: The relevance of parasite multiplication and pathoantigenicity. Acta Tropica 85: 375 390. CHILLER, T. M., M. A. SAMUDIO, AND G. ZOULEK. 1990. IgG antibody reactivity with Trypanosoma cruzi and Leishmania antigens in sera of with Chagas disease and leishmaniasis. American Journal of Tropical Medicine and Hygiene 43: 650 656. CUBA CUBA, C., W. OGUNKOLADE, M. K. HOWARD, AND M. A. MILES. 2001. Immunological selection for Leishmania (Viannia) braziliensis antigens. Annals of Tropical Medicine and Parasitology 95: 473 483. CUPOLILLO, E., G. GRIMALDI, JR., AND H. MOMEN. 1994. A general classification of New World Leishmania using numerical zymotaxonomy. American Journal of Tropical Medicine and Hygiene 50: 296 311., H. MOMEN, AND G. GRIMALDI, JR. 1998. Genetic diversity in natural populations of New World Leishmania. Memorias do Instuto Oswaldo Cruz 93: 663 668. CYSNE-FINKELSTEIN, L., F. AGUIAR-ALVES, R. M. TEMPORAL, AND L. L. LEON.1998. Leishmania amazonensis: Long-term cultivation of axenic amastigotes. Experimental Parasitology 89: 58 61. GANDON, S., V. A. JANSEN, AND M. VAN BAALEN. 2001. Host life history and the evolution of parasite virulence. International Journal of Organic Evolution 55: 1056 1062. GENESTRA, M., W. J. SOUZA, L.CYSNE-FINKELSTEIN, AND L. L. LEON. 2003. Comparative analysis of the nitric oxide production by Leishmania sp. Medical Microbiology and Immunology 192: 217 223.,, D. GUEDES-SILVA, G.M.MACHADO, L.CYSNE-FINKEL- STEIN, R. J. SOARES-BEZERRA, F. P. MONTEIRO, AND L. L. LEON. 2006. Nitric oxide biosynthesis by Leishmania amazonensis containing a high percentage of metacyclic forms. Archives of Microbiology 185: 348 354. GONçALVES, C. C., E. M. REICHE, B. A. DE ABREU, T. G. SILVEIRA, T. C. FELIZARDO, K.R.MAIA, R.COSTACURTA, E.J.PADOVESI, B.P.DIAS, S. I. JANKEVICIUS, AND J. V. JANKEVICIUS. 2002. Evaluation of antigens from various Leishmania species in a Western blot for diagnosis of American tegumentary leishmaniasis. American Journal of Tropical Medicine and Hygiene 66: 91 102. GRAHAM, R. C., AND M. J. KARNOVSKY. 1965. The histochemical demonstration of monoamine oxidase activity by coupled peroxidatic oxidation. Journal of Histochemistry and Cytochemistry 13: 604 605. GRIMALDI, G., JR., R. J. DAVID, AND D. MCMAHON-PRATT. 1987. Identification and distribution of New World Leishmania species characterized by serodeme analysis using monoclonal antibodies. American Journal of Tropical Medicine and Hygiene 36: 270 287. HOWARD, M. K., M. M. PHAROAH, F. ASHALL, AND M. A. MILES. 1991. Human urine stimulates growth of Leishmania in vitro. Transactions of Royal Society of Tropical Medicine and Hygiene 85: 477 479. JAFFE, C. L., R. SHOR, H. TRAU, AND J. H. PASSWELL. 1990. Parasite antigens recognized by with cutaneous leishmaniasis. Clinical Experimental and Immunology 80: 77 82. JOHNSTONE, A., AND R. THORPE. 1982. Basic techniques. In Immunochemistry in practice, A. Johnstone, and R. Thorpe (eds.). Blackwell Science, Oxford, U.K., p. 1 3. KWEIDER, M., J. L. LEMESRE, F., DARCY, J. P. KUSNIERZ, A. CAPRÓN, AND F. SANTORO.1987. Infectivity of Leishmania braziliensis promastigotes is dependent on the increasing expression of a 65,000-dalton surface antigen. Journal of Immunology 138: 299 305.

MATTA ET AL. DIFFERENCES OF LEISHMANIA (V.) PARASITES 515 LAINSON, R. 1997. Leishmania e Leishmaniose, com particular referência à região Amazônica do Brasil. Revista Paraense de Medicina 11: 29 40. LEON, L. L., G. M. MAChADO, L. E. DE CARVALHO, AND G. GRIMALDI, JR. 1990. Antigenic differences of Leishmania amazonensis isolates causing diffuse cutaneous leishmaniasis. Transactions of Royal Society of Tropical Medicine and Hygiene 84: 678 680. MATTA, N. E. 2005. Comparação da resposta imune a antígenos de espécies de subgênero Viannia de pacientes de áreas endêmicas de Leishmania guyanensis e L. braziliensis. Ph.D. Dissertation. Instituto Oswaldo Cruz, Rio de Janeiro, Brazil, 120 p. MCMAHON-PRATT, D., E. BENNETT, AND J. R. DAVID. 1982. Monoclonal antibodies that distinguish subspecies of Leishmania braziliensis. Journal of Immunology 129: 926 927. NAGAKURA, K., H. TACHIBANA, Y. KANEDA, AND T. NAKAE. 1986. Leishmania braziliensis: Localization of glycoproteins in promastigotes. Experimental Parasitology 61: 335 342. PEARSON, R. D., R. ROMITO, P. H. SYMES, AND J. L. HARCUS. 1981. Interaction of Leishmania donovani promastigotes with human monocyte-derived macrophages: Parasite entry, intracellular survival, and multiplication. Infection and Immunity 32: 1249 1253. REY, J. A., B. L. TRAVI, A. Z. VALENZIA, AND N. G. SARAVIA. 1990. Infectivity of the subspecies of the Leishmania braziliensis complex in vivo and in vitro. American Journal of Tropical Medicine and Hygiene 43: 623 631. RODRIGUEZ, N., A. G. HERNÁNDEZ, AND F. MERINO. 1983. Effect of a purified excreted factor from Leishmania braziliensis on macrophage activity. International Archives of Allergy and Applied Immunology 72: 206 210. ROMERO, G. A., E. ISHIKAWA,E.CUPOLILLO,C.B.TOALDO,M.V.GUERRA, G. M. PAES, V. O. MACÊDO, AND J. J. SHAW. 2002. Identification of antigenically distinct populations of Leishmania (Viannia) guyanensis from Manaus, Brazil, using monoclonal antibodies. Acta Tropica 82: 25 29. RUSSELL, D. J., AND H. WILHELM.1986. The involvement of the major surface glycoprotein (gp63) of Leishmania promastigotes in attachment to macrophages. Journal of Immunology 136: 2613 2620. SACKS, D., AND P. PERKINS. 1984. Identification of an infective stage of Leishmania promastigotes. Science 223: 1417 1419. SARAVIA, N. G., K. WEIGLE, C. NAVAS, I. SEGURA, L. VALDERRAMA, A. Z. VALENCIA, B. ESCORCIA, AND D. MCMAHON-PRATT. 2002. Heterogeneity, geographic distribution, and pathogenicity of serodemes of Leishmania viannia in Colombia. American Journal of Tropical Medicine and Hygiene 66: 738 744. TOWBIN, H., T. STACHELIN, AND J. GORDON. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proceedings of the National Academy of Sciences USA 76: 4350 4354. YAO, C., J. E. DONELSON, AND M. E. WILSON. 2003. The major surface protease (MSP or GP63) of Leishmania sp. Biosynthesis, regulation of expression, and function. Molecular and Biochemical Parasitology 132: 1 6.