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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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 chagasic mother to her offspring, but the male partner'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. 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.

Introduction

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'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's transfer of the parasite to the offspring in approximately 10% of pregnancies15; the male partner's contribution to in utero 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 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.

Protocol

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 nº 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

  1. Ensure that the research team participates in a Health System Chagas Disease Program to deliver health care to Chagas patients.
  2. 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.
  3. Deliver health care to the people in the study families for a period of five years.
  4. 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 °C.
  5. Collect 2 mL of the semen ejaculates from adult volunteer family members and proceed as described in step 3.3.

2. Growth of parasites

  1. 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.
    1. 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 °C for 3 months.
    2. Place 100 µL 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.
    3. Harvest the epimastigote isolates in the supernatant of axenic LIT medium at 27 °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's-modified essential medium (DMEM).
    4. Inoculate the confluent L6 muscle cell culture flasks with 1 x 106 ECI1-to- ECI21 T. cruzi epimastigote isolates.
    5. Grow the T. cruzi ECI1-to-ECI21 and the Berenice archetype trypomastigotes in L6 muscle cell cultures in 75 mL culture flasks.
    6. Feed cells with 15 mL of DMEM at pH 7.4 supplemented with 5% fetal bovine serum, 100 IU/mL penicillin, 100 µg/mL streptomycin, 250 nM L-glutamine, and 5% CO2 at 37 °C1.
    7. Use the positive control T. cruzi Berenice trypomastigotes to phenotype the ECI1-to-ECI21 isolates from tissue culture, as described in step 5.2.
    8. Grow the negative control Leishmania braziliensis27 in DMEM supplemented with 20% fetal bovine serum only, and use the parasite promastigotes as a negative control.
    9. Use 1 x 106 T. cruzi ECI1-to-ECI21 trypomastigotes in the supernatant from the cell culture to infect mice.
    10. Search for T. cruzi trypomastigotes in the tail blood after the first week of the infection.
    11. 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.

3. DNA extraction and PCR analyses

  1. 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.
  2. 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), and from the test blood mononuclear cells of 109 human family study subjects1,27,28.
  3. 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 °C, recover spermatozoa from the supernatant after centrifugation for 5 min at 13,000 x g, and extract the haploid DNA23.
  4. Place 1 mL of the cells in extraction buffer (10 µM NaCl, 20 µM EDTA, 1% SDS, 0.04% proteinase-K, and 1% dithiothreitol), and mix the solutions by inversion and shaking.
    1. Centrifuge the solutions for 10 min at 13,000 x g and 25 °C, and transfer the viscous supernatant to a spin column.
    2. Centrifuge the spin column for 1 min at 10,000 x g and discard the eluate; add 500 µL binding buffer to the spin column (Table of Materials), centrifuge it for 1 min at 12,000 x g, and discard the eluate.
    3. Add 600 µL washing buffer to the spin column, centrifuge it for 1 min at 12,000 x g, and discard the eluate.
    4. Repeat this step twice, and transfer the spin column to a sterile 1.5 mL micro centrifuge tube.
    5. Add 100 µL 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.
    6. 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 °C until use in the PCR analysis.
  5. 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' blood and sperm, from the Berenice T. cruzi positive control and from the L. braziliensis negative control.
    1. Prepare the PCR mixture with 10 ng template DNA, 0.4 µM of each pair of primers, 2 U of Taq DNA polymerase, 0.2 µM dNTPs, and 15 µM MgCl2 in a 25 µL final volume.
    2. Initiate the DNA amplification program at 94 °C for 30 s to denature the template, and cool the samples to 55 °C for 30 s. Then, incubate the samples at 94 °C for 90 s to extend the annealed primers. Return the temperature to 94 °C for 30 s to initiate the next cycle, and incubate the samples an additional 3 min at 72 °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 °C29.
    3. Amplify a T. cruzi DNA telomere repeat sequence annealed to the Tcz1/2 primers at both extremities.
    4. Analyze the amplification products on a 1.3% agarose gel and observe the 188-nt DNA bands on a UV-illuminator.

