Confirmation of Borrelia burgdorferi sensu stricto and Anaplasma phagocytophilum in Ixodes scapularis,Southwestern Virginia

Introduction

In the northeastern and upper midwestern regions of the United States, Ixodes scapularis (Acari: Ixodidae) is the vector for both Borrelia burgdorferi sensu stricto (s.s.) (Spirochaetales: Spirochaetaceae), the causative agent of Lyme disease (LD) in the United States, and Anaplasma phagocytophilum (Ricketsiales: Anaplamataceae), the causative agent of human granulocytic anaplasmosis (HGA). Ticks harboring B. burgdorferi have been documented from the northeastern and coastal regions of Virginia (Naldony et al. 2011), but there have been no published reports of infected ticks from the mountainous, western portion of the state, which is part of the southern Appalachian region. Indeed, the literature suggests I. scapularis ticks were historically absent from this region (Sonenshine, 1979).

The expanding number of human Lyme borreliosis cases reported each year renders accurate determination of the geographic range where B. burgdorferi poses a potential infectious threat increasingly important. The present study sought to confirm the presence and determine the prevalence of B. burgdorferi and A. phagocytophilum in I. scapularis collected from a newly endemic region of southwestern Virginia.

Material and Methods

Questing adult Ixodes spp. ticks were collected from October, 2012, through April, 2013, by standard cloth drags at three sites in Giles County and Pulaski County, Virginia. Sites selected for dragging included both public recreational areas and a low-density private residential community with mixed hardwood habitat. Site A was a public park bordered by woody edge habitat and a river, site B was a wooded, low-density residential area, and site C was a grassy and wooded area near a playground at a recreational lake. Ticks were placed in 70% ethanol at the time of collection and held at room temperature until processed for PCR.

Individual ticks were identified morphologically by comparison to standard keys (US Department of Agriculture 1976) and then dissected. Total nucleic acid was extracted as previously described. Briefly, internal tick contents were digested in a proteinase K lysis buffer; the DNA was extracted with phenol/chloroform, resuspended in 50 μL of buffer, and stored at −80°C (Halos et al. 2004). Nucleic acid extracts from each tick were individually tested by PCR to confirm tick species and for B. burgdorferi and A. phagocytophilum as previously described (Little et al. 1997, Stromdahl et al. 2003, Macaluso et al. 2003, Nadolny et al. 2011). Amplicons were purified using a commercial kit (Wizard Genomic DNA Purification Kit, Promega, Fitchburg, WI) according to manufacturer's guidelines and sequenced directly (SmartSeq, Eurofins Genomics, Huntsville, AL).

Sequence analysis and alignment were performed using MacVector software (MacVector, Inc., Cary, NC). Sequences generated from study samples were compared with published sequences using the nucleotide Basic Local Alignment Search Tool (BLASTn, National Center for Biotechnology Information, Bethesda, MD). Differences in prevalence of B. burgdorferi in ticks between collection sites and tick gender were evaluated for significance with a Pearson chi-squared test using Excel (Microsoft 2008, Redmond, WA) with significance assigned at p<0.05.

Results

A total of 356 adult ticks (202 female, 154 male) were collected from the three sites. All ticks were morphologically and molecularly identified as I. scapularis (GenBank L43862.1). B. burgdorferi s.s. was detected in 32/101 ticks (31.7%) from site A, 49/154 ticks (31.8%) from site B, and 36/101 ticks (35.6%) from site C, for a total prevalence of 32.9% (117/356). Female I. scapularis specimens were more likely (p<0.05) to be PCR positive for B. burgdorferi (75/202; 37.1%) than male specimens (42/154; 27.3%). A. phagocytophilum was detected by PCR in 3/356 ticks (0.8%), one from site A in which B. burgdorferi was not detected and two from site B co-infected with both agents. No A. phagocytophilum was detected in ticks from site C. All sequences showed between 99% and 100% identity with corresponding GenBank sequences (B. burgdorferi CP001205.1, A. phagocytophilum JN181070.1).

Discussion

This study documents B. burgdorferi s.s. in high prevalence in questing I. scapularis ticks outside of the area traditionally considered endemic for Lyme borreliosis, suggesting recent expansion of this disease system into the southern Appalachian region. Previous work documents I. scapularis on the northern portions of the Appalachian Trail, but historical, published reports of I. scapularis are lacking from the southern Appalachian region, suggesting that the population sampled in the present paper is newly established (Sonenshine 1979, Oliver and Howard 1988). The prevalence of B. burgdorferi and A. phagocytophilum in adult ticks is comparable to that previously reported from endemic areas in the northeastern United States (Adelson et al. 2004). Geographic expansion of Lyme borreliosis in other areas of North America has also been recently documented (Wang et al. 2014).

When accurate, geographic distribution patterns can aid proper diagnosis of Lyme borreliosis in humans and dogs. However, this approach can be complicated by expanding the range of ticks infected with B. burgorferi s.s. and the potential presence of other species in the B. burgdorferi sensu lato (s.l.) group in a given area, making identification of pathogens in field collected ticks important (Maggi et al. 2010). A. phagocytophilum, an agent of both human and veterinary disease, was also detected in the ticks in the present study; both pathogenic and apparently nonpathogenic variants of A. phagocytophilum have been described (Massung et al. 2003).

These data confirm that populations of I. scapularis harboring both B. burgdorferi and A. phagocytophilum are established in southwestern Virginia; additional investigations in other areas of southern Appalachia are needed. Confirmation of tick species, as made by molecular testing in the present paper, is important. Expansion of the geographic range of Ixodes affinis, which is present in more southern and coastal regions of Virginia and neighboring states, could complicate tick identifications and ecology of Lyme borreliosis in the future (Maggi et al. 2010, Naldony et al. 2011). Recent reports indicate that I. scapularis ticks have also expanded their range in neighboring West Virginia, together with increased reports of Lyme borreliosis from this region (West Virginia Department of Health and Human Resources 2012). Field research documenting the expanding geographic range of these disease agents is important to support prompt diagnosis and treatment of human and canine Lyme borreliosis.