A027 (EC031)

Spatial spread and demographic expansion of Lyme borreliosis
spirochaetes in Eurasia

Stephanie Vollmer¹, Edward J. Feil¹, Chen-Yi Chu²,³, Stephen L. Raper¹, Wu-Chun Cao², Klaus Kurtenbach¹*, Volker Fingerle⁴ and Gabriele Margos⁴,⁵

1. University of Bath, Bath, UK

2. Beijing Institute of Microbiology and Epidemiology, Beijing, China

3. PLA Institute of Disease Control and Prevention, Beijing, China

4. National Reference Centre for Borrelia, Germany

5. LMU Munich, Munich, Germany

†Deceased, March 2009.

The Lyme borreliosis (LB) group of spirochaetes currently comprises 18 named species that vary in their geographic distribution, host specificity, and ability to cause disease in humans. In Europe, the species *Borrelia afzelii* and *Borrelia garinii* are highly abundant, and are also regularly found in Asia. It has been shown that *Borrelia* species associated with birds, such as *B. garinii*, showed limited geographic structuring between European countries while, the rodent associated species, *B. afzelii*, showed extensive spatial structuring in Europe. Here, we included a total of 89 *B. afzelii* and 121 *B. garinii*, comprising strains from Europe and China, into the analyses. We used multilocus sequence analysis (MLSA) based on eight chromosomal housekeeping genes (clpA, clpX, nifS, pepX, pyrG, recG, rplB, and uvrA) to show that when the wider, inter-continental, distribution is considered, there is evidence of spatial structuring even in the bird-associated species *B. garinii*. Furthermore, our investigations into historical LB populations provided evidence for range expansions of *B. garinii* and *B. afzelii* populations in Europe in the distant past. We propose that the expansion of *B. afzelii* in Europe may be linked to rodent population expansions after the last glacial maximum.
A028 (EC033)

Borrelia DNA in king penguins in Crozet

Frédéric Schramm¹*, Michel Gauthier-Clerc², Jean-Charles Fournier³, Cathy Barthel¹,
Danièle Postic⁴, Yves Handrich³, Yvon Le Maho³ and Benoît Jaulhac¹

1. EA 7490, Faculté de Médecine, Strasbourg, France

2. Université de Strasbourg, Strasbourg, France

3. Université de Strasbourg, Institut Pluridisciplinaire Hubert Curien, Strasbourg, France

4. MIVEGEC (Maladies infectieuses et vecteurs: écologie, génétique, évolution et contrôle), UMR 5290 CNRS/IRD,
UR 244 IRD UM1 UM2, Montpellier, France

The hard tick *Ixodes uriae* parasitises an extensive range of seabird species in the circumpolar areas of both the northern and the southern hemispheres. *I. uriae* has also been shown to be infected with *Borrelia burgdorferi* sensu lato (Bbsl), the agent of Lyme borreliosis in the northern hemisphere. Fifty tick-infested adult king penguins (*Aptenodytes patagonicus* halli) breeding in Crozet Archipelago (South Indian Ocean) were examinated for Bbsl spirochetemia by *in vitro* DNA amplification. Bbsl DNA was detected from the blood of 2 out of the 50 king penguins tested, and further identified as *B. garinii*. These data represent the first report on direct Lyme borreliosis spirochetes detection from seabirds in the Southern hemisphere. Our results indicate a possible reservoir role of king penguins in the spirochete's natural maintenance and could contribute to improve our knowledge about the role of seabird species in the complex epizootiology of Bbsl and evolution of Lyme disease.
A029 (EC037)

# The presence of ticks and mice infected with *Borrelia burgdorferi* and *Ehrlichia* spp. confirm Central Mexico as an endemic zone of Lyme disease and ehrlichiosis

Gordillo-Pérez Guadalupe¹, D. Vargas Margarita², Covarrubias Yatzareth¹,
Sosa-Gutierrez Carolina¹, D. Guzmán-Cornejo Carmen³, M. Montiel Griselda³,
V. Sánchez-Cordero and Torres Javier¹

1. Unidad de Investigación Médica en Enfermedades Infecciosas, Hospital de Pediatria, Centro Medico Nacional SXXI-IMSS, Mexico City, Mexico

2. Facultad de Agrobiología, Universidad Michoacana, Uruapan, Mexico

3. Instituto de Biología, UNAM Mexico City, Mexico City, Mexico

Lyme disease (LD) is the tick-borne zoonosis more frequently reported in the USA with an incidence of 22,000 cases per year, caused by *Borrelia burgdorferi*, transmitted by *Ixodes scapularis* or *Ixodes pacificus* ticks, and maintained by *Peromyscus* mouse. A seroprevalence of 1.1% has been reported in Mexico with the Northeast and Mexico City as the zones with higher prevalence how the Mexico City 6.2 and 3.4%, respectively. We recently reported the first cases of LD in Mexico, which patients were exposed to ticks in national parks near Mexico City.

**Objective:** The aim of this study was to explore if the enzootic cycle for *Borrelia* and *Ehrlichia* is present in national parks visited regularly by families.

**Material and methods:** During the period 2009–2011, we collected mice and ticks in the forest of three parks in central Mexico. To determine the presence of *Borrelia burgdorferi* and *Ehrlichia* infections, the mice were sacrificed and tissues (vessel, articulation, and ear) were sampled, and tick's guts homogenized. One fraction was cultured in BSK II medium and another used for DNA extraction to amplify *fla* and *ospA*. *B. burgdorferi* genes or 16S RNA and gp 36 *Ehrlichia* genes.

**Results:** 24 out of 202 (11.9%) *Peromyscus* and *Neotomodon alstoni* mice were positive for *B. burgdorferi* and 28 (14%) for *Ehrlichia* sp. and 10 out 31 *Ixodes spinipalpis* and *Ixodes tovaris* ticks (30%) collected on 25 mice were positive. Amplified sequences had 95–100% homology with *B. burgdorferi* sensu stricto and 98% homology with *Ehrlichia canis* genes.

**Conclusions:** We confirmed that the enzootic cycles for Lyme disease and ehrlichiosis are present in national parks in central Mexico, and are maintained by *I. spinipalpis* or *I. tovaris* ticks and by *Peromyscus* or *Neotomodon* mice. The health system in Mexico needs to apply preventive measures to control the tick vectors.
A030 (EC038)

Contribution of tick phenology to geographical gradients in Lyme borreliosis
in Eastern and Central North America

Howard S. Ginsberg<sup>1,2,3,4,5,6</sup>, Eric L. Rulison<sup>1,2,3,4,5,6</sup>, Russell L. Burke<sup>1,2,3,4,5,6</sup>, Lorenza Beati<sup>1,2,3,4,5,6</sup>, Graham J. Hickling<sup>1,2,3,4,5,6</sup> and Jean I. Tsao<sup>1,2,3,4,5,6</sup>

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

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

3. Hofstra University, Hempstead, NY, USA

4. Georgia Southern University, Statesboro, GA, USA

5. Michigan State University, East Lansing, MI, 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 United States. Tick phenologies showed the hypothesized pattern from north to south along the east coast, with the classic northern (nymphs before larvae) pattern in Massachusetts, but with larvae before nymphs in Florida (which would disrupt the transmission dynamic) 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.
A031 (EC039)

The effects of environmental moisture on seasonal tick abundance

K. Berger<sup>1,2,3</sup>, H. Ginsberg<sup>1,2,3</sup>, K. Dugas<sup>1,2,3</sup>, L. Hamel<sup>1,2,3</sup> and T. Mather<sup>1,2,3</sup>

1. University of Calgary, Calgary, AB, Canada

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

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

Lyme borreliosis is the most commonly reported vector-borne disease in north temperate regions worldwide; however, tick population trends and disease incidence is difficult to predict. While the importance of moisture availability to tick survival is accepted, its role in tick activity and abundance is not well understood. For example, laboratory studies have demonstrated tick mortality due to low environmental moisture, whereas some field studies have found little or no correlation between the effects of weather and tick abundance. By using data from over a decade of continuous statewide tick surveillance, we assess the effects of environmental moisture on tick survival at multiple scales. We document substantial inter-annual variability in nymphal tick abundance. Comparison of 14-year nymphal tick surveillance records for the state of Rhode Island and corresponding hourly relative humidity (RH) records for the same period of time, identified periods of sub-optimal conditions affecting tick survival. We identify tick-adverse humidity events (TAHEs), periods of sub-optimal moisture availability defined by microclimatic parameters identified in a laboratory setting. Knowledge of these microclimatic parameters are required to accurately characterize the effects of weather on tick numbers, and offer the possibility of increasingly accurate predictability of tick populations and incidence of human disease. Our findings provide the basis for the development of a weather-based early warning system to increase public awareness and target tick-borne disease preventive measures.
A032 (EC040)

# Are blackbirds *Turdus merula* competent reservoirs for *Borrelia turdi* in Western Europe? A Xenodiagnostic experiment

A. C. Norte¹,²,³, I. Lopes de Carvalho¹,²,³, M. S. Núncio¹,²,³, J. A. Ramos¹,²,³ and L. Gern¹,²,³

1. Department of Life Sciences, Marine & Environmental Research Center, Coimbra University, Coimbra, Portugal

2. Center for Vector & Infectious Diseases Research (CEVDI/INSA), Lisbon, Portugal

3. Laboratory of Eco-Epidemiology of Parasites, Institute of Biology, Neuchâtel University, Neuchâtel, Switzerland

To confirm that thrushes, such as blackbirds *Turdus merula*, play a key role as reservoirs for some *Borrelia* genospecies in Europe, we performed a xenodiagnostic experiment with blackbirds captured in a mixed wood located in western Portugal where *Borrelia turdi*, an uncommon genospecies in Europe, is the most prevalent genospecies associated with birds. Four out of five birds transmitted spirochetes to xenodiagnostic ticks. Two birds transmitted *Borrelia valaisiana* to 25.7 and 10.5% of ticks, and two transmitted *B. turdi* to 6.4 and 5.4% of ticks. Our results showed that blackbirds transmit *B. valaisiana* and *B. turdi* to *I. ricinus* feeding larvae, acting as amplifying hosts for these genospecies in nature.
A034 (EC043)

Blacklegged tick feeding success on cotton mice and broadheaded skinks
in an endemic site in Florida

Jasmine Parham¹,², Genevieve C. Pang¹,², James Parmer¹,² and Jean I. Tsao¹,²

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

2. Stetson University, DeLand, FL, USA

Although *Ixodes scapularis* (i.e., blacklegged tick) populations are established across the eastern United States, Lyme disease incidence is approximately two orders of magnitude lower in the south than in the north. This disparity may be attributed to geographical differences in host community composition, as host species differ in their ability to harbor and transmit the pathogen (i.e., reservoir competence) and relative impacts on tick feeding, survival, and molting success (i.e., host-specific grooming behaviors and blood meal quality). To investigate these effects, we conducted feeding trials at a research site in northern Florida, where prior field surveys showed that larvae and nymphs feed on both *Peromyscus gossypinus* (i.e., cotton mice) and *Plestiodon laticeps* (i.e., broadheaded skinks). We also investigated interactive effects between host species and tick genetics by feeding groups of larvae and nymphs that belonged to genetic clade SI (typically found in the southeast) and AmI (found throughout the eastern United States, and predominates in the north). In order to quantify feeding success, we estimated tick attachment duration, engorgement weights, and molting success. Ticks of known genetic clade were generated in a laboratory facility at Michigan State University, although supplemental trials included ticks of other ages and genetic backgrounds. Mice and skink hosts were captured directly from the field site. Feeding trials were conducted in a facility where ticks and hosts were exposed to ambient (i.e., outdoor) light, temperatures, and relative humidity. Hosts were housed in individual pens with false bottoms suspended over trays of water. Trays, food dishes, and refugia were checked every 24 h for engorged ticks until 48 h had elapsed whereby no ticks were recovered from any host. We discovered that larval and nymphal ticks that fed on cotton mice and belonged to the AMI clade began detaching earlier than ticks fed on broadheaded skinks and belonged to the SI clade (in most cases by greater than 24 h). Nymphs belonging to the SI clade and nymphs that fed on broadheaded skinks were significantly heavier on average than nymphs belonging to the AMI clade and fed on cotton mice, respectively. Due to large variation in larval weights, observed differences were not significant, although similar trends were observed. The molting of engorged ticks is currently being monitored and will be discussed. These results suggest that the host species and tick genetics have interactive effects on feeding success and therefore influence tick population establishment and maintenance.
A035 (EC044)

Blacklegged tick drop-off timing on cotton mice and broadheaded skinks

Genevieve C. Pang¹,², Graham J. Hickling¹,² and Jean I. Tsao¹,²

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

2. University of Tennessee, Knoxville, TN, USA

The differential effects of different host species on *Ixodes scapularis* (i.e., blacklegged tick) vectors have been well established in the literature. Previous findings support host species differences in reservoir competence (i.e., their ability to harbor and transmit the pathogen to the blacklegged tick vector) as well as direct impacts on tick survival and molting success (e.g., grooming behaviors and blood meal quality). However, less is known about whether host species differ in terms of when ticks are likely to detach from the host. In nature, relatively small differences in host drop-off timing could have large impacts on a tick's probability of survival and future host-seeking probability, given the broad range of microhabitats that hosts encounter throughout a diurnal cycle. In order to determine whether differences in attachment duration and drop-off timing existed between hosts, we conducted feeding trials of larval and nymphal blacklegged ticks on two host species that differ in their activity periods. Experiments were conducted at a field site in northern Florida where there is an established population of blacklegged ticks. Hosts used in this study were wild-caught *Peromyscus gossypinus* (i.e., cotton mice) and *Plestiodon laticeps* (i.e., broadheaded skinks), both of which are parasitized by juvenile blacklegged ticks. Ticks used for this project derived from colonies reared in a laboratory facility at Michigan State University. Colonies comprised ticks collected from various locations within the range of blacklegged ticks as well as having different genetic backgrounds. Feeding trials were conducted in a facility where ticks and hosts were exposed to ambient light, temperatures, and relative humidity. Hosts were housed in individual pens with false bottoms suspended over trays of water. Trays, food dishes, and refugia were checked for engorged ticks every 6-8 h on a 24 h cycle until 48 h had elapsed whereby no ticks were recovered from any host. Our results reveal host species differences in tick detachment times. Both larvae and nymphs that fed on broadheaded skinks primarily detached between 6:00 a.m. and 2:00 p.m., whereas the majority of ticks that fed on cotton mice detached between 2:00 p.m. and 10:00 p.m. This pattern may be due in part to changes in host behaviors, physiological states, and/or abiotic conditions that occur throughout a diurnal period. This study has implications for understanding another aspect of how a host species may impact tick survivorship and future host-finding success via their effects on the distribution of ticks among microhabitats.
A036 (EC045)

Survival patterns of northern compared to southern genotypes of *Ixodes scapularis*
in North America

Howard S. Ginsberg<sup>1,2,3</sup>, Eric L. Rulison<sup>1,2,3</sup>, Isis Kuczaj<sup>1,2,3</sup> and Jean I. Tsao<sup>1,2,3</sup>

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

Survival patterns of northern and southern genotypes of *Ixodes scapularis* were studied under northern and southern
environmental conditions in laboratory experiments. Larvae of both American clade 1 (descended from ticks collected
in Wisconsin) and Southern clade 1 (from ticks collected in South Carolina) survived longer under northern than
under southern conditions. These results suggest that southern environments are less hospitable for *I. scapularis*
survival, which might be related to the predominantly northern distribution of Lyme borreliosis in North America.
American clade 1 ticks survived longer than Southern clade 1 ticks under both northern and southern conditions in
the lab. However, field data suggest higher survival of southern than northern clade ticks under field conditions in the
south. Improved survival of southern clade ticks under southern conditions in the field might result from behavioral
differences between clades, possibly related to host associations.
A037 (EP017)

Two natural reservoir hosts identified for *Borrelia miyamotoi* in Switzerland

O. Schumann, C. Schumann, C. Burri Cordinier and L. Gern

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

In Europe, at least 10 *Borrelia burgdorferi* sensu lato (sl) species have been identified in the tick *Ixodes ricinus*. Furthermore, *Borrelia miyamotoi*, a species related to the relapsing fever spirochetes, has been reported in questing *I. ricinus*. The mouse *Peromyscus leucopus* and passerine birds were identified to play a role in the maintenance of *B. miyamotoi* in USA. Nevertheless, no study was conducted to demonstrate potential reservoir hosts for *B. miyamotoi* in Europe. Here, we were interested in the role played by wild rodents in the maintenance of *B. miyamotoi* in Switzerland. In 2011 and 2012, small mammals were captured in an area where *B. miyamotoi* occurs at a prevalence of 2.4% in questing nymphs. *I. ricinus* ticks from small mammals were analyzed after the moult by PCR/RLB to identify the different *Borrelia* genospecies. In parallel, ear biopsies and blood samples were taken to confirm the presence of *Borrelia* in rodents and xenodiagnosis was used to evaluate their reservoir role. Most of the 110 captured rodents (95.5%) were infested by *I. ricinus* ticks and 8.18% were infested by *Borrelia* infected *I. ricinus* larvae. *Borrelia afzelii* (2.8%), *B. bavariensis* (1.6%), and *B. miyamotoi* (0.17%) were detected in the ticks attached to rodents. *B. miyamotoi* was observed by microscopy in one blood sample. Xenodiagnostic larvae fed on infected *Myodes glareolus* and *Apodemus flavicollis* revealed the transmission of *B. miyamotoi* with infection prevalence reaching 10–30%. This study showed that at least two rodent species are reservoir hosts for *B. miyamotoi* in Europe.
A038 (EP018)

Review of the pathogen diversity of human interest transmitted by
*Ixodes ricinus* in Switzerland

C. Burri Cordonier, M. Frötscher, P. Lehmann and L. Gern

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

The tick *Ixodes ricinus* is mainly known to transmit the virus of tick-borne encephalitis (TBE) and the etiologic agent of Lyme borreliosis (LB), the bacteria *Borrelia burgdorferi* sensu lato (sl). However, additional pathogens belonging to the genera *Rickettsia*, *Anaplasma*, and *Babesia* have been reported in this tick species. The aim of this study was to review the current situation concerning these various pathogen genera in Switzerland including recent and older data. Screening of more than 110,000 *I. ricinus* collected in more than 400 different sites allowed to identify 15 different tick-borne pathogen species: seven different *Borrelia* species, two *Rickettsia* species (*Rickettsia helvetica* and *Rickettsia monacensis*), *Candidatus Neoehrlichia* mikurensis, *Anaplasma phagocytophilum*, and three *Babesia* species (*Babesia divergens*, *Babesia microti*, and *B. venatorum*) as well as TBE virus. Globally, *B. burgdorferi* sl and *Rickettsia* spp. were the most prevalent pathogens with infection prevalence in questing ticks varying from 23 to 50% for *Borrelia* spp. and from 10 to 21% for *Rickettsia* spp. *C. Neoehrlichia* mikurensis was identified in 6% of examined ticks. These pathogens were detected in *I. ricinus* ticks from all investigated sites. In contrast, other pathogens such as TBE virus, *A. phagocytophilum*, and *Babesia* spp. were localized at restricted areas with prevalence lower than 2%. LB is frequent in Switzerland with between 7000 and 12,000 annual cases (yearly incidence 131 per 100,000 inhabitants) and TBE reaches around 100 cases every year (yearly incidence 1.6 per 100,000 inhabitants). Concerning the other tick-borne pathogens, well-documented cases do not exist, except for *C. Neoehrlichia* mikurensis, since three human cases have been recently reported.
A039 (EP020)

Temporal and spatial distribution of Lyme borreliosis in Southern US

Abha Grover¹, Erin McGregor¹, Alexandra Brown¹, Srikanth Kanameni², May Boggues³, Raul F. Medina² and Maria D. Esteve-Gassent¹

1. Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA

2. Department of Entomology, College of Agriculture and Life Sciences, Texas A&M University, College Station, TX, USA

3. Arizona State University, Tempe, AZ, 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 is 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 in southern US. To determine the distribution of LB in Texas, we collected ticks from different deer hunting stations, from veterinary clinics and animal shelters in different counties across the state. 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 *B. 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 correlates with the deer-hunting season. Other tick species such as *Amblyomma americanum* and *Amblyomma cajenenses* were found infected with *B. burgdorferi* (26.6 and 48%, respectively). Genetic markers flaB and IGR were the most effective in detecting ticks infested with *B. burgdorferi*. Our preliminary population genetic study suggests that ticks positive for both markers were mostly distributed in eastern Texas. The distribution of *Borrelia* we found in ticks correlates with the distribution of the majority of canine samples that tested positive for *B. burgdorferi* in this study and with the cases of LB in humans reported to CDC since 2000.
A040 (EP026)

Ecology of *Ehrlichia muris* in the American midwest: vector–host–pathogen associations and genetic diversity of an “emerging” human and animal health threat

Sarah A. Hamer<sup>1,2,3</sup>, Lisa Auckland<sup>1,2,3</sup>, Isis M. Kuczaj<sup>1,2,3</sup>, Ellen Stromdahl<sup>1,2,3</sup> and Jean I. Tsao<sup>1,2,3</sup>

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

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

3. US Army Public Health Command, Edgewood, MD, USA

In 2009, an *Ehrlichia muris*-like organism was for the first time associated with human disease in the United States, and in 2012 canine disease from infection was first reported. To begin to understand the ecology of the *E. muris*-like organism (hereafter *E. muris*) in the Midwestern US, we analyzed field samples collected from 2010 to 2012 at Fort McCoy army installation in west central Wisconsin, near the origin of the recently reported human infections. Samples were collected with a rigorous ecological sampling design to allow exploration of pathogen-vector-host associations and pathogen genetic diversity. We subjected 674 samples, including 199 ear biopsies from 4 small mammal species, 62 *Ixodes scapularis* adults removed from hunter-harvested deer, and 408 questing *I. scapularis*, to three separate PCR assays (citrate synthase, GroEL, and 16S rRNA) to detect and characterize *E. muris* infection. Small mammals exhibited low *E. muris* infection prevalence, with 3.4% of 79 *Peromyscus leucopus*, 1.5% of 65 *Clethrionomys gapperi*, and none of 55 shrews testing positive. The infection prevalence of questing nymphs (10.9%, *n* = 190) was double that of questing adults (5.1%, *n* = 138); suggesting that larval bloodmeal hosts may be reservoirs and associated with more infection than the nymphal bloodmeal hosts. Questing larvae (*n* = 80 pools consisting of 206 larvae) were characterized by 1.5% minimum infection prevalence indicating possible transovarial transmission. Adult ticks removed from white-tailed deer (*Odocoileus virginianus*) had a similar infection prevalence (4.8%) to the questing adults, suggesting that deer are not important reservoirs. Sequencing of the citrate synthase gene fragment revealed that 27/37 samples shared 100% homology with the *E. muris* organism found in all four positive controls, which were derived from ticks from WI at the time of the human outbreak. The remaining 10 samples contained one to four nucleotide substitutions from the common strain. In contrast, sequences for the other two genes (GroEL and 16S rRNA) were uniform across our samples and the positive controls. The ecology of *E. muris* in the American Midwest is complex, involving multiple reservoir hosts and co-infections with other pathogens in the *I. scapularis* microbial guild. Given the high abundance of *I. scapularis* in this region, the presence of *E. muris* in all three life stages, and the relatively high infection prevalence estimates in questing nymphs and adults, future studies must continue to elucidate the ecology of this “emerging” pathogen.
A041 (EP027)

Detection of *Borrelia miyamotoi* in *Ixodes* ticks and small mammals in Russia

Liudmila S. Karan¹, Nadezhda M. Kolyasnikova¹,², Kristina A. Gridneva¹,
Denis S. Sarksyan³ and Marina V. Fedorova¹

1. Central Research Institute of Epidemiology, Moscow, Russia

2. Chumakov Institute of Poliomyelitis and Viral Encephalitides of Russian Academy of Medical Sciences, Moscow, Russia

3. State Medical Academy, Izhevsk, Russia

Background: The relapsing fever group spirochete *Borrelia miyamotoi* has been found in hard-body ticks in Europe, Russia, Japan, and USA. Different species are involved in transmission of *Borrelia miyamotoi* including *Ixodes persulcatus*, *Ixodes ricinus*, *Ixodes scapularis*, *Ixodes pacificus*, and *Ixodes dentatus*. We studied the presence of *B. miyamotoi* in hard-body ticks and small mammals in Russia.

