Abstract Results

Borrelia burgdorferi in Ixodes scapularis adult female ticks, Wisconsin 2010-2013Alyssa Kruger & Dr. Lloyd Turtinen

Department of BiologyUniversity of Wisconsin - Eau Claire

Our four-year study surveyed questing adult female Ixodes scapularis tick populations from 21 west-central Wisconsin counties for the presence of Borrelia burgdorferi bacteria. This bacterium, which is transmitted by a tick bite, is responsible for Lyme disease in humans and other animals. Students in Biology 306 (Infectious Disease Ecology) and faculty in the Biology Department have collected ticks by sweeping methods or visual detection on clothing. DNA was extracted from individual ticks and processed by a real-time polymerase chain reaction (qPCR) to detect the B. burgdorferi rec A gene. Ticks positive for the bacteria were identified by the presence of a 222 base pair DNA product with a melting temperature (Tm) of 82°C. The DNA size was verified by gel electrophoresis. Some 222 base pair products were also sequenced to verify that they were the rec A gene. Three hundred forty-one ticks were assayed, and 35.7% of the ticks carried the bacterium. Generally speaking, the percentage of tick carrying the bacteria steadily increased year to year. In Eau Claire County, the percentage of ticks assayed and found to carry the bacterium changed from 27.8% in 2010 to 51.2% in 2013. The significance of these findings will be discussed.

Background

Red- Positive Control Green- Positive Sample Blue- Negative Control

Fluorescence vs. Cycle # Fluorescence vs. Temp.

Fig 4. A female deer tickhttp://bugguide.net

DNA Extraction1. Tick homogenization:

• Buffer• SDS• Proteinase K

2. DNA extraction:• Phenol• Chloroform

3. Ethanol precipitation

Funded provided by Faculty Student Research Collaboration ORSP Grants.

References

Lyme disease is a bacterial infection transmitted to humans through a deer tick bite. The most common indication of an infection is a red bulls-eye shaped rash, referred to as Erythema migrans, although this is absent in 25% of infections. Later symptoms include fever, chills, fatigue, muscle and joint aches. If left untreated, chronic arthritis and/or neurological problem can develop.1  As the number of Lyme disease cases increases, it is important to understand the ecology of the disease.2 Surveys have been conducted in other countries and regions of the United States. For example, studies conducted in Germany and in New Jersey found 35% and 43% of ticks, respectively, to be carriers of the bacteria.3,4,5 However, few to no reported studies have been conducted in Wisconsin.  Our survey was conducted using ticks collected between spring and early fall in 2010, 2011, 2012 and 2013 (Fig 1-3). To determine if a tick was carrying the bacterium, we performed DNA extraction and qPCR to amplify the rec A gene DNA. Tick samples containing DNA with a Tm of 82°C were presumed positive and verified through agarose gel-electrophoresis by the presence of a 222 bp DNA fragment representing the amplified region of the rec A gene (Fig. 4-8). Random samples were sequenced to compare to the rec A sequence (Fig. 9-10).

Materials and MethodsDNA Amplification

Fig 5. Illustration of qPCR events

Fig 6. Fluorescence increases as SYBR green binds to newly synthesized dsDNA. SYBR green only fluoresces when bound to dsDNA.

Fig 7. The Tm is determined by taking the derivative of the fluorescence as the temperature increases. The decrease in fluorescence is due to the separation of dsDNA. Tm (76°C)= primer dimers, Tm

(82°C)= product.

Agarose Gel-Electrophoresis

Fig 8. Visualization of 222 bp qPCR products separated by electrophoresis. Fragments less than 100 bp in length are the result of primer dimers. Samples 1-10 are positive for product. Highlighted samples were sent in for sequencing. An additional sample, not shown, was also sequenced.

Fig. 9 (left).6 Blast search of positive control number 1, B. burgdorferi sensu stricto strain B31 DNA (ATCC 35210), (Query). Pairwise alignment with known B. burgdorferi rec A gene sequence(sbjct).

Fig. 10 (right).7,8,9 Multiple alignment of four random sanger sequenced products. Rows 1 and 2 are positive controls, 3 and 4 are random tick samples. Purines, A & G, are shown in pink, pyrimidines, T & C, are shown in green. N indicates the base’s identity was not determined. A “-” indicates a missing base compared to other sequenced samples.

