Name: sexual transmission of american trypanosomes from males and females to naive mates
Uploaded: 2019-01-27T13:00:00.000+00:00
Duration: 13 min 55 s
Description: Watch this Scientific Journal Video about Sexual Transmission of American Trypanosomes from Males and Females to Naive Mates at JoVE.com

We acknowledge the laboratory facilities and the critical comments of Izabela Dourado, Carla Araujo, and Clever Gomes and the technical assistance of Bruno Dalago and Rafael Andrade. We are indebted to the Foundation for the Advancement of Science (FAPDF), The National Research Council, Ministry of Science and Technology (CNPq/MCT), and The Agency for Training Human Resources, Ministry of Education (CAPES/ME), Brazil, for supporting these investigations.

The Human and the Animal Research Committees of the Faculty of Medicine of the University of Brasilia approved all the procedures with human subjects and laboratory animals, respectively, in research protocols 2500.167567 and 10411/2011. The Ethics Committee of the Public Foundation Hospital Gaspar Vianna (protocol n&#186; 054/2009 and CONEP 11163/2009) approved the free consent forms for the field study, with extension to the Ministry of Health National Commission on Human Research (CONEP 2585/04). The protocol was adjusted to assess T. cruzi DNA in diploid blood mononuclear cells and in haploid gametes of semen ejaculates. The laboratory animals received humane care; the mice, subjected to heart puncture before sacrifice, were under anesthesia.
1. Recruitment of human participants
<ol>
	<li>Ensure that the research team participates in a Health System Chagas Disease Program to deliver health care to Chagas patients.</li>
	<li>Recruit human participants from families enrolled in the program, showing at least one case with fever, malaise, headache, tachycardia, and edema, the main clinical symptoms of the acute Chagas disease14.</li>
	<li>Deliver health care to the people in the study families for a period of five years.</li>
	<li>Obtain 15 mL of venous blood from the study participants on three occasions one year apart, divide the sample into three 5 mL aliquots, and store them in the refrigerator at 4 &#176;C.</li>
	<li>Collect 2 mL of the semen ejaculates from adult volunteer family members and proceed as described in step 3.3.</li>
</ol>
2. Growth of parasites
<ol>
	<li>Using the aliquot from step 1.4, mix the blood with 5 units of anticlotting sodium heparin; make a slope culture by the inoculation of blood from family participants with the diagnosis of acute Chagas disease.
	<ol>
		<li>Inoculate 5 mL of the unclotted human blood into a 50 mL screw cap tube blood-agar slant plus 5 mL of liver infusion-tryptose medium (LIT), and incubate the sample in a shaker at 27 &#176;C for 3 months.</li>
		<li>Place 100 &#181;L of the supernatant medium on top of glass slides, cover with slips and search for blood T. cruzi epimastigotes under the microscope, every four weeks.</li>
		<li>Harvest the epimastigote isolates in the supernatant of axenic LIT medium at 27 &#176;C; wash the cells in PBS pH 7.4, centrifuge at 1,000 x g for 10 min; dilute the epimastigotes in the pellet in 5 mL of Dulbecco&#39;s-modified essential medium (DMEM).</li>
		<li>Inoculate the confluent L6 muscle cell culture flasks with 1 x 106 ECI1-to- ECI21 T. cruzi epimastigote isolates.</li>
		<li>Grow the T. cruzi ECI1-to-ECI21 and the Berenice archetype trypomastigotes in L6 muscle cell cultures in 75 mL culture flasks.</li>
		<li>Feed cells with 15 mL of DMEM at pH 7.4 supplemented with 5% fetal bovine serum, 100 IU/mL penicillin, 100 &#181;g/mL streptomycin, 250 nM L-glutamine, and 5% CO2 at 37 &#176;C1.</li>
		<li>Use the positive control T. cruzi Berenice trypomastigotes to phenotype the ECI1-to-ECI21 isolates from tissue culture, as described in step 5.2.</li>
		<li>Grow the negative control Leishmania braziliensis27&#160;in DMEM supplemented with 20% fetal bovine serum only, and use the parasite promastigotes as a negative control.</li>
		<li>Use 1 x 106&#160;T. cruzi ECI1-to-ECI21 trypomastigotes in the supernatant from the cell culture to infect mice.</li>
		<li>Search for T. cruzi trypomastigotes in the tail blood after the first week of the infection.</li>
		<li>Search the nests of amastigotes in hematoxylin-eosin stained sections of the heart, skeletal muscle, and reproductive organs of the infected mice, one month thereafter.</li>
	</ol>
	</li>
</ol>
3. DNA extraction and PCR analyses
<ol>
	<li>Place 5 mL of the blood (step 1.4) aliquot in a sterile EDTA tube, and perform density gradient centrifugation for 45 min at 3,000 x g. Use a pipette to harvest the mononuclear cells from the whitish phase above the red cells, wash the white cells twice in 5 mL of PBS, pH 7.4, by centrifugation for 10 min at 1,500 x g in a different 15 mL tube, and use the cells for the DNA extraction.</li>
	<li>Extract the DNA from ECI-1 to ECI-21 T. cruzi, from the positive control Berenice T. cruzi, from the negative control L. braziliensis (step 2.1.8),&#160;and from the test blood mononuclear cells of 109 human family study subjects1,27,28.</li>
	<li>Dilute 2 mL of the sperm samples obtained in step 1.5 in DMEM (1:4, v/v); incubate for 45 min at 5% CO2 and 37 &#176;C, recover spermatozoa from the supernatant after centrifugation for 5 min at 13,000 x g, and extract the haploid DNA23.</li>
	<li>Place 1 mL of the cells in extraction buffer (10 &#181;M NaCl, 20 &#181;M EDTA, 1% SDS, 0.04% proteinase-K, and 1% dithiothreitol), and mix the solutions by inversion and shaking.
	<ol>
		<li>Centrifuge the solutions for 10 min at 13,000 x g and 25 &#176;C, and transfer the viscous supernatant to a spin column.</li>
		<li>Centrifuge the spin column for 1 min at 10,000 x g and discard the eluate; add 500 &#181;L binding buffer to the spin column (Table of Materials), centrifuge it for 1 min at 12,000 x g, and discard the eluate.</li>