Methods: Over a six-year period, *I. ricinus* (n = 1090 individuals) and *I. persulcatus* (n = 1958) were collected in different parts of Russia (Central Russia, North Caucasus, Volga River Region, Ural, and Siberia), as well as in East Kazakhstan. Small mammals were collected in the Republic of Udmurtia, Volga River Region. The most prevalent species was *Myodes glareolus* (n = 240 individuals), while 40 other mammals belonged to the *Apodemus uralensis*, *Mus musculus*, *Apodemus flavicollis*, and *Myodes rutilus* species. *B. miyamotoi* was amplified from ticks or tissue samples using specific real-time PCR, confirmed in most cases by amplicon sequencing. DNA and RNA of other tick-borne pathogens were detected using the commercial kit “AmpliSense™ TBEV, *B. burgdorferi* sl, *A. phagocytophilum*, *E. chaffeensis*/*E. muris*” (CRIE, Russia, Moscow).

Results: *B. miyamotoi* was detected 1.2% of *I. ricinus* ticks in central Russia and the North Caucasus. Higher infection rates of *I. persulcatus* were found in central Russia (1.9%, *n* = 628), Volga Region (5.7%, *n* = 544), Ural (2.9%, *n* = 481), and Siberia (3.3%, *n* = 305). *I. persulcatus* collected in east Kazakhstan had nearly the same *B. miyamotoi* infection rate (3.7%, *n* = 533). The 62% of *I. ricinus* ticks and 35% of *I. persulcatus* ticks, which were positive for *B. miyamotoi*, were also co-infected with *Borrelia* from *B. burgdorferi* sl group. The *B. miyamotoi* and *B. burgdorferi* sl infection rates of *Myodes glareolus* were 3.3 and 18.3%, respectively. Only one other species of rodents (*A. uralensis*) was found to be infected with *B. miyamotoi* (one of eight animals).

Conclusion: *B. miyamotoi* infection rates in *I. persulcatus* and *I. ricinus* ticks in Russia are relatively low in comparison to *B. burgdorferi* sl. About 40-50% patients with serologically confirmed *Ixodes* tick-borne borreliosis (ITBB) in Russia do no have an erythema migrans rash. In the Republic of Udmurtia and Yekaterinburg Region, we were able to detect *B. miyamotoi* DNA in blood samples of about half of the patients without erythema migrans (about a quarter of all ITBB patients). Because of the observed higher infection prevalence in humans than ticks, we conclude that *B. miyamotoi* is like other relapsing fever spirochetes, requires less time and/or fewer bacteria to infect humans compared to Lyme borreliosis spirochetes.
A042 (V014)

A novel relapsing fever *Borrelia* sp. and reptile-associated *Borrelia* sp. from
amblyomma geoemydae, and its difference of the transstadial transmission

Takano Ai, Chieko Sugimori, Kozue Sato, Fujita Hiromi, Kadosaka Teruki, Konnai Satoru, Tajima Tomoko, Watanabe Haruo, Ohnishi Makoto and Kawabata Hiroki

*Laboratory of Epidemiology, Joint Faculty of Veterinary Medicine, Department of Veterinary Medicine, Yamaguchi University, Yamaguchi, Japan*

We found novel relapsing fever *Borrelia* sp. (Borrelia sp. AGRF) and reptile-associated *Borrelia* sp. (Borrelia sp. tAG) from *Amblyomma geoemydae* in Japan. The *Borrelia* sp. AGRF was phylogenetically related to the hard (ixodid) bodied tick borne relapsing fever *Borrelia* spp., *Borrelia miyamotoi* and *Borrelia lonestari*. On the other hand, the *Borrelia* sp. tAG formed a monophyletic group and it was closely related to reptile-associated *Borrelia* spp. The prevalence of the *Borrelia* sp. AGRF and *Borrelia* sp. tAG were 14.2% (39/274) and 31.4% (86/274), respectively. In addition, these 14 ticks (5.1%) were co-infected with both *Borrelia* spp. The transstadial transmission of *Borrelia* sp. AGRF was occurred in the tick midgut and the salivary glands, although *Borrelia* sp. tAG was only detected in the tick midgut of *A. geoemydae*. The difference of the borrelial niche regarding the transstadial transmission might be associated with borrelial characterization. Thus, our findings may contribute to the elucidation of transstadial transmission mechanisms of *Borreliae* in ticks.
A043 (M003)

6S RNA in *Borrelia burgdorferi*

Scott Samuels¹,², Karen Wassarman¹,² and Amanda Brinkworth¹,²

1. Division of Biological Sciences, The University of Montana, Missoula, MT, USA

2. Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA

*Borrelia burgdorferi* is transmitted from its tick vector to a vertebrate host, which requires regulating the program of gene expression via an alternative sigma factor cascade in which RpoS globally controls the genes required for the different phases of the enzootic cycle. In *Escherichia coli*, 6S RNA facilitates the switch to RpoS-mediated gene expression in stationary phase by binding to RpoD-RNA polymerase holoenzyme, which sequesters RpoD from recycling and competing with RpoS as well as regulating transcription at RpoD-dependent promoters. Perhaps not surprisingly, the 6S RNA in *B. burgdorferi* (Bb6S) does not accumulate at high cell density, as in *E. coli*. Bb6S binds *in vitro* to RNA polymerase holoenzyme from both *E. coli* and *Bacillus subtilis* (with RpoD and SigA, respectively), and we are currently working on overexpressing the *B. burgdorferi* RNA polymerase to assay for binding. We have mutated the Bb6S gene and are currently evaluating its role gene expression and the enzootic cycle.
A044 (M004)

Induction of Lyme disease spirochete death by increasing RpoS expression

Linxu Chen, Qilong, Xu, Ge Yihe and Fang Ting Liang

Louisiana State University, Baton Rouge, LA, USA

RpoS, one of the two alternative σ factors in *Borrelia burgdorferi*, is tightly regulated by multiple regulators and, in turn, controls expression of essential and critical virulence determinants. Here, we show increasing RpoS expression induces spirochete death. The immediate effect of this induction was to promote bacterial division and to result in a rapid increase in cell number before causing bacterial death. Although induction led to over 99% spirochetes dead, a very small number of bacteria were able to survive after prolonged induction of more than 10 days. Interestingly, this induction of cell death did not show DNA fragmentation, the hallmark of programmed cell death. The potential biological significance of induced cell death may help *B. burgdorferi* regulate its population and maintain lifecycle.
A045 (M006)

Bacterial metabolic activity as a rheostat to control expression of
infection-associated genes

Brian Stevenson, Nicholas A. Brown, Alicia M. Chenail, Alyssa D. Antonicello and Brandon L. Jutras

*University of Kentucky, Louisville, KY, USA*

The Lyme disease spirochete controls production of its proteins during each stage of the infectious cycle, as do all other pathogenic organisms. An early hypothesis proposed that *Borrelia burgdorferi* directly senses environmental temperature to determine expression levels of the OspC and Erp infection-associated proteins. We recently demonstrated that *B. burgdorferi* actually responds to the variations in its metabolic activity that occurs at different temperatures rather than temperature per se. This new hypothesis helps explain results of other recent studies on borrelial gene expression. Moreover, evaluation of other pathogens indicates that effect of bacterial metabolism on pathogenesis-associated factors is a wide-spread phenomenon. Both *Borrelia*-specific and broadly applicable results will be presented.
A046 (M007)

Regulation of the *Borrelia burgdorferi* DnaX–EbfC operon

Nicholas A. Brown, Brandon L. Jutras, Alicia M. Chenail, Alyssa D. Antonicello
and Brian Stevenson

*University of Kentucky, Louisville, KY, USA*

*Borrelia burgdorferi* EbfC is a chromosomally encoded nucleoid-associated protein that exerts global effects of borrelial gene expression. The ebfC gene forms an operon along with dnaX, which encodes a major component of DNA polymerase. Both EbfC and DnaX are maximally expressed during periods of rapid bacterial growth, and repressed when the bacteria do not divide. Although the majority of known bacterial species also possess a dnaX–ebfC operon, essentially nothing is known about how any organism controls production of these genes. We have identified a DNA-binding protein that specifically binds near the promoter 5' of dnaX, and present results of studies on the role of this protein in borrelial production of DnaX and EbfC. Noting that all medically valuable antibiotics target bacterial metabolic targets, and that nearly all human pathogens contain a dnaX–ebfC operon, these studies may lead to development of novel, broad-spectrum antibacterial therapies.
A047 (M008)

Genome comparisons reveal conserved regulatory sequences on two constitutive plasmids in *Borrelia burgdorferi sensu lato*

Che Martin<sup>1,2,3,4,5</sup>, Sherwood Casjens<sup>1,2,3,4,5</sup>, Emmanuel Mondodin<sup>1,2,3,4,5</sup>, Claire Fraser-Liggett<sup>1,2,3,4,5</sup>, Steven Schutzer<sup>1,2,3,4,5</sup>, Benjamin Luft<sup>1,2,3,4,5</sup> and Wei-Gang Qiu<sup>1,2,3,4,5</sup>

1. *Hunter College of the City University of New York, New York, USA*

2. *University of Maryland, College Park, MD, USA*

3. *University of Utah School of Medicine, Salt Lake City, UT, USA*

4. *Stony Brook University, Stony Brook, NY, USA*

5. *New Jersey Medical School, Newark, NJ, USA*

Gene regulation plays a critical role in *Borrelia burgdorferi* especially during its transitions between the tick and vertebrate environments, but *cis*- and *trans*-regulatory elements in its genome remain largely unknown. Upon the recent completion of the genome sequences of additional twenty *B. burgdorferi* sensu lato strains, we used an evolutionary comparative approach to identify adaptively evolving genes and potential regulatory elements in their genomes. Here, we report findings from a comprehensive analysis of ORF and intergenic spacer (IGS) sequences of two constitutive plasmids lp54 and cp26. The lp54 plasmid is evolutionary more dynamic than cp26 showing frequent gains and losses of genes. Twelve lipoprotein genes on lp54 are newly discovered to be associated with adaptive divergence among *B. burgdorferi* sensu lato species. We identified over one hundred long (10 bases or more) IGS sequence blocks that are perfectly conserved across eight *B. burgdorferi* sensu lato species. These conserved IGS blocks contain known or predicted *cis*- and *trans*-regulatory elements such as promoters, ribosomal binding sites, intrinsic transcription terminators, sigma-factor binding sites, and small RNAs. Orthologous ORF and IGS sequences were made publicly available via the *Borrelia* Ortholog Retriever website at http://borreliagenome.org/orth_get/. These newly identified conserved IGS sequence blocks are prime targets for investigations into the molecular mechanisms of gene regulations in the Lyme disease pathogens.
A048 (M009)

Role of signal recognition particle docking protein (FTsY) in pathophysiology
of *Borrelia burgdorferi*

Ying-Han Lin*, S.-L. Rajasekhar Karna and J. Seshu

*The University of Texas at San Antonio, San Antonio, TX, USA*

Purpose: Lyme disease, caused by the spirochete *Borrelia burgdorferi*, is the most common vector-borne disease in the US. Signal recognition particle (SRP) docking protein FTsY is one of the key components of the cellular machinery for targeting membrane proteins. Major targets for bacterial FtsY are integral membrane proteins and there is a dearth of information on the role of FTsY in the biogenesis of borrelial membranes.

Design Methods: We over-expressed the borrelial FTsY (31 kDa) homolog (BB0076) using the pET23a system, purified to homogeneity and generated mono-specific serum in BALB/c mice. FTsY deletion mutant was generated in an infectious strain *B. burgdorferi* strain B31 and complemented a functional copy of FTsY in *cis* using gentamicin resistance cassette. We have borrelial strains with the requisite genetic elements for analysis of the role of this protein in infection using the C3H/HeN mouse model of Lyme disease. Antisera against a variety of borrelial GTPases were generated and used in determining their contribution to borrelial physiology in the absence of FTsY.

Results: Immunoblot analysis of *B. burgdorferi* propagated under either tick (pH 7.6/23°C) or mammalian conditions (pH 6.8/37°C) revealed no significant differences in the levels of borrelial FTsY. Deletion of SRP-FTsY reveled a significant reduction in the levels of major outer surface protein C (OspC) suggesting that FTsY could play a role in the adaptation of *B. burgdorferi* to vertebrate host-specific conditions. This phenotype could be due to limitation in the trafficking of OspC to the membranes thereby altering the infectivity phenotype of *B. burgdorferi*. Levels of SRP-Ffh were elevated in the mutant compared to the control strains.

Conclusion: The reduction in the levels of OspC in the mutant demonstrates that the trafficking of OspC is either reduced or there are other transcriptional effects mediated due to the absence of FTsY resulting in reduced levels of this key vertebrate host specific protein. FTsY deficient mutant will be a critical genetic tool to decipher the SRP-mediated translocation of virulence related proteins of *B. burgdorferi*.
A049 (M013)

To SN(I)P or not to SN(I)P – that is no question!

Gabriele Margos¹,², Helmut Blum³, Stefan Krebs³, Andreas Sing⁴, Bernhard Liebl⁴, Manfred Wildner⁴, Patrick Guertler⁴, Cecilia Hizo-Teufel², Reinhard K. Straubinger¹ and Volker Fingerle²,⁴

1. Department of Infectious Diseases and Zoonoses, LMU Munich, Munich, Germany

2. German National Reference Centre for Borrelia, Oberschleißheim, Germany

3. Gene Center, LMU Munich, Munich, Germany

4. Bavarian Health and Food Safety Authority, Oberschleißheim, Germany

In Europe, Lyme borreliosis is caused by several species of the *Borrelia burgdorferi* sensu lato complex including *B. burgdorferi* sensu stricto, *Borrelia afzelii*, *Borrelia garinii*, and *Borrelia bavariensis*. It has been suggested that symptoms caused by the different species vary in their late manifestations, for example *B. afzelii* was mainly associated with skin manifestations while *B. garinii* and *B. bavariensis* have been associated with neuroborreliosis. However, there are cases that seem to deviate from this principle. To understand the relationship of *Borrelia* strains that induce a wide variety of symptoms such as erythema migrans, neuroborreliosis, and arthritis, we have re-sequenced the genome of more than 25 isolates of *Borrelia burgdorferi*. Patient isolates were obtained predominantly in Bavaria, Germany between 1985 and 2010. Using an Illumina MiSeq platform, we have applied "next generation sequencing" to these samples. This technique produces sequence information for the whole genome of microorganisms and offers unprecedented power to study biological relationships of bacterial pathogens. In recent years, methods based on next generation sequencing have become more reliable (in terms of sequence length and depth) and can now be applied economically to bacterial strains. Next generation sequencing produces a huge number of short sequence reads (50–250 bp in length) that can be mapped onto a reference genome permitting the identification of single nucleotide polymorphisms (SNPs) and these can be used to analyze microevolutionary processes, i.e., within bacterial populations, as well as the long-term evolutionary history of species. Sequence reads were mapped against full genome sequences available in GenBank. In addition, a novel algorithm, termed co-phylog (Yi and Lin, 2013), was explored to infer phylogenetic relationships between closely related species. For an initial comparison we concentrated on mutations in the core genome (omitting SNPs that were potentially acquired by recombination) to understand the pattern of descent within and between species. The data also permitted estimates of divergence times.
A050 (M016)

Metal ion homeostasis regulates virulence expression in *Borrelia burgdorferi*

Peng Wang¹, Klemen Strle², Jeremy Whitson¹, Lubomira Cubonova¹, Tom Santangelo¹, John Olesik¹, Allen C. Steere² and Xin Li¹

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

2. *Massachusetts General Hospital, Boston, MA, USA*

The ferric uptake regulator (Fur) family of DNA-binding proteins represses and/or activates gene expression based on metal-dependent signal sensing. *Borrelia burgdorferi* Fur homologue, also known as *Borrelia* oxidative stress regulator (BosR), is required for spirochetal adaption to the mammalian host, during which the expression of outer surface protein A (OspA), a tick colonization factor, is repressed, whereas the expression of OspC, a mammalian host colonization factor, is activated. However, it is unclear what metal(s) or even if metal(s) regulates BosR activity in the spirochete. Here, we show that perturbations to iron and copper homeostasis, either through genetic manipulation or by adjusting metal levels in the medium, significantly affect OspC expression in *B. burgdorferi*, with iron exerting a positive effect and copper exerting a negative effect. Furthermore, the effects of these metals at the cellular level correlated with their effects at the molecular level. While Fe-bound BosR is active, Cu-bound BosR is inactive in DNA-binding. Deficiency in BosR also results in an increase in cellular Cu level. This finding that BosR controls the homeostasis of a metal which regulates its DNA-binding activity has established in the Lyme disease spirochete a regulatory paradigm that is characteristic of the Fur family of metalloregulators.
A051 (M019)

# Whole genome sequencing (WGS) of 22 *Borrelia burgdorferi* strains and variants to understand human invasiveness and enzootic proliferation

Steven E. Schutzer¹, Claire M. Fraser-Liggett², Wei-Gang Qiu³, Emmanuel F. Mongodin², John J. Dunn⁴, Benjamin J. Luft⁵ and Sherwood R. Casjens⁶

1. New Jersey Medical School, Newark, NJ, USA
2. University of Maryland, College Park, MD, USA
3. Hunter College, New York, NY, USA
4. Brookhaven National Laboratory, Upton, NY, USA
5. Stony Brook University, Stony Brook, NY, USA
6. University of Utah Medical School, Salt Lake City, UT, USA

Human Lyme disease is an emerging disease, it may also be a diverging disease. To better understand this, we determined the first sequence of type strain B31. We obtained NIH funding to increase the number of *Borrelia burgdorferi* and related strains we sequenced to 22. Due to the nature of the multiple plasmids in these isolates this was a challenging project that has been successful and is helping move the entire field forward not incrementally but large leaps. The information derived from these whole genome sequencing (WGS, determined by Sanger methods) is extremely useful for investigation into pathogenesis, safe vaccine development, and diagnostic tests for infection. Some of the “Lyme-associated agents,” even though some of the component species do not cause human disease, include at least *B. burgdorferi*, *Borrelia garinii*, *Borrelia afzelii*, *Borrelia valaisiana*, *Borrelia lusitanie*, *Borrelia japonica*, *Borrelia andersonii*, *Borrelia turdae*, *Borrelia tanukii* and *Borrelia bissettii*. WGS has proven to be a fast and cost efficient way to attack many unknown questions simultaneously in a non-anecdotal way and provide a very firm foundation for the generation of new, informed, and testable hypotheses, which would be difficult or impossible to formulate by other means. As but one example, our initial whole-genome comparison off our *B. burgdorferi* genome sequences, demonstrated that in contrast to previous assumptions that genetic changes occurred only by point mutations, we found that closely related *B. burgdorferi* bacteria frequently horizontally exchange genetic information. To facilitate discovery of genetic variability associated with human virulence among the strains, these genomes were chosen, in part, to allow three levels of phylogenetic comparisons including between-species variations, within-species (*Bb* sensu stricto) polymorphisms, and the most recent evolutionary divergence between sister clonal groups. The sequence data have been deposited in Genbank, publications, and more analyses will be forthcoming in additional publications. Comparative analyses of the chromosomes and plasmids within and between species will be presented.
A052 (M020)

Genotyping Lyme borreliosis spirochetes

Michael Reiter, Anna M. Schötta, Andreas Müller, Hannes Stockinger and Gerold Stanek

*Infection and Immunology Unit, Center for Pathophysiology, Infectiology and Immunology, Institute for Hygiene and Applied Immunology, Medical University of Vienna, Wien, Austria*

Lyme borreliosis, the most common tick borne disease in the northern hemisphere, is caused by spirochetes of the *Borrelia burgdorferi* sensu lato (sl) group, a complex that currently comprises 19 genospecies. Whereas in North America a single genospecies, *B. burgdorferi* sensu stricto, is the cause of Lyme borreliosis, in Europe also *Borrelia afzelii* and *Borrelia garinii* have been identified and confirmed to cause the disease. However, other genospecies such as *Borrelia spielmanii*, *Borrelia bisettii*, and *Borrelia valaisiana* are also suspected to cause Lyme borreliosis. Our aim is to identify *Borrelia* genospecies of clinical isolates as well as from a tick collection representative for Austria. We dispose of a large number of field collected ticks from Austria as well as numerous DNA isolates from specimens of patients who had presumably Lyme borreliosis. The latter comprise DNA extracts of the last 8 years. The origin of these extracts comprises skin samples from suspected erythema migrans, as well as cerebral spinal fluid and synovia. Our study will allow a comprehensive overview of the *B. burgdorferi* *sl* genospecies distribution in certain specimens of patients and in ticks collected in Austria. Currently, we are using a nested PCR in combination with a newly developed nested real time PCR approach for detection and identification of *B. burgdorferi* *sl* genospecies. The clinically evident cases are being correlated with the PCR results in order to learn about the sensitivity of the methods.
A053 (M023)

DipA, a pore-forming protein in the outer membrane of Lyme disease spirochetes
exhibits specificity for the permeation of dicarboxylates

S. Bergström<fnref n="1" />,<fnref n="2" />, M. Bonde<fnref n="1" />,<fnref n="2" />, M. Thein<fnref n="1" />,<fnref n="2" />, Y. Östberg<fnref n="1" />,<fnref n="2" /> and R. Benz<fnref n="1" />,<fnref n="2" />

The Lyme disease causing Spirochete *Borreliae* are highly dependent on nutrients provided by their hosts. An important route for influx of nutrients is through porins that are situated in the outer membrane. In this study, a 36 kDa protein that functions as a putative dicarboxylate-specific porin is described. The protein was found in the outer membrane of Lyme disease *Borrelia*. It was purified by hydroxyapatite chromatography from *Borrelia burgdorferi* B31 and designated DipA, for dicarboxylate-specific porin A. The dipA gene was partially sequenced and corresponding genes could be identified in the genomes of *B. burgdorferi* B31, *Borrelia garinii* PBi, and *Borrelia afzelii* PKo. DipA also has a high homology to the Oms38 porin found in relapsing fever *Borrelia*. The black lipid bilayer assay was used to characterize DipA and the protein was found to have a single-channel conductance of 50 pS in 1 M KCl, to be slightly selective for anions and was not voltage-dependent. The channel could be partly blocked by different di- and tricarboxylic anions. The results imply that DipA forms a porin specific for dicarboxylates, which may play an important role for the uptake of specific nutrients in different *Borrelia* species. Ongoing studies that will be presented aim to further understand the function of this porin and will be achieved by mutational analysis. We will also generate expression constructs for DipA for structural and physiological studies of this porin.

