Detection of zoonotic human pathogens in Ixodes scapularis in Wisconsin-2017

Ixodes scapularis, more commonly known as the blacklegged tick, is the primary vector for tick-borne diseases (TBD) in the upper Midwestern United States. It is the most important vector for Borrelia burgdorferi s.s. (primary agent of Lyme disease), Borrelia miyamotoi (tick-borne relapsing fever), Anaplasma phagocytophilum, Powassan virus, and Babesia microti, each of which represents a significant risk to human health (Lee et al. 2014, Hahn et al. 2018, Johnson et al. 2018). Over the last decade, the incidence of TBD has increased across the United States, and new tick-borne human pathogens continue to emerge (Eisen and Eisen 2018). In the upper Midwest, Borrelia mayonii and Ehrlichia muris eauclairensis represent two human pathogens only recently identified; both are vectored by I. scapularis (Pritt et al. 2009, 2016a, 2016b). Transmission of multiple pathogens by a single vector may result in co-infections that complicate our understanding of pathogen fitness, transmission success, and disease diagnosis (Grab et al. 2007). Given the limited data on the prevalence of TBD in I. scapularis throughout the upper Midwest, this cross-sectional study aimed to further investigate the presence of human pathogens in I. scapularis ticks.

Tick sampling occurred in two distant geographic regions of in Wisconsin with established populations of I. scapularis ticks; site one (WSH) was located on a private property in Washburn County in an oak dominate forest (45.741270, −91.482142), and site two (WAL) was located within a red pine stand in the South Kettle Moraine State Forest in Walworth County (42.835775, −88.617064). Questing I. scapularis ticks were collected by drag sampling the forest understory with a 1 meter² white flannel cloth attached to a wooden dowel. Sampling was conducted until a minimum of 100 nymphal ticks were collected from each location. All ticks collected were visually identified by lifecycle stage and sex in the field, and subsequently placed in RNAse inhibitor (WSH) or ethanol (WAL) for transport to the laboratory for further identification and processing.

Individual WSH I. scapularis ticks underwent mechanical disruption with a combination of 0.1 and 2.4 mm zirconium oxide beads while nymphs from WAL were isolated in 1.7mL centrifuge tubes and then bisected using an 18 gauge PrecisionGlide needle (Becton, Dickinson and Company, Franklin Lakes, NJ). Nucleic acid extraction was performed after ticks were mechanically disrupted (WSH: Magna Pure LC Total nucleic acid Kit, Roche, Indianapolis, IN; WAL: Bioline Isolate II Genomic DNA Kit, Meridian Life Science, Inc., Memphis, TN). Extracted nucleic acids were then tested using previously described real-time PCR assays for B. burgdorferi s.s. (oppA2 gene: Pritt et al. 2016a), A. phagocytophilum/Ehrlichia spp. (groEL gene: Pritt et al. 2011), and Ba. microti (18S gene: Burgess et al. 2017). Extracted nucleic acids were also tested using real-time fluorescent resonance energy transfer (FRET) PCR assays for B. miyamotoi (GlpQ: Forward primer 5’-TCCAGAACATACCTTAGAAGC- 3’; reverse primer 5’-ATCAAATCTTTCACTGAGACTTA-3’; fluorescein-labeled probe 5’-GACAATGTTCCTATTATAATGCACGACCC-fl-3’; 5’-LC640-GAAATTGACACAACCACAAATGTTGCAC-3’), and Powassan virus (NS5: forward primer 5’-ACTAGAATGGCCATGACAGAC-3’; reverse primer 5’-TCATCTCTGGTGCACATCC-3’; fluorescein-labeled probe 5’-CACAAAGGCCCAGGAACCACAGC-fl-3’; red labeled probe 5’-LC640-GGCACCAGIGTGATCATGAGAGCIGT-3’); (WSH site only because ticks were stored in ethanol at WAL). All assays were performed on the Roche LightCycler 2.0 or 480 instruments and utilize FRET probes for detection and identification of the amplified nucleic acid. A total of 239 I. scapularis nymphs and 36 adults were collected and tested. WSH contributed 115 nymphs and 36 adults and WAL contributed 124 nymphs. Five additional adult ticks were collected from WAL but were not tested due to the low sample size.

Testing identified infection with at least one of the six bacterial/protozoan pathogens in 15 of the 36 adult ticks (42%) tested from the northwestern location (WSH). Further, 39 of the 124 nymphs (31%) at the southeastern site (WAL) and 35 of the 115 nymphs (30%) at WSH were infected with at least one pathogen. Prevalence of each pathogen is reported in Table 1. Borrelia burgdorferi s.s was the most commonly detected pathogen, with highest prevalence at the northwestern site (WSH). Anaplasma phagocytophilum was the second most commonly detected pathogen with 5–9% of nymphs testing positive. Only 0.9–4% of nymphs were positive for Babesia microti. No nymphs were infected with E. muris eauclairensis or B. mayonii at WAL or WSH although both pathogens were detected in adults at WSH. Borrelia miyamotoi was found only in nymphs (both sites). Powassan virus was not detected at WSH.

