Borrelia and Babesia in wild vertebrates, ticks, and humans in Florida Kerry L.
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Transcript Borrelia and Babesia in wild vertebrates, ticks, and humans in Florida Kerry L.
Borrelia and Babesia in wild vertebrates,
ticks, and humans in Florida
Kerry L. Clark, M.P.H., Ph.D.
Associate Professor of Epidemiology
Department of Public Health
University of North Florida
Topics for Discussion
Evidence of Borrelia and Babesia species in
vertebrates and ticks
Primarily molecular data
Geographic distribution
Species distribution and infection prevalence
Vertebrates: mammals and reptiles
Ticks
Borreliosis and babesiosis case reports
Summary of present data
Major Tick-Borne Diseases in the
Southeastern USA
Rocky Mountain Spotted Fever
Human Monocytic Ehrlichiosis (HME)
Human Anaplasmosis (formerly HGE)
Lyme Borreliosis
Relapsing Fever Borreliosis?
Human Babesiosis?
Lyme Borreliosis
Lyme disease
Most common vector-borne disease in
U.S.
Over 23,700 cases reported in 2002
Lyme Disease: Major Manifestations
Skin:
Erythema migrans rash; later disseminated rash
Musculoskeletal:
Myalgias, arthralgias, recurrent arthritis in large
joints
Neurologic:
Headache, Bell’s palsy, concentration
Cardiac
Constitutional:
Flu-like symptoms, malaise, fatigue
Geographic Distribution of LD in USA
Lyme Disease in Florida
Reported Cases of Lyme Disease in Florida,
1985-2002*
(http://www.lyme.org/resources/stats/2000_04.html)
90
79
80
71
Number of Cases
70
60
55
50
56
59
54
43
40
35
30
30
28
24
17
20
6
10
2
0
1
7
0
0
1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002
*2002 data are provisional; standard case definition began in 1991.
LD Spirochete, Borrelia burgdorferi
Lyme borreliosis group pathogens
At least 11 species (B. burgdorferi s.l.)
3 confirmed pathogens
B. burgdorferi sensu stricto (USA)
B. garinii (Europe/Asia)
B. afzelii (Europe/Asia)
Other pathogenic species?
B. bissettii, B. andersonii in USA
LD Vector: Blacklegged (Deer) Tick,
Ixodes scapularis
Geographic Distribution of LD Vectors
B. burgdorferi Life Cycle
?
?
LD Seasonal Distribution (USA overall)
LD Seasonal Risk
Note: These estimates are based primarily on data from the northeastern USA
Materials and Methods
Site and habitat selection
Materials and Methods
Vertebrate and tick sampling
Methods: DNA Testing
DNA extractions
Host-seeking adult ticks
Rodent ear tissue
Raccoon, rodent, lizard blood (“Nobutos”)
Qiagen Dneasy Tissue kit
Epicentre Masterpure kit
Screening PCR for B. burgdorferi s.l. flagellin (flaB)
389-bp. nested PCR product
DNA sequencing
Results:
Borrelia burgdorferi flagellin DNA among host-seeking adult ticks collected
in northern Florida, 1999-2005
County
Site
Duval
lone star tick
4/118
3.4%
lone star tick
1/27
3.7%
lone star tick
0/35
0%
lone star tick
0/63
0%
Species total
lone star tick
19/622
3.1%
Duval
blacklegged tick
5/108
4.6%
blacklegged tick
5/108
4.6%
10/216
4.6%
Lake
Univ. North Florida
Tick species
No. positive/
No. tested
Prevalence
Alexander Springs
River Forest
St. Johns
Guana River
Univ. North Florida
St. Johns
Guana River
Species total
blacklegged tick
(Clark 2004. J. Clin. Microbiol. 42: 5076-5086)
Results: vertebrate sampling and testing
Prevalence of Borrelia burgdorferi flagellin DNA among small
mammals collected in Florida, 1999.
