r.t. trout 1 , c.d. steelman 1 , a.l. szalanski 1 , k. kvamme 2 , and p.c. williamson 3
DESCRIPTION
Genetic & Spatial Identification of Borrelia & Rickettsia Pathogens from Arkansas Ticks & Mammals. R.T. Trout 1 , C.D. Steelman 1 , A.L. Szalanski 1 , K. Kvamme 2 , and P.C. Williamson 3 1 University of Arkansas Entomology • Fayetteville, Arkansas 72701 - PowerPoint PPT PresentationTRANSCRIPT
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