INFECTION(PREVALENCE(IN(A(NOVEL(' SCAPULARIS'POPULATION(IN(NORTHERN(WISCONSIN( ( ( ( ( ( ( A(thesis(submitted(in(partial(fulfillment(of(the( requirements(for(the(degree(of(Master(of(Science( ( ( ( By(( ( ( ( MARY(LYNN(WESTWOOD( B.S.,(Wright(State(University,(2015( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( 2017( Wright(State(University(

WRIGHT'STATE'UNIVERSITY'' GRADUATE'SCHOOL' ' ' August'25th,'2017'' ' I'HEREBY'RECOMMEND'THAT'THE'THESIS'PREPARED'UNDER'MY'SUPERVISION'BY'Mary' Lynn'Westwood'ENTITLED'Infection'Prevalence'in'a'Novel'Ixodes'scapularis'Population'in' Northern'Wisconsin'BE'ACCEPTED'IN'PARTIAL'FULFILLMENT'OF'THE'REQUIREMENTS'FOR' THE'DEGREE'OF'Master'of'Science' ' '______'' Thomas'Rooney,'Ph.D.'Thesis'Director' ' ' '______'' David'Goldstein,'Ph.D.,'Chair'' Department'of'Biological'Sciences'' College'of'Science'and'Mathematics'' ' Committee'on'Final'Examination' ' ' ______'' Thomas'Rooney,'Ph.D.' ' ' ______' Jeffrey'Peters,'Ph.D.'' ' ' ______'' Megan'Rúa,'Ph.D.'' ' ' ______Robert'E.W.'Fyffe,'Ph.D.' Vice'President'for'Research'and' '''''''''''''''''''''''''Dean'of'Graduate'School' ' ' ' ' ' ABSTRACT' ' Westwood,'Mary'Lynn.'M.S.'Department'of'Biological'Sciences,'Wright'State'University,' 2017.'Infection'Prevalence'in'a'Novel'Ixodes'scapularis'Population'in'Northern'Wisconsin' ' ' ' Ixodes'scapularis'(i.e.'the'blacklegged'or'deer')'is'an'important'vector'of' emerging'human'pathogens.'Over'the'past'few'decades,'the'incidence'of'blacklegged' tick[associated'zoonotic'diseases'have'increased'in'accordance'with'an'expansion'of'the' blacklegged'tick'geographic'range.'Data'concerning'the'infection'prevalence'of' blacklegged''in'this'region'is'highly'variable'and'fragmentary.'Using'a'novel' population'of'these'ticks,'constituting'part'of'the'invasion'front'in'Vilas'County,'

Wisconsin,'I'examined'infection'prevalence'and'vector'aspects'of'their'ecology.''During' the'summer'of'2016,'I'collected'461'blacklegged'ticks'and'screened'them'using'a' standard'polymerase'chain'reaction'assay'designed'to'identify'three'emerging'zoonotic' pathogens:''burgdorferi,'Anaplasma'phagocytophilum,'and''microti.'The' overall'infection'probability'was'30%,'25%,'and'14%'respectively.'The'probability'of'co[ infection'ranged'from'2[6%'and'the'conditional'probability'of'co[infection'was'not' significant,'indicating'that'there'are'yet'to'be'detectable'relationships'between'co[ infecting'pathogens.''Blacklegged'ticks'were'found'in'two'main'vegetation'types:'Oak'and'

Northern'Hardwood.'These'vegetation'types'are'typical'blacklegged'tick'habitat'since' they'provide'an'adequate'layer'of'leaf'litter'which'is'necessary'to'prevent'desiccation.'

Overall,'my'work'suggests'that'blacklegged'ticks'in'this'region'may'be'an'important' source'of'pathogen'pressure.'This'study'aids'in'a'better'understanding'of'the'geographic'

iii range'increase'of'blacklegged'ticks'in'North'America'and'the'entomologic'risk'posed'by' these'novel'populations.''

' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' '

iv TABLE'OF'CONTENTS' Page'' I.'INTRODUCTION'AND'OBJECTIVES...... 1'' ' Zoonotic'Diseases………………………………….………...... …...... 1'' ' Ixodes'scapularis………………...... 3'' ' Lyme'Borreliosis……………………………...………………...…………...... 6'' ' Borrelia'burgdorferi...... 7' ' Anaplasma'phagocytophilum………………………………….………………………...... 8' ' Babesia'microti...... 9'' ' Co[infection…………………………………………...... 12'' ' Vilas'County...... 12' II.'METHODS...... 14'' ' Study'Area'and'Sites...... 14'' ' Tick'Collection…………………………………...... 14' ' Pathogen'Detection...... 17'' ' Data'Analysis...... 18'' III.'RESULTS...... 19''' ' Infection'Prevalence...... 19'' ' Vector'Ecology...... 19' IV.'DISCUSSION'AND'CONCLUSIONS...... 24'' ' Infection'Prevalence...... 24'' ' Vector'Ecology...... 27' ' Conclusions...... 28' V.'REFERENCES...... 30'' ' '

v LIST%OF%TABLES% Table%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%Page% 1.%Realized%reservoir%competence...... 11% 2.%Summary%of%I.#scapularis%collection%data...... 20%% 3.%Summary%of%targets%and%primers%for%pathogens...... 21% 4.%Summary%of%observed%and%expected%coIinfection%prevalence...... 22% 5.%Summary%of%conditional%probabilities…...... 23% % % Figure% 1.%VectorIborne%zoonotic%disease%dynamics...... 1% 2.%Map%of%property%including%section%numbers……………………………………………………………….16% %

vi I.# INTRODUCTION# # OVERVIEW#

# To#understand#how#pathogens#are#passed#to#humans#it#is#necessary#to# understand#the#relationship#between#ticks,#pathogens,#hosts,#and#the#environment.#Each# component#of#the#disease#system#has#the#capacity#to#shape#the#disease#dynamics#and# entomologic#risk#of#a#given#geographic#location.#The#following#sections#are#intended#to# guide#the#reader#through#the#complexity#of#blacklegged#tickFborne#disease#dynamics#and# provide#an#understanding#of#entomologic#risk#in#a#newlyFestablished#population#of# vectors.##

# ZOONOTIC#DISEASES# # # Zoonotic#diseases#(zoonoses)#are#defined#by#the#transmission#of#pathogens#from# nonFhuman##to#humans.#They#account#for#roughly#60%#of#human#diseases#and#60F

75%#emerging#human#diseases#

(Cleaveland#2001).#Emerging# diseases#are#especially#relevant#to# human#health#as#they#include# diseases#that#have#increased#in# Figure#1.#VectorFborne#disease#systems#involve#the#cycling#of# the#past#couple#of#decades#or# pathogens#between#nonFhuman#reservoir#hosts#(A)#and#vectors#(B).# When#one#of#these#pathogens#is#acquired#by#a#human#(C)#the# threaten#to#increase#in#the#near# resulting#disease#is#termed#a#.##

1 future#(Jones#et#al.#2008).#These#diseases#are#caused#by#pathogens,#such#as#bacteria,# viruses,#or#other#microorganisms.#They#may#be#transmitted#by#direct#contact#between# hosts#(ex.#through#the#bite#or#scratch#of#an#infected#),#indirect#contact#(ex.#through# contact#with#contaminated#objects#such#as#a#cage#or#aquarium),#or#through#a#vector.#A# vector#is#generally#an##species#(e.g.#fleas,#ticks,#mosquitoes,#and#mites)#that# transmits#pathogens#between#hosts.##

# The#chain#of#transmission#in#vectorFborne#zoonoses#is#often#highly#complex#due#to# the#numerous#species#involved#(LoGuidice#et#al.#2003).#Unless#a#pathogen#is#capable#of# transmitting#directly#from#the#female#to#her#offspring,#vectors#must#first#feed#from#an# infected#host#to#become#infected.#This#host,#termed#the#reservoir#host,#remains# chronically#infected#yet#asymptomatic#(Figure#1A).#Once#the#vector#acquires#the# pathogen(s)#(Figure#1B),#it#may#transfer#the#pathogen(s)#to#subsequent#hosts#(including# humans;#Figure#1C).##

Of#all#vector#species,#ticks#boast#the#highest#diversity#of#pathogens#vectored#

(McCoy#et#al.#2015).#These#pathogens#are#often#widespread#and#may#be#of#medical# concern#(McCoy#et#al.#2015).#To#better#understand#public#health#risk#posed#by#a#vector,# entomologic#risk#is#often#quantified.#Entomologic#risk#is#a#metric#that#can#be#summarized# by#numerous#factors,#one#of#the#most#important#being#the#infection#prevalence#within#a# population#of#vectors.#Determining#the#proportion#of#I.#scapularis#infected#with#B.#

