Ixodes scapularis, more commonly known as the blacklegged tick, is the primary vector of 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. 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 Wisconsin with established populations of I. scapularis ticks; site one (WSH) was located on private property in Washburn County in an oak‐dominated 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 m² 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 1.7 ml 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). Futher, 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.

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

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. 2016a, 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, 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. (2016a) 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. (2016a) 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 co‐infected with several pathogens should serve as a reminder that even a single tick bite may expose a human to multiple TBD pathogens.

肩cap舌虫（Ixodes scapularis），通常被称为黑脚tick，是美国中西部上部tick传播疾病（TBD）的主要媒介。它是Borrelia burgdorferi ss（莱姆病的主要病原体），miyamotoi的Borrelia miyamotoi（t传复发性发热），吞噬性无浆细胞， Powassan病毒和巴氏杆菌（Babesia microti）的最重要载体，它们均对人体健康构成重大威胁（Lee等人2014年，Hahn等人2018年，Johnson等人2018年）。在过去的十年中，全美国的TBD发病率上升，并且新的由tick传播的人类病原体不断出现（艾森和艾森2018）。在中西部的上部，梅氏疏螺旋体（Borrelia mayonii）和鼠埃里希毛虫（Ehrlichia muris eauclairensis）代表了两种人类病原体，它们是最近才发现的。两者均由肩cap鱼（I. scapularis）提供载体（Pritt等人2016a，2016b）。单个载体传播多种病原体可能导致共感染，这使我们对病原体适应性，传播成功和疾病诊断的理解更加复杂（Grab等人，2007年）。鉴于整个中西部中部肩cap鱼中TBD患病率的数据有限，这项横断面研究旨在进一步调查肩cap鱼壁tick中人类病原体的存在。

ick虫取样发生在威斯康星州两个遥远的地理区域，并有肩I鱼tick虫种群。第一个站点（WSH）位于沃什伯恩县（Washburn County）一片橡树为主的森林中（45.741270，-91.482142），第二个站点（WAL）位于沃尔沃思县（Walworth County）南水壶冰a州立森林的红松林中（ 42.835775，‐88.617064）。追问肩突硬蜱蜱通过用1M拖采样林下收集²白色绒布连接到木销钉上。进行采样，直到从每个位置收集到至少100个若虫tick。通过现场生命周期阶段和性别在视觉上识别收集的所有s，然后将其放入RNAse抑制剂（WSH）或乙​​醇（WAL）中，以运送到实验室进行进一步的识别和处理。

单独的WSH肩cap小tick通过0.1和2.4 mm氧化锆珠的组合进行机械破坏，而将WAL的若虫分离在1.7 ml离心管中，然后使用18号PrecisionGlide针将其一分为二（Becton，Dickinson and Company，Franklin Lakes ，NJ）。机械剔除壁虱后进行核酸提取（WSH：Magna Pure LC总核酸试剂盒，罗氏，印第安纳波利斯，印第安纳州； WAL：Bioline Isolate II基因组DNA试剂盒，Meridian Life Science，Inc。，孟菲斯，田纳西州）。然后使用先前描述的实时PCR技术对提取的核酸进行B.burgdorferi ss（oppA2基因：Pritt等人2016a），嗜A.phagocytophilum / Ehrlichia的检测spp。（groEL基因：Pritt等，2011）和Ba。microti（18S基因：Burgess等人2017）。还使用实时荧光共振能量转移（FRET）PCR分析法对宫本芽孢杆菌（GlpQ：正向引物5'-TCCAGAACATACCTTAGAAGC-3'; 反向引物5'-ATCAAATCTTTCACTGAGACTTA-3'; 荧光素标记探针5'-GACAATGTTCCTATTATAATGCACGACCC-fl-3'; 5'-LC640-GAAATTGACACAACCACAAATGTTGCAC-3'）和Powassan病毒（NS5：正向引物5'-ACTAGAATGGCCATGACAGAC-3';反向引物5'-TCATCTCTGGTGCACATCC-3';荧光素标记的探针5'-CACAAAGGCCCAGGAACCACAGC ;红色标记的探针5'-LC640-GGCACCAGIGTGATCATGAGAGCIGT-3'）; （WSH站点仅是因为壁虱在WAL储存在乙醇中）。所有测定均在Roche LightCycler 2.0或480仪器上进行，并利用FRET探针检测和鉴定扩增的核酸。总共239个肩若虫和36名成年人被收集和测试。WSH贡献了115个若虫和36个成年人，WAL贡献了124个若虫。从WAL收集了另外五个成年tick，但由于样本量少，因此未进行测试。

