Detection of Borrelia burgdorferi infected Ixodes scapularis (Acari: Ixodidae) associated with local Lyme disease transmission in Nebraska, USA, 2021

In August 2021, the Nebraska Department of Health and Human Services was notified by a local public health department of a cluster of two Lyme disease cases in patients with local exposure to wooded areas in a county located in their jurisdiction. Epidemiological investigations revealed that the two patients had similar symptom onset dates and had likely exposure to ticks at wooded sites located directly adjacent to one another. Two environmental investigations were completed in October 2021 and consisted of tick surveys at the patients' reported sites of tick exposure. 12 ticks were collected across the two surveys and identified the black‐legged tick (Ixodes scapularis). During subsequent testing of the collected ticks, spirochete bacteria were isolated, cultured and confirmed as Borrelia burgdorferi sensu stricto by PCR. In total, 7 of 12 (58.3%) I. scapularis ticks tested positive for B. burgdorferi s.s. The results of this study document the fourth known established population of I. scapularis in Nebraska and confirms the first detection of B. burgdorferi s.s. in field collected ticks from Nebraska. The epidemiological and environmental investigation data provide the first evidence for local Lyme disease transmission occurring within Nebraska. These findings highlight the need for continued surveillance of I. scapularis and its associated pathogens in Nebraska to further characterize human risk and monitor emergence into other areas of the state.

identification of the Lyme disease tick vector increased suspicion that the Lyme disease-causing bacteria, B. burgdorferi sensu stricto, could potentially be acquired in eastern Nebraska.
Historically, Nebraska has reported Lyme disease cases every year (CDC, 2022)

that meet the Council of State and Territorial
Epidemiologists adopted standardized case definition (CDC, 2022).
Through epidemiological investigations, the majority of these cases report out of state travel to known areas with endemic I. scapularis and Lyme disease transmission (e.g. Minnesota and Wisconsin). Lyme disease cases that do not report out of state travel, and thus potentially locally acquired, are followed up to determine possible sites of exposure and recent, known tick bites. If exposure sites can be determined, environmental surveys (i.e. tick dragging/flagging) are undertaken where possible. Prior to 2021, these environmental surveys did not reveal the presence of I. scapularis at the reported sites of exposure. Thus, no definitive evidence to support endemic Lyme disease transmission in Nebraska has been identified. Here we report the first known Lyme disease cases directly linked to local sites with evidence of B. burgdorferi s.s. infected I. scapularis in Nebraska.

| Ethical statement
This study is exempt from human research approval as the actions are considered disease surveillance activities and no ethics approval is required.

| Epidemiological investigations
In August 2021, the Nebraska Department of Health and Human Services was notified by a local public health department of a cluster of two Lyme disease cases in patients with reported exposure to wooded areas in a county located within their jurisdiction ( Figure 1). The first patient (Patient 1) was a 34-year-old male eastern Nebraska resident, with no previous history of a Lyme disease diagnosis. He reported frequent outdoor exposure to wooded areas at his residence but recounted no known tick attachments or outof-state travel in the 30 days prior to symptom onset. He developed a cough and sweating on 5 July 2021, presented to the emergency department on the same day, received intravenous hydration, and was released. He additionally developed arthralgia, myalgia, fatigue F I G U R E 1 Location of likely site of exposure for two locally transmitted Lyme disease cases and collection site of Borrelia burgdorferi s.s. positive Ixodes scapularis ticks within Thurston County, Nebraska. Map created using ESRI ArcGIS Pro 2.9 (ESRI, https://www.esri.com). Data from ESRI, GEBCO, DeLorme, NaturalVue and the State of Nebraska. B. burgdorferi s.s., B. burgdorferi sensu stricto.

