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Bioluminescent imaging of Trypanosoma cruzi infection in Rhodnius prolixus
Henriques Cristina,Castro Daniele P,Gomes Leonardo HF,Garcia Eloi S
Parasites & Vectors , 2012, DOI: 10.1186/1756-3305-5-214
Abstract: Background Usually the analysis of the various developmental stages of Trypanosoma cruzi in the experimentally infected vertebrate and invertebrate hosts is based on the morphological observations of tissue fragments from animals and insects. The development of techniques that allow the imaging of animals infected with parasites expressing luciferase open up possibilities to follow the fate of bioluminescent parasites in infected vectors. Methods D-luciferin (60 μg) was injected into the hemocoel of the whole insect before bioluminescence acquisition. In dissected insects, the whole gut was incubated with D-luciferin in PBS (300 μg/ml) for ex vivo bioluminescence acquisition in the IVIS Imaging System, Xenogen. Results Herein, we describe the results obtained with the luciferase gene integrated into the genome of the Dm28c clone of T. cruzi, and the use of these parasites to follow, in real time, the infection of the insect vector Rhodnius prolixus, by a non- invasive method. The insects were evaluated by in vivo bioluminescent imaging on the feeding day, and on the 7 th, 14 th, 21 st and 28 th days after feeding. To corroborate the bioluminescent imaging made in vivo, and investigate the digestive tract region, the insects were dissected. The bioluminescence emitted was proportional to the number of protozoans in regions of the gut. The same digestive tracts were also macerated to count the parasites in distinct morphological stages with an optical microscope, and for bioluminescence acquisition in a microplate using the IVIS Imaging System. A positive correlation of parasite numbers and bioluminescence in the microplate was obtained. Conclusions This is the first report of bioluminescent imaging in Rhodnius prolixus infected with trypomastigotes of the Dm28c-luc stable strain, expressing firefly luciferase. In spite of the distribution limitations of the substrate (D-luciferin) in the insect body, longitudinal evaluation of infected insects by bioluminescent imaging is a valuable tool. Bioluminescent imaging of the digestive tract infected with Dm28c-luc is highly sensitive and accurate method to track the fate of the parasite in the vector, in the crop, intestine and rectum. This methodology is useful to gain a better understanding of the parasite – insect vector interactions.
Cruzipain Promotes Trypanosoma cruzi Adhesion to Rhodnius prolixus Midgut  [PDF]
Lívia Almeida Uehara,Otacílio C. Moreira,Ana Carolina Oliveira,Patrícia Azambuja,Ana Paula Cabral Araujo Lima,Constan?a Britto,André Luis Souza dos Santos,Marta Helena Branquinha,Claudia Masini d'Avila-Levy
PLOS Neglected Tropical Diseases , 2012, DOI: 10.1371/journal.pntd.0001958
Abstract: Background Trypanosoma cruzi is the etiological agent of Chagas' disease. Cysteine peptidases are relevant to several aspects of the T. cruzi life cycle and are implicated in parasite-mammalian host relationships. However, little is known about the factors that contribute to the parasite-insect host interaction. Methodology/Principal Findings Here, we have investigated whether cruzipain could be involved in the interaction of T. cruzi with the invertebrate host. We analyzed the effect of treatment of T. cruzi epimastigotes with anti-cruzipain antibodies or with a panel of cysteine peptidase inhibitors (cystatin, antipain, E-64, leupeptin, iodocetamide or CA-074-OMe) on parasite adhesion to Rhodnius prolixus posterior midgut ex vivo. All treatments, with the exception of CA074-OMe, significantly decreased parasite adhesion to R. prolixus midgut. Cystatin presented a dose-dependent reduction on the adhesion. Comparison of the adhesion rate among several T. cruzi isolates revealed that the G isolate, which naturally possesses low levels of active cruzipain, adhered to a lesser extent in comparison to Dm28c, Y and CL Brener isolates. Transgenic epimastigotes overexpressing an endogenous cruzipain inhibitor (pCHAG), chagasin, and that have reduced levels of active cruzipain adhered to the insect gut 73% less than the wild-type parasites. The adhesion of pCHAG parasites was partially restored by the addition of exogenous cruzipain. In vivo colonization experiments revealed low levels of pCHAG parasites in comparison to wild-type. Parasites isolated after passage in the insect presented a drastic enhancement in the expression of surface cruzipain. Conclusions/Significance These data highlight, for the first time, that cruzipain contributes to the interaction of T. cruzi with the insect host.
