In vitro effects of lapachol and ß-lapachone against Leishmania amazonensis.

Leishmaniasis is a neglected disease that affects millions of people worldwide, and special attention should be given to treatment because the available drugs have limitations, which can lead to low therapeutic adherence and parasitic resistance. This study evaluated the activity of the bioactive naphthoquinones, lapachol and ß-lapachone, against Leishmania amazonensis. The cell alterations were evaluated in vitro on promastigote and amastigote forms. The lethal dose (LD50) at 24, 48, and 72 h on the promastigote's forms using lapachol was 75.60, 72.82, and 58.85 µg/mL and for ß-lapachone was 0.65, 1.24, and 0.71 µg/mL, respectively. The naphthoquinones significantly inhibited the survival rate of L. amazonensis amastigotes at 83.11, 57.59, and 34.95% for lapachol (82.28, 41.14, and 20.57 µg/mL), and 78.49, 83.25, and 80.22% for ß-lapachone (3.26, 1.63, and 0.815 µg/mL). The compounds on the promastigote's forms led to the loss of mitochondrial membrane potential, induced changes in the integrity of the membrane, caused damage to cells suggestive of the apoptotic process, and showed inhibition of tumor necrosis factor (TNF)-α and interleukin (IL)-6 production. The results showed that these naphthoquinones are promising candidates for research on new drugs with anti-Leishmania activity derived from natural products.

In vitro effects of lapachol and β-lapachone against Leishmania amazonensis

Leishmaniasis is a neglected disease that affects millions of people worldwide, and special attention should be given to treatment because the available drugs have limitations, which can lead to low therapeutic adherence and parasitic resistance. This study evaluated the activity of the bioactive naphthoquinones, lapachol and β-lapachone, against Leishmania amazonensis. The cell alterations were evaluated in vitro on promastigote and amastigote forms. The lethal dose (LD50) at 24, 48, and 72 h on the promastigote's forms using lapachol was 75.60, 72.82, and 58.85 μg/mL and for β-lapachone was 0.65, 1.24, and 0.71 μg/mL, respectively. The naphthoquinones significantly inhibited the survival rate of L. amazonensis amastigotes at 83.11, 57.59, and 34.95% for lapachol (82.28, 41.14, and 20.57 µg/mL), and 78.49, 83.25, and 80.22% for β-lapachone (3.26, 1.63, and 0.815 µg/mL). The compounds on the promastigote's forms led to the loss of mitochondrial membrane potential, induced changes in the integrity of the membrane, caused damage to cells suggestive of the apoptotic process, and showed inhibition of tumor necrosis factor (TNF)-α and interleukin (IL)-6 production. The results showed that these naphthoquinones are promising candidates for research on new drugs with anti-Leishmania activity derived from natural products.

Predominance of Nyssomyia neivai (Pinto) on the Islands of the Paraná River, Southern Brazil.

Leishmaniasis is a vector-borne disease caused by the protozoan Leishmania and it is one of the six most important infectious diseases in the world. The aim of this study was to conduct a research on the fauna and most frequented ecotopes by sandflies on islands of the Paraná River. The sandflies were collected with Falcão light traps in 10 islands in the municipalities of São Pedro do Paraná, Porto Rico, and Querência do Norte, in Paraná State, Brazil, from November 2012 to November 2014. In total, 76,145 specimens of Nyssomyia neivai, the only species found, were collected, of which 63,015 were female and 13,130, male. The largest number of sandflies was collected mainly in domiciles and peridomiciles. The islands of the Paraná River showed predominance of Ny. neivai, which is largely involved in the transmission of Leishmania and a risk of human leishmaniasis to the island inhabitants and tourists.

Calophyllum brasiliense Modulates the Immune Response and Promotes Leishmania amazonensis Intracellular Death.