2 burgdorferi,#A.#phagocytophilum,#and#B.#microti,#as#well#as#rates#of#coFinfection,#provides# valuable#data#concerning#the#pathogenicity#of#a#tick#population.##

#

IXODES#SCAPULARIS#

# Ixodes#scapularis#(i.e.#the#blacklegged#tick#or#deer#tick)#is#a#hardFbodied#tick#of#the# family#.#It#is#an#obligate,#hematophagous#ectoparasite#that#harbors#numerous# zoonotic#pathogens#(Adelson#et#al.#2004).#Blacklegged#ticks#can#be#found#throughout#the# eastern#portion#of#the#United#States#and#have#now#been#found#in#Canada,#likely#due#to# recent#migration#events#via#birds#(Scott#et#al.#2014).##

# Understanding#the#life#cycle#of#blacklegged#ticks#is#critical#in#understanding# disease#risk.#Blacklegged#ticks#acquire#one#blood#meal#per#life#stage,#and#it#is#during#one# of#their#blood#meals#that#they#are#capable#of#acquiring#a#pathogen#and#transmitting#it#to# a#subsequent#host.#In#nature,#blacklegged#ticks#have#a#2Fyear#life#cycle#that#begins#in#the# spring.#Egg#clutches#of#around#1,500#to#2,000#larval#ticks#hatch#during#the#late#spring#or# early#summer.#Once#a#tick#has#taken#a#successful#blood#meal#at#the#larval#stage#it#falls#off# its#host#and#overwinters.#The#following#spring,#the#tick#emerges#as#a#nymph#and#seeks#a# suitable#host#for#a#second#blood#meal.#Once#engorged,#the#nymph#falls#off#its#host#and# molts.#During#the#fall,#adults#emerge#and#host#seek#for#a#final#time.#Adult#females#take#a# blood#meal#while#adult#males#simply#hostFseek#to#find#a#mate.#Mating#generally#occurs#on# a#host#and#the#male#dies#postFcopulation.#Mated#and#replete,#the#female#falls#off#the#host#

3 and#overwinters#for#a#second#time.#In#the#spring,#the#adult#female#lays#her#eggs#and#the# cycle#begins#again#(Schmidt#&#Ostfeld#2001).##

# Nymphal#ticks#are#the#likely#source#behind#most#human#cases#of#blacklegged#tickF borne#disease.#This#is#generally#attributed#to#the#size#of#nymphs#whose#small#size#makes# detection#exceedingly#difficult#(Falco#et#al.#1999;#Piesman#et#al.#1987).#Larval#blacklegged# ticks#have#yet#to#take#a#blood#meal#and#are#therefore#free#from#any#zoonotic#pathogens# that#are#not#transFovarially#transmitted.#Since#many#common#blacklegged#tickFassociated# pathogens#(e.g.#Borrelia#burgdorferi,#Anaplasma#phagocytophilum,#and#Babesia#microti)# are#not#known#to#be#transFovarially#transmitted,#this#lifestage#is#considered#the#least# infective#(Schmidt#&#Ostfeld#2001).##

# Host#species#play#an#important#role#in#tickFborne#disease#dynamics.#Not#only#do# hosts#aid#in#pathogen#transmission,#they#also#contribute#to#tick#population#range# increase.#Ticks#themselves#are#not#capable#of#moving#very#far;#however,#through#the# movement#of#hosts#such#as#whiteFtailed#deer#(Odocoileus#virginianus)#and#migratory# birds,#they#are#capable#of#significant#travel#(Leighton#et#al.#2012).#Much#of#the#geographic# range#increase#of#blacklegged#tick#populations#can#be#attributed#to#the#movement#of# whiteFtailed#deer#and#migratory#birds#(Leighton#et#al.#2012).#Passive#surveillance#suggests# that#ticks#are#capable#of#averaging#425#km#mean#dispersion#distance#through#the# movement#of#migratory#birds#(Ogden#et#al.#2008).#WhiteFtailed#deer#serve#as#the#primary# host#of#adult#blacklegged#ticks#(Rand#et#al.#2003).#Unsurprisingly,#blacklegged#ticks#can#be#

4 found#in#most#regions#of#the#United#States#that#are#inhabited#by#whiteFtailed#deer#(Rand# et#al.#2003).#Importantly,#however,#whiteFtailed#deer#do#not#directly#increase#pathogen# prevalence#(LoGuidice#et#al.#2003).#Adult#ticks#are#unlikely#to#take#a#blood#meal#after# feeding#upon#a#whiteFtailed#deer#(since#this#is#generally#their#final#blood#meal)#and#are# therefore#unlikely#to#pass#on#any#pathogens#to#subsequent#hosts.#WhiteFtailed#deer#are# also#not#competent#reservoirs#for#the#most#common#blacklegged#tickFborne#pathogen,#

Borrelia#burgdorferi#(LoGuidice#et#al.#2003).#

# Competent#reservoirs#are#required#for#successful#pathogen#cycling#between#ticks# and#host#species.#One#such#reservoir#is#the#whiteFfooted#mouse#(Peromyscus#leucopus).#It# is#the#most#competent#reservoir#for#B.#burgdorferi#in#the#northeastern#and#north#central#

U.S.#(Levi#et#al.#2016).#P.#leucopus#is#found#throughout#the#eastern#twoFthirds#of#United#

States,#portions#of#southern#Canada,#and#Mexico#(Linzey#et#al.#2008).#As#a#result#of#it’s# extensive#range,#P.#leucopus#occupies#a#variety#of#habitats.#In#the#eastern#U.S.,#P.# leucopus#is#most#abundant#in#low#to#midFelevation#dry#forests#with#shrubby#understory,# but#is#also#found#in#brushy#fields.#In#contrast,#in#the#southwestern#U.S.#it#inhabits#semiF desert#vegetation#(Linzey#et#al.#2008).##

# Not#all#regions#in#which#blacklegged#ticks#can#be#found#have#high#rates#of#Lyme# borreliosis#(LB)#and#other#blacklegged#tick#vectored#pathogens.#This#may#be#due#to#a# number#of#factors#including#differences#in#hostFseeking#behavior#(i.e.#questing)#(Arsnoe#et# al.#2015),#climatic#variables#(Ogden#et#al.#2014),#biodiversity#(Schmidt#et#al.#2001),#and#

5 tick#genetics#(Qiu#et#al.#2002,#Humphrey#et#al.#2010).#Variation#in#local#habitat#and# environmental#attributes#influence#the#interaction#of#vector,#reservoir#host,#and# pathogen#ecologies.#

#

LYME#BORRELIOSIS##

# LB#is#the#most#common#vectorFborne#zoonosis#in#temperate,#Holarctic#ecozones#

(Vourc’h#et#al.#2016).#It#is#caused#by#the#bacterium#Borrelia#burgdorferi#sensu#lato.#In#the#

United#States,#an#estimated#300,000#cases#of#LB#are#contracted#annually#(CDC#MMWR,#

2011F2013).#The#current#distribution#of#confirmed#LB#cases#in#North#America#is#highly# concentrated#with#the#majority#of#cases#originating#in#the#northeast#and#upper#Midwest#

(Kugeler#et#al.#2015).#Europe#reports#an#estimated#85,000#cases#of#LB#each#year#(Lindgren# et#al.#2006),#making#it#the#most#prevalent#vectorFborne#disease#in#the#northern# hemisphere.##

# LB#has#been#found#in#over#half#of#all#U.S.#counties#(Eisen#et#al.#2016),#with#an# overwhelming#majority#of#cases#reported#from#states#in#the#northeast#and#upper# midwest#(CDC#MMWR#2011F2013).#Furthermore,#the#number#of#reported#cases#is#on#the# rise#and#the#geographic#range#of#LB#incidence#is#expanding#(CDC#MMWR#2011F2013).#The# number#of#counties#in#northFcentral#states#exhibiting#a#high#incidence#of#LB#increased# from#22#to#78,#or#by#nearly#250%#from#1993#to#2015#(Kugeler#et#al.#2015).#The#increase#in#

6 both#geographic#range#and#incidence#of#LB#constitute#a#great#public#health#risk#that# requires#continual#monitoring#and#surveillance#(Bacon#2008).##