<footnote n="1">1. Department of Molecular Biology, Umeå University, Umeå, Sweden</footnote>

<footnote n="2">2. Jacobs-University Bremen, Bremen, Germany</footnote>
A054 (M026)

Role of single lipoproteins in the formation of lipid rafts microdomains in *Borrelia*

Alvaro Toledo and Jorge L. Benach

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

*Borrelia burgdorferi*, the agent of Lyme disease, contains numerous lipoproteins as well as free cholesterol and cholesterol glycolipids, in its outer membrane. Structural, biochemical, and biophysical analysis revealed that spirochetal cholesterol glycolipids exist in microdomains in cultured and *in vivo* derived spirochetes. Mass spectrometry analysis of detergent resistance membranes isolated from *B. burgdorferi* grown in three different culture conditions (4, 33, 37°C plus blood) showed that the protein content of each fraction was similar and several lipoproteins (OspA, OspB, and OspC) that could potentially play a structural role in the formation of these domains were identified. Other proteins detected in the lipid microdomain are involved in protein and glycolipid catabolic processes, such as HtrA, P83/100, and a LysM domain protein. Several mutants (ΔOspA, ΔOspB, and ΔOspC) were analyzed to measure the impact of lipoprotein in lipid raft formation by thin layer chromatography (TLC) and transmission electronic microscopy (TEM). Lipid extracts were resolved on an HPTLC silica plate with a chloroform-methanol solvent system and stained with iodine vapor while TEM data were analyzed using the Ripley's K-function. In addition, FRET measurements on living *Borrelia* were made as previously described by LaRocca et al. (2010) using 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene *p*-toluenesulfonate [TMA-DPH (donor)] and Octadecyl Rhodamine BChloride [ODRB (acceptor)]. Fluorescence was measured as a function of increasing temperature every 5°C from 15 to 40°C. These studies provide evidence of the molecular complexity and uniqueness of the membrane of *B. burgdorferi* and prove that single deletions of lipoproteins do not affect lipid raft formation.
A055 (M027)

# Investigation of the mechanism of the requirement for P66 in *Borrelia burgdorferi* infection in mice

Laura C. Ristow, Hiromi Sato and Jenifer Coburn

Medical College of Wisconsin, Milwaukee, WI, USA

P66 is an outer membrane protein of *Borrelia burgdorferi* that has been shown to have β3 integrin-binding and channel forming activities *in vitro*. The protein is essential for *B. burgdorferi* to cause infection in mice, but not ticks. Our current focus is on determining the mechanism underlying the defect in Δp66 strains. The Δp66 strains are able to survive in a mammalian host in dialysis membrane chambers implanted into the peritoneum of rats, but are cleared from the site of inoculation in skin within 24-48 h. Live imaging of wild-type (WT) RFP expressing plus GFP expressing Δp66 strains at the site of inoculation reveal that both are motile, and both rapidly disappear from the skin at the inoculation site. The WT bacteria reappear within 5-7 days, but the mutants never reappear. Quantitative PCR data are consistent with the live imaging analyses. We are currently investigating whether ablation of either macrophages or dendritic cells can rescue the Δp66 mutants, and whether the integrin-binding activity of P66, specifically, is required for *B. burgdorferi* to cause infection.
A056 (M029)

mRNA lifetime in *Borrelia burgdorferi*

Linda Archambault, Mike Gilbert, Simmons Borchert and Paula Schlax

Bates College, Lewiston, ME, USA

*Borrelia burgdorferi*, the causative agent of Lyme disease, requires shifts in gene expression to undergo its natural enzootic cycle between tick and vertebrate hosts. mRNA degradation is an important mechanism for gene regulation, but has never been studied in *B. burgdorferi*. We are examining the half-lives of individual mRNAs to determine whether mRNA half-lives correlate with gene function, particularly functions related to the enzootic cycle. We have monitored the mRNA levels of rpoS and flaB following transcriptional arrest in *B. burgdorferi*. Approximately 3 h after transcriptional arrest, rpoS levels were significantly decreased, but still detectable. However, flaB mRNA levels remain high for at least 19 h. qRT-PCR using identical amounts of total RNA were performed and indicate that flaB Ct values increase (suggesting a decrease in relative RNA levels) but are high compared to control reactions run without template or run in the absence of reverse transcriptase. These results suggest that flab mRNA remains a significant fraction of the total RNA and may indicate that the transcript is long-lived. Long-lived fragments of artificially regulated ospC and rpoS transcripts in inducible strains following removal of the inducer IPTG from culture media was also observed.
A057 (M033)

A new method for efficient delivery of bacterial pathogens into ticks

Jenifer Coburn, Monika Casey, Lavinia Padmore and Dara Frank

Medical College of Wisconsin, Milwaukee, WI, USA

Vector-borne bacterial pathogens that are maintained in natural enzootic cycles are of considerable public health interest. The portions of the pathogen life cycles in mammalian hosts have been studied extensively in some cases, but studies in arthropod vectors lag behind. In part, this is due to the difficulties in loading arthropods with sufficient and reproducible doses of bacteria to allow selections or screens for bacterial genes that are expressed in the vector environment. We developed a method to introduce reproducible numbers of *Francisella tularensis* LVS into *Dermacentor variabilis* nymphs. Nymphal ticks are placed on mice and allowed to feed. On the day before tick drop off, LVS is introduced into the mice by retroorbital injection, and the ticks are allowed to feed to repletion. Using this method, different doses of bacteria can be introduced into the mice, and therefore into the ticks. A purMCD mutant derivative of LVS that is not infectious in cell culture or murine models of infection was introduced into the ticks, and demonstrated to be present in the nymphs at drop off but not in freshly emerged adults. A complemented mutant in which the purMCD locus was expressed under the control of an exogenous promoter survived from the molt to the adult stage. This system will allow for a "promoter trap" selection for genes that are expressed in the tick stage of the *F. tularensis* life cycle, and is likely to be adaptable to other vector-borne bacterial pathogens.
A058 (M034)

Comparative transcriptome analysis of geographical distinct virulent and attenuated *Babesia bovis* reveals different gene expression profiles

M. J. Pedroni¹,², K. S. Sondgeroth¹,², G. M. Gallago-Lopez¹,², I. Echaide¹,² and A. O. T. Lau¹,²

1. Washington State University, Pullman, WA, USA

2. Instituto Nacional de Tecnología Agropecuaria, Buenos Aires, Argentina

*Babesia bovis* is a tick-borne apicomplexan protozoan responsible for causing splenic fever globally not only in agriculturally important livestock, but also in human being, often mis-diagnosed for malaria. One unique feature of *B. bovis* is that virulent strains can cause cerebral babesiosis, a syndrome of neurovirulence clinically similar to cerebral malaria. In nature, a wide diversity of *B. bovis* virulent phenotypes exist but experimentally, attenuation can be induced *in vivo* by serial passages of virulent parental strain in a splenectomized host. The phenotypic characteristic of neurovirulence is gradually lost, and an attenuated derivative is obtained. Animals infected with the attenuated derivative are protected upon virulent parental challenge, indicating that determinants of virulence may be modulated independently of epitopes responsible for protective immunity. Mechanism that results in virulence loss is currently unknown. Using *B. bovis* as a model system, not only can one explore the virulence loss phenotype, downstream genomic manipulation is feasible. Recent genomic comparison between three virulent and attenuated *B. bovis* strain pairs indicated that there are no significant changes among the protein-coding genes shared between the pairs that could explain the divergent phenotypes although an overall attenuated strain genome reduction by all three attenuated strains was observed. These data suggest one of two phenomena: (i) differences between virulent and attenuated strain pairs lie in the non-coding regions, which influence transcriptomic variability between virulent and attenuated strains or (ii) the loss of genome content during the attenuation process is the key to attenuation. In this study, we investigated the transcriptomic profiles between two geographically unrelated *B. bovis* strain pairs and reports that though shared differential gene expressions are found, the overall transcriptomic signatures are distinct, suggesting multiple routes to attenuation.
A059 (V007)

# Inactivation of the *Borrelia burgdorferi* BBA66 gene causes a tick transmission defect resulting in attenuated mouse infection

Kevin S. Brandt¹,², Toni G. Patton¹,², Christi Nolder¹,², Dawn R. Clifton¹,², James A. Carroll¹,² and Robert D. Gilmore¹,²

1. Division of Vector Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO, USA

2. Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

Lyme borreliosis, or Lyme disease in humans, is caused by the tick borne pathogen, *Borrelia burgdorferi*. The spirochete exists in a natural transmission cycle colonizing *Ixodes* spp. ticks that acquire the organism after feeding on infected reservoir hosts, most commonly small mammals. Transmission of *B. burgdorferi* from infected nymphal ticks to a host during the bloodmeal feeding process is the key event for human infection. Defining the tick/pathogen/host interactions that facilitate vector transmission is of significant interest in developing strategies to interrupt the enzootic cycle of Lyme borreliosis. The impact of the *B. burgdorferi* surface localized immunogenic lipoprotein BBA66 on vector and host infection was evaluated by inactivating the encoding gene, bba66, by insertional mutagenesis, and characterizing the mutant phenotype throughout the mouse-tick-mouse natural cycle. The BBA66-deficient mutant isolate remained infectious in mice by needle inoculation of *in vitro* cultured organisms, but differences in spirochete burden and pathology in the tibiotarsal joint were observed relative to the parental wild-type (WT) strain. *Ixodes scapularis* larvae successfully acquired the BBA66-mutant following feeding on infected mice, and the organisms persisted in these ticks through the molt to nymphs. A series of tick transmission experiments (n = 7) demonstrated that the ability of mutant-infected nymphs to infect laboratory mice was significantly impaired compared to mice fed upon by WT-infected ticks (45 vs 85%, respectively; *p* value 0.0082). Trans-complementation of the mutant with an intact copy of bba66 restored the WT infectious phenotype in mice via tick transmission. Additionally, a statistical analysis of the number of BBA66 mutant-colonized ticks that fed to repletion as a correlation for mouse infectivity was assessed. Mice were more likely to become infected with the mutant isolate when more than four ticks fed to completion. These results suggest a role for BBA66 in facilitating *B. burgdorferi* dissemination and transmission from tick vector.
A060 (V008)

# Analysis of the *Borrelia burgdorferi* transcriptome in fed nymphs reveals a unique expression profile

Radha Iyer¹, Melissa J. Caimano², Justin D. Radolf²,³ and Ira Schwartz¹

1. Department of Microbiology and Immunology, New York Medical College, Valhalla, NY, USA

2. Department of Medicine, University of CT Health Center, Farmington, CT

3. Department of Genetics and Developmental, University of CT Health Center, Farmington, CT

**Aim:** To characterize and compare the transcriptional profiles of *Borrelia burgdorferi* (Bb) in fed nymphs, in a mammalian host-like environment (dialysis membrane chambers [DMC]) and *in vitro* at 37°C.

**Methods:** Unfed nymphs infected with Bb isolate B31 5A4 were fed to repletion on naive mice. Total RNA was isolated from a pool of five Bb-infected replete nymphs. After DNase treatment and depletion of ribosomal RNA, bacterial mRNA was amplified using the Ambion Message Amp II kit. Amplified BbRNA was hybridized to Bb strain B31-based whole genome microarrays and expression profiles were characterized and analyzed using GeneSpringGX V12.0 software. Using the same methodology, transcriptional profiles were examined for Bb cultivated in DMC and *in vitro* following temperature-shift (37°C). Microarray data were validated by real time RT-PCR of a selected subset of genes.

**Results:** We identified 704 Bb transcripts in fed nymphs, 894 transcripts in DMC, and 840 transcripts *in vitro*. Comparison of transcriptomes revealed 364 gene transcripts that were expressed under all three conditions; these transcripts, 66% of which are chromosomally encoded, presumably constitute the “core transcriptome” that is necessary for Bb survival. In addition, we identified 127 transcripts (94 chromosomal and 33 plasmid-encoded) that were expressed only in fed nymphs; expression of 242 transcripts was detected only in Bb grown in DMCs (88% plasmid-encoded). These genes presumably require either tick- or mammalian host-specific signals for expression. Gene-by-gene comparison of normalized expression data sets identified 106 and 292 transcripts that were differentially expressed (*p* < 0.05) in fed nymphs and DMCs, respectively, compared to spirochetes grown *in vitro*.

**Conclusion:** To our knowledge, this is the first report describing Bb global gene expression profiles from *in vivo* samples containing limited copies of pathogen. The data demonstrate that Bb has a dynamic transcriptome that likely changes throughout the enzootic cycle in response to temporal and host-specific environmental or physiological signals. This method should also be suitable for characterizing the transcriptomes in tissues of experimentally infected mammals.
A061 (V013)

The tick midgut protein Clone19 plays a vital role in the migration of *Borrelia burgdorferi* from the midgut to the salivary glands during a bloodmeal

Sukanya Narasimhana¹,², Jeroen Coumou¹,², Kathleen DePonte¹,², Joppe W. R. Hovius¹,² and Erol Fikrig¹,²

1. Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA

2. Center for Experimental and Molecular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands

The causative agent of Lyme disease, *Borrelia burgdorferi*, is transmitted to humans by the bite of *Ixodes* ticks. It takes approximately 24-48 h before *B. burgdorferi* migrates from the tick's midgut to the salivary glands from where actual transmission to the host may occur. We have recently identified a protein, designated as Clone19 that interacts with *B. burgdorferi* and is located in the tick midgut. We used yeast surface display technology for this identification, where we expressed an *Ixodes scapularis* midgut RNA library, which was probed with *B. burgdorferi* membrane proteins. By studying expression profiles by RT-PCR we established that Clone19 was expressed to a higher extent in the tick midgut than in the salivary glands during the course of a blood meal. Inhibition of Clone19 - by dsRNA silencing - in *B. burgdorferi*-infected ticks that were fed on mice resulted in lower *B. burgdorferi* loads in the salivary glands and significantly lower *B. burgdorferi* numbers in the skin of these mice. These findings were partially corroborated in mice immunized with rabbit anti-Clone19 IgG. After confirming *in vitro* that recombinant Clone19 binds to viable *B. burgdorferi*, we are currently investigating the interaction between Clone19 and *B. burgdorferi*. Recent preliminary data show a difference in *B. burgdorferi* outer membrane expression in the absence of Clone19. Further characterization of Clone19 and knowledge on the binding of *B. burgdorferi* to Clone19 will provide insight into the mechanisms exploited by *B. burgdorferi* to migrate through the tick during a blood meal. This might lead to new strategies to prevent transmission.
A062 (P020)

The HtrA protease of *Borrelia burgdorferi* degrades outer membrane protein BmpD and chemotaxis phosphatase CheX

James L. Coleman¹,², Jameson T. Crowley², Alvaro M. Toledo² and Jorge L. Benach²

1. *N. Y. S. Department of Health, Stony Brook University, Stony Brook, NY USA*

2. *Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY USA*

The high temperature requirement A (HtrA) family of serine proteases is found in all cells from prokaryotes to primates and is a key player in protein quality control. In prokaryotes, HtrA proteins function in the periplasm to degrade or remodel damaged or improperly folded membrane proteins. A unifying feature of this family is the presence of a proteolytic domain (Ser-His-Asp catalytic triad) and either one or two C-terminal PDZ domains that mediate protein-protein interactions. *Escherichia coli* DegP, the prototypical HtrA protease, is a bi-functional molecule, which, in addition to degrading misfolded proteins, functions as a chaperone during a protein folding stress response. *Borrelia burgdorferi*, the spirochetal agent of Lyme disease, codes for a single HtrA homolog, HtrABb (BB0104), which displays 41% amino acid identity to DegP. Recombinant HtrABb exhibited physical and biochemical similarities to DegP, in that it formed a trimeric structure as its fundamental unit and degraded casein via its catalytic serine198 (a catalytic site mutant, HtrABbS198A, did not exhibit proteolytic activity *in vitro*). HtrABb and DegP have some important differences as well. Native HtrABb occurred in both membrane-bound and soluble forms. In addition, despite its homology to DegP, HtrABb could not complement an *E. coli* DegP deletion mutant. Lyme disease patients, as well as infected mice and rabbits, developed a robust antibody response to HtrABb, suggestive of a role in the immunopathogenesis of Lyme disease. In co-immunoprecipitation studies, a number of potential binding partners for HtrABb were identified, as well as two specific proteolytic substrates, basic membrane protein D (BmpD/BB0385) and chemotaxis signal transduction phosphatase CheX (BB0671). HtrABb may function in regulating outer membrane lipoproteins and in modulating the chemotactic response of *B. burgdorferi*.
A063 (P021)

**BobA, from the Lyme disease spirochete *Borrelia burgdorferi*, is surface exposed and binds human fibronectin via multiple independent sites**

Robert A. Gaultney, Tammy Gonzalez, Angela M. Floden and Catherine A. Brissette

*University of North Dakota, Grand Forks, ND, USA*

The eukaryotic protein fibronectin (Fn) is a common target for bacterial pathogens, including staphylococcal and streptococcal species important in causing common infections. The Lyme disease spirochete, *Borrelia burgdorferi*, has several known fibronectin-binding proteins, thereby suggesting importance of this host factor in a Lyme disease infection. Recently, another borrelial protein, BB0347, was identified as having homology to other known fibronectin-binding proteins. To analyze the potential for this protein in a mammalian infection, we created recombinant BB0347 and analyzed its binding to fibronectin *in vitro*. We were also able to determine the expression of this protein by *B. burgdorferi* in culture conditions via RT/QRT-PCR and BB0347-specific Western blotting. Additionally, we cloned peptides from the full-length protein into expression vectors to determine the location of any potential Fn-binding sites. The potential importance of BB0347 was also examined by (1) determining the sub-cellular location of the protein, (2) analyzing the known cell-binding domain of fibronectin for stearic hindrance upon treatment with BB0347, and (3) examining the immunogenicity of the bacterial protein in a mouse infection. BB0347 was found to bind *Fn* *in vitro*, and multiple fibronectin-binding sites facilitate this binding. Additionally, the borrelial protein is expressed by the bacterium in culture, and the expression is at least partially dependent on temperature. BB0347 was also seen to be located on the outer membrane of *B. burgdorferi* and did not interfere with the binding of fibronectin-CBD-specific antibodies to the host protein. Lastly, mice injected with live *B. burgdorferi* formed antibodies against BB0347, suggesting a potential for therapies that target this protein in a Lyme disease patient. Finally, due to its specific properties, we have decided to name the protein borrelial outer-membrane binding protein A (BobA).
A064 (P041)

Role of magnesium (Mg²⁺) in the patho-physiology of *Borrelia burgdorferi*

Trever C. Smith¹,², Tricia Van Laar¹,², S.-L. Rajasekhar Karna¹,² and J. Seshu¹,²

