ROLE OF SYMBIONTS IN TRANSMISSION OF PATHOGENS BY TICKS

• Sponsoring Institution: State Agricultural Experiment Station
• Project Status: COMPLETE
• Funding Source: STATE
• Reporting Frequency: Annual
• Accession No.: 1010305
• Project Start Date: Jul 12, 2016
• Project End Date: Jun 30, 2021
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108

Performing Department
Entomology

Non Technical Summary
Symbionts of blood-sucking insects have been shown to control pathogen acquisition and transmission bypriming the immune system of insects, and to provide essential nutrients, but nearly nothing is known for ticksymbionts. In order to implement paratransgenic strategies to control tick-borne diseases, it is necessary tounderstand the role of symbionts in tick biology, and how symbionts regulate the response of ticks to tickbornepathogens. We will elucidate the role of tick symbionts in provision of nutrients lacking in blood,priming the tick immune system, and their interactions with tick-borne pathogens to identify new strategies forthe prevention of tick-borne diseases.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
722	3120	1130	100%

Knowledge Area
722 - Zoonotic Diseases and Parasites Affecting Humans;

Subject Of Investigation
3120 - Spiders, mites, ticks, and other arthropods;

Field Of Science
1130 - Entomology and acarology;

Keywords

ticks

arthropod vector

rickettsia

blacklegged tick

tick control

paratransgenesis

Goals / Objectives
The goal of this project is to determine role of Rickettsiabuchneri in blacklegged ticks and assess its suitability for paratransgenesis. We will make a thorough analysis of the tripartite relationship between the black-legged tick, Ixodesscapularis, its symbiont, R. buchneri, and two pathogens that it transmits, Borrelia burgdorferi and Anaplasma phagocytophilum. First, we will identify the contributions of the symbiont to tick development and reproduction, as well as its role in the vectorial capacity of I. scapularis. Second, we will generate R. buchneri mutants using a random mutagenesis approach to insert a replaceable cassette. Mutants that are colonization competent will then be selected for recombinase-mediated cassette exchange with sequences encoding antimicrobial factors.Aim 1) Determine R. buchneri role in a) tick nutrition, fertility/fitness, and b) in exclusion of other rickettsiae (e.g., Rickettsia monacensis), and c) susceptibility to A.phagocytophilum (Ap) and B.burgdorferi (Bb). This will delineate the role of R. buchneri as a nutritional symbiont and as a symbiont that regulates tick microbial interactions.Aim 2) Prepare R. buchneri transposon mutants using a Himar1 construct flanked by mismatched loxP sequences for subsequent replacement using Recombinase Mediated Cassette Exchange (RMCE). Select mutants able to colonize ticks. This will demonstrate that aposymbiotic ticks can be reconstituted with a modified symbiont.