Calophyllum brasiliense is a plant from the Brazilian rain forests and has been used in folk medicine for the treatment of various diseases, including leishmaniasis. This infectious disease depends on the Leishmania sp. and the host immune response. C. brasiliense antileishmanial activity is well known, but the effects on immune response remain to be investigated. This study showed the leishmanicidal and immunomodulatory effects of a 30 µg/mL of hydroalcoholic extract of C. brasiliense in murine macrophages before and after Leishmania (Leishmania) amazonensis infection. The semiquantitative cytokine RNA expression was determined by RT-PCR and the anti-Leishmania activity was measured by infection index (IF). Hydroalcoholic extract of C. brasiliense reduced more than 95% of IF when used before and after Leishmania infection, with 3 and 24 h of treatment (p < 0.05). C. brasiliense inhibited or reduced significantly (p < 0.05) the TNF-α, IL-1ß, IL-18, and IL-10 mRNA expression. The antileishmanial and anti-inflammatory effects showed the potential of C. brasiliense as an alternative therapy for leishmaniasis and it must be investigated.

Phototoxic effect of aluminium-chlorine and aluminium-hydroxide phthalocyanines on Leishmania (l.) amazonensis.

This study investigated the activity of photosensitive phthalocyanines on promastigotes and amastigotes of Leishmania (L.) amazonensis. Aluminum phthalocyanine chloride (AlPcCl), Aluminum phthalocyanine hydroxide (AlPcOH) and zinc phthalocyanine (PcZn) were tested in the presence (matte red LED, potency of 2.5-2.3 µW for 30 min) and absence of light against L. amazonensis promastigotes and the parasite viability was evaluated after 24, 48 and 72 h. The amastigote forms were treated with AlPcCl and AlPcOH, following the same lighting protocols described for the promastigote forms, being evaluated after 24 h. Cytotoxicity to human erythrocytes and peritoneal macrophages was also evaluated. The results showed that AlPcCl and AlPcOH in the presence of light have antileishmania activity, with leishmanistatic effects on promastigotes and amastigotes of L. amazonensis, without causing cytotoxicity to peritoneal macrophages and erythrocytes. The concentrations that inhibited 50% of the promastigote forms after 24 h of light exposure were 0.21 ±â€¯0.08 µM for AlPcCl and 0.23 ±â€¯0.06 µM for AlPcOH. In 48 h and 72 h after the treatment, the IC50 of AlPcCl was 0.13 ±â€¯0.02 and 0.12 ±â€¯0.03 µM and for AlPcOH was 0.14 ±â€¯0.01 µM and 0.11 ±â€¯0.01 µM, respectively. PcZn showed no activity on promastigotes of L. amazonensis. This study showed a substantial photodynamic activity of the phthalocyanines AlPcCl and AlPcOH against intracellular amastigotes forms of L. amazonensis after irradiation, presenting IC50 values of 0.62 ±â€¯0.06 µM and 0.92 ±â€¯0.12 µM, respectively. These results support the possibility of using photodynamic therapy for the treatment of cutaneous leishmaniasis.

Association between HLA-C*04 and American cutaneous leishmaniasis in endemic region of southern Brazil.

Leishmaniasis is a parasitic infectious disease with global repercussions. American cutaneous leishmaniasis (ACL) is endemic in southern Brazil and its pathogenesis varies according to parasite species, immune response, and host genetics. In terms of immunogenetics, many host genes, including HLA (human leukocyte antigen), could be involved in susceptibility to and protection against ACL. Accordingly, the aim of this study was to investigate the association between HLA class I genes (HLA-A, -B, and -C) and ACL in an endemic region of southern Brazil. The allele frequencies of 186 patients diagnosed with ACL and 278 healthy individuals were compared. HLA class I (HLA-A, -B, and -C) typing was carried out by PCR-SSO using Luminex technology. The results revealed an association between the HLA-C*04 allele and the patient study group, in which it appeared more frequently than in the control group [21.5 vs 13.49% (P = 0.0016 and Pc = 0.0258; OR = 1.7560; 95%CI = 1.2227-2.5240)], thereby suggesting an increased susceptibility to ACL. Additional allelic groups such as HLA-A*02, HLA-B*35, HLA-B*45, HLA-C*01, and HLA-C*15 were also implicated; however, further investigation is necessary to confirm their association with ACL. Therefore, the results obtained in this study demonstrate the involvement of HLA class I genes in the susceptibility or resistance to ACL, with significant association between HLA-C*04 and ACL susceptibility.