1. South Texas Center for Emerging Infectious Diseases (STCEID), Center for Excellence in Infection Genomics,

The University of Texas at San Antonio, San Antonio, TX, USA

2. Department of Biology, The University of Texas at San Antonio, San Antonio, TX, USA

Lyme disease (LD) is caused by the spirochetal bacterium *Borrelia burgdorferi* (Bb) and is the most prevalent arthropod-borne disease in the US. The life cycle of Bb relies on the colonization of either a tick vector or vertebrate host. In order to adapt to these two hosts, Bb has developed a complex network of genetic and metabolic strategies to rapidly respond to signals received from its host-specific environments. One such strategy is the metal ion dependent transport and intracellular signaling that facilitates adaptive gene expression unique to these hosts. Bioinformatic analysis of the Bb genome revealed the presence of a potential magnesium transporter (bb0380) known as MgtE adjacent to a gene annotated as protein kinase C 1 inhibitor (bb0379). The presence of histidine triad motif (HIT) in BB0379 indicated that this protein could function as a nucleotide binding protein. By utilizing reverse transcription PCR, we showed that MgtE and BB0379 are co-transcribed in Bb under laboratory growth conditions (pH 7.6/320C). To determine the significance of BB0380 and BB0379 in patho-physiology of Bb, we over-expressed and purified recombinant MgtE and BB0379 in *Escherichia coli* and generated anti-sera in mice. Immunoblot analysis revealed that MgtE expression is higher under conditions mimicking the unfed-tick (pH 7.6/230 C) than fed-tick (6.8/370 C). Increasing the pH of the media was found to up-regulate levels of MgtE independent of temperature. Chelation of the growth medium resulting in reduced concentrations of divalent cations was found to increase the levels of MgtE with a concomitant decrease in the levels of SodA, BosR, and OspC. Both mgtE and bb0379 were deleted in Bb strain B31-A3 by replacement with streptomycin resistance marker under the control of a borrelial promoter. The *in vitro* and *in vivo* phenotypic analysis of this mutant will help determine the contributions of these genes to the patho-physiology of Bb.
A065 (D011)

A homologue of tick salivary lectin pathway inhibitor (TSLPI) in *Ixodes ricinus*
protects *Borrelia burgdorferi* sensu lato from killing by human complement

A. Wagemakers¹, J. Coumou¹, G. A. Oei², E. Herkes¹, T. J. Schuijt¹, A. Nijhof³, S. Narasimhan⁴,
E. Fikrig⁴, T. van der Poll¹, A. D. Bins¹ and J. W. Hovius¹

1. Center for Experimental and Molecular Medicine, Academic Medical Center, Amsterdam, Netherlands

2. Department of Medical Microbiology, Academic Medical Center, Amsterdam, Netherlands

3. Freie Universität Berlin, Berlin, Germany

4. Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA

TSLPI is a salivary protein in *Ixodes scapularis* and has been shown to inhibit the host lectin complement pathway by preventing mannose binding lectin (MBL) binding. TSLPI plays an important role in transmission of *Borrelia burgdorferi* sensu stricto from *I. scapularis* to the murine host as well as acquisition from the host to the tick. *Ixodes ricinus* is the main vector for *B. burgdorferi* sensu lato in Europe. With RT-PCR based techniques we identified a TSLPI homologue in *I. ricinus* salivary glands with 93% homology at the DNA and 89% at the protein level. Moreover, a Western blot with SGE from *I. ricinus* female adult ticks (fed to repletion) probed with *I. scapularis* rTSLPI-rabbit antiserum demonstrated the native protein. A BLAST search and a phylogenetic tree indicate that the TSLPI proteins are related to numerous *I. scapularis* and *Ixodes pacificus* proteins including Salp14 (a known Factor Xa inhibitor). To confirm functional homology with *I. scapularis* TSLPI we produced recombinant *I. ricinus* TSLPI in a *Drosophila* expression system and showed that *I. ricinus* rTSLPI protects *B. burgdorferi* sensu stricto and *B. garinii* from killing by human complement. In addition, *I. ricinus* rTSLPI inhibited MBL-dependent complement activation over time in a dose-dependent manner. We have generated *I. ricinus* rTSLPI mutants lacking potential N-glycosylation sites to assess whether these are involved in binding to the carbohydrate recognition domain of MBL and the complement inhibitory effect. To conclude, we have identified a homologue of *I. scapularis* TSLPI in the main European vector of Lyme borreliosis, *Ixodes ricinus*, which protects *Borrelia burgdorferi* sensu lato from killing by the lectin complement pathway at the tick bite site. Our current and future studies aim to elucidate the mechanism underlying TSLPI's MBL-inhibitory function and whether vaccination against TSLPI could prevent *Borrelia* infection.
A066 (D054)

Sensitivity of different *Borrelia* genospecies to dog serum complement

J. Nepereny and V. Vrzal

Bioveta, a.s., Komenskeho, Ivanovice na Hane, Czech Republic

Lyme disease is a chronic multisystem infectious disease that is the most common arthropod-borne infectious disease both in Europe and in the United States. The disease is caused by a group of spirochetes collectively known as *Borrelia burgdorferi* sensu lato. This group of microorganisms is composed of three closely related, most pathogenic subspecies – *Borrelia burgdorferi* sensu stricto, *Borrelia afzelii*, and *Borrelia garinii*. While *B. burgdorferi* sensu stricto is the cause of virtually all Lyme diseases in North America, *B. garinii* and *B. afzelii* prevail in Europe. The existing problems in diagnosis and treatment of Lyme disease and inability to effectively control and reduce the distribution of *Borrelia* vectors gave rise to an urgent need to manufacture a vaccine that would be capable to effectively immunize susceptible species of domestic animals, especially dogs, against infection with *Borrelia burgdorferi* sensu lato. Protective efficiency against each genomospecies of *Borrelia* in a vaccine must be proved by a challenge test on target species prior to registration and approval by state authority. Infection of target species has to be performed via infected ticks. The European Pharmacopoeia contains no special monograph describing efficacy testing of vaccines against borreliosis for veterinary use. Totally, 22 different *Borrelia* strains were tested for their sensitivity to dog serum complement. The borreliol immobilization, bleb formation, and bacteriolysis under dark field microscope were observed, number of live *Borrelia* cells after 8 days of cultivation was calculated and color change of suspension during cultivation was measured. Complement mediated killing of *Borrelia* in OspA and OspC antibodies-free dog serum is important for selection of *Borrelia* strains for preparation of monospecifically and artificially infected ticks for the challenge test. The aims of our work were to select suitable *Borrelia* strains for use in the challenge experiments in mice and dogs. These selected strains must unambiguously invoke specific clinical and serological changes, typical for Lyme borreliosis. Accurate mastered method of the challenge test and the efficacy assessment of vaccine against Lyme borreliosis will be used to prepare a draft monograph and its submission for approval to the European Pharmacopoeia.
A068

# Whole genome sequencing of *Ixodes ricinus*, the European Lyme disease vector

Wibke Jochum, Anna L. Reye and Claude P. Muller

National Public Health Laboratory/CRP Santé, Institute of Immunology, Luxembourg, Luxembourg

*Ixodes ricinus* is the most common tick species and most important vector of human and animal pathogens in western Europe. In contrast to extensive studies in *Ixodes scapularis*, the most important vector of *Borrelia* in North America, information about the most important western European tick *I. ricinus* is mostly limited to epidemiology and ecology. In the last decade, genomics has become a central discipline of biomedical research and genomic approaches are also increasingly applied to study hematophagous arthropods. Data from an *I. ricinus* transcriptome shotgun sequencing of salivary glands are available on NCBI and the genome of *Ixodes scapularis* has already been sequenced in a multiannual genome project as community effort by several investigators. In this study, the genome of its European counterpart, *I. ricinus*, has been sequenced. The sequence has been generated by second generation sequencing using the Illumina Hi Seq 2000 (Illumina, San Diego, USA of Ambry Genetics, Aliso Viejo, USA). DNA was sheared and DNA fragment ends were repaired and phosphorylated using the Klenow fragment, T4 DNA polymerase, and T4 polynucleotide kinase. Then, an adenine was added to the 3' end of the blunted fragments and ligation of illumina Adapters was performed. Gel purification was used to size select the ligated products, which were then PCR amplified using illumina paired-end primers. The library size and concentration were determined using an Agilent Bioanalyzer. Data analysis is based on more than 700,000,000 obtained sequences probably yielding an average six-fold coverage. Sequences will be aligned to references of *I. scapularis* (whole genome contigs) and *I. ricinus* (salivary gland transcriptome library). If necessary, (partial) de novo analysis will be performed. After genome annotation, all sequence data will be made available to public databases such as NCBI or VectorBase. These genome data will provide the basement for identification of *I. ricinus* genes and molecules that will facilitate understanding of physiological processes and tick-host as well as tick-pathogen interaction.
A069 (P008)

# Pathogenesis of Lyme neuroborreliosis: the response of oligodendrocytes to *Borrelia burgdorferi*

Geetha Parthasarathy and Mario T. Philipp

Division of Bacteriology and Parasitology, Tulane National Primate Research Center, Covington, LA, USA

Lyme neuroborreliosis (LNB) is a form of Lyme disease that affects both the central and peripheral nervous systems, with debilitating outcomes. In a rhesus model of LNB, we have previously shown that brains of rhesus macaques that were inoculated with *Borrelia burgdorferi* exhibit inflammatory signs, as evidenced by release of inflammatory mediators, and by oligodendrocyte and neuronal cell death. *In vitro* analysis of this phenomenon indicated that while *B. burgdorferi* can induce inflammation and apoptosis of oligodendrocytes per se, microglia are required for neuronal apoptosis. We hypothesized that the inflammatory milieu elicited by the bacterium in microglia or oligodendrocytes contributes to the apoptosis of neurons and glial cells, respectively, and that downstream signaling events in NFkB and MAPK pathways play a role in these phenotypes. To test these hypotheses in oligodendrocytes, of which little is known, several key pathway inhibitors were used to determine their effect on inflammation and apoptosis, as induced by *B. burgdorferi*. Inhibition of the ERK pathway in the presence of *B. burgdorferi* had the most profound effect on reducing inflammation, followed by JNK, p38, and NFkB pathways indicating that while multiple pathways are involved in eliciting inflammation, the MEK/ERK pathway is predominantly utilized. In addition to eliciting inflammation, low doses of *B. burgdorferi* also elicited an upregulation of total p53 protein, and suppression of the ERK pathway mitigated this effect. While inhibition of p53 had a minimal effect in reducing inflammation, suppression of the ERK pathway or p53 reduced apoptosis as measured by active caspase-3 activity and TUNEL positivity. Thus it is likely that while inflammation and apoptosis in oligodendrocytes are predominantly mediated through the MEK/ERK pathway, they are also two divergent arms of this pathway.
A070 (P009)

Early type I interferon response in mouse skin correlates with
*Borrelia burgdorferi* dissemination

Andrea C. Love, Ira Schwartz and Mary M. Petzke

New York Medical College, Valhalla, NY, USA

*Borrelia burgdorferi* is the extracellular spirochetal agent of Lyme disease, the most common tick-transmitted infection in the United States. Inoculation of *B. burgdorferi* into the skin can lead to the establishment of localized or disseminated infection. Previous work in this laboratory demonstrated that *Borrelia burgdorferi* induces the production of type I interferon (IFN) by human peripheral blood mononuclear cells (PBMCs) *ex vivo*. This response was found to be dependent on *B. burgdorferi* genotype and plasmid content. Based on this observation *ex vivo*, we assessed type I IFN expression during early infection *in vivo*. C3HeJ mice were intradermally needle inoculated with *B. burgdorferi* of varying genotypes. Skin snips were collected from the site of inoculation 24 h later and real-time RT-PCR was used to assess transcript levels for IFN-β and the IFN-responsive genes *mx1*, *ifit1*, *ifit3*, *irf7*, *excl10*, *stat2* and *gbp1*. Infection with B515, an RST1 isolate that induces high levels of IFN-α in human PBMCs and disseminates in mice, led to significant upregulation in transcript levels of type I IFN-responsive genes in 24-h mouse skin snips (*p* < 0.01, all). Conversely, B331, an RST3 isolate that does not disseminate in mice or induce IFN-α in human PBMCs, and B31-4, a lab strain lacking all linear plasmids, were unable to elicit this effect. Live spirochetes were cultured from 24-h skin snips from all B515-infected mice, as well as from 3 of 4 B331-infected mice, indicating that lack of a type I IFN response in the latter group was not due to spirochete clearance. Further, none of the mice infected with B31-4 or B331 developed disseminated infection, whereas 5 of 6 B515-infected mice did (*p* < 0.001). This indicates a link between the ability of *B. burgdorferi* to induce type I IFNs and capacity to disseminate, suggesting that there are inherent differences in the interactions of different *B. burgdorferi* genotypes and the host at the site of infection.
A071 (P010)

Conditional deletion of MyD88 during either acute or chronic phases
of *Borrelia burgdorferi* infection exacerbates murine Lyme borreliosis

Alexia A. Belperron, Jialing Mao and Linda K. Bockenstedt

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

Toll-like receptor (TLR) recognition of *Borrelia burgdorferi* (Bb) is a key factor controlling pathogen burden in Lyme borreliosis. Previous studies have shown that Bb infection of mice deficient in TLR2 or the TLR intracellular adaptor molecule MyD88 develop up to 100-fold increases in spirochete burdens relative to wildtype (WT) mice. In this study, we generated conditional MyD88 mutant mice (C57BL6 MyD88fl/-Esr1cre/wt[B6Cre+]) that can be rendered MyD88 deficient by tamoxifen treatment to determine whether MyD88-dependent immunity influences pathogen burdens after spirochetes have disseminated and adaptive immune responses have been generated. WT B6 mice, Cre+ mutants, and Cre-littermates were inoculated intradermally with 10e4 Bb914, a transformant of Bb strain 297 constitutively expressing GFP under the control of the flab promoter. Groups of mice were injected intraperitoneally with tamoxifen administered in oil daily for 10 days or with vehicle only during the acute dissemination phase (within 3 weeks) or the persistent phase (12 weeks) of infection. Splenic macrophages from tamoxifen-treated Cre+ mice produced lower levels of TNFα upon *in vitro* stimulation with Bb lysate and mice treated early in infection had increased IgG1 and reduced IgG2a reactivity on immunoblot, consistent with reduction in MyD88 expression. The majority of Cre+ mice treated with tamoxifen during the acute or chronic phase of infection were bacteremic on the last day of treatment, whereas Bb was cultured from the blood of only one of the WT and Cre-control mice. Bacteremia associated with MyD88-deficiency was transient, however, because none of the Cre+ mice had positive blood cultures when analyzed 10 days after completion of tamoxifen treatment. Pathogen burdens in ear tissues of Cre+ mice rendered MyD88-deficient were increased, as assessed 10 days after completion of tamoxifen treatment by qPCR for Bb DNA and by visualization of higher spirochete numbers in ear skin with two-photon intravital microscopy. Tamoxifen treatment of chronically infected Cre+ mice also resulted in elevated pathogen burdens in the heart and ear tissues, without effects on Bb-specific IgG isotypes. Preliminary results show that conditional deletion of MyD88 also results in increased amounts of GFP deposits at cartilaginous sites in the ear and knee joints in comparison to WT or sham-treated Cre+ mice. These results demonstrate that MyD88-dependent immunity is not only necessary to control pathogen burdens and eliminate spirochete antigenic remnants when Bb first establishes infection, but also during a persistent phase when spirochetes have colonized tissues and Bb-specific IgG titers are high.
A072 (P011)

Tick-specific borrelial antigens appear to be up-regulated in American but not European patients with Lyme arthritis, a late manifestation of Lyme borreliosis

Xin Li¹, Klemen Strle²,³, Peng Wang¹, David I. Acosta¹, Gail A. McHugh², Nikhil Sikand², Franc Strle³ and Allen C. Steere²

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

2. *Massachusetts General Hospital, Boston, USA*

3. *University Medical Center Ljubljana, Ljubljana, Slovenia*

*Borrelia burgdorferi* (Bb) sensu lato, the etziologic agent of Lyme borreliosis, adapts to distinct environments in the mammalian host and the tick vector by differential gene expression. As a result, infected mice are not exposed to and rarely make antibodies to the set of antigens that are preferentially expressed in the tick, including outer surface protein A (OspA), Borrelia iron and copper-binding protein A (BicA), and OspD. Surprisingly, however, antibodies to OspA and BicA have been noted in American patients with Lyme arthritis. Here, we examined serum samples from 210 American patients and 66 European patients with a range of early or late manifestations of Lyme borreliosis and found that only American patients with Lyme arthritis commonly had antibody responses to OspA, BicA, and OspD. This suggests that infection with American but not European *Borrelia* strains often leads to concerted up-regulation or de-repression of tick-specific spirochetal antigens in these patients.
A073 (P012)

CspA orthologs of serum-resistant Lyme disease spirochetes are multifunctional complement inhibitory molecules

Claudia Hammerschmidt¹,²,³, Arno Koenigs¹,²,³, Corinna Siegel¹,²,³, Teresia Hallström¹,²,³, Christine Skerka¹,²,³, Reinhard Wallich¹,²,³, Peter F. Zipfel¹,²,³ and Peter Kraiczy¹,²,³

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

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

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

Many important human pathogens overcome the destructive effects of complement by recruitment of fluid-phase complement regulators. Particularly, *Borrelia burgdorferi*, *Borrelia afzelii*, and *Borrelia spielmanni* resist complement-mediated killing by binding of CFH and FHL-1. Recently, the CFH and FHL-1-binding CspA protein has been identified as the key molecule of *B. burgdorferi* conferring resistance to complement-mediated killing. Owing to their property to bind these regulators, it has been suggested that CspA of *B. afzelii* and *B. spielmanni* is responsible for serum resistance, but so far closer examination of the CspA proteins of *B. afzelii* and *B. spielmanni* is lacking. Here, we investigate and compare the different binding capacities of the CspA orthologs for CFH and plasminogen and further look at their ability to inhibit complement by either binding these fluid-phase regulators or independently of these regulatory molecules. A serum-sensitive *B. garinii* strain was used to generate transformed spirochetes, ectopically producing CspA orthologs to examine their function in serum resistance in vivo. Recombinant CspA orthologs were used to analyze the inhibitory effect on complement activation and to assess their potential to inactivate C3b by binding of complement regulator factor H (CFH) and plasminogen. Irrespective of their species origin, all three CspA orthologs impart resistance to complement-mediated killing when produced ectopically in a serum-sensitive *B. garinii* strain. All three CspA orthologs simultaneously bound CFH and plasminogen. CspA orthologs differ in regard to their capacity to inactivate C3b via bound plasmin(ogen) and inhibit formation of the membrane attack complex (MAC). CspA of *B. afzelii* binds plasmin(ogen) and inhibits the MAC more efficiently than CspA of *B. burgdorferi* and *B. spielmanni*. Taken together, CspA orthologs of serum-resistant Lyme disease spirochetes act as multifunctional evasion molecules that inhibit formation of the MAC and inactivate C3b by binding CFH and plasmin(ogen).
A075 (P019)

An ex vivo skin model to study the role of skin dendritic cells in the early
immunopathogenesis of Lyme borreliosis

Lauren M. K. Mason¹, Tom van der Poll¹, Teunis B. H. Geijtenbeek² and Joppe W. R. Hovius¹

1. Center for Experimental and Molecular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands

2. Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands

*Borrelia burgdorferi* sensu lato, the causative agent of Lyme borreliosis, is transmitted into the skin of the host during a tick bite. In the skin, two different types of dendritic cell (DC); epidermal Langerhans cells (LCs) and dermal dendritic cells (DDCs), are among the first cells of the immune system to come into contact with *Borrelia*. These cells patrol the skin for potential pathogens, which are recognized via pattern recognition receptors (PRR), upon which they become mature and migrate out of the skin. In draining lymph nodes, DCs prime T cells launch an adaptive immune response against the invading pathogen. Thus, DCs form a crucial link between the innate and adaptive immune system. It is of paramount importance to study DC subsets in their natural environment. Therefore, in order to study the role of skin DCs in the early immunopathogenesis of Lyme borreliosis, we set up a human *ex vivo* model using full-thickness donor skin. *B. burgdorferi* sensu stricto strain B31 was injected intradermally into the skin, mimicking the transmission of *Borrelia* via a tick bite. Subsequently, we studied the migration of LCs and DDCs out of the skin 24-72 h after inoculation, the activation profile of migrated cells by flow cytometry and measured cytokine production. We found a partially dose-dependent significant increase in the migration of both LCs and DDCs in response to injection with spirochetes over time, compared to injection with PBS. A preliminary experiment revealed that inhibition of Toll-like receptor (TLR)-2 – the TLR largely responsible for the recognition of *Borrelia* – with a blocking antibody inhibited the increase in migration. DCs migrated from skin inoculated with high numbers of spirochetes also expressed slightly, but significantly higher levels of the maturation marker CD83 and produced higher levels of multiple pro-inflammatory cytokines. No difference was observed in the expression of HLA-DR or CD86. Finally, we were able to culture *Borrelia* from inoculated skin 48 h after inoculation, demonstrating that the DCs are interacting with live spirochetes in this model. In conclusion, we have shown that inoculation of human skin with *Borrelia* enhances migration of LCs and DDCs out of the skin. The migrated cells are more mature and produce higher levels of pro-inflammatory cytokines than cells migrated from skin injected with PBS. This model is currently being applied as a new tool in further studies on the role of these cells in the immunopathogenesis of Lyme borreliosis.
A076 (P023)

Differential adhesion of Lyme disease spirochetes to human keratinocytes

S. Bigelmayr, C. Hammerschmidt, A. Koenigs and P. Kraiczy

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

Upon entry into the human host, spirochetes have developed numerous strategies to successfully colonize host tissues and survive in an unfavorable environment. Adhesion to human cells is thought to be a key step for the establishment of an infection causing multiple clinical symptoms including serious neurological as well as long-term dermatological manifestations. Infection of the various tissues depends on the ability of spirochetes to bind to different cell types. In this study, we examined the ability of Lyme disease spirochetes belonging to five distinct human pathogenic genospecies (*Borrelia burgdorferi*, *Borrelia afzelii*, *Borrelia garinii*, *Borrelia spielmanii*, and *Borrelia javariensis*) and two genospecies with disputed pathogenic potential (*Borrelia lusitaniae* and *Borrelia valaisiana*) to bind to human keratinocytes. Among the genospecies analyzed, *B. valaisiana* and *B. spielmanii* showed the strongest attachment while *B. javariensis*, *B. garinii*, and *B. afzelii* displayed moderate binding, suggesting a role of these five genospecies in mediating infection of the human skin. By contrast, *B. burgdorferi* and *B. lusitaniae* showed no binding at all. Furthermore, intraspecies differences have been observed among *B. garinii*, *B. javariensis*, *B. afzelii*, and in particular *B. valaisiana*. Recently, it has been shown that complement regulator-acquiring surface proteins (CRASPs) bind to components of the extracellular matrix. To further assess the role of these infection-associated borrelial outer surface proteins in mediating interactions with human cells, *B. garinii* cells producing distinct CRASPs originally derived from *B. burgdorferi* were screened for binding to keratinocytes. Interestingly, binding capacity for human keratinocytes increased up to four-fold when testing a *B. garinii* strain producing CRASP-4 but not CRASP-1, CRASP-2, or CRASP-3 compared to wild-type *B. garinii* cells lacking CRASPs. Taken together, these data provide evidence that distinct borrelial genospecies differ in their ability to attach to human keratinocytes and, in addition, support a role of certain CRASPs as potential adhesins of Lyme disease spirochetes.
A077 (P027)

