# Inferring tick movements at the landscape scale by SNP genotyping

Olivier Plantard, Elsa Quillery

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Olivier Plantard, Elsa Quillery. Inferring tick movements at the landscape scale by SNP genotyping. 13TH INTERNATIONAL CONFERENCE ON LYME BORRELIOSIS AND OTHER TICK BORNE DISEASES, Aug 2013, Boston, United States. hal-02744498

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13TH INTERNATIONAL CONFERENCE
ON LYME BORRELIOSIS AND OTHER
TICK BORNE DISEASES

Boston, MA, USA, 18-21 August 2013

<figure><img src="image_2.png" /></figure>

13th International Conference on
Lyme Borreliosis and other Tick Borne
Diseases Abstracts

ISBN: 978-2-88919-408-7

DOI: 10.3389/978-2-88919-408-7
# Summary of the Thirteenth International Conference on Lyme Borreliosis and Other Tick-Borne Diseases

Linda K. Bockenstedt and Linden Hu

Within this issue of Frontiers, are abstracts from the Thirteenth International Conference on Lyme and Other Tick-Borne Diseases (ICLB), held August 18–21, 2013 in Boston, Massachusetts. ICLB is one of the preeminent conferences in the world for clinical, epidemiological, and pathogenetic studies of tick-borne diseases. The first conference was held in November, 1983, at Yale University in New Haven, CT, and focused on Lyme borreliosis, which had recently been identified as an *Ixodes* tick-transmitted spirochetal infection. Since then, the conference has been held every 2–3 years alternating between sites in the United States and in Europe. Although Lyme borreliosis remains the most common vector borne-disease in the United States and in Europe, the subject matter of the conference has expanded to include other important agents of human disease that are transmitted by *Ixodes* sp. ticks including but not limited to *Anaplasma*, *Ehrlichia*, *Babesia* species, relapsing fever spirochetes, and tick borne encephalitis virus. The Thirteenth ICLB conference brought together some of the most knowledgeable investigators in the world along with their trainees to share scientific discoveries relevant to infections transmitted by *Ixodes* ticks. Attendees came from a diversity of backgrounds including physicians, clinical and laboratory-based researchers, veterinarians, ecologists, and entomologists from academia, industry, and government, representing 26 countries in North America, Europe, and Asia. There were over 250 participants presenting 220 posters.

The conference opened with a celebration honoring the careers of two physician scientists – Stephen Malawista<fnref n="1" /> and Allen Steere – whose investigation of childhood arthritis ultimately led to the discovery of Lyme disease, the first *Ixodes* tick-borne disease identified in the United States. Malawista, whose scientific career was dedicated to the study of inflammation and blood leukocytes in human disease, especially in gout and Lyme disease, was Chief of the Section of Rheumatology at Yale. Steere, a CDC-trained epidemiologist, had just begun his rheumatology postdoctoral fellowship training and was poised to “get to the bottom” (his own words) of what was causing Lyme arthritis, a research endeavor to which he has devoted his entire career. To honor them both, a poster entitled “A Newspaper Survey of The Discovery of Lyme Disease” was displayed to chronicle the remarkable epidemiologic efforts that culminated in the identification of an emerging *Ixodes* tick-borne spirochetal infection the United States.

As can be seen in the abstracts that were presented orally and in poster format, the conference covered a range of topics, from ecology and epidemiology, to new information about the basic biology of *Lyme Borrelia* and other tick-borne pathogens, to disease pathogenesis and host immunity, to clinical presentations, diagnostics, interventions, and outcomes. Three themes in particular were highlighted during the plenary sessions: (1) ways to curtail the spread of tick-borne diseases; (2) approaches to the question of *Lyme Borrelia* persistence in humans after treatment; and (3) priorities for future research of tick-borne diseases.

The opening plenary lecture was delivered by the entomologist Durland Fish, who described environmental strategies to limit the spread of tick-borne diseases. Although such measures can be effective, he cautioned the audience on the need to act quickly, as their implementation is easier before enzootic cycles have been firmly established. On-going active surveillance is essential for monitoring the potential threat of tick-borne diseases. New strategies for disrupting existing zoonoses are critical, as we learned from Paul Mead of the Centers of Disease Control and Prevention that the estimated annual incidence of Lyme disease in the United States is about 300,000 cases. In addition to Lyme disease, *Ixodes* ticks transmit other pathogens of human concern, including *Anaplasma phagocytophilum*, *Babesia* species, tick-borne encephalitis virus, and the newly emergent relapsing fever spirochete *Borrelia* miyamotoi. Regarding the latter, Platonov and our Russian colleagues reported the range of clinical and epidemiologic features 128 *Borrelia* miyamotoi cases – the largest documented series in the world.

A special plenary session was devoted to the issue of bacterial persistence. James Collins from Boston University gave a scintillating presentation on bacterial “persisters,” defined as dormant cells within a genetically homogeneous bacterial population that are tolerant to the effects of antibiotics. Excessive use of antibiotics may actually drive bacteria to a state of persistence, contrary to a popular lay notion that long-term therapy is the solution to their eradication. A panel discussion ensued in which Mark Klempner reviewed the clinical trial data showing lack of efficacy of extended courses of antibiotics for people with post-treatment Lyme disease syndrome; Stephen Barthold discussed the results of animal studies suggesting *Lyme Borrelia* persistence; Linda Bockenstedt explained the importance of using tick-borne infection (ideally with a single nymphal tick) rather than cultured inocula to study *Lyme Borrelia* persistence in animal models if the goal is to gain insight into human disease; Monica Embers reported preliminary results of a study using tick-transmitted infection in the rhesus macaque *Lyme borreliosis* model; and

<footnote n="1">¹ Malawista passed away on September 18, 2013, after a five-year battle with metastatic melanoma.</footnote>
Brian Fallon raised questions about the etiology of neurocognitive complaints in people with post-treatment Lyme disease syndrome and how best to study them. Although no answers were provided, the group discussion provided a forum for researchers to clarify issues and identify concerns.

The conference concluded with summaries from each of the session chairs regarding future directions in the field. The general lack of funding for training the next generation of ecologists/entomologists in the field of tick-borne diseases was raised, particularly in light of the anticipated retirement of many long-standing leaders in this area. The need for better prevention strategies for tick-borne diseases was highlighted, with some calling for re-release of the LymeRx vaccine and others suggesting that drastic measures may be necessary if outbreaks of Powassan virus become more prevalent now that the virus has expanded to *Ixodes* sp. ticks that transmit Lyme disease. Finally, the need to conduct studies in humans to gain more information about human biology and how it impacts the outcome from tick-borne diseases was conveyed.

Overall, the conference was considered a great success. The feedback from attendees was uniformly positive, which Linden and I can only attribute to the quality of the science and the participation of the attendees themselves. We are grateful for the opportunity to continue this biannual tradition, started in 1983 by Steere, and look forward to the next conference in Vienna, Austria in 2015.
# Inferring tick movements at the landscape scale by SNP genotyping

Elsa Quillery and Olivier Plantard*

INRA (Institute National de la Recherche Agronomique), Paris, France

Assessment of tick movements is particularly important for the understanding of the eco-epidemiology of tick-borne-diseases and for the design of control methods against this vector and transmitted pathogens. Tick dispersal is mainly due to host movements. However, because of the high diversity of *Ixodes ricinus* hosts, to which is associated a wide range of dispersal abilities (ranging from a few meters for rodents to hundred of kilometers for birds) and of the unknown relative contribution of the different host species in tick feeding, estimating tick dispersal is a particularly challenging task. Moreover, capture-mark-recapture methods cannot be used for those small animals with their growth interrupted by molting. Thus, population genetics – through the assessment of gene flow – provides a particularly valuable tool to estimate tick dispersal. Unfortunately, the microsatellite loci – considered as the most powerful markers for populations genetics studies due to their hypervariable and co-dominant features – developed until now for *I. ricinus* exhibit heterozygous deficiency and null alleles that are harmful for any population genetics investigations. To circumvent this caveat, we have developed a set of 384 single nucleotide polymorphisms (SNPs). The genotyping of those bi-allelic markers is unambiguous and straightforward and the large number of loci allows a wide coverage of the polymorphism in the whole *I. ricinus* genome. Because of the scarcity of genomic resources for *I. ricinus*, we have conducted a partial sequencing of its genome to isolate SNPs. Among the 1.2 millions of reads obtained (average length 540 bp), SNPs were isolated using an original bioinformatic method. A set of three primers have been designed for each individual SNP locus. Finally, a Fluidigm array was used to allow the genotyping of individual ticks on 384 different SNPs. Five hundred questing nymphs – individually georeferenced – collected in a landscape (including forests, hedges, and pastures) from western France were genotyped. The analysis of the variability of tick populations with landscape genetics tools will be discussed. Moreover, each genotyped tick is also analyzed to identify the host used for its previous blood meal, and *Anaplasma phagocytophilum*, *Borrelia bugdorferi*, as well as *Babesia divergens* are looked for and characterized molecularly. It is thus possible to investigate potential associations of tick genotypes with particular hosts, pathogens, or biotope. This is the first study reporting the development of primers allowing the genotyping of SNPs in *I. ricinus* and of landscape genetics of any ticks.
# Tick adverse moisture events (TAMEs) determine nymphal blacklegged tick encounter risk

Kathryn Berger¹*, Howard Ginsberg², Katherine Dugas¹, Lutz Hamel³ and Thomas Mather¹

1. Center for Vector-Borne Disease, University of Rhode Island, Kingston, RI, United States

2. USGS Pawtucket Wildlife Research Center, Coastal Field Station, University of Rhode Island, Kingston, RI, United States

3. Department of Computer Sciences, University of Rhode Island, Kingston, RI, United States

*Ixodes scapularis* nymphs, the primary Lyme borreliosis vector stage in the eastern United States, displays sensitivity to conditions of low environmental moisture, with increasing tick mortality demonstrated under longer exposures to low relative humidity conditions in the laboratory. However, field studies examining the relationships between environmental moisture, tick encounter risk, and Lyme borreliosis incidence have generated conflicting results. We examined the relationship between tick adverse moisture events (TAMEs), defined for nymphal *I. scapularis* as >8 h of continuous <82% near-ground relative humidity (RH) and tick encounter risk using 14 years of tick surveillance data from Rhode Island (USA). The total number of TAMEs occurring in June of each year was negatively related to total seasonal nymphal *I. scapularis* encounter risk during that year, suggesting that extended lower humidity periods may serve to reduce nymphal blacklegged tick populations. Linear regression of the total number of TAMEs during June successfully predicted total seasonal nymphal tick encounter risk for the same year (*P* ≤ 0.027). Furthermore, the number of TAMEs occurring during June was positively related to the ratio of tick counts collected in early season compared to late season (*P* = 0.040), suggesting that the significant TAME population effect was due to tick mortality. Multiple regression of a four-parameter model incorporating June humidity and degree of winter severity on the 2 year life-cycle of *I. scapularis* did not improve the analysis (*P* = 0.660). Results provide insight into the means by which environmental moisture influences tick survival. Moreover, findings offer the possibility of increasingly accurate predictability of tick encounter risk and improved targeting for tick-borne disease preventive measures.
# Genetic variability of *Anaplasma phagocytophilum* and its implication in the ecology of anaplasmosis in Central Europe

M. Derdáková¹,²*, I. Baráková¹,³, L. Pangrácová², G. Carpi³,⁴, M. Stanko¹,², H. Hauffe³, L. Mošanský² and A. Rizzoli³

1. Institute of Zoology, SAS, Bratislava, Slovakia

2. Parasitological Institute, SAS, Hlinkova, Košice, Slovakia

3. Fondazione Edmund Mach, San Michele all'Adige, Trento, Italy

4. The Pennsylvania State University, University Park, PA, USA

*Anaplasma phagocytophilum* is the causative agent of granulocytic anaplasmosis of both medical and veterinary importance. In Europe, *A. phagocytophilum* is transmitted by the Ixodid ticks, mostly *Ixodes ricinus*, however, other tick species such as *Ixodes trianguliceps* may contribute to the enzootic cycle of this bacterium. It is maintained in nature by several vertebrate hosts. Genetic analyses of *A. phagocytophilum* strains can reveal ecological forces that have shaped their genetic diversity, such as the extent of the pathogen vector- and host-specialization. In order to understand the ecology of this pathogen in Central Europe, we have analyzed and compared the genetic variability of different *A. phagocytophilum* strains from questing and feeding ticks collected from vegetation and different vertebrate hosts (roe deer, rodents, birds, sheep, and dogs), as well as blood and biological samples of some vertebrate hosts (rodents and birds), from several sites in two regions of Central Europe (Slovakia and Northern Italy). *A. phagocytophilum* was detected in questing and host feeding *I. ricinus* ticks from all studied sites, as well as from feeding *I. trianguliceps* on rodents and rodents' ear and spleen biopsies. Prevalence of *A. phagocytophilum* in areas with rodents was significantly much lower than in areas without rodents. In areas where *I. trianguliceps* ticks were absent, we did not detect *A. phagocytophilum* in rodents. Phylogenetic analysis based on four genetic loci of *A. phagocytophilum* positive samples have shown that *A. phagocytophilum* genotypes in questing *I. ricinus* and feeding *I. ricinus* from ungulates, birds, and dogs were distinct from genotypes found in rodents and feeding *I. trianguliceps*. The Msp4, DOV, and GroEL sequences of *A. phagocytophilum* genotypes showed considerable heterogeneity but none of the positive questing *I. ricinus* tick was found infected with the rodent genotype that was identical to the genotype detected in *I. trianguliceps*. The GroEL sequences from rodents were closely related to the homologous sequences found in previous studies in questing *I. persulcatus*. Our study from central Europe confirms the previous findings from UK that *A. phagocytophilum* strains have specific associations with two vectors and different reservoir hosts. Unlike in the US, *A. phagocytophilum* genotypes that are associated with rodents are probably transmitted solely by *I. trianguliceps* ticks, therefore these strains may be not of risk for humans, considering the narrow host selection behavior and feeding preference of this tick species.
# Trends in news reports about ticks and the infections they transmit: analysis of The NY Times archives, 1870–2012

Andrew K. Eschner

*Field Veterinary Services, Merial Limited, Duluth, GA, USA*

Ticks and their associated pathogens/maladies have plagued man and beast for thousands of years. While there may be instances of occasional references pertaining to ticks in historical writings, first-hand descriptive accounts of how ticks have affected people, their livestock, and even their pets are rare, if not absent, from our collective conscience prior to the industrial age. The digital-age has transformed many areas of modern life and one of these is the opportunity to copy (scan), store, and quickly sort through large quantities of recorded information. In that vein, The New York Times has digitized a large portion of their US newspaper archives dating back to the 1860s rendering them "searchable" by key words for analysis and reflection. This author became fascinated by the possibility to mine these digital archives of newspaper references and articles pertaining to how ticks have impacted American society over the past 150 years. The intent of this brief oral abstract will be to trace the arc of human concern/observation relating to ticks in our society. By means of a year-by-year sweep of daily life in the newspaper since the 1860s, this paper will report on how ticks have contributed to human concern, anxiety, and financial loss, be it in the agricultural setting (cattle), in new disease discoveries (cattle and human), and their consequences on human/companion animal health.
Positional cloning identifies β-glucuronidase as a key regulator of murine
Lyme arthritis and rheumatoid arthritis

Kenneth K. C. Bramwell¹,²*, Ying Ma¹,², John H. Weis¹,², Paul Allen¹,², James F. Zachary²,³,
Cory Teuscher²,⁴ and Janis J. Weis¹,²

1. University of Utah, Salt Lake City, UT, USA

2. Washington University, St. Louis, MO, USA

3. University of Illinois, Champaign, IL, USA

4. University of Vermont, Burlington, VT, USA

Lyme disease is the most prevalent arthropod-borne illness in the US. Up to 60% of patients develop inflammatory arthritis following infection. Host genetics play a key role, as inbred mouse strains exhibit consistent differences in Lyme arthritis severity. Our lab previously described maximal linkage of disease (LOD, 10.2) severity to the *Bbaa2* locus on mouse chromosome 5. Recently, we developed 15 advanced congenic mouse lines carrying sub-intervals of *Bbaa2* from susceptible C3H on an otherwise uniform resistant B6 genetic background. This led to the positional cloning of a novel disease regulator, the lysosomal enzyme β-glucuronidase (*Gusb*). C3H mice harbor a coding-non-synonymous point mutation in the *Gusb* gene and are *Gusb* hypomorphs, exhibiting a 90% enzymatic deficiency. Two sub-strains of CBA mice carrying the hypomorphic or wild type *Gusb* alleles develop severe Lyme arthritis or are protected, respectively. A *Gusb-null* mouse strain on the B6 background develops severe Lyme arthritis, while heterozygotes are protected. Likewise, heterozygous congenic mice carrying one copy of the C3H *Gusb* deficiency allele are also protected. Transgenic overexpression of *Gusb* in C3H mice to correct the defect led to a profound and highly significant reduction in Lyme arthritis severity, indicating that *Gusb* is a key regulator in this strain. *Gusb* congenic mice also develop more severe disease in the KBxN model of rheumatoid arthritis, suggesting a conserved role. Experiments with radiation chimeras indicate that the lysosomal pool of *Gusb*, not serum level, regulates disease severity and that residual joint resident cells primarily mediate the effect. Severe deficiencies in *Gusb* and many other lysosomal enzymes are known to manifest themselves clinically as lysosomal storage diseases with joint involvement, implying a shared mechanism of pathogenesis.
008

# The role of integrins in vesicular trafficking and immune responses to *Borrelia burgdorferi*

Tanja Petnicki-Ocwieja*, Meghan Marre, Aurelie Kern and Linden T. Hu

Division of Geographic Medicine and Infectious Diseases, Tufts Medical Center, Boston, MA, USA

Host cell responses are tailored to the infecting microbe. Although a relatively simple concept, a continuing area of research has been the elucidation of how a handful of immune receptors generate such a wide range of inflammatory signatures. Cell biology has been shown to be crucial in customizing these responses. Depending on cellular location, signaling results in marked differences in cytokine profile. Our studies with *Borrelia burgdorferi* have allowed us to contribute to a shifting paradigm in our understanding of the role of cellular compartmentalization in Toll-like receptor (TLR2) signaling. In our current work, we explore the relevance of specific motifs within cell surface molecules and the role of intracellular trafficking molecules to define internalization and sorting into intracellular compartments. We show that the β1 integrin affects *B. burgdorferi* dependent TLR2 signaling and internalization of the pathogen through the NPXY motifs in its cytoplasmic domain. Mutations in the tyrosine residue in two of these motifs significantly decrease signaling of *B. burgdorferi* and TLR2 ligands. In addition, we show that this decrease is due to a defect in internalization of *B. burgdorferi* and TLR2-ligands attached to beads. Thus, the NPXY motif of the β1 integrin serves as an internalization signal for TLR2 ligands and *B. burgdorferi*. Upon internalization of *B. burgdorferi* or TLR2 ligand-beads, adaptor protein complexes in the endosomal vesicular pathway play an important role in inflammatory responses induced by these ligands, as cells from knockout mice show a significant decrease in either TLR2 ligand-beads or *B. burgdorferi*-induced signaling. The mode of internalization is crucial to proper trafficking since free TLR2 ligand is not affected in the same way as *B. burgdorferi* or TLR2 ligand-beads. Our study is the first to identify a role for the integrin NPXY motif in the internalization of *B. burgdorferi* and TLR2 ligands and describe vesicular trafficking of *B. burgdorferi* and TLR2.
Regulation of *Borrelia burgdorferi*-induced arthritis by TAM receptors

Diana L. Vesely*, Alexia A. Belperron, Carmen Booth, Carla V. Rothlin and
Linda K. Bockenstedt

Yale University School of Medicine, New Haven, CT, USA

Toll-like receptor (TLR) mediated recognition of *Borrelia burgdorferi* (Bb) components initiate Bb-induced inflammatory responses, which include production of type 1 IFN. In the murine model of Lyme borreliosis, arthritis susceptibility in C3H mice has been linked to robust type 1 IFN expression levels and neutralization of its effects ameliorates disease. Although much is known about the mechanisms through which Bb incites inflammation, relatively little is known about the endogenous pathways that regulate the severity of the inflammatory response to Bb, particularly in the disease-quiescent phase of infection. Recent studies have shown that the Tyro3/Axl/Mer (TAM) family of receptor tyrosine kinases plays a critical role in clearance of apoptotic cell debris and in reducing inflammation associated with TLR stimulation and type 1 IFN cytokine production. In this study, we used *mer*<sup>-/-</sup> and *axl*<sup>-/-</sup> *mer*<sup>-/-</sup> mice to assess effects of these deficiencies on Bb infection and arthritis. Bone marrow-derived dendritic cells (DCs) and macrophages (DMs) from B6 and C3H mice exhibit increased expression of *axl* and *mer* mRNA within 24 h of *in vitro* stimulation with Bb lysate. TAMs exert their anti-inflammatory effects through induction of *socs1/3* expression, and Bb-induced *socs* expression as assessed by qRT-PCR was impaired in DMs and DCs from *axl*<sup>-/-</sup> *mer*<sup>-/-</sup> mice. Although B6 *mer*<sup>-/-</sup> mice infected for 14, 21, or 46 days had equivalent arthritis incidence and severity as B6 WT mice, B6XC3H (F1) *mer*<sup>-/-</sup> mice exhibited significantly enhanced arthritis at 21 days of infection. No differences in pathogen burden assessed by qPCR for Bb *recA* or Bb-induced antibody titers were found. Effects of TAM deficiency on Bb infection were more apparent in *axl*<sup>-/-</sup> *mer*<sup>-/-</sup> mice, with arthritis present in 12/31 B6.129 *axl*<sup>-/-</sup> *mer*<sup>-/-</sup> joints and 0/27 B6.129 control joints by histopathology at 21 days of infection. Arthritis persisted in B6.129 *axl*<sup>-/-</sup> *mer*<sup>-/-</sup> mice at least until 42 days of infection, the latest time point examined. Two-photon intravital imaging of the patellar entheses at this late time point revealed enhanced deposition of amorphous GFP deposits in infected *axl*<sup>-/-</sup> *mer*<sup>-/-</sup> joints (4 of 5 mice examined) in comparison to infected WT joints (0 of 6 mice examined). Taken together, these findings indicate that the TAM pathway may serve to limit Bb-induced inflammation and to facilitate clearance of Bb antigenic remnants that may be deposited in the entheses during infection.
010

# Dysregulation of CD4+CD25HI+ T cells in the synovial fluid of patients with antibiotic-refractory Lyme arthritis

Elise E. Drouin*, Nalini K. Vudattu, Klemen Strle and Allen C. Steere

Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

**Background:** Lyme arthritis, a late stage manifestation of Lyme borreliosis, results from infection with the tick-borne spirochete *Borrelia burgdorferi* (Bb). Lyme arthritis usually resolves after spirochetal killing with antibiotics (antibiotic-responsive arthritis). However, some patients have persistent synovitis that lasts for months or years despite >3 months of antibiotic therapy (antibiotic-refractory arthritis). This latter outcome is postulated to result from either persistent infection, retained spirochetal antigens, or infection-induced autoimmunity. Regardless of the antigenic driver(s) behind this illness, we postulate that this subgroup of patients is unable to properly down-regulate their immune response leading to immune dysregulation and persistent synovitis.