# The#increase#in#LB#incidence#can#be#attributed#to#more#than#just#the#range# expansion#of#Ixodes#spp.#ticks.#Human#influence#on#the#environment,#including#forest# fragmentation#and#the#creation#of#peridomestic#habitats,#allow#for#an#increase#in#human# activity#in#tickFfriendly#habitats#(Hamer#2010).#Coupled#with#an#increase#in#surveillance# and#heightened#awareness#of#tickFborne#disease#risk,#this#has#led#to#an#overall#increase#in# tickFborne#disease#incidence#in#recent#years.##

# #

BORRELIA#BURGDORFERI#

# Borrelia#burgdorferi#sensu#lato#(s.l.),#is#an#obligate#bacteria#of#the#phylum#

Spirochaetes.#At#least#19#genospecies#(i.e.#subdivisions)#of#Borrelia#have#been#described#

(Skotarczak#2014).#They#are#collectively#referred#to#as#the#Borrelia#burgdorferi#species# complex#(or,#B.#burgforferi#s.l.).#These#genospecies#are#described#as#having#unique# antigen#structures#and#display#varying#organotropic#and#pathogenic#potential#(Skotarczak#

2014).#They#generally#vary#in#geographic#range;#however,#several#species#overlap#and#can# be#found#to#simultaneously#coFinfect#a#vector#or#host#(Rauter#&#Hartung#2005;#Skotarczak#

2014).#In#North#America,#B.#burgdorferi#sensu#stricto#(s.s.)#and#the#recently#described#B.# mayonii#are#the#only#known#causative#agents#of#LB#(Pritt#et#al.#2016;#Tilly#et#al.#2008).#

Both#B.#burgdorferi#s.s.#and#B.#mayonii#are#vectored#by#blacklegged#ticks#in#the#eastern#

7 half#of#the#United#States.#Along#the#Pacific#coast,#B.#burgdorferi#s.s.#is#vectored#by#the# western#blacklegged#tick#(Ixodes#pacificus).#In#Europe,#the#genospecies#B.# burgdorferi#sensu#strictu#(s.s.),#B.#garinii,#and#B.#afzelii#are#the#recognized#etiological# agents#of#LB#(Tilly#et#al.#2008)#and#are#vectored#by#I.#persulcatus#and#I.#ricinus#(Mannelli#et# al.#2012).#In#Asia,#B.#afzelii#and#B.#garinii#are#vectored#by#I.#persulcatus#(Masuzawa#2004).##

# The#most#competent#reservoir#for#B.#burgdorferi#in#the#northeastern#and#north# central#U.S.#is#the#whiteFfooted#mouse#(Peromyscus#leucopus)#(Levi#et#al.#2016;#see#Table#

1).#P.#leucopus#is#generally#abundant#in#many#LB#endemic#areas,#such#as#the#northeast# and#upper#Midwest#U.S#(Levi#et#al.#2016).#The#overlap#of#blacklegged#tick#and#P.#leucopus# range#allows#for#pathogen#amplification#in#these#regions.##

#

ANAPLASMA#PHAGOCYTOPHILUM#

# Human#Granulocytic##(HGA)#is#caused#by#the#intracellular#bacterium#

Anaplasma#phagocytophilum.#Signs#of#HGA#typically#appear#within#one#or#two#weeks# upon#being#bitten#by#an#infected#tick,#though#diagnosis#is#often#difficult#as#a#result#of#the# large#range#of#potential#symptoms#(CDC#MMWR,#2006).#Most#commonly,#infected# individuals#will#experience#a#combination#of#fever,#headache,#muscle#pain,#malaise,#chills,# nausea,#abdominal#pain,#cough,#and#confusion#(CDC#MMWR,#2006).#In#severe#cases,# individuals#have#presented#difficulty#breathing,#hemorrhage,#renal#failure,#or#neurological# problems#(CDC#MMWR,#2006).##

8 # No#evidence#has#been#documented#supporting#the#role#of#transovarial# transmission#of#A.#phagocytophilum#in#blacklegged#ticks#(Dunning#et#al.#2006).#Therefore,# for#I.#scapularis#to#acquire#the#bacterium,#it#can#be#assumed#that#they#must#feed#upon#an# infected#host.#A#variety#of#small#mammals#have#been#implicated#as#competent#reservoirs# of#A.#phagocytophilum,#one#of#those#being#P.#leucopus#(see#Table#1).#Multiple#bird#species# can#also#act#as#competent#reservoirs#for#the#bacterium#(Keesing#et#al.#2012).###

# A.#phagocytophilum#is#vectored#by#blacklegged#ticks#the#northeast#and#upper#

Midwest#United#States,#and#by#I.#pacificus#in#northern#California#(Keesing#et#al.#2012).#

HGA#was#first#recognized#as#a#human#disease#in#the#1990’s,#though,#since#the#CDC#began# reporting#annual#cases#in#1999,#the#number#of#reported#cases#has#risen#steadily#(CDC#

MMWR,#2011F2013).#Nationally,#there#were#348#cases#of#HGA#reported#in#2000,#and#

1,761#cases#were#reported#in#2010.#In#2011,#the#CDC#reclassified#HGA#as#a#Nationally#

Notifiable#Disease.#Over#the#course#of#two#years,#from#2011#to#2013,#nearly#fourF thousand#cases#of#HGA#were#reported#nationwide#(CDC#MMWR,#2011F2013).##

#

BABESIA#MICROTI#

# #is#caused#by#the#protozoan#blood#parasite#Babesia#microti.#Though# many#individuals#infected#with#B.#microti#remain#asymptomatic,#certain#cases#can# become#severe#and#even#lifeFthreatening.#Complications#from#infection#can#lead#to#low# and#unstable#blood#pressure,#severe#hemolytic#anemia,#a#very#low#platelet#count,#

9 disseminated#intravascular#coagulation#(resulting#in#blood#clots#and#bleeding),# malfunction#of#vital#organs,#and#in#some#cases,#death#(Joseph#et#al.#2011).###

# According#to#available#evidence,#there#does#not#appear#to#be#transovarial# transmission#of#B.#microti#(Gray#et#al.#2002,#Gray#et#al.#2010,#Uilenberg#2006,#Oliveira#et# al.#1979).#Like#B.#burgdorferi#and#A.#phagocytophilum,#P.#leucopus#is#a#common#and# competent#reservoir#host#for#B.#microti,#though#it#was#also#found#that#a#variety#of#other# small#mammal#species#can#act#as#a#reservoir#(Hersh#2014)#(see#Table#1).#B.#microti#shares# the#same#vector#as#A.#phagocytophilum#and#B.#burgdorferi#in#the#northeast#and#north# central#United#States;#all#three#pathogens#are#vectored#by#I.#scapularis.#

# Babesiosis#became#nationally#notifiable#in#1998#(CDC#MMWR,#2011F2013),#and# was#reported#7,746#times#nationwide#from#2011#to#2013.#The#expansion#of#tick# populations#into#regions#previously#uninhabited#allows#for#greater#contact#between#ticks# and#humans,#heightening#the#risk#of#infection#(Piesman#et#al.#2008).#Babesiosis#is#of# growing#concern,#especially#in#the#northeastern#United#States,#where#certain#regions# have#seen#a#large#increase#in#incidence#over#the#past#decade#(Joseph#et#al.#2011).#

#

# # # # # #

10 Table#1.#Realized#reservoir#competence#of#several#common#hosts#for#B.#burgdorferi,#A.#phagocytophilum,# and#B.#microti.#Realized#reservoir#competence#is#a#metric#generally#used#to#describe#the#propensity#of#a# host#to#infect#a#feeding#vector.#This#can#be#quantified#by#measuring#the#infection#prevalence#for#a#given# pathogen#of#xenodiagnostic#ticks#after#having#fed#from#an#infected#host#species#(Brunner#2008).## # Realized#Reservoir# Pathogen# Reservoir#species# Citation# Competence#(%)# WhiteFfooted#mouse#(Peromyscus#leucopus)# 92.1# # Eastern#chipmunk#(Tamias#striatus)# 55# LoGuidice#et#al.#2003# B.#burgdorferi# ShortFtailed#shrew#(Blarina#brevicauda)# 41.8# # WhiteFtailed#deer#(Odocoileus#virginianus)# 4.6# # WhiteFfooted#mouse#(Peromyscus#leucopus)# ~12# # A.#phagocytophilum# Eastern#chipmunk#(Tamias#striatus)# ~14# Keesing#et#al.#2012# ShortFtailed#shrew#(Blarina#brevicauda)# ~14# # WhiteFfooted#mouse#(Peromyscus#leucopus)# ~30# # B.#microti# Masked#shrew#(Sorex#cinereus)# ~25# Hersh#et#al.#2012# Raccoon#(Procyon#lotor)# ~22# # #