Immune Responses in Broiler Chicks Fed Propolis Extraction Residue-supplemented Diets.

This study was conducted to evaluate the effect of inclusion of propolis extraction residue in the feed of broilers from 1 to 21 d of age on phagocytic activity of macrophages, cutaneous basophil hypersensitivity response to phytohemagglutinin, antibody production against Newcastle disease, lymphoid organ weight and hematological profile and to determine the optimal level of inclusion. 120 chicks, reared in metabolism cages until 21 days of age, were distributed in a completely randomized design, with five treatments (0%, 1%, 2%, 3%, and 4% of propolis residue) and six replications. The relative weight of thymus and monocyte percentage were affected by propolis residue, with a quadratic response (p<0.05) and lowest values estimated at 2.38% and 2.49%, respectively. Changes in relative weight of cloacal bursa and spleen, percentage of lymphocyte, heterophil, basophil, eosinophil, and heterophil:lymphocyte ratio, antibody production against Newcastle disease, phagocytic activity of macrophages and the average number of phagocytosed erythrocytes were not observed. The nitric oxide production with regard to positive control (macrophages+erythrocytes) decreased linearly (p<0.05) with increased doses of propolis residue. The remaining variables of nitric oxide production (negative control - macrophages, and difference between the controls) were not affected by propolis residue. The cutaneous basophil hypersensitivity response to phytohemagglutinin as determined by the increase in interdigital skin thickness exhibited a quadratic response (p<0.05), which predicted a lower reaction response at a dose of 2.60% of propolis residue and highest reaction response after 43.05 hours of phytohemagglutinin injection. The inclusion of 1% to 4% of propolis extraction residue in broiler diets from 1 to 21 days of age was not able to improve the immune parameters, despite the modest changes in the relative weight in thymus, blood monocyte percentage, nitric oxide concentration, and interdigital reaction to phytohemagglutinin.

Efficacy of components from leaves of Calophyllum brasiliense against Leishmania (Leishmania) amazonensis.

Leishmanicide potential of Calophyllum brasiliense leaves on promastigote and amastigote of Leishmania (Leishmania) amazonensis is evaluated. The LD(50) of dichloromethane extract and hexane fraction for promastigotes was respectively 40 microg/ml and 20 microg/ml. In mouse peritoneal macrophages infected with Leishmania amastigotes the Infection Index decreased respectively 100% and 84.2% in 80 microg/ml and 40 microg/ml concentrations of dichloromethane extract. Hexane fraction decreased infection index respectively by 98.7% and 91.3% within the same concentrations. It was found that pretreatment with dichloromethane extract or with hexane fraction of experimentally infected BALB/c mice decrease the volume of the lesions by L. (L.) amazonensis. Moreover, animals treated topically also revealed healing lesions. Besides, the parasite load in the animals' popliteal lymph nodes was significantly reduced in treated animals, showing that plant components actually control infection. Results show that crude extract and hexane fraction of C. brasiliense reveal a significant in vitro and in vivo leishmanicide activity.

Cell migration induced by Leishmania (Leishmania) amazonensis, Leishmania (Leishmania) major and Leishmania (Viannia) braziliensis into the peritoneal cavity of BALB/c mice

In American cutaneous leishmaniasis, the initial infection phase is characterized by recruitment of neutrophils and monocytes. The migration of these cells in response to the presence of Leishmania in the peritoneum of affected animals remains unclear. The objective of this study was to investigate cell migration to the peritoneum of BALB/c mice after infection with Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis and Leishmania (Leishmania) major. Initially, Leishmania spp. was intraperitoneally inoculated in five groups of six animals each and the cell migration was analyzed 0, 3, 6, 12, 24 and 48 hours after infection. Different cell counts were performed with a staining kit and showed a higher percentage of polymorphonuclear than mononuclear cells in all three species studied. The total cell count revealed peak migration in L. (L.) amazonensis and L. (L.) major at six hours, and in L. (V.) braziliensis at 12 hours. These results suggest that factors released from different cell types probably act by attracting polymorphonuclear cells, with the peak migration most likely depending on the species of Leishmania inoculated into the host.