**Methods:** Immune dysregulation in refractory patients was assessed by comparing the phenotype, frequency, and function of CD4+ T effector (Teff) and T regulatory (Treg) cell populations in the synovial fluid (SF) of these two patient groups. Samples from antibiotic-responsive patients (N = 15) were obtained during infection, whereas those with antibiotic refractory patients (N = 16) were obtained during the post-antibiotic period when immune dysregulation is thought to play a critical role. Immune cells were analyzed using flow cytometry and suppression and cytokine assays.

**Results:** Significant differences between the two patient groups were found in the CD4+CD25hi+ population, a population of cells usually composed primarily of Treg cells. Refractory patients had substantially fewer Foxp3-positive Treg cells and an enrichment of Foxp3-negative Teff cells in the CD25hi+ cell population was compared with responsive patients. Additionally, CD4+CD25hi+ cells in refractory patients had significantly greater expression of GITR and OX-40, two co-receptors that augment T cell function. Suppression assays showed that CD4+CD25hi+ T cells in patients with refractory arthritis did not effectively suppress proliferation of CD4 + CD25-negative T cells or the secretion of the inflammatory cytokines IFN- and TNF, whereas those from patients with responsive arthritis did. Finally, in refractory patients, higher ratios of CD25hi+Foxp3-negative/CD25hi+Foxp3-positive cells correlated directly with longer durations of arthritis.

**Conclusion:** Patients with antibiotic-refractory arthritis had lower frequencies of Treg cells in SF and greater expression of T cell co-receptors that are known to enhance Teff resistance to suppression compared to patients with antibiotic-responsive arthritis. These lower frequencies of Treg were unable to effectively suppress Teff cells leading to immune dysregulation and persistent synovitis. Additionally, in refractory patients, higher Teff to-Treg cell ratios in SF were associated with longer post-antibiotic durations of arthritis. These data suggest that the immune responses in these patients were highly augmented leading to immune dysregulation and an antibiotic-refractory Lyme arthritis outcome.
# Delineation of *Borrelia burgdorferi* dissemination kinetics and persistence within murine skin via intravital microscopy

J. P. Lavik*, P. Sekar, V. Shukla, A. L. Nestor-Kalinoski and R. M. Wooten

University of Toledo College of Medicine, Toledo, OH, USA

After transmission via tick-bite, *Borrelia burgdorferi* (Bb) must rapidly detect its new host and adapt such that it can disseminate and persist in the face of potent innate and adaptive immune responses. There is a great interest in identifying these critical adaptations that allow immune evasion, but as an obligate parasite, *in vitro* analyses have provided conflicting results. Thus, there is a great need to develop a more accurate system for assessing spirochete-host interactions *in vivo*. To address this, we have developed a confocal microscopy-based system that allows us to visualize fluorescent, virulent spirochetes within intact murine skin and in real-time. The goal of this study is to use these techniques to accurately describe Bb motility behaviors during early and late infection, the environments preferentially inhabited by the bacterium, and to observe interactions with skin-resident immune cells. Cutaneous inoculation of fluorescent Bb into murine ears revealed that a population of bacteria appeared to fragment and die within minutes of inoculation while other bacteria endured. Spirochete numbers were constant until 48–72 h post-inoculation, when rapid proliferation was observed and the bacteria began to disseminate from the injection site. Maximum Bb numbers were observed at day 5 post-infection followed by a 96-fold drop in numbers by day 12, which correlates with the induction of Bb-specific antibodies. Viable spirochetes disseminate through the dermis and appear to preferentially inhabit collagen-associated strata. Two major motility patterns were observed: backward-forward and extended run. Velocity measurements indicated that during early dissemination, Bb could achieve speeds of 1262 µm/min, while during late dissemination the bacteria exhibited only backward-forward motility with velocities of 237 µm/min. Parallel experiments using mouse strains possessing fluorescent immune cell subpopulations indicated that Bb did interact with Langerhans cells, but no phagocytosis events were observed. Other antigen-presenting cells were observed to contact and internalize Bb, and neutrophils migrated into infection foci within 3–6 h and also internalized substantial numbers of bacteria. However, the majority of spirochetes were able to escape, as their speeds were 40–100 times greater than the fastest immune cells. These findings suggest that Bb motility/chemotaxis is critical for pathogenesis and immune evasion at both early and late stages of infection, and that Bb displays constant motility through all phases of infection. Expansion of this imaging model is anticipated to allow true delineation of host-Bb interactions vital for Lyme disease progression and may subsequently reveal key targets for preventative and curative therapies.
013

Blood donor screening for *Babesia microti*: implications for blood safety

Erin Moritz*, Stephanie Johnson, Colleen Winton, Laura Tonnetti, Rebecca Townsend, Gregory Foster, David Krysztof, Patricia Devine, Philip Molloy, Edward Brissette, Victor Berardi and Susan Stramer

American Red Cross, IMUGEN, Inc., Imugen, Norwood, MA, USA

American Red Cross, Washington, DC

Background: *Babesia microti* is a transfusion-transmissible intraerythrocytic parasite; incidence is increasing in the US and no FDA-licensed screening test is available. We conducted blood donor screening for *B. microti* using investigational antibody and DNA tests in selected counties of Massachusetts and Connecticut.

Methods: Arrayed fluorometric immunoassay (AFIA) and real-time PCR (IMUGEN, Norwood, MA, USA) were performed on EDTA-anticoagulated whole blood samples obtained from individuals presenting to donate at selected blood drives in MA and CT between June and November of 2012. The AFIA utilized native *B. microti* as an antigen substrate with reactivity defined as fluorescence at >1:128 dilution. Repeat AFIA-reactive (pos) samples were tittered to endpoint and tested by research Western blots. DNA was extracted using an automated magnetic bead isolation/ purification system. PCR primers/probes targeted the *B. microti* 18S ribosomal RNA gene; the 50% detection limit is 10 piroplasms/mL. PCR-reactive samples were retested 3 times and those having ≥1 reactive result were considered positive. Initial PCR-reactive samples retesting reactive <3 times were termed inconclusive (inc). PCR-negative samples were tested using an enhanced sensitivity probit function PCR (ePCR). Quantitative PCR was used to determine parasite load; hamster pairs were inoculated to determine infectivity. Reactive donors were invited into a follow-up study for repeat testing and completion of a risk factor questionnaire.

Results: Of 19,625 donors screened, 100 (0.51%) tested pos/inc for *B. microti* antibodies and/or DNA, including five AFIA-neg/PCR-pos, 13 AFIA-pos/PCR-pos, and 80 AFIA-pos/PCR-neg. Two were inconclusive. Four PCR-neg units tested ePCR positive. Parasite loads were 13–870,000 piroplasms/mL; 11/16 hamster pairs developed signs of B. microti infection. Follow-up samples (117 from 82 donors) were obtained, confirming past/current infection; tick exposure (38/71, 53%); and time spent outdoors (68/71, 96%) were reported frequently. Recipient tracing of transfused red cells from prior donations from infected donors revealed 2/8 AFIA-pos recipients, but both reported other risk factors.

Conclusion: Blood donor screening in endemic areas is not only feasible, but also results in the removal of likely-infectious products from the blood supply.
014

Borrelia burgdorferi antigen epitope mapping and development of
a multi-peptide seroassay for Lyme disease

Paul M. Arnaboldi¹,², Mariya Sambir¹,² and Raymond J. Dattwyler¹,²*

1. *Biopeptides Corp.*, New York, NY, USA

2. *New York Medical College*, New York, NY, USA

The two-tier system for the serodiagnosis of Lyme disease was established in the mid-1990s to address non-specificity inherent in whole or lysed *Borrelia burgdorferi*-based immunoassays. This was meant to be temporary until more specific technologies were developed; yet, despite a great deal of research, this system is still in use. Whole *B. burgdorferi* protein antigens, natural and recombinant, contain both epitopes unique to *B. burgdorferi* and epitopes that are similar to those from other bacteria. Peptide-based assays, such as the C6 assay, circumvent issues of nonspecificity by elimination of nonspecific epitopes. While the efficacy of a single-peptide based serological assay is limited by both antigenic variation and the variability inherent in the human immune response, a multi-peptide assay containing specific epitopes from several different *B. burgdorferi* antigens would avoid this limitation while maintaining superior specificity and sensitivity compared to current assays. We performed epitope mapping of the *B. burgdorferi* antigens OspC type K, OspC type A, FliLB, DbpA, DbpB, BmpA, OppA2, BBG33, LA-7, RecA, p66, Bbk32, OspF, p35, CRASP2, and ErpP. Overlapping peptide libraries consisting of 15 AA peptides overlapping by 10 AA were probed with sera from eight patients with clinically active Lyme disease, each of which had multiple well-defined bands on a commercial Lyme immunoblot strip. Multiple epitopes were detected for each antigen. Thirty-six peptides containing epitopes that were detected by a minimum of six of eight patient sera were further evaluated for reactivity with multiple serum samples derived Lyme disease patients presenting to Westchester Medical Center, Gundersen Lutheran Medical Center, or StonyBrook Medical Center. Sera from patients with rheumatoid arthritis and syphilis or from healthy individuals from Lyme disease endemic (southern New York) or non-endemic (New Mexico) areas were used as controls. Peptides from OspC type K (1), OppA2 (2), FliLB (2), Bbk32 (1), OspF (2),p35 (1), ErpP (1), DbpA (2), RecA (1), BmpA (1), and a previously identified modified IR6/FlaB chimera performed well in the initial analysis and are being further evaluated in combination. One such mixture, consisting of peptides derived from OspC, BmpA, OppA, FliLB, VlsE, and FlaB detected seropositivity in 50% of early LD sera obtained from patients upon first presentation with erythema migrans, while C6, the most specific and sensitive antigen, currently detected only 42%. We continue to evaluate peptide combinations with the goal of developing a seroassay containing five or more antigen targets.
015

Rapid point-of-care test for Lyme disease

Victor Kovalenko¹,²*, Ludmila Reshetnikova¹,², Gary P. Wormser¹,² and Andrew E. Levin¹,²

1. *Diagnostic Innovations, Immunetics, Inc., Boston, MA, USA*

2. *New York Medical College, New York, NY, USA*

**Introduction:** The primary diagnostic approach for Lyme disease is based on serology, however, no serologic test is currently available in a rapid, point-of-care format. The ability to obtain accurate serologic results in a primary clinical setting would improve turnaround time for diagnosis, potentially enable resolution of uncertain cases, and reduce unnecessary antibiotic prescription where the diagnosis can be rapidly excluded.

**Methods:** A rapid test in a lateral flow format using a novel highly sensitive colorimetric detection technology was developed, using the C6 peptide augmented by an OspC-derived C10 peptide as antigens. The rapid test detects all antibody classes, takes less than 10 min to carry out from start to finish, and yields brightly colored test and control bands that can be read by eye. The test was evaluated on a panel of 207 serum specimens from patients with Lyme disease confirmed by positive skin or blood culture, of which 173 were from patients presenting with erythema migrans, and the remainder from patients with symptoms of disseminated infection including neuroborreliosis and Lyme arthritis. Rapid test results were compared with results from standard two-tier testing based on C6 ELISA and Lyme Western blots.

**Results:** Serum samples drawn from 59 patients within 1 week after appearance of single erythema migrans lesions were detected with 85% sensitivity by the rapid test vs. 49% sensitivity by C6 ELISA. For 29 multiple erythema migrans patient sera, sensitivities were 100% for the rapid test vs. 85% for the C6 ELISA. Sera from 22 patients with neuroborreliosis were detected with 100% sensitivity by the rapid test vs. 86% by C6 ELISA. Overall sensitivity among 173 patients with erythema migrans was 89% for the rapid test vs. 73% for the C6 ELISA. Analytical sensitivity of the rapid test was equal or better than that of separate ELISAs using C6 and C10 peptides. Specificity of the rapid test in 180 healthy blood donor sera was 100%.

**Conclusions:** The C6/C10 Lyme rapid test has demonstrated high sensitivity in detection of Lyme patient sera, significantly exceeding that of a comparable ELISA. The test may be especially useful in detection of very early stage Lyme disease where other assays are challenged in sensitivity. As a point of care test, the C6/C10 rapid test could offer clinicians an effective tool to enable more accurate in-office diagnosis of Lyme disease and may prove a suitable alternative to the standard two-tier testing algorithm.
# Laboratory diagnosis of Lyme neuroborreliosis – a comparison of three commercial CSF anti-*Borrelia* antibody assays

Anna J. Henningsson¹,²*, Malin Christiansson¹,², Ivar Tjernberg¹,², Sture Löfgren¹,² and Andreas Matussek¹,²

1. Department of Clinical Microbiology, Ryhov County Hospital, Jönköping, Sweden

2. Department of Clinical Chemistry, Kalmar County Hospital, Kalmar, Sweden

**Background:** Lyme neuroborreliosis (LNB) is the most common manifestation of disseminated borreliosis in Europe. The diagnosis is usually based on the medical history, clinical findings, pleocytosis in cerebrospinal fluid (CSF) together with anti-*Borrelia* antibody analyses in paired CSF, and serum samples. The aim of this study was to compare the widely used IDEIA Lyme neuroborreliosis test (Oxoid) using purified native flagellum as single test antigen, with two newly developed tests using several recombinant antigens for the laboratory diagnosis of LNB.

**Materials and Methods:** Serum and CSF from 199 patients investigated for suspected LNB during 2003–2007 were analyzed with the IDEIA Lyme neuroborreliosis (Oxoid), VIDAS Lyme IgG (bioMérieux), and recomBead Borrelia IgM/IgG (Mikrogen) assays. Intrathecal antibody indices (AI) were calculated as recommended by the manufacturers. Fifty-six of the patients had confirmed LNB, i.e., CSF pleocytosis and anti-*Borrelia* antibodies detected in CSF by Lyme Borreliosis ELISA Kit 2nd Generation (DAKO), the routine method used for laboratory diagnosis of Lyme borreliosis 2003–2007 at the Department of Clinical Microbiology, Jönköping, Sweden. Furthermore, the LNB patients were clinically evaluated regarding symptoms and response to antibiotic therapy, and they have been well characterized in previous studies. Forty-two patients had CSF pleocytosis but no anti-*Borrelia* antibodies detected by the DAKO test. The remaining 101 patients had no CSF pleocytosis and no anti-*Borrelia* antibodies detected with the DAKO assay in the CSF, and served as controls.

**Preliminary Results:** The IDEIA Lyme neuroborreliosis test performed with an overall sensitivity (both IgM and IgG AI) of 89% and a specificity of 100%. The VIDAS Lyme IgG test that measures anti-*Borrelia*-IgG AI only showed a sensitivity of 80% and a specificity of 99%. An overall sensitivity (IgM and IgG AI taken together) of 98% and a specificity of 100% were achieved by the recomBead Borrelia test.

**Conclusions:** We conclude that all three assays evaluated in this study performed very well regarding the specificity. However, the preliminary data suggest an improved diagnostic sensitivity with the recomBead Borrelia test as compared to the other two assays.
017

# Proteomic analysis of cerebrospinal fluid distinguishes post-treatment Lyme syndrome as a neurologic entity

Steven E. Schutzer¹*, Thomas E. Angel², Tao Liu², Jonas Bergquist³, Brian A. Fallon⁴ and Richard D. Smith⁴

1. New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ, USA

2. Pacific Northwest National Laboratory, Richland, WA, USA

3. Uppsala University, Uppsala, Sweden

4. Columbia University, New York, NY, USA

The etiology of persisting symptoms, particularly fatigue and cognitive abnormalities, after *Borrelia burgdorferi* infection (CDC-criteria) remains unknown. Its existence as a biologic entity, independent of persisting infection, is controversial. To determine if there is a biological evidence for such symptoms, termed neurologic post-treatment Lyme syndrome (nPTLS), we performed proteomic analysis of cerebrospinal fluid (CSF) because it is akin to a "liquid window" of the brain, reflecting its microenvironment and the symptoms are referable to the central nervous system (CNS). We wanted to determine if the CSF proteome of nPTLS had (1) abnormal proteins and (2) proteins distinguishing nPTLS from normal and other diseases with fatigue and cognitive abnormalities. Our published normal CSF-proteome reference database (2586 proteins; 56% only in CSF, not plasma) allowed this. We used high-abundant protein separation, fractionation, and quantitative ultra-sensitive mass spectrometry (2D-LC-MS/MS) to characterize CSF from (a) nPTLS (n = 30) meeting CDC and NIH-trial criteria for Lyme initially, antibody to *Borrelia*, but no PCR or culture evidence of a persisting post-treatment infection, (b) normal (n = 200), (c) CDC-defined chronic fatigue syndrome (CFS) (n = 45), and (d) other neurologic diseases controls (n = 20). These methods have not been applied to Lyme CSF before. Quantitative differences in protein abundance revealed group-specific differences (p-value < 0.05) (Will show in figures/tables, with full protein names). CSF proteomes of each group contained 2500-2800 identifiable proteins; ~25% were group-specific. nPTLS had 2768 non-redundant proteins - 692 were nPTLS-specific. Both groups, and individuals within the groups, could be distinguished from the other groups by their specific CSF proteins (p < 0.01). Pathway analysis (Ingenuity) demonstrated data could be used for pathogenetic hypothesis generation. A few examples: Complement proteins (C1S, C4B, C1QB, C1QC) were significantly increased (p = 0.005) in nPTLS CSF compared to the others. CDK5-signaling pathway was significantly enriched (p = 0.00009) for proteins in CFS not nPTLS. Some proteins were decreased in both compared to healthy normals. However, quantitative distinguishing differences were found. In neural networks, e.g., axonal guidance, proteins in nPTLS were increased relative to CFS (ADAM23, EPHA7, PFN1, ROCK1). Our data demonstrate that nPTLS has abnormal presence of CSF proteins establishing that it is a neurologic entity and distinguishing it from other conditions with similar features even in the absence of microbiologic evidence of a persisting infection (suggesting it is related to a host response). CSF proteins found during this discovery phase may prove useful to develop and validate host biomarkers for this condition.
018

Metabolic biomarkers and biosignatures for improved diagnosis of Lyme disease

Claudia R. Molins¹*, Laura V. Ashton², Seababrata Mahapatra², Gary P. Wormser³,
Martin E. Schriefer¹ and John T. Belisle²

1. Division of Vector-Borne Diseases, CDC, Fort Collins, CO, USA

2. Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO, USA

3. Department of Medicine, New York Medical College, Valhalla, NY, USA

Antibody-based diagnostics (EIA and WB) for Lyme disease have been widely used in laboratory confirmation for over 20 years. However, substantial limitations that include lack of specificity, sensitivity that is dependent upon the stage of infection, and the inability to distinguish between current and past infections persist. Additionally, results are often interpreted subjectively due to a lack of assay standardization. As an alternative to assessment of antibody profiles, we have applied metabolomics platforms for the discovery of biomarkers and biosignatures that could be used as diagnostics of infection, disease staging, prognostics, and indicators of cure. Specifically, we have analyzed by liquid chromatography–mass spectrometry (LC–MS) the metabolic profiles of human sera collected from patients diagnosed with Lyme disease and that fit into stage-specific categories of early localized Lyme, early disseminated Lyme, late Lyme, or cured Lyme. As control groups, sera from healthy individuals living in regions endemic and non-endemic for Lyme disease were also analyzed. Furthermore, sera from individuals with rheumatoid arthritis were included as controls against late Lyme disease. The LC–MS analysis of human sera resulted in the resolution of several thousand small molecule metabolites. The metabolic profiles of individual groups were compared using the Agilent Mass Profiler Professional software package. A defined set of metabolites with statistically different (p < 0.05) abundances between the following groups were repeatedly observed: early Lyme disease vs. healthy endemic controls, early Lyme disease vs. late Lyme disease, late Lyme disease vs. healthy controls, and late Lyme disease vs. rheumatoid arthritis. Additionally, overlapping metabolites that differentiate early Lyme disease from healthy endemic controls were identified within two separate analyses of two sample sets representing early Lyme disease. The identities of the small molecule metabolites that comprise the various biosignatures are being determined. To further assess the specificity of the biosignatures, sera from look-alike disease and syndromes (syphilis, infectious mononucleosis, multiple sclerosis, severe periodontitis, and fibromyalgia) are being analyzed.
# Toward diminishing the Lyme *Borrelia* threat from nature: a prospective five-year field study report

Luciana Meirelles Richer<sup>1,2,3</sup>, Godefroy Devevey<sup>1,2,3</sup>, Dustin Brisson<sup>1,2,3</sup>, Richard S. Ostfeld<sup>1,2,3</sup>, Nordin Zeidner<sup>1,2,3</sup> and Maria Gomes-Solecki<sup>1,2,3*</sup>

