The trypanosoma cruzi agent of Chagas disease may produce long-lasting symptomatic infections that may break open into clinical recognized pathology. The finding of the Trypanosoma cruzi nests in the seminiferous tube of a boy that died of Chagas disease suggests the research protocol to unravel the possible sexual transmission of the Chagas parasites. Deliver health care for the period of five years and recruit the families, showing participants with clinical symptoms of the acute Chagas disease, ACG.
Obtained 15 ml of venous blood of family participants on three occasions one year apart, divided in three, five mil aliquots and store at four Centigrades. Collected two ml of the serum ejaculate from adult volunteers show the T.cruzi in the blood of the ACG cases to validate the molecular assays. Isolate the T.cruzi from the ACG blood aliquot is stored in sterile 10 ml tube with anticoagulant.
Inoculate five ml of the blood into 50 ml tube slants plus five ml of liver infusion tryptose medium and incubate in a shaker at 27 Centigrades for three months. Search microscopy for the T.cruzi in 100 microliter of the supernatant on the glass slide. Harvest the flagellates from the medium centrifuged at 1, 000 x G for 10 min and washed in PBS pH 7.4.
Inoculate 75 ml L6 muscle cell culture flask each with 10 to the sixth T.cruzi isolates. Feed the culture cells with 15 ml of Dulbecco Minimal Essential Medium, DMEM at 37 Centigrades. Grow the parasite isolates and the positive control Berenice T.cruzi.
Grow the T.cruzi relative Leishmania braziliensis in DMEM as a negative control. Use 10 x 10 to the sixth tissue culture derived T.cruzi trypomastigotes to infect mice. Search for the nests of the T.cruzi in the sections of the muscles and organs of reproduction after one month.
Use the blood aliquots in the EDTA Corning sterile 10 ml tube and collect the white cells by density gradient centrifugation at 3, 000 x G for 45 min. Wash the cells twice with PBS and centrifuge at 1, 500 x G for 10 min at a different tube. Extract the DNA from the isolate sent from the Berenice T.cruzi from L.braziliensis from 109 family subjects and from the spermatozoa from 21 participants as in the step 3.3 of the protocol.
Measure the DNA concentrations on 0.8%agarose gel and absorbed at 206 nanometers. Use the T.cruzi TCZ one dash two primers anneal to the 188 nucleotide probe in the PCR analysis. Make the PCR mix with 10 nanogram template DNA, 0.4 micromolar of each pair of primers, two units stock DNA polymerase, 0.2 micromolar DNA-TPs, and 5 micromolar magnesium chloride in a 25 microliter final volume.
Conduct the DNA amplification as in step 3.5.2 and show the DNA bands in the gel. Use the T.cruzi 188 nucleotide probe anneals to date primers label with alpha 32P dATP and separate the PCR product on a 1.3%agarose gel and transfer to a positively charged nylon membrane. Wash the membrane and now expose to X-rays field for variable periods.
Grow the clones selected from the X-rays field and sequence commercially with the TCZ one dash two primers. Conduct a three independent immunofluorescence assays and triplicate one to 100 serum dilution in PBS from 109 study participants. Centrifuge the five ml aliquot of the enclosed blood at 1, 000 x G for 30 min and store the serum at minus 20 Centigrades.
Run independent immunofluorescence serum dilutions from the study samples obtained on three occasions one year apart. Use 5 microliter of the formally incubated T.cruzi amastigotes or L.braziliensis promastigotes, 10 parasites per microliter in PBS suspensions onto glass slide and let it dry overnight, as in step 5.3.3. Incubate the parasite on the glass with 100 microliters of the one to 100 serum dilutions and cover with slip in a moist chamber for one hour at 37 Centigrades, then wash twice with PBS.
Incubate the air-dried glass slides with one to 1, 000 dilution of a fluorescein labeled rabbit antibody to human IgG, wash and dry as well. Mount the glass slide with a cover slip and examine it under the UV light microscope. Detect the T.cruzi and the L.braziliensis soluble antigen one microgram in 100 microliter of carbonate buffer pH 9.6.
