R.T. TroutR.T. Trout11, C.D. Steelman, C.D. Steelman11, A.L. Szalanski, A.L. Szalanski11, , K. KvammeK. Kvamme22, and P.C. Williamson, and P.C. Williamson33

11University of Arkansas Entomology • Fayetteville, Arkansas 72701University of Arkansas Entomology • Fayetteville, Arkansas 7270122University of Arkansas Anthropology • Fayetteville, Arkansas 72701University of Arkansas Anthropology • Fayetteville, Arkansas 7270133University of North Texas Health Science Center • Ft. Worth, Texas University of North Texas Health Science Center • Ft. Worth, Texas

76107-269976107-2699

Genetic & Spatial Identification of Genetic & Spatial Identification of Borrelia Borrelia & & Rickettsia Rickettsia PathogensPathogensfrom Arkansas Ticks & Mammalsfrom Arkansas Ticks & Mammals

Tick - Host Associations

Larvae

NymphAdult

• Several Species• Variable Symptoms • Infection variable • Cases

– Misdiagnosed– Under reported

Borrelia Pathogens

Ixodes scapularis Amblyomma americanum

B. burgdorferi

B. lonestari

• Spotted fevers• Several Species• Variable Symptoms

– Fever, edema, spreading rash

• Infection variable • Cases

Dermacentor variabilis Ixodes scapularisAmblyomma americanum

Rickettsia Pathogens

Arkansas Tick-borne Diseases• Changing State

– Fragmented habitat– top-10 growing

metropolitan areas (Anonymous 2001)

• 1996: military cases – R. rickettsii– R. typhus– B. burgdorferi

(Warner et al. 1996)

Borrelia in Arkansas

26 cases of LD in

Arkansas, since 2002

Rickettsia in Arkansas

231 cases of RMSF

in 45 Ctys., since 2002

ObjectivesObjectives

• Genetically identify bacteria in Arkansas by examining ticks & hosts using molecular techniques

– Borrelia pathogens – Rickettsia pathogens

• Identify potential risk areas using geographic information systems

Outline

• General Methods for the Project

• Results–Tick Collections

–Borrelia from Ticks & Hosts

–Rickettsia from Ticks

–Co-infection

• Summary & Future Direction

Methods: Tick CollectionsLyme disease risk increases in areas with

Ab positive canines & ticks on white-tailed deer (Daniels et al. 1993)

Tick DNA- Ethanol Vials:

Qiagen DNEasy Protocol

Host DNA- Whatman FTA Cards: Protocol

4 Reactions1 Tick DNA (16S mtDNA)

1 Borrelia (flaB)2 Rickettsia (gltA & rompB)

Methods: Extraction & PCR

Methods: PCR

• Tick DNA Reaction– 16S mt DNA ribosomal RNA

(Black & Piesman 1994)

• Borrelia genus specific reaction– flaB gene

(Barbour et al. 1996)

• Rickettsia genus specific reactions– gltA

(Kollars & Kengluecha 2001)– rompB

(Regnery et al. 1991)

16S tick

mtDNA

Methods: Sample Verification

Sequenced at UNT Health Science Center, Ft. Worth TX

GenBank MapIncidence

gltA rompB flaB

Methods: Host Distribution

247 Clinics35 Participated

(14%)

Moran’s I:Deer- 0.46 (1.19) Pet- 0.01 (0.47)

Methods: GIS Mapping

Results!

IDW2

BufferMask

Results: Tick Collections

Canines– 1498 ticks from 173 canines– 8 ± 4.0 ticks/host (range 1-100)

Felines– 84 ticks from 16 felines– 5 ± 2.4 ticks/host (range 1-17)

Deer– 1581 ticks from 250 deer– 6 ± 0.3 ticks/host (range 0-32)

Results: Tick Collections

AmblyommaA. americanum

A. maculatumD. variabilis

I. scapularisR. sanguineus

Total

Tick Species

0

180

360

540

720

900

Ticks from CaninesTicks from FelinesTicks From Deer

1200

1400

1600

Tota

l No.

Tic

ks C

oll.

AmblyommaA. americanum

A. maculatumD. variabilis

I. scapularisR. sanguineus

Total

Tick Species

0

180

360

540

720

900

Ticks from CaninesTicks from FelinesTicks From DeerTicks From Deer Tested1200

1400

1600

Tota

l No.

