<i>Trypanosoma cruzi</i>, Etiological Agent of Chagas Disease, Is Virulent to Its Triatomine Vector <i>Rhodnius prolixus</i> in a Temperature-Dependent Manner

<div><p>It is often assumed that parasites are not virulent to their vectors. Nevertheless, parasites commonly exploit their vectors (nutritionally for example) so these can be considered a form of host. <i>Trypanosoma cruzi</i>, a protozoan found in mammals and triatomine bugs in the Americas, is the etiological agent of Chagas disease that affects man and domestic animals. While it has long been considered avirulent to its vectors, a few reports have indicated that it can affect triatomine fecundity. We tested whether infection imposed a temperature-dependent cost on triatomine fitness. We held infected insects at four temperatures between 21 and 30°C and measured <i>T</i>. <i>cruzi</i> growth <i>in vitro</i> at the same temperatures in parallel. <i>Trypanosoma cruzi</i> infection caused a considerable delay in the time the insects took to moult (against a background effect of temperature accelerating moult irrespective of infection status). <i>Trypanosoma cruzi</i> also reduced the insects’ survival, but only at the intermediate temperatures of 24 and 27°C (against a background of increased mortality with increasing temperatures). Meanwhile, <i>in vitro</i> growth of <i>T</i>. <i>cruzi</i> increased with temperature. Our results demonstrate virulence of a protozoan agent of human disease to its insect vector under these conditions. It is of particular note that parasite-induced mortality was greatest over the range of temperatures normally preferred by these insects, probably implying adaptation of the parasite to perform well at these temperatures. Therefore we propose that triggering this delay in moulting is adaptive for the parasites, as it will delay the next bloodmeal taken by the bug, thus allowing the parasites time to develop and reach the insect rectum in order to make transmission to a new vertebrate host possible.</p></div

Growth of cultures of <i>Trypanosoma cruzi</i> epimastigotes kept under different temperatures.

<p>(A) growth through time (live parasites counted in a flow cytometer after staining with fluorescein diacetate). (B) growth rates with temperature. Error bars are standard errors obtained from two replicates.</p

Effects of <i>Trypanosoma cruzi</i> infection on second instar <i>Rhodnius prolixus</i> nymphs over four temperatures.

<p>Insects were fed a blood meal at day 0 and were offered no further food. (A-D) time required to moult from second to third instar. (E-H) survival at 30, 60 and 90 days post-blood meal. P values indicated in each graph represent statistical significances of comparisons of infected versus uninfected control insects, using Tukey HSD <i>post hoc</i> tests from nested ANOVA analyses.</p

Sensitivity and specificity of the immunofluorescent assay in the diagnosis of <i>Leishmania</i> infected dogs.

a<p>[true positives/(true positives+false negatives)] (percentage).</p>b<p>[true negatives/(true negatives+false positives)] (percentage).</p>c<p>Symptomatic and asymptomatic.</p

Magnetic microspheres flow cytometry characterization and evaluation.

<p>Reactivities of representative symptomatic (A) and asymptomatic (B) sera tested to different defined combinations of <i>Li</i>cTXNPx and rk39 antigens. Results are expressed as the percentage of positive microspheres. The levels of IgG antibodies anti-rK39 (C) and anti-<i>Li</i>cTXNPx (D) were measured in sera of symptomatic (S), asymptomatic (AS1), asymptomatic PCR+ (AS2), <i>Leishmania</i>-negative but presenting other clinical conditions (OP) and <i>Leishmania</i> negative healthy dogs from non-endemic areas (N). Results are expressed as the percentage of positive microspheres.</p

Optimization of the experimental conditions.

<p>(A) Unspecific binding of serum antibodies to uncoated magnetic microspheres. Positive serum sample is represented in red, while negative serum sample in blue. (B) BFS as blocking agents (blue for positive serum and black for negative). (C) Positive and negative serum samples diluted from 1∶100 to 1∶6400. (D) PMSI-8.07Ni-NTA coated with rK39 and PMSI-1.7Ni-NTA coated with <i>Li</i>cTXNPx in the presence of a positive serum or a negative serum and PMSI-8.07Ni-NTA coated with <i>Li</i>cTXNPx and PMSI-1.7Ni-NTA coated with rK39 in the presence of a positive serum or a negative serum. (E) PMSI-8.07Ni-NTA coated with rK39 and PMSI-1.7Ni-NTA coated with <i>Li</i>cTXNPx formed populations of different sizes as consequence of microspheres aggregation. For further analysis, only the non-aggregated populations (black line) were used. (F) Percentage of aggregated microspheres is higher in PMSI-1.7Ni-NTA than in PMSI-8.07Ni-NTA microspheres.</p