		<li>Add 600 &#181;L washing buffer to the spin column, centrifuge it for 1 min at 12,000 x g, and discard the eluate.</li>
		<li>Repeat this step twice, and transfer the spin column to a sterile 1.5 mL micro centrifuge tube.</li>
		<li>Add 100 &#181;L of TE buffer, incubate the tube at room temperature for 2 min, and then centrifuge the tube for 1 min at 12,000 x g. The buffer in the microcentrifuge tube contains the DNA.</li>
		<li>Measure DNA concentrations by running aliquots on a 0.8% agarose gel and by reading the absorbance at 260 nm with a spectrophotometer. Store the DNA samples at -20 &#176;C until use in the PCR analysis.</li>
	</ol>
	</li>
	<li>Use T. cruzi Tcz1/2 primers annealed to the specific 188-nt specific telomere sequence probe29 and run the PCR with DNA from the family study subjects&#39; blood and sperm, from the Berenice T. cruzi positive control and from the L. braziliensis negative control.
	<ol>
		<li>Prepare the PCR mixture with 10 ng template DNA, 0.4 &#181;M of each pair of primers, 2 U of Taq DNA polymerase, 0.2 &#181;M dNTPs, and 15 &#181;M MgCl2 in a 25 &#181;L final volume.</li>
		<li>Initiate the DNA amplification program at 94 &#176;C for 30 s to denature the template, and cool the samples to 55 &#176;C for 30 s. Then, incubate the samples at 94 &#176;C for 90 s to extend the annealed primers. Return the temperature to 94 &#176;C for 30 s to initiate the next cycle, and incubate the samples an additional 3 min at 72 &#176;C. At the end of the 32nd cycle, cool the samples for 10 min at room temperature, and store them in the refrigerator at 4 &#176;C29.</li>
		<li>Amplify a T. cruzi DNA telomere repeat sequence annealed to the Tcz1/2 primers at both extremities.</li>
		<li>Analyze the amplification products on a 1.3% agarose gel and observe the 188-nt DNA bands on a UV-illuminator.</li>
	</ol>
	</li>
</ol>
4. Southern hybridization
NOTE: Southern hybridization was used to discard most of the false positive PCR amplicons in the agarose gel.
<ol>
	<li>Subject the PCR amplification products from uninfected controls, from Chagas case positive controls, from 109 test samples of diploid DNA and from haploid DNA of 21 study family participants to Southern hybridizations.</li>
	<li>Employ the T. cruzi 188-nt DNA-specific telomere sequence probe annealed to the Tcz1/2 primers shown; label the probe with [&#945;-32P] 2&#39;-deoxyadenosine triphosphate (dATP) using a random primer labeling kit, and analyze the amplification products on a 1.3% agarose gel at 60 V overnight at 4 &#176;C.</li>
	<li>Transfer the gel to a positively charged nylon membrane using the capillary method overnight.</li>
	<li>Hybridize the DNA bands transferred to the nylon membrane with the radiolabeled 188-nt probe, which binds the EcoR1&#160;digests of the genomic DNA in 25 &#181;L of the enzyme-specific buffer for variable periods.</li>
	<li>Wash the membrane twice for 15 min at 65 &#176;C with 2x SSC and 0.1% SDS.</li>
	<li>Expose the X-ray films to the nylon membrane and autoradiograph the bands on the membrane for one week.</li>
	<li>Grow the clones selected from PCR and Southern hybridization with the T. cruzi PCR amplification products, which are hybridized with the specific radiolabeled DNA probe.</li>
	<li>Commercially sequence the clones using the Tcz1/2 primers set annealed to the T. cruzi-specific telomere footprint2,23.</li>
</ol>
5. Immunological assays
NOTE: The sensitivity and specificity of the indirect immunofluorescence (IIF) and of the enzyme-linked immunosorbent assay (ELISA) were assessed in the serum from six Chagas patients with demonstrable parasitemia and from six Chagas-free, deidentified serum bank samples. The assays conducted with the double serum dilutions in PBS, pH 7.4, revealed that the IIF at 1:100 dilutions and the ELISA optical densities (ODs) at 0.150 and above separated the positive from the negative results.
<ol>
	<li>Use 5 mL of the unclotted blood aliquot (step 1.4) kept at room temperature for 1 h, and centrifuge it at 1,500 x g for 30 min to separate the serum in the supernatant.</li>
	<li>Perform IIF and ELISA in triplicate 1:100 serum dilutions to detect the T. cruzi and L. braziliensis antigens as described previously28.</li>
	<li>IIF
	<ol>
		<li>Conduct the IIF assay in triplicate serum dilutions of the 109 study subjects&#39; samples obtained on three occasions one year apart.</li>
		<li>Place 5 &#181;L suspensions of the formalin 1% -treated T. cruzi epimastigotes or L. braziliensis promastigotes onto glass slides; let the parasites dry overnight in a hood at room temperature and store the glass slides at -20 &#176;C until use.</li>
		<li>Place 20 &#181;L of the patients&#39; serum dilutions on top of glass slides coated with 5 &#181;L of the formalin-killed (10 parasites/&#181;L) T. cruzi epimastigotes or L. braziliensis promastigotes.</li>
		<li>Incubate the glass slide covered with a slip for 1 h in a moist chamber at 37 &#176;C and wash thrice with PBS.</li>
		<li>Incubate the air-dried glass slide with a 1:1,000 dilution of a fluorescein-labeled rabbit antibody (Table of Materials) to human IgG for 1 h at 37 &#176;C; wash and dry the slide.</li>
		<li>Mount the slide with a cover slip and examine it under an UV light microscope. 
		NOTE: A positive exam is an apple-green T. cruzi epimastigote silhouette, shown in the video.</li>
	</ol>
	</li>
	<li>ELISA
	<ol>
		<li>Run an ELISA to detect the T. cruzi and the L. braziliensis soluble antigens (1 &#181;g/100 &#181;L in 0.1 M carbonate buffer, pH 9.6) on coated microplate wells.</li>
		<li>Incubate the 1:100 serum dilutions in triplicate coated wells for 2 h at room temperature.</li>
		<li>Wash the plates thrice with PBST (PBS containing 0.5% Tween-20), pH 7.4, solution before drying.</li>
		<li>Incubate the plates with 50 &#181;L of a 1:1,000 dilution of rabbit anti-human IgG antibody for 90 min at 37 &#176;C.</li>