Tic

ks C

oll.

Results: Tick Collections

All ticks from pets were tested but

Only 56% of the ticks collected from deer

were tested

Ixodes scapularis Amblyomma americanum

Results: Borrelia

0180360540720900

N Positive N Tested

120014001600

01530456075100

125150

To

tal

No

. T

icks C

oll

.

AmblyommaA. americanum

A. maculatumD. variabilis

I. scapularisR. sangineus

Total

Tick Species

060

120180240300500650800

Results: Borrelia in Ticks

57%

Ticks from Canines

Ticks from Deer

12% 24% 24% 10% 36%24%

43%38% 33%

37%37%

23%

0% 27% 0% 34% 0%34% 20%

42%

Ticks from Felines

Results: Borrelia in Ticks

0

16

32

48

64

80

N. Positive B. burgdorferi B. lonestari

100

130

160

No

. Id

en

tifi

ed

0

4

8

12

16

20

A. Aa Am Dv Is Rs0

6

12

18

24

30100

165

230

A Aa Am Dv Is Rs

Tick Species

0

20

40

60

80

100150

205

260

Canines

Deer

Felines

Total

AmblyommaA. americanum

A. maculatumD. variabilis

I. scapularisR. sanguineus

Total

Tick Species

0

10

20

30

40

50

No

. Id

en

tifi

ed

B. burgdorferi

B.l MO2002-V1

B.l. NC/MD

B. l. TX076

B.l. Scc26

B.l. Aa115

Results: Borrelia species in ticks

613 ticks positive3 B. burgdorferi

5 B. lonestari strains22 SSc26

38 M2002-V121 Tx/0644 Nc/Md1 AA115

*487 ticks not sequenced

Canines Felines Deer

Tick Species

0

12

24

36

48

60

N Positive N Tested

100

225

350

Tota

l No.

Tic

ks C

oll.

Results: Borrelia spp. in Hosts

15%4%

25%

Host Animal

Pet Animals Deer

Host Animal

0

5

10

15

20

25

N. P

osi

tive

Sam

ple

s

B. burgdorferi

B. lonestari

Not Sequenced

Results: Borrelia in Hosts

N = 1

N = 21

N = 3

N = 14

Results: Borrelia Incidence

Ticks from Pets

(n = 355 / 1585 ticks)

Ticks from Deer

n = 259 / 695 ticks)

Results: Borrelia Incidence

Blood from Pets

(n = 9/174 hosts)

Blood from Deer

(n = 55/220 hosts)

Results: Borrelia Incidence IDW2

Legend

Pet Ticks! Positive

( Negative

Pet Ticks IDW

0 - 0.131

0.1311 - 0.2895

0.2896 - 0.448

0.4481 - 0.9997

Legend

Deer Ticks# Positive

* Negative

Deer Ticks IDW

0 - 0.1901

0.1902 - 0.492

0.4921 - 0.794

0.7941 - 0.9999

Ticks from Pets

(n = 355 / 1585 ticks)

Ticks from Deer

n = 259 / 695 ticks)

Legend

Pets! Positive

( Negative

Pet IDW

0 - 0.02585

0.02586 - 0.05937

0.05938 - 0.09288

0.09289 - 0.9865

Legend

Deer# Positive

* Negative

Deer IDW

0 - 0.1344

0.1345 - 0.3418

0.3419 - 0.5492

0.5493 - 0.9989

Results: Borrelia Incidence IDW2

Blood from Pets

(n = 9/174 hosts)

Blood from Deer

(n = 55/220 hosts)

Results: Borrelia Incidence IDW2

Map Algebra: (4 maps) * State Mask

Results: Borrelia Risk Areas

2007 Cases (n=8)Benton, Carroll, Crawford, Saline, Washington Co.

Legend

County

State B Risk Multiplied

Value

High : 4.70847

Low : 0

Results: Borrelia Risk Areas

Legend

City

County

State B Risk Multiplied

Value

High : 4.70847

Low : 0

Results: Borrelia Risk Areas

Legend

City

County

State B Risk Multiplied

Value

High : 4.70847

Low : 0

Dermacentor variabilis Ixodes scapularisAmblyomma americanum

Results: Rickettsia

Results: Rickettsia in Ticks

0180360540720900

N Positive N Tested

120014001600

01530456075100

130160

To

tal

No

. T

icks C

oll

.

