Ixodes affinis Neumann is a hard-bodied (ixodid) tick known to be a competent vector for Borrelia burgdorferi, the agent of Lyme disease, and agents of other human diseases (Keirans et al. 1999). Ixodes affinis has been reported in Florida, Georgia, and South Carolina and throughout coastal North Carolina (Clark et al. 1998, Harrison et al. 2010). Harrison et al. (2010) indicated that I. affinis was established throughout the coastal plain of North Carolina up to the Virginia border and suggested that I. affinis might occur in Virginia.
Ixodes affinis is morphologically very similar to Ixodes scapularis Say, the primary vector for B. burgdorferi in the eastern United States. Although all active stages of I. affinis can be morphologically distinguished from I. scapularis (Durden and Keirans 1996, Keirans and Clifford 1978, Oliver et al. 1987), field-collected specimens are often damaged and difficult to identify. The distributions of these Ixodes spp. overlap in some areas, which can make visual distinction very difficult, particularly in juvenile stages (Keirans et al. 1996). Understanding the role of the two species in disease vector ecology provides an incentive to confirm the identity of any Ixodes species ticks in areas where ranges overlap (Goddard 1992). Traditionally I. affinis adults are reported to actively quest during the hot summer months when I. scapularis adults are dormant (Harrison et al. 2010). I. affinis are not known to bite humans, but are considered more important than the human biting vector, I. scapularis, in maintaining the enzootic spirochete cycle among mammalian host species (Oliver 1996, Oliver et al. 2003). I. affinis feed on many common species of mammals and as such may amplify the prevalence of B. burgdorferi and B. bissettii in these reservoir hosts (Clark 2004). Potentially this would increase the number of infected animals that in turn could transmit the pathogen to I. scapularis.
As part of an ongoing study to document the presence and abundance of tick vectors of infectious diseases in southeastern Virginia, individual Ixodes spp. ticks were identified by morphological criteria and confirmed using molecular methods. This paper presents evidence of established populations of I. affinis in Virginia and discusses potential implications for the spread of B. burgdorferi infections in mammalian host reservoirs.
Adult and nymphal ticks were collected from nine sites throughout southeastern Virginia using flags made of white denim attached to dowel rods and dragged through vegetation. The sites sampled represent a variety of habitat types, including forests and grasslands, from the Atlantic Ocean beaches to James City County, VA (Figure 1). All ticks found on flags were removed with forceps and placed in vials labeled with the time and date of collection, as well as the sampling location, temperature, and weather. Ticks were identified to the lowest taxonomic level possible using morphological criteria (Keirans and Clifford, 1978) before being frozen at –80° C. In order to ensure accurate identification of the Ixodes spp., molecular techniques were employed.
Ixodes ticks collected from the same site on the same day were pooled into groups of up to four adults and eight nymphs. DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen Inc., Valencia, CA) according to the manufacturer's protocol and stored at –20° C until processing. Samples were originally pooled to test for the presence of B. burgdorferi and other pathogens. Because multiple Ixodes species were not expected in the region, all pools were sequenced to determine species only after one pool suggested the presence of I. affinis: subsequently two individual ticks were sequenced. Our continued sampling efforts will sequence ticks individually that cannot be morphologically identified to species level. Species identification was confirmed by sequencing. A 454 bp fragment of the tick mitochondrial 16S ribosomal RNA gene was amplified on an iCycler (BioRad Laboratories, Hercules, CA) using primers 16S-1 (5′-GTCTGAACTCAGATCAAGT- 3′) (Macaluso et al. 2003) and 16S+1 (5′- CTGCTCAATGATTTTTTAAATTGCTGT-3′). All PCR products for sequencing were purified by using Wizard PCR Preps DNA Purification System (Promega, Madison, WI), and sequencing reactions were performed by using the BigDye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) as described by the manufacturer and using 16S primers. Sequence similarities were identified by a BLAST search (http://blast.ncbi.nlm.nih.gov).
Tick species identification
All Ixodes ticks collected were tentatively identified morphologically using microscopic examination before pooling; molecular methods were used to confirm species. A total of 94 Ixodes spp. ticks were collected during the summer of 2010. DNA was extracted and a fragment of the 16S rRNA was amplified from each pool. Sequencing analysis revealed that of the 55 Ixodes pools tested, 23 pools shared maximum identities with the I. scapularis 16S ribosomal RNA gene while 32 pools, including four nymphal pools, shared maximum identities with the I. affinis ribosomal RNA gene. Maximum identities ranged from 93% to 100% for I. affinis and I. scapularis. In addition to pooled samples, two individual I. affinis were sequenced and a BLAST search revealed a 97% maximum identity to Ixodes affinis 16S ribosomal RNA gene (accession number U95879.1). An alignment showed these individually sequenced samples to be 99.8% identical to the consensus sequence of the I. affinis pools.