		<li>Wash the plates thrice with PBST solution and allow them to dry at room temperature.</li>
		<li>Incubate the plates with 100 &#181;L of 1:1,000 dilutions of alkaline phosphatase-conjugated goat anti-rabbit IgG (Table of Materials) for 90 min at 37 &#176;C.</li>
		<li>Wash the plates thrice with PBST, add the substrate p-nitro phenyl phosphate, and wait for color development.</li>
		<li>Read the ODs at 630 nm in a multimode plate reader.</li>
		<li>Run the triplicate dilutions of the test serum from the study population and plot the ODs to identify the specific T. cruzi antibody titers.</li>
	</ol>
	</li>
</ol>
6. Assessments of immune tolerance
NOTE: A chicken model system was used to test T. cruzi infections after the first week of embryo development.
<ol>
	<li>Inoculate chicken fertile eggs with 100/10 &#181;L T. cruzi trypomastigotes harvested from tissue culture medium; inoculate mock control eggs with 10 T. cruzi trypomastigotes per &#181;L culture medium. Seal the hole with tape.</li>
	<li>Incubate 20 T. cruzi-infected eggs and an equal number of mock control fertile eggs at 37 &#176;C and 65% humidity for 21 days.</li>
	<li>Keep the chicks that hatch in the incubator for 24 h and, thereafter, at 32 &#176;C in hoods with temperature control for three weeks.</li>
	<li>Grow the chicks hatched from T. cruzi-inoculated eggs and from mock control eggs to the adult stage in a positive air pressure room at 24 &#176;C, in individual cages placed in separate aisles.</li>
	<li>Challenge all the adult chicks thrice at six months of age with 107 formalin-killed trypomastigotes injected subcutaneously, weekly, according to the scheme in the video.</li>
	<li>Draw blood from a wing vein of chickens hatched from the T. cruzi-inoculated eggs and from the mock controls four weeks after the last immunization to obtain the serum.</li>
	<li>Use the chicken serum to detect the specific T. cruzi antibody by IIF and ELISA as described in step 5 of the protocol.</li>
</ol>
7. Infection of mice with T. cruzi from Chagas patients&#39; semen ejaculates
<ol>
	<li>Use the semen ejaculates from an adult PCR+ Chagas disease patient (step 1.5) and from an adult PCR- Chagas-free individual.</li>
	<li>Use two groups of 12 one-month-old BALB/c naive mice kept in hoods under positive air pressure at 24 &#176;C and fed food ad libitum.</li>
	<li>Instill the human Chagas-positive semen aliquots (100 &#181;L) into the peritoneum and equal amounts into the vagina of group-A mice.</li>
	<li>Instill the control Chagas-free semen aliquots (100 &#181;L) into the peritoneum and an equal amount into the vagina of group-B mice.</li>
	<li>Sacrifice the experimental mice under anesthesia five weeks after the semen instillations and subject the tissue sections to staining with hematoxylin-eosin.</li>
	<li>Use microscopy to search for T. cruzi trypomastigotes and amastigotes in the heart, skeletal muscle, and reproductive organs in groups of mice.</li>
</ol>
8. Transmission of the T. cruzi infection by intercourse
<ol>
	<li>Use 10 male and female six-week-old BALB/c mice in the experiments.</li>
	<li>Inoculate five male and five female mice intraperitoneally with 1 x 105&#160;T. cruzi trypomastigotes from the tissue culture.</li>
	<li>Breed the mice until three months of age: group I will be formed by five T. cruzi-infected female mice and five control noninfected male mates; group II will be formed by five T. cruzi-infected male and five control noninfected female mates. Group III will be formed by five male and five female control naive uninfected mates.</li>
	<li>House each breeding pair in one cage placed inside a safe box with a 5 mm grid and lock-in door to prevent escape.</li>
	<li>Feed the mice chow and water ad libitum. Raise a total of 70 weaning progenies in groups II and I for at least six weeks.</li>
	<li>Draw blood by heart puncture from the parental (FO) and progeny (F1) mice under anesthesia, sacrifice the mice, and submit sections of the heart, skeletal muscle, and reproductive organs for pathological analysis.</li>
</ol>
9. Assessment of immune privilege
<ol>
	<li>Obtain tissues from T. cruzi-infected and naive control mice (step 8.6), and cut the paraffin-embedded samples into 4 &#181;m thick sections.</li>
	<li>Remove the paraffin and dehydrate the sections onto glass slides with several changes of xylene and graded washes with 100% to 70% ethanol, for 1 min each.</li>
	<li>Incubate the tissue sections with 0.05% saponin once, followed by three distilled water washes at room temperature.</li>
	<li>Block the tissue sections with 5% nonfat powdered milk for 45 min. Wash the slides in 0.1 M PBST and incubate the sections with the Chagas mouse anti-T. cruzi serum or with the control uninfected mouse serum at a 1:20 dilution for 2 h.</li>
	<li>Wash the slides thrice for 5 min in PBST and dry at room temperature before incubation with a 1:1,000 dilution of alkaline phosphatase-conjugated rabbit anti-mouse IgG.</li>
	<li>Rinse the slides in PBST and add 100 &#181;L of 3,3&#39; diaminobenzidine for a 5 min incubation, followed by three washes with PBST and counterstaining with Harris hematoxylin for 30 s.</li>
	<li>Wash the slides in distilled water for 5 min, dehydrate them in 70%, 80%, 90%, and 100% ethanol for 1 min, and mount them in buffered glycerin.</li>
	<li>Examine the slides with a bright-field light microscope, and capture photo images with a microcamera with software and an analyzer program.</li>
	<li>Document the immune privilege of the parasite in the absence of inflammatory reactions in the reproductive organs.</li>
</ol>
10. Statistical analyses
<ol>
	<li>Use Biomedical Edit for sequence analysis, perform alignments with BLAST, and determine the E-value statistical significance (p &#60; 0.05).</li>
	<li>Perform a one-way analysis of variance (ANOVA) and the Tukey test to compare the OD means plus or minus standard deviations.</li>
</ol>