Incubate with 100 microliter of the one to 100 serum dilution in triplicate quartered wells. Wash the plates twice with PBS-Tween solutions and wait to dry. Incubate with 100 microliter of the one to 1, 000 dilutions of alkaline phosphatase conjugate, goat anti-rabbit IgG.
Wash the plates twice with PBS-Tween, add the substrate para-nitrophenylphospate and wait for the color development. Read the optical densities at 630 nanometers in a multi-mode plate reader. Transilluminate fertile chicken eggs and make a hole in the shell at their bubble.
Inoculate 100 trypomastigotes on each shell culture medium in the mock control eggs. Seal the hole with adhesive tape. Incubate the eggs at 37 Centigrades, 65%humidity for 21 days.
Grow the chicks hatched from live control eggs group A from the mock controls, group B and from the T.cruzi inoculated eggs, group C until their adult life. Challenge the chickens of group B and C twice with 10 x 10 to the seventh formally incubated trypomastigote weekly. Obtain two ml of the venous blood from a wing vein of the A, B, and C chickens five weeks after challenge.
Separate the serum and run immunofluorescence analyzer to detect the T.cruzi antibody. Secure animal welfare with excellent housing, food, and water supply. Use the semen ejaculate from an adult, and a APCR positive Chagas disease case, and from a control subject as in step 1.5 of the protocol.
Instill 100 microliter aliquots of semen into the peritoneal and into the vagina of mice. The sexual transmission of the T.cruzi infection in parental and progeny mice. The footprint of the Trypanosoma cruzi from the acute Chagas disease showed by the nDNA-PCR products that form in advance with the radio labeled probe.
The phenotype of the T.cruzi revealed with the immunofluorescence assay. The anti-T. cruzi antibody does not recognize L.braziliensis.
Graphic representation of the ELISA and of the nDNA-PCR assays. Groups one and two respectively, negative and positive control. Group three, positive nDNA-PCR and the specific antibody assays.
Group four with the T.cruzi infections detected by the nDNA-PCR but in the absence of the specific antibody. Group five, infection free participants. The heredograms and mapping of the family population showed discrepancies among the ratios of the anti-T.
cruzi antibody and those of the nDNA-PCR assays. Open is square and circle, negative male and female. Red with anti-T.
cruzi antibody and nDNA-PCR positive. Black, positive nDNA-PCR alone. The immune tolerance in chickens hatched from T.cruzi inoculated eggs.
A, mock control, B, live controlled chickens challenged with the T.cruzi antigen. C, chickens hatched from the T.cruzi inoculated eggs after challenge with the specific antigen. The infectivity of the T.cruzi showed upon semen instills into the peritoneal cavity and into the vagina.
The mice sacrificed three weeks of these stills showed the amastigote nest in the heart, in the skeletal muscle, and in the vas deferens and in the uterine tube. The sexual transmission of the T.cruzi infections in the mouse model system. The T.cruzi infected males and females, transmitted the infection to naive mates.
The T.cruzi infection vertically transferred from the parental to the progeny mice. The Southern blotting revealed the parasite anginate band in a majority of the F1 litters. The immunoperoxidase stain T.cruzi amastigotes showed engoniablast and in the interstitial of the epididymitis and into the lumen of seminiferous tubes of the progeny mice.
The parasitological demonstration of the protozoan in 21 acute cases of Chagas disease was crucial to validate the T.cruzi footprint in all the infected subjects. The discrepancies among the ratios of the parasite antibody assays and those of the nucleic acids tests revealed the majority of the footprints in the antibody-free patients. The broad difference well explains in the chicken model reflected to the T.cruzi after the first week of the embryo development.
The immune tolerance adult chickens showed the parasite footprints but with the absence of the specific antibody. The vital anti-cruzi in Chagas patient ejaculates initiated widespread infections upon instills into the mouse vagina and as well into the peritoneal cavity. The pathology showed the T.cruzi amastigote nests in the heart and skeletal muscles and in the vas deferens and the uterine tube.
The breeding of Chagas mice with naive mates revealed the infected females and males transmitted the T.cruzi to the uninfected naive mates and the majority of their litters acquired the infection vertically transferred to the progeny. The family-based protocol addressed the nucleic acids assay in order to perform reliable blood transfusions and epidemiology inquiries and to elucidate the potential ongoing sexually transmitted Chagas disease.
