Detection of Rickettsia montanensis in Dermacentor variabilis in Northern Wisconsin

Introduction

D ermacentor variabilis (Say) (Acari: Ixodidae) can be found throughout most of the continental United States and is important for its ability to vector important pathogens such as Rickettsia rickettsii and Francisella tularensis, the etiological agents of Rocky Mountain spotted fever and tularemia, respectively (Duncan et al, 2021). D. variabilis can also vector Rickettsia montanensis, which was once considered to be nonpathogenic, but recent reports have shown can cause subclinical and clinical infections in both humans as well as animals (McQuiston et al, 2012, Moncayo et al, 2016, Snellgrove et al, 2021).

In addition, R. montanensis has been shown to inhibit co-infection of R. rickettsii and may contribute to limiting its prevalence (Macaluso et al, 2002). R. montanensis has been reported in some states, mainly along the east coast, the Upper Midwest, and Canada (Dergousoff et al, 2009, Hecht et al, 2019, Lippi et al, 2021). In this study, we wish to report the initial detection and prevalence rate of R. montanensis in D. variabilis located in northern Wisconsin.

Materials and Methods

The collections were conducted in Ashland County, located in Northern Wisconsin where the land cover is primarily mesic northern hardwood forest. Tick collections were conducted using tick drags at two locations located on private property within Ashland County, Wisconsin, in May of 2022. Four 15-meter transects were established at each collection site; two transects were established along trails and two in interior sections along the edge of grassy fields that were under canopy cover. Each of these transects were sampled three times on the same day between 10 am and 2 pm, 1 day for each collection site. Ticks were sexed and identified to species using morphological features (Lindquist et al, 2016).

A random subset of ticks identified as D. variabilis from each collection site were homogenized using mortar and pestle or bead mill. DNA extraction and isolation were performed using QIAGEN Gentra Puregene Tissue Kit and protocols. All DNA were screened for a 1.2 kb amplicon of the 190-kDA antigen/outer membrane protein A (ompA) gene using PCR (Regnery et al, 1991). PCR was conducted using 12.5 μL of Thermo Scientific™ DreamTaq™ Hot Start PCR Master Mix, 8.5 μL water, 0.1 μM of each forward and reverse primers (Rr.190.4442p, Rr.190.5664n), and 3.0 μL of sample DNA.

Samples were then run with a 2-min hot start at 95°C for 2 min, followed by 40 cycles of denature (94°C for 45 s), anneal (52°C for 45 s), and extension (72°C for 1 min). A 10-min extension was run after the final cycle. Gel electrophoresis was conducted using a 1% agarose gel with TAE buffer and prestained with GelGreen (Biotium, CA). Negative controls were included with both the PCR amplification and gel electrophoresis. Gels were observed using blue LED transilluminator and samples with banding in the 1.2 kb range were prepared for DNA sequence analysis. Amplified fragments were purified using the QIAGEN PCR Purification Kit and submitted for Sanger sequencing using the forward primer (Functional Biosciences, Madison, WI).

Results

A total of 52 D. variabilis were collected from site 1 and a 24 from site 2. Gel electrophoresis identified four (n = 16) possible positive samples for Rickettsia spp., all of which came from collection site 2. Of these samples, one was unable to be identified. The other three amplified fragments were identified as 99.84% identical to R. montanensis strains (GenBank acc. nos. CP003340.1 and U83447.1), with one nucleotide difference. All three samples contained the same single difference.

Discussion

Predictive modeling by Lippi et al (2021) indicated that the probability of R. montanensis infected D. variabilis in Wisconsin is low, and nearly nonexistent in northern Wisconsin; however, those authors acknowledged that under sampling across the full range of D. variabilis habitats may inhibit the accuracy of modeling and that more data are needed to understand the full range of this tick and potential pathogen. Although this report is based on a relatively small sample size, a 19% positive rate for R. montanensis is consistent with prevalence rates reported for the upper Midwest and parts of Canada (Dergousoff et al, 2009, Hecht et al, 2019).

The importance of monitoring the presence and prevalence of R. montanensis lies in a better understanding of the range of infected D. variabilis, the potential of R. montanensis to inhibit R. rickettsia, and the ability of R. montanensis to infect and cause disease in humans and animals. Human cases of spotted fever rickettsiosis (SFR) from Wisconsin are reported frequently to the CDC; however, cases are often not confirmed. With cases of SFR existing in the United States and Canada, and the low prevalence rates of R. rickettsii (Dergousoff et al, 2009, Hecht et al, 2019), it is important that we monitor other potentially pathogenic rickettsiae bacteria and evaluate their role in causing disease. Further studies of R. montanensis that are more robust in sample size and over a greater geographical range in Wisconsin are needed to give a better understanding of this potential tick-borne pathogens range and prevalence.