<ol>
	<li>Araujo, P. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+transmission+of+American+trypanosomiasis+in+humans:+a+new+potential+pandemic+route+for+Chagas+parasites.">Sexual transmission of American trypanosomiasis in humans: a new potential pandemic route for Chagas parasites.</a> Mem&#243;rias do Instituto Oswaldo Cruz. 112 (6), 437-446 (2017).</li><li>Teixeira, A. R. L., Nitz, N., Bernal, F. M., Hetch, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parasite+Induced+Genetically+Driven+Autoimmune+Chagas+Heart+Disease+in+the+Chicken+Model.">Parasite Induced Genetically Driven Autoimmune Chagas Heart Disease in the Chicken Model.</a> Journal of Visualized Experiments. (65), 3716 (2012).</li><li>Kalil-Filho, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Globalization+of+Chagas+Disease+Burden+and+New+Treatment+Perspectives.">Globalization of Chagas Disease Burden and New Treatment Perspectives.</a> Journal of the American College of Cardiology. 66 (10), 1190-1192 (2015).</li><li>Nunes, M. C. P., Dones, W., Morillo, A., Encina, J. J., Ribeiro, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chagas+Disease:+An+Overview+of+Clinical+and+Epidemiological+Aspects.">Chagas Disease: An Overview of Clinical and Epidemiological Aspects.</a> Journal of the American College of Cardiology. 62 (9), 767-776 (2013).</li><li>Pinazo, M. J., Gascon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chagas+disease:+from+Latin+America+to+the+world.">Chagas disease: from Latin America to the world.</a> Reports in Parasitology. 2015 (4), 7-14 (2015).</li><li>Kessler, D. A., Shi, P. A., Avecilla, S. T., Shaz, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Results+of+lookback+for+Chagas+disease+since+the+inception+of+donor+screening+at+New+York+Blood+Center.">Results of lookback for Chagas disease since the inception of donor screening at New York Blood Center.</a> Transfusion. 53 (5), 1083-1087 (2013).</li><li>Klein, N., Hurwitz, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Globalization+of+Chagas+Disease:+A+Growing+Concern+in+Nonendemic+Countries.">Globalization of Chagas Disease: A Growing Concern in Nonendemic Countries.</a> Epidemiology Research International. , (2012).</li><li>P&#233;rez-Molina, J. A., Norman, F., L&#243;pez-V&#233;lez, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chagas+disease+in+non-endemic+countries:+epidemiology,+clinical+presentation+and+treatment.">Chagas disease in non-endemic countries: epidemiology, clinical presentation and treatment.</a> Current Infectious Diseases Report. 14 (3), 263-274 (2012).</li><li>Hotez, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chagas+disease:+&amp;#34;the+new+HIV/AIDS+of+the+Americas&amp;#34;.">Chagas disease: &amp;#34;the new HIV/AIDS of the Americas&amp;#34;.</a> PLoS Neglected Tropical Diseases. 6, e1498 (2012).</li><li>Schmunis, G. A., Yadon, Z. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chagas+disease:+a+Latin+American+health+problem+becoming+a+world+health+problem.">Chagas disease: a Latin American health problem becoming a world health problem.</a> Acta Tropica. 115 (1-2), 14-21 (2010).</li><li>Franco-Paredes, C., Bottazzi, M. E., Hotez, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Unfinished+Public+Health+Agenda+of+Chagas+Disease+in+the+Era+of+Globalization.">The Unfinished Public Health Agenda of Chagas Disease in the Era of Globalization.</a> PLoS Neglected Tropical Diseases. 3 (7), e470 (2009).</li><li>Teixeira, A. R. L., Vinaud, M., Castro, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Emerging Chagas Disease. In: Chagas Disease: - A Global Health Problem. 3, 18-39 (2009).</li><li>Lee, B. Y., Bacon, K. M., Bottazzi, M. E., Hotez, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+economic+burden+of+Chagas+disease:+a+computational+simulation+model.">Global economic burden of Chagas disease: a computational simulation model.</a> Lancet Infectious Diseases. 13 (4), 342-348 (2013).</li><li>Teixeira, A. R. L., Hecht, M. M., Guimaro, M. C., Sousa, A. O., Nitz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+Chagas+Disease:+Parasite+Persistence+and+Autoimmunity.">Pathogenesis of Chagas Disease: Parasite Persistence and Autoimmunity.</a> Clinical Microbiology Review. 24 (3), 592-630 (2011).</li><li>Murcia, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+factors+and+primary+prevention+of+congenital+Chagas+disease+in+a+nonendemic+country.">Risk factors and primary prevention of congenital Chagas disease in a nonendemic country.</a> Clinical Infectious Diseases. 56 (4), 496-502 (2013).</li><li>Chagas, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+human+trypanosomiasis.+Morphology+and+lifecycle+of+Schizotrypanum+cruzi,+the+cause+of+a+new+human+disease.">New human trypanosomiasis. Morphology and lifecycle of Schizotrypanum cruzi, the cause of a new human disease.</a> Mem&#243;rias do Instituto Oswaldo Cruz. 1, 159 (1909).</li><li>Vianna, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+to+the+study+of+the+Pathology+of+Chagas+disease.">Contribution to the study of the Pathology of Chagas disease.</a> Mem&#243;rias do Instituto Oswaldo Cruz. 3, 276 (1911).</li><li>Teixeira, A. R. L., Roters, F., Mott, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+Chagas+disease.">Acute Chagas disease.</a> Gazeta M&#233;dica da Bahia. 3, 176-186 (1970).</li><li>Teixeira, A. R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+and+Control+of+Chagas+Disease+&#8211;+An+Overview.">Prevention and Control of Chagas Disease &#8211; An Overview.</a> International STD Research, Reviews. 7 (2), 1-15 (2018).</li><li>Rios, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+sexual+transmission+support+the+enzootic+cycle+of+Trypanosoma+cruzi?.">Can sexual transmission support the enzootic cycle of Trypanosoma cruzi?.</a> Mem&#243;rias do Instituto Oswaldo Cruz. 113 (1), 3-8 (2018).</li><li>Lenzi, H. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypanosoma+cruzi:+compromise+of+reproductive+system+in+acute+murine+infection.">Trypanosoma cruzi: compromise of reproductive system in acute murine infection.</a> Acta Tropica. 71 (2), 117-129 (1998).</li><li>Carvalho, L. O. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypanosoma+cruzi+and+myoid+cells+from+seminiferous+tubules:+Interaction+and+relation+with+&#64257;brous+components+of+extra+cellular+matrix+in+experimental+Chagas&#8217;+disease.">Trypanosoma cruzi and myoid cells from seminiferous tubules: Interaction and relation with &#64257;brous components of extra cellular matrix in experimental Chagas&#8217; disease.</a> International Journal of Experimental Pathology. 90 (1), 52-57 (2009).