Infecção experimental de Calomys callosus (Rengger, 1830), (Cricetidae - Rodentia) a quatro espécies de parasitos

Neste trabalho foram estudados exemplares do roedor, Calomys callosus, nascidos em laboratório, a infecções experimentais com quatro parasitos: Plasmodium berghei, Leishmania mexicana amazonensis, Schistosoma mansoni e Hymenolepsis nana. A positividade das infecções foi de 80% para os três primeiros parasitos e 0 para H. nana. C. callosus é um roedor de excelente adaptação em laboratório e de fácil manuseio. Acredita-se que, de acordo com os resultados obtidos neste trabalho, este animal poderia ser um bom modelo experimental de laboratório para certos agentes patogênicos.

Patogenia da leishmaniose cutânea experimental: a importância da necrose na eliminação dos parasitos das lesões

Um estudo histopatológico e ultraestrutural das lesões da leishmaniose cutânea causada pela Leishmania mexicana amazonensis em duas cepas isogênicas de camundongo, uma susceptível (Balb/c) e outra resistente (A/J), demonstrou que os amastigotas ficavam bem preservados nos vacúolos parasitóforos dos macrófagos, igualmente em ambas as cepas. A reação de imunofluorescência revelou antigenos parasitários no interior e na membrana dos macrófagos de maneira idêntica para ambas as cepas. A diferença ocorria quando os macrófagos apareciam destruídos e as leishmanias ficavam livres ou fagocitadas por polimorfonucleares, neutrófilos e eosinófilos. Estes parasitos exibiam então graus variáveis de nítidas alterações degenerativas. No camundongo resistence, a necrose, de tipo caseoso ou fibrinóide, era mais disseminada e mais freqüente que no animal susceptível. Os achados observados indicaram que as leishmanias não são destruídas no interior dos macrófagos e sim fora deles, especialmente quando fagocitadas por leucócitos polimorfonucleares. A necrose apareceu como o mecanismo mais saliente através do qual o hospedeiro elimina os parasitos das lesões, sendo a mesma um aspecto importante da reação de hipersensibilidade tardia que ocorre nos animais resistentes.

The use of different concentrations of leishmanial antigen in skin testing to evaluate delayed hypersensitivity in american cutaneous leishmaniasis

Three concentrations of Leishmania mexicana amazonensis sonicated whole promastigote antigen (30, 9.6 and 3 ug N in 0.1 ml) wereprepared and 0.1 ml of each inoculated intradermally intopatients who live in one endemic leishmaniasis region in Brazil. Patients were divided into groups with active cutaneous leishmaniasis (ACL), healed cutaneous leishmaniasis (HCL), mucosal leishmaniasis (ML), and Controls (C). Skin reactions were recorded by measuring induration 48 hours after inoculation. Skin tests using 9.6 ugN/0.1 mlyielded the best diagnostic resultssince 97% of 30 patients with active lesions (cutaneous or mucosal) and 83% with HCL showed reactions of 5 mm orgreater as compared with 4% Controls. Tests using 30ug N/O. 1 ml causedan unacceptable levei of skin reactions with necrosis (10% of ACL patients tested and 17% of HCL, respectively). Tests using 3 ug N/O. 1 ml were less sensitive since only 87% of patients with active lesions and 68% with HCL had reactions of 5mm orgreater. The 3 ug N/O. 1 ml dose was utilized to ask the questions whether skin delayed hypersensitivity decreased with time after the initial lesion and whether mucosal involvement is associated with enhaced hypersensitivity to leishmanial antigen. Decreased delayed hypersensitivity was noted only in those patients who had an initial lesion more than 30 years ago. The mean induration of the reaction in 10 patients with ML was 11.3 mm ± 7.15, in 41 patients with HCL, 9.27 mm ± 6.78 and in 20patients with ACL 10. 7 mm ± 6.10 mm. The percent of patients with 5 mm orgreater induration was ML 80%, HCL 71%, ACL 90%. Thus, we could not confirm an association between enhanced delayed hypersensitivity and mucosal involvement in leishmaniasis.

Observações sobre calazar em Jacobina, Bahia. VI - Investigações sobre reservatórios silvestres e comensais