Project Methods
AIM 1. To elucidate the symbiotic contributions of the I. scapularis symbiont, R. buchneri, we will take advantage of the tools that we have developed to transform R. buchneri, and the membrane feeding system (19) to feed and introduce symbionts and pathogens into ticks (16, 28). Our collection of tick cell lines from I. scapularis, our culture isolate of its rickettsial symbiont, R. buchneri, our Himar1 transposon and shuttle plasmid constructs coupled with our combined expertise ensure that the studies proposed here are possible. The concepts to be examined are novel, and have potential to establish a new paradigm of the regulation of tick-borne pathogen transmission to humans.Role of R. buchneri and potential for paratransgenesis: This will be done by determining the role of R. buchneri in a) I. scapularis nutrition, fertility/fitness, and b) susceptibility to Ap and Bb, and c) exclusion of other rickettsiae (R. monacensis). This will delineate the role of R. buchneri as a nutritional symbiont and as a symbiont that regulates tick-microbial interactions. Rationale: In order to exchange the wt symbiont for a paratransgenesis candidate, it is necessary to elucidate the contribution of R. buchneri to I. scapularis biology. R. buchneri occurs in nearly all of females suggesting that it provides a vital service to ticks (9). Nevertheless, ticks cleared of the symbiont produce viable offspring (prelim. data). The ability of I. scapularis to survive without R. buchneri allows us to observe phenotypic effects associated with the presence or absence of the symbiont. We will compare wt with aposymbiotic I. scapularis to elucidate effects on fitness/reproductive success and resistance to infection by Ap (human isolate HGE2; 29) and Bb (tick isolate B31; 30). R. peacockii and R. montanensis interfere with transovarial transmission of a second Rickettsia species (31, 32). To determine if R. buchneri can exclude superinfection of I. scapularis ovaries, we will evaluate transovarial maintenance of R. monacensis, a species of undetermined pathogenicity associated with I. ricinus, in aposymbiotic ticks. I. scapularis can be infected with Bb and R. monacensis by capillary- or membranefeeding (18, 19, 33). If we find that larvae readily acquire Ap, Bb and R. monacensis via membrane feeding, we will use this approach, Experimental approach: Aposymbiotic I. scapularis: We have produced aposymbiotic ticks by injecting engorged females with ciprofloxacin, and offspring are largely free of R. buchneri DNA. Because not all ticks are cleared of symbionts, ticks will be PCR tested post hoc for presence of the rickettsial gltA gene 46, and positive ticks excluded from data analysis. Offspring from untreated females will be controls. We will focus on F1 females because F1 adults are essentially free of R. buchneri [prelim. data], and development of progeny (F1) from treated females is similar to progeny from wt females. F2 ticks may also be included in experiments.Changes in fitness of aposymbiotic I. scapularis: Fitness, measured by feeding, molting and reproductive success, will be compared between wt and aposymbiotic ticks. Aposymbiotic and wt female ticks will be fed in the presence of males using the membrane feeding system (19). Weight of engorged females, egg mass and hatch rate will be measured (21). Resulting larvae and nymphs will be fed on hamsters, and the proportion that engorge and molt will be compared for each group. After each molt, a representative number of ticks will be tested for the gltA gene as a proxy of R. buchneri presence.Nutrient provision by R. buchneri: Reduced fitness in aposymbiotic tsetse flies can be rescued by addition of nutrients provided by symbionts (23, 24, 34). Because R. buchneri encodes folate, ubiquinone and biotin, we expect that aposymbiotic ticks will experience a nutrient deficit affecting fitness. We will try to rescue tick fitness by supplementing blood meals offered via membrane feeder with these cofactors. We will also offer extracts from cultured and purified R. buchneri.Bb and Ap Susceptibility of aposymbiotic I. scapularis: Aposymbiotic F1 larvae will be compared to wt larvae from sham-injected mothers. Larvae will be exposed to Ap, Bb and R. monacensis by membrane feeding. After molting, 10 nymphs from each group will be tested by qPCR to determine the proportion of each group infected, and the quantity of bacteria present as follows: For Ap, we will use primers that target a unique region of ank; for Bb, qPCR will target the Bb-specific recA gene 35, 36, for R. monacensis 37, we will design primers to amplify a fragment of f1pep1, coding for peptidase S9, which is absent in R. buchneri; for R. buchneri, we will target lgrB that codes for linear gramicidin synthase subunit B and is unique to this species. Tick DNA will be amplified using primers targeting mitochondrial 16S rDNA 38 as a quality control. Infected nymphs and adults will be tested for transstadial and transovarial transmission.Bacterial maintenance and global expression of immune-related genes:Aposymbiotic and wt (control) F1 nymphs infected previously with Ap, Bb or R. monacensis will be fed on the membrane feeder to produce adults. Females will be housed with wt males, membrane-fed, and allowed to oviposit. We will conduct pilot studies to determine the number of ticks needed to obtain statistically significant results, and we already know that 100% of wt ticks acquire infection with Bb. Fitness of engorged ticks will be quantified as described above, and a subset of eggs from 10 ticks will be tested by qPCR to determine transovarial transmission. Preliminary RNAseq data from aposymbiotic females indicated dysregulation of the immune system. We will use this approach to examine the immune response to Ap and Bb in aposymbiotic and wt ticks.Functional analysis of the R. buchneri antibiotic synthesis genes: Sequence analysis did not reveal promoters between genes, suggesting co-transcription (39). We will move the gene clusters to our shuttle vector so they can be manipulated, e.g., by removal of the repressor and increasing promoter strength. Due to the presence of putative resistance and drug transporter genes in these gene clusters, it may be necessary to study these genes in E. coli. We will prepare extracts from cultured rickettsiae at the time of peak gene/operon expression (or shortly thereafter), and test them in antibiotic inhibition assays with bacteria known to be sensitive to erythromycin, gramicidin, kanamycin, gentamicin, istamycin, using these antibiotics as controls (40, 41). Extracts will also be tested against rickettsiae in culture. These tests will reveal whether R. buchneri produce functional antibiotics.