American cutaneous leishmaniasis in dogs from an endemic urban area in Cianorte municipality, Paraná State, Brazil

American cutaneous leishmaniasis (ACL) was investigated in dogs from an urban endemic area in Cianorte, Paraná state, Brazil. Of 169 studied dogs, none presented suspected ACL lesions. Eleven animals (6.6 percent) had anti-Leishmania braziliensis antibodies (titers > 40) detected by the immunofluorescent antibody test (IFAT) while four (2.4 percent) showed L. braziliensis-complex DNA by the polymerase chain reaction (PCR). Although no associations were found between IFAT or PCR results and age, sex, origin, free-roaming animals or length of residence at the address, the majority of IFAT- or PCR-positive dogs were from the urban area of the city and were allowed to roam freely beyond their neighborhood. The presence of anti-Leishmania braziliensis antibodies and L. braziliensis-complex DNA in dogs from this urban area near a native-forest park indicates the importance of following up on these dogs to confirm the ACL diagnosis.

Ocorrência de leishmaniose tegumentar em cães de área endêmica no Estado do Paraná / Occurrence of cutaneous leishmaniasis in dogs of endemic area, Paraná State

Investigou-se a infecção por parasitas do complexo Leishmania braziliensis em 50 cães da cidade de Maringá, PR, onde ocorreram casos humanos de leishmaniose tegumentar americana (LTA), entre agosto e dezembro de 2006. Os casos humanos de LTA ocorreram nos anos de 2003 a 2004. Nenhum animal apresentou lesão, mas 12 (24,0 por cento) tinham sorologia e/ou PCR positivas, e a positividade tanto da IFI como da PCR foi de 14,0 por cento. Trinta e cinco animais eram de residências, e oito deles (22,8 por cento) tiveram testes laboratoriais para LTA positivos. Os outros 15 eram cães errantes, sendo que quatro deles (26,7 por cento) apresentaram PCR positiva. A presença de animais com infecção assintomática por Leishmania em área onde a LTA é endêmica pode ser um indicador do potencial de sua transmissão para o homem e uma referência para a implantação de medidas de controle e prevenção da doença.

The infection by Leishmania braziliensis complex was studied in 50 dogs from Maringá, PR, where American Cutaneous Leishmaniasis (ACL) in humans have been detected. Data were collected from August to December 2006 in an area in which ACL cases in humans were reported from 2003 to 2004. Indirect immunofluorescent test (IIF) and polymerase chain reaction (PCR) were applied. No lesions were found in the animals, although 12 (24.0 percent) had positive IIF and/or PCR. Positiveness was 14.0 percent for IIF and PCR. Lab tests showed that eight (22.8 percent) out of the 35 home animals were ACL positive. The other 15 animals were stray dogs, out of which four (26.7 percent) were PCR positive. The appearance of asymptomatic Leishmania-infected dogs in an ACL endemic area may be an indicator of ACL transmission potential for humans and a reference for the establishment of control measures and disease prevention.

Aspectos epidemiológicos da Leishmaniose Tegumentar Americana na região Noroeste do Estado do Paraná / Epidemiological aspects of American Cutaneous Leismaniasis in the northwest region of the state of Paraná

No Estado do Paraná, a primeira notificação de leishmaniose tegumentar americana ocorreu em 1917 e a partir de 1980 observou-se um aumento do número de casos, mantendo-se endêmica e acometendo pessoas de todas as faixas etárias e em ambos os sexos. Este estudo teve como objetivo realizar um levantamento epidemiológico sobre a ocorrência de LTA em pacientes atendidos no Laboratório de Ensino e Pesquisa em Análises Clínicas da Universidade Estadual de Maringá (LEPAC/UEM). Foi realizado um estudo retrospectivo e descritivo em base de dados secundário de 1986 a 2005, com 1656 pacientes segundo as variáveis: sexo, idade, ocupação, procedência, local de moradia, forma clínica e diagnóstico. A maioria dos pacientes era do sexo masculino (72,6%) e adquiriu a infecção no Estado do Paraná (97,8%), residia em área urbana (64,3%) dos quais 51,3% adquiriu a infecção durante atividades de lazer. O diagnóstico da maioria dos pacientes foi estabelecido nos dois primeiros meses de evolução das lesões (54,0%) e apresentando a forma cutânea da doença (88,9%). Uma parcela (34,4%) significativa dos pacientes que residiam em área rural adquiriu a infecção no domicílio ou peridomicílio. O estudo mostra a predominância da forma cutânea da leishmaniose tegumentar americana e sugere a atividade de lazer e o ambiente do domicilio como fatores preditivos importantes para a infecção.

