A cardiomyocyte mannose receptor system is involved in Trypanosoma cruzi invasion and is down-modulated after infection.
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Mannosyl binding sites were detected "in vitro" on cardiomyocytes (CM) surface using horseradish peroxidase (HRP) as the ligand. Binding assays revealed a specific recognition system, which was time- and concentration-dependent. The binding required physiological pH and was inhibited by EDTA and trypsin treatments. HRP binding was reduced by pre-incubations with low concentrations of D-mannose. Ultrastructural analysis of the endocytic process was followed using HRP coupled to colloidal gold particles (HRP-Au). The tracer was found within caveolae characterizing early steps of the receptor-mediated endocytosis. The addition of 10 mM D-mannose to the interaction medium blocked Trypanosoma cruzi uptake by CM. The labeling of CM with a subsaturating concentration of HRP-Au before their infection showed, by ultrastructural studies, that its association with trypomastigote forms occurred frequently near to HRP-gold particles that could also be seen to comprise the parasitophorous vacuole. After infection of CM with T. cruzi, a considerable reduction on HRP binding was noticed. Binding was almost completely restored by treating the infected cultures with the trypanocidal drug Nifurtimox. Our "in vitro" findings suggest that cardiomyocyte's mannose receptors localized at the sarcolemma mediates T. cruzi recognition and can be down-modulated by parasite infection. (+info)
Evaluation of antigen detection and antibody detection tests for Trypanosoma evansi infections of buffaloes in Indonesia.
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Two Ag-ELISAs, an IgG-specific antibody detection ELISA (IgG ELISA) and a card agglutination test (CATT) for the detection of Trypanasoma evansi infections in buffaloes in Indonesia, were compared. Diagnostic sensitivity estimates were obtained by testing sera from 139 Indonesian buffaloes which had been found to be infected by parasitological tests. Diagnostic specificity was estimated by testing sera from 263 buffaloes living in Australia. Response-operating characteristic curves were constructed, and optimal ELISA cut-off values, which minimized the number of false-negative and false-positive results, were chosen. The IgG ELISA had the highest sensitivity (89%) and the CATT had the highest specificity (100%). There was a significant difference between the sensitivities (71 and 81%), but not between the specificities (75 and 78%), of the two Ag-ELISAs. The four tests were further compared by calculation of post-test probabilities of infection for positive and negative test results using a range of prevalence values, and likelihood ratios. The results suggested that the CATT was the best test to 'rule-in' infection (i.e. the highest probability of infection in test-positive animals) and the IgG ELISA was the best test to 'rule-out' infection (i.e. the lowest probability of infection in test-negative animals). (+info)
Characterization of a vacuolar pyrophosphatase in Trypanosoma brucei and its localization to acidocalcisomes.
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Inorganic pyrophosphate promoted the acidification of an intracellular compartment in permeabilized procyclic trypomastigotes of Trypanosoma brucei, as measured by acridine orange uptake. The proton gradient generated by pyrophosphate was collapsed by addition of nigericin or NH(4)Cl. Pyrophosphate-driven proton translocation was stimulated by potassium ions and inhibited by KF, by the pyrophosphate analogs imidodiphosphate and aminomethylenediphosphonate (AMDP), and by the thiol reagent p-hydroxymercuribenzoate at concentrations similar to those that inhibit the plant vacuolar H(+)-pyrophosphatase (PPase). The proton translocation activity had a pH optimum around 7.5 and was partially inhibited by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (10 microM) and unaffected by bafilomycin A(1) (40 nM), concanamycin A (5 nM), sodium o-vanadate (500 microM), oligomycin (1 microM), N-ethylmaleimide (100 microM), and KNO(3). AMDP-sensitive pyrophosphate hydrolysis was detected in both procyclic and bloodstream trypomastigotes. Measurements of acridine orange uptake in permeabilized procyclic trypomastigotes in the presence of different substrates and inhibitors suggested the presence of H(+)-ATPase, H(+)-PPase, and (ADP-dependent) H(+)/Na(+) antiport activity in the same compartment. Separation of bloodstream and procyclic trypomastigote extracts on Percoll gradients yielded fractions that contained H(+)-PPase (both stages) and H(+)/Na(+) exchanger (procyclics) activities but lacked markers for mitochondria, glycosomes, and lysosomes. The organelles in these fractions were identified by electron microscopy and X-ray microanalysis as acidocalcisomes (electron-dense vacuoles). These results provide further evidence for the unique nature of acidocalcisomes in comparison with other, previously described, organelles. (+info)
Metalloproteases in Trypanosoma rangeli-infected Rhodnius prolixus.
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Protease activities in the haemolymph and fat body in a bloodsucking insect, Rhodnius prolixus, infected with Trypanosoma rangeli, were investigated. After SDS-polyacrylamide gel electrophoresis containing gelatin as substrate, analysis of zymograms performed on samples of different tissues of controls and insects inoculated or orally infected with short or long epimastigotes of T. rangeli, demonstrated distinct patterns of protease activities: (i) proteases were detected in the haemolymph of insects which were fed on, or inoculated with, short epimastigotes of T. rangeli (39 kDa and 33 kDa, respectively), but they were not observed in the fat body taken from these insects; (ii) protease was also presented in the fat bodies derived from naive insects or controls inoculated with sterile phosphate-saline buffer (49 kDa), but it was not detected in the haemolymph of these insects; (iii) no protease activity was observed in both haemolymph and fat bodies taken from insects inoculated with, or fed on, long epimastigotes of T. rangeli. Furthermore, in short epimastigotes of T. rangeli extracts, three bands of the protease activities with apparent molecular weights of 297, 198 and 95 kDa were detected while long epimastigotes preparation presented only two bands of protease activities with molecular weights of 297 and 198 kDa. The proteases from the insect infected with T. rangeli and controls belong to the class of either metalloproteases or metal-activated enzymes since they are inhibited by 1,10-phenanthroline. The significance of these proteases in the insects infected with short epimastigotes of T. rangeli is discussed in relation to the success of the establishment of infection of these parasites in its vector, R. prolixus. (+info)
Procedurally similar competitive immunoassay systems for the serodiagnosis of Babesia equi, Babesia caballi, Trypanosoma equiperdum, and Burkholderia mallei infection in horses.