1. *The University of Tennessee Health Sciences Center, Memphis, TN, USA*

2. *The University of Pennsylvania, Philadelphia, PA, USA*

3. *The Cary Institute for Ecosystem Studies, Millbrook, NY, USA*

High prevalence of ticks infected with *Borrelia burgdorferi* has been linked to increased incidence of Lyme disease and areas of high prevalence-high risk have been mapped recently. Preventing the vector from acquiring or transmitting a pathogen is a useful strategy for preventing Lyme disease in humans. Subcutaneous injection of wildlife reservoirs with soluble OspA was previously shown to reduce vector infection prevalence with *B. burgdorferi* the year following intervention. Over the past 10 years, we developed and tested an OspA-based oral reservoir targeted vaccine (RTV) against *B. burgdorferi* and embarked on a comprehensive multi-year analysis to evaluate the effect of this baited vaccine on Lyme borreliosis over time. In Dutchess County, New York, seven plots were included in the field trial: four plots (NY1-NY4) were treated with RTV; three control plots were not treated. Between April and September, from 2007 to 2011, we deployed a total of 75,000 RTVs in Sherman traps and captured 3791 individual white-footed mice for an average deployment of ~20 oral RTVs per mouse. The average number of recaptures between highest and lowest for all grids/all years ranged from 4.3 to 8.6. In contrast to the controls, we observed a decline in NIP in all treated plots: NY1 2007-2011 (53, 51, 43, 34, 14%); NY2 2008-2011 (38, 25, 36, 35%); NY3 2009-2011 (47, 26, 25%) and NY4 2009-2011 (58, 26, 18%). Differences are statistically significant in the two plots that received RTV for the longest periods of time (NY1 2007-2011 $\chi^2$, *p* < 0.0001 and NY2 2008-2011 $\chi^2$, *p* = 0.0069). In NY1, the overall difference between proportions between the first (2007) and the last (2011) year of the study was 45.70% (95% CI, 27.21-64.19%). In NY2, the overall difference between proportions between the first (2008) and the last year (2011) was 14.83% (95% CI, -2.63 to 32.29%). Accounting for declines in NIP validated by time, the difference between the highest (NY1, 45.70%) and lowest (NY2, 14.83%) reduction in NIP leads to a projection of 30.87% decrease in Lyme disease incidence in human populations over 4-5 years following sustained deployment of RTV in the field, as per Ginsberg's P1 = 1 - (1 - k1)n. Here, we report information that enables strategic implementation of a reservoir targeted vaccine by public health officials targeting hot zones for tick infection prevalence that would diminish the *Lyme Borrelia* threat from nature and could reduce Lyme disease cases by one third.
020

Efficacy of a single peridomestic application of acaricide to prevent Lyme and other tick-borne diseases

Alison Hinckley<sup>1,2,3,4*</sup>, James Meek<sup>1,2,3,4</sup>, Julie Ray<sup>1,2,3,4</sup>, Sara Niesobecki<sup>1,2,3,4</sup>,
Katherine Feldman<sup>1,2,3,4</sup>, Erin Jones<sup>1,2,3,4</sup>, Bryon Backenson<sup>1,2,3,4</sup>, Nadia Thomas<sup>1,2,3,4</sup>,
Wilson Miranda<sup>1,2,3,4</sup>, Ashley Kay<sup>1,2,3,4</sup> and Paul Mead<sup>1,2,3,4</sup>

1. Centers for Disease Control and Prevention, Atlanta, GA, USA

2. Connecticut Emerging Infections Program, Yale School of Public Health, New Haven, CT, USA

3. Maryland Department of Health and Mental Hygiene, Baltimore, MD, USA

4. New York State Department of Health, New York, NY, USA

Background: In the Northeastern USA, tick-borne diseases (TBDs) are a major public health concern and identifying effective prevention measures has been challenging. A single springtime application of acaricide has been shown to kill 95% of ticks in controlled studies. Although residential use of pesticides to control ticks is widespread in certain areas, the efficacy of residential pesticides to prevent human TBDs is unknown. Through a TickNET collaboration with the Centers for Disease Control and Prevention and Emerging Infections Programs in Connecticut, Maryland, and New York we sought to evaluate the efficacy of a single residential acaricide application to prevent TBDs.

Methods: In 2011 and 2012, we conducted a randomized, blinded, placebo-controlled trial in select areas of CT, MD, and NY. Participants were consenting adults from freestanding houses with >2 inhabitants and a yard. An enrollment survey was administered to measure select risk factors for TBDs and demographics. Households were randomly assigned to receive a single application of commercially available acaricide (bifenthrin) or placebo on their yard in spring, according to industry standards. Tick drags were conducted 2-3 weeks post-treatment on a 10% sample to verify treatment efficacy. Surveys regarding numbers of ticks attached/ crawling were administered at 1-4 months post-treatment. A final survey to capture self-reported TBDs was administered at 5 months. Self-reported TBDs were validated by medical record review.

Results: A total of 2646 participating households were enrolled over 2 years. Properties ranged from 1/2 to 5 acres in size. Post-treatment tick drags indicated a 75.8% reduction in ticks on treated vs. placebo properties in 2011 and a 48.4% reduction in 2012. However, the number of ticks encountered by participants was similar between the treated and placebo groups at 1-4 months post-treatment in 2011. Similarly, there was no difference in the number of cases of TBD. Analysis of 2012 data is pending.

Discussion: Although acaricide application was effective at reducing tick abundance in both years, data from 2011 indicate no reduction in tick encounters or disease. Reasons for this may include (1) periodomestic exposure is not a principle source of TBD risk; (2) the highest risk in the peridomestic environment may occur in untreated areas (e.g., gardens); or (3) the efficacy of acaricide treatments need to be significantly higher to affect TBD risk. These preliminary results highlight the importance of human studies for understanding the efficacy of tick-borne disease prevention efforts.
021

# Potential for antibiotic creams to prevent Lyme disease spirochete transmission

Joseph Piesman* and Andrias Hojgaard

Centers for Disease Control and Prevention, Atlanta, GA, USA

Although oral antibiotic formulations have been used to prophylactically prevent Lyme disease in clinical trials, the practice of prophylactically treating tick bite via oral doxycycline may not be widely practiced. As an alternative, the use of antibiotic creams placed directly onto the site where a tick was detected and removed may be attractive to residents of tick endemic regions. In the laboratory over the last decade, we have tried to develop an animal model to test optimal regimes for delivering antibiotics prophylactically. Using oral doxycycline, our results have been mixed varying from 23 to 74% efficacy; in addition, the oral doxycycline must be applied right at the time of tick removal. Accordingly, we tested cream formulations that could be applied to the site where ticks were allowed to attach and were removed from mice in a laboratory model. Doxycycline essentially showed no efficacy in this model. But, a 4% azithromycin cream was 100% effective blocking spirochete transmission in a single application when applied to the tick bite site. Moreover, the azithromycin cream was 100% effective when applied up to 72 h after tick removal. We hope to extend these observations that are confirmatory to success with azithromycin cream (albeit using different application regimes) in animal models in Europe.
# Meta-analysis of the clinical presentation and epidemiological features of 128 *Borrelia miyamotoi* cases from Russia

Alexander E. Platonov<sup>1,2,3*</sup>, Denis S. Sarksyan<sup>1,2,3</sup>, Ludmila S. Karan<sup>1,2,3</sup>, Victor V. Maleev<sup>1,2,3</sup>, Durland Fish<sup>1,2,3</sup> and Peter J. Krause<sup>1,2,3</sup>

1. Central Research Institute of Epidemiology, Moscow, Russia

2. State Medical Academy, Izhevsk, Russia

3. The Yale School of Public Health, New Haven, CT, USA

The borreliosis caused by *Borrelia miyamotoi* (Bmt) and transmitted by *Ixodes* ticks is a new human infection first described in Russia (Platonov et al., 2011). Recently, a few human cases of Bmt infection have been identified in the USA (Krause et al., 2012; Gugliotta et al., 2012). We summarize the clinical and epidemiologic features of Bmt infection in 128 cases in Russia in order to provide tools for a more effective search of the disease wherever Lyme disease is endemic. The search and laboratory confirmation of acute Bmt cases were carried out according to a previously published report (Platonov et al., 2011). Patients were enrolled after being bitten by *Ixodes persulcatus* ticks in Central Russia (Republic of Udmurtia and Sverdlovsk Region) in 2009–2012 between May 1 and July 30 that coincides with the peak period of tick activity. They developed flu-like symptoms between 11and 16 days after tick bite. Bmt DNA was found in their blood samples at admission. About 80% of the patients were adults (20–65 years) with a preponderance of males. Clinical symptoms consisted of fever (≥38°C), weakness, headache, and chills in nearly all the patients. Erythema migrans rashes were observed only in 4% of patients. More than that 60% of patients had signs of dysfunction in one or more organs, including the liver (jaundice with increased ALT and AST), kidney (proteinuria, elevated blood creatinine and urea levels), heart (myoglobin in blood, abnormal echocardiography), and lung (abnormal chest X-ray findings). Meningeal signs with normal cerebrospinal fluid were noted in 10% of patients. About half the patients had signs of hypercoagulation (mild initial signs of DIC). Most cases of Bmt infection were mild. Recurrent episodes of fever were observed in about 10% of cases with two or three episodes occurring before the start of antibiotic therapy. No serious sequelae of Bmt infection were noted in our patients, although follow up was limited. Serious neurologic sequelae and severe disease in pregnant women with adverse fetal outcome have been observed with other relapsing fever borreliosis. Additional studies will be needed to assess the global health burden of Bmt infection.
# Long-term clinical outcome after Lyme neuroborreliosis in childhood

Barbro H. Skogman¹,²*, Kajsa Glimåker³, Maria Nordwall ³, Magnus Vrethem ³, Lars Ödkvist³ and Pia Forsberg ³

1. Clinical Department of Pediatrics, Falun General Hospital, Falun, Sweden

2. Center for Clinical Research in Dalarna, Falun, Sweden

3. Faculty of Health Sciences, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden

**Objectives:** To determine long-term clinical outcome in children with confirmed Lyme neuroborreliosis and to evaluate persistent subjective symptoms compared to a control group.

**Patients and Methods:** After a median of 5 years, 84 children with confirmed Lyme neuroborreliosis underwent a neurological re-examination, including a questionnaire. Medical records were analyzed and a control group (n = 84) was included.

**Results:** The total recovery rate was 73% (n = 61). Objective neurological findings, defined as "definite sequelae," were found in 16 patients (19%). The majority of these children had persistent facial nerve palsy (n = 11), but other motor or sensory deficits occurred (n = 5). Neurological signs and/or symptoms defined as "possible sequelae" were found in another seven patients (8%), mainly of sensory character. Nonspecific subjective symptoms were reported by 35 patients (42%) and 32 controls (38%) (ns). Affected daily activities or school performance were reported to the same extent in both groups (23 vs 20%, ns).

**Conclusion:** The long-term clinical recovery rate was 73% in children with confirmed Lyme neuroborreliosis. Persistent facial nerve palsy occurred in 13% whereas other motor or sensory deficits were found in another 14%. Neurological deficits did not affect daily activities or school performance more often among patients than controls and should be considered as mild. Furthermore, nonspecific subjective symptoms such as headache, fatigue, memory, or concentration problems were reported as often among patients as controls, and should not be considered as sequelae after Lyme neuroborreliosis.
# Elevated levels of IL-23 in a subset of patients with post-Lyme disease symptoms following erythema migrans

Klemen Strle¹*, Daša Stupica², Elise E. Drouin¹, Allen C. Steere¹ and Franc Strle²

1. Division of Rheumatology, Allergy and Immunology, Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

2. Department of Infectious Diseases, University Medical Center Ljubljana, Ljubljana, Slovenia

**Background:** Erythema migrans (EM) and its associated symptoms typically resolve with 10–21 days of oral antibiotic therapy. However, about 10% of patients have subjective symptoms after antibiotic treatment, termed post-Lyme disease symptoms. Here, we investigated whether particular immune responses at the time of infection, or thereafter, correlate with the development of post-Lyme symptoms.

**Methods:** In previous European studies (Am. J. Med. 2010;123:79–86; Clin. Infect. Dis. 2012;55:343–350), 510 patients with EM were treated with 10–15 days of antibiotics and re-evaluated over 12 months. Of the 510 patients, 62 (12%) had post-Lyme symptoms. For this study, sera were available from 45 of the 62 patients. For comparison, sera were randomly selected from 41 patients whose symptoms resolved with antibiotic therapy. Serum levels of 23 cytokines and chemokines, representative of innate, and adaptive TH1, TH2, and TH17 immune responses, were assessed at each time point in both patient groups.

**Results:** Significant differences between groups were found for the TH1-associated chemokines, CXCL9 and CXCL10, and the TH17-associated cytokine, IL-23. Of the 86 patients, 85 had detectable levels of CXCL9, 84 of CXCL10, and 41 of IL-23. When stratified by culture results prior to treatment, the 39 patients with negative *Borrelia* cultures had significantly higher levels of CXCL9 and CXCL10, but lower levels of IL-23, than the 47 culture-positive patients, suggesting that TH1-type immune responses were more effective in spirochetal killing. When stratified according to post-Lyme symptoms, the levels of IL-23, but not CXCL9 or CXCL10, were significantly higher prior to therapy in patients who developed post-Lyme symptoms (*P* = 0.04), and these levels remained significantly elevated at subsequent time points. Moreover, of the 41 patients with detectable IL-23 levels, 25(61%) had post-Lyme symptoms, and all seven patients with levels >230 ng/ml had such symptoms. Because all patients became culture negative after antibiotics and because antibody responses to *Borrelia* VlsE C6 peptide declined similarly after treatment in patients with or without post-Lyme symptoms, persistent infection was not a likely cause of these symptoms. In contrast, antibody responses to human endothelial cell growth factor (ECGF), the first known autoantigen in Lyme disease, were more common in patients with post-Lyme symptoms (*P* = 0.07). Furthermore, antibody responses to ECGF, but not to VlsE C6 peptide, correlated directly with IL-23 levels (*R* = 0.5, *P* = 0.02).

**Conclusion:** Our findings indicate that a subset of patients has untoward TH17-type immune responses associated with the development of post-Lyme symptoms. These observations offer a new paradigm for the study of patients with post-Lyme disease symptoms.
# Safety and immunogenicity of a novel multivalent OspA vaccine against Lyme borreliosis in healthy adults: a double-blind, randomized, dose-escalation phase I/II clinical trial

Gerald Aichinger¹, Nina Wressnigg¹, Eva-Maria Pöllabauer¹, Daniel Portsmouth³,
Alexandra Löw-Baselli¹, Sandor Fritsch², Ian Livey³, Brian A. Crowe³, Michael Schwendinger³,
Peter Brühl³, Andreas Pilz³, Thomas Dvorak², Julia Singer², Clair Firth², Benjamin Luft⁴,
Bernhard Schmitt⁵, Markus Zeitlinger⁶, Markus Müller⁶, Herwig Kollaritsch⁶,
Maria Paulke-Korinek⁶, Meral Esen⁷, Peter G. Kremsner⁷, Hartmut J. Ehrlich and
P. Noel Barrett³

1. Vaccine R&D, Baxter BioScience, Vienna, Austria

2. Global R&D, Baxter BioScience, Vienna, Austria

3. Vaccine R&D, Baxter BioScience, Orth/Donau, Austria

4. Stony Brook University Medical Center, New York, USA

5. Health Center Mainz, Mainz, Germany

6. Medical University of Vienna, Vienna, Austria

7. University of Tübingen, Institute of Tropical Medicine, Tübingen, Germany

**Background:** Lyme borreliosis (LB) is caused by *Borrelia burgdorferi* sensu stricto (s.s.) in the US and by multiple *Borrelia* species in Europe and Asia. No human vaccine is available.

**Methods:** The safety and immunogenicity of a multivalent vaccine containing protective epitopes from *Borrelia* outer surface protein (OspA) serotypes 1–6, formulated with or without aluminum hydroxide, was investigated in a study in Austria and Germany. Three hundred *Borrelia*-seronegative adults aged 18–70 years were equally randomized to receive three doses of either 30, 60, or 90 µg, 28 days apart, and a booster 9–12 months after the first immunization. In an extension of this study, 350 seronegative and seropositive subjects were enrolled to receive a similar dosing schedule of either 30 or 60 µg adjuvanted vaccine. Primary endpoints were the frequency and severity of reactions and the antibody responses to OspA serotypes 1–6, as determined by ELISA. The induction of biologically active antibodies was assessed by surface binding and bacterial killing assays. The study is registered with ClinicalTrials.gov, number NCT01504347.

**Results:** Adverse reactions were predominantly mild, and no vaccine-related serious adverse events were reported. The frequency of systemic reactions and the risk for moderate or severe systemic reactions was significantly lower for adjuvanted compared to non-adjuvanted formulations [risk ratio (RR) 0.54, *P* < 0.001; RR = 0.35, *P* = 0.034, respectively]. Both 30 and 60 µg doses were well tolerated in seronegative and seropositive subjects. All doses and formulations induced substantial mean IgG antibody titers against OspA serotypes 1–6 after the primary (range 6944–17,321) and booster (19,056–32,824) immunizations. Post-booster antibody titers against all serotypes were significantly increased in adjuvanted compared to non-adjuvanted formulations (*p* < 0.0001–0.0025). In seronegative subjects, the 30 µg adjuvanted formulation induced the highest post-booster antibody titers [GMT range from 26,143 (95%CI 18,906–36,151) for OspA 2 to 42,381 (31,288–57,407) for OspA 5]. However, in seropositive subjects the 60 µg adjuvanted formulation induced significantly higher post-booster antibody titers [GMT range from 28,735 (95%CI 21,530–38,351) for OspA 1 to 42,381 (95%CI 32,991–54,442) for OspA 5] compared with the 30 µg adjuvanted formulation [GMT range from 12,653 (95%CI 8659–18,489) for OspA 1 to 17,485 (95%CI 12,470–2518) for OspA 5]. Vaccine-induced antibodies bound to and promoted the killing of *Borrelia* strains representing all major human pathogenic species.

**Conclusions:** The novel multivalent OspA vaccine is a promising candidate vaccine to prevent LB in the US and Europe, and possibly globally.
# Epidemiology of Lyme borreliosis in Europe – what does trend analysis tell us?

C. Klier¹, B. Liebl¹, M. Wildner¹, S. O'Connel², A. Sing¹ and V. Fingerle¹*

1. *Bavarian Health and Food Safety Authority, National Reference Centre for Borrelia, Oberschleißheim, Germany*

2. *Lyme Borreliosis Unit, Health Protection Agency Microbiology Laboratory, Southampton General Hospital, Southampton, UK*

Lyme Borreliosis (LB), caused by spirochetes of the *Borrelia burgdorferi* s.l. complex, is the most common tick-borne infectious disease in the northern hemisphere mainly affecting skin, nervous system, or joints. Concise data on the epidemiology of LB in European countries are rare. This review aimed at providing current data on the incidence of LB in Europe. Data were collected by literature search in PubMed, grey literature such as national health registries, as well as personal contacts. Data on LB incidence of 32 European countries were obtained. Incidences among European countries do vary not only due to geographic location but also due to data acquisition (voluntary or mandatory reporting, studies, sentinels, varying enrolment criteria). Geographic distribution shows lowest incidences of LB in southwest Europe (Portugal <0.7 × 10⁵, 2007) and along the Mediterranean coast. Incidences increase northward (Sweden 69 × 1105, 1995) as well as from Western Europe (England and Wales 1.8 × 10⁵, 2011) towards Central Europe (Switzerland 156 × 10⁵, 2011). The highest incidence was reported in Slovenia (242 × 10⁵, 2010). Data on Neuroborreliosis (NB) are available from 10 European countries with national incidences ranging from 0.6 × 10¹ in France to 11 × 10⁵ in Sweden. Time series of incidences reveal major trends: countries with (I) ongoing upward trend such as Estonia, (II) an increase until 2006 followed by a stable phase like Germany, (III) undulating incidences such as Lithuania, and (IV) stable incidence such as Denmark or Norway. Data on the incidence of LB in Europe are heterogeneous and hard to compare due to the various approaches regarding data collection, enrolment criteria, case definitions, or the type and year of study. For the purpose of comparable data, well designed sentinel or prospective studies should be instituted. Although there is increase of LB in some countries over the past decade data do not support the hypothesis that LB is on a rise all over Europe.
Similar Lyme disease risk in contrasting host diversity settings

Maria Diuk-Wasser*, Sarah States, Robert Brinkerhoff, Corrine Folsom O'Keefe and Tanner Steeves

Yale School of Public Health, New Haven, CT, USA

Biodiversity's role in buffering against human infectious disease has been proposed as an ecosystem service widely applicable to a number of infectious diseases. The dilution effect hypothesizes that increased host diversity reduces pathogen transmission by reducing direct or indirect (through vectors) contact between infected and susceptible hosts. Biodiversity may also influence disease risk if pathogens can adapt, or specialize, to different host species. Lyme disease, caused by the bacterium *Borrelia burgdorferi* and transmitted by *Ixodes scapularis* ticks, is a common zoonotic disease in the northeastern United States. A wide range of taxa are competent hosts, but vary in their transmission efficiencies, with *Peromyscus leucopus* thought to be the most competent host. The dilution effect predicts that host communities dominated by *P. leucopus* represent the highest risk to humans, measured as the density of host-seeking *I. scapularis* nymphs. Human risk may also be increased in mouse dominated communities if there is positive selection for *B. burgdorferi* strains that have high persistence in mice (specialized to mouse hosts) and cause disseminating infection in humans. A comparative study of a host species-poor community dominated by *P. leucopus* (Block Island, RI) and a host species-rich community (Mansfield, CT) allowed us to evaluate support for the dilution effect and host specialization hypotheses. We examined three specific predictions of these hypotheses: (1) *B. burgdorferi* nymphal infection prevalence and density of infected nymphs is higher in the species-poor than species-rich community, (2) *B. burgdorferi* genotype diversity is lower in the species-poor than the species-rick community, and (3) "*P. leucopus*-adapted" strains are more prevalent in the species-poor community. We collected mammal-derived larvae and questing nymphs from 2010 and 2011 field seasons and sequenced *B. burgdorferi* DNA to estimate genotype diversity and richness. We found no differences in nymphal infection prevalence and density of infected nymphs between the two communities, providing no evidence for the dilution effect. We also found high amounts of genetic variation in the species-poor community, refuting the host specialization hypothesis. Additionally, proportions of disseminating *B. burgdorferi* strains found primarily in *P. leucopus*, were similar between species-poor and species-rich communities. We found little evidence for either the dilution effect or host specialization theories, suggesting that more research is needed to elucidate the complex factors driving *B. burgdorferi* infection and diversity patterns.
# Predicting the rate of Lyme disease spread in Canada