A. Aa Am Dv Is Rs Total

Tick Species

0255075

100125200275350

33%

Ticks from Canines

Ticks from Deer

40%37% 32 3% 40%

9%

37%56%

0%

37%38%

26%

39%40%

0%14%

0%10%

31%

11%

Ticks from Felines

Results: Rickettsia in TicksTick

Pos/Tested (%)

R. amblyommii R. montanensis Other ?

A.42/106 (40%)

5 1 0 0

Aa268/715 (37%)

86 01 conorii

2 tarasevichiae1 rickettsii

2

Am65/166 (39%)

43 0 3 parkeri 1

Dv10/212(5%)

1 1 0 0

Is129/340 (38%)

2 12 0 19

Rs31/356(9%)

3 0 0 0

Total545/1895

(29%)140 14 7 22

Results: Rickettsia Incidence

Ticks from Pets

(n = 425/1580 ticks)

Ticks from Deer

(n = 120/315 ticks)

Results: Rickettsia Incidence

Legend

Deer Ticks

No. Pos. Genes

# 2 positive

# 1 positive

* negative

County

DrTickIDW

Value

High : 1.99623

Low : 0

Legend

Pet Ticks

No. Pos. Genes

! 2 positive

! 1 positive

( negative

County

VT Tick IDW

0 - 0.1534

0.1535 - 0.4351

0.4352 - 0.7169

0.717 - 1.996

Ticks from Pets

(n = 425/1580 ticks)

Ticks from Deer

(n = 120/315 ticks)

Results: Rickettsia Incidence

Legend

Pet Ticks

No. Pos. Genes

! 2 positive

! 1 positive

( negative

Deer Ticks

No. Pos. Genes

# 2 positive

# 1 positive

* negative

County

Risk Multiplied

Value

High : 3.98494

Low : 6.43956e-009

2007 Cases (n=121): Benton, Cleburne, Crawford, Faulkner, Hot Spring, Pope, Pulaski, Van Buren, White (>4 cases)

Results: Rickettsia Risk Areas

Legend

City

County

Risk Multiplied

Value

High : 3.98494

Low : 6.43956e-009

Results: Rickettsia Risk Areas

Legend

City

County

Risk Multiplied

Value

High : 3.98494

Low : 6.43956e-009

Results: Co-infectionTick

Rickettsia Pos/Tested

(%)

Borrelia Pos/Tested

(%)Canines Felines Deer

A.42/106 (40%)

11/110 (10%)

55

(1 B.l. & R. a.)0

Aa268/715 (37%)

178/719 (25%)

56(6 B.l. & R. a.) (1 B.l. & R. p.)

0 4

Am65/166 (39%)

51/178 (29%)

8 09

(1 B. l. & R. p.)

Dv10/212(5%)

20/213 (14%)

0 1 0

Is129/340 (38%)

252/693 (36%)

19 0 35

Rs31/356(9%)

91/362 (25%)

4 0 1

Total545/1895

(29%)613/2275

(27%)92 6 49

Results: Co-infection

Borrelia Risk Map Rickettsia Risk Map

Map Algebra: Add the 2 Maps

Legend

County

Tick Risk Multiplied

Value

High : 5.88434

Low : 0.00481565

Results: Co-infection

Results: Co-infection

Legend

City

County

Tick Risk Multiplied

Value

High : 5.88434

Low : 0.00481565

SummaryBorrelia Rickettsia

• All Tick Species Positive

• All Hosts Positive• Borrelia burgdorferi &

Borrelia lonestari

• All Tick Species Positive

• R. amblyommii, R. montanensis, R. parkeri, R. rickettsii

SummaryBorrelia Rickettsia

Future DirectionDetermine Population Structure of Aa & Is

Conduct Field Work in Designated Risk Areas

Identify the Pathogens in Humans

Determine Symptoms & Pathogenesis

Determine Vector Competency…

Identify Environmental Variables with Risk Areas

Acknowledgements

• Arkansas Veterinary Medical Association

• University of Arkansas

– Dr. D. Steelman

– Dr. A. Szalanski

– Dr. K. Loftin

– Dr. K. Kvamme

• University of North Texas

– Phillip Williamson

– Peggy Billingsly

– Janel

• Veterinary Entomology Laboratory– J. Shen

• Insect Genetics Laboratory– J. McKern– M. Tolivar

• Friends and Family