A surface protease of Lyme disease bacteria degrades host ECM components
and induces inflammatory cytokines

Theresa M. Russell, Mark J. Delorey and Barbara J. B. Johnson

Centers for Disease Control and Prevention, Division of Vector-Borne Diseases, Fort Collins, CO., USA

*Borrelia burgdorferi* is deposited into the skin during the feeding of an infected tick. Systemic infection develops as *Borrelia* migrates through tissue extracellular matrices (ECM) to colonize various parts of the body. The paradigm in Lyme disease research has been that *B. burgdorferi* does not produce proteases capable of damaging host tissues. Consequently, borrelial migration and the inflammation-related pathologies observed in Lyme disease have been attributed to an exuberant host inflammatory response to the bacteria. Here, we identify and characterize a surface-exposed protease of *B. burgdorferi* (BbHtrA) that is expressed in human disease and conserved within the major Lyme disease spirochetes. BbHtrA degrades fibronectin and numerous proteoglycans found in skin, joints, and neural tissues. BbHtrA degradation of fibronectin releases highly pro-inflammatory fibronectin fragments FnIII13-14 and Fnf-29. These bioactive fragments may amplify the inflammatory processes triggered by the presence of the bacteria itself. When this hypothesis was tested directly by exposing chondrocytes to BbHtrA in vitro, inflammatory cytokines (sICAM-1 and IL-6) and chemokines (CXCL1, CCL1, CCL2, CCL5, and IL-8) that are hallmarks of Lyme disease were induced. Furthermore, these responses required the proteolytic activity of the protease. Together, these data suggest that *B. burgdorferi* has the capacity to degrade the host ECM and thereby participate directly in its own dissemination. Additionally, BbHtrA proteolytic activity may exacerbate the host inflammatory responses observed in Lyme disease.
A078 (P028)

# Pyruvate protects *Borrelia burgdorferi* from hydrogen peroxide killing and enhances infectivity in a murine model of Lyme disease

Bryan Troxell and Frank Yang

Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA

*Borrelia burgdorferi*, the causative agent of Lyme disease, encounters reactive oxygen species (ROS) stress during its enzootic cycle in the arthropod and mammalian host. Although *B. burgdorferi* lacks known ROS-defensive enzymes such as peroxidase or catalase, this pathogen was thought to be resistant to H₂O₂ due to the near absence of intracellular iron which prevents DNA damage due to the Fenton reaction. In this report, we demonstrate that pyruvate, a potent H₂O₂ scavenger, can promote *B. burgdorferi* resistance to H₂O₂. When extracellular pyruvate is absent, *B. burgdorferi* is sensitive to even a low dose of enzymatically generated H₂O₂ by glucose oxidase (GOX). H₂O₂ killing coincided with a disruption of the intracellular NAD+/NADH balance, suggesting that the killing is at least partially due to a redox imbalance. Furthermore, we showed that pyruvate could enhance the infectivity of *B. burgdorferi* in a murine model, through the reduction of ROS generated by human neutrophils. Our findings suggest that pyruvate, in addition to its classical role in metabolism, serves as an important H₂O₂ scavenger for organisms that lack enzymatic defense against H₂O₂. Extracellular pathogens may exploit the metabolite pyruvate, present in blood and tissues, to defend against H₂O₂ during infection.
A079 (P031)

# Heterogeneity of *Borrelia burgdorferi* ss population and its involvement in *Borrelia* virulence: study on a murine model

Nathalie Boulanger¹,², Aurélie Kern¹,², Gilles Schnell¹,², Amandine Boeuf¹,², Laurence Sabatier¹,², Sylvie De Martino¹,² and Benoît Jaulhac¹,²

1. EA7290, virulence bactérienne précoce: groupe Borréliose de Lyme, Facultés de médecine et de pharmacie, Université de Strasbourg, Strasbourg, France

2. Laboratoire de Spectrométrie de Masse BioOrganique, Institut Pluridisciplinaire Hubert Curien, Strasbourg, France

*Borrelia* is responsible for multisystemic disorder in humans, with an initial cutaneous inflammation, the erythema migrans then disseminated manifestations in the skin, the joint, and the central nervous system. Since the skin seems to be a key organ in the development of Lyme borreliosis and might define the organotropism, we compared the skin inflammation of different clinical isolates (human pathotypes) of *Borrelia burgdorferi* sensu stricto in C3H/HeN mouse. All the pathotypes showed an intense multiplication in the skin at day 7, pointing out the role of the skin as an amplifying site for *Borrelia* during the transmission. We found differences in their dissemination to the target organs in mice, more interestingly a very different inflammatory gene profile in the infected skin. In addition to host factors, *Borrelia* infecting strains might be constituted of a heterogenous population of bacteria that might influence its virulence in humans. To better investigate this hypothesis, we carried out a cloning on BSK-modified agar medium of *Borrelia burgdorferi* ss, strain 297. Among the different clones, we selected one for its high inflammatory profile in the skin and its speed of dissemination in mice. A precise proteomic analysis by electrophoresis and mass spectrometry revealed specific proteins for the virulent clone studied, not detected in the native strain. We further investigated the kinetics of the expression of these specific proteins in the mouse. Such an approach could constitute an original strategy to identify *Borrelia* proteins essential in the transmission and development of the bacteria in the vertebrate host.
A080 (P033)

Autophagy modulates *Borrelia burgdorferi*-induced production of IL-1beta

Kathrin Buffen¹, Marije Oosting¹, Theo S. Plantinga¹, Thirumala-Devi Kanneganti²,
Mihai G. Netea¹ and Leo A. B. Joosten¹

1. Department of Medicine, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands

2. Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA

*Borrelia burgdorferi* sensu lato is the causative agent of Lyme disease. Recent studies have shown that recognition of the spirochete is mediated mainly by TLR2 and NOD2, which have been associated to autophagy. Since IL-1beta is linked to the pathogenesis of Lyme disease, we examined the role of autophagy for the production of IL-1beta in response to *B. burgdorferi*. Our results demonstrate activation of autophagy in GFP-LC3 expressing HeLa cells shortly after exposure to *B. burgdorferi*. Human PBMCs treated with autophagy inhibitors showed an increased IL-1beta and IL-6 production in response to *Borrelia* exposure, while TNFalpha production was unchanged.

Autophagy induction against *B. burgdorferi* was dependent on reactive oxygen species (ROS) since cells from patients with chronic granulomatous disease (CGD), which are defective in ROS production, also produced elevated IL-1beta compared to healthy controls. Further, the enhanced production of the proinflammatory cytokines was due to the elevated mRNA expression in the absence of autophagy. Since several studies indicated a link between high concentrations of IL-1beta and pathogenic Th17 cells leading to increased joint damage in rheumatoid arthritis (RA) or psoriasis patients, we hypothesize that the induction of autophagy could be beneficial during the chronic phase of Lyme disease leading to a decreased IL-1beta production and inhibition of Th17 cells. In summary, we have elucidated the link between *B. burgdorferi* recognition and an important regulatory mechanism of inflammation by autophagy in Lyme disease. These findings may reveal novel therapeutic targets to treat Lyme disease patients in the future.
A081 (P034)

Interferon-γ secreted by *Borrelia burgdorferi* exposed CD56+ NK(T) cells promotes keratinocyte-derived β-defensins and psoriasis (S100A7) production

Rosa Pascual Domingo¹, Marije Oosting¹, Kathrin Buffen¹, Ellen van de Boogaard², Joost Schalkwijk², Mihai G. Netea¹ and Leo A. B. Joosten¹

1. Department of Medicine, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands

2. Department of Dermatology, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands

*Borrelia burgdorferi* sensu lato is the causative agent of Lyme disease. It has been shown that *Borrelia*-derived antigens can stimulate CD56+ NK(T) cells to produce several proinflammatory cytokines. Since NK(T) cells are abundantly present in the skin of humans, we investigated whether NK(T) cells derived cytokines can activate keratinocytes to produce proinflammatory mediators. This interaction between NK(T) cells and keratinocytes may be of importance in the development of the erythema migrans (EM) that occurs after infection of humans with *B. burgdorferi*. Our results demonstrated that CD3–CD56+ NK cells are the main producers of *B. burgdorferi*-induced IFN-γ production by human primary PBMCs rather than CD3+ CD56+ NK-T cells. Apart from IFN-γ, NK(T) cells produced high levels of both IL-1β and IL-6 after *in vitro* exposure to *B. burgdorferi*. It reveals that CD56+ NK(T) cells isolated from Lyme disease patients produce more IFN-γ than NK(T) cells from healthy individuals, when exposed to *B. burgdorferi*. To investigate the cross-talk between *B. burgdorferi* activated NK(T) cells and human keratinocytes, we performed *in vitro* experiments, in which keratinocytes were exposed to culture supernatants of *B. burgdorferi* stimulated NK(T) cells. After exposure to these NK(T) cell supernatants for 48 h, the mRNA expression of hBD2, hBD3, and psoriasis was determined in the keratinocytes. The mRNA expression of hBD-2 and psoriasis was strongly upregulated in keratinocytes and to a lesser extent hBD3. It has been demonstrated that psoriasis is involved in skin inflammation and therefore psoriasis might contribute to the development of EM. In addition, both hBD2 and hBD3 have been linked to chemotaxis of proinflammatory cells, such as monocytes and macrophages, and therefore may support skin inflammation. Future research is warranted to identify the precise role of NK(T) cells originated IFN-γ in the development of EM. Novel insights in the cross-talk between NK(T) cells and keratinocytes may result in a better understanding of the mechanisms behind the formation of the classical erythema migrans after infection with *B. burgdorferi*.
A083 (P045)

# Understanding barriers to *Borrelia burgdorferi* dissemination during infection using massively parallel sequencing

Erin B. Troy<sup>1,2,3</sup>, Tao Lin<sup>1,2,3</sup>, Lihui Gao<sup>1,2,3</sup>, David W. Lazinsky<sup>1,2,3</sup>, Andrew Camilli<sup>1,2,3</sup>,
Steven J. Norris<sup>1,2,3</sup> and Linden Hu<sup>1,2,3</sup>

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

2. Department of Pathology and Laboratory Medicine, University of Texas Medical Center, Houston, TX

3. Department of Molecular Biology and Microbiology, Tufts University Medical School, Boston, MA, USA

*Borrelia burgdorferi* is an invasive spirochetal pathogen that can cause acute and chronic infections in the skin, heart, joints, and central nervous system of infected mammalian hosts. Following transmission into a mammalian host through the bite of an *Ixodes scapularis* tick, the bacteria establish infection at the inoculation site and then quickly disseminate to distal tissues initiating long-term colonization. In this process, *B. burgdorferi* encounters multiple potential barriers to infection including adapting to environmental changes in nutrients, pH, and temperature, breaking through tissue barriers to invasion and dissemination, and evading host immune responses. Little is currently understood about which barriers have the largest effect on *B. burgdorferi* infection. A population bottleneck is an event in which the size of a population is temporarily, stochastically reduced. Bottlenecks during infection represent points at which the pathogen experiences a barrier to establishing and maintaining infection. To identify population bottlenecks during *B. burgdorferi* infection of a murine host, we adapted a quantitative massively parallel sequencing strategy, Tn-seq, for studies of *B. burgdorferi* virulence. This technology allowed us to quantitatively measure the frequency of individual *B. burgdorferi* strains within a mixed population of transposon mutants. Tn-seq is sensitive and reproducible. Individual mutants present at frequencies as low as 0.1% could be reliably detected within a population. This is a versatile technique that can be used to answer an array of open questions in *B. burgdorferi* biology. Both wild type mice and mice lacking the toll-like receptor adapter molecule MyD88 were infected with a pool of infectious *B. burgdorferi* transposon mutants with insertions in the same gene. At multiple time points post-infection, bacteria were isolated from the mice and the composition of the *B. burgdorferi* populations at the injection site and in distal tissues determined. We identified a population bottleneck at the site of infection that significantly altered the composition of the population. The magnitude of this bottleneck was reduced in MyD88/- mice indicating a role for innate immunity in limiting early establishment of *B. burgdorferi* infection. There was not a significant bottleneck during the colonization of distal tissues suggesting that founder effects are limited and there is not a strict limitation on the number of organisms able to initiate populations at distal sites. The observed lack of host barriers to disseminated infection may account for the environmental and clinical observations that mammalian hosts can be infected with multiple strains of *B. burgdorferi* simultaneously.
A084 (M012)

Macrophage inflammatory response to *Borrelia burgdorferi* is diminished in the presence of mouse sera compared to sera from deer and humans

Ping He¹,², Robert Smith¹,², Fatima Sammy¹,², Tian Lin¹,², Laura Beasley-Topliffe¹,², Young Kwak¹,², Bruce Cahill¹,², Susan Elias¹,², Charles Lubelczyk¹,² and Shaw Warren¹,²

1. *Infectious Disease Unit, Massachusetts General Hospital, Charlestown, MA, USA*

2. *Vector-borne Disease Laboratory, Maine Medical Center Research Institute, South Portland, ME, USA*

Lyme disease is maintained in the northeastern United States in enzootic cycles of *Borrelia burgdorferi* sensu stricto (Bb ss) by *Ixodes scapularis* and vertebrate reservoir hosts. Resistance to infection (ability of an infected host to limit microbial burden) and resilience to infection (ability to limit disease) may determine which hosts are effective (reservoir competent). White-footed mice (*Peromyscus leucopus*) remain Bb-infected for months whereas white-tailed deer (*Odocoileus virginianus*) appear highly resistant to infection and do not contribute to maintenance of the enzootic cycle. One hypothesis for differences in resistance and resilience among species is that proteins in serum modulate the response of macrophages to Bb differently. We investigated whether macrophage inflammatory response to Bb was diminished in the presence of mouse sera relative to sera from deer, humans, and other species. Mouse bone marrow-derived macrophages (BMDMs) or human peripheral blood mononuclear cells (PBMCs) were stimulated with Bb or Pam3Cys in the presence of either mouse, human, deer or calf serum. Mouse serum suppressed Bb- and Pam3Cys-induced TNF more than human, calf, or deer serum. Bb also induced significantly less TNF after incubation in mouse whole blood compared to in human whole blood. These data are consistent with the hypothesis that persistent infection of mice with low accompanying morbidity could be at least in part due to a relative decrease in the response to Bb by immune cells in mouse tissues or blood compared with those of deer or humans, with consequently less secondary inflammation that would lead to clearance of the organism.
A085 (M024)

Borrelia miyamotoi infection of *Peromyscus leucopus* and *Mus musculus*

Alan G. Barbour, Azadeh S. Estabragh and Arash G. Langeroudi

University of California Irvine, Irvine, CA, USA

*Borrelia miyamotoi* and *Borrelia lonestari* are two species in the relapsing fever (RF) cluster of the genus *Borrelia*, but they are transmitted by hard ticks instead of soft. The role of these and closely related species, like *B. theileri*, in human disease was doubted until recent reports from Russia and the United States provided evidence of *B. miyamotoi* infection in humans as well as their reservoir species. *B. miyamotoi* infection is of particular public health concern, because its reservoirs and tick vectors, such as *Ixodes scapularis*, are the same as those for Lyme disease species and other pathogens in North America, Europe, and Asia. However, little is known about the biology and pathogenesis of this species in either rodents, or the mammals, or humans. We previously found that the genetic diversity of *B. miyamotoi* appears to be more limited than for Lyme disease species, but this was based on only a few loci. Further characterization of the genome of *B. miyamotoi* and the identification of markers for genotyping are underway. We also previously reported that in natural infections of the white-footed mouse *Peromyscus leucopus*, *B. miyamotoi* and *B. burgdorferi* occupy different niches, with *B. miyamotoi* behaving like other RF species in reaching higher densities in the blood and *B. burgdorferi* reaching higher densities in the skin. Further studies of *B. miyamotoi* during experimental and natural infection are in progress. These include investigation of innate and adaptive immune responses in *P. leucopus* and the laboratory mouse *Mus musculus* and further characterization of the courses and extents of infection. The results may provide for further understanding of the ecology of *B. miyamotoi* and the pathogenesis and immunity of infection in reservoir hosts and in humans.
A086 (M032)

A mathematical model of the population dynamics of *Borrelia burgdorferi*
in Lyme disease

Sebastian C. Binder, Arndt Telschow and Michael Meyer-Hermann

Helmholtz Centre for Infection Research, Westfalian Wilhelms-University Muenster, Technical University
Braunschweig, Braunschweig, Germany

Although studied extensively in human patients and animal models, the intra-host dynamics of the tick-borne bacteria of the genus *Borrelia* are not fully understood. *Borrelia* exhibits a remarkably high infectivity, despite being killed efficiently *in vitro* by human phagocytic cells. In experimentally infected mice, the first immune response almost clears the infection at the injected skin site. However, the bacterial population recovers ~1 week post infection and reaches an even larger concentration before it stabilizes at lower levels. We developed a mathematical model describing the bacterial growth to explain the bacterial dynamics, including a simple phenomenological model of innate and adaptive immune processes. According to our model, the peculiar dynamics at the infection site as measured in published experimental studies cannot be explained by bacterial migration processes or a limitation in the capacity of phagocytic cells alone. The mathematical model predicts that *Borrelia* recovers from the strong initial immune response by adapting to the host environment and that this adaptation process is critical to the pathogen's survival inside its host at the very first stage of the disease.
A087 (V005)

Factor XA-induced activation of Factor V is of paramount importance in initiating
the coagulation system: lessons from a tick salivary protein

Tim J. Schuijt¹,³, Kamran Bakhtiari², Sirlei Daffre³, Kathleen DePonte³, Simone J. H. Wielders⁴,
J. Arnoud Marquart², Joppe W. Hovius¹, Tom van der Poll¹, Erol Fikrig³, Gerry A. F. Nicolaes⁴,
Joost C. M. Meijers² and Cornelis van 't Veer¹

1. Center for Experimental and Molecular Medicine, University of Amsterdam, Amsterdam, Netherlands

2. Department of Experimental Vascular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, AZ, Netherlands

3. Yale University, New Haven, CT

4. Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands

Background: Generation of active procoagulant cofactor FVa to the enzyme FXa to form prothrombinase complex is a pivotal initial event in blood coagulation and has been the subject of investigative effort, speculation, and controversy. The current paradigm assumes that FV activation is initiated by limited proteolysis by traces of (meizo) thrombin.

Methods and Results: Recombinant tick salivary protein TIX-5 was produced and anticoagulant properties were studied using plasma, whole blood, and purified systems. We here report that anticoagulant tick salivary protein TIX-5 specifically inhibits FXa-mediated FV activation involving the B-domain of FV and show that FXa activation of FV is pivotal for plasma and blood clotting. In line, tick feeding is impaired on TIX-5 immune rabbits displaying the *in vivo* importance of TIX-5.

Conclusions: Our data elucidate a unique molecular mechanism by which ticks inhibit the host's coagulation system. Based on our data, we propose a revised blood coagulation scheme wherein direct FXa-mediated FV activation occurs in the initiation phase during which thrombin-mediated FV activation is restrained by fibrinogen and inhibitors.
A088 (V009)

The role(s) of RNA sensing TLRs and adapter molecules in the host defense to *Borrelia burgdorferi* and in *B. burgdorferi* RNA-mediated type I IFN induction

Amy A. Devlin, Priya Jayaraman and Jennifer C. Miller

Department of Microbiology, North Carolina State University, Raleigh, NC, USA

It is well established that both TLR2 and MyD88 assume critical roles in host defense to *Borrelia burgdorferi*. TLR2-/- mice contain hundreds additional *B. burgdorferi*, whereas MyD88-/- mice harbor thousands more spirochetes within their ankle joints when compared with the bacterial burden present within wild-type mouse ankle joints. These data suggest that other TLR(s) may participate in the host defense to *B. burgdorferi*. We recently discovered that *B. burgdorferi* RNA is a potent immunostimulatory ligand for mouse bone marrow-derived macrophages (BMDMs). Based on this finding, we examined whether TLR7, a MyD88-dependent receptor for single-stranded RNA, was required for clearance of *B. burgdorferi* from mouse ankle joints. Equivalent numbers of *B. burgdorferi* were detected by quantitative real-time PCR within the ankle joints of wildtype and TLR7-/-C57BL/6 (B6) mice sacrificed at 4 weeks post-infection. *B. burgdorferi*-specific serum IgG levels at 4 weeks post-infection also did not differ between wildtype and TLR7-/- animals. Type I IFN-stimulated gene transcript (ISG) levels were also assessed within B6 and TLR7-/- mouse joints at 1-week post-*B. burgdorferi* infection. In accordance with a previous report, B6 ankle joint cells did not induce the Type I ISG Oasl2. However, Oasl2 transcription was markedly upregulated within TLR7-/- ankle joints. Type I ISG transcript levels were induced to similar levels in wildtype B6 and TLR7-/- BMDMs following stimulation with either live *B. burgdorferi* or *B. burgdorferi* RNA. Taken together, our data indicate that TLR7 is not required for *Borrelia burgdorferi* clearance from mouse ankle joints or for Type I IFN-responsive gene induction. Current studies are examining the role(s) of the TLR adapter molecules MyD88 and TRIF in the Bb RNA-mediated BMDM Type I IFN response.
B001 (V002)

Neural correlates of depression in PTLDS

James R. Moeller¹, Andrew E. Levin², John G. Keilp¹, Harold A. Sackeim³,⁴ and Brian A. Fallon¹

1. Department of Psychiatry, New York State Psychiatric Institute, Columbia University, New York, NY, USA

2. Immunetics Inc., Boston, MA, USA

3. Department of Psychiatry, Columbia University, New York, NY, USA

4. Department of Radiology, Columbia University, New York, NY, USA

Depressive symptoms, common among patients with post-treatment Lyme disease syndrome (PTLDS), have been described by some investigators as subjective-likely to have little relationship to Lyme disease. Neurobiological data from a treatment study of post-treatment Lyme disease syndrome (with encephalopathy) (ePTLDS) allow a further examination of depressive symptoms in terms of neural activity and Bb-specific antibody reactivity in serum and the intrathecal space.