Nick H. Ogden¹,²*, Patrick A. Leighton¹,² and L. Robbin Lindsay¹,²

1. Public Health Agency of Canada, Ottawa, ON, Canada

2. University of Montreal, Montréal, QC, Canada

Identifying invading tick populations provides early warning for emerging tick-borne diseases that are spreading their geographic range. But how fast do tick-borne pathogens invade after ticks become established? Surveillance data for the tick *Ixodes scapularis* and the agent of Lyme disease *Borrelia burgdorferi* in southern Canada, where these species are invading, revealed a space-time cluster of ticks of low *B. burgdorferi* infection prevalence in southern Quebec signaling the location where tick populations became established beginning in 2004. The cluster disappeared in 2009 indicating a 5 year gap between tick and *B. burgdorferi* invasion. Simulations of a model of *I. scapularis* populations and *B. burgdorferi* transmission identified numbers of immigrating ticks rather than host density and diversity as key determinants of the speed pathogens invade after ticks become established. Greater numbers of immigrating infected nymphs would be expected in central compared to eastern Canada because in source populations in Midwestern USA, nymphal and larval ticks are active in spring when migratory birds can carry ticks north. In northeastern USA, tick populations that are sources for immigrating ticks for eastern Canada, nymphs but few larvae are active in spring. Consequently, we hypothesized that a 5 year gap would occur between tick and *B. burgdorferi* invasion in eastern Canada, but a much shorter gap would occur in central Canada. Consistent with this hypothesis, analysis of surveillance data revealed clusters of ticks with low infection prevalence of >5 years duration in locations in eastern Canada where *I. scapularis* is invading, but a non-significant cluster of only 3 years duration in regions of central Canada where *I. scapularis* is invading. We have identified the speed at which *B. burgdorferi* invades following *I. scapularis* invasion, and that synchrony of larval and nymphal tick activity in spring is a key factor determining the gap between tick and pathogen invasion. This has immediate application in interpreting imminence of Lyme disease risk when surveillance identifies emerging tick populations in Canada and general application in predicting of the speed of invasion of emerging tick-borne pathogens elsewhere.
# The risk of acquiring *Borrelia* infection after a tick bite – a prospective follow-up study

Peter Wilhelmsson¹, Linda Fryland², Pontus Lindblom¹, The TBD STING-Study Group, Johanna Sjöwall³,⁴*, Dag Nyman⁵, Jan Ernerudh², Christina Ekerfelt ², Pia Forsberg³,⁴ and Per-Eric Lindgren¹

1. Division of Medical Microbiology, Linköping University, Linköping, Sweden

2. Division of Clinical Immunology, Linköping University, Linköping, Sweden

3. Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden

4. Clinic of Infectious Diseases, University Hospital, Linköping, Sweden

5. Åland Group, Borrelia Research, Åland, Finland

No previous study has, to our knowledge, investigated all the possible risk factors for the risk of developing Lyme borreliosis (LB), such as rate of *Borrelia* infections in tick population, frequency of human contacts with tick biotopes, length of tick exposure, *Borrelia* species, and number of spirochetes, individual susceptibility to infection, gender and age simultaneously. Therefore, a prospective epidemiological study, the tick-borne diseases (TBD) STING-study, was conducted, which aimed to investigate the clinical outcome and the serological responses in newly tick-bitten individuals and correlate the clinical data with findings in the ticks. The ticks that had bitten humans, blood samples, and questionnaires were collected from newly tick-bitten humans in Sweden and at the Åland Islands, Finland. The participants were followed-up after 3 months with collection of additional blood samples, ticks and questionnaires. The ticks and the feeding-time were analyzed with a USB-microscope, while the tick nucleic acid was analyzed for detection and quantification of both the *Borrelia* 5S–23S IGS and the 16S–23S IGS rRNA genes with two different PCR assays. The blood samples were screened for anti-*Borrelia*-specific antibodies with two different ELISA assays. Seroconversion, i.e., development of new antibodies, was defined as change from sero-negative to sero-positive, or a minimum two-fold increase in OD/index-values. The study subjects were considered to have a probable current *Borrelia* infection if their seroconversions were verified in a strip-immunoassay. Approximately one fourth of the 2154 ticks collected from 1568 study subjects harbored *Borrelia* spirochetes. However, only 51 study subjects (3.3%) showed seroconversion during the study period, and only 22 subjects (1.4%) were diagnosed with clinical LB.

Self-experienced symptoms possibly associated with LB were reported by 15% of the study subjects, but only 45% attended health care due to the experienced symptoms. Thirty-eight percent of all ticks were removed within 24 h.

The study subjects who seroconverted after bite by a *Borrelia*-infected tick removed their ticks later compared to non-seroconverted study subjects bitten by a *Borrelia*-infected ticks (median 62 and 31 h, respectively; *p* < 0.001).

The number of spirochetes in the removed ticks did not differ between seroconverted and non-seroconverted subjects. In conclusion, the risk of developing LB after a bite by a *Borrelia*-infected tick is small, and does not seem to be correlated to the number of spirochetes in the tick. Asymptomatic infections are more common than diagnosed LB among those who become infected with *Borrelia* after a tick-bite from a *Borrelia*-infected tick.
030

# Nymphal blacklegged ticks are not the vector of locally acquired Lyme disease in southeastern states: evidence from tick phenologies and human biting data

Graham Hickling¹*, James Matthias², Abelardo Moncayo³, Ellen Stromdahl⁴ and Jean Tsao⁵

1. The University of Tennessee, Knoxville, TN, USA

2. Florida Department of Health, Tallahassee, FL, USA

3. Tennessee Department of Health, Communicable and Environmental Diseases Services, Nashville, TN, USA

4. U.S. Army Public Health Command, Edgewood, MD, USA

5. Michigan State University, East Lansing, MI, USA

In the northeastern and midwestern United States, the main vector of Lyme disease is the nymphal life stage of the blacklegged tick (*Ixodes scapularis*). One line of evidence for this conclusion is the strong association between disease onset and the timing of nymphal host-seeking, which in the north peaks from late spring to early summer. A second line of evidence is that the majority of *Borrelia*-infected ticks removed from humans in northern states by the Department of Defense's tick test program are nymphs. The incidence of Lyme disease cases reported from southeastern states is two orders of magnitude lower than in northern states, and peaks in summer. Unlike in the north, Lyme case onset dates in the Southeast correlate poorly with nymphal black-legged tick activity, which peaks earlier in the year as a consequence of warm spring temperatures. Furthermore, almost all blacklegged ticks removed from humans in the southeast are adults. We conclude that the summer peak in Lyme disease reports in southeastern states results from (i) northern-acquired cases diagnosed following summer travel to the south and (ii) mis-diagnosis of non-Lyme rashes and infections vectored by abundant, summer-active lone star ticks (*Amblyomma americanum*). Based on the human-biting data, we predict that the (low) numbers of locally acquired Lyme disease cases in southern states will have onset dates coinciding with the winter activity of adult blacklegged ticks rather than the activity of blacklegged nymphs. Analysis of recent Florida Lyme disease case data that distinguished local versus out-of-state infections supports this prediction.
031

OspC phyletic type influences mammalian host range

Richard T. Marconi*, Christopher Earnhart and Delacy Rhodes

Virginia Commonwealth University, Richmond, VA, USA

OspC is an essential virulence factor of the Lyme disease spirochetes. While the precise function of OspC remains elusive, recent evidence suggests that it participates in interactions with host/tick derived ligands that are required for successful transmission. Phylogenetic analyses have revealed the existence of more than 30 genetically stable ospC phyletic types. It has been postulated that ospC diversity may have evolved to allow for expansion of the permissive host range of the Lyme disease spirochetes. Consistent with this, only a limited subset of ospC phyletic types have been demonstrated in isolates recovered from humans. To directly determine if ospC type influences infectivity, a panel of transgenic strains expressing different ospC genes (types A, E, F, H, M, Pko, Pbi, MOD-1, MOK-3a, and MOS-1b) in a *Borrelia burgdorferi* B31 genetic background were generated, characterized, and tested for their ability to establish infection in mice. Interestingly, the introduction of a type Pko ospC gene into the B31 background eliminated infectivity. This observation is consistent with earlier studies that demonstrated that strain Pko does not infect mice and instead preferentially infects other mammalian species. In addition, using a canine infectivity model, we sought to determine if the ospC genotype of strains that infect dogs differs from that reported for humans. *Ixodes scapularis* ticks collected in Rhode Island were fed on purpose bred dogs, tissue biopsies were collected, and the ospC genotype of the infecting strains determined. ospC genotypes rarely recovered from human Lyme disease patients from the same geographic region were found to predominate in canine infections. Collectively, the data presented here indicate a correlation between OspC type and the ability to establish infection in specific mammalian species.
# Genomic characterization of lone star virus, a novel bunyavirus in the *Amblyomma americanum* tick

Andrea Swei¹,²,³, Brandy J. Russell¹,²,³, Samia N. Naccache¹,²,³, Narayanan Veerarghavan¹,²,³, Mark A. Pilgard¹,²,³, Barbara J. B. Johnson¹,²,³ and Charles Y. Chiu¹,²,³*

1. University of California, San Francisco, CA, USA

2. Centers for Disease Control and Prevention, Atlanta, GA, USA

3. San Francisco State University, San Francisco, CA, USA

Bunyaviridae is the largest family of viruses and infect a variety of plant, insect, and animal hosts. Recently, emerging tick-borne bunyaviruses have been discovered in association with acute febrile illness in humans, including the severe fever with thrombocytopenia syndrome virus (SFSTV) in China and heartland virus in the United States. Here, we used unbiased next-generation ("deep") sequencing to characterize the genome of lone star virus (LSV), an unclassified bunyavirus originally isolated from the lone star tick *Amblyomma americanum*. Cultures of LSV in a human (HeLa) and monkey (Vero) cell line demonstrated cytopathic effect within 72 h of incubation, and vacuolization was observed in infected Vero, but not HeLa, cells. In-depth analysis of ~7.6 million shotgun sequencing reads for viral sequences (~2 h on a 64-core computational server) and de novo assembly of the LSV genome (~6 h) was performed using an in-house developed rapid cloud computing-based pipeline for pathogen discovery in metagenomic data generated from clinical/environmental samples. The genome of LSV is a highly divergent phlebovirus in the family Bunyaviridae, sharing <31% amino acid identity with any other virus, and situated in a well-supported phylogenetic clade that includes the tick-borne SFSTV and Heartland viruses. Genomic characterization of LSV is an important step in the development of diagnostic tools to assess the risk of arbovirus transmission by *A. americanum*, a proven disease vector with an expanding geographic range and associated with southern tick-associated rash illness (STARI), which is clinically like erythema migrans but of unknown etiology. This study also underscores the utility of unbiased deep sequencing analyses in investigation of clinical samples for vector-borne pathogens of potential clinical and public health significance.
# Anaplasma phagocytophilum AnkA and HDAC1 recruitment in epigenetic modulation of host gene transcription

Kristen E. Rennoll-Bankert, Sara H. Gilmore and J. Stephen Dumler*

*The Johns Hopkins University School of Medicine, Baltimore, MD, USA*

*Anaplasma phagocytophilum* (Aph) is *Rickettsia* of neutrophils transmitted by *Ixodes* spp. ticks and is the cause of human granulocytic anaplasmosis. Aph infection is associated with differential transcription of many neutrophil genes at a time when cellular function dramatically changes to promote bacterial survival and dissemination. In Aph infection, downregulation of many host defense genes occurs when histone 3 (H3) is deacetylated at their promoters. Cellular histone deacetylase (HDAC) expression and activity increase with Aph infection in HL-60 cells and overexpression promotes infection whereas pharmacologic inhibition and siRNA silencing of HDAC1 abrogate infection. Aph Ankyrin A (AnkA) is secreted via the T4SS, enters neutrophil nuclei, and binds to AT-rich DNA as at the CYBB promoter, where it silences transcription. AT-rich binding regions and repression of transcription are attributes of matrix attachment region binding proteins, like SATB1 that recruit HDAC1 for transcriptional silencing. We hypothesized that AnkA decreases transcription at CYBB by (i) endogenous HDAC activity; (ii) acting as a transcriptional repressor; and/or (iii) recruiting histone deacetylases. To address this hypothesis, recombinant AnkA (rAnkA) was tested for HDAC activity by enzymatic assay. We also used DNA pull-down of a biotinylated CYBB promoter and co-immunoprecipitation (co-IP) with AnkA mab to identify whether HDAC1 is associated with AnkA bound to DNA/chromatin in lysates from *A. phagocytophilum*-infected HL-60 cells or cells transfected and expressing AnkA. rAnkA lacks histone deacetylase activity *in vitro*. HDAC1 was bound to the CYBB promoter when AnkA was also bound, but not in uninfected cells that lack AnkA in DNA pull-down assays. Co-IP using AnkA mab also precipitated HDAC1, but not in cells lacking AnkA. The absence of endogenous HDAC in AnkA cannot explain CYBB H3 deacetylation. However, co-localization of HDAC1 and AnkA in DNA pull-down assays and the physical interaction of AnkA and HDAC1 in co-IP support AnkA recruitment of HDAC1, promoting H3 deacetylation, a closed chromatin configuration and gene silencing. While transcriptional changes often occur with intracellular pathogens that introduce secreted proteins into the host nucleus, this is the first example of a prokaryotic protein that directly binds host DNA and recruits chromatin modifying complexes to alter transcription for at least one locus that governs microbe survival. Whether AnkA or other Aph nuclear effectors contribute to an altered transcription program and function in neutrophils will be important to further investigate.
# The lipid microdomains of *Borrelia*

Jorge L. Benach

Department of Molecular Genetics and Microbiology, Center for Infectious Diseases, Stony Brook University, Stony Brook, NY, USA

The presence of lipid microdomains that contain cholesterol-glycolipids on the outer membrane of the *Borrelia* (in both the Lyme disease and relapsing fever species) is important for structural, nutritional, and pathogenesis reasons. At the ultrastructural level, the lipoproteins OspA and OspB can be seen associated with the discrete microdomains on the outer membrane. Disruption or binding of the lipoprotein by antibodies results in a destabilization of the outer membrane. At the structural level, blind docking analyses have shown that the binding of both OspA and OspB to two of the cholesterol-glycolipids (microdomain forming lipids) could occur via the cleft within the exposed parts of the lipoproteins. It has been long known that cholesterol is required as a component of liquid medium (BSK) to grow *Borrelia* and this is provided as free cholesterol and as a complex with serum lipoproteins and in serum contaminants of albumin. This would suggest that *Borrelia* has the ability to incorporate free cholesterol directly from the medium. *Borrelia* can also acquire cholesterol directly from eukaryotic cell membranes following attachment in what appears to be lipid-lipid interactions. In a similar manner, *Borrelia* also can exchange its cholesterol glycolipids with cells either by direct attachment or by release of outer membrane vesicles charged with the lipids. This two-way exchange not only serves nutritional roles for the spirochetes, but also a means of transferring antigens to the surface of the cells. The cholesterol glycolipids are immunogenic and can elicit the development of antibodies that are cross-reactive with host molecules. Antibodies to the cholesterol glycolipids of *Borrelia* recognize surface molecules in eukaryotic cells and this cross reactivity could have an additive effect to the immunopathogenesis of Lyme disease. The exchange of cholesterol glycolipids and the response to the cholesterol glycolipids derived from the spirochetes on cells or matrix could also be an important aspect of the pathogenesis of the Borrelioses.
# The *Borrelia burgdorferi* adhesin DbpA is bifunctional, binding to extracellular matrix to foster tissue colonization and to the host complement regulatory protein C4BP to promote bloodstream survival

Yi-Pin Lin<fnref n="1" />*, Jon Blevins<fnref n="2" /> and John M. Leong<fnref n="1" />

*Borrelia burgdorferi sensu lato*, the causative agent of Lyme disease, can infect the skin at the site of the tick bite and may subsequently spread via the bloodstream to the heart, nervous system, and joints, leading to multisystemic disease. Complement serves as a first line of immune defense in the bloodstream and pathogenic *Borreliae* produce surface proteins (e.g., Erp or Csp) that bind to host complement regulatory proteins such as Factor H to diminish complement activation and promote serum survival. *B. burgdorferi* produces two surface lipoproteins, decorin binding protein A (DbpA) and B (DbpB) that promote spirochetal binding to the extracellular matrix (ECM) components decorin and dermatan sulfate. A *B. burgdorferi* dbpBA mutant that is incapable of producing either DbpB or DbpA is unable to establish mammalian infection. In this study, we investigated the activities of DbpA that are required for colonization. Expression of wildtype DbpA, but not DbpA C11, which lacks the C-terminal 11 amino acids of DbpA and is incapable to bind to either decorin or dermatan sulfate, promoted stable murine colonization by *B. burgdorferi* dbpBA, indicating that the binding activity of DbpA is critical for colonization. Interestingly, unlike the parental *B. burgdorferi* dbpBA mutant, derivatives that ectopically produce either DbpA or DbpA ΔC11 were detected in the bloodstream at 3 days post-infection and were resistant to human serum *in vitro*. Further, pull-down assays of human serum and quantitative binding analyses using recombinant DbpA revealed that DbpA recognizes the complement regulatory protein C4-binding protein (C4BP) with high affinity (KD = 0.82 ± 0.03 µM). These findings suggest that DbpA is a bifunctional protein that binds ECM to promote tissue colonization and C4BP to protect the spirochete from immune clearance at early stages of infection.

<footnote n="1">1. Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA, USA</footnote>

<footnote n="2">2. Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR, USA</footnote>
**Borrelia burgdorferi infection in *Ixodes ricinus* ticks increases their chances to find a host**

C. Herrmann* and L. Gern

*Laboratory of Eco-Epidemiology of Parasites, Institute of Biology, University of Neuchâtel, Emile Argand, Neuchâtel, Switzerland*

An increasing number of studies suggest that vector-borne parasites are able to alter phenotypic traits in their arthropod vectors in a way that it would enhance microorganism transmission. Ticks transmit pathogens causing human health concerns throughout the world, in particular *Borrelia burgdorferi sensu lato* (s.l.), the agent of Lyme disease. The vector of *B. burgdorferi* s.l. in Europe, *Ixodes ricinus*, is very sensitive to desiccation. During questing for hosts on vegetation, ticks experience desiccating conditions so that they have to regularly move to the litter zone to rehydrate. Moving down and up, vegetation reduces tick energy reserves, which is detrimental for tick survival. In addition, rehydration periods in the litter layer decrease the chances to find a host by reducing questing time. Therefore, a better resistance to desiccation would increase tick lifespan as well as the chances to find a host and subsequently transmit pathogens. This led us to investigate whether *Borrelia* infection increases *I. ricinus* survival under hot and dry conditions and decreases their need for humidity. Results showed (1) that under desiccating conditions *Borrelia*-infected nymphs and adults survived better (43 and 50% survival, respectively) than their uninfected counterparts (28 and 38% survival, respectively) and (2) that *Borrelia*-infected ticks moved less (36%) to an environment favorable for maintaining water balance than uninfected individuals (51%). This corroborated our hypothesis suggesting higher tolerance to desiccation in *I. ricinus* ticks that are infected by *B. burgdorferi* spirochetes. We then investigated whether the higher tolerance to desiccation in infected ticks was related to higher energy reserves. We observed that, in fact, *Borrelia* infection in *I. ricinus* nymphs was associated with higher lipid content (reflecting tick energy reserves). For a given size *Borrelia*-infected ticks harbored 12% more fat than uninfected individuals. Hence, *I. ricinus* ticks benefit from *Borrelia* infection by an increased lifespan (more fat and more resistance to desiccation) and by an increased questing period (less need to move to the litter zone). In fine, *Borrelia* infection enhances tick chances to find a host and to subsequently transmit the pathogen. Although the underlying mechanisms leading to our results remain to be fully understood, our observations contribute significantly to the acknowledgement of the influence of *B. burgdorferi* s.l. spirochetes on phenotypic traits of their tick vector *I. ricinus* optimizing their transmission to the next host.
Transmission of dynamics of *Borrelia turicatae*

William Boyle and Job Lopez*

*Mississippi State University, College Station, MS, USA*

Relapsing fever spirochetes are a globally neglected pathogen and very little is known regarding the events involved with vector colonization and transmission. Our lab studies the *Borrelia turicatae*-Ornithodoros turicata model of relapsing fever spirochetes, and a unique characteristic of pathogen-host interactions when compared to other tick-borne disease is the rapidity of transmission. Depending on the developmental stage, *O. turicata* will feed to repletion within 5-60 min. Given the duration of the bloodmeal, relapsing fever spirochetes have evolved mechanisms to establish long-term infections within the salivary glands and are consequently transmitted in a relatively short period of time. With very little known regarding salivary gland colonization by *B. turicatae*, we have commenced studies to quantify spirochete densities within the tissues and to assess the rapidity of transmission. Time trials were performed where we determined transmission frequencies between ticks that were allowed to attach onto mice for 10 s when compared to ticks that were allowed to engorge (a process that that was completed within 5-10 min). Our findings characterize spirochete densities within the salivary glands and indicate that *B. turicatae* is transmitted within seconds of attachment.
# Inability to utilize certain carbohydrates results in loss of fitness for
*Borrelia burgdorferi* in the enzootic cycle

Arianna Corona*, Radha Iyer, Christopher Pappas, Dionysios Liveris and Ira Schwartz

New York Medical College, Valhalla, NY, USA

**Background:** During the life cycle of *Borrelia burgdorferi*, nutrient availability is an important factor that contributes to the modulation of gene expression for survival in mammals and the tick vector. Glycerol 3-phosphate dehydrogenase (GlpD) is responsible for channeling glycerol utilization to the glycolytic pathway. GlpD mutants were able to complete the tick-mouse cycle, but showed decreased fitness in unfed nymphs and delayed transmission to naïve mice. We aimed to identify the alternate carbohydrate that the GlpD mutant utilizes to survive the tick-mammal cycle in the absence of glycerol utilization. A candidate source is chitobiose, the monomer of chitin, which is found in the tick cuticle. ChbC is a component of the chitobiose transporter required for uptake of chitobiose.