The first notification of American cutaneous Leishmaniasis (LTA) in the state of Paraná, Brazil, occurred in 1917 and an increasing number of cases has been reported since 1980. This parasitic skin disease, spread by the bite of infected sandflies, is still an endemic problem, with recurrence in both sexes and in all age groups. The objective of this study was to perform a survey on the occurrence of American cutaneous Leishmaniasis in patients attended at the Teaching and Research Clinical Analysis laboratory at the State University of Maringá (LEPAC/UEM). A retrospective and descriptive study was carried out, based on secondary data (1986-2005) on 1656 patients, relating to their sex, age and occupation and the origin, clinical forms and positive diagnosis of the disease. Most of the patients were male (72.6%), lived in the urban area (64.3%) and acquired the infection during outdoor leisure activities (51.3%) in the State of Paraná (97.8%). The cutaneous form of the infection predominated (88.9%) and the diagnosis was made in the first two months of development of the lesions (54.0%). A significant part (34.4%) of the patients who lived in the rural area acquired the infection inside or close to their homes. The study shows the predominance of the cutaneous form of American cutaneous Leishmaniasis and suggests that leisure activity and housing conditions could be useful predictive factors for the infection.

Serological survey for Leishmania sp. infection in wild animals from the municipality of Maringá, Paraná state, Brazil

Leishmania sp. infection was investigated in wild animals from the Ingá Park, in the municipality of Maringá, Paraná state, Brazil, where American cutaneous leishmaniasis (ACL) is an endemic disease. Sixty-five mammals, comprising Didelphis albiventris, Cerdocyon thous, Lycalopex vetulus, Cebus apella, Dasyprocta azarae, Dasypus novemcinctus, Procyon cancrivorus and Nasua nasua, were captured. Blood samples were collected for parasite cultivation. Antibodies were investigated by direct agglutination test (DAT) using Leishmania (Viannia) braziliensis as antigen. Flagellates were observed in blood cultures of 14 (35.9 percent) Didelphis albiventris. Anti-Leishmania antibodies were detected in 31 (51.6 percent) specimens of Cerdocyon thous, Lycalopex vetulus, Cebus apella, Dasyprocta azarae, Procyon cancrivorus and Nasua nasua. These results suggest that Cerdocyon thous and Lycalopex vetulus (crab-eating fox), Cebus apella (capuchin monkey), Dasyprocta azarae (agouti), Procyon cancrivorus (crab-eating raccoon) and Nasua nasua (coati) play an important role in the ACL transmission cycle in the northwestern region of Paraná, Brazil.

Leishmania (Viannia) braziliensis: New primers for identification using polymerase chain reaction.

The objective of this study was to develop specific primers for Leishmania (Viannia) braziliensis species identification using PCR. The designed primers (LBF1 and LBR1) were evaluated for sensitivity and specificity using various L. (V.) braziliensis serodemes and various Leishmania species and also using Trypanosoma cruzi. A specific fragment of 536bp was detected from 50ng of DNA in a crude extract derived from L. (V.) braziliensis. The DNA fragment was not detected when DNA from other Leishmania species or from T. cruzi was used as template in the PCR. Furthermore, when tested with DNA from cutaneous leishmaniasis the designed primers and reaction gave positive results. Taking into consideration that the primers LBF1 and LBR1 could specifically identify L. (V.) braziliensis, they could be considered for use in L. (V.) braziliensis diagnosis and epidemiological studies.

American cutaneous leishmaniasis in horses from endemic areas in the north-central mesoregion of Paraná state, Brazil.