Durante os anos de 1982 a 1986, a investigação sobre mamíferos comensais e silvestres, da periferia da cidade de Jacobina, Bahia, mostrou, ao lado do escasso número de exemplares, uma reduzida variedade específica dessa fauna. Capturou-se apenas 11 espécies, entre as quais, predominou o Didelphis albiventris, que abrangeu 44% dos 213 espécimens capturados. Entre os 193 com exames já concluídos, 84 eram exemplares de D. albiventris e 2 estavam infectados pela Leishmania donovani senso lato, 1 por L. mexicana amazonensis, 1 por L. braziliensis, subespécie e 3 por Trypanosoma cruzi Também foram observadas formas suspeitas de serem amastigotas de leishmanics, nos esfregaços de órgãos de 3 exemplares de Dasyprocta aguti, 1 Cercomys cunicularius - e 1 Oryzomys eliurus. 0 restante dos exemplares, inclusive 14 de Lycalopex vetulus, estava negativo para flagelados. Apesar de reforçado por outros indicadores epidemiológicos, como a predominância específica, a freqüência domiciliar, a atratividade para a vetora Lutzomyia longipalpis, e a concomitância com casos humanos nos mesmos locais, o índice de 2,3% de infecção natural do Didelphis albiventris, não autoriza a conclusão definitiva de ser o marsupial o mais importante reservatório natural da leishmaniose visceral em Jacobina.

American cutaneous leishmaniasis: presentation and problems of patient management

We report our experience with the diagnosis and treatment of 60 patients with American cutaneous leishmaniasis. They were infected in Panama (55), Brazil (4) or Colombia (I). Among 35 patients with a 3 week exposure in Panama, the mean maximum incubation period was 33 days (range 4-81 days). Diagnosis was delayed an average of 93 days after onset of skin lesions, due to the patient's delay in seeking medical attention (31 days), medical personnel's delay in considering the diagnosis (45 days), and the laboratory's delay in confirming the diagnosis (17 days). Forty-four patients (73%) developed ulcers typical of cutaneous leishmaniasis. Sixteen additional patients (27%) had atypical macular, papular, squamous, verrucous or acneiform skin lesions that were diagnosed only because leishmanial cultures were obtained. Of the 59 patients treated with pentavalent antimonial drugs, only 34 (58%) were cured after the first course of treatment. Lesions which were at least 2 cm in diameter, ulcerated, or caused by Leishmania braziliensis were less likely to be cured after a single course of treatment than were lesions smaller than 2 cm, nonulcerated or caused by Leishmania mexicana or Leishmania donovani.

Características epidemiológicas da leishmaniose tegumentar americana em uma região endêmica do Estado da Bahia: III. Fauna flebotomínica

A fauna flebotomínica da região de Três Braços, uma área endêmica de leishmaniose cutânea-mucosa localizada no sudeste do Estado da Bahia, na região cacaueira, é muito variada. Foram identificadas 30 espécies de Lutzomyia em 13.535 exemplares coletados entre os anos de 1976 e 1984. Lu. withmani foi a espécie altamente predominante no ambiente peridoméstico e no interior das residências, com percentuais de 99,0 e 97,5, respectivamente. Na floresta, as espécies predominantes foram Lu. ayrozai e Lu. yuilli, aparecendo Lu. whitmani com apenas 1,0% do total de exemplares examinados. Lu. flaviscutellata, vetor comprovado da Leishmania mexicana amazonensis, foi também coletada em baixos índices. Lu. wellcomei, vetor da L. braziliensis braziliensis na Serra dos Carajás, Pará, Brasil, não foi encontrada na região de Três Braços onde o parasito causando infecções humanas é predominantemente L. b. braziliensis. Embora não se tenha encontrado infecção natural por promastigotas em 1.832 fêmeas de diversas espécies examinadas, discute-se a possibilidade de Lu. whitmani ser um vetor da L.b. braziliensis na região, mantendo, provavelmente, a transmissão entre o cão e o homem.

Seroepidemiological Studies of Cutaneous Leishmaniasis in the Campeche State of México

Seroepidemiological studies of cutaneous leishmaniasis were carried out on 169 individuals in a rural area of the Campeche state of México. Fifty showed cutaneous lesions suggestive of leishmaniasis, 70% were parasite positive and 96% skin test positive. An overall 40% positivity to skin test with Montenegro's antigen was found. Most of the affected individuals were males from 11 to 30 years-old. Antibodies were determined by immunofluorescent antibody test (IFA) and by Western blot. Two antigen preparations were used, one from a Leishmania mexicana strain which produced localized cutaneous leishmaniasis (LCL) and the other from a diffuse cutaneous leishmaniasis (DCL). In the general population from the area of study 19% gave positive IFA tests with DCL antigen and 20% with LCL antigen while for the patients 67% gave positive IFA tests with DCL and 71% with LCL. By Western blot analysis most of the patients recognized more antigens in the DCL than in the LCL strain. In the DCL strain 78% of patients recognized a 105 kDa, 34% a 139 kDa, 28% a 117 kDa and 26% a 205 kDa MW antigen. In the LCL strain 40% of patients recognized a 205 kDa and 22% a 175 kDa antigens

Purification and Partial Characterization of Trypanosoma cruzi Triosephosphate Isomerase