**Methods:** A glpD-chbC double mutant was constructed by disrupting the glpD gene in a chbC mutant strain. The double mutant strain's ability to survive and be transmitted to naïve mice was assessed and compared to wild type and the respective single mutant strains during the tick-mouse cycle.

**Results:** The glpD-chbC double mutant and single mutants were infectious in needle inoculated mice and had spirochete densities in fed larvae comparable to wild type. The double mutant had significantly lower spirochete load in flat nymphs relative to wild type (*p* < 0.05). In fed nymphs, the chbC mutant had significantly lower spirochete loads compared to wild type (*p* < 0.05). Additionally, the chbC and glpD-chbC mutant strains both required a longer duration of tick attachment for spirochetal transmission to naïve mice. Interestingly, whereas a GlpD mutant could successfully infect all mice after 72 h of tick feeding, only one of eight mice could be infected by ticks containing glpD-chbC mutants under similar conditions.

**Conclusion:** The data suggest that glpD mutants are not utilizing chitobiose to survive in the nutrient poor flat nymph. Currently, the identity of the alternative carbohydrate source is not known. The simultaneous disruption of both glpD and chbC appears to have a more severe phenotype for transmission than disruption of either gene alone. Additionally, the data suggest that utilization of chitobiose plays a role in the feeding nymph and is required for subsequent transmission of the spirochete to the mammalian host.
# Microrna MIR-146A is a critical suppressor of arthritis in the mouse model of Lyme disease

Robert Lochhead*, Ying Ma, John Weis and Janis Weis

Department of Pathology, University of Utah, Salt Lake City, UT, USA

Lyme disease is caused by infection with *Borrelia burgdorferi*, a tick-borne spirochete, is endemic to temperate regions in the northern hemisphere, and is the most common vector-borne disease in the United States. Often, infection leads to acute arthritis in humans. Our lab uses the mouse model of Lyme disease to study the molecular mechanisms of arthritis development. MicroRNAs (miRNAs) are small, non-coding RNAs that are involved in posttranscriptional gene regulation. miRNAs act as inhibitors of translation, are capable of targeting many different genes, and are important in regulating many cellular processes, including immune response to pathogens. Abnormal miRNA expression and function has been linked in humans to a variety of immune disorders such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). However, the importance of miRNAs in bacterial infections and inflammation is just beginning to be appreciated, and no studies have been published on how miRNAs regulate immune response to *Borrelia* infection. To identify miRNAs important to suppression of Lyme arthritis, we performed an unbiased screen of miRNA expression during infection with *B. burgdorferi* in the arthritis-susceptible C3H and IL10-/- (B6) mouse strains, as well as in the arthritis-resistant B6 mouse strain. This screen identified several candidate miRNAs differentially regulated during host response to this pathogen. These included many that have been identified previously as being important regulators of immune function. Among these, miR-146a was highly up-regulated in all three strains. Previous studies have shown that miR-146a suppresses NF-kB signaling, and we and others have demonstrated that TLR2/MyD88 dependent activation of NF-kB is a major regulator of immune response in mice and humans, but determining its role in arthritis development has remained elusive. We hypothesized that this miRNA was acting as an immune suppressor during infection with *B. burgdorferi*. Furthermore, we predicted that arthritis-resistant B6 mice lacking miR-146a would harbor fewer bacteria and have more severe arthritis. We therefore infected age-matched miR-146a-/- (B6) mice with *B. burgdorferi*, along with WT B6 controls. As predicted, miR-146a-/- mice developed significantly more severe arthritis at four weeks post-infection. Infected joints also contained significantly fewer bacteria in infected joints, as measured by *B. burgdorferi*-specific 16S rRNA. These data showed that increased arthritis observed in miR-146a-/- mice is due to a defect in host immune regulation, rather than host defense. We used fluorescence-activated cell sorting (FACS) followed by qRT-PCR to identify cellular sources of miR-146a in infected joint tissue. We found that miR-146a was highly enriched in both the myeloid and lymphoid cell fractions, but was not detectible in endothelial and synovial fibroblast-enriched cell fractions. Additionally, joints from infected miR-146a-/- mice contained higher numbers of myeloid cells at two weeks post-infection, suggesting that these are important effecter cells during arthritis development. Consistent with NF-kB dysregulation, mice had significantly higher levels of NF-kB-induced cytokines, such as IL6, and elevated transcript levels of arthritis-promoting chemokines, such as CXCL1 and CXCL10. These data show that miR-146a is critical in regulating NF-kB signaling during infection, and is the first known model to directly link NF-kB dysregulation to arthritis development. This novel model provides the field a much-needed system for elucidating the role of NF-kB in Lyme Arthritis development, and provides a valuable therapeutic target for individuals suffering from Lyme Arthritis and other related immune disorders. Finally, this study highlights the critical role of miRNAs in regulating host response to *B. burgdorferi* infection and inflammation.
Human TLR10 is a pattern recognition receptor with a suppressive function

Marije Oosting<fnref n="1" />,<fnref n="2" />*, Theo S. Plantinga<fnref n="1" />,<fnref n="2" />, Judith M. Bolscher<fnref n="1" />,<fnref n="2" />, Rachel Vestering-Stenger<fnref n="1" />,<fnref n="2" />, Anja Garritsen<fnref n="1" />,<fnref n="2" />, Hans van Eenennaam<fnref n="1" />,<fnref n="2" />, Patrick Sturm<fnref n="1" />,<fnref n="2" />, Bart-Jan Kullberg<fnref n="1" />,<fnref n="2" />, Gijs G. M. Zom<fnref n="1" />,<fnref n="2" />, Ferry A. Ossendorp<fnref n="1" />,<fnref n="2" />, Gosse J. Adema<fnref n="1" />,<fnref n="2" />, Jos W. M. van der Meer<fnref n="1" />,<fnref n="2" />, Mihai G. Netea<fnref n="1" />,<fnref n="2" /> and Leo A. B. Joosten<fnref n="1" />,<fnref n="2" />

Toll-like receptor (TLR) 10 is the only pattern recognition receptor without known ligand specificity and biological function. TLR10 is a part of the genetic locus that encompasses TLR1, TLR2, and TLR6. TLR2 is biologically active as a homodimer and as heterodimers formed with TLR1 or TLR6. Since TLR10 also dimerizes with TLR2, we reasoned that TLR10 may also exert its biological function as a heterodimer with TLR2. When TLR10 on human peripheral blood mononuclear cells (PBMC) was blocked with a specific neutralizing antibody, we found increased cytokine production after stimulation with Pam3Cys or *Borrelia burgdorferi*. These data point to an inhibitory role for TLR10 in conjunction with TLR2. This was further established using monocyte-derived dendritic cells exposed to the same antibody. We found upregulation of sPam3Cys-induced cytokine responses when the antibody was added. Next, we transfected HEK293 cells, either with TLR2 alone or with TLR2 and TLR 10. When cells co-transfected with TLR2 and TLR10 were exposed to TLR2 ligands (Pam3Cys or *Borrelia burgdorferi*), cytokine production was inhibited compared to cells transfected with TLR2 alone. This effect was found to be specific for TLR2, as TLR5-induced cytokine production was not affected by co-transfection with TLR5 and TLR10. Further evidence for the inhibitory role of TLR10 was obtained with RNA silencing of TLR10 in PBMC and in monocyte-derived macrophages; here too, we found enhanced cytokine production when these cells were exposed to TLR ligands. The inhibitory effect of TLR10 on cytokine production is mediated by the p38 MAPK-pathway. Finally, individuals bearing TLR10 polymorphisms display an increased capacity to produce cytokines upon stimulation with TLR2, but not TLR4 ligands in a gene-dose-dependent manner. These results demonstrate that the biological function of the orphan receptor TLR10 is to downregulate TLR2-mediated cytokine responses and establish TLR10 as the first TLR with a suppressive function.

<footnote n="1">1. Department of Medicine, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands</footnote>

<footnote n="2">2. Nijmegen Institute of Infection, Inflammation, and Immunity (N4i), Radboud University Nijmegen Medical Center, Nijmegen, Netherlands</footnote>
Human TLR8 is activated upon recognition of *Borrelia burgdorferi* RNA in the
phagosome of human monocytes

Jorge L. Cervantes¹*, Carson J. La Vake¹, Caitlin O'Connell⁴, Paulo Verardi⁴ and
Juan C. Salazar¹,²,³

1. Department of Pediatrics, University of Connecticut Health Center, Farmington, CT, USA

2. Department of Immunology, University of Connecticut Health Center, Farmington, CT, USA

3. Division of Pediatric Infectious Diseases, CCMC, Hartford, CT, USA

4. Department of Pathobiology and Veterinary Science, University of Connecticut, Storrs, CT, USA

Phagocytosed *Borrelia burgdorferi* (Bb) induces innate immune signals in human monocytes which include transcription of IFN-β. We have previously reported that in response to phagocytosed Bb, TLR8 co-operates with TLR2 in the induction of NF-κB mediated cytokines, while TLR8 is self-amplifying and solely responsible for induction of IFN-β, through IRF-7. Aiming to dissect these phagosomal signaling processes and to identify the ligands, which trigger these responses in humans, we first utilized HEK.293 cells stably transfected with human TLR2, TLR-7, or TLR-8, along with an NFk-B reporter. We show that borrelial lysates and lipidated ipoproteins are internalized and activate human TLR2 in HEK.293 cells. We also demonstrate that Bb RNA activates human TLR8. On the other hand, TLR8 was not activated by Bb DNA, nor by Vaccinia virus DNA, which like Bb is A-T rich. Neither RNA nor DNA induced activation of human TLR7 in HEK.293 cells. We then stimulated highly purified human monocytes with live Bb (MOI 10:1) and equivalent amounts of Bb DNA or RNA. We observed that delivery of Bb RNA and DNA into the phagosomes elicits transcription of IFN-β through IRF7. Using confocal microscopy, we show that TLR8 colocalizes with internalized Bb-RNA bound to its fluorescent delivery vehicle (PEI) and in both early (EEA1) and late endosomes (LAMP-1). RNA staining of internalized live Bb confirms that once internalized the bacterium is confined to the phagosome. Utilizing fluorescent dextran particles and GFP-Bb as well as ethynyluridine-stained Bb RNA, we provide evidence that once phagocytosed, neither Bb nor its RNA leak into the cytosol. Initially associated with recognition of ssRNA of viral origin, our findings demonstrate that phagosomal TLR8 activation occurs upon recognition of bacterial RNA. We conclude that Bb RNA constitutes the TLR8 ligand responsible for eliciting type I IFN signals in human monocytes and these responses take place solely from within the phagosome of human monocytes.
**Borrelia bavariensis** sp. nov. resist complement-mediated killing by a novel immune evasion mechanism

Claudia Hammerschmidt¹,²,³,⁴, Arno Koenigs¹,²,³,⁴, Teresia Hallström¹,²,³,⁴, Volker Fingerle¹,²,³,⁴, Christine Skerka¹,²,³,⁴, Reinhard Wallich¹,²,³,⁴, Peter F. Zipfel¹,²,³,⁴ and Peter Kraiczy¹,²,³,⁴*

1. Institute of Medical Microbiology and Infection Control, University Hospital of Frankfurt, Frankfurt, Germany

2. Leibniz-Institute for Natural Products Research and Infection Biology, Jena, Germany

3. National Reference Center for Borrelia, Oberschleißheim, Germany

4. Institute of Immunology, University of Heidelberg, Heidelberg, Germany

Spirochetes of the *Borrelia burgdorferi sensu lato* complex differ in their resistance to human complement. So far, resistance directly correlates with the ability of *Borreliae* to bind host-derived complement regulators of the alternative pathway (AP), factor H, and factor H-like protein-1 via genetically distinct complement regulator-acquiring surface proteins (CRASPs). In the present study, we investigated *Borrelia bavariensis* to gain deeper insight into the molecular mechanism of immune evasion of this neuroinvasive *Borrelia* species. The contribution of complement regulators of the AP, classical pathway (CP), and terminal pathway in facilitating complement resistance of *B. bavariensis* was examined by employing serum adsorption assays. Interestingly, none of the serum-resistant *B. bavariensis* isolates tested were able to bind complement regulators in detectable amounts. Additionally, two CspA orthologous proteins of *B. bavariensis*, BGA66 and BGA71, were analyzed regarding their intrinsic regulatory activity of the CP and AP by performing hemolytic assays as well as their capacity to inhibit formation of the membrane attack complex. These functional analyses revealed that BGA71 was able to inhibit activation of the CP, while BGA66 blocked activation of both, AP and CP. To examine the mode of action of BGA66 and BGA71 *in vivo*, a serum-sensitive *B. garinii* strain lacking all CRASPs was transformed with individual shuttle vectors harboring the entire BGA66 or BGA71 encoding genes. Employing growth inhibition assays, survival of transformants in non-immune human serum (NHS) was analyzed. Spirochetes producing either BGA66 or BGA71 on their surface were able to survive in the presence of NHS, indicating that both proteins exhibit complement inhibitory activity. Our data reveal a novel immune evasion strategy of *B. bavariensis* that is independent from binding of complement regulators. Moreover, the molecular mechanism involves direct interaction of BGA66 and BGA71 with complement components, resulting in complete protection of *B. bavariensis* from complement-mediated killing.
# The role of DBPA and B adhesins of *Borrelia burgdorferi sensu lato* in the adhesion of *Borrelia* to endothelial cells

Jemiina Salo*, Jaatinen Annukka, K. Viljanen Matti and Hytönen Jukka

Department of Medical Microbiology and Immunology, University of Turku, Turku, Finland

**Objectives:** Lyme borreliosis (LB) is an infectious disease caused by the *Borrelia burgdorferi sensu lato* spirochetes. Systemic dissemination of LB from the initial infection focus on the various target organs is thought to occur via the hematogenous route. However, the molecular mechanisms mediating the interaction between the spirochetes and the vascular endothelial cells remain incompletely understood. There are three major *Borrelia* genospecies, *Borrelia burgdorferi sensu stricto* (Bbss), *Borrelia garinii* (Bg) and *Borrelia afzelii* (Ba) causing disease in humans. Decorin binding proteins (DbpA and B; Dbps) of the three genospecies differ in the amino acid sequence, and our earlier results show that they, indeed, have different binding properties to decorin potentially leading to differences in dissemination and tissue tropism of the bacteria. Interestingly, we have shown that under stationary incubation, DbpA of Bg showed a weak adherence to decorin, whereas under flow the same protein bound to decorin with high affinity. The present study aims at clarifying the role DbpA and B adhesins of different genospecies in the adherence of the spirochetes to human endothelial cells.

**Methods:** A DbpA and B deficient knock out strain was modified to express dbpAB operon of Bg, Ba or Bbss. HUVECs isolated from umbilical cords were cultured in plastic chamber slides. Fluorescently labeled bacteria were allowed to adhere to the cells. The interaction of *Borrelia* with endothelial cells was recorded and quantified with a confocal microscope.

**Results:** Our results showed that adherence of *Borrelia* to HUVECs under stationary incubation was DbpA and/or B dependent. The strains expressing DbpA and B of Bg and Bbss exhibited clear adhesion to the cells, while the binding of the strain with Dbps of Ba was low. As control, the DbpA and B deficient knock out strain did not bind to the HUVECs.

**Conclusion:** Dbps of Bbss, Bg, and Ba contribute differently to HUVEC adhesion of *Borrelia* under stationary incubation. Experiments addressing adhesion of the strains to HUVECs under flow are on-going. We are also working on the identification of the receptor molecules of endothelial cells.
## Lipoprotein succession in *Borrelia burgdorferi*: similar but distinct roles for OspC and VlsE at different stages of mammalian infection

Kit Tilly*, Aaron Bestor and Patricia Rosa

Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT, USA

The spirochete *Borrelia burgdorferi* alternates between tick and mammalian hosts, requiring major changes in gene expression and protein production to adapt to these diverse niches. Among the adaptations observed is a shift among the major outer surface lipoproteins OspA, OspC, and VlsE at different stages of the infectious cycle. We hypothesize that this series of outer surface proteins carry out a basic but essential function, and that OspC and VlsE fulfill this requirement during early and persistent stages of mammalian infection, respectively. Previous work by other investigators suggested that several *B. burgdorferi* outer-surface lipoproteins, including OspA and VlsE, could substitute for OspC at the initial stage of mouse infection, when OspC is transiently but absolutely required. In this study, we assessed whether vlsE and ospA could restore infectivity to a *B. burgdorferi* ospC mutant and found that neither protein effectively compensated for the absence of OspC during early infection. In contrast, we determined that the requirement for OspC extended throughout infection if VlsE was not available. Such OspC-producing (VlsE-deficient) spirochetes can persist in SCID mice, where OspC is not a target of an acquired immune response. Together these results indicate that OspC can substitute for VlsE when the capacity for antigenic variation is irrelevant, but these two abundant lipoproteins are most likely optimized for their related but specific roles during early and persistent infection of mammals by *B. burgdorferi*.
# Role of the DNA mismatch repair protein mutS in VlsE recombination and infectivity of *Borrelia burgdorferi*

Tao Lin*, Lihui Gao and Steven J. Norris

Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, TX, USA

Antigenic variation is a common mechanism of immune evasion, a process by which organisms change their antigenic proteins and thereby gain a selective advantage over individuals that retain parental antigenic determinants, resulting long term of persistent infection. The 35 kDa surface-exposed lipoprotein VlsE undergoes antigenic variation during *Borrelia burgdorferi* infection in mammalian hosts and humans, and is believed to play a major role in the pathogenesis of *B. burgdorferi* and infection. Little is known about the role of biological factor in VlsE recombination. Previous studies indicated that inactivation of ruvA and ruvB genes caused reduced infectivity phenotype and greatly diminished rate of vlsE recombination in *B. burgdorferi*. To assess the roles of potential trans-acting factors in vlsE recombination, we screened transposon mutants of genes known to be involved in DNA replication, recombination, and repair for their effects on infectivity and vlsE recombination including those in BB0797 (mutS), BB0098 (mutS-II), and BB0211 (mutL). Mutation in gene mutS exhibited an intermediate infectivity phenotype in C3H/HeN mice, a mutant in mutS-II was noninfectious in both immunocompetent and severe combined immunodeficiency (SCID) mice. PCR-RFLP results indicated that mutS had reduced vlsE RFLP variability. Analysis of 272 vlsE sequences from *B. burgdorferi* clones indicated that the mutS mutant exhibited a reduced rate of vlsE recombination. The mutS mutant had delayed clearance of parental sequence relative to the parental strain (5A18NP1) and fewer unique vlsE sequences, the mutS mutant was full infectious in C3H/scid mice, indicating that failure to evade the immune response is the basis for compromised infectivity of mutS mutant in immunocompetent. These data provide evidence that MutS is another protein important in vlsE recombination, antigenic variation, and infectivity in *B. burgdorferi*.
Defining the compartmentalization of the *Borrelia burgdorferi* B31 lipoproteome

Shiyong Chen, Alexander S. Dowdell, Christina B. Azodi, Maxwell D. Murphy, Ariel J. Dunn and Wolfram R. Zückert*

*Department of Microbiology, Molecular Genetics and Immunology, University of Kansas School of Medicine, Kansas City, KS, USA*

The current list of known virulence factors of the Lyme disease agent *Borrelia burgdorferi* is dominated by lipoproteins, but the compartmentalization of this class of peripherally anchored membrane proteins within the spirochetal has not been explored comprehensively. To do so, we set out to clone all predicted lipoprotein genes of the *Borrelia burgdorferi sensu stricto* type strain B31 into a pBSV2-derived expression plasmid that provided a 5' flaB promoter for constitutive expression and a 3' hexahistidine tag for protein detection. The resulting plasmid library was then used to transform the B31 clone B31e2, and an established protocol of proteolytic shaving, membrane fractionation, and Western immunoblotting were used to localize the recombinantly expressed lipoproteins. Seventy-seven lipoproteins were localized to the bacterial surface, while 29 were localized to the periplasm. Six of the latter were detected in the inner membrane, and six in the periplasmic leaflet of the outer membrane. Expression of 13 lipoproteins could not be detected, suggesting either C-terminal processing or protein instability; lack of a hexahistidine-tag Western immunoblot signals in a mutant lacking the C-terminal protease CtpA support the latter. Five lipoprotein genes could not be cloned. Ultimately, the results of this study will be crucial in defining the spirochetal cell envelope as well as canonical lipoprotein localization determinants. Furthermore, they may aid in the identification and characterization of new vaccine candidates.
# Use of nonelectrolytes reveals the channel size and oligomeric structure of the *Borrelia burgdorferi* P66 porin

Mari Bonde¹,²,³*, Iván Bárcena-Uribarri¹,²,³, Ingela Nilsson¹,²,³, Roland Benz¹,²,³, Jenifer Coburn¹,²,³ and Sven Bergström¹,²,³

1. Department of Molecular Biology, Umeå University, Umeå, Sweden

2. Biophysics, Jacobs University, Bremen, Germany

3. Immunology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI, USA

P66 is an outer membrane protein found in the Lyme disease spirochete *Borrelia burgdorferi*. The protein forms pores in artificial lipid bilayers with an extremely high single channel conductance of 11 nS in 1 M KCl. In this study, single-channel conductance experiments in the presence of nonelectrolytes of known hydrodynamic radii were performed. This was done to investigate the actual diameter of the P66 channel. Nonelectrolytes with a hydrodynamic radii equal or below 0.34 nm could pass through the pore. Neutral molecules with larger radii could only partially fill the channel or were not able to enter it. From these results, the P66 channel was predicted to be ≤1.9 nm with a 0.8 nm diameter constriction site. When analyzing the complex formed by P66 with PEG 400, PEG 600 or maltohexaose blockage of one P66 single-channel conductance unit was found to occur in about eight subconductance states. This indicates that the P66 channel could be an oligomer of around eight individual channels. BlueNative PAGE and immunoblot analysis confirmed this possible organization of P66 and revealed a protein complex of ~460 kDa. A second dimension SDS PAGE showed that P66 is the only component of this pore-forming protein complex. Ongoing studies aim to obtain the crystal structure of the P66 protein. Large amount of P66 protein purified from a P66 over expressing B31 A3 *B. burgdorferi* strain has been obtained in milligram quantity, which will be further used for structural and biochemical analysis.
**Borrelia burgdorferi** oxidative stress regulator BosR directly represses lipoproteins primarily expressed in the tick during mammalian infection