</li><li>Hecht, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inheritance+of+DNA+transferred+from+American+trypanosomes+to+human+hosts.">Inheritance of DNA transferred from American trypanosomes to human hosts.</a> PLoS One. 12, e9181 (2010).</li><li>Alarcon, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presencia+de+epimastigotes+de+Trypanosoma+cruzi+en+el+plasma+seminal+de+ratones+con+infecci&#243;n+aguda.">Presencia de epimastigotes de Trypanosoma cruzi en el plasma seminal de ratones con infecci&#243;n aguda.</a> Bolet&#237;n Malariolog&#237;a y Salud Ambiental. 51, 237 (2011).</li><li>Teixeira, A. R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypanosoma+cruzi+in+the+Chicken+Model:+Chagas-Like+Heart+Disease+in+the+Absence+of+Parasitism.">Trypanosoma cruzi in the Chicken Model: Chagas-Like Heart Disease in the Absence of Parasitism.</a> PLoS Neglected Tropical Diseases. 5 (3), e1000 (2011).</li><li>Guimaro, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Autoimmune+Chagas-Like+Heart+Disease+by+Bone+Marrow+Transplantation.">Inhibition of Autoimmune Chagas-Like Heart Disease by Bone Marrow Transplantation.</a> PLoS Neglected Tropical Diseases. 8 (12), e3384 (2014).</li><li>Oliveira, C. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leishmania+braziliensis+isolates+differing+at+the+genome+level+display+distinctive+features+in+BALB/c+mice.">Leishmania braziliensis isolates differing at the genome level display distinctive features in BALB/c mice.</a> Microbes and Infection. 6 (11), 977-984 (2004).</li><li>Mendes, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposure+to+mixed+asymptomatic+infections+with+Trypanosoma+cruzi,+Leishmania+braziliensis+and+Leishmania+chagasi+in+the+human+population+of+the+greater+Amazon.">Exposure to mixed asymptomatic infections with Trypanosoma cruzi, Leishmania braziliensis and Leishmania chagasi in the human population of the greater Amazon.</a> Tropical Medicine, International Health. 12, 629 (2007).</li><li>Moser, D. R., Kirchhoff, L. V., Donelson, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Trypanosoma+cruzi+by+DNA+amplification+using+the+polymerase+chain+reaction.">Detection of Trypanosoma cruzi by DNA amplification using the polymerase chain reaction.</a> Journal of Clinical Microbiology. 27 (7), 1477-1482 (1989).</li><li>Da Silva, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Sexual transmission of Trypanosoma cruzi in mus musculus [MsD thesis]. , (2013).</li><li>Ribeiro, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+sexual+transmission+support+the+enzootic+cycle+of+Trypanosoma+cruzi?.">Can sexual transmission support the enzootic cycle of Trypanosoma cruzi?.</a> Mem&#243;rias do Instituto Oswaldo Cruz. 113 (1), 3-8 (2018).</li><li>Billingham, R. E., Brent, L., Medawar, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actively+acquired+tolerance+of+foreign+cells.">Actively acquired tolerance of foreign cells.</a> Nature. 172 (4379), 603-606 (1953).</li><li>Burnet, F., Fenner, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The production of Antibodies. , (1949).</li><li>Hasek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parabiosis+of+birds+during+their+embryonic+development.">Parabiosis of birds during their embryonic development.</a> Chekhoslovatskaia Biology. 2 (1), 29-31 (1953).</li><li>Coura, J. R., Junqueira, A. C. V., Fernades, O., Valente, S. A., Miles, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+Chagas+Disease+in+Amazonian+Brazil.+Trends+Parasitology.">Emerging Chagas Disease in Amazonian Brazil. Trends Parasitology.</a> Trends Parasitology. 18 (4), 171-176 (2002).</li><li>Teixeira, A. R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+Chagas+disease:+Trophic+network+and+cycle+of+transmission+of+Trypanosoma+cruzi+from+palm+trees+in+the+Amazon.">Emerging Chagas disease: Trophic network and cycle of transmission of Trypanosoma cruzi from palm trees in the Amazon.</a> Emerging Infectious Diseases. 7 (1), 100112 (2001).</li><li>Hazebroek, M., Dennert, R., Heymans, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Idiopathic+dilated+cardiomyopathy:+possible+triggers+and+treatment+strategies.">Idiopathic dilated cardiomyopathy: possible triggers and treatment strategies.</a> Netherland Heart Journal. 20 (7-8), 332-335 (2012).</li><li>Arimura, T., Hayashi, T., Kimura, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+etiology+of+idiopathic+cardiomyopathy.">Molecular etiology of idiopathic cardiomyopathy.</a> Acta Myologica. 26 (3), 153-158 (2007).</li><li>Dec, W. G., Fuster, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Idiopathic+Dilated+Cardiomyopathy.">Idiopathic Dilated Cardiomyopathy.</a> New England Journal of Medicine. 33, 1564-1575 (1994).</li><li>Niederkorn, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=See+no+evil,+hear+no+evil,+do+no+evil:+the+lessons+of+immune+privilege.">See no evil, hear no evil, do no evil: the lessons of immune privilege.</a> Nature Immunology. 7 (4), 354-359 (2006).</li><li>Smith, B. E., Braun, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ+cell+migration+across+Sertoli+cell+tight+junctions.">Germ cell migration across Sertoli cell tight junctions.</a> Science. 338 (6118), 798-802 (2012).</li><li>Garth, A., Wilbanks, J., Streilein, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluids+from+immune+privileged+sites+endow+macrophages+with+the+capacity+to+induce+antigen-specific+immune+deviation+via+a+mechanism+involving+transforming+growth+factor-&#946;.">Fluids from immune privileged sites endow macrophages with the capacity to induce antigen-specific immune deviation via a mechanism involving transforming growth factor-&#946;.</a> European Journal of Immunology. 22 (4), 1031-1036 (1992).</li><li>Meng, J., Anne, R., Greenlee, C., Taub, J., Braun, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sertoli+Cell-Specific+Deletion+of+the+Androgen+Receptor+Compromises+Testicular+Immune+Privilege+in+Mice.">Sertoli Cell-Specific Deletion of the Androgen Receptor Compromises Testicular Immune Privilege in Mice.</a> Biology of Reproduction. 85 (2), 254-260 (2011).</li><li>Fujisaki, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+Treg+cells+providing+immune+privilege+to+the+haematopoietic+stem-cell+niche.">In vivo imaging of Treg cells providing immune privilege to the haematopoietic stem-cell niche.</a> Nature. 474 (7350), 216-219 (2011).</li><li>Wood, K. J., Sakaguchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+T+cells+in+transplantation+tolerance.">Regulatory T cells in transplantation tolerance.</a> Nature Review Immunology. 3 (3), 199-210 (2003).</li></ol>

