Comparative evaluation of phenol and thimerosal as preservatives for a candidate vaccine against American cutaneous leishmaniasis.

For decades thimerosal has been used as a preservative in the candidate vaccine for cutaneous leishmaniasis, which was developed by Mayrink et al. The use of thimerosal in humans has been banned due to its mercury content. This study addresses the standardization of phenol as a new candidate vaccine preservative. We have found that the proteolytic activity was abolished when the test was conducted using the candidate vaccine added to merthiolate (MtVac) as well as to phenol (PhVac). The Montenegro's skin test conversion rates induced by MtVac and by PhVac was 68.06% and 85.9%, respectively, and these values were statistically significant (p < 0.05). The proliferative response of peripheral mononuclear blood cells shows that the stimulation index of mice immunized with both candidate vaccines was higher than the one in control animals (p < 0.05). The ability of the candidate vaccines to induce protection in C57BL/10 mice against a challenge with infective Leishmania amazonensis promastigotes was tested and the mice immunized with PhVac developed smaller lesions than the mice immunized with MtVac. Electrophoresis of phenol-preserved antigen revealed a number of proteins, which were better preserved in PhVac. These results do in fact encourage the use of phenol for preserving the immunogenic and biochemical properties of the candidate vaccine for cutaneous leishmaniasis.

Systemic and compartmentalized immune response in canine visceral leishmaniasis.

Human visceral leishmaniasis (VL) and canine visceral leishmaniasis (CVL) are the most important emerging diseases with high prevalence in Latin American countries and are mainly caused by Leishmania (L.) chagasi (Syn=L. infantum). CVL has a great impact on Brazilian public health because domestic dogs are the most important VL peri-domicile reservoirs in both urban and peri-urban areas. Our findings highlight the complexity of cellular immunological events related to the natural infection from dogs by L. chagasi, additionally correlating major peripheral blood phenotypic markers with clinical status and tissues parasite density. Our main results demonstrated that lower frequency of circulating B cells and monocytes are important markers of severe CVL, whereas increased levels of CD8+ lymphocytes appear to be the major phenotypic feature of asymptomatic disease. Determination of the isotypes patterns during CVL demonstrated that asymptomatic dogs and those with low parasitism are associated with an increase of IgG1, while the symptomatic dogs and those with high parasitism are associated with an increase of IgG, IgG2, IgM, IgA and IgE immunoglobulins. Pioneer findings obtained by our group showed a correlation between clinical status of CVL with degree of tissue parasite density. This data demonstrated that asymptomatic dogs presented low parasitism while symptomatic dogs are associated with high parasite load in various tissues such as skin, bone marrow and spleen. We have also investigated the association between tissue parasitism and CVL clinical forms. Regardless of clinical status, skin and spleen are the major sites of high parasite density during ongoing CVL. Furthermore, we demonstrated that bone marrow and spleen parasite density are the most reliable parasitological markers to decode the clinical status of CVL. In this article, we have reviewed some aspects of the histopathological and immunological events occurring in natural and experimental L. chagasi/L. infantum infection, pointing out the main L. chagasi-parasitized tissue. We have discussed the importance of the association between parasite density, immunological/histopathological aspects and clinical status of the CVL, their current applications, challenges for the future and potential opportunities in CVL research.

Advances in flow cytometric serology for canine visceral leishmaniasis: diagnostic applications when distinct clinical forms, vaccination and other canine pathogens become a challenge.

We have previously reported the applicability of flow cytometry anti-fixed Leishmania infantum chagasi promastigotes IgG (FC-AFPA-IgG) as a novel serological device for laboratorial diagnosis of CVL. Herein, we validate throughout a blind study applied into a broader range of coded sera samples that FC-AFPA-IgG at serum dilution 1:8192 have an outstanding performance to discriminate the serological reactivity of canine visceral leishmaniasis (CVL, n=64) and Leishmune vaccines (VAC, n=62) and non-infected controls (NI, n=25). Moreover, we have evaluated the performance of indirect immunofluorescence antibody test (IFAT) and the crude-antigen enzyme-linked immunosorbent assay (ELISA) in parallel with FC-AFPA-IgG, to discriminate the seroreactivity of NI, CVL and VAC. Our data demonstrated that both ELISA and FC-AFPA-IgG showed similar performance to detect the seronegativity in 100% of NI, whereas FC-AFPA-IgG displayed better performance to exclude seropositivity in 100% of VAC. The high kappa agreement indexes observed suggested similar performance between these two serological testes when distinct clinical forms of CVL become a challenge. Furthermore, the FC-AFPA-IgG applied at sera dilution 1:8192 showed a remarkable performance to discriminate CVL from other co-endemic canine infections with high co-negativity in dogs infected with Trypanosoma cruzi and Leishmania braziliensis (86% and 84%, respectively). In conclusion, the data presented here re-emphasize the applicability of FC-AFPA-IgG as an innovative methodology able to discriminate post-infection imunomediated seroreactivity from that triggered by prophylactic immunization with minor cross-reactivity with other relevant canine pathogens, which may contribute as a supplementary assay for the CVL immunodiagnosis.