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11 COFINFECTION#

# B.#burgdorferi,#A.#phagocytophilum,#and#B.#microti#are#capable#of#coFinfecting#a# single#tick#and#share#similar#symptoms#at#the#onset#of#illness#which#makes#accurate# diagnosis#and#subsequent#treatment#difficult#(Steiner#et#al.#2008).#CoFinfection#also# increases#the#severity#of#illness#in#infected#individuals#(Hersh#et#al.#2014).#In#a#recent# study,#more#than#50%#of#individuals#diagnosed#with#chronic#Lyme#disease#were#found#to# be#coFinfected#with#at#least#one#additional#pathogen.#For#Babesia#and#Anaplasma,#coF infection#was#found#in#32.3%#and#4.8%#of#cases,#respectively#(Johnson#et#al.#2014).#

Geographical#location#and#prevalence#of#pathogens#within#reservoir#hosts#and#vectors#all# play#a#role#in#the#risk#of#human#coFinfection.#Potential#microbial#interaction#within#vectors# and#hosts#may#also#influence#risk#of#coFinfection#(Steiner#et#al.#2008).#Determining#rates# of#coFinfection#allows#for#a#greater#understanding#of#entomologic#risk#and#can#guide# public#health#awareness.##

#

VILAS#COUNTY#

# Vilas#County#is#located#on#the#outer#range#of#the#geographic#expansion#of#Lyme# disease#and#other#blacklegged#tickFvectored#pathogens#(Emerging#Infectious#Diseases#

2015,#Dahlgren#2011,#Wisconsin#Division#of#Public#Health#2015).#From#2008#to#2014,#the#

Wisconsin#Department#of#Health#Services#reported#3,296#cases#of#HGA#statewide,#with#

49#cases#reported#in#Vilas#County#(Wisconsin#Division#of#Public#Health#2015).#During#the#

12 same#time,#Babesiosis#was#reported#249#times#in#Wisconsin,#with#5#of#those#cases#having# been#acquired#in#Vilas#County#(Wisconsin#Division#of#Public#Health#2016).#This#region#will# be#the#focus#of#our#study.#Our#specific#aims#include#1)#to#determine#the#proportion#of#

Ixodes#scapularis#ticks#infected#with#Borrelia#burgdorferi,#Anaplasma#phagocytophilum,# and#Babesia#microti#at#Dairymen’s#Club#in#Vilas#County,#Wisconsin,#and#2)#to#characterize# the#vector#ecology#in#this#novel#geographic#location.##

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13 II.# METHODS#

STUDY#AREA##

# This#study#was#conducted#on#a#2,500Fhectare#private#property#located#in#the#

Northern#Highlands#region#of#Vilas#County,#Wisconsin#(46°9’#N,#89°51’#W).#The#property,# owned#by#the#nonFprofit#outdoors#club,#Dairymen’s#Inc.,#is#used#primarily#for#lowFimpact# outdoor#recreational#activities#and#land#stewardship#initiatives#(Rooney#2009;#Albert#

1995).#The#climate#in#northern#Wisconsin#is#continental,#indicating#significant#annual# variation#in#temperature#(average#minimum#temperature#during#winter#is#F20°#C#and# average#maximum#temperature#of#32°#C#during#summer)#and#moderate#rainfall#(annual# precipitation#ranges#from#550F780#mm#annually)#(Albert#1995).#The#property#contains# oligotrophic#drainage#lakes,#wetlands,#and#forest#(Rooney#2009).#Pleistocene#glacial# deposits#helped#to#shape#the#region,#forming#moraines#and#outwash#plains#(Rooney#

2009).#Spodosol#soil#in#this#region#varies#from#loams#to#loamy#sands#(Albert#1995).##

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TICK#COLLECTION##

# Blacklegged#ticks#were#collected#June#5F8#2016.#Collections#were#performed# during#the#midFmorning#to#afternoon#in#order#to#optimize#sampling#effort.#Weather# conditions#during#tick#collection#were#fair#(i.e.#no#rain#and#temperatures#ranging#from#11# to#14°#C).#Ticks#were#collected#using#a#standard#tickFdrag#method#(DiukFWasser#et#al.#

2006).#Briefly,#a#1m2#white#flannel#cloth#was#dragged#along#vegetation#and#ticks#were#

14 collected#from#the#cloth#approximately#every#20#paces#to#ensure#optimal#collection#(DiukF

Wasser#et#al.#2006).#The#total#number#of#ticks#collected#per#transect#was#counted#and#the# mean#number#of#ticks,#as#well#as#a#standard#error#(SE),#was#computed#per#trail.#Collection# was#performed#along#a#total#of#38#100#m#transects,#but#also#outside#of#the#transects#to# augment#the#sample#size#for#pathogen#screening.#Tick#collection#outside#of#the#transects# was#done#randomly#in#areas#with#high#tick#density.##

Tick#collections#were#performed#in#northern#mesic#forest#(northern#hardwoods)# and#dry#mesic#forest#(white#pine,#red#oak,#and#red#maple)#vegetation#types#(Curtis#1959).#

Trails#surveyed#included#Honeytree,#Long#Birch,#Cottontail,#Mink,#Raccoon,#Alpine,#and#

Bobcat.#Blacklegged#ticks#were#identified#to#species#and#stage#via#morphology#and# preserved#in#individual#glass#vials#filled#with#7.5#mL#70%#ethanol.#Trail#name#and# management#section#number#were#noted#at#the#beginning#of#each#transect.#A#summary# of#average#tick#collections#per#transect#is#located#in#Table#2.##

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15 Figure#2.#Map#of#property.#

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16 PATHOGEN#DETECTION#

# Ticks#were#stored#in#vials#filled#with#70%#ethanol#at#room#temperature#until#DNA# extraction,#approximately#25#weeks#after#collection.#DNA#extraction#was#performed#using# a#DNeasy®#Blood#and#Tissue#Kit#(Qiagen,#Valencia,#CA).#The#extraction#protocol#was# optimized#to#digest#the#tick#exoskeleton#by#incubating#the#ticks#overnight#in#lysis#buffer# and#proteinase#K#at#56°#C.#Pathogen#primers#for#each#pathogen#were#adopted#from#the# literature#(Prusinski#et#al.#2014).#Each#25#µl#PCR#reaction#contained#4#µl#of#DNA#template# and#21#µl#of#Mastermix#comprised#of#1.25#µl#of#each#forward#and#reverse#primer,#12.5#µl#

2X#Taq#polymerase,#and#6#µl#ddH2O.#Amplification#conditions#included#denaturing#for#5# minutes#at#95°#C,#followed#by#35#cycles#of#denaturation#at#95°#C#for#30#seconds,#primer# annealing#at#60°#C#for#30#seconds,#extension#at#72°#C#60#seconds,#and#a#final#extension# step#for#10#minutes#at#72°#C#(Eppendorf#Mastercycler®,#Hauppage,#NY).#Amplification# products#were#then#run#on#2%#agarose#gel#stained#with#SYBR®#Green.#Products#were# visualized#under#ultraviolet#(UV)#light,#presence#or#absence#for#each#pathogen#was# determined,#and#then#images#were#digitally#captured.#The#target#genes,#product#size,#and# primers#for#each#pathogen#are#included#in#Table#2.##

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17 DATA#ANALYSIS#

# The#infection#prevalence#for#B.#burgdorferi,#A.#phagocytophilum,#and#B.#microti# was#determined#by#calculating#the#ratio#of#singular#infection#to#the#total#number#of#ticks# tested#(461).#CoFinfection#prevalence#was#determined#by#calculating#the#ratio#of#each#coF infection#scenario#to#the#total#sample.#CoFinfection#prevalence#was#calculated#for#all# possible#combinations:#B.#burgdorferi#+#A.#phagocytophilum,#B.#burgdorferi#+#B.#microti,#A.# phagocytophilum#+#B.#microti,#and#B.#burgdorferi#+#A.#phagocytophilum#+#B.#microti.#95%# confidence#intervals#(CI)#were#calculated#for#each#estimate#of#infection#prevalence.#The# conditional#probability#of#coFinfection#(i.e.#P#(B|A)#=#P(A#and#B)/P(A),#or,#the#probability#of# an#infection#(B),#given#that#another#infection#(A)#has#already#occurred)#was#calculated#for# each#coFinfection#scenario#(Table#5).#To#check#for#significance,#a#chiFsquare#test#for# goodness#of#fit#was#performed#for#each#coFinfection#scenario.#