The authors declare that they have no competing financial interests.

Herein, we discuss a family-based research protocol that answered the question of whether human Chagas disease stems from sexually transmitted intraspecies T. cruzi infections. Early studies could not provide evidence of the sexual transmission of T. cruzi infections, probably because the available data and information on Chagas disease were obtained separately from the individual3,4,5,<sup class="xr...

The protozoan parasite Trypanosoma cruzi belonging to the family Trypanosomatidae undergoes trypomastigote and amastigote life cycle stages in mammalian hosts and exists as epimastigotes in the insect-vector&#39;s (Reduviid: Triatominae) gut and in axenic culture. In recent decades, several studies have shown the presence of Chagas disease in countries on four continents considered triatomine bug free1,2,3,4,5,6,7,8,9,10,11,12,13; the dispersion of American trypanosomes was initially attributed to Latin American immigrants to the Northern Hemisphere, but the possibility that some are autochthonous cases of Chagas disease can no longer be denied3,4,5,6,7,8,9,10,11,12,13,14. The only recognizable endogenous source of T. cruzi transmission has been ascribed to the chagasic mother&#39;s transfer of the parasite to the offspring in approximately 10% of pregnancies15; the male partner&#39;s contribution to in utero&#160;infections through semen ejaculates has remained unrecognized.
Over one century ago, investigators16,17 observed intracellular T. cruzi amastigotes in the theca cells of the ovary and in the germ line cells of the testicles of acute cases of Chagas disease. The nests and clumps of T. cruzi trypomastigotes and amastigotes in theca cells of the ovary, in goniablasts and in the lumen of seminiferous tubules (Figure 1) of fatal acute Chagas disease cases develop immune privilege in the organs of reproduction in the absence of inflammatory infiltrates18,19. In recent decades, a few experimental studies have shown nests of the round amastigote forms of T. cruzi in the seminiferous tubule, epididymis, and vas deferens as well as in the uterus, tubes and ovary theca cells of acutely infected mice1,20,21,22. Furthermore, in the course of family studies to document the transfer of protozoan&#160;mitochondrial DNA from parental Chagas patients to their descendants, T. cruzi nuclear DNA (nDNA) was verified in human haploid germ line cells23, and parasite life cycle stages were observed in the ejaculates of chagasic mice24. These findings are in agreement with reports on the immune tolerance attained by the progeny of T. cruzi-infected hosts in the absence of the specific antibody1,25,26. Additionally, epidemiological reports that suggested the spread of endemic Chagas disease to the other continents3,4,5,6,7,8,9,10,11,12,13 are now supported by experimental studies showing that Chagas disease can be transmitted sexually1. The present investigation presents an epidemiologic family study protocol and shows that T. cruzi infection propagates by sexual intercourse.

<table><tbody><tr><td>BCIP and NBT redox system</td><td>Sigma-Aldrich</td><td>681 451 001</td><td></td></tr><tr><td>Blood DNA Purification columns</td><td>Amersham Biosciences</td><td>27-9603-01</td><td></td></tr><tr><td>d-ATP, [&amp;alpha;-&lt;sup&gt;32&lt;/sup&gt;P], 250 &amp;micro;Ci.</td><td>&amp;nbsp; Perkin Elmer&amp;nbsp;&amp;nbsp;</td><td>BLU012H</td><td></td></tr><tr><td>DNA, Solution Salt Fish Sperm</td><td>AMRESCO</td><td>064-10G</td><td></td></tr><tr><td>dNTP Set, 100 mM Solutions</td><td>GE Healthcare</td><td>28-4065-51</td><td></td></tr><tr><td>&lt;em&gt;Eco&amp;nbsp;&lt;/em&gt;RI</td><td>Invitrogen</td><td>15202-021</td><td></td></tr><tr><td>Goat anti-human IgG- alkaline phosphatase conjugated</td><td>Southern Biotech</td><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 2040-04</td><td></td></tr><tr><td>Goat anti-human IgG- FITC conjugated</td><td>Biocompare</td><td>MB5198020</td><td></td></tr><tr><td>Hybond &amp;ndash; N+ nylon membrane</td><td>GE Healthcare</td><td>RPN303B</td><td></td></tr><tr><td>Hybridization oven</td><td>Thomas Scientific</td><td>95-0031-02</td><td></td></tr><tr><td>Micro imaging software cell Sens software</td><td>Olympus, Japan</td><td></td><td></td></tr><tr><td>Molecular probes labeling System</td><td>Invitrogen</td><td>700-0030</td><td></td></tr><tr><td>&lt;em&gt;Nsi&lt;/em&gt; I</td><td>Sigma-Aldrich</td><td>R5584 1KU</td><td></td></tr><tr><td>Plasmid Prep Mini Spin Kit</td><td>GE Healthcare</td><td>28-9042-70</td><td></td></tr><tr><td>Plate reader&amp;nbsp;</td><td>Bio-Tek GmBH</td><td>2015</td><td></td></tr><tr><td>Rabbit anti-chicken IgG-alkaline phosphatase conjugated</td><td>Sigma-Aldrich</td><td>A9171</td><td></td></tr><tr><td>Rabbit anti-chicken IgG-FITC conjugated</td><td>Sigma-Aldrich</td><td>F8888</td><td></td></tr><tr><td>Rabbit anti-mouse IgG- alkaline phosphatase conjugated</td><td>Sigma Aldrich</td><td>A2418&amp;nbsp;</td><td></td></tr><tr><td>Rabbit anti-mouse IgG-FITC conjugated</td><td>Biorad</td><td>MCA5787</td><td></td></tr><tr><td>Spin Columns for radio labeled DNA purification, Sephadex G-25, fine</td><td>Sigma-Aldrich</td><td>G25DNA-RO&amp;nbsp;</td><td></td></tr><tr><td>Taq DNA Polymerase Recombinant</td><td>Invitrogen</td><td>11615-010</td><td></td></tr><tr><td>Thermal cycler system</td><td>Biorad, USA</td><td>1709703</td><td></td></tr><tr><td>Vector Systems</td><td>Promega</td><td>A1380</td><td></td></tr></tbody></table>

sexual transmission of american trypanosomes from males and females to naive mates