Progress 07/12/16 to 06/30/21

Outputs
Target Audience: Our reports are for a target audience that includes scientists and public health officials that can implement and apply this new knowledge to the control of ticks and tick borne pathogens. Effective control of ticks is a regional and national human and animal health priority and we provide information that can be used by regional and national public health officials. This project examines new ways to reduce the prevalence of ticks and tick-borne disease agents around homes, farms, recreational areas and military bases. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project provided training and professional development for undergraduate (3), graduate (1) and postdoctoral (2) students and career research scientists (2). How have the results been disseminated to communities of interest? Our results were disseminated to colleagues in the scientific community via papers published in open access scientific journals available online at journal web sites and presentations made at national and international scientific meetings. These scientific meetings are attended by public health, military, homeland security officials, scientists from the CDC and state public health departments. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Rickettsia buchneri is the principal symbiotic bacterium of the medically significant tick, Ixodes scapularis. We completed our quantification of the growth of R. buchneri naturally occurring in I. scapularis ticks throughout their life cycle. This rickettsia has been detected primarily in the ovary of adult female ticks and is vertically transmitted, but its tissue-tropism in other life stages and function in regard to tick physiology is limited. We determined the tissue tropism of R. buchneri in female I. scapularis ticks via RNA fluorescent in situ hybridization and visualized strong positive signals of R. buchneri surrounding the nucleus of the developing oocytes. The tissue tropism of R. buchneri in I. scapularis and in vitro growth dynamics data strengthens the contemporary understanding of R. buchneri as a transovarially transmitted, non-pathogenic endosymbiont. Furthermore, the tissue localization of R. buchneri elucidates its physiological specialization and how it impacts pathogen transmission and vector competence of this medically important tick vector. Ticks are obligate hematophagous arthropods and must tolerate starvation during off?host periods. Macroautophagy is a well-conserved self-eating mechanism of cellsurvival and is essential for recycling cellular contents during periods of starvation, stress, and injury in organisms. Our observations showed that autophagy (Atg) family genes are highly conserved in ticks and function in autophagy pathway induced by amino acid starvation. These results also provide valuable insight for further autophagy-related research as a new strategy for blocking the transmission of tickborne pathogens. We also determined if tick cells are permissivefor Wolbachia infection. Ixodes scapularis, Ixodes ricinus and Rhipicephalus microplus cell lines were inoculated with four strains of Wolbachiaderived from diverse insect taxa (planthopper, mosquito and sand fly). Bacterial growth was monitored by microscopy and confirmed by PCR. All four Wolbachia strains successfully infected two or more cell lines and were maintained through serial passage in tick cells for up to 14 months. We concluded that cultured tick cells are capable of being infected with, and supporting long-term growth of, multiple Wolbachia strains in vitro. However, this does not prove that ticks can harbour Wolbachia within their cells in vivo; further studies are needed to determine whether or not ticks naturally harbor Wolbachia in the absence of parasites, and if ticks can be experimentally infected with these bacteria.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Wang XR, Burkhardt NY, Kurtti TJ, Oliver JD, Price LD, Cull B, Thorpe CJ, Thiel MS, Munderloh UG. Mitochondrion-Dependent Apoptosis Is Essential for Rickettsia parkeri Infection and Replication in Vector Cells. mSystems. 2021 Mar 16;6(2):e01209-20. doi: 10.1128/mSystems.01209-20. PMID: 33727398; PMCID: PMC8546998
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Thorpe CJ, Wang XR, Munderloh UG, Kurtti TJ. Tick Cell Culture Analysis of Growth Dynamics and Cellular Tropism of Rickettsia buchneri, an Endosymbiont of the Blacklegged Tick, Ixodes scapularis. Insects. 2021 Oct 27;12(11):968. doi: 10.3390/insects12110968. PMID: 34821769; PMCID: PMC8626015.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Cull B, Burkhardt NY, Wang X-R, Thorpe CJ, Oliver JD, Kurtti TJ, Munderloh UG. 2022. The Ixodes scapularis Symbiont Rickettsia buchneri Inhibits Growth of Pathogenic Rickettsiaceae in Tick Cells: Implications for Vector Competence. Front. Vet. Sci., 06 January 2022 | https://doi.org/10.3389/fvets.2021.748427
• Type: Book Chapters Status: Published Year Published: 2022 Citation: Munderloh UG, Kurtti TJ. 2022. Climate and other global factors at the zoonotic interface in America: Influence on diseases caused by tick-borne pathogens. Pp. 318-325. IN. Climate, Ticks and Disease, ed. P. Nuttall. CABI Climate Change Series.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Our reports are for a target audience that includes scientists and public health officials that can implement and apply thisnew knowledge to the control of ticks and tick borne pathogens. Effective control of ticks is a regional and national human and animal healthpriority and we provide information that can be used by regional and national public health officials. This project examinesnew ways to reduce the prevalence of ticks and tick-borne disease agents around homes, farma, recreational areas and militarybases. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided training and professional development for undergraduate (3), graduate (1) and postdoctoral (2) studentsand career research scientists (2). How have the results been disseminated to communities of interest?Our results were disseminated to colleagues in the scientific community via papers published in open access scientificjournals available online at journal web sites and presentations made at national and international scientific meetings.These scientific meetings are attended by public health, military, homeland security officials, scientists from the CDC andstate public health departments. What do you plan to do during the next reporting period to accomplish the goals?We will examine the tissue tropism ofR. buchneriin immature (embryos, larvae and nymphs)Ixodes scapularisby transmission electron microscopy.TheR. buchnerigenome includes genes known to be involved with the production of antibiotics.We will continue studies examiningwhether theR. buchneriantibiotic genes are functional and whether they exert antibiotic effects on bacterial pathogens (Borreliaand Anaplasma) that are associated withI. scapularis.