**Methods:** The sample, drawn from an NINDS-funded study of ePTLDS, consists of 25 patients (age 45 ± 13 years and 56% female). Beck Depression Inventory (BDI) self-report assessed depression. Bb-specific antibody measure was the C6 ELISA (serum, CSF and intrathecal antibody Index – Immunetics C6-AI). The measure of neural activity is regional glucose metabolism (rCMRglu-PET). Functional regional interactions associated with depression are modeled using Principal Component Maps of covarying regional metabolism (PC-maps). Changes in nodal interactions associated with differences in intrathecal antibody reactivity are modeled as ANOVA interactions between PC-maps and C6-AI. To conserve statistical power, ANOVA is restricted to the three PC-maps that account for the largest patient variance in rCMRglu-PET.

**Results:** C6-AI was elevated in 15 patients and non-elevated in 10. Group mean BDI scores are comparable, as are each group's range of scores from mild to severe. *p* < 0.005 for the ANOVA prediction of BDI based on the PC Maps and C6-AI results (*R*² = 0.68). PC-Maps 2 and 3 have significant main effects (*p* < 0.005 and 0.01, respectively) and significant interactions with C6-AI (*p* < 0.05 and 0.005, respectively). Patient activity in PC-Map 2 is correlated with depression severity in patients with non-elevated C6-AI (*r* = 0.80); but not in patients with elevated C6-AI (*r* = 0.24). The reverse is true of PC-Map 3: *r* = 0.04 in patients with non-elevated C6-AI; and *r* = 0.66, for elevated C6-AI. PC-Map 2 reveals strong functional interactions between amygdala, nucleus accumbens, midbrain areas (PAG and VTA), brain stem (NTS), hypothalamus, hippocampus, thalamus, and anterior cingulate Cg25. PC-Map 3 reveals a different functional pattern of interactions, mainly between amygdala, PAG and thalamus. (PC-Map correlations with neuropsychological tests and secondary self-reports are non-significant.)

**Conclusion:** The findings indicate that depressive symptoms reported by ePTLDS patients have objective neural correlates and suggest that the Bb-specific immune status plays an important role in mediating the brain activation pattern for depression. Moreover, the identified correlates of ePTLDS depression represent prominent features of the animal and human neural networks of major depressive disorder. As new results, the next step is replication.
B002 (V010)

Clinical and immunological characteristics in relation to different *Borrelia burgdorferi* s.l. species in patients with Lyme neuroborreliosis

Paula Gyllemark¹, Andreas Matussek², Pia Forsberg³, Jan Ernerudh³ and Anna J. Henningsson²

1. Department of Infectious Diseases, Ryhov County Hospital, Jönköping, Sweden

2. Department of Infectious Diseases, Ryhov County Hospital, Sweden

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

**Background:** In Europe, Lyme neuroborreliosis (LNB) is most often caused by *Borrelia garinii*, but other *Borrelia* species have also been isolated from cerebrospinal fluid (CSF) of LNB patients. Time to recovery varies despite antibiotic treatment, possibly due to variations in pathogenicity between *Borrelia* species, as well as divergences in the host inflammatory response. The immune response within CNS in LNB is mainly of the T helper (Th)1 type with involvement of B cells, supported for example by high levels of the B cell-associated chemokine CXCL13. Furthermore, there are indications of an intrathecal Th17 response in some LNB patients, as mirrored by elevated CSF-IL-17 levels. The aims of this study were: (1) to assess the intrathecal and systemic adaptive immune response in LNB patients by measuring several Th1-, Th2-, Th17-, and B cell-related chemokines and cytokines; (2) to identify clinical and laboratory parameters related to clinical outcome; (3) to try to establish the causative *Borrelia* species and relate the findings to clinical and immunological data.

**Methods:** Two hundred two patients investigated for suspected LNB during 2007–2009 were included and stratified into four groups: (1) 59 with positive *Borrelia*-specific antibody index (AI) and CSF pleocytosis (confirmed LNB); (2) 21 with positive *Borrelia*-specific AI without CSF pleocytosis (possible late LNB); (3) 21 with negative *Borrelia*-specific AI but with CSF pleocytosis and clinical symptoms strongly suggestive of LNB (possible early LNB); (4) 101 without *Borrelia*-specific AI or pleocytosis (non-LNB controls). Clinical data were collected from medical charts. Serum and CSF will be analyzed during spring 2013 for CXCL8, CXCL10, CXCL13, CCL20, CCL22, IL-17, BAFF, and APRIL by Luminex technology and ELISA. AI will be assessed for several *Borrelia* species-specific antigens using the recomBead *Borrelia* kit for Luminex. CSF will be analyzed for *Borrelia*-specific nucleic acid using PCR.

**Preliminary Results:** Dominating symptoms in group 1 were headache, facial palsy, fatigue, and radicular pain. Group 2 had generally more unspecific symptoms of longer duration, e.g., fatigue, headache, and paresthesia. Group 3 consisted mainly of children presenting with recent onset of facial palsy and/or meningitis. Most of the patients in groups 1–3 recovered within 1–3 months, altogether 10% had symptoms lasting more than 6 months despite treatment.

**Conclusion:** Post-treatment symptoms lasting over 6 months were associated with higher age, longer-lasting symptoms prior to treatment and headache, tiredness, vertigo and paresthesia. Further analyses may reveal whether immunological parameters or *Borrelia* species are associated with clinical outcome.
B003 (D002)

# Identifying barriers to chemical-based tickborne disease prevention measures: a survey of health fair participants in Fairfield County, Connecticut

Jennifer Reid¹, Karen Thompson² and Neeta Connally²

1. Department of Ridgefield Health, Ridgefield, CT, USA

2. Western Connecticut State University, Danbury, CT, USA

**Background:** Chemical measures, including the use of repellents, rodent-targeted bait boxes, and residential acaricidal sprays, are among the myriad recommended prevention measures aimed at preventing tickborne diseases. Despite published data suggesting such measures are effective at reducing tick populations and/or preventing human tick bites, the use of chemical measures is not widespread. A survey was conducted to help identify potential barriers to the use of such chemically based tickborne disease prevention measures.

**Methods:** Participants at health fairs throughout Fairfield County, CT, were asked to complete an anonymous survey regarding the effectiveness, safety, and cost of using chemical measures for tickborne disease prevention. Respondents were queried about their beliefs pertaining to the use of DEET vs. "natural" repellents, permethrin-treated clothing, residential acaricide sprays, and rodent-targeted bait boxes.

**Results:** A total of 216 attendees at 10 health fairs completed the survey. Of these, 88% were concerned about a family member acquiring a tickborne disease and 77% practiced some form of tickborne disease prevention. Most respondents were aware that people use DEET-based repellents (84%), residential acaricides (79%), and botanical "natural" repellents (71%) as a means of preventing tickbites. In contrast, fewer participants were aware that permethrin clothing spray and rodent-targeted bait boxes could be used for Lyme disease prevention (41 and 29%, respectively). Less than half of the respondents agreed that any of the chemical measures were safe to be used around adults, children, and pets. Less than half of the respondents agreed that the measures were safe for the environment. With the exception of DEET-based repellent use, the majority of respondents were unsure if these measures were effective for preventing tick encounters.

**Discussion:** This survey assessed beliefs of health-seeking individuals from a highly disease-endemic region. Most respondents were concerned about risk for tickborne diseases and were aware that repellents and residential yard sprays were used for prevention. Most respondents, however, also questioned the safety and efficacy of these measures. The majority of respondents were not aware of use, efficacy, or safety of permethrin clothing sprays and rodent bait boxes. The data suggest the need for increased education and research regarding chemical measures for tickborne disease prevention.
B004 (D007)

Nanopolymer encapsulation of a vaccinia virus-vectored reservoir-targeted vaccine for Lyme disease enhances safety and delivery

Aurelie Kern<fnref n="1" />, Willa Zhou<fnref n="2" />, Mekki Bensaci<fnref n="1" />, Qiaobing Xu<fnref n="2" /> and Linden Hu<fnref n="1" />

The incidence of Lyme disease is steadily increasing in the United States and in Europe. The development of strategies to control this infection remains an important challenge. In this context, vaccination of reservoir hosts is an approach that may help decrease carriage of *Borrelia burgdorferi* and thus reduce incidence of human disease. We have previously reported protective efficacy of a vaccinia-virus expressing OspA vaccine. This vaccine, administered orally to *Peromyscus* mice as a single dose, confers protection against both transmission of *B. burgdorferi* to mice as well as protection against acquisition of spirochetes by larval ticks feeding on infected mice. Although other virally vectored reservoir targeted vaccines have been released into the environment with little environmental impact, distribution of replication competent viruses does pose an exposure risk for humans and non-target animals. In addition, much of the viral dosage administered with the vaccine is killed by gastric acid in the stomach. To overcome these issues, we are developing vaccines encapsulated with pH nanopolymers designed to degrade at specific locations in the digestive tract. We have now shown that encapsulated virus can be successfully inactivated by the nanopolymer coating and can released in its infectious form after incubation in fluid in the correct pH. Animal studies are on-going and we anticipate that we will have data to present at the conference on the efficacy of different nanopolymer encapsulated vaccines by the time of the conference.

<footnote n="1">1. Division of Geographic Medicine and Infectious Diseases, Tufts Medical Center, Boston, MA, USA</footnote>

<footnote n="2">2. Department of Biomedical Engineering, Tufts University, Medford, MA, USA</footnote>
B005 (D008)

Tick bite prophylaxis: a pilot survey to understand health care provider practices

Anna Perea, Paul Mead and Alison Hinckley

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

Background: In a recent CDC survey of over 2000 primary care providers, >30% reported prescribing tick bite prophylaxis in the prior year. To better understand current practices, we surveyed health care providers (HCPs) to determine how frequently and for what reasons they prescribed tick bite prophylaxis. Additionally, we sought to determine how often health care providers prescribed prophylaxis when they felt it was not indicated.

Methods: We included four questions regarding tick bite prophylaxis in the DocStyles 2012 survey, a computer-administered questionnaire of 3149 U.S. HCPs (primary care physicians, pediatricians, and nurse practitioners). We combined responses with information on provider demographics and practice type. Responses of HCPs in 14 Lyme disease highly-endemic states, defined as those in which incidence exceeding the 2011 national average of 7.8 cases/100,000 population, were compared with those of HCPs in other states.

Results: Of the 1485 respondents, 56% reported prescribing tick bite prophylaxis at least once in the previous year, including 74% of HCPs in highly-endemic states and 48% in other states. The most common reasons for prescribing prophylaxis in both highly endemic and non-highly endemic areas were "to prevent Lyme disease" (87 and 69%, respectively); "patients request it" (37 and 43%, respectively); and "to prevent other tickborne diseases" (21 and 35%, respectively). Of HCPs who provided prophylaxis in the previous year, 45% did so even when they felt it was not indicated. Given a hypothetical scenario involving a patient with an attached tick, 38% of HCPs from highly endemic states and 15% from other states would prescribe a single dose of doxycycline as prophylaxis; 19% from highly endemic states and 27% from other states would prescribe a full course of doxycycline.

Conclusion: A large proportion of HCPs prescribe tick bite prophylaxis for diseases other than Lyme disease, and in areas where Lyme disease is rare. Despite their specificity, tick bite prophylaxis guidelines may be difficult for providers to implement. HCPs may not be prepared to identify an *Ixodes scapularis* tick at a specific life stage; identify the degree of engorgement in the absence of tick attachment estimates; or estimate the local rate of *I. scapularis* infection with *Borrelia burgdorferi*. Additionally, patients may not want to adopt a "wait and see" approach, particularly if they lack health insurance or have a high co-pay. More information is needed regarding the efficacy of tick bite prophylaxis for diseases other than Lyme disease.
B006 (D009)

**An immunoinformatics-based "genes to vaccines" approach for accelerated anti-tick vaccine discovery**

Wendy Shattuck¹, Megan Dyer¹, Lenny Moise²,³, Frances Terry³, Rakesh Tiwari⁴, Keykavous Parang⁴, Jose Ribeiro⁵ and Thomas Mather¹

1. Center for Vector-Borne Disease, The University of Rhode Island, Kingston, RI, USA

2. Institute for Immunology and Informatics, The University of Rhode Island, Kingston, RI, USA

3. Epivax, Inc., Providence, RI, USA

4. Department of Biomedical and Pharmaceutical Sciences, The University of Rhode Island, Kingston, RI, USA

5. NIH-NIAID, Bethesda, MD, USA

Tickborne diseases (TBDs) are a world-wide concern and constitute serious public health problems in vital need of solutions. Ticks can cause significant disease in humans and domestic animals as they carry more than 20 emerging or Category A–C pathogens. Additionally, many species of ticks are infected with and are capable of transmitting multiple agents, even in a single bite. Rather than target each individual pathogen transmitted by ticks, development of a broad-spectrum vaccine against the tick itself could prevent tickborne pathogen transmission, and such an anti-tick vaccine approach could represent a major milestone for improving public health. It is well established that ticks and other arthropod vectors manipulate host immune responses by secreting proteins from their salivary glands during blood feeding, while previously tick-bitten hosts, including humans, "fightback" by expressing an acquired immunity to these proteins. Experimental and epidemiological evidence shows that such naturally-acquired resistance to tick feeding (example, acquired tick resistance) through various cellular immune mechanisms can effectively inhibit tick blood feeding and significantly diminish or even prevent pathogen transmission and infection. Using a genes-to-vaccine platform to accelerate vaccine candidate discovery, we present data on immunoinformatic-derived peptides mined from the *Ixodes scapularis* salivary gland transcriptome as anti-tick vaccine candidates. Peptide epitopes were selected for their predicted ability to stimulate cell-mediated immunity, ability to enhance robust antibody-mediated immunity in humans, as well as for their demonstrated HLA binding capacity. Immunization in a novel HLA transgenic mouse model and subsequent tickborne pathogen challenge is providing *in vivo* validation of candidate peptide immunogenicity, and is identifying potential immune correlates of protection against *Borrelia burgdorferi* transmission.
B007 (D015)

N-terminally disulfide-bridged OspC protects mice from infection with *Borrelia burgdorferi* B31 better than its monomeric homologue

Diane G. Edmondson¹,²*, Sabitha Prabhakaran¹,²*, Christian Probst¹,², Wolfgang Schlumberger¹,², Winfried Stöcker¹,², Steven J. Norris¹,² and Lars Komorowski¹,²

1. Laboratory Medicine, Department of Pathology, UTHealth Medical School, Houston, TX, USA

2. Institute of Experimental Immunology, EUROIMMUN AG, Luebeck, Germany

**Introduction:** OspC, a plasmid-encoded outer membrane protein with high inter-strain diversity, has been studied as a candidate antigen for the development of a functional Lyme vaccine by several groups. In prior studies, the use of recombinant OspC has generally led to measurable protection against *Borrelia* expressing the autologous sequence in mouse models. However, most approaches failed to produce long-lasting protection or cross-protection against strains expressing other OspC variants, and in some studies no protection was observed. To overcome these limitations, chimeric fusion proteins of different OspC variants have been constructed, but immunoprotection studies have not been reported. The analysis of sera from OspC-immunized mice showed IgG reactivity against a major non-conserved peptide epitope in the central exposed part of OspC ("loop 5") that was not associated with protection in animals.

**Materials and Methods:** In this study, N-terminally (Cys19) disulfide-bridged dimeric OspC (with a Cys131Ser point mutation and an N-terminal His tag) and a comparable monomeric OspC (Cys19Gly) based on the sequence of *Borrelia burgdorferi* B31 were expressed in *Escherichia coli*. The proteins were used to immunize C3H/HeN mice in combination with alum; four immunizations at 2-week intervals were used in most experiments. At the end of the immunization schedule, the mice were needle-inoculated with *B. burgdorferi* B31, clone 5A4. Two weeks postinoculation, mice were sacrificed and tissues were analyzed for viable bacteria by culture experiments. Different quantities of OspC were tested. Serum samples were collected at different time points and analyzed by Western blot using a B31 membrane extract and ELISA using recombinant OspC.

**Results:** Both the dimeric and monomeric forms of OspC produced strong pan-IgG responses against the immunizing protein. After four immunizations with the dimer (0.1–10 µg per injection), nearly all animals (28/30, 93%) were protected from infection without any adverse effects. In contrast, a low proportion of mice immunized with monomeric OspC in this same dose range were protected against infection (3/30, 10%). Nodules appeared at the injection site when ³¹¹0 µg of the recombinant proteins were injected. Immunized animals developed mainly IgG1 against OspC, whereas the anti-OspC response in infected animals was characterized by high IgG3 levels.

**Discussion:** N-terminally disulfide-bridged OspC was much more effective than the monomeric OspC homologue in inducing a protective immune response against needle-inoculated *B. burgdorferi* B31. Additional investigations are needed to determine immunization efficacy in tick inoculation and cross-strain protection, and to uncover the mechanism of this increased protective activity.
B008 (D017)

# Use of topical antimicrobial agents for preventing dissemination of *Borrelia burgdorferi* (BB) in a mouse-infection model of Lyme disease

Charles Pavia¹,², Maria Plummer¹,² and Alice O’Connor¹,²

1. NYIT College of Osteopathic Medicine, Old Westbury, NY, USA

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

**Background:** Based on recently published reports including our own, discrepancies exist on whether topical application of antibiotics at the site of tick attachment would be a useful option for preventing cutaneous and extracutaneous Lyme disease (LD). We hypothesize that by modifying the murine animal model for LD, using a hairless but immunocompetent mouse strain (in an attempt to optimize antimicrobial penetration through the skin), it will possibly be better to determine minimally effective treatment regimens, given topically or by other parenteral routes, for preventing Bb infection.

**Methods:** Separate groups of 6–12 weeks old SKH (hairless) mice were infected intradermally (i.d.) in the abdominal area with 100,000 culture-grown Bb, of human isolate strain BL206. One hour and 24 h later, the following agents were applied topically over the site of inoculation: (i) a 10% povidone/1% iodine (PVI) solution; (ii) an erythromycin ointment; and (iii) a tetracycline gel formulation. Another group of similarly infected mice received two intramuscular or i.d. injections (given 18–24 h apart) of either saline or ceftriaxone (CTX: 50 mg/kg/dose), starting 1 day after borrelial inoculation. Two to 4 weeks after treatment, cultures of the urinary bladder and skin tissue from the ear were established in BSK medium in order to determine the presence or absence of Bb in both treated and untreated control mice.

**Results:** It was found that topical applications of PVI, erythromycin, and tetracycline were unable to prevent Bb dissemination to the urinary bladder or ear. *In vitro* studies also showed that PVI was rapidly borreliacidal at a dilution of up to 1:200 from what was applied to the inoculation site. In addition, the two-dosage regimen of CTX was 100% effective in sterilizing the urinary bladders and ear skin of the Bb-infected mice, and such treatment did not cause any renal damage based on histopathologic analyses. It is also noteworthy that, consistent with past studies, none of the mice developed any rashes at the Bb-inoculation site.

**Conclusion:** Our findings indicate that the topical application of various antimicrobial preparations, that are acceptable for use in humans, was unable to prevent syringe-transmitted infection in a mouse model of LD, but short-course treatment with CTX that was given by injection was curative.
B009 (D020)

Investigation of a vaccine targeting Lyme borreliosis in Europe

Pär Comstedt, Markus Hanner, Wolfgang Schüler, Andreas Meinke and Urban Lundberg

Intercell AG, Vienna, Austria

In Europe, the three *Borrelia* species, *Borrelia afzelii*, *Borrelia burgdorferi*, and *Borrelia garinii* are the main species causing the most common tick-borne zoonosis, Lyme borreliosis. The heterogeneity between the *Borrelia* species and within *B. garinii* makes the search for conserved antigens providing broad protection challenging. By applying a genomic approach relying on human antibodies, we have identified 122 antigenic *Borrelia* proteins associated with Lyme borreliosis, including already known and published protective antigens. Using several *in vitro* assays, we narrowed down the selection of vaccine candidates. These candidates plus some selected from the literature have been analyzed in our mouse protection models, challenging mice either subcutaneously or using laboratory infected ticks. From these initial studies, we have identified one promising candidate that shows some heterogeneity between different *Borrelia* strains. Therefore, we are investigating a multi-valent vaccine composition to achieve a satisfactory theoretical coverage against the different *Borrelia* species causing Lyme borreliosis in Europe.
B010 (D027)

Dementia-like syndromes in patients with Lyme neuroborreliosis

W. Kristoferitsch¹, F. Aboul-Enein¹, J. Jecel², H. Rausschka², M. Rainer³,
G. Stanek⁴ and P. Fischer³

1. Department of Neurology, SMZ-Ost Donauspital, Vienna, Austria

2. Department of Neurology, SMZ-Ost Donauspital, Vienna, Austria

3. Department of Psychiatry, SMZ-Ost-Donauspital, Vienna, Austria

4. Institute for Hygiene and Applied Immunology, Medical University, Vienna, Austria

Objective: To evaluate the frequency and characteristics of dementia-like syndromes in adult patients with Lyme neuroborreliosis (LNB).

Methods: A retrospective single-center study at the neurological and psychiatric departments of a tertiary health facility over a period of 9 years. Diagnostic classification of LNB was established according to EFNS guidelines (1). All patients with dementia-like syndromes and LNB showed pleocytoisis and intrathecal *Borrelia burgdorferi*-specific antibody synthesis in their cerebrospinal fluid. They were diagnosed and treated by one or more of the authors.

