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

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 free^{1,2,3,4,5,6,7,}

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 hu....

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 individual^3,4,5,

Materials

List of materials used in this article

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