Bacterial pathogens were found in the Lone Star Tick in Central Nebraska.

A Field Study to Determine the Presence of Rickettsia Bacterial Pathogens in The Lone Star Tick in Central Nebraska.

By David Johnson

Nebraska Academy of Science

Central City High School

Chelle Gillan- Central City High School

This study was performed due to my interest in the outdoor activities, and the time I spend with my family and pets outdoors. I received the original idea for this project from my veterinarian when he gave a presentation involving the Lone Star Tick at an event. The goal of the project was to determine the presence of  dangerous pathogens in my area that have the potential to affect local people. The information found could provide much needed data on both the spread of the tick and the pathogens that it currently carries bringing much needed public awareness. This project can also help in informing the public that the Lone Star Tick is in fact far more of a danger than many other ticks in the US due to its aggressive nature.

Question: Are lone star ticks containing Rickettsia bacterial pathogens present near the Platte River in Merrick and Hall County?

Hypothesis:

The lone star tick will be found in Merrick and Hall Counties, in central Nebraska.

Bacterial pathogens in the Spotted Fever group will be found in Lone Star ticks collected from near the Platte River in Merrick and Hall County.

Independent Variables: location within central Nebraska (county, river system,  particular parks, trails, camps, private land)

Dependent Variables:  Bacterial Pathogens (present or absent)

Twelve by twelve inch tick traps were created out of corrugated cardboard that was lined with duct tape, with half of the sticky side of the tape exposed and facing up.  Ten traps were taken to each of the designated locations at a time of day when it was still a relatively cool temperature (18-27 ℃).  The traps were  placed in a shaded area within leaf cover on the ground 20 feet apart and approximately 90 grams of dry ice was placed on the center of the piece of cardboard. After two hours the traps were collected and the ticks were removed and placed into vials containing 70% ethanol. The vials containing the ticks were stored in a freezer at -18 °C until DNA analysis took place. To accomplish DNA analysis, ticks were transferred to extractor vials containing beads and marked using an identification system that allowed for individual identification of each tick. The vials were placed in a homogenizer  machine, so they could be crushed, allowing easier extraction of the DNA. A digestive solution was then used to further break down the tick, separating the raw DNA from other organic and inorganic material.  Washing solution was added to the vial multiple times to wash the extra organic and inorganic material away from the tick to allow for a purer DNA sample. APCR reaction was run that included a series of cycles that activated the primers and amplified the DNA. Once the PCR reaction was completed a fluorescent dye was added to the sample to allow visualization of the DNA after gel electrophoresis A fresh 96 sample electrophoresis gel was prepared for all of the tick samples. The samples were then placed in the individual gel wells, and the gel was placed on the electrophoresis board. An electrical current was run through the sample to separate the DNA fragments by weight. The gel was placed in  a transilluminator  that used UV rays to make the DNA bands visible. The DNA bands of the gel were  compared to a chart of known pathogen DNA weights to identify which samples contained a tick pathogen.

Figure 1 above depicts the mean, standard deviation, standard error, and the range, maximum and minimum of the number of ticks collected in traps at each location. Each of the locations had ten separate trials for a total of 30 trials. Camp Augustine was found to have the highest number of ticks per trap at an average of 7.6, followed by Bader Park at 1.7 and Dark Island Trail at 0.8 ticks per trap. Camp Augustine exhibited a standard deviation of 30.9, followed by Bader Park at a standard deviation of 3.8, and the Dark Island Trail was the lowest with a standard deviation of 1.7. The standard error of all locations was the same at 1.1. Camp Augustine exhibited the highest range of 14 followed by Bader Park with a range 6 and the Dark Island Trail last with a range of 3.

Figure 2 shows the average number of ticks collected from each location, and the results of the Anova test.  The lone star tick was found to be significantly more prevalent at Camp Augustine than at Bader or the Dark Island Trail with no significant difference between the Dark Island Trail and Bader.

Figure 3 shows the pathogen rate of the lone star tick in three different locations. Each of the locations were tested for the same pathogens, which were spotted fever group, Ehrlichia chaffeensis, Ehrlichia ewingii, and Borrelia lonestari. Bader Park was found to have no pathogens, though it did have the smallest number of ticks collected. One tick collected from the Dark Island Trail tested positive for SFG, which was the most common of the three pathogens.  Camp Augustine had the largest number of samples and the highest pathogen rate. Five of the lone star ticks collected at Camp Augustine were found to have SFG rickettsia. Three separate ticks were each found to have one of the following pathogens: Ehrlichia chaffeensis, Ehrlichia ewingii, Borrelia lonestari.

Figure 4 shows the rate of prevalence of pathogens in the ticks collected during the study. SFG was the most common pathogen found with the other three pathogens Ehrlichia chaffeensis, Ehrlichia ewingii, and Borrelia lonestari all sharing the same rate of prevelance.

Figure 5 shows the number of adult ticks collected at different locations per trap. Camp Augustine shows the overall most consistent number of ticks collected per trap. With the exception of Trap 4, there were ticks collected at every trap at this location. At Bader, ticks were most commonly collected at the traps set closest to the river. The ticks at the Dark Island Trail were collected from below the trail bridge and next to the lakes located along the river.

I would first like to thank Dr. Lee, of the Central City Veterinary Clinic, for helping me with my study idea.  I would also like to thank Dr. Sundstrom and the rest of Dr. Little’s lab staff at Oklahoma State University for sharing their tick expertise with me. I would especially like to thank Dr. Luedtke of the University of Nebraska at Kearney for allowing me to test the ticks for pathogens in his lab and teaching me the steps of PCR. I would also like to thank my mom for help with tick collection. Lastly, I would like to thank my teacher Mrs. Gillan for taking the time to help develop my research plan and other assistance on my paper, poster, and presentation.

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There are several improvements that could be made to the study. I would increase the number of test locations and get assistance so that I could test all locations on the same day. I would also include other species of ticks to get a better understanding of the types of ticks in the local area and the danger that they present. There were three very important findings from this study.  This first was the large abundance of lone star ticks collected from Camp Augustine, which is a Boy Scout camp that serves thousands of guests each year.  The second important finding was the presence of several bacterial pathogens in the ticks that were collected. These pathogens have the potential to cause serious human diseases.  Lastly, and most importantly, according to Dr. Susan Little, of OSU, the SFG pathogen rates in my study of approximately 5%, matched the rates found in studies of other leading edge locations in Kansas and Oklahoma. This is compared to a typical infection rate of approximately 50% in well-established populations. This type of research is very important so that we are aware of dangers we face from pathogens and where they are most heavily located. My study contains the first data of this nature collected in Central Nebraska.