The influence of copper, selenium and zinc on the response to the Montenegro skin test in subjects vaccinated against American cutaneous leishmaniasis.

We evaluated the relationship between the trace elements copper, zinc and selenium and the response to the Montenegro skin test (MST) in 172 volunteers vaccinated against American cutaneous leishmaniasis. The MST diameter was categorized as negative and in quartiles of positive response, constituting five groups. Trace element serum levels were analyzed by coupled plasma atomic emission spectrometry and hydride generation atomic absorption spectrometry, with study subjects classified into two groups depending on low or high levels of trace elements observed. MST-positive subjects had an MST diameter (mean+/-SD) of 10.35+/-4.64mm, with copper, selenium and zinc serum levels of 1433.7+/-665.7microg/l, 88.6+/-39microg/l and 999.2+/-366microg/l, respectively. The MST diameter was significantly different in the selenium groups only. The selenium levels also differed with the quartiles of the MST diameters of individuals testing positive (P<0.05). Our findings suggest that trace elements, particularly selenium, should be measured in future vaccine trials so that vaccine immunogenicity and response can be assessed and compared between different studies.

Histopathology, parasite density and cell phenotypes of the popliteal lymph node in canine visceral leishmaniasis.

While enlargement of popliteal lymph nodes (LN) is frequently described in canine visceral leishmaniasis (CVL), there are few histopathologic studies of lymph nodes during this chronic immunopathological condition. Besides a detailed histopathologic analysis, we have characterized the parasite load and major immunophenotypic features of the LN in Leishmania (Leishmania) chagasi-infected dogs. Our major histopathological findings highlight that hypertrophy/hyperplasia of LN cortical and medullary zones was the principal characteristic observed in asymptomatic dogs (AD), whereas atrophy of LN cortical zone was predominant in symptomatic animals (SD). The LN parasite density detected by anti-Leishmania immunohistochemical assay or expressed as Leishman Donovan Units was also highly correlated with the skin parasitism, the most reliable parameter to decode the clinical status of CVL. The major LN immunophenotypic changes during ongoing CVL were an increased frequency of T-lymphocytes, particularly CD8+ T-cells, up-regulation of MHC-II expression by lymphocytes and decreased levels of CD21+ B-cells. Our findings further demonstrated that changes in the LN B-lymphocyte compartment exhibited a negative correlation with the skin parasite load. Conversely, we also showed evidence for a positive association between skin parasitism and LN T-cell-mediated immunity, suggesting that T-cells, especially CD8+ lymphocytes, may have a Type-2 immunological profile in this lymphoid tissue in response to CVL.

Histopathological and immunohistochemical investigations of the hepatic compartment associated with parasitism and serum biochemical changes in canine visceral leishmaniasis.

The immunopathological evaluation of the hepatic compartment associated with parasitism and biochemical findings are essential for understanding the genesis of hepatomegaly in canine visceral leishmaniasis (CVL). Three clinical groups of dogs naturally infected with Leishmania chagasi [i.e., asymptomatic (AD, n=12), oligosymptomatic (OD, n=12) and symptomatic (SD, n=17)] were assessed and compared with a group of non-infected dogs (NID, n=11). Intense reaction of the Kupffer cells, capsule and portal inflammation, and the presence of intralobular granulomas, were observed in the different clinical groups. Dogs in the SD group presented a higher frequency of parasitism compared with the AD group. Inflammatory alterations were more intense in the SD group and were associated with parasitism. Our results indicated an association between histological liver changes and the progression of biochemical alterations according to progression of clinical forms of CVL, and the direct relationship between clinical symptoms and frequency of hepatic parasitism.