Results: Forty-six patients with definite LNB were identified. The vast majority of patients (n = 40) were diagnosed with Banwarth's syndrome (BS), a well-characterized painful radiculoneuritis. Subacute meningoencephalitis (n = 1), facial palsy without other symptoms of BS (n = 1) and acrodermatitis chronica atrophicans-associated neuropathy (n = 1) were other rare diagnoses. Three patients (6.5%) at the age of 71, 77, and 80, however, presented with a predominant dementia-like syndrome (MMSE 17, 22, and 16, respectively). None of them showed signs or symptoms of BS. Disease duration prior to admission was 3, 6, and 12 months. Their initial diagnosis was either Alzheimer disease or normal pressure hydrocephalus. All the three patients had severe deficits in their episodic memory, disorientation (n = 3), acalculia (n = 2), and agraphia (n = 2) favoring a diagnosis of dementia. Rapid disease progression in combination with weight loss (n = 3), gait disturbance (n = 3), tremor (n = 2), urinary incontinence (n = 2), headache and vomiting (n = 2), and predominant attention deficits (n = 3), especially, were reasons to question the diagnosis of primary dementia. Complete remission or significant improvement of the dementia-like syndrome was achieved in all patients with a single course of 2 g ceftriaxone i.v. daily over 2-4 weeks. There were no relapses.

Conclusion: Dementia-like syndromes as predominant manifestation of LNB are rare. They made up for 6.5% of adult patients with LNB in our hospital population. In elderly patients, they may be misdiagnosed as primary dementia or as idiopathic normal pressure hydrocephalus. Therapeutic response to a 2- to 4-weeks course on ceftriaxone was excellent.

Reference:

1. Mygland A, Ljøstad U, Fingerle V, Rupprecht T, Schmutzhard E, Steiner I, et al. EFNS guidelines on the diagnosis and management of European Lyme neuroborreliosis. *Eur J Neurol* (2010) **17**:8–16.
B011 (D034)

Bring back Lymerix!

Sam Telford

Tufts University, Medford, MA, USA

Lymerix is the only mode of intervention for which there is peer reviewed evidence for efficacy in reducing risk of the population level. It was withdrawn from the market due to financial considerations but those considerations have changed with the greatly expanded population at risk. Lymerix was safe and effective and is a tool of public health utility that could be used until second generation vaccines are developed, tested, and approved. I will review the reasons for reviving Lymerix, and what it would take to do so.
B012 (D039)

# The immunogenic properties of candidate DNA vaccines against CCHF

T. A. Panova, O. V. Nosareva, A. E. Nesterov, S. V. Seregin and V. S. Petrov

The State Research Center of Virology and Biotechnology VECTOR, Koltsovo, Novosibirsk Oblast, Russia

Crimean-Congo hemorrhagic fever virus (CCHFV) is viral zoonotic agent distributed widely in over 30 countries in Eurasia and Africa, causes severe disease in humans with a fatality rate of approximately 30%. The causative agent is a tick-borne virus, belonging to the family Bunyaviridae, genus *Nairovirus* that is transmitted to humans through infected tick bites, or by direct contact with viremic humans or animals. The epidemiology of CCHF reflects the geographic distribution of *Ixodid* ticks, belonging to the genus *Hyalomma*. The number of reported cases has increased in recent years, possibly due to climatic change and human perturbations of biocenoses that may have led to the migration of tick vectors. There is currently no commercially available vaccine for CCHF. Therefore, the aim of this study was to test the immunogenic properties of two candidate DNA-vaccines against CCHFV carrying the genes of structural proteins, plasmids pV3N, and pV3GN, obtained earlier in our organization. To study the immune response was performed twice by intramuscular immunization of mice of each of the plasmids and their mixture. In our study, cellular (methods ELISPOT and cell proliferation assay) and humoral (IEA) immune response were evaluated.

Antigen-specific antibodies were detected in all experimental groups, except for the control group, after immunization. Numbers of cytokine-secreted cells were measured in commercial ELISPOT kits. The obtained results demonstrate that the immune response was induced in the T-cell type. The positive value for antigen-specific proliferation cultures of splenocytes stimulated *in vitro* with antigen of CCHFV was seen for group immunized mixture pV3N and pV3GN by the end of the observation. Our results show that immunization of mice plasmids pV3N, pV3GN, and their mixture leads to the formation of cellular and humoral immune response, and the use of a mixture of plasmids is the most promising as a candidate vaccine.
B013 (D041)

Cutaneous lymphoproliferative diseases associated with *Borrelia burgdorferi* in patients with malignant B cell lymphoma

Heidelore Hofmann¹, J. Ellwanger¹, A. Todorova¹, W. Stolz² and V. Fingerle³

1. Klinik für Dermatologie, Technische Universität München, München, Germany

2. Klinikum Schwabing, Abteilung Dermatologie, Munich, Germany

3. Nationales Referenzzentrum für Borrelien, Bayerisches Landesamt für Gesundheit, München, Germany

We here report on three patients with malignant B cell lymphoma who show *Borrelia burgdorferi* (Bb) associated cutaneous lymphoproliferative diseases responding to antibiotic therapy. High anti-Bb-IgG antibodies in all cases, detection of Bb by PCR in two (one of which also by culture) combined with substantial regression after antibiotic therapy argues for an important yet undefined role of Bb in pathogenesis of such rare manifestations. Case 1: a 58-year-old female patient with chronic lymphatic B cell leukemia (B-CLL) since 5 years developed cutaneous nodular leukemic infiltrates (nose, ear, both nipples), and blurry redness and edema of the face. Skin biopsy revealed B-CLL infiltrates. Bb serology showed strong IgG reactivity [immunoblot: p83, p58, p41, p21, DpbA, VlsE, (p43, p30,OspC)] and Bb sensu stricto was detected by PCR and cultivation from several skin biopsies. After ceftriaxone therapy of 2 g for 21 days, nodular skin infiltrates improved remarkably, but Bb DNA remained detectably. After a second 21 days course of ceftriaxone, the remaining skin infiltrates resolved and Bb DNA was not detectable afterward. B-CLL remains stable since 2 years. Case 2: a 64-year-old male patient was treated for intra-abdominal follicular lymphoma (three cycles of CHOP followed by eight cycles Rituximab as maintenance therapy) with good response. Ten months after start of therapy, he developed erythematous cutaneous infiltrates (face, ears, and dorsal hands) and complained about double vision, paresthesias, and diminished muscular power. Histology showed an interstitial granulomatous dermatitis with a dense lympho-histiocytic infiltrate with complete depletion of CD 20+ lymphocytes. *Borrelia* serology revealed strong IgG reactivity (immunoblot: p83, p58, p43, p41, OspC, Osp17, DbpA, p14, VlsE). PCR from skin biopsies (ear lobes, forehead) revealed DNA from both Bb s.s. and *Borrelia afzelii* as shown by RFLP. The patient recalled a tick bite with an insufficiently treated (7 days Doxycyclin 100 mg) Erythema migrans in 1997. Skin infiltrates, musculoskeletal pains, and visual symptoms disappeared after ceftriaxone 2 g for 21 days. The retroperitoneal lymphoma is in complete remission since 2 years. Case 3: a 76-year-old male patient was treated for Non-Hodgkin Lymphoma with paraproteinemia with two cycles of Fludara and four cycles of Fludara/ Rituximab. During the last cycle, he developed erythematous infiltrates in his face and lower legs, looking very similar to the second patient. *Borrelia* serology showed very high IgG antibodies. He recalled a large erythema after tick bite 3 years ago, without antibiotic treatment. Skin infiltrates responded very well to ceftriaxon 2 g for 21 days.
B014 (D051)

Effectiveness of recombinant OSP-A Lyme vaccine in clinical practice

M. Vázquez¹,², M. L. Cartter¹,² and E. D. Shapiro¹,²

1. Yale School of Medicine, New Haven, CT, USA

2. Connecticut Department of Public Health, Hartford, CT, USA

Background: In 1998, a recombinant OSP-A vaccine (LYMErixTM) for Lyme disease (LD) was approved in the U.S. for use in persons aged 15–70 years. A pre-licensure randomized clinical trial using strict diagnostic criteria indicated an efficacy of 49–76%, depending on the number of doses received. In 2002, the manufacturer voluntarily discontinued production of this vaccine. There has been no direct assessment of the effectiveness of this vaccine as it was used in clinical practice.

Objective: To assess the effectiveness of Lyme vaccine in Connecticut.

Design/Methods: We conducted a matched case–control study (up to two controls per case) of the effectiveness of Lyme vaccine. Cases of LD were identified from an active surveillance system of the CT Department of Public Health. Cases were classified as: definite LD – cases that met the national surveillance case definition for LD; Possible LD – cases that met most criteria but were missing some information (e.g., size of the erythema migrans rash); unlikely to have LD – all others. Controls were persons without LD selected using sequential digit dialing and were matched to the cases by age and area of residence. All medical records were reviewed to determine vaccination status and pertinent medical information. The effectiveness of Lyme vaccine was estimated from the matched odds ratios.

Results: We enrolled 869 age-eligible persons (median age: 48 years) reported to have Lyme disease from 2000 to 2003 (cases) and 1128 matched controls. Based on clinical and laboratory data, 64% of cases had definite LD, 14% had possible LD, and 18% were unlikely to have LD (4% had incomplete information). At least one matched control was enrolled for 709 cases (82%). Overall, 44/709 cases (6%) and 73/1128 controls (6%) had received at least one dose of Lyme vaccine. The vaccine's overall effectiveness was 1% (95% CI: -47 to 34%), *P* = NS. The vaccine was 37% effective (95% CI: -7.3 to 63%; *P* = 0.08) against definite cases of LD and 0% effective (95% CI: -422 to -12%; *P* = 0.02) for cases unlikely to have LD. However, the effectiveness of the vaccine for definite cases of LD who had received at least three doses of the vaccine was 58% (95% CI: 15–79%), *P* = 0.02. This estimate was unchanged after adjusting for potential confounders.

Conclusion: We conclude that the effectiveness of Lyme vaccine was poor in preventing all reported cases of LD, but that it was effective in preventing definite cases of LD after three doses of the vaccine. Misclassification of cases of LD is common and can affect the assessment of effectiveness of Lyme vaccine in clinical practice.
B015 (D052)

# Clinical characteristics associated with *Borrelia burgdorferi* sensu lato skin culture results in patients with erythema migrans

Franc Strle¹, Lara Lusa², Eva Ružić-Sabljić³, Vera Maraspin¹, Stanka Lotrič Furlan¹,
Jože Cimperman¹, Katarina Ogrinc¹ and Daša Stupica¹

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

2. Institute for Biostatistics and Medical Informatics, Ljubljana, Slovenia

3. Faculty of Medicine, Institute of Microbiology and Immunology, Ljubljana, Slovenia

**Background:** The diagnosis of erythema migrans (EM) is primarily clinical, but the most reliable diagnosis also includes the isolation of *Borreliae* from the skin lesion. It is not yet clear how the clinical features in EM patients with a positive *Borrelia* skin culture result differ from those in whom isolation of *Borrelia* from skin was not successful.

**Methods:** To gain insight into predictors for a positive *Borrelia* EM skin culture, we assessed the differences in basic demographic, epidemiologic, and clinical data in 608 culture-positive patients and 501 culture-negative adult patients with the clinical diagnosis of solitary EM according to Slovenian case definition criteria for EM.

**Results:** In comparison to patients with negative skin culture result, those with a positive *Borrelia* spp. culture were older (OR = 1.10, *p* = 0.03), had more often an interval >2 days between tick bite and the onset of EM (OR = 2.74, *p* < 0.001), were more likely to have an EM diameter ³5 cm (OR = 3.00, *p* = 0.02), and the lesion was more often located on the extremities (OR = 1.64; *p* = 0.001). In contrast, a positive culture result was not associated with the recollection of the tick bite at the site of EM, the expansion of skin lesions, or central clearing. Restricted cubic splines and univariate logistic regression analyses revealed that the probability of a positive culture increased markedly with rising size of the largest diameter of the lesion up to ~15 cm and decreased slowly thereafter. A patient with a 15-cm EM lesion had almost threefold greater odds for a positive skin culture than patients with a 5-cm lesion. Multiple regression analysis showed that patients with an interval of at least 2 days between tick bite and the onset of EM had threefold greater odds for EM positivity than patients who reported tick bite but had no time interval between the bite and the onset of the lesion.

**Conclusion:** Several clinical characteristics, including some of those used in EM case definitions, were associated with a positive *Borrelia* spp. skin culture result. The findings are limited to adult European patients with solitary EM which was caused predominantly by *Borrelia afzelii*, and may not be applicable for other causative agents or different clinical situations.
B016 (P004)

# Quantitative detection of *Borrelia burgdorferi sensu lato* in skin samples of erythema migrans patients: correlation of results with clinical findings

D. Stupica¹, L. Lusa², M. O'Rourke³, A. Traweger⁴, I. Livey⁵ and F. Strle¹

1. University Medical Center Ljubljana, Ljubljana, Slovenia

2. Faculty of Medicine Ljubljana, Ljubljana, Slovenia

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

4. Institute for Tendon and Bone Regeneration, SCI-TReCS, Paracelsus Medical University Salzburg, Salzburg, Austria

5. Vaccine R&D, Baxter Innovations, Orth an der Donau, Austria

**Objective:** Limited data are available on the correlation of *Borrelia* burden detected in skin biopsies retrieved from patients with erythema migrans (EM) and the course of disease and treatment outcome. The aim of the presented study was to determine if *Borrelia* burden in skin biopsies from EM lesions correlates with pretreatment clinical course of the disease and treatment outcome.

**Methods:** Skin biopsies taken from 121 adult patients with solitary EM lesions were tested by culture and quantitative PCR (qPCR) for the presence of *Borrelia burgdorferi sensu lato*. In addition, epidemiological, clinical, and microbiological evaluations were performed at baseline, as well as 14 days, 2, 6, and 12 months after commencing treatment with either amoxicillin or cefuroxime axetil.

**Results:** A total of 94 from 121 (77.7%) samples were positive for *B. burgdorferi* sensu lato by qPCR testing and 65 of 118 (55.1%) biopsies yielded a positive culture, of which 96.8% were typed as *Borrelia afzelii* and 3.2% as *Borrelia garinii*. Based on the qPCR data, the median number of spirochetes per biopsy was significantly higher in culture-positive versus culture-negative specimens. The association between *Borrelia* burden in skin biopsies and the pretreatment course of the disease (represented by demographic, epidemiological, clinical or laboratory characteristics) as well as treatment outcome (primarily assessed as percentage of patients with complete clinical response) was found not to be statistically significant.

**Conclusion:** Based on these results, we assume that neither *Borrelia* skin culture results nor *Borrelia* burden in skin biopsy specimens significantly influence the pretreatment course of the disease or treatment outcome in EM patients.
B017 (P016)

Tick-mediated *Borrelia burgdorferi* infection of nonhuman primates for assessment
of antibiotic efficacy

Monica E. Embers, Nicole R. Hasenkampf, Britton J. Grasperge, Lara A. Doyle-Meyers, Amanda C. Tardo and Mary B. Jacobs

Tulane National Primate Research Center, Covington, LA, USA

Introduction: With over 30,000 new cases reported annually, Lyme disease is the most common tick-borne infection in North America. The causative agent, *Borrelia burgdorferi*, can chronically infect humans, causing rash, arthritis, carditis, and neurological dysfunction. A proportion of Lyme disease patients experience symptoms after antibiotic treatment and the etiology of those symptoms are a matter of debate. Our prior results have demonstrated that *B. burgdorferi* spirochetes, administered by injection, could persist in nonhuman primates (NHP) after antibiotic therapy. We aimed to test this again following tick-mediated infection.

Methods: Ten macaques were tick-inoculated with *B. burgdorferi* and five were treated 4 months later with a 28-day course of doxycycline. Assessment for infection includes serology and xenodiagnosis.

Results: All animals developed mild erythema at the sites of the tick bites, but only one developed a bona fide erythema migrans lesion. Months later, ticks fed upon monkeys for xenodiagnosis was efficient, but generated local papular lesions, indicating a moderate level of anti-tick immunity. We have begun the assessment of xenodiagnostic ticks for infection and the comprehensive serological analysis.

Conclusion: Nonhuman primates exhibit variable local responses to tick-feeding and divergent antibody responses following infection with the identical strain of *B. burgdorferi*. A better understanding of diverse responses in an outbred population may help to improve the diagnosis of Lyme disease.
B018 (P022)

Immune profiling in Lyme disease: serum chemokines as markers for acute infection and clinical outcomes

Mark J. Soloski, Hong Kai Ji, George Wu, Katie Wagner, William H. Robinson, Lauren A. Crowder and John N. Aucott

Johns Hopkins School of Medicine, Johns Hopkins Bloomberg School of Public Health, Stanford University School of Medicine, Stanford, CA, USA

Lyme disease is an immune-mediated inflammatory disease initiated by infection with the tickborne spirochete *Borrelia burgdorferi*. Chemokines are key effector molecules that orchestrate the migration of innate and adaptive immune cells and direct them to sites of tissue injury and inflammation. We have conducted a 2-year prospective cohort study of patients with early, untreated Lyme disease from a mid-Atlantic suburban population. Patients were seen at seven study visits between time of diagnosis (acute disease) and 2 years post-treatment follow-up. We have measured the levels of key chemokines in serum samples derived from a cohort of patients with early Lyme disease as well as age- and gender-matched controls. In particular, the chemokines CXCL9 (MIG) and CXCL10 (IP-10) are increased during early acute Lyme disease (p < 0.0005). Following antibiotic treatment, the levels of CXCL9 and CXCL10 are reduced, and remained low through 2 years of follow-up. Other chemokines principally involved in the movement of innate immune cells (ex.CXCL8/IL-8) were not significantly altered in acute Lyme disease. Consistent with the role of CXCL9/CXCL10 in attracting immune T cells to the site of infection, CXCR3+ expressing CD4 and CD8 T cells are reduced in the blood of early acute Lyme disease (p = 0.003 and p = 0.046, respectively) and the extent of the decrease correlated with chemokine levels. The levels of CXCL9/10 did not relate to the size or number of skin lesions, but surprisingly high levels of serum CXCL9/CXCL10 were associated with elevated liver enzymes levels (p = 0.016). In addition, acute Lyme patients who went on to develop Post Treatment Lyme Disease Syndrome (PTLDS) do not display decreased levels of CXCR3+ T cells during acute disease. Collectively, these results indicate that the levels of chemokines and chemokine receptor expression level patterns may prove to be informative biomarkers for Lyme disease and predictive of disease outcomes.
B019 (P042)

Elevated IFNα activity in patients with a history of Lyme disease and persistent
cognitive deficits

Elzbieta Jacek<sup>1,2,3</sup>, Brian A. Fallon<sup>1,2,3</sup>, Mary K. Crow<sup>1,2,3</sup>, Gary P. Wormser<sup>1,2,3</sup>
and Armin Alaedini<sup>1,2,3</sup>

1. Columbia University Medical Center, New York, NY, USA

2. Hospital for Special Surgery, New York, NY, USA

3. New York Medical College, Valhalla, NY, USA

Following antibiotic treatment for Lyme disease, some patients report persistent or relapsing symptoms of pain, fatigue, and/or cognitive deficits. Factors other than active infection, including immune abnormalities, have been suggested, but few clues regarding mechanism have emerged. IFNα is a cytokine that is produced in response to inflammation by a variety of cells. Several studies have shown a correlation between increased IFN activity and neurocognitive impairment in humans and in animal models. We carried out an analysis of serum IFNα activity in 19 *Borrelia burgdorferi* seropositive patients with a history of Lyme disease and persistent objective memory impairment. Control specimens included sera from 11 borrelial seropositive individuals with a history of Lyme disease who were symptom free, as well as 20 healthy individuals without serologic evidence or history of Lyme disease. IFNα activity was measured by detection of serum-induced changes in specific target genes, using a functional cell-based assay and quantitative real-time PCR. Sera from the patient cohort induced significantly higher expression of IFIT1 and IFI44 target genes than those from healthy controls, indicating increased IFNα activity. The increase in IFNα activity yields novel clues regarding the mechanism contributing to the ongoing neuropsychiatric symptoms in patients with a history of Lyme borreliosis.
B020 (P043)

Clinical and histological disease patterns in the skin in Lyme borreliosis

E. Aberer

Department of Dermatology, Medical University of Graz, Graz, Austria

Lyme borreliosis can behave like a chameleon presenting with different clinical disease spectra similar to syphilis and Lupus erythematosus. The classical clinical symptoms erythema migrans (EM), *Borrelia* lymphocytoma (BL), and acrodermatitis chronica atrophicans (ACA) are clearly defined. Histologically, EM shows a perivascular dermatitis although different clinical variants can be observed as a homogenous erythema as seen in *Borrelia garinii* infection or anular lesions mostly observed after *Borrelia afzelii* infection. In clinically resolving EM lesions, papules and nodules can arise, histologically characterized as granulomas, lymphocytoma, interstitial myositis of the arrector pili muscle or as an infiltrative lymphadenosis benign cutis. BL presents as a polyclonal B- and T-cell proliferation and in ACA a T-cell-mediated infiltrate with plasma cells is typical, however, leading to severe damage of all skin structures including the epidermis, the collagenous, and elastic tissue in the latter disease. A similar tissue reaction like in ACA can also occur as an acute event, similar to early morphea, with collagen degeneration. In chronic *Borrelia* infection skin sclerosis, fibroid nodules and anetodermas are observed. EM can also present a Köbner phenomenon for the outbreak of psoriasis, in *B. afzelii*-induced morphea lesions, reversible Churg-Strauss granulomas were observed after contact with mineral wool. Granuloma anulare like lesions with molecular detection of *Borrelia burgdorferi* were described. An acute diffuse reversible sclerosis of the trunk was seen in a patient with Raynaud's phenomenon. Cutaneous calcinosis in EM area was observed in a patient with dermatomyositis. *Borrelia* can induce partly reversible malignant proliferations including a *B. afzelii*-induced marginal cell lymphoma. In summary, *B. burgdorferi* can induce local tissue destructions leading to atrophy or sclerosis, granulomatous tissue reactions and benign and malign lymphoproliferative disorders.
B021 (P044)

Disorders of blood coagulation in patients with acute *Borrelia miyamotoi* infection

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

1. Central Research Institute of Epidemiology, Moscow, Russia

2. State Medical Academy, Izhevsk, Russia

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

Introduction: The borreliosis caused by *Borrelia miyamotoi* (Bmt) is a new human infection first described in Russia. In addition to high fever (38°C), fatigue, headache, and other flu-like symptoms, about 60% of Bmt patients have signs of mild or moderate organ dysfunction, including the liver, kidney, heart, and lung. *Borrelia* secretes no known toxins, so we hypothesized that disorders of blood microcirculation might be responsible, at least in part for organ dysfunction.

