Prevalence and Coinfection of Three Tick-Borne Pathogens in Questing Adult Blacklegged Ticks Ixodes scapularis (Vilas County,Wisconsin)

Introduction

I xodes scapularis (i.e.,the blacklegged tick or deer tick) is an obligate blood-feeding ectoparasite that harbors multiple pathogens that cause disease, including Lyme disease (Steiner et al. 2008). Lyme disease is the most commonly reported tick-borne disease in Wisconsin, where its incidence has increased severalfold since the early 1990s (Kugeler et al. 2015). In addition to Borrelia burgdorferi (the Lyme disease spirochete), blacklegged ticks harbor additional pathogens. These include Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis (HGA), and Babesia microti, the causative agent of babesiosis. All three pathogens are capable of coinfecting a single tick, and can cause influenza-like symptoms in infected individuals with an onset of 1–3 weeks after initial infection (Steiner et al. 2008). The similarity of symptoms at the onset of infection, coupled with the lack of awareness of tick-borne pathogen diversity, can lead to mis- or underdiagnosis (Steiner et al. 2008).

We estimated the prevalence of adult questing ticks carrying zoonotic pathogens in Vilas County, a popular outdoor recreation destination in Wisconsin. The blacklegged tick population probably established in the early 2000s (Lee et al. 2013), so this study represents the first screening of a novel population. We performed PCR assays to detect B. burgdorferi sensu lato, A. phagocytophilum, and B. microti. In addition, we estimated coinfection prevalences, as coinfection has been shown to increase the severity of illness in infected individuals (Hersh et al. 2014, Diuk-Wasser et al. 2016).

Materials and Methods

We collected adult questing blacklegged ticks from June 5 to 8, 2016, in Vilas County, Wisconsin, near the town of Boulder Junction (46°7′ N, 89°4′ W). Only adult stage ticks were collected as nymphal and larval stages were not largely present in the population at the time of collection. We used a standard tick-drag method along grassy hiking trails in forest vegetation and stored ticks at ambient temperature in vials filled with 70% ethanol. DNA extraction was performed using a DNeasy^® Blood and Tissue Kit (Qiagen, Valencia, CA). Primers for each pathogen were adopted from the literature (Prusinski et al. 2014, Table 1). Each 25 μL PCR reaction contained 4 μL of DNA template and 1.25 μL (10 nM) of each forward and reverse primer, 12.5 μL 2 × Promega Go Taq™ Master Mix, and 6 μL ddH₂O. Amplification conditions included denaturing for 5 min at 95°C, followed by 35 cycles of denaturation at 95°C for 30 s, primer annealing at 60°C for 30 s, extension at 72°C for 60 s, and a final extension step for 10 min at 72°C (Eppendorf Mastercycler^®, Hauppage, NY). Amplification products were then run on 2% agarose gels stained with SYBR^® Green. Presence or absence was determined by visualizing gels under ultraviolet light; the presence of a band indicated a positive infection.

We determined the infection prevalence for B. burgdorferi, A. phagocytophilum, and B. microti by calculating the ratio of individual ticks testing positive to the total number of ticks tested (n = 461). Coinfection prevalence was determined by calculating the ratio of each coinfection scenario to the total sample. We computed 95% confidence intervals using Wilson's method (Brown et al. 2001) for each estimate of infection prevalence.

Results and Discussion

The probability that a black-legged tick contained any of the three pathogens examined was 47.9–57.1%. The singular infection prevalence was 14.2–20.5% for B. burgdorferi, 11.4–17.2% for A. phagocytophilum, and 4.6–8.4% for B. microti.

The probability that a tick contained at least two pathogens was 11.4–17.2%. Coinfection prevalence was 4.6–8.4% for B. burgdorferi and A. phagocytophilum, 1.5–3.7% for A. phagocytophilum, and B. microti, and 2.1–4.8% for B. burgdorferi and B. microti. The probability of tick coinfection with all three pathogens was 0.9–2.6%.

Our observed infection prevalence of B. burgdorferi is about half the estimated infection prevalence 30.0–35.7% in the state of Wisconsin (Lee et al. 2014, Turtinen et al. 2015). However, our observed infection prevalence of A. phagocytophilum was two- to threefold greater than the estimated infection prevalence of 5.4% observed in blacklegged ticks collected from northern Wisconsin counties (Murphy et al. 2017). Although our findings vary from previously reported averages for both B. burgdorferi and A. phagocytophilum, they fall within observed spatial variation (Lee et al. 2014, Turtinen et al. 2015, Murphy et al. 2017). The infection prevalence of B. microti was in line with the estimated infection prevalence of 5% observed in southwestern Wisconsin blacklegged ticks (Kowalski et al. 2015).

Conclusion

Coinfected ticks can vector multiple pathogens and exacerbate symptoms of tick-borne infections (Hersh et al. 2014). Indeed, studies examining coinfection in ticks are lacking, and since coinfection is regarded as the rule rather than the exception (Moutailler et al. 2016), our study represents a valuable contribution to tick-borne pathogen reporting efforts. This is made especially important as our study population is recently established, indicating that even novel populations are capable of harboring pathogens at rates equal to, or higher than, neighboring populations (Kowalski et al. 2015, Murphy et al. 2017).

Finally, although awareness of Lyme borreliosis by physicians and patients has grown in recent decades, HGA and babesiosis are not as well recognized. As a result, patients with influenza-like symptoms after a tick bite will test negative for Lyme borreliosis if they have contracted HGA or babesiosis. Additional blacklegged tick-borne pathogens may also be harbored by Wisconsin tick populations, yet go largely undetected due to low rates of surveillance. For example, one recent study screened individuals who presented with Lyme borreliosis in Wisconsin for Powassan virus and found high rates of coinfection (Frost et al. 2017). Our results show that coinfection is common in our study area and, if not properly recognized, leads to a greater risk of underdiagnosis. The use of diagnostic tick-borne pathogen panels, rather than an individual pathogen test, is warranted in patients with suspected tick exposure in regions where coinfection is known or suspected.