Geographic and temporal occurrence of I. affinis collections
Twenty-eight adult and four nymphal tick pools confirmed as I. affinis were collected during the summer of 2010 from eight of the sampled sites representing six independent cities and counties in southeastern Virginia (Figure 1). Ticks from a variety of habitats were collected from three sites in the city of Virginia Beach, and one site each in the city of Chesapeake, city of Portsmouth, Northampton County, James City County and Isle of Wight County (Table 1). I. affinis specimens were collected between April and November, 2010, with the greatest number between mid-May and mid-June (Figure 2). There was considerable overlap in the times of year that both species were active with the exception of late August. To determine temporal distribution of I. affinis, all sites were pooled. While it is known that tick seasonal dynamics vary with habitat type, we believe that pooling the sites in this way provides a broad picture of I. affinis activity in the Hampton Roads region. Although I. affinis was collected from sites representing grassland and numerous forest types, our most productive collection site bordered the Great Dismal Swamp in Chesapeake and represented an old-field habitat undergoing secondary succession.
Table 1. Results of sequencing tests for Ixodes spp. ticks collected using flags at each study site over the summer of 2010. All ticks were adults except as noted at one site. Basic habitat information for each site is also provided. See Figure 1 for identification and location of each site.
Successional old field (grassland)
Mixed pine-deciduous forest
Successional old field (grassland)
Mixed pine-deciduous forest
Mixed pine-deciduous forest
Pools with I. affinis present
Total Ixodes spp. pools
Total Ixodes spp. ticks
Our results confirm the suggestion by Harrison et al. (2010) that I. affinis has extended its range north beyond the North Carolina border and into the coastal plain of southeastern Virginia. The discovery of both adults and nymphs indicates that breeding populations of I. affinis are now established in Virginia. Ixodes affinis was uncommon in North Carolina prior to 1988 (Harrison et al. 2010), which suggests a recent Northward expansion into Virginia only in the last two decades. It is plausible that I. affinis crossed from North Carolina to Virginia through one of the numerous potential hosts that utilize coastal plain habitats in both states (Webster et al. 1985). Two important hosts linked to I. affinis abundance in Georgia and South Carolina are the cotton mouse and cotton rat (Durden and Oliver 1999, Clark et al. 2001, Oliver et al. 2003). These and related species are abundant in southeastern Virginia and are common at the Chesapeake site where 11 of the 12 pools of adult ticks contained at least one I. affinis.
The discovery of I. affinis in Virginia has implications for the disease vector ecology of ticks in the genus Ixodes in Virginia. Summer time activity of I. affinis adults has been a key behavioral trait that differentiates them from I. scapularis, which has been reported to quest more actively in the cooler months (Clark et al. 1998, Goddard 1992). While our report found that I. affinis peak in mid-May to mid-June, the continued questing activity of I. affinis until November and the persistent activity of I. scapularis in late spring and early summer complicates the distinction of species by their seasonal questing behaviors. Because of the difficulty of differentiating between I. affinis and I. scapularis relying on morphology alone, it is possible that I. affinis is more widespread in Virginia than we report. The northward expansion of this tick throughout the coastal plain of the southeastern United States suggests that areas further north that share coastal plain characteristics may also be vulnerable to invasion by this tick.
Although I. affinis rarely bites humans and thus plays little if any role in direct transmission of pathogens to humans, I. affinis has been reported to be more important than I. scapularis in maintaining the enzootic cycles of B. burgdorferi and B. bissettii (Harrison et al. 2010, Maggi et al. 2010, Oliver 1996, Oliver et al. 2003) in the southeastern United States. The discovery of established populations of I. affinis along with I. scapularis in areas of Virginia with many competent host species could have implications for the amplification of B. burgdorferi and B. bissettii cycles in wild animal hosts. Continued monitoring of the tick populations is needed to determine what effect a second competent B. burgdorferi vector may have on Lyme borreliosis in southeastern Virginia.
This paper serves to document widespread populations of I. affinis in a variety of habitats throughout southeastern Virginia. Adult and nymph I. affinis in Virginia appear to be actively questing throughout spring, summer, and autumn in this region. These populations were discovered through comprehensive surveys of tick populations at over 15 sites in southeastern Virginia. As we continue to collects ticks from around this region, Ixodes ticks will be individually sequenced if necessary for identification to species. Continuations of our sampling efforts in this region will provide us with more information about the ecology of I. affinis at the northern extent of its range, including which habitats support the most abundant adult and nymph populations and how habitat impacts life history parameters and interactions with I. scapularis.
We thank field assistants Brandon Rowan and Ryan Wright for their help in collecting ticks during the summer of 2010. This project was supported by Grant Number K25AI067791 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases or the National Institutes of Health.