Antigenicity of a whole parasite vaccine as promising candidate against canine leishmaniasis.

Human visceral leishmaniasis, one of the most important zoonoses, is caused by the protozoa Leishmania chagasi (syn. L. infantum) and is present as a fatal disease common in South America and Europe where dogs and wild canids are the main reservoirs. A vaccine against visceral leishmaniasis would be an important tool in the control of this disease in dogs. Although the current strategies for vaccination against leishmaniasis are based on the use of recombinant antigens, killed vaccines are still attractive in terms of stability of their biochemical composition and antigenicity, cost, and safety. Here we evaluate the immunogenicity of a whole parasite vaccine as a promising candidate against canine leishmaniasis, demonstrated by cellular reactivity, changes in the cellular profile of the peripheral blood and by the differential production of immunoglobulins. Our results showed that immunization elicited mainly a strong cellular reactivity and increase in T-lymphocytes, particularly the subpopulation CD8(+) that would be related to the control of tissue parasitism. In addition, a higher production of anti-Leishmania total IgG, characterized by mixed isotypes profile (IgG1 and IgG2), was demonstrated.

Mixed cytokine profile during active cutaneous leishmaniasis and in natural resistance.

Most studies on immune response in human cutaneous leishmaniasis evaluate patients with active disease in comparison with healthy uninfected controls or patients that have had the lesions healed, however, little is known about the immune response associated with natural resistance. In this paper we evaluate the cytokine expression patterns of T-cells and the plasmatic levels of nitrite and nitrate in patients with localized cutaneous leishmaniasis (LCL) as well as endemic non-infected individuals with positive (MST) and negative (NI) Montenegro skin test, without previous history of leishmanial lesions. Our results demonstrated an increased number of IFN-gamma+ and TNF-alpha+ T-cells and high level of plasma nitrite and nitrate in LCL patients. Moreover, we have observed that early in infection (LCL equal/less than 60 days of lesion evolution), Leishmania patients present predominance in IL-4+ and IL-10+ T-lymphocytes. However, this is a transitory phenomenon, since patients with older lesions (LCL more than 60 days of lesion evolution) show a predominant Type-1 immune profile, suggesting that disease development may depend on a transient deregulation of T-cell response, during the initial phase of infection. Interestingly MST displayed a basal mixed Type-0 cytokines profile. However, the low frequency of IL-4+ T-cells, high IFN-gamma+/IL-10+ cell ratio as well as elevated nitrite and nitrate plasma levels observed in MST, suggested that despite basal levels of cytokines, a high proportion of Type-1 over Type-2 cytokines would count to prevent parasite growth and lesion development.

Comparison of polymerase chain reaction with other laboratory methods for the diagnosis of American cutaneous leishmaniasis: diagnosis of cutaneous leishmaniasis by polymerase chain reaction.

An evaluation of 5 laboratory methods for diagnosing American cutaneous leishmaniasis (ACL) was carried out on patients from an endemic area of Brazil. From 164 patients presenting cutaneous lesions, and suspected to have ACL, 133 (81.1%) were confirmed for the disease by Montenegro skin test (MST) and/or parasitologic examination (PE). In both groups of patients, the positivity of polymerase chain reaction (PCR) was similar to that of immunofluorescence assay and enzyme-linked immunosorbent assay, and higher than that of MST and PE (P < .05). In the group of patients suspected to have ACL, PCR presented the same positivity as PE and MST together. No correlation between positivity of the laboratory methods and clinical or epidemiologic aspects was observed. Our data confirmed the value of PCR as an alternative laboratory method for diagnosing ACL, especially for those patients with negative PE and MST.

Immuno-biochemical evaluations of phenol and thimerosal as antigen preservatives in Montenegro skin test.

