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The Lyme borreliosis (LB) cycle, involving Ixodes scapularis and Borrelia burgdorferi sensu stricto (Bbss), is well documented in the northeastern US, where LB is becoming increasingly prevalent. In coastal North Carolina, I. affinis has been shown to have a higher incidence of Bbss than I. scapularis. My objectives were, to assess changes in prevalence of Bbss in Ixodes spp. along a transect from Virginia to Florida, and to assess the value of dogs and mesomammals as sentinels for spread of Borrelia burgdorferi sensu lato. Ixodes spp. were collected at sites from 37.4o N to 30.0o N in 2011-12. Veterinary clinics from Virginia, to Florida were asked to collect ticks from dogs, and mesomammals were livetrapped in South Carolina and checked for ticks. Positive samples were identified to species by nested PCR and sequenced using the 16S-23S rRNA intergenic spacer genes. A subset of ticks was subjected to 12/16S rRNA genetic procedures. Overall, 46 ticks were positive for Bbsl. Species identify of 22 Borrelia DNA samples sequenced was 54% Bbss, 32% B. bissettii, and 13% B. miyamotoi. All Bbsl-positive I. scapularis for which 12S analysis was completed belonged to the ‘American’ lineage of ticks. Genetic clustering (FST values) was evident among I. scapularis positive ticks, indicating an association between infection and haplotype. A total of 370 Ixodes spp. ticks were collected from dogs and mammals. Only four were Borrelia-positive (one I. affinis from a white-tailed deer in Virginia and two I. affinis from dogs in North and South Carolina respectively). No I. scapularis or I. affinis ticks were obtained from mesomammals. The pronounced prevalence gradient, whereby Bbsl was not found in ticks on vegetation south of 35.2˚N cannot be explained by an absence of vectors. Ixodes affinis was collected in low numbers yet comprised half of the infected ticks identified, highlighting the importance of this species for sylvatic cycles of Bbsl. Our furthest-south record of a Borrelia-infected tick was from a dog, demonstrating that dogs can indeed be useful as sentinels for LB. Findings indicate that the risk of locally-acquired Bbss infection in the coastal southeast is at present low.