Peng Wang¹, Zhihui Cheng¹, Michael R. Zianni¹, Poonam Dadhwal², Yasuko Rikihisa¹,
Fang Ting Liang² and Xin Li¹*

1. *The Ohio State University, Columbus, OH, USA*

2. *Louisiana State University, Baton Rouge, LA, USA*

Differential gene expression is a key strategy adopted by the Lyme disease spirochete, *Borrelia burgdorferi*, for adaptation and survival in the mammalian host and the tick vector. Outer surface protein A (OspA) is expressed during tick colonization and repressed in the mammal. The *Borrelia* oxidative stress regulator (BosR) is a DNA-binding protein belonging to the ferric uptake regulator (Fur) family. Here, we show that BosR is required for OspA repression in the mammal. Furthermore, BosR directly binds to the ospA promoter through recognition of palindromic sequences homologous to those recognized by other Fur homologues, but with a less stringent requirement for the spacer length. A total of 156 *B. burgdorferi* genes have been identified to have one or more putative BosR-binding sites in their promoter region. Remarkably, 12 of the 19 genes which were previously identified in a genome-wide microarray study to be >fivefold repressed in the mammal are among this putative BosR regulon. Footprint analysis using *B. burgdorferi* genomic DNA showed that promoter regions containing putative BosR-binding sites were protected by BosR from DNase I digestion. These data suggest that BosR directly represses transcription of many genes, which are down-regulated in the mammal.
Persistence of *Borrelia burgdorferi*, the Lyme disease spirochete, within its arthropod vector involves three distinct genetic programs

Melissa J. Caimano<sup>1,2*</sup>, Star Ems<sup>1,2</sup>, Anna Allard<sup>1,2</sup>, Amit Luthra<sup>1,2</sup>, Christine Diethmaier<sup>1,2</sup>, Robert Gilmore<sup>1,2</sup> and Justin D. Radolf<sup>1,2</sup>

1. University of Connecticut Health Center, Farmington, CT, USA

2. Lyme Borreliosis Laboratory, CDC, Fort Collins, CO, USA

Following acquisition from an infected mammalian host during the larval blood meal, *Borrelia burgdorferi* (Bb) must adhere to rapidly differentiating midgut epithelial cells, resist deleterious substances within the lumen, and alter its metabolic machinery to utilize alternative carbon sources. During transmission, spirochetes presumably must cope with these same bloodmeal-associated stressors, while traversing the midgut and salivary glands in order to gain access to the mammalian host. A major conundrum in Lyme disease research is how acquisition and transmission can elicit ostensibly similar protective/physiologic adaptations, yet evoke such markedly different spirochete behaviors. Spirochetes lacking the alternative σ factor RpoS are impaired in network formation, the initial non-motile phase of biphasic dissemination. Using site-directed and Tn-mediated mutagenesis, we now have identified nine RpoS-upregulated genes (cdr,mcp45, a04, a24, a33, a34, a64, a72, and b09) involved in infection via tick bite, results which collectively support our conceptualization of RpoS-dependent gene expression as a “Go” signal for tick-to-mammal transmission. In contrast, spirochetes within acquiring ticks remain associated predominantly with the luminal surfaces of the midgut and, importantly, do not form networks. Bb within feeding larvae are in an RpoS-OFF state, thereby allowing for de-repression of ospA, the prototypical tick-phase borrelial gene. OspA-deficient Bb survive the larval blood meal, but do not persist through the molt, enabling us to conclude that the RpoS-OFF state contributes to the induction of a tick-adaptive “Stay” signal. Spirochetes lacking the Hk1/Rrp1 two-component system (TCS), which signals via synthesis of c-di-GMP, are destroyed within the midguts of feeding larvae. Structural modeling of the Hk1 periplasmic sensor points to tick- and/or host-derived neurotransmitters as potential activating ligands. Side-by-side comparison of Δhk1, Δrrp1, and Δglp Bb revealed that Hk1/Rrp1-dependent gene products aside from those involved in glycerol uptake and utilization are required to counter noxious substances associated with tick-feeding; we postulate that this occurs via remodeling of the borrelial cell envelope. The requirement for c-di-GMP signaling within feeding nymphs demonstrates that the blood meal-associated stressors that Bb encounters during acquisition also are present during transmission. In summary, the tick phase of Bb’s enzootic cycle appears to involve three distinct genetic programs: RpoS-ON, RpoS-OFF, and Hk1/Rrp1-ON, which provide Go, Stay, and blood meal-survival signals, respectively.
# Bpur, a novel *Borrelia burgdorferi* nucleic acid-binding protein, has global impacts on the proteome and Lyme spirochete infectivity

Brandon L. Jutrasa*, Alicia M. Chenaila, Dustin Carrollb, M. Clarke Millerc, Ashutosh Verma, Haining Zhub and Brian Stevenson

University of Kentucky, Lexington, KY, USA

Lyme disease spirochete, *Borrelia burgdorferi*, persists in nature through infectious cycles between vertebrate hosts and tick vectors. Precise regulation of bacterial gene and protein expression is critical for all borrelial infection processes. We have identified and biophysically characterized a novel *B. burgdorferi* nucleic acid-binding protein, which we named Bpur. Intriguingly, Bpur is highly similar in both sequence and structure to the PUR-domain proteins of humans and other eukaryotes, which are crucial cellular regulatory factors. ChIP-seq and RIP-seq demonstrate that *B. burgdorferi* Bpur binds to both RNA and DNA in live bacteria. We produced a mutant of *B. burgdorferi* in which Bpur is locked into an “ON” state and, using next-generation RNA-sequencing and proteomic analyses, observed significant changes in the levels of over 50 mRNAs and proteins. In addition, the Bpur-ON mutant is significantly impaired in ability to infect mammals. Given these effects, it is not surprising that Bpur expression is tightly regulated, in part, by a self-regulatory mechanism at the post-transcriptional level. Taken together, these features make Bpur an attractive target for development of novel anti-borrelial therapies. Furthermore, since most eukaryotes produce PUR domain proteins, these results provide not only new information on gene regulation in the Lyme spirochete, but on critical aspects of protein-nucleic acid interactions spanning all forms of life.
A001 (EC001)

Gradients in blacklegged tick abundance and infection prevalence with *Borrelia burgdorferi* correlated with distribution of Lyme borreliosis throughout the eastern U.S.A.

Jean I. Tsao¹*, Lorenza Beati², Russell Burke³, Howard Ginsberg⁴, Graham Hickling⁵ and Nicholas Ogden⁶

1. Michigan State University, East Lansing, MI, USA

2. Georgia Southern University, Statesboro, GA, USA

3. Hofstra University, Hempstead, NY, USA

4. USGS Patuxent Wildlife Research Center, Laurel, MD, USA

5. The University of Tennessee, Knoxville, TN, USA

6. Université de Montreal, Montreal, QC, Canada

In the eastern U.S., a striking contrast exists between the distributions of Lyme borreliosis and its vector tick. Whereas most human cases occur in the North, the blacklegged tick is found throughout the region. Ticks infected with the agent of Lyme borreliosis are, however, rarely found in the South. Several hypotheses have been proposed to explain the North–South gradients in Lyme borreliosis and infected ticks. Over the last 3 years, our research team has worked to test these hypotheses by integrating field and laboratory data with modeling studies. We obtained ecological data at nine replicate field sites throughout eastern U.S. and tested three major classes of hypotheses: (1) vertebrate host community composition effects on maintenance cycles of *Borrelia burgdorferi*; (2) climate factors on tick abundance, seasonal activity, and questing behavior; and (3) tick genetic factors affecting the same. Here, I will present the results of geographic variation in tick abundance and infection prevalence to test the hypothesis that blacklegged tick populations are too scarce in the South to maintain *B. burgdorferi*. Blacklegged ticks were present at all our sites, despite great variation in habitat type ranging from swampy lowlands to mixed uplands. Peak questing adult abundance varied markedly, from <10 ticks to ~90 ticks per 1000 m². The density of questing adults was highest in the North, but the abundance at our Florida site ranked higher than our Massachusetts and New Jersey sites. Nymphs were dragged at all sites, but rarely from southern sites. Questing adults were present everywhere, so these results confirm strongly the hypothesis that nymphs in the South rarely host-seek above the leaf litter, and so pose little risk of transmitting pathogens to humans. This would be the case even if they were infected with *B. burgdorferi*; our assays also confirm that adult infection prevalence (range 0–40%) is lowest in the South, although 3% of questing adults at our Florida site were infected. This widely distributed survey for both adult and nymphal ticks shows that: (1) blacklegged ticks are present throughout the South; (2) their densities in the South are generally much lower than in the North, although certain habitats support dense populations; and (3) infection with *B. burgdorferi* is far more prevalent in the north (even though some southern populations are more abundant that some highly-infected northern populations). Additional factors – including vertebrate host composition, host-preference, and host-seeking phenology – may contribute to *B. burgdorferi* maintenance in blacklegged ticks.
A002 (EC003)

Comparison of phenology and pathogen infection rates of *Ixodes scapularis* removed from soldiers at Camp Ripley, MN; Ft. McCoy, WI; and Ft. Indiantown Gap, PA; 1997–2012

Ellen Y. Stromdahl¹, Robyn Nadolny¹,², Shane M. Hall¹, Mary A. Vince¹ and Chad Elkins¹

1. U.S. Army Public Health Command, Edgewood, MD, USA

2. Old Dominion University, Norfolk, VA, USA

Two different observations sparked this comparison among *Ixodes scapularis* removed from Soldiers and submitted to the Department of Defense Human Tick Test Kit Program. First, the adult life stage of *I. scapularis* was more frequently received from Camp Ripley, MN, in the summer months (June and July) than from other U.S. installations. Second, the novel human pathogen, Ehrlichia species "Wisconsin" or "EML" was detected in *I. scapularis* removed from Soldiers only from MN and WI, but not from any other installations within the range of the tick. We compared the phenology and pathogen infection rates of *I. scapularis* removed from Soldiers at Camp Ripley, MN; Ft. McCoy, WI; and Ft. Indiantown Gap, PA. Many variables among the installations are very similar. The three facilities comprise large acreages of brush and woods that provide excellent tick habitat. Anthropophilic tick species *I. scapularis* and *Dermacentor variabilis* are abundant. The Soldiers typically deploy for 2-weeks training sessions and rarely leave post at each installation; they are dressed in identical uniform while training. We compared the abundance of *I. scapularis* adults vs. nymphs removed from Soldiers in the month of June. From 1997 to 2010, for example, there were 55% adults and 45% nymphs (n = 428) from Camp Ripley; 19% adults and 81% nymphs (n = 220) from Ft. McCoy; and 7% adults and 93% nymphs (n = 147) from Ft. Indiantown Gap. We also compared the infection prevalences of *Anaplasma phagocytophilum*, *Babesia microti*, *Borrelia burgdorferi*, and "EML." From 1997 to 2010, greater prevalences of infections of all pathogens were reported from Camp Ripley. Infection with "EML" was reported only from Camp Ripley and Ft. McCoy. Prevalence of pathogen coinfection was also greater at Camp Ripley. These three locations vary in annual temperature cycles, and seasonal synchronicity of *I. scapularis* life stages – in this case, adults and nymphs – may be driven by the differences in the amplitude of the annual cycle of maximum temperature (differences in annual temperature cycles – more extreme vs. milder seasonal climates). This seasonal synchronicity of life stages also affects pathogen infection prevalence (Gatewood et al., 2009).
A003 (EC005)

Blacklegged tick development and survivorship in the eastern United States

Genevieve C. Pang, Jean I. Tsao and Graham Hickling

Michigan State University, East Lansing, MI, USA

In the eastern United States, Lyme borreliosis cases are caused by blacklegged ticks (*Ixodes scapularis*) that are infected with the bacterium *Borrelia burgdorferi*. Lyme borreliosis incidence is about two orders of magnitude lower in the South than the Northeast and upper Midwest. Unpublished results from multi-state surveys indicated that, although blacklegged tick populations are established in southern states, overall population densities are lower compared with the northern states. This difference in Lyme borreliosis risk may be partly due to regional differences in blacklegged tick abundance and activity via their effects on tick-host encounter rates and pathogen transmission and maintenance. We investigated the geographic variation in developmental timing and survivorship of blacklegged ticks in Wisconsin, Rhode Island, Tennessee, and Florida. In order to infer the relative roles of local adaptation and environment, we also examined how development timing and survivorship changed when ticks were transplanted among regions. Pairs of blacklegged tick adults were collected from field sites and fed on laboratory rabbits in the fall of 2011 and spring of 2012. Engorged females produced in the fall were returned to their site of origin in the following winter. Engorged females produced in the spring were redistributed via a reciprocal transplant design across sites in Wisconsin, Rhode Island, Florida, and Tennessee. Engorged females were housed individually in plastic tubes within field enclosures that allowed for airflow, precipitation, and exposure to ambient light. Enclosures were monitored every 2-3 weeks to determine oviposition timing, egg mass survivorship, hatch timing, and larval survivorship. The development timing and survivorship of spring-fed engorged females and subsequent egg masses converged within each site, regardless of female origin. In among-site comparisons, the developmental transitions occurred more rapidly at the southern sites. In the northern sites, the developmental timing of oviposition, egg hatch, and larval questing activity appeared to converge for fall and spring engorged females, although fall-fed female survivorship was lower. However, in Florida, the fall-fed females developed earlier than the spring-fed females and survivorship of the two groups was comparable. Due to a lack of available questing adults, this particular comparison could not be made for the Tennessee site. These results suggest that regional differences in abiotic factors play a key role in observed regional variation in development and survivorship of this species. Improved understanding of the underlying abiotic factors driving blacklegged tick activity and survivorship will contribute to improved regional models for predicting future Lyme borreliosis risk.
A004 (EC006)

Investigating the importance of deer for Lyme disease ecology:
a natural experiment presented by lake Michigan Islands

Jennifer Sidge¹,², Erik Foster³ and Jean Tsao⁴,⁵

1. College of Veterinary Medicine, MSU, East Lansing, MI, USA

2. Comparative Medicine and Integrative Biology, MSU, East Lansing, MI, USA

3. Michigan Department of Community Health, Lansing, MI, USA

4. Department of Fisheries and Wildlife, MSU, East Lansing, MI, USA

5. Large Animal Clinical Sciences, MSU CVM, East Lansing, MI, USA

Lyme disease is emerging across North America as the blacklegged tick, *Ixodes scapularis* invades new areas. How the tick spreads and becomes established is of great interest for public health. White-tailed deer are believed to be the most important hosts for adult *I. scapularis* and critical for its spread and maintenance, but few opportunities exist to investigate tick and pathogen dynamics in their absence. Two Lake Michigan Islands, one with deer and one without, presented just this opportunity. These two Islands are 5 km apart from one other and ~19 km from the mainland. We compared the abundance of ticks on North and South Manitou Islands (Sleeping Bear National Lakeshore) with three mainland sites. We hypothesized that in the absence of deer, ticks, and the Lyme disease pathogen *Borrelia burgdorferi* would be detected at low levels due to dispersal by migratory birds, whereas no larvae should be detected. In 2011, we surveyed for ticks and pathogen by trapping rodents from June to September, and we surveyed for questing ticks from June to October. Sites were visited approximately once per month. We set rodent traps along 6250 m² transects for a total of 300 trap nights per visit. We also dragged for questing ticks along these transects, sampling 3000 m² per visit. We preserved all ticks collected off of hosts and the vegetation in 70% ethanol. We also collected one 2 mm ear punch per mouse per visit, which was also stored in 70% ethanol. In the laboratory, all ticks were identified to species and were assayed for infection with *B. burgdorferi* by targeting the 16S gene using quantitative PCR. Blacklegged ticks were found at all sites and relative abundance of rodents was relatively higher on Islands compared to mainland sites. Questing adult tick relative densities were the highest on the Island with deer. Although tick density was the lowest on the deer-less Island (~75 times lower than on the island with deer) and *B. burgdorferi* was not detected, contrary to our prediction, we found larvae attached to hosts, suggesting the possibility of a self-sustaining tick population in the absence of deer. Continued sampling in 2012 confirmed this finding. This intriguing result suggests the need to examine the roles of alternative mammal hosts (coyotes, rabbits) as well as of birds for maintaining Lyme disease ticks and risk on these Islands.
A005 (EC007)

Apparent range expansion of *Ixodes scapularis* in Virginia – inferences from Lyme disease case data and vector phylogenetic analysis

R. Jory Brinkerhoff, Will F. Gilliam, Rebecca Kelly and David Gaines

Department of Health (DG), University of Richmond, Richmond VA, USA

Lyme disease, caused by the bacterium *Borrelia burgdorferi* and transmitted in the eastern United States by the blacklegged tick (*Ixodes scapularis*) is increasing in incidence and expanding geographically. Recent environmental modeling efforts based on extensive field collections of host-seeking *I. scapularis* predicted a coastal distribution of ticks in mid-Atlantic states as well as an elevational limit of 510 m. However, human Lyme disease cases are increasing most dramatically at higher elevation locations in Virginia, a state that is experiencing rapid emergence of this disease. Our goal was to explore the apparent incongruity between human case data and predicted and observed *I. scapularis* distribution. We hypothesized that density of *B. burgdorferi*-infected ticks should be highest in locations associated with high human disease incidence if epidemiological data represent endemic cases. However, if ticks are limited by factors associated with higher elevations, we would expect higher densities of infected ticks in locations with fewer Lyme disease cases, suggesting that cases at high elevations are either misdiagnosed or represent allochthonous exposure. To test this hypothesis, we sampled *I. scapularis* nymphs at four field sites along an elevational gradient in central Virginia. Each site was visited four times to capture temporal variation in nymphal host-seeking activity. We used PCR amplification of the ospC gene to determine *B. burgdorferi* infection status in individual ticks. We found significantly higher densities of infected ticks at our highest elevation site and overall low densities at lower elevation sites. Phylogenetic analysis of tick mtDNA gene sequences suggests that the ticks at our western and central Virginia sampling sites are more closely related to northern populations than to southern-variant *I. scapularis*, potentially indicating recent population expansion from the North rather than a re-emergence of southern populations. Our results suggest that: (1) the range of *I. scapularis* is increasing to the south, mirroring that of observed Lyme disease cases in humans, (2) *I. scapularis* may not be limited by elevation, at least along the southern Lyme disease expansion front, and (3) Lyme disease cases at higher elevation sites likely represent local exposure and enzootic maintenance of *B. burgdorferi* at these sites. Specific mechanisms to account for *I. scapularis* range expansion have not been identified but may include dispersal and movement of the mammalian and avian host species. Ultimately, identifying the environmental drivers of tick occurrence and expansion will be critical to understanding changes in human risk to tick-borne diseases.
A006 (EC008)

Revive, a surveillance program for tick and tick-borne diseases: molecular detection
of *B. burgdorferi* and *Rickettsia* spp.

I. Lopes de Carvalho, A. S. Santos, M. M. Santos-Silva, R. de Sousa, M. S. Núncio and
Revive Workgroup

Centre for Vectors and Infectious Diseases Research, National Institute of Health, Porto, Portugal

To assess the risk posed by ticks and tick-borne pathogens to Public Health is essential to have reliable data on tick
population, namely main species, status, distribution, and the changing trends in tick distribution and abundance.
Since 2011, the Ministry of Health requested to the National Institute of Health to implement and coordinate a
nationwide surveillance program. During 2012, ticks were collected all over the year from hosts and by flagging
the vegetation. Through the engagement of several health agencies, more than >10.000 ticks from 848 records
across mainland Portugal have been submitted, representing 13 tick species *Dermacentor marginatus*; *Dermacentor
reticulatus*; *Haemaphysalis punctata*; *Hyalomma lusitanicum*; *Hyalomma marginatum*; *Ixodes canisuga*; *Ixodes
hexagonus*; *Ixodes ricinus*; *Ixodes ventalloi*; *Rhipicephalus annulatus*; *Rhipicephalus bursa*; *Rhipicephalus pusillus*;
*Rhipicephalus sanguineus*. The majority of ticks sent to the laboratory were *R. sanguineus* (69%), followed by
*R. pusillus* (16.4%) and *H. marginatum* (9.7%). The other tick species were collected in occasionally, such as
*I. ricinus* with only 0.3%. Sixty-five ticks were collected on humans and *R. sanguineus* was the most frequent (41%)
followed by *I. ricinus* (20%). All the human ticks and about 5% of the ticks collected from vegetation or in hosts
were analyzed for the presence of *Rickettsia* spp. and *Borrelia* spp. *B. lusitaniae* and seven rickettsial species were
detected, namely *R. aeschlimannii*, *R. conorii Malish*, *R. helvetica*, *R. massilae*, *R. raoulti*, and *R. slovaca*. Some ticks
species were found to be only infected with a bacterial agent, as for example *H. marginatum* with *R. aeschlimannii*,
and *D. reticulatus* with *R. slovaca*, when others such as *R. sanguineus* and *I. ricinus* were infected with three different
agents (*R. conorii Malish*, *R. massilae* and *B. lusitaniae*, and *R. helvetica*, *R. monacensis*, *B. lusitaniae*, respectively).
In the future, other tick-borne pathogens will be included in the program. Due to the high density of *Hyalomma* spp.
present in Portugal and the recent detection of Crimeia Congo Haemorrhagic Fever (CCHF) in Spain, the surveillance
of this virus will be implemented during this year. REVIVE program has already produced valuable results and
will be maintained for at least the next 4 years that will allow to observe the trends in the Portuguese tick fauna
and pathogens. Another important output of this network is to raise awareness about tick-borne diseases among the
populations and the healthcare providers as medical doctors and nurses.
A007 (EC010)

Coinfection and population dynamics of *Borrelia bissettii* and *Borrelia burgdorferi* in a tick-murine borreliosis model