Montenegro skin test (MST) represents the main complementary diagnostic test for tegumentary leishmaniases (TL) in endemic regions. Most antigen formulations used for the MST contain thimerosal as preservative. The Food and Drug Administration (FDA), however, recommended reducing or eliminating thimerosal from vaccines and other biological reagents and the Agência Nacional de Vigilância Sanitária (ANVISA) in Brazil, prohibited the use of mercurial compounds in immunobiologicals. In the search for an alternative stabilizer, phenol and thimerosal were tested as antigen preservatives in MST. Formulations were tested when fresh and after a 12-month storage at 4 degrees C in TL confirmed mice and human patients, and were evaluated for protein constitution by SDS-PAGE, Western blot and anti-gp63 ELISA. In mice, a decrease in the diagnostic effectiveness in merthiolate formulation was observed after a 12-month storage. SDS-PAGE, Western blot and anti-gp63 ELISA analyses showed a degradation of antigen proteins in both formulations after 12-month storage and that phenol-preserved antigen was quantitatively and qualitatively better than the merthiolate-preserved one. In patients, the average of induration diameter was larger in fresh antigens (p<0.05). However, storage time did not jeopardize their diagnostic capacity. No non-specific reactions produced by phenol or merthiolate were observed neither in humans nor in mice. Phenol could be a good alternative to replace the merthiolate in MST, and despite the proteolytic activity, antigens remain viable for at least 12 months.

Isotype patterns of immunoglobulins: hallmarks for clinical status and tissue parasite density in Brazilian dogs naturally infected by Leishmania (Leishmania) chagasi.

The role of anti-leishmanial immune response underlying the susceptibility/resistance during canine visceral leishmaniasis (CVL) has been recognized throughout ex vivo and in vitro investigations. Recently, we demonstrated that immunoglobulin levels (Igs), as well as the parasite load are relevant hallmarks of distinct clinical status of CVL. To further characterize and upgrade the background on this issue, herein, we have evaluated, in Leishmania (Leishmania) chagasi naturally infected dogs, the relationship between tissue parasitism (skin, bone marrow, spleen, liver and lymph node), the CVL clinical status (asymptomatic (AD), with no suggestive signs of the disease; oligosymptomatic (OD), with maximum three clinical signs-opaque bristles; localized alopecia and moderate loss of weight; symptomatic (SD), serologically positive with severe clinical signs of visceral leishmaniasis), and the humoral immunological profile of anti-Leishmania immunoglobulins (IgG, IgG1, IgG2, IgM, IgA and IgE). Our major statistically significant findings revealed distinct patterns of tissue parasite density within L. chagasi-infected dogs despite their clinical status, pointing out the spleen and skin as the most relevant sites of high parasitism during ongoing CVL. Parasite density of bone marrow and spleen were the most reliable parasitological markers to decode the clinical status of CVL. Moreover, the parasite density of bone marrow better correlates with most anti-Leishmania Igs reactivity. Additionally, a prognostic hallmark for canine visceral leishmaniasis was found, highlighting strong correlation between IgG1 and asymptomatic disease, but with IgA, IgE and IgG2 displaying better association with symptomatic disease. The new aspects of this study highlighted pioneer findings that correlated the degree of tissue parasite density (low (LP), medium (MP) and high (HP) parasitism) with distinct patterns of anti-Leishmania Igs reactivity. In this scope, our data re-enforce the anti-Leishmania IgG but with IgA reactivity as the better marker for overall tissue parasitism. The association between clinical status, Ig profile and the tissue parasitism support a novel investigation on the impact of humoral immune response and susceptibility/resistance mechanism during ongoing CVL.

Parasite density and impaired biochemical/hematological status are associated with severe clinical aspects of canine visceral leishmaniasis.

We have performed a detailed investigation in 40 dogs naturally infected with Leishmania infantum (syn. chagasi), subdivided into three groups: asymptomatic (AD = 12), oligosymptomatic (OD = 12) and symptomatic (SD = 16), based on their clinical features. Twenty non-infected dogs (CD) were included as control group. Serological analysis, performed by IFAT and ELISA, demonstrated higher antibodies titers in SD in comparison to the AD. A positive correlation was found between parasite density in the spleen and skin smears as well as the bone marrow parasitism with clinical status of the infection. We observed that the progression of the disease from asymptomatic to symptomatic clinical form was accompanied by intense parasitism in the bone marrow. It is likely that this led to the impaired biochemical/hematological status observed. Finally, we believe that the follow-up of these parameters could be a relevant approach to be used as markers during therapeutic and vaccine evaluations.

Immunotherapy, immunochemotherapy and chemotherapy for American cutaneous leishmaniasis treatment.