Comparison of polymerase chain reaction on fresh tissue samples and fecal drops on filter paper for detection of Trypanosoma cruzi in Rhodnius prolixus
Dorn, PL;Flores, J;Brahney, B;Gutierrez, A;Rosales, R;Rodas, A;Monroy, C;
Memórias do Instituto Oswaldo Cruz , 2001, DOI: 10.1590/S0074-02762001000400010
Abstract: pcr detection of trypanosoma cruzi in rhodnius prolixus using fresh tissue or fecal drops on filter paper showed comparable results: 38.7% infection rate using the fresh tissue sample and 37.9% by dried fecal drop.
Comparison of polymerase chain reaction on fresh tissue samples and fecal drops on filter paper for detection of Trypanosoma cruzi in Rhodnius prolixus  [cached]
Dorn PL,Flores J,Brahney B,Gutierrez A
Memórias do Instituto Oswaldo Cruz , 2001,
Abstract: PCR detection of Trypanosoma cruzi in Rhodnius prolixus using fresh tissue or fecal drops on filter paper showed comparable results: 38.7% infection rate using the fresh tissue sample and 37.9% by dried fecal drop.
Trypanosoma cruzi Immune Response Modulation Decreases Microbiota in Rhodnius prolixus Gut and Is Crucial for Parasite Survival and Development  [PDF]
Daniele P. Castro, Caroline S. Moraes, Marcelo S. Gonzalez, Norman A. Ratcliffe, Patrícia Azambuja, Eloi S. Garcia
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0036591
Abstract: Trypanosoma cruzi in order to complete its development in the digestive tract of Rhodnius prolixus needs to overcome the immune reactions and microbiota trypanolytic activity of the gut. We demonstrate that in R. prolixus following infection with epimastigotes of Trypanosoma cruzi clone Dm28c and, in comparison with uninfected control insects, the midgut contained (i) fewer bacteria, (ii) higher parasite numbers, and (iii) reduced nitrite and nitrate production and increased phenoloxidase and antibacterial activities. In addition, in insects pre-treated with antibiotic and then infected with Dm28c, there were also reduced bacteria numbers and a higher parasite load compared with insects solely infected with parasites. Furthermore, and in contrast to insects infected with Dm28c, infection with T. cruzi Y strain resulted in a slight decreased numbers of gut bacteria but not sufficient to mediate a successful parasite infection. We conclude that infection of R. prolixus with the T. cruzi Dm28c clone modifies the host gut immune responses to decrease the microbiota population and these changes are crucial for the parasite development in the insect gut.
Towards an understanding of the interactions of Trypanosoma cruzi and Trypanosoma rangeli within the reduviid insect host Rhodnius prolixus
Azambuja, Patrícia;Ratcliffe, Norman A.;Garcia, Eloi S.;
Anais da Academia Brasileira de Ciências , 2005, DOI: 10.1590/S0001-37652005000300004
Abstract: this review outlines aspects on the developmental stages of trypanosoma cruzi and trypanosoma rangeli in the invertebrate host, rhodnius prolixus. special attention is given to the interactions of these parasites with gut and hemolymph molecules and the effects of the organization of midgut epithelial cells on the parasite development. the vector insect's permissiveness to t. cruzi, which develops in the vector gut, largely depends on the host nutritional state, the parasite strain and the molecular interactions with trypanolytic compounds, lectins and resident bacteria in the gut. t. rangeli invades the hemocoel and once in the hemolymph, can be recognized and activates the defense system of its insect vector, i.e., the prophenoloxidase system, phagocytosis, hemocyte microaggregation, superoxide and nitric oxide activity and the eicosanoid biosynthesis pathway. taken together, these findings not only provide a better understanding of the interactions parasite - insect vector, but also offer new insights into basic physiological processes involved in the parasites transmission.
Towards an understanding of the interactions of Trypanosoma cruzi and Trypanosoma rangeli within the reduviid insect host Rhodnius prolixus  [cached]
Azambuja Patrícia,Ratcliffe Norman A.,Garcia Eloi S.
Anais da Academia Brasileira de Ciências , 2005,
Abstract: This review outlines aspects on the developmental stages of Trypanosoma cruzi and Trypanosoma rangeli in the invertebrate host, Rhodnius prolixus. Special attention is given to the interactions of these parasites with gut and hemolymph molecules and the effects of the organization of midgut epithelial cells on the parasite development. The vector insect's permissiveness to T. cruzi, which develops in the vector gut, largely depends on the host nutritional state, the parasite strain and the molecular interactions with trypanolytic compounds, lectins and resident bacteria in the gut. T. rangeli invades the hemocoel and once in the hemolymph, can be recognized and activates the defense system of its insect vector, i.e., the prophenoloxidase system, phagocytosis, hemocyte microaggregation, superoxide and nitric oxide activity and the eicosanoid biosynthesis pathway. Taken together, these findings not only provide a better understanding of the interactions parasite - insect vector, but also offer new insights into basic physiological processes involved in the parasites transmission.