Brian Leydet and Fang-Ting Liang

Louisiana State University, Baton Rouge, LA, USA

Lyme Borrelia is the most common vector borne disease in the United States and is caused by spirochetes from the *Borrelia burgdorferi sensu lato* (s.l.) complex. Seven species of Lyme Borrelia have been described in the United States, yet *B. burgdorferi sensu stricto* (s.s.) is widely considered the main cause of human Lyme borreliosis. The most common vector for Lyme spirochetes is hard tick from theIxodidea family. Several species of ticks in the United States are competent vectors of Lyme Borrelia; however, the most common vectors for human Lyme are *Ixodes scapularis* and *Ixodes pacificus*. Many other ticks circulate Borrelia in cryptic enzootic cycles. *Borrelia bissettii* is a Lyme Borrelia spirochete that is endemic to multiple regions in the US and Europe. Past studies show that certain strains of *B. bissettii* cause pathology in murine hosts, while more recently publications implicate *B. bissettii* inhuman disease. Because *B. bissettii* circulates between ticks and small rodents, in a similar fashion to *B. burgdorferi s.s.*, we assessed the ability of *I. scapularis* to transmit *B. bissettii* in a murine transmission model under both single and dual infection scenarios. Utilizing culture and quantitative PCR, we monitored the population of borreliae in the tick from larval acquisition to flat adult and measured infection loads in various murine tissues. Additionally, by monitoring growth, this study demonstrates competition between *B. burgdorferi s.s.* and *B. bissettii* in traditional spirochete media. We show that *I. scapularis* can acquire *B. bissettii* and will maintain the infection trans-stadially at numbers significantly less than *B. burgdorferi s.s.*, yet it is an incompetent vector in a murine-model. Spirochete loads in some murine tissues were significantly higher under single infection when compared to dual infection. Under dual infection, we also characterize the heterogeneous Borrelia population numbers in multiple murine tissues and describe differential acquisition of Lyme Borrelia in ticks. Description of this atypical Borrelia infection in ticks may help elucidate the biological processes undergone by the closely related human pathogenic *B. burgdorferi s.s.* in its tick vector, and expounds our understanding on the ecology of *B. bissettii* a potential emerging human pathogen.
A008 (EC011)

Vector ecology of Lyme disease in New York State: a phylogeographic approach

Melissa Prusinski¹, Camilo E. Khatchikian², Melissa Stone¹,³, P. BryonBackenson¹,
Ing-Nang Wang³, Michael Z. Levy⁴ and Dustin Brisson²

1. Bureau of Communicable Disease Control, New York State Department of Health, New York, NY, USA

2. Department of Biology, University of Pennsylvania, Philadelphia, PA, USA

3. Department of Biological Sciences, State University of New York at Albany, Albany, NY, USA

4. School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

In New York State (excluding New York City), the number of diagnosed Lyme disease cases has steadily increased over the past three decades, corresponding with expansion of the geographic range of *Ixodes scapularis*. The major goal of our group is to investigate the complex interactions between *Borrelia burgdorferi*, *I. scapularis*, and their natural environment that have potentially contributed to the increased geographic range of Lyme disease in New York. Twenty-seven locations in eight counties were surveyed for *I. scapularis* to determine tick abundance, and prevalence and distribution of tick-borne pathogens, over a 4-years period. A total of 11,204 *I. scapularis* (3300 nymphs, 7904 adults) were individually screened for *B. burgdorferi* using polymerase chain reaction, and the frequency and geographic distribution of *B. burgdorferi* genotypes were analyzed. Pathogen prevalence varied geographically and temporally during the time period examined, and was related to measurements of tick density. We used statistical models to evaluate the temporal and spatial heterogeneity of *I. scapularis* populations and to assess the biotic and abiotic factors that influenced species distribution and population growth, utilizing empirical data documenting growth of the tick population. Tick population density increased annually across the geographic range and was positively correlated with mild temperatures, low precipitation, low forest cover, and high urbanization. We also used population genetic tools to infer tick demographic history and migration patterns. We determined that both geographic range and tick population densities are increasing, primarily through progressive local migration events from southern populations to adjacent northern locations. Heterogeneities were found in the distribution and frequency of *B. burgdorferi* major ospC groups, with ospC group K (associated with disseminated Lyme disease in humans) occurring most frequently. Our studies can lead to a mechanistic understanding of how the environmental factors in an ecosystem determine the rate and direction of dispersal of a pathogen and its vector, influencing the corresponding geographic range of human Lyme disease. Our long-term goal is to elucidate the mechanisms contributing to human Lyme disease risk that could be targeted by ecological control strategies or educational campaigns.
A009 (EC012)

Borrelia species DNA in questing *Amblyomma maculatum* (gulf coast ticks) from Mississippi

Jung Keun Lee, Whitney Crow Smith, Flavia Girao Ferrari and Andrea Varela-Stokes

Mississippi State University, Mississippi State, MS, USA

In the United States, *Ixodes scapularis* and *Ixodes pacificus* are primary vectors for the Lyme disease agent, *Borrelia burgdorferi sensu stricto*. Within the United States, the majority of Lyme disease cases are reported from the Northeast and upper Midwest. Few cases are reported elsewhere in the country, including the South, though the region falls within the geographic range of *I. scapularis*. In fact, in the South, the true extent of Lyme disease is under some dispute. The occurrence of Lyme disease in this region is also complicated by the existence of a Lyme disease-like illness, "southern tick-associated rash illness" (STARI). STARI is linked to the bite of *Amblyomma americanum*, the most common tick species in the South, but the agent of STARI, if any, has not been identified. *A. americanum* may harbor *Borrelia lonestari*, an apparently non-pathogenic Borrelia, but is not a competent vector for *B. burgdorferi sensu stricto*. The recent detection of *B. burgdorferi* and *B. lonestari* DNA in *Amblyomma maculatum* removed from deer and canids in Arkansas led to our current investigation of Borrelia in questing *A. maculatum*. In this study, DNA extracts of 306 unfed adult *A. maculatum*, collected from vegetation in Mississippi during 2009 and 2010, were used as template for nested PCR amplification of the *flaB* gene using Borrelia-wide primers. Amplicons were bidirectionally sequenced, and sequences were compared to those in the NCBI database. Of 306 tested tick extracts, two produced amplicons with closest identity (89 and 95%) to *Borrelia turcica*, a species first described nearly a decade ago from Hyalomma ticks in Turkey. Two additional tick extracts produced amplicons with sequences that had 100% identity to *Borrelia hermsii*, and a fifth sequence had 100% identity to *B. burgdorferi*. As *B. hermsii* and *B. burgdorferi* DNA has been used as a positive control in our laboratory, these may represent contaminants. In order to confirm these results and acquire additional information about the unknown Borrelia species identified in the two tick extracts, we will amplify a portion of the 16S rRNA gene using Borrelia genus-wide primers. However, additional experiments will be necessary to evaluate the role of the unknown Borrelia species in human disease. While the infection rate of *A. maculatum* with this uncharacterized Borrelia was quite low (<1%), adult *A. maculatum* can bite humans, possibly adding another layer of complexity to diagnosis of Lyme disease in the South.
A010 (EC013)

Frequency of rickettsiales infections in ticks collected in different regions of México

Sosa-Gutierrez Carolina¹, Vargas-Sandoval Margarita², Torres Javier¹
and Gordillo-Pérez Guadalupe¹

1. Unidad de Investigacion Medica de Enfermedades Infecciosas y Parasitarias, Centro Medico Nacional SXXI, IMSS1, Mexico City, Mexico

2. Facultad de Agrobiología, Universidad Michoacana de San Nicolás de Hidalgo, Uruapan, Michoacán, Mexico

Tick-borne rickettsial diseases (TBRD) are emerging zoonotic diseases caused by obligate intracellular bacteria and transmitted by *Ixodidae* ticks. This group is integrated by *Anaplasma phagocytophilum*, *Ehrlichia chaffeensis*, *Ehrlichia canis*, and *Rickettsia rickettsii*. The distribution of the vector has been described in parts of Asia, Africa, Europe, and America. Six rickettsiae species are pathogens to humans. The transmission of these pathogens to human has been increased due to recreational activities during the Spring and Summer seasons, and the introduction of residential houses in forest areas. In Mexico, a case of ehrlichiosis and anaplasmosis, and epidemic outbreaks of Rickettsiosis have been reported, but the prevalence of TBRD and of infected vectors is unknown.

Objective: To determine the frequency of *A. phagocytophilum*, *E. chaffeensis*, *E. canis*, and *R. rickettsii* in ticks of Mexico.

Materials and Methods: DNA was extracted from 323 ticks collected from 1997 to 2012 in Northwest, Northeast, Central, and Southwest states of Mexico. Infections were determined in extracted DNA by PCR amplification with 16S genes to each agents and CS 78-323 to R. rickettsii.

Results: Eighty-nine (27.5%) ticks were positive for rickettsial infections, 44 (13.6%) for *A. phagocytophilum*, 40 (12.4%) for *E. canis*, 4 (1.2%) for *E. chaffeensis*, and 4 (1.2%) for *R. rickettsii*. The infected ticks were identified as *Rhipicephalus sanguineus* in 74 (83%), *Ixodes scapularis* in 4 (4.5%), *Dermacentor* spp. in 4 (4.5%), *Amblyomma cajennense* in 3 (3.3%), *Boophylus microplus* in 2 (2.2%), *Haemaphisalis leporis-palustris* in 1 (1.1%), and *A. dissimile* in 1 (1.1%). Coinfection with *A. phagocytophilum* and *E. canis* was identified in 3 (3.3%) ticks. *A. phagocytophilum* was more frequent in the Center and East of Mexico, *E. canis* in the Northwest, and *E. chaffeensis* and *R. rickettsii* in the Center of the country.

Conclusion: We report for the first time *A. phagocytophilum* and *E. chaffeensis* infection in ticks from Mexico. *R. sanguineus* was the tick most frequently infected with *Rickettsia*, although its role as a competent vector for *A. phagocytophilum* and *E. chaffeensis* is unknown. Clinicians must consider rickettsial infections in febrile and hemorrhagic cases from zones endemic for infected ticks.
A011 (EC014)

Recent population growth and range expansion of blacklegged ticks in
New York State

Camilo E. Khatchikian¹, Melissa Prusinski², Melissa Stone²,³, P. Bryon Backenson²,
Ing-Nang Wang³, Erica Foley¹, Stephanie N. Seifert¹, Michael Z. Levy¹ and Dustin Brisson¹

1. University of Pennsylvania, Philadelphia, PA, USA

2. New York State Department of Health, New York, NY, USA

3. University at Albany, Albany, NY, USA

Blacklegged tick (*Ixodes scapularis*) populations have recently been discovered in areas of the Northeastern United
States once thought to be devoid of this species. The establishment of these populations may have resulted from either
in situ growth of existing populations that maintained very low densities or by migration and subsequent colonization.
We used Bayesian phylogeographic and demographic approaches to infer the origin and demographic history of
recently detected blacklegged tick populations in the Northeastern United States. The data and results indicate that
the newly detected tick populations are not the product of in situ population growth from an existing low-density
population, but from recent colonization resulting in a geographic range expansion. This expansion in the geographic
range proceeded primarily through progressive local migration events from southern populations to adjacent northern
locations, although long-distance migration events were detected. Demographic phylogeographic inferences that
both the geographic range and the population densities are increasing. The results of these analyses address critical
questions concerning this medically important disease vector, including the history of the populations, the role of
local and long-distance dispersal in their establishment, and the connectedness among new and old populations. These
results may be critical to the development of monitoring and disease control policies in the near future.
A012 (EC015)

Prevalence and distribution of deer tick virus in cervids, Maine, USA

C. B. Lubelczyk¹, S. P. Elias¹, A. Bosco-Lauth², S. Robinson³, M. R. Renell¹, L. B. Smith¹, P. W. Rand¹, E. H. Lacombe¹, S. D. Sears³, G. Ebel², R. P. Smith Jr.¹ and J-P. Mutebi⁴

1. Maine Medical Center Research Institute, South Portland, ME, USA

2. Colorado State University, Department of Veterinary Medicine, Fort Collins, CO, USA

3. Maine Center for Disease Control and Prevention, Augusta, ME, USA

4. US Centers for Disease Control and Prevention, Arbovirus Disease Branch, Fort Collins, CO, USA

Powassan virus (POW) and deer tick virus (DTV) are closely related tick-borne flaviviruses that circulate in North America. The number of POWV and DTV human infection reported to the CDC has increased in recent years suggesting increased circulation of these viruses. In 2010, we examined sera from harvested white-tailed deer *Odocoileus virginianus* and moose *Alces americana* and screened for DTV antibodies. Our aim was to find out if deer and moose are useful sentinels for DTV activity especially since they are frequently parasitized by *ixodid* ticks, the vectors for POWV and DTV. Sera from deer were collected in Maine from 13 counties statewide while moose sera were collected from three northern counties. The sera were screened for neutralizing antibody using a novel recombinant DTV-WNV chimeric virus. In white-tailed deer, 59/328 (18%) contained antibodies to DTV in 12 counties surveyed. In moose, 12/146 (8.2%) samples from three counties showed DTV antibodies. When examined at the county level, no association between antibody-positive deer and the current distribution of *Ixodes scapularis*, the vector of DTV, was noted. Overall, DTV antibody-positive sera were more prevalent in southern counties with established *I. scapularis* populations versus northern counties where the distribution is emergent [25.7% (19/74) in south vs. 15.7% (40/254) in north, chi-square test, P = 0.05]. Our results suggest that transmission of DTV is occurring across a wide geographic area in northern New England, despite a lack of recognized human cases in the region.
A013 (EC016)

Climate change by 2060 and range expansion of deer ticks
(*Ixodes scapularis*) in Maine

Susan Elias¹, Eleanor Lacombe¹, Charles Lubelczyk¹, Peter Rand¹, Robert Smith Jr.¹,
Sean Birkel², Kirk Maasch², June Ficker³, Megan Saunders⁴,⁵, Sara Robinson⁵ and
Stephen Sears⁵

1. Maine Medical Center Research Institute, South Portland, ME, USA

2. Climate Change Institute, The University of Maine, Orono, ME, USA

3. Wells Reserve, Wells, ME, USA

4. University of Southern Maine, Portland, ME, USA

5. Maine Center for Disease Control, Augusta, ME, USA

Over the past two decades, Lyme and other tick-borne diseases have increased with the range expansion of the
deer tick (*Ixodes scapularis*) in Maine. However, there has been insufficient accumulation of degree days (DD) in
northern counties and at higher elevations to permit larval hatch prior to immobilizing temperatures. In this study,
we examined the likelihood that by 2060 *I. scapularis* will be able to complete its life cycle statewide and expand its
range given climate warming. A time series of adult and nymphal deer tick submissions from the public to the Maine
Medical Center Vector-borne Disease Laboratory (1989–2010), a time series of larval burdens on resident songbirds
(1989–2010), and weather data from Portland allowed us to examine correlations between deer tick abundance and
weather. On a monthly basis, adult *I. scapularis* were positively correlated with early (November–December) and
late (March) winter temperatures and negatively correlated with snowfall and snow depth, and July nymphs were
positively correlated with rainfall and negatively correlated with maximum temperature in July. August larvae were
negatively correlated with Julian day at which 1241 DD >6°C were accumulated. The University of Maine Climate
Change Institute used the Weather Research and Forecasting model to simulate climate over the northeastern U.S.
at 24 km resolution for 2060, configured to use lateral boundary input from the global circulation model used in the
Intergovernmental Panel on Climate Change Fifth Assessment Report. The models indicated that the 2060 annual
temperature climates of northern, central, and southern Maine will be like present central Maine, southern Maine, and
Massachusetts, respectively, and that annual rainfall in Maine will increase. We predict that by 2060, given suitable
habitat and hosts: (a) milder winters (warmer with less snow) will increase the length of the questing season for
adult *I. scapularis* particularly in northern and central Maine, likely resulting in increased questing and fecundity,
(b) greater July rainfall is likely to prevent mortality through dessication; and (c) sufficient accumulation of DD >6°C
across the state will permit timely eclosion statewide, so the life cycle of *I. scapularis* can be completed statewide.
Increased incidence in Lyme disease is expected due to increases in *I. scapularis* abundance particularly in the
northern six counties if warming and increased precipitation is accompanied by: (a) changes in forest cover types,
(b) increases in suburbanization, and (c) peridomestic overabundance of deer.
A014 (EC017)

Stability of tularemia natural foci on Martha's Vineyard

H. K. Goethert and S. R. Telford

*Cummings School of Veterinary Medicine, Tufts University, Medford, MA, USA*

Type A tularemia has been endemic to Martha's Vineyard, MA, USA, for the last 12 years, with a median of five cases reported each year. More than half of the cases comprise primary pneumonic exposure, but the fomites remain unidentified. American dog ticks appear to stably maintain the agent, with 2-4% of host seeking adult ticks containing evidence of infection each year in some foci that we study. Host blood meal analyses indicate that the meadow vole feeds most subadult dog ticks, but these hosts are extremely scarce in our study sites, with typical densities of fewer than 5/ha. Because the agent of tularemia has been consistently present over the last decade, the relative absence of rodent hosts appears to be compensated for by diverse mechanisms such as transovarial transmission, co-feeding, enhanced longevity of dog ticks, as well as ancillary vectors such as deerflies. Tularemia ecology on Martha's Vineyard directly contrasts with that of the sympatric deer tick transmitted infection, particularly Lyme disease, with respect to the contribution of chronically infected rodent hosts to the stability of transmission.
A015 (EC019)

# Dominance of *Borrelia lusitaniae* in mountain ecosystem in Northern Slovakia
(Central Europe)

Veronika Taragelová¹, Lenka Mydlová¹, Diana Selyemová¹, Radovan Václav¹,
Branislav Petko² and Markéta Derdáková¹,²

1. Institute of Zoology, SAS, Bratislava, Slovakia

2. Institute of Parasitology, SAS, Košice, Slovakia

Lyme borreliosis (LB) is the most prevalent tick-borne disease in Europe and USA. This disease is caused by spirochetes of the complex *Borrelia burgdorferi sensu lato* (s.l.) and is transmitted to humans by ticks of the genus *Ixodes*. *Borrelia afzelii*, *Borrelia garinii*, and *Borrelia valaisiana* are the most common European genospecies. On the contrary, *Borrelia lusitaniae* predominates in countries of the Mediterranean, such as Portugal, Morocco, and Tunisia. In Slovakia, its prevalence is low and restricted to only a few sites. The aim of our research was to study the expansion of ticks into higher altitudes in mountain ecosystem and its infection with the *B. burgdorferi s.l.* genospecies. Questing ticks were collected by flagging in three altitudes: 600, 800, and 1000 m above sea level (a. s. l.) during 2004, 2006–2011. Genomic DNA of ticks was isolated by alkaline hydrolysis method. Presence of borreliae was tested by specific PCR and further genotyped by RFLP and/or sequencing. Tick abundance was highest at 600 m a. s. l, and lowest at 1000 m a. s. l. Total infection prevalence of *B. burgdorferi* s.l. was 29%. The infection prevalence of borreliae decreased from 38.5 to 4.4% from 600 to 1000 m a. s. l. *B. lusitaniae* was the most frequent genospecies (>60% of positive ticks) during all studied years (2004, 2006–2007, and 2009–2011), with the exception in 2008 when *B. afzelii* predominated (62%). In this year, *B. lusitaniae* was detected in 24.1% of positive ticks. Our study confirmed the spread of *I. ricinus* ticks into the higher altitudes – into new habitats represented by mixed and coniferous forests. Noteworthy, our study site in mountains represented natural focus of *B. lusitaniae*, which is usually associated with lizards and dry xerothermic habitats of Iberian Peninsula. Why is occurrence of *B. lusitaniae* long term and dominant in our study site? Climatic conditions, composition of vegetation and hosts are fundamentally different in comparison with dry habitats of Mediterranean. Further studies are currently undertaken to unravel the ecological associations of this endemic focus of *B. lusitaniae*.
A016 (EC020)

Emergence of *Borrelia burgdorferi*-infected *Ixodes scapularis* and *Peromyscus leucopus* in Ohio

Peng Wang and Xin Li

*The Ohio State University, Columbus, OH, USA*

Currently, Ohio is considered by the Centers for Disease Control and Prevention (CDC) to be non-endemic for Lyme disease, the most common vector-borne disease in the United States. However, according to a passive surveillance program at Ohio Department of Health, the number of *Ixodes scapularis*, the tick that transmits Lyme disease, has increased sharply since 2009. All the three active stages (larva, nymph, and adult) of *I. scapularis* were found in a central Ohio area surveyed in this study. Furthermore, seven nymphal and eight adult *I. scapularis* tested positive for genomic DNA of *Borrelia burgdorferi*, the causative agent of Lyme disease. Antibodies to *B. burgdorferi* antigens were detected in 2 of 10 (20%) captured *Peromyscus leucopus* as well as 41 of 355 (11.5%) dogs residing in Ohio. Collectively, these data indicate that the enzootic life cycle of *B. burgdorferi* is beginning to establish in Ohio.
A017 (EC021)

# Analysis of *Borrelia burgdorferi sensu stricto* strains from the Southeastern United States: relatedness to strains from endemic and non-endemic North American and European localities

Golovchenko Maryna¹,², Rudenko Natasha¹,², Grubhoffer Libor¹,² and James H. Oliver Jr.¹,²

1. Biology Centre, Institute of Parasitology, České Budějovice, Czech Republic

2. The James H. Oliver Jr. Institute of Arthropodology and Parasitology, Georgia Southern University, Statesboro, GA, USA

The first expanded analysis of *ospC* alleles of *Borrelia burgdorferi sensu stricto* from the southeastern United States demonstrated that *ospC* genotypes commonly associated with human Lyme disease in endemic European and North American regions were detected in *B. burgdorferi* strains isolated from non-human biting tick *Ixodes affinis* and rodent hosts in southeastern United States. We found that 30% of southeastern United States isolates were *ospC* allele L strains, a type previously considered to be exclusively European. Majority of the samples were cultured from *I. affinis*, ticks that usually do not bite humans and from ear clips or bladders of two major reservoir hosts of Borrelia in southeastern United States, *Peromyscus gossypinus* and *Sigmodon hispidus*. *OspC* allele L shared the frequency of distribution with *ospC* allele B strains (30.2% each). Two other ospC alleles detected among southeastern strains were alleles G and H (28.3 and 11.3%, respectively). It was believed that *ospC* allele L very rarely, if ever, causes human disease. Analyzed in this study, *B. burgdorferi ospC* allele L strains showed the ability to disseminate in two of the most common natural reservoir hosts in the south, the cotton mouse (*P. gossypinus*) and cotton rat (*S. hispidus*). The limited distribution of both primary reservoir hosts of *B. burgdorferi s.s. ospC* type L strains suggests that globally rare *ospC* allele L might be limited largely to the southeastern United States. Detection of the invasive ospC type B in 30% of samples in strains from the southeastern United States raises the question whether LD risk to humans in this region has been overlooked, or if the geographic distribution of the LD spirochete has evolved over time. The previous analysis of non-human biting *I. affinis* from southeastern United States revealed that they are heavily infected with *B. burgdorferi* (33–35%). Most probably, maintenance vectors, such as *I. affinis* could have significant impact in LD dynamics, helping to maintain high levels of *B. burgdorferi* in reservoir hosts that are later fed upon by bridge vectors often biting human. Following analysis of *B. burgdorferi* strains is necessary to clarify the molecular epidemiology of Borrelia, to evaluate spirochete-vector-host associations, and possible disease risk to humans in a geographic region where the presence of LD is controversial.
A018 (EC022)

Ecology of *Borrelia burgdorferi sensu lato* in diverse habitats of Slovakia

M. Derdáková¹,², V. Tarageľová¹, J. Koči³, E. Ondrisková³, M. Chvostáč⁴, T. Vaculová⁴,
D. Selyemová¹, R. Václav¹, L. Pekárik¹ and B. Peťko²

1. Institute of Zoology, SAS, Bratislava, Slovakia

2. Institute of Parasitology, SAS, Košice, Slovakia

3. Institute of Virology, SAS, Bratislava, Slovakia

4. Faculty of Natural Sciences, Commenius University, Bratislava, Slovakia

In the Central Europe, *Borrelia burgdorferi s.l.* is represented by diverse spectrum of genospecies. Since different clinical manifestation and specific host associations has been assigned to different genospecies, it is crucial to exactly identify borrelia circulating in natural foci. We have analyzed genetic variability and ecological associations of *B. burgdorferi s.l.* indifferent habitats of Slovakia. More than 5500 questing *Ixodes ricinus* ticks were sampled from 10 diverse habitats including mountain spruce forest, lowland deciduous forest, xerothermic steppe, suburban forest, urban park, game reserve, and woodland-farmland ecotone. Moreover, at two sites (urban and rural) host feeding ticks collected from birds, lizards, and rodents were analyzed as well. About 1221 birds, 114 rodents, and 9 lizards were captured. In total, 21.6% of questing ticks were infected with *B. burgdorferi s.l.* The overall prevalence between different sites varied from 7.1 to 46%. Significant differences were observed within single site between different years. *Borrelia afzelii*, *Borrelia garinii*, and *Borrelia valaisiana* were detected at each studied site as the most prevalent with the few exceptions. In sub-mountain area of central Slovakia, *Borrelia lusitaniae* constantly predominated.