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18 #

III.# RESULTS#

# The#overall#probability#for#infection#with#any#of#the#three#pathogens#was#52%#

(242/461=52%#±#4.5%#CI).#The#singular#infection#prevalence#was#29%#(134/461=29%#±#

4.1%#CI)#for#B.#burgdorferi,#25%#(116/461=25%#±#4.0%#CI)#for#A.#phagocytophilum,#and#

14%#(66/461=14%#±#3.2%#CI)#for#B.#microti.#CoFinfection#prevalence#was#8.2%#

(38/461=8.2%#±#2.5%#CI)#for#B.#burgdorferi#and#A.#phagocytophilum,#4.3%#(20/461=4.3%#

±#1.8%#CI)#for#A.#phagocytophilum#and#B.#microti,#and#3.5%#(16/461=3.5%#±#1.7%#CI)#for#

B.#burgdorferi#and#B.#microti#(Table#4).#The#probability#of#coFinfection#with#all#three# pathogens#was#1.7%#(8/461=1.7%#±#1.2%#CI)#(Table#3).#None#of#the#coFinfection#scenarios# were#found#to#be#significant#(Table#5).##

# 121#ticks#were#collected#within#the#transects#and#an#additional#340#ticks#were# collected#outside#of#the#transects#(Table#5).#In#total,#461#blacklegged#ticks#were#collected#

(nymphs:#n=20,#adults:#n=441).#Long#Birch#trail#had#the#highest#average#I.#scapularis# collection#per#transect#(15#ticks#per#100#m#transect)#(Table#2).##

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19 Table#2.#Summary#of#I.#scapularis#collection#data.#Each#transect#measured#100#m2.#Northern#dryFmesic# forest#is#generally#characterized#by#eastern#white#pine#(Pinus#strobus)#and#red#pine#(Pinus#resinosa)#trees# and#irregular#glacial#topography.#Soil#types#include#loamy#sands#or#sands.#Mesic#forest#is#generally# dominated#by#sugar#maple#(Acer#saccharum)#trees#and#glacial#till#plains#and#moraines.#Soils#types#in#mesic# forest#are#typically#loamy.# # Trail# Vegetation# ##Transects#/trail# ##Ticks#collected/transect#(±#SE)# Honeytree## Mesic# 6# 0.67#(±#0.33)# Long#Birch# Mesic# 10# 15.00#(±#6.78)# Cottontail# DryFmesic# 2# 1.00#(±#1)# Mink# DryFmesic# 6# 1.33#(±#0.49)# Raccoon# DryFmesic# 8# 5.13#(±#1)# Alpine# DryFmesic# 8# 0.75#(±#0.44)# Bobcat# DryFmesic# 4# 0.00#(±#0)# #

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20 Table#3.#Target#genes#and#primers#for#I.#scapularisFassociated#pathogens#(Prusinski#et#al.#2014).#

Pathogen# Target# Product# Primers# Gene# Size# B.#burgdorferi# ospA# 417#bp# OspA1#F#(5qFAATAGGTCTAATATTAGCCTTAATAGCF3q)# and#OspA2R#(5qFTCAAGTCTGGTTCCGTCTGCTCF3q)# A.#phagocytophilum# p44# 334#bp# MSP3#F#(5qFCCAGCGTTTAGCAAGATAAGAGF3q)#and# MSP3R#(5qFGCCCAGTAACAACATCATAAGCF3q)# B.#microti# 16SRlike# 238#bp# Bab1#(5qFCTTAGTATAAGCTTTTATACAGCF3q)#and# Bab4#(5qFATAGGTCAGAAACTTGAATGATACAF3q)# # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # #

21 Table#4.#Observed#and#expected#coFinfection#prevalence#of#B.#burgdorferi,#A.#phagocytophilum,#and#B.# microti#with#associated#ChiFSquare#values.# ### Observed# Expected# ChiFSquare# Pathogens# Infection# Infection# (df#=#1)# Prevalence# Prevalence# 38/461# P(B.#burgdorferi#∩#A.#phagocytophilum)# 34/461#(7.4%)# 1.02# (8.2%)# 20/461# P(A.#phagocytophilum#∩#B.#microti)# 17/461#(3.7%)# 1.08# (4.3%)# 16/461# P(B.#burgdorferi#∩#B.#microti)# 15/461#(3.2%)# 0.13# (3.5%)# 8/461# P(B.#burgdorferi#∩#A.#phagocytophilum∩#B.#microti)# F# F# (1.7%)# 242/461# P(Any#Pathogen)# F# F# (52.5%)# # # # # # # # # # # # # # # # # # # # # # # # # # # # #

22 Table#5.#Conditional#probability#of#coFinfection#for#each#given#infection#scenario.# ## Pathogens# Conditional#Probability# P(B.#burgdorferi#|#A.#phagocytophilum)# 0.328# P(B.#burgdorferi#|#B.#microti)# 0.242# P(A.#phagocytophilum|#B.#burgdorferi)# 0.284# P(A.#phagocytophilum#|#B.#microti)# 0.303# P(B.#microti#|#B.#burgdorferi)# 0.119# P(B.#microti#|#A.#phagocytophilum)# 0.172# #

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23 IV.$ DISCUSSION$

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INFECTION#PREVALENCE#

# Entomologic#risk#is#the#summation#of#numerous#factors,#one#of#the#most# important#being#the#infection#prevalence#within#a#population#of#vectors.#Determining#the# proportion#of#ticks#infected#with#B.#burgdorferi,#A.#phagocytophilum,#and#B.#microti,#as# well#as#rates#of#coFinfection,#provides#valuable#data#concerning#the#pathogenicity#of#I.# scapularis#populations#in#northeastern#Wisconsin.#This#study#shows#that#even#novel# populations#of#blacklegged#ticks#are#capable#of#harboring#numerous#pathogens.##

# Our#study#found#an#overall#infection#prevalence#of#B.#burgdorferi#of#29%#which# closely#resembles#what#has#been#found#in#previous#studies.#One#recent#study#showed#an# overall#infection#prevalence#for#B.#burgdorferi#of#35.7%#in#the#state#of#Wisconsin,#and#a#

33%#prevalence#specifically#for#Vilas#County#(Turtinen#et#al.#2015).#Their#sample#size#for#

Vilas#County#was#very#limited,#however,#with#only#three#adult#female#ticks#collected#and# screened#(Turtinen#et#al.#2015).#The#aforementioned#study,#performed#from#2010#to#

2013,#found#highly#variable#B.#burgdorferi#infection#prevalence#over#the#course#of# sampling.#From#2010#to#2013,#the#infection#prevalence#increased#from#approximately#

21%#to#50%#throughout#the#entire#state.#It#is#uncertain#if#this#apparent#increase#in# prevalence#can#be#attributed#to#an#actual#increase,#biased#sampling,#or#high#yearly# variability#(Turtinen#et#al.#2015).##

24 # Another#study,#performed#in#multiple#Wisconsin#counties#from#2009#to#2013,# found#an#average#B.#burgdorferi#infection#prevalence#of#30%#(Lee#et#al.#2014).#Results# from#this#study#also#showed#high#yearly#variability#in#B.#burgdorferi#infection#prevalence.#

There#was#also#noted#variability#depending#upon#the#location#sampled#(Lee#et#al.#2014).#

Variability#may#be#influenced#by#numerous#factors#including#the#availability#of#suitable# hosts#and#habitat,#as#well#as#weather#conditions#(Lee#et#al.#2014).#The#ticks#in#our#study# were#collected#during#one#sampling#season,#so#we#are#unable#to#compare#data#across# years.#However,#following#findings#from#other#studies,#it#is#reasonable#to#hypothesize#that# variation#in#infection#prevalence#over#time#and#space#would#be#detected.##

# Our#study#found#an#overall#infection#prevalence#for#A.#phagocytophilum#of#25%.#

When#compared#with#one#2015#study#which#found#an#infection#prevalence#of#5.4%#in# questing#nymphal#blacklegged#ticks,#our#detection#of#A.#phagocytophilum#appears# substantially#greater#(Murphy#et#al.#2017).#Approximately#70%#of#all#cases#of#HGA#occur# in#Wisconsin#and#Minnesota,#and#recent#evidence#suggests#the#incidence#of#HGA#might# be#much#greater#in#Wisconsin#than#Minnesota’s#12.3#cases#per#million#(CDC#MMWR#

2006).#Our#finding#that#this#novel#blacklegged#tick#population#carries#a#relatively#high# prevalence#of#A.#phagocytophilum#is#in#agreement#with#estimates#of#high#incidence#of#