Impacts
What was accomplished under these goals? Rickettsia buchneriis the principal symbiotic bacterium of the medically significant tick,Ixodesscapularis. We completed our quantification of the growth ofR. buchnerinaturally occurring inI. scapularisticks throughout their life cycle. This rickettsia has been detected primarily in the ovary of adult female ticks and is vertically transmitted, but its tissue-tropism in other life stages and function in regard to tick physiology is limited. We determined the tissue tropism ofR. buchneriin femaleI. scapularisticks via RNA fluorescent in situ hybridization andvisualizedstrong positive signals ofR. buchnerisurrounding the nucleus of the developing oocytes. The tissue tropism ofR. buchneriinI. scapularisand in vitro growth dynamics data strengthens the contemporary understanding ofR. buchnerias a transovarially transmitted, non-pathogenic endosymbiont. Furthermore, the tissue localization ofR. buchnerielucidates its physiological specialization andhow it impacts pathogen transmission and vector competence of this medically important tick vector. Ticks are obligate hematophagous arthropods and must tolerate starvation during off?host periods. Macroautophagy is a well-conserved self-eating mechanism of cell survival and is essential for recycling cellular contents during periods of starvation, stress, and injury in organisms. Our observations showed that autophagy (Atg) family genes are highly conserved in ticks and function in autophagy pathway induced by amino acid starvation. These results also provide valuable insight for further autophagy-related research as a new strategy for blocking the transmission of tickborne pathogens. We also determined if tick cells are permissive forWolbachiainfection.Ixodes scapularis,Ixodes ricinusandRhipicephalus micropluscell lines were inoculated with four strains ofWolbachiaderived from diverse insect taxa (planthopper, mosquito and sand fly). Bacterial growth was monitored by microscopy and confirmed by PCR. All fourWolbachiastrains successfully infected two or more cell lines and were maintained through serial passage in tick cells for up to 14 months. We concluded that cultured tick cells are capable of being infected with, and supporting long-term growth of, multipleWolbachiastrainsin vitro. However, this does not prove that ticks can harbourWolbachiawithin their cellsin vivo; further studies are needed to determine whether or not ticks naturally harborWolbachiain the absence of parasites, and if ticks can be experimentally infected with these bacteria.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Khoo JJ, Kurtti TJ, Husin NA, Beliavskaia A, Lim FS, Zulkifli MMS, Al-KhafajiAM, Hartley C, Darby AC, Hughrd GL, AbuBakar S, Makepeace B, Bell-Sakyi L. 2020. Isolation and propagation of laboratory strains and a novel flea-derived field strain of Wolbachia in tick cell lines. Microorganisms 8, 988 https://doi.org/10.3390/microorganisms8070988
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Madhav M, Brown G, Morgan JAT, Asgari S, McGraw EA, Munderloh UG, Kurtti TJ, James P. 2020. Wolbachia successfully replicate in a newly established horn fly, Haematobia irritans irritans (L.) (Diptera: Muscidae) cell line. Pest Manag Sci 76: 2441-2452DOI 10.1002/ps.5785
• Type: Journal Articles Status: Awaiting Publication Year Published: 2020 Citation: Oliver JD, Price LD, Burkhatdt NY, Heu CC, Khoo BS, Thorpe CJ, Kurtti TJ, Munderloh UG. 2020. Growth dynamics and antibiotic elimination of symbiotic Rickettsia bucheri in the tick Ixodes scapularis (Acari: Ixodidae). Appl Environ Microbiol doi:10.1128/AEM.01672-20
• Type: Theses/Dissertations Status: Published Year Published: 2020 Citation: Thorpe CJ. 2020. Growth Dynamics and Tissue Tropism of Rickettsia buchneri, The Endosymbiont of The Blacklegged Tick, Ixodes scapularis. Masters Thesis. 75 pages.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Wang X-R, Kurtti TJ, Oliver JD, Munderloh UG. 2020. The identification of tick autophagy-related genes in Ixodes scapularis responding to amino acid starvation. Ticks Tick-borne Dis. 11: 101402 https://doi.org/10.1016/j.ttbdis.2020.101402