American cutaneous leishmaniasis (ACL) is an endemic disease in the north-west of Paraná state, Brazil, where it occurs in humans and dogs. Few studies have been undertaken on the occurrence of the disease in other domestic animals and horses. In this study we investigated the infection of horses by Leishmania in ACL-endemic rural areas. Direct agglutination test (DAT) and polymerase chain reaction (PCR) were employed. Fifty-five horses from rural areas in the municipalities of Doutor Camargo, Ourizona, São Jorge do Ivaí, Ivatuba and Santa Fé (Agua do O) were analysed. DAT-detected antibody titres varied from 10 to 640, and 42 (76.3%) animals presented titres > or =20. PCR analyses led to the detection of Leishmania (Viannia) DNA in the blood samples of three (7.1%) of 42 animals. The presence of anti-L. (V.) braziliensis antibodies and Leishmania (Viannia) DNA in horses from endemic areas suggests their participation in the ACL transmission cycle.

Detection of Leishmania (Viannia) DNA in blood from patients with American cutaneous leishmaniasis.

The aim of the present study was to investigate the presence of Leishmania (Viannia) subgenus DNA in peripheral blood from patients with cutaneous lesions due to American cutaneous leishmaniasis. The buffy coats from 68 blood samples were analyzed by polymerase chain reaction using the MP1L/MP3H primers. The parasite DNA was detected in 2 (3.4%) out of 59 patients who had amastigotes present in samples taken from lesions. The presence of Leishmania (Viannia) DNA in the blood of these patients indicates hematogeneous parasite dissemination.

Comparação entre imunofluorescência indireta e aglutinação direta para o diagnóstico sorológico da leishmaniose tegumentar americana em cães errantes / Comparison between indirect immunofluorescence and direct agglutination for the serologic diagnosis of American cutaneous leishmaniasis in stray dogs

Estudaram-se 92 cães errantes com o objetivo de comparar a imunofluorescência indireta (IFI) e a aglutinação direta (AD) na investigação da leishmaniose tegumentar americana (LTA) canina, no período de outubro de 1999 a novembro de 2001. Os animais foram examinados quanto à presença de lesões e submetidos à intradermorreação de Montenegro (IDRM) e à pesquisa de anticorpos anti-Leishmania por IFI e AD. A IFI apresentou sensibilidade de 78,9 por cento e especificidade de 93,8 por cento (título >40), e a AD sensibilidade de 57,9 por cento e especificidade de 68,8 por cento (título >80). Dois cães apresentavam lesão, mas a pesquisa do parasito foi negativa. A IDRM, realizada em 89 cães, foi positiva em três (3,4 por cento). Trinta e quatro cães (37,0 por cento) foram positivos para a IFI, 61 (66,3 por cento) para a AD e 69 (75,0 por cento) para a IFI e/ou a AD. Dos cães com a IFI positiva, sete eram de Floresta, 13 de Maringá, 12 de Leópolis, um de São Jorge do Ivaí e um (50,0 por cento) de Campo Mourão. Os resultados sugerem o contato prévio desses animais com o parasito.

Ninety-two stray dogs were studied in order to compare indirect immunofluorescence (IIF) and direct agglutination (DA) in the investigation of American cutaneous leishmaniasis (ACL), from October 1999 to November 2001. The dogs were evaluated for the presence of lesions, and submitted to the Montenegro skin test (MST) and the search for anti-Leishmania antibodies by IIF and DA. IIF showed 78.9 percent sensitivity and 93.8 percent specificity (titers >40), and DA presented 57.9 percent sensitivity and 68.8 percent specificity (titers >80). In two dogs there were lesions, but the parasite was not detected. The MST was positive in 3 (3.4 percent) out of 89 dogs. Thirty-four (37.0 percent) dogs showed anti-Leishmania antibodies through IIF, 61 (66.3 percent) through DA, and 69 (75.0 percent) through IIF and/or DA. Of the dogs with positive IIF, 7 came from the municipal areas of Floresta, 13 of Maringá, 12 of Leópolis, 1 of São Jorge do Ivaí and 1 (50.0 percent) of Campo Mourão. These data suggest previous contact of these animals with the parasite.

Isolation of Leishmania (Viannia) braziliensis glycolipid antigens and their reactivity with mAb SST-1, specific for parasites of Viannia subgenus.