The enzyme triosephosphate isomerase (TPI, EC 5.3.1.1) was purified from extracts of epimastigote forms of Trypanosoma cruzi. The purification steps included: hydrophobic interaction chromatography on phenyl-Sepharose, CM-Sepharose, and high performance liquid gel filtration chromatography. The CM-Sepharose material contained two bands (27 and 25 kDa) with similar isoelectric points (pI 9.3-9.5) which could be separated by gel filtration in high performance liquid chromatography. Polyclonal antibodies raised against the porcine TPI detected one single polypeptide on western blot with a molecular weight (27 kDa) identical to that purified from T. cruzi. These antibodies also recognized only one band of identical molecular weight in western blots of several other trypanosomatids (Blastocrithidia culicis, Crithidia desouzai, Phytomonas serpens, Herpertomonas samuelpessoai). The presence of only one enzymatic form of TPI in T. cruzi epimastigotes was confirmed by agarose gel activity assay and its localization was established by immunocytochemical analysis. The T. cruzi purified TPI (as well as other trypanosomatid' TPIs) is a dimeric protein, composed of two identical subunits with an approximate mw of 27,000 and it is resolved on two dimensional gel electrophoresis with a pI of 9.3. Sequence analysis of the N-terminal portion of the 27 kDa protein revealed a high homology to Leishmania mexicana and T. brucei proteins

Molecular and biochemical characterisation of Trypanosoma cruzi phosphofructokinase

The characterisation of the gene encoding Trypanosoma cruzi CL Brener phosphofructokinase (PFK) and the biochemical properties of the expressed enzyme are reported here. In contradiction with previous reports, the PFK genes of CL Brener and YBM strain T. cruzi were found to be similar to their Leishmania mexicana and Trypanosoma brucei homologs in terms of both kinetic properties and size, with open reading frames encoding polypeptides with a deduced molecular mass of 53,483. The predicted amino acid sequence contains the C-terminal glycosome-targeting tripeptide SKL; this localisation was confirmed by immunofluorescence assays. In sequence comparisons with the genes of other eukaryotes, it was found that, despite being an adenosine triphosphate-dependent enzyme, T. cruzi PFK shows significant sequence similarity with inorganic pyrophosphate-dependent PFKs.

Chemistry, cytotoxicity and antileishmanial activity of the essential oil from Piper auritum

Leishmaniasis is one of the most important parasitic infections, but current treatments are unsatisfactory due to their toxicity, cost and resistance. Therefore, the development of new antileishmanial compounds is imperative. Many people who live in endemic areas use plants as an alternative to treat the disease. In this paper, we characterised the essential oil from Piper auritum, evaluated its cytotoxicity and determined its antileishmanial activity. The chromatogram obtained by gas chromatography revealed 60 peaks and we found that safrole was the most abundant compound, composing 87% of the oil. The oil was active against the promastigotes of Leishmania major, Leishmania mexicana, Leishmania braziliensis and Leishmania donovani with a favourable selectivity index against peritoneal macrophages from BALB/c mice. The Piper-oil inhibited the growing of intracellular amastigotes of L. donovani with an IC50 value of 22.3 ± 1.8 μg/mL. This study demonstrates the usefulness of the essential oils as a promising alternative to treat leishmaniasis.

Descripción del ARN de transferencia mitocondrial para Serina (UCN) de Lutzomyia columbiana (Diptera, Psychodidae)

Lutzomyia columbiana es un flebotomíneo considerado como vector sospechoso de Leishmania mexicana y Leishmania braziliensis en Colombia. Este insecto pertenece al grupo verrucarum, que incluye algunos taxones isomórficos, lo que ha estimulado la búsqueda de marcadores moleculares que permitan, además de diferenciar las especies, estudiar sus relaciones de parentesco. En este artículo se describe por primera vez la estructura putativa del ARN de transferencia mitocondrial para serina que reconoce el codón UCN (ARNtSer) de Lu. columbiana. El ADN genómico fue extraído, amplificado y secuenciado a partir de seis especímenes colectados con cebo humano. La estructura secundaria del ARNtSer fue inferida con el programa tRNAscan-SE 1.21. El gen ARNts consistió de 67 pares de bases (pb), encontrándose un solo haplotipo en los seis individuos secuenciados. El ARNtSer de Lu. columbiana mostró 7 apareamientos intracatenarios en el brazo aceptor del aminoácido, 3 en el brazo dihidrouridina (DHU), 5 en el brazo del anticodón y 5 en el brazo ribotimidina-pseudouridina-citosina (TøC). El tamaño de las lupas correspondió a 5 nucleótidos en la DHU, 7 en la anticodón, 4 en la variable y 7 en la TøC. Lu. columbiana se distingue del resto de especies de Lutzomyia y Phlebotomus secuenciadas a la fecha por la presencia de una guanina en la posición nucleotídica 64, que produce un apareamiento no canónico tipo uracilo-guanina en el brazo aceptor. Se necesitan más estudios para confirmar la utilidad del ARNtSer como marcador molecular para la discriminación de especies de flebotomíneos.