Methods: Twenty-four patients with Lyme disease (LD) and 28 Bmt patients treated in Izhevsk City Hospital (Russia) in 2010–2011 were enrolled in the study. Laboratory confirmation of acute LD and Bmt cases was performed according to a previously published report (Platonov et al., 2011). On admission, the Bmt patients were subjected to standard coagulation testing. In addition, the blood microcirculation was studied by slit lamp microscopy.

Results: Most of the 28 Bmt patients had some coagulation pathway abnormalities: activated partial thromboplastin time <24 s (54%); international normalized ratio (INR) <0.7 (36%) and INR >1.3 (18%); thrombin time (TT) <13 s (25%) and TT >18 s (18%); antithrombin III level £75% of normal value (50%); fibrinogen level >4 g/L (26%); euglobulin lysis time greater than the upper normal limit for age (21%); and the presence of d-dimers (43%). Platelet counts were <150,000/mL of blood in 12 patients (43%). All the Bmt patients had at least one abnormal coagulation parameter of the eight that were tested. Eighteen (64%) of the Bmt patients had marked coagulopathy with three to six abnormal laboratory findings. In contrast, all the eight parameters were normal in 17 (71%) patients with LD. The other seven LD patients had only one or two abnormal coagulation parameters (p < 0.001 in comparison with Bmt patients). Microscopic examination of eye capillary blood flow revealed pathological findings that included aggregates of erythrocytes and platelets and obstructed and/or sinuous capillaries in 22 (79%) of the Bmt patients, but none of the LD patients. A total of 14 Bmt patients had both coagulation and microcirculatory abnormalities. Eleven of them also had transient signs of organ dysfunction.

Conclusion: Disorders of blood microcirculation in Bmt patients may contribute to organ dysfunction.
B022 (P047)

Cerebral sequestration associated with neurovirulence is reduced after serial
in vivo passage of *Babesia bovis*

Kerry Sondgeroth, Terry McElwain, Annie Chen-Allen, Andy Allen, Glen Scoles and Audrey Lau

Washington State University, Pullman, WA, USA

Severe neurological signs develop during acute infection by virulent strains of *Babesia bovis* in cattle, similar to those produced in humans suffering from Plasmodium falciparum malaria. Neurovirulence is postulated to be associated with cytoadherence of infected erythrocytes to cerebral capillary endothelium and subsequent capillary sequestration. Serial passage of virulent strains in cattle results in attenuated derivatives that do not cause neurologic disease. We evaluated *in vivo* whether attenuated strains derived by serial passage lose the property of cerebral sequestration by examining brain biopsies at different times during acute infection. We then tested whether subsequent tick passage would result in reversion to neurovirulence with an associated change in cytoadherence ability. Cerebral biopsies of spleen intact animals inoculated intravenously with a parental virulent or derived attenuated strain of *B. bovis* were evaluated for capillary sequestration at two different time points: (1) at the onset of clinical babesiosis; and (2) during severe clinical disease. In animals infected with the virulent parental strain, there was a significant increase in sequestration between the first and second biopsy time points. The passaged derivative strain, while still capable of sequestration at a significantly reduced level, did not change between the first and second sampling. Necropsy examination confirmed the second time point findings and demonstrated that sequestration identified at necropsy reflects pathologic changes occurring in live animals. Tick passage of virulent *B. bovis* did not alter the clinical or pathologic phenotype, resulting in acute, severe babesiosis with cerebral sequestration in calves exposed to infected larvae. However, animals exposed to larvae infected with the passaged derivative strain did not develop acute babesiosis, nor was any sequestration observed in cerebral capillaries. Interestingly, although sequestration in the brain did not occur following tick passage of the attenuated derivative strain, qPCR analysis of multiple tissues showed no significant differences in the level of parasitemia between animals infected with the virulent or attenuated derivative. Serial *in vivo* passage of this virulent *B. bovis* strain in splenectomized animals results in a significant decrease in sequestration and neurovirulence when compared to the parental strain. In addition, not only does the derived attenuated strain maintain tick transmissibility, it does not revert to virulence following tick passage. The lack of clinical signs and sequestration after passage within the tick, despite similar parasite tissue levels, is striking and suggests that there are other factors that mediate cerebral sequestration leading to neurovirulence.
B023 (EC034)

# Symptomatology and clinical picture of post-treatment Lyme disease syndrome

Kathleen B. Kortte, Lauren A. Crowder and John N. Aucott

Johns Hopkins School of Medicine, Baltimore, MD, USA

**Introduction:** After antibiotic treatment for Lyme disease, a subset of individuals report persistent symptoms associated with functional decline, termed Post-Treatment Lyme Disease Syndrome (PTLDS). One challenge in rectifying the PTLDS literature is the lack of consistent methodology for measuring the symptoms of PTLDS. The current study incorporates structured, standardized instruments to capture the symptom picture of individuals with early Lyme disease.

**Methods:** Sixty-six patients with clinically diagnosed untreated Lyme disease with active erythema migrans were enrolled in a prospective cohort study. Patients were treated with 3 weeks of doxycycline and followed for 6 months. At each visit, a physical examination, interval history, clinical symptoms, and self-administered questionnaires were performed. Additionally, at visits 2 and 3, standardized neurocognitive testing was completed. At 6 months post-treatment, an operationalized definition based on the IDSA criteria for PTLDS was used to group these patients according to symptom persistence and SF-36 health function scores. Eight patients met criteria for PTLDS (PTLDS+). Twenty-six matched controls were also evaluated at two visits 6 months apart.

**Results:** At the time of diagnosis (pretreatment), both the PTLDS+ and PTLDS- groups differed from controls on symptoms of pain (*p* < 0.0005) and somatic depressive symptoms (*p* < 0.05). However, there were no statistically significant group differences on fatigue or affective depressive symptoms. Immediately post-treatment, the PTLDS+ group differed from both the PTLDS- and controls on symptoms of pain (*p* < 0.0005), and somatic depressive symptoms (*p* < 0.01), but not on symptoms of fatigue or affective depressive symptoms. Finally, at 6 months post-treatment, the PTLDS+ group was statistically significantly different from the PTLDS- group and controls on measures of fatigue (*p* < 0.05), pain (*p* < 0.01), somatic depressive (*p* < 0.0005), and affective depressive symptoms (*p* < 0.01). On the neurocognitive testing, we found no significant differences in cases and controls.

**Conclusion:** There is a distinct pattern of symptoms for individuals who meet criteria for PTLDS at 6 months post-treatment that can distinguish these individuals from the PTLDS- group and controls. Specifically, PTLDS+ shows a pattern of pain and somatic depressive symptoms at 4 weeks post-treatment. And then by 6 months post-treatment, PTLDS+ have notable fatigue, affective and somatic pain, and affective and somatic depressive symptoms that are distinct from PTLDS- and controls. Interestingly, there is no evidence of true persistent cognitive decline or major depression in the PTLDS+ or PTLDS- groups.
B024

European Lyme disease transmission model: an essential tool to study vaccine
candidates against human borreliosis

Radek Šíma, Ondřej Hajdušek, Adéla Harcubová and Petr Kopáček

*Biology Centre, Institute of Parasitology, Academy of Sciences of the Czech Republic, Ceske Budejovice, Czech Republic*

Lyme disease is an emerging vector-borne disease of temperate climates with a concurrent distribution spanning North America and Eurasia. It is caused by *Borrelia* species collectively known as *Borrelia burgdorferi* sensu lato, primarily transmitted by *Ixodes* spp. ticks. An intensive effort has been put toward elucidation the host-pathogen interactions during *Borrelia* infection. The main aim of this endeavor was to find out factors that could be used as vaccinogens to prevent *Borrelia* transmission. Better understanding of the mechanisms of Lyme disease transmission can be approached by using laboratory animal models. Present transmission models testing the *Borrelia* vaccine candidates employ *Ixodes scapularis* nymphs – *B. burgdorferi* sensu stricto – mice system. Our aim was to develop a transmission model concerning main European tick species *Ixodes ricinus* and European Lyme disease agents (*Borrelia afzelii*, *Borrelia garinii*, *B. burgdorferi* sensu stricto) to test candidate tick antigens studied and characterized in our laboratory. Infectious, low-passage *B. burgdorferii* isolates were grown in a culture and injected subcutaneously into mice. Noninfected *I. ricinus* larvae were fed on *Borrelia* infected mice to generate infected nymphs. Presence of *B. burgdorferi* spirochetes within the tick nymphs was tested using nested PCR amplifying a part of 16S–23S intergenic region. The infection rates in nymphs were evaluated by quantitative real-time PCR. To examine *Borrelia* infection in mice, biopsies from different organs (ear, skin, heart, urinary bladder, and joint) were analyzed. Biopsies were first screened using nested PCR protocol to detect *Borrelia* DNA, and positive samples were further quantified using quantitative real-time PCR. Our results showed that urinary bladder is the most reliable tissue for *Borrelia* detection during persistent murine infection (at least 4 weeks post infection). However, additional examination of heart muscle and/or joints is recommended for precise detection of spirochetal infection in mice. Optimized transmission model was further employed to test tick immune genes influencing *Borrelia* dissemination within the tick. Current knowledge of tick immunity in relation to *Borrelia* infection will be presented.

**Acknowledgments:** This work was supported by the project Postdok BIOGLOBE (CZ.1.07/2.3.00/30.0032) co-financed by the European Social Fund and the state budget of the Czech Republic and by the Grants 13-12816P (GA CR), 13-27630 P (GA CR) and Project FR-TI3/156 from Ministry of Industry and Trade, Czech Republic.
# The first case of meningoencephalitis by the relapsing fever spirochete *Borrelia miyamotoi* in Europe

J. W. Hovius, B. de Wever, M. Sohne, M. C. Brouwer, J. C. Coumou, A. Wagemakers, G. A. Oei, H. Knol, N. Narasimhan, C. J. Hodiamont, S. Safari, H. M. Horlings, E. Fikrig, H. Sprong and M. H. J. van Oers

The Academic Medical Center (AMC), University of Amsterdam, Amsterdam, Netherlands

We here describe a case of meningoencephalitis in an immunocompromised patient with a history of non-Hodgkin lymphoma and rituximab therapy caused by the relapsing fever spirochete *Borrelia miyamotoi*. A 70-year-old man had experienced many tick-bites in the Netherlands while hiking nearby his recreational house in the dunes. He presented with cognitive slowing, memory deficits, and a disturbed gait, which had gradually developed over several months and were progressive over the last few weeks prior to presentation. Upon neurological examination, there was a distinct bradyphrenia and the patient scored 26 of 30 points on the mini mental state examination. Vital signs were normal and body temperature was 36.4°C. Cranial MRI showed no abnormalities, but repeated lumbar punctures showed cerebrospinal fluid (CSF) pleocytosis. Extensive microbiological, pathological, and haematological diagnostic testing initially failed to reveal the cause of this chronic meningitis. In light of the frequent tick exposure and an obvious relation in time with recent tick bites, he was treated with ceftriaxone intravenously for 2 weeks for a possible Lyme neuroborreliosis. Over a period of several weeks, the patient fully recovered. Supported by the recent evidence of the presence of *B. miyamotoi* in *Ixodes ricinus* ticks across Europe and a recent case report in the *NEJM* on *B. miyamotoi*-induced meningoencephalitis in a similar patient in the USA, we retrospectively considered *B. miyamotoi* as the causative agent. Indeed, in stored pre-treatment cerebrospinal fluid (CSF), we showed the direct presence of motile spirochetes by dark field microscopy. In addition, a 16S rDNA pan-relapsing fever *Borrelia* quantitative (q)PCR and a qPCR targeting the *B. miyamotoi* flagellin gene proved to be positive in two separate pre-treatment CSF samples and a pre-treatment blood sample. Interestingly, 2.2% of 352 questing *I. ricinus* nymphal ticks from the vicinity of the patient's recreational house were positive for *B. miyamotoi* by qPCR. Amplification and sequencing of the *glpQ* and *p66* genes confirmed *B. miyamotoi* as the causative agent and revealed 100% identical sequences in ticks and the patient's clinical samples. ELISA and Western blot did not reveal anti-GlpQ antibodies in blood and CSF. This case represents the second documented case of *B. miyamotoi*-induced meningoencephalitis in the world and the first in Europe. It underscores that physicians worldwide confronted with immunocompromised patients from *Ixodes* tick-endemic areas with a meningoencephalitis should consider *B. miyamotoi* as a potential causative agent and should be aware that regular diagnostic tests for *Borrelia burgdorferi* will most likely overlook this diagnosis. Whether *B. miyamotoi* is also able to cause neurological symptoms in immunocompetent patients requires further investigation.
B026 (D003)

Serum antibody responses of patients with southern tick-associated rash illness (STARI)

Mark A. Pilgard, Mark J. Delorey, Paul S. Mead, Martin S. Schriefer and Barbara J. B. Johnson

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

STARI is a provisional name of an illness of unknown etiology associated with the bite of lone star ticks (*Amblyomma americanum*), a species that is most common in the south and southeastern United States. To study the cause of STARI, patients were enrolled in an IRB-approved study to collect clinical specimens including serum. The enrollment criteria were the presence of a physician-diagnosed erythema migrans (EM)-like rash (³5 cm) and possible exposure to ticks in the southern U.S. or, if outside this region, an attached lone star tick at the rash site. Here, we evaluated STARI patients for evidence of exposure to *Borrelia burgdorferi* sensu lato or to *Borrelia lonestari* by serology. Antibody responses (IgM and IgG) in acute- and convalescent-phase (A/C) serum samples of STARI patients were evaluated by ELISA against antigens from 10 strains of *Borrelia*. *B. burgdorferi* sensu lato whole cell antigens were prepared from representative strains isolated from ticks or rodents in the southern U.S.: *Borrelia americana* SCW-30E, *B. americana* SCW-33, *Borrelia andersonii* SI-10, *Borrelia bissettii* FD-1, *B. burgdorferi* MI-4, *B. burgdorferi* SCW-6, and *Borrelia carolinensis* SCW-14. Antibody responses to *B. lonestari* were evaluated using recombinant glycerophosphodiester-phosphodiesterase (GlpQ) antigen from *B. lonestari* and a surrogate whole-cell antigen from *Borrelia miyamotoi* FR64b (Japan), the species most closely related to *B. lonestari* that is available in pure culture. In addition, standard two-tiered serology for Lyme disease was performed using antigens from strain B31. Serum samples from two types of control populations were studied using the same experimental ELISAs: healthy blood donors and early Lyme disease (EM) patients. Serum from healthy blood donors was obtained at two points in time separated by 24–37 days. Paired A/C serum samples from patients with culture-confirmed Lyme disease were obtained from the CDC reference collection. ELISA OD differences between the first and second sample were calculated for each individual in the three populations. To compare the distributions of ELISA OD differences among the three populations, an ordered heterogeneity test will be employed. The test orders the groups according to mean ranks and correlates the observed ranks to the possible expected ranks. Preliminary analysis compared the distributions of OD differences for antibody responses of STARI patients and healthy blood donors to GlpQ (*B. lonestari*). Results from the two-sample Kolmogorov–Smirnov test (*p* = 0.83) and equivalence test of distribution means (*p* < 0.001) indicate that responses of these two populations to GlpQ are the same.
B027 (D012)

Comparison of two beads immunoassays for serological diagnosis of
Lyme borreliosis

R. Lienhard¹,², A. Resenterra¹,² and O. Péter¹,²

1. *ADMED Microbiologie, La Chaux-de-Fonds, Switzerland*

2. *Service of Infectious Diseases, Central Institute of Valais Hospital, Sion, Switzerland*

Objective: The two beads immunoassays were evaluated and compared for performance of the serological diagnosis of Lyme borreliosis

Methods: Multianalyt™ *Borrelia burgdorferi* IgG and IgM (Virion/Serion, Würzburg) on BD FacsCanto II (Becton Dickinson) and Multimetrix *Borrelia* IgG and IgM (Progen, Heidelberg) assays on Luminex were evaluated and compared with our in-house Western blots. We performed both assays according to manufacturer's instructions. We evaluated sensitivity with a panel of sera from patients presenting an erythema migrans (EM) (n = 25), with early disseminated stage of neuroborreliosis (NB) (n = 30) confirmed by a specific intrathecale antibody synthesis, as well as with late disseminated stages (LD) arthritis or acrodermatitis chonica atrophicans (n = 20). Specificity was challenged with 80 sera from blood donors (DON) living around the city of La Chaux-de-Fonds, a low endemic area for Lyme borreliosis (JuraMountains).

Results: Specificity of IgM, IgG, and cumulative IgM or IgG (G + M) obtained for Multianalyt (MA) and Multimetrix (MM) were respectively 91.3, 88.8, and 82.5% for MA, and 83.8, 78.8, and 63.8% for MM. To compare, we obtained 92.5, 92.5, and 86.3% with our immunoblots. Included are true positive revealing seroprevalence in asymptomatic donors and a false-positive rate. The rates of equivocal results obtained in G + M were as high as 20% for MM and 11.3% for MA. For EM positive, results obtained for IgM, IgG and G + M were 68, 48, and 72% (MA) and 76, 68 and 88% (MM), respectively. For NB, they were 33.3, 80, and 83.3% (MA) and 66.7, 96.7, and 100% (MM), respectively. All LD tested were positive in IgG for both the assays. Equivocal results could be as high as 24 or 16% for IgG in EM, respectively, for MA and MM, but special focus was made on true positive results. These assays are considered as confirmatory tests and results should be positive or negative. Equivocal results are useless for confirmation.

Conclusion: Globally, the assays showed good to higher sensitivity than our confirmatory blots. However, we are concerned about the too low specificity that will generate too many false positive results on clinically poorly defined borreliosis cases. Assay evaluation in a country should be presented before commercialization to reflect local epidemiology and determine essential test values as seroprevalence, specificity, and sensitivity. Confirmatory assays for Lyme borreliosis are to be very specific (>95%) to be useful.
B028 (D014)

Study on the cross reactivity of anti-*Borrelia* and treponemal antibodies

H.-J. Hagedorn¹, A. Kraminer-Hagedorn² and D. Muenstermann¹

1. *Labor Krone GbR, Bad Salzuflen, Germany*

2. *Spirolab GmbH, Bad Salzuflen, Germany*

Due to the antigenic relationship of *Treponema* and *Borrelia*, false positive results in *Borrelia* antibody tests can occur in samples from syphilis patients. In Germany, there is an actual discussion about the need to introduce a mandatory syphilis testing for all positive *Borrelia* antibody findings, to rule out nonspecific results induced by treponemal antibodies. The relevance of this requirement was the topic of this study.

Samples: First, 50 selected samples with borrelial antibodies (panel 1) and 50 samples with treponemal antibodies (panel 2) were regardless of the results tested with all diagnostic tests used in this study. Following 85 samples from the syphilis routine laboratory (panel 3) were included in the study as well.

Tests: Liaison® *Borrelia* IgG CLIA (DiaSorin), recomWell *Borrelia* IgG/IgM-EIA (Mikrogen), Enzygnost® Lyme link Vlse IgG and Borreliosis IgM (Siemens), anti-*Borrelia* plus VlsE-ELISA IgG and anti-*Borrelia*-ELISA IgM (Euroimmun), *Borrelia* ViraStripe® IgG/IgM(Viramed), recomLine *Borrelia* IgG/IgM (Mikrogen), Architect Syphilis TPCMIA (Abbott), TPPA (Fujirebio), IgG- and 19S-IgM-FTA-ABS-Test, RPR (Biokit), Treponema + VDRL Virablot® IgG/IgM (Viramed), recomLine Treponema IgG/IgM (Mikrogen).

Results: Specificity of syphilis tests in 50 samples with *Borrelia* antibodies (panel 1): Architect Syphilis TP CMIA 100%, TPPA 98%, IgG-FTA-ABS 96%, 19S-IgM-FTA-ABS 100%, Treponema + VDRL Virablot IgG/IgM 100%, recomLine Treponema IgG/IgM 100%. For the 50 syphilis samples (panel 2), IgG antibodies against the borrelial antigens p83/100, p58, p43, p39, OspA, OspC, p18, p17 and p14 could not be detected. In addition, in the recomLine *Borrelia* IgG assay, all VlsE antibody findings were negative. Using the ViraStripe® *Borrelia* IgG test in six samples, antibodies against p30, and in two different samples VlsE antibodies, were demonstrated. The recomLine *Borrelia* IgM showed no false positive result. In the ViraStripe® *Borrelia* IgM, five samples showed OspC antibodies of questionable specificity. In more than 50% of the syphilis samples, IgG p41-antibodies with a slightly lower frequency but partially strong reactivity IgG-p41 antibodies were demonstrated. Specificity of *Borrelia* IgG-tests in 135 syphilis samples (panels 2 and 3) were as follows: recomWell *Borrelia* IgG ELISA 99.3%, LiaisonBorrelia IgG CLIA 98.5%, Enzygnost Lyme link VlsE IgG ELISA 100%, and anti-*Borrelia* + VlsE-ELISA IgG 64.7%. Specificity of *Borrelia* IgM tests in 135 syphilis samples (panels 2 and 3) were: recomWell *Borrelia* IgM ELISA 96.9%, Enzygnost Borreliosis IgM ELISA 95.5%, and anti-*Borrelia*-ELISA IgM 93.8%.

Conclusion: Samples positive for borrelial antibodies usually do not lead to false positive syphilis antibody findings. Inadequate specificity is a problem, when syphilis samples are tested with an assay based on *Borrelia* whole cell antigen preparations, but not when recombinant antigens are used or when in whole cell antigen assays a preabsorption step for the elimination of potential crossreacting antibodies is integrated. Using the two-tier diagnostic procedure, the specificity of the anti-*Borrelia* screening results is usually clarified. Therefore, an additional routine application of a syphilis test is not necessary. However, a routine syphilis testing without consent of the patient is also an ethical problem.