测试从西北地区（WSH）中检测出36个成年tick中的15个（42％）中有至少6种细菌/原生动物病原体之一被感染。此外，东南部站点（WAL）的124个若虫中有39个（31％），而WSH站点的115个若虫中有35个（30％）感染了至少一种病原体。表1中报告了每种病原体的患病率。伯氏疏螺旋体是最常见的病原体，在西北地区（WSH）患病率最高。吞噬性无浆细胞是第二常见的病原体，若虫为阳性的占5-9％。只有0.9-4％若虫阳性巴贝田鼠。没有若虫感染了鼠埃里希氏大肠杆菌或马其芽孢杆菌在WAL或WSH，尽管在WSH的成年人中都检测到了两种病原体。仅在若虫（两个地点）中都发现了宫状疏螺旋体。在WSH未检测到Powassan病毒。

在两个位置检测到十个共感染的tick。在WSH，成年s中的共感染发生率更高（11％），若虫中则为3％。伯氏疏螺旋体是最常见的共同感染病原体（表2），其次是吞噬曲霉。

在对威斯康星州两个地点的调查中，我们检测到六种人类TBD病原体和五种不同频率的不同共感染。若虫中最常见的壁虱传播病原体伯氏疏螺旋体和嗜吞噬菌A.的患病率分别为13–24％和5–9％。患病率的结果类似于威斯康星州和明尼苏达州的先前报告（Lee等人2014年，Pritt等人2016a，Hahn等人2018年，Johnson等人2018年）。新兴病原体，宫本芽孢杆菌，马其顿芽孢杆菌，Ba。microti和E. muris eauclairensis它们的检出率通常与中西部地区上报的相似（Barbour等，2009 ; Pritt等，2016 ; Johnson等，2018）。例如，Barbour等。（2009年）发现，从威斯康星州12个地点收集到的若虫平均有1.9％被B. miyamotoi感染。Pritt等。（2016a）报告称，2014年在威斯康星州西北部收集的267例肩cap鱼中，有5.2％感染了马氏杆菌，与本文报道的结果相似。然而，普里特等。（2016年）还发现在该位置的81个若虫中有3.7％感染了这种病原体。在我们的研究中，没有发现任何一处若虫感染了美人双歧杆菌，这表明寄主社区或其他时空条件下的生态变化可能会影响若虫的感染率。

当前研究中的两个站点位于该州的西北（WSH）和东南（WAL）象限，相距约523公里（325英里）。几种病原体的地理范围未知。在这两个地方都发现了小巴贝虫和宫本芽孢杆菌，这表明每个地方的范围将涵盖该州的大部分地区。

该研究有三个明显的局限性。首先，在威斯康星州仅采样了两个地理站点，并且只进行了几天。其次，分析了相对较少的壁虱，并且在东南部（WAL）只有大量的若虫。第三，不一致的tick存储限制了我们检测病原体全谱的能力。无论如何，这些数据增加了我们对威斯康星州tick传播病原体患病率的认识。我们认识到在单个时间点进行采样的局限性，并建议采用纵向模型来更好地衡量将来的变化。

总之，这项研究证明了美国中西部上部地区的肩I鱼I中的病原体流行和共感染。这些发现强调了在临床评估该地区与tick传播疾病相符的综合征时，需要考虑多种TBD药物。同时感染成年病原体和成虫的s虫的检测应该提醒人们，即使是一口tick虫也会使人暴露于多种TBD病原体。