The first choice of treatment for American cutaneous leishmaniasis is the pentavalent antimonial drug. Although it has been shown that this treatment is mostly effective and indicated, some disadvantages should be taken into account such as side effects, long term treatment inconveniences and counter-indication for patients suffering from cardiopathy, nephropathy; yet, aging, pregnancy and other conditions. With the advent of the vaccine anti-American cutaneous leishmaniasis as a prophylactic measure, studies on therapy using the vaccine associated or not with other drugs have been performed by many investigators and it is currently among the alternative treatments and prevention measures for American cutaneous leishmaniasis. In conclusion, the association between antimony and vaccine (immunochemotherapy) showed the same cure rate when compared with the standard treatment (100%) and it was also able to reduce the salt volume in 17.9% and treatment length from 87 to 62 days, decreasing side effects.

Identification and antimicrobial susceptibility of micro-organisms recovered from cutaneous lesions of human American tegumentary leishmaniasis in Minas Gerais, Brazil.

An evaluation of the microbiota present in cutaneous ulcers from 31 patients with a clinical and parasitological diagnosis of American tegumentary leishmaniasis (ATL) was carried out by the standard filter paper disc technique, including antimicrobial susceptibility of the bacterial isolates. Microbial examination indicated that 21 patients (67.7%) were contaminated with one to four bacteria and some of them also with yeast. A total of 142 micro-organisms were isolated. Staphylococcus aureus was the most frequently recovered bacterium (95.2% of positive patients) and was found to produce type B (70% of the staphylococcal isolates) and type C (50%) enterotoxins as well as toxic shock syndrome toxin (60%). Proteus mirabilis (33.3% of the positive patients), Streptococcus pyogenes (19.0 %), H(2)S-negative Proteus species (19.0%), Klebsiella oxytoca (14.3%), Enterobacter species (9.5%), Peptostreptococcus species (9.5%), Pseudomonas species (4.8%), Prevotella bivia (4.8%), Escherichia coli (4.8%), Streptococcus agalactiae (4.8%), Bacteroides fragilis (4.8%), Candida albicans (9.5%) and Candida tropicalis (4.8%) were also isolated. Surprisingly, Staph. aureus isolates were susceptible to almost all tested drugs, although some of them were resistant to penicillin (69%) and ampicillin + sulbactam (68%). Concerning obligate anaerobes, all the Gram-negative isolates (25% of the total) were resistant to metronidazole. The results of the present study show that microbial secondary contaminants, particularly Staph. aureus, should be considered in the diagnosis and treatment of ATL lesions.

Risk factors for mucosal manifestation of American cutaneous leishmaniasis.

A case-comparison study was carried out to identify risk factors for mucosal manifestations of American cutaneous leishmaniasis (ACL) in southeast Brazil, using a series of 2820 patients, diagnosed with ACL between 1966 and 1999. The significant factors independently associated with mucosal leishmaniasis were: gender, age, nutritional status and length of disease. Mucosal leishmaniasis occurred 1.7 times more frequently among males than females; twice as often in individuals older than 22 years compared with the younger group; almost four times as often in individuals with severe malnutrition compared with those who were well nourished; and almost four times more frequently in individuals reporting the disease for more than 4 months compared with those reporting a shorter duration of the disease. Among individuals older than 22 years the risk of mucosal leishmaniasis increased significantly (from 1.9 to 9.6) as the nutritional status decreased, when compared with younger and well-nourished patients. The characteristics herein described and correlated with severe forms could be used as diagnostic markers as part of clinical screening in areas endemic for ACL.

Establishment of a microplate assay for flow cytometric assessment and it is use for the evaluation of age-related phenotypic changes in canine whole blood leukocytes.

The effectiveness of flow cytometric assays for canine use is still requiring standardization. Despite several studies using purified mononuclear cells, no methodology or reference ranges are available for immunophenotyping of whole blood leukocytes (WBL). Fresh and pre-fixed WBL were used to identify cell-subsets, (Thy-1(+)/CD5(+)/CD4(+)/CD8(+)/CD21(+) and CD14(+)) and measure MHC-II, CD45RA/CD45RB expression. We described here an efficient method for fast quantification of canine-WBL, using pre-fix in a microplate assay, which allows long-term sample storage prior to phenotyping. Decreased percentage of CD5(+)-T-cells within the lymphocyte-gate and increased percentage of CD21(+)-B-cells were observed in young animals, which led to higher T/B cell ratios in middle-aged dogs. Lower numerical counts of Thy-1(+), CD4(+), CD8(+) and CD21(+) lymphocyte were observed when compared to young animals. In addition, we identified an age-related decline of MHC-II/CD45RA expression by lymphocytes. We proposed an improved method for phenotyping of canine peripheral blood mononuclear cells (PBMC) that has significant use for researchers and veterinary clinicians. The hematological changes of senescence previously identified on PBMC could be adequately reproduced on features identified by whole blood. Furthermore, this study supplies normal range references as baseline standards for clinical purposes, besides specific immunological parameters to monitor canine aging process.