HGA#in#the#state#of#Wisconsin.##

# Our#study#found#an#infection#prevalence#of#14%#for#B.#microti.#In#2015,#a#study# performed#in#southwestern#Wisconsin#found#5%#of#questing#I.#scapularis#were#infected#

25 with#B.#microti#(Kowalski#et#al.#2015).#Similar#to#our#findings#regarding#A.# phagocytophilum,#our#data#suggest#a#relatively#high#infection#prevalence#compared#to# published#data.#This#may#be#due#to#spatial#and#temporal#variation#in#tickFborne#pathogen# infection#prevalence,#which#has#been#noted#in#various#other#studies#(e.g.#Turtinen#et#al.#

2015,#Lee#et#al.#2014).###

# Initially,#it#was#surprising#that#there#were#no#significant#relationships#found# between#coFinfecting#pathogens.#Recent#studies#suggest#that#there#is#indeed#a# relationship#between#B.#burgdorferi#and#B.#microti.#B.#microti#has#been#estimated#to#have# a#basic#reproductive#rate#(R0)#of#<1#(Davis#&#Bent#2011).#This#implies#that#without#the#aid# of#facilitation,#B.#microti#should#not#be#able#to#persist#in#nature.#This#is#contrary#to#what# has#been#described#in#surveillance#data.#Indeed,#as#noted#earlier#and#further#supported# by#this#study,#B.#microti#is#present#within#vector#populations#and#is#increasing#in#its# geographic#range#and#incidence.#This#increase#has#been#attributed#to#facilitation#by#B.# burgdorferi#(DiukFWasser#et#al.#2016).#The#spread#of#B.#microti#has#been#confined#to#LBF endemic#areas,#and#thus#locations#situated#along#the#invasion#front#of#blacklegged#ticks# may#present#a#delay#in#facilitation#due#to#ecological#bottlenecks.#This#study#was# conducted#in#an#area#that#has#only#recently#been#invaded#by#blacklegged#ticks,#so#it#is# possible#that#in#upcoming#years#the#effects#of#facilitation#will#become#apparent.#Future# studies#in#this#region#of#northern#Wisconsin#may#help#to#elucidate#what#ecological#drivers# are#necessary#for#B.#microti#emergence#and#persistence#(DiukFWasser#et#al.#2016).#

26 VECTOR#ECOLOGY#

# A#recent#genetic#study#suggests#that#the#increase#of#geographic#range#of# blacklegged#ticks#is#due#to#range#expansion#rather#than#just#population#growth#

(Khatchikian#2015).#This#implies#that#either#the#ticks#are#adapting#to#novel#environments,# or#the#environment#itself#is#changing#(Khatchikian#2015).#Our#findings#regarding# blacklegged#tick#habitat#suitability#mirror#those#of#previous#studies.#Ticks#were#found#in# dry#mesic#and#mesic#forests,#which#provide#an#adequate#layer#of#leaf#litter#to#provide# cover#from#desiccation.#Ticks#were#collected#alongside#trails#in#areas#with#grass#present# amongst#the#leaf#litter#which#provides#a#scaffold#for#ticks#to#quest#(i.e.#hostFseek).##

# Even#in#areas#with#adequate#host#populations,#tick#presence#and#abundance# varies#depending#upon#environmental#factors#(Guerra#et#al.#2002).#Environmental#factors# correlated#with#tick#presence#and#abundance#include#deciduous,#dry#to#mesic#forests#and# alfisolFtype#soils#of#sandy#or#loamFsand#textures#overlying#sedimentary#rock.#The#absence# of#ticks#and#tick#abundance#is#correlated#with#grasslands,#conifer#forests,#wet#to# wet/mesic#forests,#acidic#soils#of#low#fertility#and#a#clay#soil#texture,#and#Precambrian# bedrock#(Guerra#et#al.#2002).##We#found#that#the#continental#climate#of#northern#

Wisconsin#is#habitable#to#blacklegged#ticks#and#soil#and#vegetation#in#this#region#is# conducive#to#blacklegged#tick#colonization#and#emergence.###

#

# ##

27 CONCLUSIONS#

# The#results#of#this#study#inform#both#ecological#theory#and#public#health# awareness.#The#incidence#of#blacklegged#tickRassociated#pathogens#has#been#on#the#rise# over#the#past#decade#(Dahlgren#et#al.#2011;#Krause#2003)#and#obtaining#data#which# demonstrate#the#infection#prevalence#of#newly#established#populations#is#critical#in# understanding#entomologic#risk.#Because#Vilas#County#is#located#at#an#edge#of#the# expanding#range#of#blacklegged#ticks#(Emerging#Infectious#Diseases#2015;#Dahlgren#2011;#

Wisconsin#Division#of#Public#Health#2015),#this#study#provides#valuable#data#in#marking# the#spread#of#blacklegged#ticks#which#are#highly#relevant#to#public#health#in#the#United#

States.#The#relatively#high#infection#prevalence#within#this#novel#population#of#ticks#also# underscores#the#importance#of#competent#reservoir#species#in#the#amplification#of# pathogen#spread.#Many#of#the#reservoirs#for#B.#burgdorferi,#A.#phagocytophilum,#and#B.# microti#have#been#established#on#our#study#site#for#decades.#Each#piece#of#the#tickFborne# disease#system,#hosts,#vectors,#and#pathogens,#must#be#present#for#pathogen# amplification.##

# The#recent#spread#of#blacklegged#ticks#into#novel#geographic#regions#has#been# directly#implicated#in#the#increase#of#disease#transmission#to#humans#(Hamer#et#al.#2010;#

Khatchikian#et#al.#2012).#The#ticks#collected#in#this#survey#were#obtained#from#alongside# trails#located#on#a#property#that#is#used#primarily#for#outdoor#recreational#activities,#such#

28 as#hiking#and#jogging.#This#highlights#the#potential#for#tickFhuman#interaction#and#the# need#to#characterize#novel#invasion#fronts.##

# Our#data#suggest#that#blacklegged#ticks#are#likely#colonizing#novel#environments# which#mirror#established#ecological#constraints.#This#helps#to#inform#those#monitoring# the#spread#of#I.#scapularis.#Further#studies#which#include#screening#reservoir#hosts#for# blacklegged#tickRassociated#pathogens#could#further#help#develop#theory#concerning#the# invasion#biology#of#this#disease#system.#

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29 V.# REFERENCES#

1.#Adelson,#M.E.,#Rao,#R.V.S.,#Tilton,#R.C.,#Cabets,#K.,#Eskow,#E.,#Fein,#L.,#Occi,#J.L.#and# Mordechai,#E.,#2004.#Prevalence#of#Borrelia#burgdorferi,#Bartonella#spp.,#Babesia#microti,# and#Anaplasma#phagocytophila#in#Ixodes#scapularis#ticks#collected#in#Northern#New# Jersey.#Journal#of#Clinical#Microbiology,#42(6),#pp.2799F2801.# # 2.#Albert,#D.#A.,#1995.#Regional#landscape#ecosystems#of#Michigan,#Minnesota#and# Wisconsin:#a#working#map#and#classification.#General#technical#report#NCF178.#U.S.#Forest# Service#North#Central#Forest#Experiment#Station.#St.#Paul,#Minnesota.# # 3.#Arsnoe,#I.#M.,#Hickling,#G.#J.,#Ginsberg,#H.#S.,#McElreath,#R.,#Tsao,#J.I.,#2015.#Different# Populations#of#Blacklegged#Tick#Nymphs#Exhibit#Differences#in#Questing#Behavior#That# Have#Implications#for#Human#Lyme#Disease#Risk.#PLoS#ONE.#10(5):#e0127450.## # 4.#Bacon,#R.#M.,#Kugeler,#K.#J.,#Griffith,#K.#S.,#Mead,#P.#S.,#2008.#Surveillance#for#Lyme# disease#F#United#States,#1992–2006.#Morbidity#and#Mortality#Weekly#Report.#56:1–9.# # 5.#Brunner,#J.L.,#LoGiudice,#K.#and#Ostfeld,#R.S.,#2008.#Estimating#reservoir#competence#of# Borrelia#burgdorferi#hosts:#prevalence#and#infectivity,#sensitivity,#and#specificity.#Journal# of#medical#entomology.#45(1):#139F147.# # 6.#Cleaveland,#S.#C.,#Laurenson,#M.#K.,#Taylor,#L.#H.,#2001.#Diseases#of#humans#and#their# domestic#mammals:#pathogen#characteristics,#host#range#and#the#risk#of# emergence.#Philosophical#Transactions#of#the#Royal#Society#of#London#BFBiological# Sciences.#356:#991–999.# # 7.#CDC#MMWR,#2006.#Diagnosis#and#Management#of#Tickborne#Rickettsial#Diseases:# Rocky#Mountain#Spotted#Fever,#Ehrlichioses,#and#Anaplasmosis#FFF#United#States.# Morbidity#and#Mortality#Weekly#Report.# # 8.#CDC#MMWR,#2011F2013.#Centers#for#Disease#Control#and#Prevention:#notifiable# diseases#and#mortality#tables#MMWR.#Morbidity#and#Mortality#Weekly#Report.# # 9.#Curtis,#J.T.#1959.#The#vegetation#of#Wisconsin.#University#of#Wisconsin#Press,#Madison.# # 10.#Dahlgren,#F.,#Mandel,#E.,#Krebs,#J.,#Massung,#R.,#McQuiston,#J.,#2011.#Increasing# incidence#of##chaffeensis#and#Anaplasma#phagocytophilum#in#the#United#States,# 2000–2007.#The#American#Journal#of#Tropical#Medicine#and#Hygiene.#85:#124–131.##