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Our reports are for a target audience that includes scientists and public health officials that can implement and apply this new knowledge to the control of ticks and tick borne pathogens. Effective control of ticks is a regional and national health priority and we provide information that can be used by regional and national public health officials. This project examines new ways to reduce the prevalence of ticks and tick-borne disease agents around homes, recreational areas and military bases. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided training and professional development for undergraduate (3), graduate (1) and postdoctoral (1) students and career research scientists (3). How have the results been disseminated to communities of interest?Our results were disseminated to colleagues in the scientific community via papers published in open access scientific journals available online at journal web sites and presentations made at national and international scientific meetings. Thesescientific meetings are attended by public health, military, homeland security officials, scientists from the CDC and state public health departments. We presented some of our research results at this year'sNational Entomological Society of America meetingin St LouisMO. What do you plan to do during the next reporting period to accomplish the goals?We will continue toevaluate the tissue tropism ofR. buchneriin adult female ticks via fluorescent in situ hybridization (FISH). A fluorescent DNA probe, which targetsbacterial 16S ribosomal RNA, will be applied to dissected whole-organs and examined with laser-confocal microscopy. This will be done to detect other bacteria in blacklegged ticks.

Impacts
What was accomplished under these goals? The biological role of the symbiontRickettsia buchneriin the blacklegged tickIxodes scapularisremains undefined. Additionally, the quantification and tissue tropism ofR. buchneriin field-collected MinnesotaI. scapularisticks remains unexplored. Our primary objective was to molecularly detect and quantify the number ofR. buchnericitrate synthase (gltA) gene copies throughout each flat and engorged life stage ofI. scapularisticks collected from a military base, Camp Ripley, Minnesota.Our secondary objective was to evaluate the tissue tropism ofR. buchneriin adult female ticks via fluorescent in situ hybridization (FISH). A fluorescent DNA probe, which targetsRickettsia23S ribosomal RNA, was applied to dissected whole-organs and examined with laser-confocal microscopy. As expected, we detected strong positive signals ofR. buchneriin the ovaries, but negative signals in the salivary glands, midgut, seminal receptacle, and synganglion.We continued our studiesto compare the growth dynamics ofR. buchneriin vitro and in the tick. We quantified the growth ofthe symbiontin vitro utilizing novel fluorescent plate reader method and QPCR. Wecompared growth ofsymbiont in vitro with its growth in blacklegged ticks.The number ofR. buchnericitrate synthase gene copies in cell cultures sampled over time was quantified by qPCR, and compared togltAcopy numbers in Camp Ripley collected ticks. In vitro qPCR results confirmed the doubling time of transformedR. buchneriin ISE6, anIxodesscapulariscell line, was approximately six days. In vitro experiments using a fluorescent plate reader quantitatively confirmed the slow growth rate of the transformant. The presence ofR. buchneriinI. scapularisadult males was highly variable. Possible explanations for the variability ofR. buchneriin nymphs and males, are stress coupled with autophagy, and age.R. buchneriwas detected in all femaleI scapularisadults, but variably in males, possibly because males are smaller in size and do not take a blood meal.Detection, quantification, and tissue localization ofR. buchneriaids in the elucidation of its biological role and how it impacts symbiosis, disease transmission and, vector competence ofblacklegged ticks.