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Yearly Prevalence of Borrelia burgdorferi in Adult Female Ticks

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Analysis and Conclusions The average prevalence of ticks carrying the B. burgdorferi bacteria from the years 2010 to 2013 was 35.7%. Sequencing confirmed rec A identity of products.

The Central region (Chippewa, Clark, Marathon and Eau Claire Counties) had the highest prevalence: 79/203 (38.9%) during this 3-year period. During this time period, individual counties with the highest prevalence rates were Chippewa 12/18 (66.7%), Dunn 24/54 (44.4%) and Eau Claire 62/170 (36.5%). Chippewa country had a significantly higher prevalence rate compared to Eau Claire County (p=0.0251) (Pearson’s Chi squared test with Yates’ continuity correction).

There was an increase in prevalence from year to year. In 2010, the prevalence from all regions was 11/51 (21.6%) and in subsequent years increased to 32.4% (2011), 40.9% (2012) and 51.2% (2013). When data from only regions with similar 3-year prevalence rates (Central, Southwest, West Central) were compared to normalize tick sampling area bias, it showed a significant increase in prevalence between years 2010 and 2013 (p=0.0165). However, due to inconsistent sampling (sampling design), the significance of these findings remains questionable.

1. Centers for Disease Control and Prevention, (2013). Lyme disease. Retrieved from website: http://www.cdc.gov/lyme/signs_symptoms/index.html2. Lyme Disease. http://www.dhs.wisconsin.gov/communicable/Tickborne/Lyme/DataandStatistics.htm3. Pietila , J. et. al. Journal of Clinical Microbiology. 4. Rauter, C. et. al. (2002) Journal of Clinical Microbiology. Vol. 40 No. 15. Varde, S. et. al. (1998) Emerg Inf. Dis. Vol. 4 No. 16. Altschul S.F. et. al. (1990) J. Mol. Biol. Vol. 215 No. 37. Waterhouse, A.M., et. al. (2009) Bioinformatics Vol. 25 No. 98. Larkin M., et. al. Bioinformatics (2007) Vol. 23 No. 219. Goujon M., et. al. Nucleic acids research (2010) Vol. 41

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Prevalence of Borrelia burgdorferi in Adult Female

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2010-2013 Overall Prevalence

Figure 3. Overall prevalence rate of ticks harboring Borrelia burgdorferi, the Lyme disease causing bacteria, from 2010 through 2013. * number of ticks harboring B. burgdorferi, + total number of sampled ticks, § decimal.

Sequencing Data

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Fig 1. The number of carrier and non-carrier ticks in 2010, 2011, 2012 and 2013. Southwest- Buffalo, Trempealeau, La Crosse, Vernon, Richland. Northern-Bayfield, Sawyer, Washburn, Vilas, Oneida. West Central- Polk, Barron, Dunn, St. Croix , Pierce. Eastern- Brown, Manitowoc. Central- Chippewa, Clark, Marathon, Eau Claire.

Fig 2. The number of carrier and non-carrier ticks from 2010-2013. Southwest- Buffalo, Trempealeau, La Crosse, Vernon, Richland. Northern-Bayfield, Sawyer, Washburn, Vilas, Oneida. West Central- Polk, Barron, Dunn, St. Croix , Pierce. Eastern- Brown, Manitowoc. Central- Chippewa, Clark, Marathon, Eau Claire. Overall prevalence from all regions, 2010-2013, is 35.7%

2010 2011 2012 2013

Bayfield0/3 (0.0)

Manitowoc1/8 (0.13)

Sawyer1/1 (1.0)

Washburn1/4 (0.25)

Vilas 1/3 (0.33)

Oneida0/1 (0.0)

Brown0/2 (0.0)

Barron2/11 (0.18)

Polk0/1 (0.0)

St. Croix0/3 (0.0)

Dunn24/54 (0.44)

Pierce0/4 (0.0)

Chippewa12/18 (0.67)

Eau Claire62*/170+ (0.36)§

Clark2/5 (0.4)

Marathon3/10 (0.3)

Buffalo4/4 (1.0)

Trempealeau 0/4 (0.0)

La Crosse 5/7 (0.71)

Vernon2/23 (0.09)

Richland2/5 (0.4)

30.2%

25.0%

35.6%