30 # 11.#Davis,#S.,#Bent,#S.#J.,#2011.#Loop#analysis#for#pathogens:#niche#partitioning#in#the# transmission#graph#for#pathogens#of#the#North#American#tick#Ixodes#scapularis.#Journal# Theoretical#Biology.#269:#96–103.# # 12.#DiukFWasser,#M.#A.,#Gatewood,#A.#G.,#Cortinas,#M.#R.,#YaremychFHamer,#S.,#Tsao,#J.,# Kitron,#U.,#Hickling,#G.,#Brownstein,#J.S.,#Walker,#E.,#Piesman,#J.#and#Fish,#D.,#2006.# Spatiotemporal#patterns#of#hostFseeking#Ixodes#scapularis#nymphs#(:#Ixodidae)#in#the# United#States.#Journal#of#medical#entomology.#43(2):#166F176.# # 13.#DiukFWasser,#M.#A.,#Vannier,#E.,#Krause,#P.#J.,#2016.#Coinfection#by#Ixodes#tickFborne# pathogens:#ecological,#epidemiological,#and#clinical#consequences.#Trends#in# Parasitology.#32.1:#30F42.# # 14.#Dunning#Hotopp,#J.#C.,#Lin,#M.,#Madupu,#R.,#Crabtree,#J.,#Angiuoli,#S.#V.,#Eisen,#J.,#2006.# Comparative#genomics#of#emerging#human##agents.#PLoS#Genetics.#2:#e21.# # 15.#Eisen,#R.#J.,#Eisen,#L.,#Beard,#C.#B.,#2016.#CountyFScale#Distribution#of#Ixodes#scapularis# and#Ixodes#pacificus#(Acari:#Ixodidae)#in#the#Continental#United#States.#Journal#of#medical# entomology.#53(2):#349F386.# # 16.#Falco,#R.C.,#McKenna,#D.F.,#Daniels,#T.J.,#Nadelman,#R.B.,#Nowakowski,#J.,#Fish,#D.#and# Wormser,#G.P.,#1999.#Temporal#relation#between#Ixodes#scapularis#abundance#and#risk# for#Lyme#disease#associated#with#erythema#migrans.#American#Journal#of# Epidemiology,#149(8),#pp.771F776.# # 17.#Gray,#J.,#von#Stedingk,#L.#V.,#Gürtelschmid,#M.,#Granström,#M.,#2002.#Transmission# studies#of#Babesia#microti#in#Ixodes#ricinus#ticks#and#gerbils.#Journal#of#Clinical# Microbiology.#40:#1259–63.## # 18.#Gray,#J.,#Zintl,#A.,#Hildebrandt,#A.,#Hunfeld,#K.#P.,#Weiss,#L.,#2010.#Zoonotic#babesiosis:# overview#of#the#disease#and#novel#aspects#of#pathogen#identity.#Ticks#and#TickFBorne# Diseases.#1:#3–10.## # 19.#Guerra,#M.,#Walker,#E.,#Jones,#C.,#Paskewitz,#S.,#Cortinas,#M.R.,#Stancil,#A.,#Beck,#L.,# Bobo,#M.#and#Kitron,#U.,#2002.#Predicting#the#risk#of#Lyme#disease:#habitat#suitability#for# Ixodes#scapularis#in#the#north#central#United#States.#Emerging#Infectious#Diseases.#8(3):# 289.## #

31 20.#Hamer,#S.A.,#Tsao,#J.I.,#Walker,#E.D.#and#Hickling,#G.J.,#2010.#Invasion#of#the#Lyme# disease#vector#Ixodes#scapularis:#implications#for#Borrelia#burgdorferi# endemicity.#EcoHealth.#7(1):#47F63.# # 21.#Hersh,#M.H.,#Ostfeld,#R.S.,#McHenry,#D.J.,#Tibbetts,#M.,#Brunner,#J.L.,#Killilea,#M.E.,# LoGiudice,#K.,#Schmidt,#K.A.#and#Keesing,#F.,#2014.#CoFinfection#of#blacklegged#ticks#with# Babesia#microti#and#Borrelia#burgdorferi#is#higher#than#expected#and#acquired#from#small# mammal#hosts.#PLoS#One.#9(6):#p.e99348.# # 22.#Hersh#M.H.,#Tibbetts,#M.,#Strauss,#M.,#Ostfeld,#R.S.,#Keesing,#F.,#2012.#Reservoir# Competence#of#Wildlife#Host#Species#for#Babesia#microti.#Emerging#Infectious#Diseases.# 18(12):#1951F1957.## # 23.#Humphrey,#P.#T.,#Caporale,#D.#A.,#Brisson,#D.,#2010.#Uncoordinated#phylogeograph#of# Borrelia#burgdorferi#and#its#tick#vector,#Ixodes#scapularis.#Evolution.#64(9):#2653–2663.## # 24.#Johnson,#L.,#Wilcox,#S.,#Mankoff,#J.#and#Stricker,#R.B.,#2014.#Severity#of#chronic#Lyme# disease#compared#to#other#chronic#conditions:#a#quality#of#life#survey.#PeerJ.#2:#p.e322.# Jones,#K.E.,#Patel,#N.G.,#Levy,#M.A.,#Storeygard,#A.,#Balk,#D.,#Gittleman,#J.L.#and#Daszak,#P.,# 2008.#Global#trends#in#emerging#infectious#diseases.#Nature,#451(7181),#pp.990F993.# # 25.#Joseph,#J.#T.,#Roy,#S.#S.,#Shams,#N.,#Visintainer,#P.,#Nadelman,#R.#B.,#Hosur,#S.,#2011.# Babesiosis#in#Lower#Hudson#Valley,#New#York,#USA.#Emerging#Infectious#Diseases.#17:# 843–7.## # 26.#Khatchikian,#C.E.,#Prusinski,#M.A.,#Stone,#M.,#Backenson,#P.B.,#Wang,#I.N.,#Foley,#E.,# Seifert,#S.N.,#Levy,#M.Z.#and#Brisson,#D.,#2015.#Recent#and#rapid#population#growth#and# range#expansion#of#the#Lyme#disease#tick#vector,#Ixodes#scapularis,#in#North# America.#Evolution.#69(7):#1678F1689.# # 27.#Keesing,#F.,#Hersh,#M.H.,#Tibbetts,#M.,#McHenry,#D.J.,#Duerr,#S.,#Brunner,#J.,#Killilea,#M.,# LoGiudice,#K.,#Schmidt,#K.A.#and#Ostfeld,#R.S.,#2012.#Reservoir#competence#of#vertebrate# hosts#for#Anaplasma#phagocytophilum.#Emerging#infectious#diseases.#18(12):#2013.# # 28.#Kowalski,#T.J.,#Jobe,#D.A.,#Dolan,#E.C.,#Kessler,#A.,#Lovrich,#S.D.#and#Callister,#S.M.,# 2015.#The#emergence#of#clinically#relevant#babesiosis#in#southwestern#Wisconsin.#WMJ.# 114(4):#152F157.# #