Publications

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Our reports are for a target audience that includes scientists and public health officials that can implement and apply this new knowledge to the control of ticks and tick borne pathogens. Effective control of ticks is a regional and national health priority and we provide information that can be used by regional and national public health officials. This project examines new ways to reduce the prevalence of ticks and tick-borne disease agents around homes, recreational areas and military bases. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided training and professional development for undergraduate (2), graduate (1) and postdoctoral (1) students and career research scientists (3). How have the results been disseminated to communities of interest?Our results were disseminated to colleagues in the scientific community via papers published in open access scientific journals available online at journal web sites and presentations made at national and international scientific meetings. These scientific meetings are attended by public health, military, homeland security officials, scientists from the CDC and state public health departments. We presented some of our research results at the National Entomological Society of America meetingin Vancouver, Canada What do you plan to do during the next reporting period to accomplish the goals?Growth parameters ofR. buchneritransformant in ISE6 cell line andR. buchneriwildtype inIxodes scapulariscell lines are currently being evaluated. Additional reference genes to evaluate and quantifyR. buchnerivia qPCR are in development.

Impacts
What was accomplished under these goals? The goal of this project is to determine role ofRickettsiabuchneriin blacklegged ticks and assess its suitability for paratransgenesis. We will make a thorough analysis of the tripartite relationship between the black-legged tick,Ixodesscapularis, its symbiont,R. buchneri, and two pathogens that it transmits,Borrelia burgdorferiandAnaplasma phagocytophilum. First, we will identify the contributions of the symbiont to tick development and reproduction, as well as its role in the vectorial capacity ofI. scapularis. Second, we will generateR. buchnerimutants using a random mutagenesis approach to insert a replaceable cassette. Mutants that are colonization competent will then be selected for recombinase-mediated cassette exchange with sequences encoding antimicrobial factors. Aim 1)DetermineR. buchnerirole in a) tick nutrition, fertility/fitness, and b) in exclusion of other rickettsiae (e.g.,Rickettsia monacensis), and c) susceptibility toA.phagocytophilum(Ap) andB.burgdorferi(Bb). This will delineate the role ofR. buchnerias a nutritional symbiont and as a symbiont that regulates tick microbial interactions. Aim 2)PrepareR. buchneritransposon mutants using a Himar1 construct flanked by mismatched loxP sequences for subsequent replacement using Recombinase Mediated Cassette Exchange (RMCE). Select mutants able to colonize ticks. This will demonstrate that aposymbiotic ticks can be reconstituted with a modified symbiont.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Ch�vez, ASO, Herron, MJ, Nelson, CM, Felsheim, RF, Oliver, JD, Burkhardt, NY, Kurtti, TJ, Munderloh, UG. 2018. Mutational analysis of gene function in the Anaplasmataceae: Challenges and perspectives. Ticks Tick Borne Diseases. https://doi.org/10.1016/j.ttbdis.2018.11.006
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Murgia, MV, Bell-Sakyi, L, de la Fuente, J, Kurtti, TJ, Makepeace, B, Mans, B, McCoy, KD, Munderloh, UG, Plantard,O, Rispe, C, Valle,MR, Tabor, A, Thangamani, S, Thimmapuram, J, Hill, CA. 2018. Meeting the challenge of tick-borne disease control: A proposal for 1000 Ixodes genomes. Ticks Tick Borne Diseases. 10:213-218. https://doi.org/10.1016/j.ttbdis.2018.08.009