Cytokine Profiling in Chagas Disease: Towards Understanding the Association with Infecting <i>Trypanosoma cruzi</i> Discrete Typing Units (A BENEFIT TRIAL Sub-Study)

<div><p>Background</p><p>Chagas disease caused by the protozoan <i>Trypanosoma cruzi</i> is an important public health problem in Latin America. The immunological mechanisms involved in Chagas disease pathogenesis remain incompletely elucidated. The aim of this study was to explore cytokine profiles and their possible association to the infecting DTU and the pathogenesis of Chagas disease.</p><p>Methods</p><p>109 sero-positive <i>T. cruzi</i> patients and 21 negative controls from Bolivia and Colombia, were included. Flow cytometry assays for 13 cytokines were conducted on human sera. Patients were divided into two groups: in one we compared the quantification of cytokines between patients with and without chronic cardiomyopathy; in second group we compared the levels of cytokines and the genetic variability of <i>T. cruzi.</i></p><p>Results</p><p>Significant difference in anti-inflammatory and pro-inflammatory cytokines profiles was observed between the two groups cardiac and non-cardiac. Moreover, serum levels of IFN-γ, IL-12, IL-22 and IL-10 presented an association with the genetic variability of <i>T.cruzi</i>, with significant differences in TcI and mixed infections TcI/TcII.</p><p>Conclusion</p><p>Expression of anti-inflammatory and pro-inflammatory cytokines may play a relevant role in determining the clinical presentation of chronic patients with Chagas disease and suggests the occurrence of specific immune responses, probably associated to different <i>T. cruzi</i> DTUs.</p></div

Cytokine Signatures with Frequency of Subjects with High Cytokine Levels.

<p>The diagrams were plotted using the global median of each MFI cytokine index as the cut-off mark to identify higher levels. The ascendant frequency of high cytokine producers at the with (CARD) and without Chagas cardiomyopathy (NON-CARD) was demonstrated by bar graphics. The ascendant frequency of high cytokine producers of the CONTROL group was used to generate the reference cytokine signature curves that were applied to identify changes in the overall cytokine signature from all other groups.</p

Immune Response of Calves Vaccinated with <i>Brucella abortus</i> S19 or RB51 and Revaccinated with RB51

<div><p><i>Brucella abortus</i> S19 and RB51 strains have been successfully used to control bovine brucellosis worldwide; however, currently, most of our understanding of the protective immune response induced by vaccination comes from studies in mice. The aim of this study was to characterize and compare the immune responses induced in cattle prime-immunized with <i>B</i>. <i>abortus</i> S19 or RB51 and revaccinated with RB51. Female calves, aged 4 to 8 months, were vaccinated with either vaccine S19 (0.6–1.2 x 10¹¹ CFU) or RB51 (1.3 x 10¹⁰ CFU) on day 0, and revaccinated with RB51 (1.3 x 10¹⁰ CFU) on day 365 of the experiment. Characterization of the immune response was performed using serum and peripheral blood mononuclear cells. Blood samples were collected on days 0, 28, 210, 365, 393 and 575 post-immunization. Results showed that S19 and RB51 vaccination induced an immune response characterized by proliferation of CD4⁺ and CD8⁺ T-cells; IFN-ɣ and IL-17A production by CD4⁺ T-cells; cytotoxic CD8⁺ T-cells; IL-6 secretion; CD4⁺ and CD8⁺ memory cells; antibodies of IgG1 class; and expression of the phenotypes of activation in T-cells. However, the immune response stimulated by S19 compared to RB51 showed higher persistency of IFN-ɣ and CD4⁺ memory cells, induction of CD21⁺ memory cells and higher secretion of IL-6. After RB51 revaccination, the immune response was chiefly characterized by increase in IFN-ɣ expression, proliferation of antigen-specific CD4⁺ and CD8⁺ T-cells, cytotoxic CD8⁺ T-cells and decrease of IL-6 production in both groups. Nevertheless, a different polarization of the immune response, CD4⁺- or CD8⁺-dominant, was observed after the booster with RB51 for S19 and RB51 prime-vaccinated animals, respectively. Our results indicate that after prime vaccination both vaccine strains induce a strong and complex Th1 immune response, although after RB51 revaccination the differences between immune profiles induced by prime-vaccination become accentuated.</p></div

Comparative Cytokine Signatures of NON-CARD and CARD.

<p>The diagram is a comparison between the signing of the NON-CARD (▪) and CARD (▪). There is a switch between pro-inflammatory with the boxes in solid line (IL-6, IL-2, IL-9 and IL-12) and anti-inflammatory with the boxes in dashed line (IL-5,IL-13 and IL-10) cytokines; the CARD have higher frequencies of proinflammatory cytokines and lower levels of inflammatory cytokines, whereas the opposite occurs in the case of NON-CARD.</p