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>

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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. 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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.

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">BCIP and NBT redox system</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:102px;">681 451 001</td>
			<td style="width:65px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Blood DNA Purification columns</td>
			<td style="width:183px;">Amersham Biosciences</td>
			<td style="width:102px;">27-9603-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">d-ATP, [&#945;-32P], 250 &#181;Ci.</td>
			<td style="width:183px;">&#160; Perkin Elmer&#160;&#160;</td>
			<td style="width:102px;">BLU012H</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">DNA, Solution Salt Fish Sperm</td>
			<td style="width:183px;">AMRESCO</td>
			<td style="width:102px;">064-10G</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:34px;width:336px;">dNTP Set, 100 mM Solutions</td>
			<td style="width:183px;">GE Healthcare</td>
			<td style="width:102px;">28-4065-51</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Eco&#160;RI</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:102px;">15202-021</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Goat anti-human IgG- alkaline phosphatase conjugated</td>
			<td style="width:183px;">Southern Biotech</td>
			<td style="width:102px;">&#160;&#160;&#160;&#160;&#160;&#160; 2040-04</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Goat anti-human IgG- FITC conjugated</td>
			<td style="width:183px;">Biocompare</td>
			<td style="width:102px;">MB5198020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Hybond &#8211; N+ nylon membrane</td>
			<td style="width:183px;">GE Healthcare</td>
			<td style="width:102px;">RPN303B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Hybridization oven</td>
			<td style="width:183px;">Thomas Scientific</td>
			<td style="width:102px;">95-0031-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Micro imaging software cell Sens software</td>
			<td style="width:183px;">Olympus, Japan</td>
			<td style="width:102px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Molecular probes labeling System</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:102px;">700-0030</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Nsi I</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:102px;">R5584 1KU</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Plasmid Prep Mini Spin Kit</td>
			<td style="width:183px;">GE Healthcare</td>
			<td style="width:102px;">28-9042-70</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Plate reader&#160;</td>
			<td style="width:183px;">Bio-Tek GmBH</td>
			<td style="width:102px;">2015</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Rabbit anti-chicken IgG-alkaline phosphatase conjugated</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:102px;">A9171</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Rabbit anti-chicken IgG-FITC conjugated</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:102px;">F8888</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Rabbit anti-mouse IgG- alkaline phosphatase conjugated</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td style="width:102px;">A2418&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Rabbit anti-mouse IgG-FITC conjugated</td>
			<td style="width:183px;">Biorad</td>
			<td style="width:102px;">MCA5787</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Spin Columns for radio labeled DNA purification, Sephadex G-25, fine</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:102px;">G25DNA-RO&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Taq DNA Polymerase Recombinant</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:102px;">11615-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Thermal cycler system</td>
			<td style="width:183px;">Biorad, USA</td>
			<td style="width:102px;">1709703</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Vector Systems</td>
			<td style="width:183px;">Promega</td>
			<td style="width:102px;">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 ...

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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

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.

Lyme disease is the most commonly-reported vector-borne disease in North America. The causative agent, Borrelia burgdorferi is a spirochete bacterium transmitted by Ixodid ticks. Transmission and detection of infection in animal models is optimized by the use of tick feeding, which we describe here.

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the  attachment and blood feeding of Ixodes species ticks on ...

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 ...

Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly synthesized allergic, inflammatory and immunoregulatory mediators. MCs&#8217; mediators affect multiple tissues and organs culminating in allergic and immune responses. The synthesis, storage and release of the MC mediators are highly regulated. The pre-formed mediators are packed in cytoplasmic secretory granules&#160;(SG) that fuse with the plasma membrane and release their content by regulated exocytosis. We present a protocol, based on the co-expression of a gene of interest with a reporter gene that is targeted to the SGs and is released in a regulated fashion alongside the endogenous SG mediators. The protocol enables high resolution four dimensional confocal analyses of the MC SGs and monitoring their timeline from biogenesis to triggered exocytosis. Thus, using this protocol for screening genes of interest for their phenotypic and functional impact allows deciphering the molecular mechanisms that govern the biogenesis and exocytosis of the MC SGs and identifying the regulators involved. Thereby, further insights into the cellular mechanisms that account for MCs function in health and disease should be provided.

The goal of the present&#160;protocol was to develop a method that will allow functional genomic analyses of mast cell secretion. The protocol is based on quantitative assessment of the release of a fluorescent reporter gene cotrasfected with the gene of interest and real time&#160;analyses of the secretory granule&#39;s morphology.

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly ...

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, Helicoverpa armigera densovirus (HaDV2), in a lepidopteran species, the cotton bollworm, Helicoverpa armigera (Hubner). Here, we describe the protocols that are currently used to study the effect of HaDV2 on its host. First, we establish a HaDV2-free cotton bollworm colony from a single breeding pair. Then, we orally inoculate some neonate larval offspring with HaDV2-containing filtered liquid to produce two colonies with the same genetic background: one HaDV2-infected, the other uninfected. A protocol to compare life table parameters (e.g., larval, pupal, and adult periods and fecundity) between the HaDV2-infected and -uninfected individuals is also presented, as are the protocols for determining the host-tissue distribution and transmission efficiency of HaDV2. These protocols would also be suitable for investigating the effects of other orally transmitted viruses on their insect hosts, lepidopteran hosts in particular.

Here, we present a protocol to investigate the host-tissue distribution, transmission mode, and effect on the host fitness of a densovirus within a lepidopteran species, the cotton bollworm. This protocol can also be used for studying the interaction between other orally-transmitted viruses and their insect hosts.

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

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. ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...