American trypanosomiasis is transmitted to humans by triatomine bugs through the ingestion of contaminated food, by blood transfusions or accidently in hospitals and research laboratories. In addition, the Trypanosoma cruzi infection is transmitted congenitally from a&#160;chagasic mother to her offspring, but the male partner&#39;s contribution to in utero contamination is unknown. The findings of nests and clumps of amastigotes and of trypomastigotes in the theca cells of the ovary, in the goniablasts and in the lumen of seminiferous tubules suggest that T. cruzi infections are sexually transmitted. The research protocol herein presents the results of a family study population showing parasite nuclear DNA in the diploid blood mononuclear cells and in the haploid gametes of human subjects. Thus, three independent biological samples collected one year apart confirmed that T. cruzi infections were sexually transmitted to progeny. Interestingly, the specific T. cruzi antibody was absent in the majority of family progeny that bore immune tolerance to the parasite antigen. Immune tolerance was demonstrated in chicken refractory to T. cruzi after the first week of embryonic growth, and chicks hatched from the flagellate-inoculated eggs were unable to produce the specific antibody.&#160;Moreover, the instillation of the human semen ejaculates intraperitoneally or into the vagina of naive mice yielded T. cruzi amastigotes in the epididymis, seminiferous tubule, vas deferens and uterine tube with an absence of inflammatory reactions in the immune privileged organs of reproduction. The breeding of T. cruzi-infected male and female mice with naive mates resulted in acquisition of the infections, which were later transmitted to the progeny. Therefore, a robust education, information and communication program that involves the population and social organizations is deemed necessary to prevent Chagas disease.

This research, conducted according to the protocol, aimed to detect acute cases of Chagas disease by clinical and parasitological exams. Venous blood samples were subjected to direct microscopic examination and in vitro culture for parasite growth. Twenty-one acute cases of Chagas disease showed T. cruzi in blood. The research protocol secured the isolation of T. cruzi ECI1-to ECI21 from acute Chagas disease, and the DNA samples exhibited positive DNA footprints in the r...

Watch this Scientific Journal Video about Sexual Transmission of American Trypanosomes from Males and Females to Naive Mates at JoVE.com

Sexual Transmission of American Trypanosomes from Males and Females to Naive Mates

The Trypanosoma cruzi agent of Chagas disease produces long-lasting asymptomatic infections that abruptly develop into clinically recognized pathology. The following research protocol describes a short-run family-based epidemiological study to unravel the T. cruzi infection transmitted sexually from parent to progeny.

American trypanosomiasis is transmitted to humans by triatomine bugs through the ingestion of contaminated food, by blood transfusions or accidently in ...

sexual-transmission-american-trypanosomes-from-males-females-to-naive

Research

JoVE Journal

Immunology and Infection

15.1K Views.  University of Brasilia. The Trypanosoma cruzi agent of Chagas disease produces long-lasting asymptomatic infections that abruptly develop into clinically recognized pathology. The following research protocol describes a short-run family-based epidemiological study to unravel the T. cruzi infection transmitted sexually from parent to progeny.

Video: Sexual Transmission of American Trypanosomes from Males and Females to Naive Mates

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3 and humans4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10.

Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique&#39;s relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

In this protocol, fluorescently labeled T. cruzi&#160;were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy.

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

Developmental Biology

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites.
Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection.
Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches.

This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. However, effective malaria parasite targets for vaccine and chemotherapy development are still limited. This is largely due to the unavailability of relevant and practical in vivo infection models for human Plasmodium species, most notably for P.&#160;falciparum and P. vivax. Therefore, rodent malaria species have been extensively used as practical alternative in vivo models for malaria vaccine, drug targeting, immune response, and functional characterization studies of conserved Plasmodiumspp. genes. Indeed, rodent malaria models have proven to be invaluable, especially for exploring mosquito transmission and liver stage biology, and were indispensable for immunological studies. However, there are discrepancies in the methods used to evaluate the phenotypes of transgenic and wild-type asexual and sexual blood-stage parasites.&#160;Examples of these discrepancies are the choice of an intravenous vs. intraperitoneal infection of rodents with blood-stage parasites and the evaluation of male gamete exflagellation. Herein, we detail standardized experimental methods to evaluate the phenotypes of asexual and sexual blood stages in transgenic parasites expressing reporter-gene or wild-type rodent malaria parasite species. We also detail the methods to evaluate the phenotypes of malaria parasite mosquito stages (gametes, ookinetes, oocysts, and sporozoites) inside Anopheles mosquito vectors. These methods are detailed and simplified here for the lethal and non-lethal strains of P. berghei and P. yoelii but can also be applied with some adjustments to P. chabaudi and P. vinckei rodent malaria species.

Due to the striking similarities of the life cycle and biology of rodent malaria parasites to human malaria parasites, rodent malaria models have become indispensable for malaria research. Herein, we standardized some of the most important techniques used in the phenotypic analysis of wild-type and transgenic rodent malaria species.

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. ...

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers (Diethylaminoethyl-cellulose Columns)

This method allows the separation of trypanosomes, parasites responsible for animal and human African trypanosomiasis (HAT), from infected blood. This is the best method for diagnosis of first stage HAT and furthermore this parasite purification method permits serological and research investigations.
HAT is caused by Tsetse fly transmitted Trypanosoma brucei gambiense and T. b. rhodesiense. Related trypanosomes are the causative agents of animal trypanosomiasis. Trypanosome detection is essential for HAT diagnosis, treatment and follow-up. The technique described here is the most sensitive parasite detection technique, adapted to field conditions for the diagnosis of T. b. gambiense HAT and can be completed within one hour. Blood is layered onto an anion-exchanger column (DEAE cellulose) previously adjusted to pH 8, and elution buffer is added. Highly negatively charged blood cells are adsorbed onto the column whereas the less negatively charged trypanosomes pass through. Collected trypanosomes are pelleted by centrifugation and observed by microscopy. Moreover, parasites are prepared without cellular damage whilst maintaining their infectivity.
Purified trypanosomes are required for immunological testing; they are used in the trypanolysis assay, the gold standard in HAT serology. Stained parasites are utilized in the card agglutination test (CATT) for field serology. Antigens from purified trypanosomes, such as variant surface glycoprotein, exoantigens, are also used in various immunoassays. The procedure described here is designed for African trypanosomes; consequently, chromatography conditions have to be adapted to each trypanosome strain, and more generally, to the blood of each species of host mammal.
These fascinating pathogens are easily purified and available to use in biochemical, molecular and cell biology studies including co-culture with host cells to investigate host-parasite relationships at the level of membrane receptors, signaling, and gene expression; drug testing in vitro; investigation of gene deletion, mutation, or overexpression on metabolic processes, cytoskeletal biogenesis and parasite survival.