Specific glycolipids (GLs) from Leishmania (Viannia) braziliensis promastigotes were isolated and purified. A monoclonal antibody directed to carbohydrate epitopes of these GLs was produced. mAb SST-1 recognizes a low molecular weight GL as established by solid-phase radioimmunoassay and HPTLC immunostaining, and does not cross-react with lipophosphoglycan isolated from L. (V.) braziliensis promastigotes. An indirect immunofluorescence study indicated that the antigenic GLs are present at the L. (V.) braziliensis promastigote surface. SST-1 reacted with promastigotes of L. (V.) naiffi and L. (V.) guyanensis, but not with species in the L. Leishmania subgenus i.e. L. (L.) amazonensis, L. (L.) chagasi, or L. (L.) major. All L. (V.) braziliensis serodemes tested were reactive with SST-1. These results indicate that SST-1 recognizes specific GLs expressed by species of the Viannia subgenus, and will be particularly useful for identification of L. (V.) braziliensis.

Immunolocalization of Leishmania (Viannia) braziliensis membrane antigens recognized by mAbs SST-2, SST-3, and SST-4.

The immunolocalization of Leishmania (Viannia) braziliensis stage-specific antigens recognized by mAbs was analysed by transmission electron microscopy. The antigen recognized by mAb SST-2 was present at the surface of promastigotes, including the flagellum and flagellar pocket. The reactivity of SST-2 with isolates of different serodemes showed a pronounced microheterogeneity in terms of the number of reactive bands within the low molecular weight range from 24 to 33 kDa. The 180 kDa glycoprotein recognized by mAb SST-3 was present only in the flagellar membrane. SST-3 also recognized multiple discrete bands from 160 to 200 kDa, as observed in several serodemes. In contrast, mAb SST-4, which recognizes a 98 kDa antigen, showed weak labelling on the promastigote surface by transmission electron microscopy and indirect immunofluorescence. Based on Western blotting, indirect immunofluorescence, and solid-phase radioimmunoassay, the antigens recognized by mAbs SST-2, SST-3 and SST-4 were present in all L. (V.) braziliensis analysed, from 7 different serodemes.

Genetic variation in populations of Leishmania species in Brazil.

The population structure of strains of Leishmania (Viannia) braziliensis sensu lato from Pará State and Paraná State in Brazil, of L. (V.) shawi and L. (Leishmania) amazonensis from Pará State, and the relationships of type strains of the subgenera L. (Viannia) and L. (Leishmania) were examined by the random-amplified polymorphic deoxyribonucleic acid (RAPD) technique. Four different primers (M13-40, QG1, L15996 and delta gt11R) were used. The bands were analysed using the neighbor-joining (NJ) and unweighted pair-group method with arithmetic averages (UPGMA) algorithms of the MEGA package. The topology of the NJ and UPGMA trees was very similar but they were not always identical. Both trees differentiated the standard strains of the different species. Strains from the same location were grouped together only in the UPGMA phenogram of the M13-40 primer. L. (V.) braziliensis isolates from Paraná State were genetically closer to those from Paragominas, Pará State than to those from the Amazonian regions of Carajás in Pará State and Peru. The relationship was not dependent on geographical distance. It is postulated that the groups arose from different origins, in which the Amazonian stocks were related to Psychodopygus sand flies while the Paraná strains originated from a gene pool transmitted by Lutzomyia sand flies such as Lutzomyia (Nyssomyia) whitmani. Transmission by Ps. complexus in Paragominas is considered to be a secondary adaptation from the Lutzomyia leishmanial gene pool. Although the vectors of L. (V.) braziliensis are poorly known in the Amazon region, there is strong evidence that the major vectors are all Psychodopygus spp. There was a high degree of genetic variability amongst the L. (V.) shawi strains and there was no clear grouping according to the strains' origins. The genetic variability amongst L. (L.) amazonensis strains from the same locations was much lower but they formed 2 groups which coincided with their origin. Our results support the clonal population structure of Leishmania isolates and suggest that their distribution is related to the origin of the gene pool as well as to present vector and reservoir movements.