4. Southern hybridization

NOTE: Southern hybridization was used to discard most of the false positive PCR amplicons in the agarose gel.

  1. 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.
  2. Employ the T. cruzi 188-nt DNA-specific telomere sequence probe annealed to the Tcz1/2 primers shown; label the probe with [α-32P] 2'-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 °C.
  3. Transfer the gel to a positively charged nylon membrane using the capillary method overnight.
  4. Hybridize the DNA bands transferred to the nylon membrane with the radiolabeled 188-nt probe, which binds the EcoR1 digests of the genomic DNA in 25 µL of the enzyme-specific buffer for variable periods.
  5. Wash the membrane twice for 15 min at 65 °C with 2x SSC and 0.1% SDS.
  6. Expose the X-ray films to the nylon membrane and autoradiograph the bands on the membrane for one week.
  7. 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.
  8. Commercially sequence the clones using the Tcz1/2 primers set annealed to the T. cruzi-specific telomere footprint2,23.

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.

  1. 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.
  2. Perform IIF and ELISA in triplicate 1:100 serum dilutions to detect the T. cruzi and L. braziliensis antigens as described previously28.
  3. IIF
    1. Conduct the IIF assay in triplicate serum dilutions of the 109 study subjects' samples obtained on three occasions one year apart.
    2. Place 5 µL 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 °C until use.
    3. Place 20 µL of the patients' serum dilutions on top of glass slides coated with 5 µL of the formalin-killed (10 parasites/µL) T. cruzi epimastigotes or L. braziliensis promastigotes.
    4. Incubate the glass slide covered with a slip for 1 h in a moist chamber at 37 °C and wash thrice with PBS.
    5. 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 °C; wash and dry the slide.
    6. 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.
  4. ELISA
    1. Run an ELISA to detect the T. cruzi and the L. braziliensis soluble antigens (1 µg/100 µL in 0.1 M carbonate buffer, pH 9.6) on coated microplate wells.
    2. Incubate the 1:100 serum dilutions in triplicate coated wells for 2 h at room temperature.
    3. Wash the plates thrice with PBST (PBS containing 0.5% Tween-20), pH 7.4, solution before drying.
    4. Incubate the plates with 50 µL of a 1:1,000 dilution of rabbit anti-human IgG antibody for 90 min at 37 °C.
    5. Wash the plates thrice with PBST solution and allow them to dry at room temperature.
    6. Incubate the plates with 100 µL of 1:1,000 dilutions of alkaline phosphatase-conjugated goat anti-rabbit IgG (Table of Materials) for 90 min at 37 °C.
    7. Wash the plates thrice with PBST, add the substrate p-nitro phenyl phosphate, and wait for color development.
    8. Read the ODs at 630 nm in a multimode plate reader.
    9. Run the triplicate dilutions of the test serum from the study population and plot the ODs to identify the specific T. cruzi antibody titers.

6. Assessments of immune tolerance

NOTE: A chicken model system was used to test T. cruzi infections after the first week of embryo development.

  1. Inoculate chicken fertile eggs with 100/10 µL T. cruzi trypomastigotes harvested from tissue culture medium; inoculate mock control eggs with 10 T. cruzi trypomastigotes per µL culture medium. Seal the hole with tape.
  2. Incubate 20 T. cruzi-infected eggs and an equal number of mock control fertile eggs at 37 °C and 65% humidity for 21 days.
  3. Keep the chicks that hatch in the incubator for 24 h and, thereafter, at 32 °C in hoods with temperature control for three weeks.
  4. 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 °C, in individual cages placed in separate aisles.
  5. 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.
  6. 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.
  7. Use the chicken serum to detect the specific T. cruzi antibody by IIF and ELISA as described in step 5 of the protocol.