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers Diethylaminoethyl-cellulose Columns

This method of trypanosome separation from blood depends on their surface charge being less negative than mammalian blood cells. Infected blood is placed and treated on an anion-exchanger column. This method, the most fitting diagnostic for African trypanosomiasis, provides purified parasites for immunological, biological, biochemical, pharmaceutical and molecular biology investigations.

This method allows the separation of trypanosomes, parasites responsible for animal and human African trypanosomiasis (HAT), from infected blood. This is ...

Detection of Extravascular Trypanosoma Parasites by Fine Needle Aspiration

Fine Needle Aspiration (FNA) is a routine diagnostic procedure essential to both medical and veterinary practices. It consists of the percutaneous aspiration of cells and/or microorganisms from palpable masses, organs or effusions (fluid accumulation in a body cavity) using a thin needle similar to the regular needle used for the venous puncture. The material collected by FNA is in general highly cellular, and the retrieved aspirate is then smeared, air dried, wet-fixed, stained and observed under a microscope. In the clinical context, FNA is an important diagnostic tool that serves as a guide to the appropriate therapeutic management. Because it is simple, fast, minimally invasive and requires limited investment in the laboratory and human resources, it is extensively used by veterinary practitioners, mostly in domestic, but also in farm animals. In studies using animal models, FNA has the advantage that it can be performed repeatedly in the same animal, enabling longitudinal studies through the collection of cells from tumors and organs/tissues over the course of the disease. In addition to routine microscopy, retrieved material can also be used for immunocytochemistry, electron microscopy, biochemical analysis, flow cytometry, molecular biology or in vitro assays. FNA has been used to identify the protozoan parasite Trypanosoma brucei in the gonads of infected mice, opening the possibility for a future diagnosis in cattle.

Detection of Extravascular Trypanosoma Parasites by Fine Needle Aspiration

Fine Needle Aspiration is a technique, whereby cells are obtained from a lesion or organ using a thin needle. Aspirated material is smeared, stained and examined under a microscope for diagnosis or used for molecular biology, cytometry or in vitro analysis. It is cheap, simple, quick and causes minimal trauma.

Fine Needle Aspiration (FNA) is a routine diagnostic procedure essential to both medical and veterinary practices. It consists of the percutaneous ...

AI Program for Trypanosomiasis Detection and Classification

Superior Auto-Identification of Trypanosome Parasites by Using a Hybrid Deep-Learning Model

Trypanosomiasis is a significant public health problem in several regions across the world, including South Asia and Southeast Asia. The identification of hotspot areas under active surveillance is a fundamental procedure for controlling disease transmission. Microscopic examination is a commonly used diagnostic method. It is, nevertheless, primarily reliant on skilled and experienced personnel. To address this issue, an artificial intelligence (AI) program was introduced that makes use of a hybrid deep learning technique of object identification and object classification neural network backbones on the in-house low-code AI platform (CiRA CORE). The program can identify and classify the protozoan trypanosome species, namely Trypanosoma cruzi, T. brucei, and T. evansi, from oil-immersion microscopic images. The AI program utilizes pattern recognition to observe and analyze multiple protozoa within a single blood sample and highlights the nucleus and kinetoplast of each parasite as specific characteristic features using an attention map.
To assess the AI program&#39;s performance, two unique modules are created that provide a variety of statistical measures such as accuracy, recall, specificity, precision, F1 score, misclassification rate, receiver operating characteristics (ROC) curves, and precision versus recall (PR) curves. The assessment findings show that the AI algorithm is effective at identifying and categorizing parasites. By delivering a speedy, automated, and accurate screening tool, this technology has the potential to transform disease surveillance and control. It could also assist local officials in making more informed decisions on disease transmission-blocking strategies.

Author Spotlight: AI-Driven Trypanosome Species Detection from Microscopic Images 

Worldwide medical blood parasites were automatically screened using simple steps on a low-code AI platform. The prospective diagnosis of blood films was improved by using an object detection and classification method in a hybrid deep learning model. The collaboration of active monitoring and well-trained models helps to identify hotspots of trypanosome transmission.

Trypanosomiasis is a significant public health problem in several regions across the world, including South Asia and Southeast Asia. The identification of ...

Bioengineering

Ultrastructural Expansion Microscopy in In Vitro Life Cycle of Trypanosoma cruzi

Ultrastructural Expansion Microscopy in Three In Vitro Life Cycle Stages of Trypanosoma cruzi

We describe here the application of ultrastructure expansion microscopy (U-ExM) in Trypanosoma cruzi, a technique that allows increasing the spatial resolution of a cell or tissue for microscopic imaging. This is performed by physically expanding a sample with off-the-shelf chemicals and common lab equipment.
Chagas disease is a widespread and pressing public health concern caused by T. cruzi. The disease is prevalent in Latin America and has become a significant problem in non-endemic regions due to increased migration. The transmission of T. cruzi occurs through hematophagous insect vectors belonging to the Reduviidae and Hemiptera families. Following infection, T. cruzi amastigotes multiply within the mammalian host and differentiate into trypomastigotes, the non-replicative bloodstream form. In the insect vector, trypomastigotes transform into epimastigotes and proliferate through binary fission.The differentiation between the life cycle stages requires an extensive rearrangement of the cytoskeleton and can be recreated in the lab completely using different cell culture techniques. 
We describe here a detailed protocol for the application of U-ExM in three in vitro life cycle stages of Trypanosoma cruzi, focusing on optimization of the immunolocalization of cytoskeletal proteins. We also optimized the use of N-Hydroxysuccinimide ester (NHS), a pan-proteome label that has enabled us to mark different parasite structures.

This study shows a detailed protocol to perform ultrastructure expansion microscopy in three in vitro life cycle stages of Trypanosoma cruzi, the pathogen responsible for Chagas disease. We include the optimized technique for cytoskeletal proteins and pan-proteome labeling.

We describe here the application of ultrastructure expansion microscopy (U-ExM) in Trypanosoma cruzi, a technique that allows increasing the spatial ...

Sexual Transmission of American Trypanosomes from Males and Females to Naive Mates

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Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers (Diethylaminoethyl-cellulose Columns)

Detection of Extravascular Trypanosoma Parasites by Fine Needle Aspiration

Superior Auto-Identification of Trypanosome Parasites by Using a Hybrid Deep-Learning Model

Ultrastructural Expansion Microscopy in Three In Vitro Life Cycle Stages of Trypanosoma cruzi