CD4+, CD8+ and CD4- CD8- T cell-subsets can confer protection against Leishmania m. mexicana infection

We studied the role of CD4+, CD8+, CD4- CD8- T cells and IgG anti-Leishmania after infection or vaccination in the CBA/ca mouse. Mice were either infected with L. m. mexicana promastigotes or vaccinated with parasite-membrane antigens incorporated into liposomes. Successfully vaccinated mice were used as cell-donors in adoptive transfer experiments. Naive, syngeneic recipients received highly-enriched CD4+, CD8+ or CD4- CD8- T cells from those two set of donors and challenged with live parasites. Our results showed that, both CD4+ and CD8+ T cells from infected or vaccinated donors conferred significant disease-resistance to naive recipients. In addition, adoptive transfer of CD4- CD8- T cells from vaccinated donors significantly delayed lesion growth in recipient mice. We concluded that vaccination of CBA mice correlates with the induction of protective CD4+, CD8+ and CD4- CD8- T cells and the synthesis of IgG anti-Leishmania.

Assessment of PCR in the detection of Leishmania spp in experimentally infected individual phlebotomine sandflies (Diptera: Psychodidae: Phlebotominae)

DNA amplification by the polymerase chain reaction (PCR) was applied in the investigation of the presence of Leishmania (Kinetoplastida: Trypanosomatidae) parasites in single phlebotomine sandflies. Three phlebotomine/parasite pairs were used: Lutzomyia longipalpis/Leishmania chagasi, Lutzomyia migonei/Leishmania amazonensis and Lutzomyia migonei/Leishmania braziliensis, all of them incriminated in the transmission of visceral or cutaneous leishmaniasis. DNA extraction was performed with whole insects, with no need of previous digestive tract dissection or pooling specimens. The presence of either mouse blood in the digestive tract of the sandflies or the digestive tract itself did not interfere in the PCR. Infection by as few as 10 Leishmania sp. per individual were sufficient for DNA amplification with genus-specific primers. Using primers for L. braziliensis and L. mexicana complexes, respectively, it was possible to discriminate between L. braziliensis and L. amazonensis in experimentally infected vectors (L. migonei).

Immunocytochemical identification of leishmania and Trypanosoma cruzi amastigotes in situ with homologous and heterologous polyclonal antibodies

The unlabelled antibody peroxidase-antiperoxidase method was used to study the immunocytochemical properties of Leishmania and Trypanosoma cruzi amastigotes in situ after tissues had been submitted to different fixation procedures. Antisera were obtained from rabbits chronically infected with different strains of T. cruzi or immunized with L. mexicana amazonensis and L. braziliensis guyanensis, and were applied on 5 µm thick sections. T. cruzi antigens were well stained by the three anti-T. cruzi sera and the two anti-heis.hmama.sera at optimum dilution between 1:1,000 and 1:2,000, regardless the parasite strain. Differently, the leishmanial antigens were revealed by Leishmania sera only at low dilutions (between 1:60 -1:160), whereas the anti-T. cruzi sera, at these low dilutions, gave rather weak stainings. Although there is no clear explanation for this immunocytochemical "reverse-monodirectional" cross-reactivity between Leishmania and T. cruzi, the present results show that polyclonal antibodies agains Leishmania species, when used for immunocytochemical detection of these parasites in situ, react more strongly with T. cruzi amastigotes than with the homologous amastigotes.

Studies on the relationship between lectin binding carbohydrates and different strains of Leishmania from the New World

The culture forms of L. mexicana pifanoi (LRC L-90), L. mexicana mexicana (LRC L-94, M-379); L. braziliensis braziliensis (LRC L-77, L-1, M-2903, H-LSS) and L. mexicana amazonensis (H-JMMO, M-JOF, H-21, H-PLL,M-1696) were tested with the following lectins: Canavalia ensiformis, Ricinus communis-120, Axinella polypoides, Phaseolus vulgaris, Evonymus europaeus, lotus tetragonolobus, Dolichos biflorus, Aaptos papillata II, Laburnum alpinum, Ulex europaeus, Arachis hypogaea and Soja hispida. All examined strains of Leishmania were agglutinated by C. ensiformis, R. communis-120 and A. popypoides. No agglutination reactions were observed with P. vulgaris, D.biflorus, A. papillata II, E. europaeus and L. tetragonolobus. Only L. m. pifanoi and the L. m. amazonensis strains H-JMMO and MJOF showed agglutination reactions with S. hispida, U. europaeus, L. alpinum and A. hypogaea, while L. m. mexicana (LRC L-94; M-379) strains, L. b. braziliensis H. LSS, LRC L-77; L-1; M-2903 and the L. m. amazonensis strains, H-PLL, H-21, M-1696 showed no agglutination reactions with these four lectins.