Table 1.

Summary of all tick-borne pathogens tested in adult and nymphal ticks collected from both study sites in 2017.

	Washburn County (WSH)		Walworth County (WAL)
	Adults	Nymphs	Nymphs
B. burgdorferi s.s.	52.8% (19/36)	24.3% (28/115)	12.9% (16/124)
B. mayonii	5.6% (2/36)	0.0 % (0/115)	0.0% (0/124)
B. miyamotoi	0.0% (0/36)	2.6% (3/115)	0.8% (1/124)
Ba. microti	2.8% (1/36)	0.9% (1/115)	4.0% (5/124)
E. muris eauclairensis	5.6% (2/36)	0.0 % (0/115)	0.0% (0/124)
A. phagocytophilum	5.6% (2/36)	5.2% (6/115)	8.9% (11/124)
Powassan virus	0.0% (0/36)	0.0 % (0/115)	NA*

NA*: Samples were not tested

Ten coinfected ticks were detected at the two sites. Coinfection occurred more often in adult ticks (11%) versus 3% in nymphs at WSH. Borrelia burgdorferi s.s was the most common co-infecting pathogen (Table 2), followed by A. phagocytophilum.

Table 2.

Summary of disease pathogen coinfections detected in adult and nymphal ticks collected from both study sites in 2017.

		Washburn County (WSH)		Walworth County (WAL)
		Adults	Nymphs	Nymphs
B. burgdorferi	E. muris eauclairensis	2	0	0
B. burgdorferi	Ba. microti	1	1	1
B. burgdorferi	A. phagocytophilum	1	2	0
B. miyamotoi	A. phagocytophilum	0	1	0
Ba. microti	A. phagocytophilum	0	0	1
% ticks with coinfection =		11% (4/36)	3% (4/115)	2% (2/124)

In this survey of two sites in Wisconsin, we detected six human TBD pathogens and five different co-infections of varying frequency. Prevalence of the most common tick-borne pathogens, B. burgdorferi s.s. and A. phagocytophilum, in nymphs was 13–24% and 5–9%, respectively. The prevalence results were similar to prior reports from Wisconsin and Minnesota (Lee et al. 2014, Pritt et al. 2016, Murphy et al. 2017, Hahn et al. 2018, Johnson et al. 2018). The emerging pathogens, B. miyamotoi, B. mayonii, Ba. microti and E. muris eauclairensis were also detected at rates that were generally similar to those reported from the upper Midwest (Barbour et al. 2009, Pritt et al. 2016, Murphy et al. 2017, Johnson et al. 2018). For example, Barbour et al. (2009) found an average of 1.9% of nymphs collected from 12 locations in Wisconsin were infected with B. miyamotoi. Pritt et al. (2016) reported that 5.2% of 267 adult I. scapularis collected in northwestern Wisconsin in 2014 were infected with B. mayonii, again similar to the results reported herein. However, Pritt et al. (2016) also found 3.7% of 81 nymphs were infected with this pathogen at the location. We found no nymphs infected with B. mayonii at either site in our study, suggesting that ecological variations in host communities or other temporal and spatial conditions may affect nymphal infection rates.

The two sites in the current study were located in the northwestern (WSH) and southeastern (WAL) quadrants of the state and were separated by approximately 523 km (325 miles). The geographic range for several of the pathogens is not known. Babesia microti and B. miyamotoi were found at both locations, suggesting that the range for each will encompass most of the state.

There were three notable limitations to this study. First; only two geographic sites were sampled in Wisconsin and only over a period of a few days. Second, a relatively small number of ticks were analyzed and only nymphs were present in large numbers at the southeastern location (WAL). Third, inconsistent tick storage limited our ability to detect the full spectrum of pathogens. Regardless, these data add to our knowledge of the prevalence of tick-borne pathogens in Wisconsin. We recognize the limitations of sampling at a single point in time and suggest a longitudinal model to better measure variation in the future.

In conclusion, this study demonstrates pathogen prevalence and coinfections in I. scapularis ticks among regions of the upper Midwestern United States. These findings underscore the need for consideration of multiple TBD agents when clinically evaluating patients for syndromes consistent with tick-borne illness in this region. The detection of both adult and nymphal ticks coinfected with several pathogens should serve as a reminder that even a single tick bite may expose a human to multiple TBD pathogens.