Impacts
• We detected Borrelia burgdorferi s.s. for the first time in Ixodes scapularis ticks field collected from Nebraska.
• We provide the first definitive evidence of locally transmitted Lyme disease in Nebraska.
• Timely epidemiological investigations of suspected Lyme disease cases in combination with environmental surveillance for I. scapularis is needed to identify further emergence of I. scapularis in Nebraska to better describe human Lyme disease risk in the state. and neck pain with symptoms persisting until late August. On 28   (Sonenshine, 1993). Patient 1's residence consisted of a farmstead with an open pasture surrounded by deciduous wooded areas along its perimeter. The area directly adjacent to Patient 1's residence, where Patient 2 had daily occupational exposure, was a heavily wooded upland deciduous forest. All but two collected ticks (n = 10) were placed into vials containing 95% ethanol while the remaining two were kept alive for attempted culturing of B. burgdorferi s.s. All ticks were taken to the Nebraska Public Health Environmental Laboratory (Lincoln, NE) for morphological identification (Keirans & Litwak, 1989). To test for the presence of B. burgdorferi s.s., the two live ticks were sent to Creighton University (Omaha, NE) where isolation in Barbour-Stoenner- Kelly II (BSK II) media (Barbour, 1984) and PCR testing (Bunikis et al., 2011) were conducted. The 10 remaining ticks stored in 95% ethanol were sent to the Centers for Disease Control (CDC) Division of Vector-Borne Diseases Branch (Ft. Collins, CO) where real-time PCR was conducted (Graham et al., 2018)

| RE SULTS
Across the two surveys, 12 ticks were collected and identified as I. scapularis, confirming the fourth known established population in Nebraska (Nielsen et al., 2020). Spirochetes were isolated and cultured from one of two live ticks submitted to Creighton University.
The isolate was confirmed as B. burgdorferi s.s. by PCR using primer sets specific to B. burgdorferi's repertoire of linear and circular plasmids (Bunikis et al., 2011). The isolate was found to harbour 20 of 21 plasmids, missing only 1p21 which is frequently absent in high-infectivity clones of B. burgdorferi (Iyer et al., 2003;Purser & Norris, 2000). Of the 10 ticks submitted to the CDC, B. burgdorferi s.s. was detected in six of the ticks with no additional pathogens detected. In total, 7 of 12 (58.3%; 95% CI = 30.9-82.5) I. scapularis ticks tested were positive for B. burgdorferi s.s.

| DISCUSS ION
This report documents the fourth known established population of I. scapularis in Nebraska (Nielsen et al., 2020)  Additionally, we provide further evidence of I. scapularis's continued emergence into Nebraska and its expansion westward (Eisen et al., 2016). Prior to the detection of established I. scapularis populations in 2019, only individual tick specimens were documented (Cortinas and Spomer, 2014;Eisen et al., 2016) in the state. We also successfully demonstrated the effectiveness of using epidemiological investigations to guide environmental surveillance efforts to identify potential locations of I. scapularis. In states where I. scapularis is expanding its range or may potentially expand to, using epidemiological data may prove useful in identifying areas of potential risk for local Lyme disease acquisition and help guide tick surveillance efforts.
Presently, the full extent of local human health risk in Nebraska due to the detection of I. scapularis is unknown, but of increased concern. These findings confirm the need for expanded tick surveillance programs in Nebraska and beyond. Tick surveillance programs not only identify tick species distribution in their state, but when combined with pathogen testing can also inform public health officials of pathogen presence and prevalence (Eisen & Paddock, 2021). This information can then be used to properly educate residents and healthcare providers on specific tick-borne disease risk in their area and guide public health prevention measures.

ACK N O WLE D G E M ENTS
We thank the Winnebago Tribe of Nebraska and local property owners for access to tick survey sites. We also thank Sarah Maes and Maria Rizzo from the CDC Division of Vector-Borne Diseases for pathogen testing and sample sequencing of submitted specimens, and Jennifer Parmeley for creating the map of Nebraska.

CDC Epidemiology and Laboratory Capacity for Prevention and
Control of Emerging Infectious Diseases.

CO N FLI C T O F I NTE R E S T S TATE M E NT
None.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.