Trypanosoma cruzi TcSMUG L-surface Mucins Promote Development and Infectivity in the Triatomine Vector Rhodnius prolixus  [PDF]
Marcelo S. Gonzalez ,Marcela S. Souza,Eloi S. Garcia,Nadir F. S. Nogueira,Cícero B. Mello,Gaspar E. Cánepa,Santiago Bertotti,Ignacio M. Durante,Patrícia Azambuja,Carlos A. Buscaglia
PLOS Neglected Tropical Diseases , 2013, DOI: 10.1371/journal.pntd.0002552
Abstract: Background TcSMUG L products were recently identified as novel mucin-type glycoconjugates restricted to the surface of insect-dwelling epimastigote forms of Trypanosoma cruzi, the etiological agent of Chagas disease. The remarkable conservation of their predicted mature N-terminal region, which is exposed to the extracellular milieu, suggests that TcSMUG L products may be involved in structural and/or functional aspects of the interaction with the insect vector. Methodology and Principal Findings Here, we investigated the putative roles of TcSMUG L mucins in both in vivo development and ex vivo attachment of epimastigotes to the luminal surface of the digestive tract of Rhodnius prolixus. Our results indicate that the exogenous addition of TcSMUG L N-terminal peptide, but not control T. cruzi mucin peptides, to the infected bloodmeal inhibited the development of parasites in R. prolixus in a dose-dependent manner. Pre-incubation of insect midguts with the TcSMUG L peptide impaired the ex vivo attachment of epimastigotes to the luminal surface epithelium, likely by competing out TcSMUG L binding sites on the luminal surface of the posterior midgut, as revealed by fluorescence microscopy. Conclusion and Significance Together, these observations indicate that TcSMUG L mucins are a determinant of both adhesion of T. cruzi epimastigotes to the posterior midgut epithelial cells of the triatomine, and the infection of the insect vector, R. prolixus.
Susceptibilidade de Rhodnius neglectus, Rhodnius robustus e Triatoma infestans (Hemiptera, Reduviidae, Triatominae) à infec??o por duas cepas de Trypanosoma cruzi (Kinetoplastidae, Trypanosomatidae) utilizando xenodiagnóstico artificial
Martins, Luciamáre Perinetti Alves;Rosa, Jo?o Aristeu da;Castanho, Roberto Esteves Pires;Sauniti, Guilherme Lopes;Medeiros Júnior, Hermano;
Revista da Sociedade Brasileira de Medicina Tropical , 2000, DOI: 10.1590/S0037-86822000000600007
Abstract: the susceptibility of 3rd instar nymph of triatominae rhodnius neglectus, r. robustus and triatoma infestans to trypanosoma cruzi y and amjm strains was verified using artificial xenodiagnosis. after the accomplishment of the xenodiagnosis, the faeces of the triatominae were analyzed on two-day intervals from day 5 until day 31 post infection, using the abdominal compression technique. the results showed differences in the susceptibility of the triatominae for the two strains studied, and the optimal period reading differed from day 11 to day 19 for the y strain and from day 11 to day 15 for the amjm strain. for the y strain, all three triatominae species showed good susceptibility, whereas in the amjm strain, the highest susceptibility was observed with r. neglectus, followed by t. infestans and r. robustus.
Susceptibilidade de Rhodnius neglectus, Rhodnius robustus e Triatoma infestans (Hemiptera, Reduviidae, Triatominae) à infec o por duas cepas de Trypanosoma cruzi (Kinetoplastidae, Trypanosomatidae) utilizando xenodiagnóstico artificial
Martins Luciamáre Perinetti Alves,Rosa Jo?o Aristeu da,Castanho Roberto Esteves Pires,Sauniti Guilherme Lopes
Revista da Sociedade Brasileira de Medicina Tropical , 2000,
Abstract: A susceptibilidade de ninfas de 3o estádio de Rhodnius neglectus, R. robustus e Triatoma infestans às cepas Y e AMJM de Trypanosoma cruzi foi verificada utilizando xenodiagnóstico artificial. Para a leitura do xenodiagnóstico, as fezes dos triatomíneos foram examinadas a cada dois dias, a partir do 5o até o 31o dia pós infec o, pela técnica de compress o abdominal. Os resultados mostraram diferen as na susceptibilidade dos triatomíneos para as duas cepas estudadas e o período ótimo de leitura variou do 11o ao 19o dias para a cepa Y e do 11o ao 15o dias para a cepa AMJM. Também, p de-se concluir que para a cepa Y, as três espécies de triatomíneos demonstraram boa susceptibilidade, enquanto para a cepa AMJM, a melhor susceptibilidade foi observada com R. neglectus, seguida pelo T. infestans e R. robustus.
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