Identifying Chagas Disease Reservoirs with PCR and Next-generation DNA Sequencing

For this project, students Nicolas Means from Oklahoma State University and Darlisha Owens from Grambling State University, teamed up with Dr. Travis Glenn in the Department of Environmental Health Science and Dr. Nicole Gottdenker in Veterinary Medicine to use next-generation DNA sequencing to identify disease reservoirs.

Nicolas J. Means1, Darlisha Owens2, Troy Kieran3, Travis C. Glenn3, Nicole Gottdenker3

1Oklahoma State University, 2 Grambling State University

3University of Georiga

American trypanosomiasis (Chagas Disease) is a zoonotic vector-borne disease caused by the protozoan parasite Trypanosoma cruzi,  and is an important cause of morbidity and mortality in Latin America. T. cruzi circulates between reservoir hosts (wild and domestic mammals) and hematophagous triatomine insect vectors. Humans are susceptible to the disease once infected with the parasite by contact with the infected insect vector, ingestion of food or drink contaminated with the pathogen, transplacental transmission, or by transfusion with infected blood or tissue transplants. Research has shown that blood meal analysis, via standard PCR and sequencing, are capable of identifying host reservoirs down to the species level, but these techniques are limited because: 1) they often cannot identify multiple blood meals within a vector, 2) they cannot be used to simultaneously detect vector infection with trypanosomes or coinfection with other pathogens, and 3) they may require a relatively large amount of vertebrate reservoir DNA, which may be degraded in the insect vector. The objective of this study is to standardize next generation sequence methodologies for simultaneous blood meal species identification and trypanosome infection within kissing bugs, Rhodnius pallescens, a triatomine vector of Chagas disease.

To prepare samples for next generation sequencing, we had to quantify the amount of DNA extracted from R. pallescens, normalize the DNA concentrations, and optimize the PCR conditions for each portion of the next generation Taggimatrix technique.  The samples were put in three different groups consisting of high, medium, and low concentration DNA.  For optimization of the PCR, there were a series of tests with a known insect and vertebrate. Conditions such as number of cycles, temperature and time changed throughout each experiment. We then used the optimized PCR conditions on the DNA from the three groups (high, medium and low) to obtain amplification of vertebrate and trypanosome DNA. From the tests, we found that 30% of the new samples that were collected had the trypanosome parasite within the DNA of the insect while 68% showed vertebrate DNA within the blood meal.

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