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Procedurally similar competitive enzyme-linked immunoassay (cELISA) methods were developed for the serodiagnosis of Babesia equi and Babesia caballi (piroplasmosis), Trypanosoma equiperdum (dourine), and Burkholderia mallei (glanders) infections in horses. Apparent test specificities for the B. equi, B. caballi, T. equiperdum, and B. mallei cELISAs were 99.2%, 99.5%, 98.9%, and 98.9%, respectively. Concordances and kappa values between the complement fixation (CF) and the cELISA procedures for the serodiagnosis of B. equi, B. caballi, T. equiperdum, and B. mallei infections in experimentally exposed horses were 76% and 0.55, 89% and 0.78, 97% and 0.95, and 70% and 0.44, respectively. The cELISA method may be a technically more reproducible, objective, and convenient approach for piroplasmosis, dourine, and glanders serodiagnosis in qualifying animals for international movement and disease eradication programs than the CF systems currently in use. Use of the cELISA method also obviated the problems associated with testing hemolyzed or anticomplementary sera. (+info)
The occurrence of Trypanosoma evansi in buffaloes in Indonesia, estimated using various diagnostic tests.
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The prevalence and incidence of Trypanosoma evansi infections in village buffaloes in Central Java were estimated using parasitological tests, two antigen-detection ELISAs (2G6 Ag-ELISA and Tr7 Ag-ELISA), an antibody-detection ELISA (IgG ELISA) and a card agglutination test (CATT). Of 2387 village buffaloes tested in five districts, 4 % (95 % confidence interval [CI]: 3 %, 5 %) were positive with the microhaematocrit test (MHCT), 58 % (95 % CI: 56 %, 60 %) were positive with the 2G6 Ag-ELISA and 70 % (95 % CI: 68 %, 72 %) were positive with the Tr7 Ag-ELISA. An increasing prevalence with age was found and the proportion of positive buffaloes was highest in the over 84 months-old age-group (68 %) with the 2G6 Ag-ELISA and in the 37-60 months-old age-group (78 %) with the Tr7 Ag-ELISA. Parasitaemic buffaloes were found in more than half of the villages visited. Corrected village-specific prevalence values obtained with the two Ag-ELISAs ranged from 0% to over 100%, and prevalence differed significantly (P < or = 0.0001) between villages in four of the five districts. Overall, 10% of buffaloes tested in markets were found to be parasitaemic and 39, 56 and 47 % were found positive with the 2G6 Ag-ELISA, IgG ELISA and CATT, respectively. Incidence rates varied according to the test used and ranged from 0.22 (95 % CI: 0.09, 0.44) to 0.44 (95 % CI: 0.24, 0.76), per animal-year at risk, in two villages. The results highlight the importance of using validated diagnostic tests to obtain accurate estimates of prevalence and incidence. These parameters are needed, for example in mathematical models, for the development and evaluation of different control strategies for T. evansi infections in buffaloes. (+info)
Trypanosomes of non-human primates from the National Centre of Primates, Ananindeua, State of Para, brazil.
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Trypanosome infections were sought in 46 non-human primates captured principally in Amazonian Brazil. Twenty-two (47.8%) were infected with four Trypanosoma species: T. cruzi, T. minasense, T. devei and T. rangeli. These preliminary results confirmed the high prevalence and diversity of natural infections with trypanosomes in primates from Brazilian Amazon and were the first formal record of simian infections with trypanosomes in the State of Acre. The presence of T. cruzi-like and T. rangeli-like parasites are recorded in four new hosts. (+info)
Hypotension in rabbits infected with Trypanosoma brucei.
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1 Blood pressures and heart rates of 12 anaesthetized rabbits chronically infected with T.brucei were measured (average infection time 39 days (range 25-67). The systolic BP was 31.4 +/- 5.7 mmHg, the diastolic BP 25.0 +/- 7.2 mmHg, and the heart rate 120.5 +/- 24.2 beats/minute. Two rabbits were already hypotensive 10 days after infection. In 12 anaesthetized control rabbits, the systolic BP was 66.2 +/- 7.3 mmHg (mean +/- s.e.), the diastolic BP 60.2 +/- 7.3 mmHg, and the heart rate 116.3 +/- 15.9 beats/minute. 2 The intravenous injection of 3 X 10(8) disintegrated trypanosomes into infected rabbits lowered the blood pressure by 41.4 +/- 22.0%. Pretreatment of two rabbits with aprotinin prior to administration of parasites prevented the fall in blood pressure. 3 Injection of 3 X 10(8) live trypanosomes complexed with hyperimmune sera produced a fall of 68.3 +/- 38.4% in the systolic BP of normal rabbits. Disintegrated or live trypanosomes, or hyperimmune sera alone had no effect. Pretreatment of animals with aprotinin prior to administration of the immune complex abolished the fall in BP. 4 The results suggest that the profound hypotension in chronic trypanosomiasis is caused by complex formation of trypanosomes with antibody. Since it can be prevented by pretreatment with aprotinin, it is likely that activation of plasma kallikrein with a subsequent release of plasma kinins contributes to this effect. (+info)