Microbial Community Assessment of Lone Star Ticks from Athens, GA

Sydney Barosko, a student from Michigan State University, worked in the lab of Dr. Travis Glenn to examine pathogen diversity in local lone star ticks.

Abstract: In the world of infectious diseases, ticks play an important role as a vector in transmitting pathogens to humans, companion animals, livestock, and wildlife. Amblyomma americanum (Lone Star ticks) are known to transmit the pathogens that cause ehrlichiosis, babesiosis, Q fever, and rickettsial diseases. Few studies have been done on the pathogen diversity of Lone Star tick individuals. We used microbial 16S amplification followed by Illumina sequencing of the 16S amplicons to characterize microbial communities in A. americanum collected near Athens, GA. We examined differences in 16S sequences:  1) when two different Taq DNA polymerases were used for amplification (one with high fidelity and the other with more tolerance for low-quality samples and primer mismatches) and, 2) between 19 male and 18 female A. americanum ticks. We focused on three genera of microbes with known pathogenic strains:  Coxiella, Rickettsia, and Ehrlichia. We did not find any significant differences between the communities when amplified with the different Taq DNA polymerases nor in the infection rate of males vs. females infected with Ehrlichia or Rickettsia.  Male vs. female infection rates did, however, differ for Coxiella. The proportion of Coxiella in the microbiome was much higher in females than in males. Our work demonstrates that 16S microbiome sequencing can be an effective tool in characterizing pathogens in ticks and builds a foundation for larger-scale surveys in the future.


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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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