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Our reports are for a target audience that includes scientists and public health officials that can implement and apply this new knowledge to the control of ticks and tick borne pathogens. Effective control of ticks is a regional and national health priority and we provide information that can be used by regional and national public health officials. This project examines new ways to reduce the prevalence of ticks and tick-borne disease agents around homes, recreational areas and military bases. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided training and professional development for 2 undergraduate and one graduate student and 3 research scientists. How have the results been disseminated to communities of interest?Our results were disseminated to colleagues in the scientific community via papers published in open access scientific journals available online at journal web sites and presentations made at national and international scientific meetings. These national and international scientific meetings are attended by public health, military, homeland security officials, scientists from the CDC and state public health departments. We presented some of our research results at the 9th Tick and Tick-borne Pathogen Conference in Cairns Australia. What do you plan to do during the next reporting period to accomplish the goals?We will examine the ability of the Lyme disease spirochete to replicate, invade tick cells infected with R. buchneri. The gene products from the antibiosis operons will also be cloned and evaluated for their efficacy against tick-transmitted bacteria.

Impacts
What was accomplished under these goals? Rickettsia buchneri, a rickettsial symbiont of the North American blacklegged tick Ixodes scapularis, is widespread and maintained in nature via transovarial transmission; there is no evidence for horizontal transmission. We sequenced the genome of R. buchneri and found that it includes two gene clusters known to be involved with the production of antibiotics, similar to those produced in other bacteria. The first cluster contains genes for polyketide and other non-ribosomal peptide synthesis enzymes. The second cluster genes are similar to those involved in aminoglycoside antibiotic synthesis, as well as genes coding for putative antibiotic exporters and an antibiotic resistance factor. We hypothesize that products from these R. buchneri genes play a role in the ability of the blacklegged tick to acquire, maintain and transmit tick borne pathogenic bacteria. This project period we examined if these genes are transcribed and if antibiosis is involved in the exclusion of tick-borne bacteria by the symbiont. We examined the transcription of 9 selected genes in both clusters by reverse-transcriptase PCR and determined that they were actively transcribed. This raised the question whether the antibiotic gene clusters are functional and their gene products are involved with the interactions of R. buchneri with other bacteria associated with I. scapularis. We developed an in vitro system to test the hypothesis of R. buchneri intracellular antibiosis against other intracellular tick borne bacteria. We used Ixodes cell lines to propagate R. buchneri wild type and transformants in tick cells. Rickettsia buchneri interfered with the invasion and growth of tick cells by other rickettsia and anaplasma. We demonstrated the interfering effect of R. buchneri on the infectivity and replication of Rickettsia monacensis and Anaplasma phagocytophilum. Cultures of Ixodes cells infected with R. buchneri demonstrated anti-rickettsial and anti-anaplasma activity. Fluorescent microscopy revealed that rickettsiae and anaplasma transfoemed to express a red fluorescent protein did not replicate in tick cells infected with R. buchneri. Infectivity of rickettsiae or anaplasma for tick cells infected with R. buchneri was reduced by 3-5 orders of magnitude.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Kurtti T, Burkhardt N, Felsheim R, Munderloh U. 2017. Is antibiosis involved in the exclusion of tick borne bacteria by the Ixodes scapularis symbiont Rickettsia buchneri? 9th Tick-borne Pathogen Conference, Cairns Australia, 27 August-1 September 2017
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Kurtti TJ, Burkhardt NY, Heu CC, Munderloh UG. 2016. Fluorescent protein expressing Rickettsia buchneri and Rickettsia peacockii for tracking symbiont-tick cell interactions. Veterinary Sciences 3, 34; doi:10.3390/vetsci3040034

Progress 07/12/16 to 09/30/16

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Nothing was accomplished from September to November 2016.

Publications