Importance of Lutzomyia longipalpis in the dynamics of transmission of canine visceral leishmaniasis in the endemic area of Porteirinha Municipality, Minas Gerais, Brazil.

A study of Lutzomyia longipalpis (Lutz and Neiva, 1912) (Diptera: Psychodidae), the primary vector of American visceral leishmaniasis (AVL), and the canine form of the disease, was carried out in Porteirinha. The city is situated in the northern part of the Brazilian State of Minas Gerais and is an endemic area of AVL. Systematic phlebotomine captures were performed in seven districts with previously reported cases of canine visceral leishmaniasis, during 2 years (January 2000--December 2001). A total of 2328 specimens of L. longipalpis were captured. The association between the local climate variables and the population density of L. longipalpis was evaluated and rainfall was determined to be a major factor, with increased populations during the rainy season (October--March). At the same time period, blood samples from every dog domiciled in the same seven districts, in total 14,077 animals, were analyzed for infection by viscerotropic Leishmania using indirect immunofluorescence assay (IFA). Accumulated incidence rates of canine VL per district varied from 3.40 to 14.34 for the 2-year period. A positive correlation between the population density of L. longipalpis and the canine cases of visceral leishmaniasis in Porteirinha was observed.

[Aspects of the ecology and behaviour of phlebotomines in endemic area for visceral leishmaniasis in State of Minas Gerais]. / Aspectos da ecologia e do comportamento de flebotomíneos em área endêmica de leishmaniose visceral, Minas Gerais.

Studies on the behavioral and feeding habits of some species of phlebotominae sand flies have contributed to the comprehension of the epidemiology of leishmaniasis. In the present work, systematic captures were performed monthly in the municipality of Porteirinha (MG) using 28 light traps (CDC) from January to December 2002. Fourteen different species of phlebotomine were captured in a total of 1,408 specimens. The highest percentage of individuals (53.3%) was collected in the peridomicile against 46.7% in the intradomicile. Lutzomyia longipalpis was the predominant species in that region. The blood feeding of 38 females of this species from the field was analyzed by precipitin reaction. The results indicated that Lutzomyia longipalpis is an opportunist (65.1%) species that feeds on a wide variety of vertebrates in nature.

Short report: quantification of leishmaniavirus RNA in clinical samples and its possible role in pathogenesis.

Leishmaniavirus (LRV) is a double-stranded RNA virus that infects the protozoa Leishmania and has been identified in numerous strains of Leishmania braziliensis and L. braziliensis guyanensis. In general, the species of Leishmania dictates disease manifestation except in the case of L. braziliensis, which is capable of causing either cutaneous or mucocutaneous leishmaniasis. We wanted to determine 1) the quantity of LRV RNA present in a clinical sample and 2) if infection with LRV was associated with a specific disease manifestation. A real-time reverse transcriptase-polymerase chain reaction assay was used to assay clinical samples for the presence of LRV. Of 47 samples tested, 12 positive samples were obtained from patients with cutaneous lesions, lesions in the process of scarring, and cutaneous scars. This is the first study to examine the prevalence of LRV RNA within a small cohort from Brazil.

Detection and characterization of leishmania antigens from an American cutaneous leishmaniasis vaccine for diagnosis of visceral leishmaniasis.

Antigens were isolated from vaccines against American Cutaneous Leishmaniasis (ACL) and their reactivity tested against nine different groups of human sera and two groups of dog sera. These antigens react specifically with human and dog visceral leishmaniasis sera when compared to sera from non-infected individuals. Sera from humans from endemic areas of ACL before, or one year after, vaccination, and ACL patients treated and cured by immunotherapy with polyvalent vaccine, did not display significant differences of reactivity to these antigens. In contrast, they displayed a significantly higher reactivity to the antigens when compared to sera from healthy humans from non-endemic areas. No sera reactivity was observed with patients carrying Chagas' disease or tuberculosis. These antigens are polysaccharides aggregates and present molecular masses ranging from 90 to over 200 KDa. These data suggest the use of these antigens for sero-diagnosis of human and canine visceral leishmaniasis.