32 29.#Kugeler,#K.J.,#Farley,#G.M.,#Forrester,#J.D.#and#Mead,#P.S.,#2015.#Geographic# Distribution#and#Expansion#of#Human#Lyme#Disease,#United#States.#Emerging#infectious# diseases.#21(8):#1455F1457.# # 30.#Lee,#X.,#D.#Coyle,#D.#Johnson,#M.#Murphy,#M.#McGeehin,#R.#Murphy,#K.#Raffa,#and#S.# Paskewitz.#2014.#Prevalence#of#Borrelia#burgdorferi#and#Anaplasma#phagocytophilum#in# Ixodes#scapularis#(Acari:#Ixodidae)#nymphs#collected#in#managed#red#pine#forests#in# Wisconsin.#Journal#of#Medical#Entomology.#51:#694F701.# # 31.#Leighton,#P.A.,#Koffi,#J.K.,#Pelcat,#Y.,#Lindsay,#L.R.#and#Ogden,#N.H.,#2012.#Predicting#the# speed#of#tick#invasion:#an#empirical#model#of#range#expansion#for#the#Lyme#disease#vector# Ixodes#scapularis#in#Canada.#Journal#of#Applied#Ecology.#49(2):#457F464.# # 32.#Levi,#Taal,#et#al.#"Quantifying#dilution#and#amplification#in#a#community#of#hosts#for# tickFborne#pathogens."#Ecological#Applications#26.2#(2016):#484F498.# # 33.#Linzey,#A.V.,#Matson,#J.#&#Timm,#R.#2008.#Peromyscus#leucopus.#The#IUCN#Red#List#of# Threatened#Species#2008.## # 34.#LoGiudice,#K.,#Ostfeld,#R.#S.,#Schmidt,#K.#A.,#&#Keesing,#F.,#2003.#The#ecology#of# infectious#disease:#effects#of#host#diversity#and#community#composition#on#Lyme#disease# risk.#Proceedings#of#the#National#Academy#of#Sciences.#100(2):#567F571.# # 35.#McCoy,#K.D.,#Léger,#E.#and#Dietrich,#M.,#2013.#Host#specialization#in#ticks#and# transmission#of#tickFborne#diseases:#a#review.#Frontiers#in#cellular#and#infection# microbiology.#3.# # 36.#McNabb,#S.J.,#Jajosky,#R.A.,#HallFBaker,#P.A.,#Adams,#D.A.,#Sharp,#P.,#Worshams,#C.,# Anderson,#W.J.,#Javier,#A.J.,#Jones,#G.J.,#Nitschke,#D.A.#and#Rey,#A.,#2008.#Summary#of# notifiable#diseasesFFUnited#States,#2006.#MMWR.#Morbidity#and#mortality#weekly# report,#55(53):#1F92.# # 37.#Murphy,#D.S.,#Lee,#X.,#Larson,#S.R.,#Johnson,#D.K.H.,#Loo,#T.#and#Paskewitz,#S.M.,#2017.# Prevalence#and#Distribution#of#Human#and#Tick#Infections#with#the#Ehrlichia#murisFLike# Agent#and#Anaplasma#phagocytophilum#in#Wisconsin,#2009–2015.#VectorFBorne#and# Zoonotic#Diseases.#17(4):#229F236.# # 38.#Ogden,#N.H.,#StFOnge,#L.,#Barker,#I.K.,#Brazeau,#S.,#BigrasFPoulin,#M.,#Charron,#D.F.,# Francis,#C.M.,#Heagy,#A.,#Lindsay,#L.,#Maarouf,#A.#and#Michel,#P.,#2008.#Risk#maps#for#range#

33 expansion#of#the#Lyme#disease#vector,#Ixodes#scapularis,#in#Canada#now#and#with#climate# change.#International#journal#of#health#geographics,#7(1),#p.24.# # 39.#Ogden,#N.H.,#Radojevic,#M.,#Wu,#X.,#Duvvuri,#V.R.,#Leighton,#P.A.#and#Wu,#J.,#2014.# Estimated#effects#of#projected#climate#change#on#the#basic#reproductive#number#of#the# Lyme#disease#vector#Ixodes#scapularis.#Environmental#health#perspectives.#122(6):#631.# # 40.#Oliveira,#M.R.#and#Kreier,#J.P.,#1979.#Transmission#of#Babesia#microti#using#various# species#of#ticks#as#vectors.#Journal#of#Parasitology.#65(5):#816F817.# # 41.#Orloski,#K.A.,#Hayes,#E.B.,#Campbell,#G.L.#and#Dennis,#D.T.,#2000.#Surveillance#for#Lyme# disease—United#States,#1992–1998.###MMWR.#Morbidity#and#mortality#weekly# report,#CDC#Surveillance#Summaries.#1F11.# # 42.#Piesman,#J.#and#Eisen,#L.,#2008.#Prevention#of#tickFborne#diseases.#Annual#Review#of# Entomology.#53:#323F343.# # 43.#Piesman,#J.,#Mather,#T.N.,#Sinsky,#R.J.#and#Spielman,#A.,#1987.#Duration#of#tick# attachment#and#Borrelia#burgdorferi#transmission.#Journal#of#clinical#microbiology,#25(3),# pp.557F558.# # 44.#Prusinski,#M.#A.,#et#al.#"Prevalence#of#Borrelia#burgdorferi#(spirochaetales:# Spirochaetaceae),#anaplasma#phagocytophilum#(:#Anaplasmataceae),#and# Babesia#microti#(Piroplasmida:#Babesiidae)#in#Ixodes#scapularis#(Acari:#Ixodidae)#collected# from#recreational#lands#in#the#hudson#valley#region,#new#york#state."#Journal#of#medical# entomology#51.1#(2014):#226F236.# # 45.#Qiu,#W.G.,#Dykhuizen,#D.E.,#Acosta,#M.S.#and#Luft,#B.J.,#2002.#Geographic#uniformity#of# the#Lyme#disease#spirochete#(Borrelia#burgdorferi)#and#its#shared#history#with#tick#vector# (Ixodes#scapularis)#in#the#northeastern#United#States.#Genetics.#160(3):#833F849.# # 46.#Rand,#P.,#C.#Lubelczyk,#G.#Lavigne,#S.#Elias,#M.#Holman,#E.#Lacombe,#R.#Smith.#2003.# Deer#density#and#the#abundance#of#Ixodes#scapularis#(Acari:#Ixodidae).#Journal#of#Medical# Entomology.#40(2):#179F184.# # 47.#Rooney,#T.P.,#2009.#High#whiteFtailed#deer#densities#benefit#graminoids#and# contribute#to#biotic#homogenization#of#forest#groundFlayer#vegetation.#Plant#Ecology.# 202:103F111.# #

34 48.#Schmidt,#K.A.#and#Ostfeld,#R.S.,#2001.#Biodiversity#and#the#dilution#effect#in#disease# ecology.#Ecology.#82(3):#609F619.# # 49.#Scott,#J.#D.,#Scott,#C.#M.,#&#Anderson,#J.#F.#(2014).#The#establishment#of#a#blacklegged# tick#population#by#migratory#songbirds#in#Ontario,#Canada.#Journal#of#Veterinary#Science# &#Medicine.#2(5).# # 50.#Steiner,#F.E.,#Pinger,#R.R.,#Vann,#C.N.,#Grindle,#N.,#Civitello,#D.,#Clay,#K.#and#Fuqua,#C.,# 2008.#Infection#and#coFinfection#rates#of#Anaplasma#phagocytophilum#variants,#Babesia# spp.,#Borrelia#burgdorferi,#and#the#rickettsial#endosymbiont#in#Ixodes#scapularis#(Acari:# Ixodidae)#from#sites#in#Indiana,#Maine,#Pennsylvania,#and#Wisconsin.#Journal#of#medical# entomology.#45(2):289F297.# # 51.#Turtinen,#L.W.,#Kruger,#A.N.#and#Hacker,#M.M.,#2015.#Prevalence#of#Borrelia# burgdorferi#in#adult#female#ticks#(Ixodes#scapularis),#Wisconsin#2010–2013.#Journal#of# Vector#Ecology.#40(1):#195F197.# # 52.#Uilenberg,#G.,#2006.#Babesia:#a#historical#overview.#Veterinary#Parasitology.#138:#3F10.## # 53.#Vourc’h,#G.,#Abrial,#D.,#Bord,#S.,#Jacquot,#M.,#Masseglia,#S.,#Poux,#V.,#Pisanu,#B.,#Bailly,# X.#and#Chapuis,#J.L.,#2016.#Mapping#human#risk#of#infection#with#Borrelia#burgdorferi# sensu#lato,#the#agent#of#Lyme#borreliosis,#in#a#periurban#forest#in#France.#Ticks#and#tickF borne#diseases.#7(5):#644F652.# # 54.#Zoonotic#Diseases.#Centers#for#Disease#Control#and#Prevention,#18#Oct.#2013.## # # #

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