7. Infection of mice with T. cruzi from Chagas patients' semen ejaculates

  1. Use the semen ejaculates from an adult PCR+ Chagas disease patient (step 1.5) and from an adult PCR- Chagas-free individual.
  2. Use two groups of 12 one-month-old BALB/c naive mice kept in hoods under positive air pressure at 24 °C and fed food ad libitum.
  3. Instill the human Chagas-positive semen aliquots (100 µL) into the peritoneum and equal amounts into the vagina of group-A mice.
  4. Instill the control Chagas-free semen aliquots (100 µL) into the peritoneum and an equal amount into the vagina of group-B mice.
  5. Sacrifice the experimental mice under anesthesia five weeks after the semen instillations and subject the tissue sections to staining with hematoxylin-eosin.
  6. Use microscopy to search for T. cruzi trypomastigotes and amastigotes in the heart, skeletal muscle, and reproductive organs in groups of mice.

8. Transmission of the T. cruzi infection by intercourse

  1. Use 10 male and female six-week-old BALB/c mice in the experiments.
  2. Inoculate five male and five female mice intraperitoneally with 1 x 105 T. cruzi trypomastigotes from the tissue culture.
  3. 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.
  4. House each breeding pair in one cage placed inside a safe box with a 5 mm grid and lock-in door to prevent escape.
  5. 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.
  6. 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.

9. Assessment of immune privilege

  1. Obtain tissues from T. cruzi-infected and naive control mice (step 8.6), and cut the paraffin-embedded samples into 4 µm thick sections.
  2. 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.
  3. Incubate the tissue sections with 0.05% saponin once, followed by three distilled water washes at room temperature.
  4. 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.
  5. 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.
  6. Rinse the slides in PBST and add 100 µL of 3,3' diaminobenzidine for a 5 min incubation, followed by three washes with PBST and counterstaining with Harris hematoxylin for 30 s.
  7. 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.
  8. Examine the slides with a bright-field light microscope, and capture photo images with a microcamera with software and an analyzer program.
  9. Document the immune privilege of the parasite in the absence of inflammatory reactions in the reproductive organs.

10. Statistical analyses

  1. Use Biomedical Edit for sequence analysis, perform alignments with BLAST, and determine the E-value statistical significance (p < 0.05).
  2. Perform a one-way analysis of variance (ANOVA) and the Tukey test to compare the OD means plus or minus standard deviations.

Results

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...

Discussion

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,

Disclosures

The authors declare that they have no competing financial interests.

Acknowledgements

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.

Materials

NameCompanyCatalog NumberComments
BCIP and NBT redox systemSigma-Aldrich681 451 001
Blood DNA Purification columnsAmersham Biosciences27-9603-01
d-ATP, [α-32P], 250 µCi.  Perkin Elmer  BLU012H
DNA, Solution Salt Fish SpermAMRESCO064-10G
dNTP Set, 100 mM SolutionsGE Healthcare28-4065-51
Eco RIInvitrogen15202-021
Goat anti-human IgG- alkaline phosphatase conjugatedSouthern Biotech       2040-04
Goat anti-human IgG- FITC conjugatedBiocompareMB5198020
Hybond – N+ nylon membraneGE HealthcareRPN303B
Hybridization ovenThomas Scientific95-0031-02
Micro imaging software cell Sens softwareOlympus, Japan
Molecular probes labeling SystemInvitrogen700-0030
Nsi ISigma-AldrichR5584 1KU
Plasmid Prep Mini Spin KitGE Healthcare28-9042-70
Plate reader Bio-Tek GmBH2015
Rabbit anti-chicken IgG-alkaline phosphatase conjugatedSigma-AldrichA9171
Rabbit anti-chicken IgG-FITC conjugatedSigma-AldrichF8888
Rabbit anti-mouse IgG- alkaline phosphatase conjugatedSigma AldrichA2418 
Rabbit anti-mouse IgG-FITC conjugatedBioradMCA5787
Spin Columns for radio labeled DNA purification, Sephadex G-25, fineSigma-AldrichG25DNA-RO 
Taq DNA Polymerase RecombinantInvitrogen11615-010
Thermal cycler systemBiorad, USA1709703
Vector SystemsPromegaA1380

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