*B. burgdorferi sensu stricto* was not detected at every site, but it was commonly found in urban parks from both western and eastern Slovakia. At these sites, *Borrelia bavariensis* and *Borrelia spielmanii* were detected. Borrelia positive ticks in urban habitats harbored mostly *B. garinii* and *B. valaisiana* assigning blackbird population an essential role for circulation of borrelia in towns. The lowest prevalence of Borrelia was found at urban area with very low density of rodents where deer supplemented the blood meal for the *I. ricinus* nymphs and only 3.3% of adults were positive. The infection prevalence in ticks feeding on hosts was 25% in ticks from birds with *B. garinii* and *B. valaisiana*, 13% in ticks from rodents with *B. afzelii* and 26% in ticks from lizards with *B. lusitaniae* predominant.

Further analysis of positive samples by RFLP, SSCP, and sequencing revealed considerable interspecific diversity including presence of two different genotypes of *B. lusitaniae* and *B. spielmanii*. Our results confirmed that local habitat structure and host composition have the highest impact on the occurrence of *B. burgdorferi s.l.* and its genetic variability.
A019 (EC024)

# Spatial and temporal distribution of Lyme disease-infected ticks in the Texas–Mexico border region

Teresa P. Feria¹, Guadalupe Gordillo-Perez², Ana L. Cavazos¹, Margarita Vargas-Sandoval⁶, Alexandra Brown³, Abha Grover³, Javier Torres², Raul Medina³, Ivan Castro-Arellano⁴, Adalberto Pérez de Leon⁵ and Maria D. Esteve-Gassent³

1. The University of Texas-Pan American, Edinburg, TX, USA

2. Centro Médico Nacional SXXI, Distrito Federal, Mexico

3. Texas A&M University, College Station, TX, USA

4. Texas State University San Marcos, San Marcos, TX, USA

5. USDAARS Livestock Insects Research Laboratory, Kerrville, TX, USA

6. Facultad de Agrobiología, Universidad Michoacana de San Nicolás de Hidalgo, Uruapan, 60090 Michoacan, Mexico

Lyme disease (LD) is the most prevalent arthropod-borne infection in the United States, with 33,097 cases of LD reported to the Centers for Disease Control and Prevention (CDC) in 2011. The disease is transmitted to a mammalian host by infected *Ixodes* ticks that carry *Borrelia burgdorferi*. There has been little effort to understand the current and future distribution of *Ixodes* ticks, and no study has focused on the Southern US and in particular in the US–Mexico border. We forecast the present and future distribution of infected *Ixodes scapularis* ticks in the US–Mexico border region of South of Texas and the Northeast of Mexico (Coahuila, NuevoLeon, and Tamaulipas). We correlate geographic data with climatic variables using a maximum entropy approach. Three different general circulatory models were used (CCCMA, CSIRO, and HANDLEY) and two IPCC scenarios (A2 and B2). One hundred models were developed and evaluated via cross validation, dividing the present data into 60 and 40% using the Area under the Curve (AUC) in an ROC plot. The final model and AUC are the average of the 100 models. Binary maps were created to assess stable habitat for *I. scapularis* in the future. The final models had AUC >0.80, which indicate that the models are robust. Our findings show suitable habitat now and in the future on the east and central parts of Texas and Northern Mexico. Consequently, these areas should be monitored to control *I. scapularis* tick infections and to assess the risk for humans to acquire Lyme disease in this geographical area. Further studies are currently being done to determine the reservoirs involved in the maintenance of this zoonotic pathogen in Southern US.
A020 (EC025)

Distribution of *Borrelia burgdorferi* in humans, dogs, and ticks in Southern USA

Ivan Castro-Arellano¹*, Abha Gorver², Erin McGregor², Alexandra Brown², May Boggues³
and Maria D. Esteve-Gassent²

1. Department of Biology, Texas State University, San Marcos, TX, USA

2. College of Veterinary Medicine and Biomedical Sciences, Texas A&M, College Station, TX, USA

3. School of Mathematical and Statistical Sciences, Arizona State University, Tempe, Arizona, USA

Lyme Borreliosis (LB) is the most prevalent arthropod borne disease in the US with 33,097 cases reported to CDC in 2011. In 2009, the case definition of LB was revised, and currently, the CDC differentiates between confirmed and probable cases for this disease. Thus, since 2009, in Texas, the ratio of probable versus confirmed cases has been repetitively 2:1. This can be attributed to different causes, from lack of testing to the presence of genetically distinct Borrelia species, strains, and/or *Ixodes scapularis* biotypes particular to Southern US. To determine the distribution of LB in Texas, we collected ticks from deer hunting stations, veterinary clinics, and animal shelters in several counties across the state. In addition, we evaluated the sero-prevalence of Lyme disease-infected dogs in Texas during the year 2011–2012. The Texas Department of State Health Services provided with the cases of confirmed human Lyme disease in the State from the year 2000 to 2012 to compare the spatial distribution with the samples studied in this project. All ticks (574 samples) were identified to species and their DNA was purified individually. Each tick sample was tested by PCR utilizing primers specific to five genetic markers [intergenic region 16SrRNA-23SrRNA (IGR), flaB, p66, ospC, and ospA], in order to determine which *Borrelia burgdorferi* strains are circulating in Texas and how they are distributed across the state. Positive PCR results were confirmed by sequencing. Sequences were used in population genetic studies. In addition, serum from 750 dogs suspected to carry Borrelia, was evaluated by ELISA and Western blot to determine their LB status. We collected *I. scapularis* ticks from 11 Texan counties. Fifty percent of the collected *I. scapularis* were infected with *B. burgdorferi senso stricto*. Most of the infected *I. scapularis* ticks and canine samples were collected from East and Central Texas from November through March, which overlaps with an increase of outdoor activities (e.g., deer-hunting season). In addition, the distribution of infected humans and dogs co-localize around recreational areas where Borrelia infected ticks were found. Other tick species such as *Amblyomma americanum* and *Amblyomma cajenenses* were also found to be infected with *B. burgdorferi* (26.6 and 48%, respectively). Clearly, infected ticks are present in southern US and likely follow different phenology timing as compared to more northern sites. We are currently evaluating potential wildlife reservoir species and their role to maintain the pathogen in the environment.
A021 (EC026)

Borrelia burgdorferi infection of *Peromyscus leucopus*, a major reservoir of Lyme disease

Vanessa J. Cook, Elisabeth Baum and Alan G. Barbour

University of California Irvine, Irvine, CA, USA

Background: The white-footed mouse *Peromyscus leucopus* serves as a reservoir for *Borrelia burgdorferi* and as a host for its tick vector in eastern North America. *B. burgdorferi* appears to be well adapted to its reservoir, as evidenced by little or no disability of their hosts and serial infections of *P. leucopus* by different *B. burgdorferi* strains. But because *P. leucopus* populations are genetically diverse, they may differ in their innate and adaptive immune responses to infection.

Methods: Laboratory-reared *P. leucopus* were infected with different *B. burgdorferi* strains, which were representative of the diversity of the species in the northeast US. Infection after 4–5 weeks was characterized by the following:
(1) analysis of antibody responses to various antigens, including OspC, VlsE, and BBK07/12 proteins;
(2) measurement of cytokine, chemokine, and acute phase reactants in the blood; and (3) quantitative assessment by PCR of spirochete burdens in different tissues.

Results: The experimental infections of the animals revealed both strain specific antibody responses and differences in responses between individual animals. Spirochetes were present in various tissues, but the overall burdens differed by *B. burgdorferi* strain and by individual mouse. In spite of disseminated infection, levels of selected cytokines and chemokines were not substantially elevated in infected animals compared to controls.

Conclusion: These findings could not only contribute in further understanding the ecology of *B. burgdorferi* in North America but also aid in the development and implementation of a field-based vaccine directed at *P. leucopus*.
A022 (EC027)

Tick-borne pathogens in *Ixodes ricinus* ticks collected from autumn migratory birds and breeding birds in Switzerland

E. Lommano¹, C. Dvořák², L. Vallotton³,⁴, L. Jenni⁵ and L. Gern¹

1. Laboratory of Eco-Epidemiology of Parasites, University of Neuchâtel, Neuchâtel, Switzerland

2. Vallorbe, Switzerland

3. Natural History Museum of Geneva, Genève, Switzerland

4. Jaman's Group, Lausanne, Switzerland

5. Swiss Ornithological Institute, Sempach, Switzerland

Birds are important in the ecology and circulation of tick-borne pathogens. They can disperse infected ticks over long distances but they can also act as reservoir hosts for tick-borne infectious agents. From 2007 to 2010, autumn migrating birds were captured at the Col de Jaman (VD), a pre-alpine pass situated at 1512 m above sea level and breeding birds were captured at two sites on the Swiss Plateau. A total of 4558 birds belonging to 71 species were caught and 1205 ticks were collected on them. Each tick was analyzed individually by reverse line blotting and real-time PCR for the presence of various tick-borne pathogens: *Borrelia* spp., *Rickettsia* spp., *Anaplasma/Ehrlichia*, and tick-borne encephalitis virus (TBEV). Altogether, 11.4% of birds (22 species) were infested and 39.8% of them (15 species) were carrying infected ticks. Bird species belonging to the genus *Turdus* were the most frequently infested with infected ticks. Eleven tick-borne pathogens were identified in bird-feeding ticks: six *Borrelia* species (*Borrelia garinii*, *Borrelia valaisiana*, *Borrelia afzelii*, *Borrelia bavariensis*, *Borrelia miyamotoi* and *Borrelia burgdorferi* ss), two *Rickettsia* species (*Rickettsia helvetica* and *Rickettsia monacensis*), *Anaplasma phagocytophilum*, TBEV, and *Candidatus Neoehrlichia mikurensis*. *Borrelia* spp. (19.5%) and *R. helvetica* (10.5%) were predominantly detected whereas *A. phagocytophilum* (2%), *R. monacensis* (0.4%), and TBEV (0.2%) were only sporadically observed. Interestingly, *C. Neoehrlichia* mikurensis was identified in a few ticks (3.3%), mainly from chaffinches. Results suggested that the common blackbird (*Tarquinio merula*) and the tree pipit (*Anthus trivialis*) might act as reservoir hosts for *A. phagocytophilum* and *R. helvetica*, respectively. Our study emphasizes the role of birds in the natural cycle of tick-borne pathogens that are of human medical and veterinary relevance in Europe.
A023 (EC028)

Contribution of tick phenology to geographical gradients in Lyme borreliosis in
eastern and central North America

Howard S. Ginsberg¹,²,³,⁴,⁵,⁶, Eric L. Rulison¹,²,³,⁴,⁵,⁶, Genevieve Pang¹,²,³,⁴,⁵,⁶, Russell L. Burke¹,²,³,⁴,⁵,⁶, Lorenza Beati¹,²,³,⁴,⁵,⁶, Graham J. Hickling¹,²,³,⁴,⁵,⁶ and Jean I. Tsao¹,²,³,⁴,⁵,⁶

1. USGS Patuxent Wildlife Research Center, Laurel, MD, USA

2. University of Rhode Island, Kingston, RI, USA

3. Michigan State University, East Lansing, MI, USA

4. Hofstra University, Hempstead, NY, USA

5. Georgia Southern University, Statesboro, GA, USA

6. University of Tennessee, Knoxville, TN, USA

Lyme Borreliosis is the most prevalent vector-borne zoonosis in North America, with major foci in the northeastern and north central United States, but with lower prevalence of infection in the southern states. We tested the hypothesis that the shorter active season in the north results in a two-year life cycle, with the older nymphal stage of *Ixodes scapularis* (which had been infected the previous year), active earlier in the season than the younger uninfected larval stage, whereas the longer active season in the southern U.S. disrupts this efficient transmission pattern. We sampled immature *I. scapularis* from 2010 to 2012 by flagging and dragging and from host animals at nine sites in the northeastern, north central, southeastern and south central U.S. Tick phenologies showed the hypothesized pattern from north to south along the east coast, with the classic northern (nymphs before larvae) pattern in Massachusetts, with larvae before nymphs during the summer in Florida (which would disrupt the transmission dynamic) but with some additional immature activity in early spring, and with intermediate patterns at mid-Atlantic sites. However, east-west patterns were also evident, with broad overlap of larval and nymphal phenologies in Wisconsin, and a northern pattern in Tennessee. Therefore, phonological patterns apparently contribute strongly to Lyme prevalence at some sites, but other factors are clearly important and likely predominate elsewhere.
A024 (EC029)

# Diversity of *Borrelia burgdorferi* sensu lato in the southeastern United States: the history and enzootiology

Rudenko Natasha¹,²,³, Golovchenko Maryna¹,²,³, Grubhoffer Libor¹,²,³ and James H. Oliver, Jr.¹,²,³

1. Biology Centre, Institute of Parasitology, České Budějovice, Czech Republic

2. The James H. Oliver Jr. Institute of Arthropodology and Parasitology, Statesboro, GA, USA

3. Georgia Southern University, Statesboro, GA, USA

Lyme disease in the southeastern United States received little attention due to the presumably low abundance of *Ixodes scapularis* in this region and low prevalence of Lyme disease spirochete in major tick vector. The low prevalence of *Borrelia burgdorferi* in *I. scapularis* does not necessary mean the low prevalence or absence of the pathogen in the region, taking into consideration the existence of “cryptic” maintenance cycles of the spirochete in the absence of classic *I. scapularis/Peromyscus leucopus* transmission. Migration of infected vertebrate hosts may have a larger impact on the contemporary expansion of pathogen population than the movement of tick vectors in this region. By maintaining the spirochetes in foci where classic transmission cycle is absent, the migrating infected hosts are increasing the risk of contracting Lyme disease to humans. Cultivation of *B. burgdorferi* sensu lato from tick vectors and diverse host specimens collected in southeastern part of the United States represent a collection of more than 300 isolates from Texas, Missouri, Rhode Island, South Carolina, Georgia, and Florida. The regions of samples origin are the part of the Atlantic Flyway, one of the major bird migration routes that follow the Atlantic coast of North America. The collection includes 152 strains from six hard tick species, 131 from three rodent host species, 13 from eight bird species and a group of isolates with confirmed multiple spirochete species originated from different sources. Previous analysis of isolates revealed a presence of multiple *Borrelia bissettii* and *Borrelia kurtenbachii* strains, recently described *Borrelia carolinensis* and *Borrelia americana*, highly diverse *B. burgdorferi* sensu stricto strains and multiple *Borrelia andersonii* strains. Comparative analysis of *B. burgdorferi* sensu stricto strains collected in Lyme disease endemic and non-endemic European and North American regions revealed close relatedness of geographically distinct populations. We found that spirochete genotypes, commonly associated with human Lyme disease in endemic European and North American regions, were detected among *B. burgdorferi* strains isolated from non-human biting tick *Ixodes affinis* and rodent hosts in southeastern USA. We discovered that some *ospC* types, previously known only from Europe, are widely distributed in the southeastern USA, a finding that confirms the trans-oceanic migration of *Borrelia* species.
A025 (EC030)

Transmission dynamics of *Borrelia* bacteria in a bird tick community

Dieter Heylen¹,², Hein Sprong¹,² and Erik Matthysen¹,²

1. University of Antwerp, Antwerp, Belgium

2. National Institute for Public Health and Environment (RIVM), Bilthoven, Netherlands

We examined the *Borrelia burgdorferi* sensu lato circulation in a tick community consisting of three species (*Ixodes ricinus*, *Ixodes frontalis*, *Ixodes arboricola*) with contrasting ecologies, but sharing a common host: the great tit (*Parus major*), one of the most common birds of European gardens and woodlands. While this bird is the principal host for the specialized nidicolous tick *I. arboricola*, it is one of the many hosts for the generalists *I. frontalis* and *I. ricinus*. We found evidence that all the three tick species have a potential to contribute to the maintenance of *Borrelia* infections in *P. major*. Field data show that the birds hosted *Borrelia*-infected larvae of both *I. frontalis* and *I. ricinus*, indicating the facilitation of *Borrelia* transmission by the songbird host. The low, but significant numbers of *Borrelia* in unfed *I. arboricola* ticks collected from bird nest boxes, provide the first field data showing that it is competent in maintaining *Borrelia* over long periods of time. Aside from the known avian genospecies (*Borrelia garinii* and *Borrelia valaisiana*), several less dominant genospecies were observed in all three ticks, including mammalian genospecies and a first record of *Borrelia turdi* for north-western Europe. In a laboratory experiment, we show that *P. major* selectively facilitates the transmission of different *Borrelia* genospecies. In this experiment, we imitated the natural situation during the bird's post-fledging period, in which *Borrelia*-naïve juvenile birds are repeatedly exposed to infected *I. ricinus* nymphs. Birds developed systemic infection of the avian genospecies, as suggested by the strong increase with infestation order in the number of infected ticks. On the other hand, birds showed a very low competence to facilitate the transmission of mammalian genospecies (*Borrelia afzelii*, *B. burgdorferi sensu stricto*, and *Borrelia spielmanii*) as shown by the decrease in infected fed ticks, although a low number of birds remained permissive for *B. afzelii*. In a final section, we present preliminary data of a laboratory experiment, investigating the vector competence of *I. frontalis* and *I. arboricola* for *B. burgdorferi* sensu lato. These data may complete the answer on the question whether *Borrelia* transmission cycles can be maintained by bird-specific ticks using *P. major* as host, and bridged by *I. ricinus* to other hosts.
A026 (EC032)

Identification of hosts by determining natural isotope signatures in unfed
*Ixodes ricinus* ticks

Olaf Schmidt¹,²,³,⁴,⁵,⁶, Hans Dautel¹,²,³,⁴,⁵,⁶, Lise Gern¹,²,³,⁴,⁵,⁶, Dieter Heylen¹,²,³,⁴,⁵,⁶,
Helge Kampen¹,²,³,⁴,⁵,⁶, Jason Newton¹,²,³,⁴,⁵,⁶ and Jeremy Gray¹,²,³,⁴,⁵,⁶

1. University College Dublin, Dublin, Ireland

2. bIS Insect Services GmbH, Berlin, Germany

3. University of Neuchâtel, Neuchâtel, Switzerland

4. University of Antwerp, Antwerp, Belgium

5. Friedrich-Loeffler-Institut, Greifswald, Germany

6. NERC Life Sciences Mass Spectrometry Facility, Bristol, UK

In a previous proof-of-concept study using laboratory animals, it was shown that ratios of the naturally occurring stable isotopes, 15N/14N and 13C/12C in host (gerbil, rabbit) blood were faithfully replicated in the tissues of ticks that had fed on them. Determination of natural isotope ratios in unfed ticks, therefore, has potential for identification of the hosts that they fed on in the previous stage. In the present study, larvae and nymphs of *Ixodes* were fed on a range of natural hosts, namely rodents (*Apodemus sylvaticus* and *Myodesglareolus*), ruminants (*Ovis aries* and *Bos taurus*), and birds (tit mice, *Parus major* and *Cyanistes caeruleus*). The natural isotope ratios in the resulting nymphs and adults were determined by elemental analysis-isotope ratio mass spectrometry, and the results examined for differences in host origins. It was found that the isotope signatures in ticks had the expected trophic shift from the signature of the relevant host bloods, so that it was possible to discriminate between ticks of different host origin. However, although the previous study suggested that it might be possible to age ticks as a result of changing natural isotope ratios over time, the present study suggested that this effect only applies to the first few weeks after the moult, during which time the ticks excrete most of the blood meal remnants from their previous blood meal. The results from this experiment suggest that it will be possible to detect and differentiate animal groups (e.g., rodents, ruminants, birds) in field-caught questing ticks.