No. of PCR positive animals/no. tested (%) of each species
Virginia Flying
County
Site opossum squirrel†
Duval
UNF
1/1
1/1
(100)
(100)
St. Johns Guana
0/1
**
River
(0)
Total
1/2
1/1
(50)
(100)
Golden
mouse†
2/2
(100)
1/1
(100)
3/3
(100)
†
= New host record
(Clark 2004. J. Clin. Microbiol. 42: 5076-5086)
Additional data:
Duval Big Talbot Island
Raccoon
0/17
Rice Cotton Cotton Wood
rat mouse
rat
rat
Total
**
22/25
2/2
**
28/31
(88)
(100)
(90)
3/3
9/10
9/13 1/2
23/30
(100) (90)
(69) (50)
(77)
3/3
31/34 11/15 1/2 51/61
(100) (91)
(73) (50)
(84)
Neighbor-Joining tree based on
390-bp of the flagellin gene
amplified from Florida small
mammals and ticks. The tree was
rooted with relapsing fever group
Borrelia spp. Bootstrap values
are percentages of 1,000
replications. Florida B.
burgdorferi strains were 98-99%
similar to other USA strains of
either B. burgdorferi sensu stricto
or B. bissettii (Clark 2004. J. Clin.
Microbiol. 42: 5076-5086).
72
99
FLGR11.Is(FL)
AA15pool(FL)
RET.KC9.On(FL)
FLTP1.Is(FL)
56
AA4Pool(FL)
FLCL3.Ia(FL)
54
RET.KC14.Gv(FL)
25 RET.KC1.Pg(FL)
FLNF26.Ia(FL)
52
B.b.ss.JD1.Is(MA)
18
RET.KC19.Dv(FL)
40
B.b.ss.SCI2.Pg(GA)
91
Bb.ss.SM1.Pg(GA)
B.b.ss.B31.Is(NY)
Bb.ss.HB19.HP(US)
56
67
B.b.ss.MI2.Pg(FL)
B.a.MOK3a.Id(MO)
80
B.a.21123.Id(US)
99
67 B.a.19857.Sf(US)
B.b.25015.Is(NY)
RET.FL42.Sh(FL)
67
74
RET.FL27.Sh(FL)
B.b.MI9.Pg(FL)
71
B.b.SCGT8a.I.m./N.f.(SC)
63
B.b.SCGT10.Nf(SC)
74
B.sp.MI8.Sh(FL)
52
B.garinii
B.afzelii
B.lonestari.Aa(TX)
B.miyamotoi
Results: lizard sampling and testing
Prevalence of B. burgdorferi s.l. flagellin (flaB) gene DNA among lizards from
Florida and South Carolina
Number of PCR positive animals/number tested (%) of each species
Broadheaded
skink
Brown
anole
Fence
lizard
Glass
lizard
Scrub
lizard
Green
anole
Ground
skink
Racerunner
Fivelined
skink
Gecko
Total
Florida
8/18
(44)
2/4
(50)
3/9
(33)
1/1
(100)
6/14
(43)
7/17
(41)
5/7
(71)
2/11
(18)
3/8
(38)
0/3
(0)
37/92
(40)
South
Carolina
13/18
(72)
NT
NT
1/1
(100)
NT
22/33
(67)
1/1
(100)
NT
12/15
(80)
NT
49/68
(72)
Total
21/36
(58)
2/4
(50)
3/9
(33)
2/2
(100)
6/14
(43)
29/50
(58)
6/8
(75)
2/11
(18)
15/23
(65)
0/3
(0)
86/160
(53.8)
(Clark et al. 2005. Appl. Environ. Microbiol. 71: 2616-2625)
FL204.As.Florida
17
Unrooted neighbor joining tree
based on 389-bp of the flagellin
gene amplified from Florida and
South Carolina lizards. Bootstrap
values are percentages of 1,000
replications. Lizard B. burgdorferi
s.l. strains were ~98-99% similar
to other USA strains of B.
andersoni, B. bissettii, and B.
burgdorferi sensu stricto. B.
lonestari was included as an
outgroup (Clark et al. 2005. Appl.
Environ. Microbiol. 71: 2616-2625)
25
FL71.Ac.Florida
47
23
39
FL66.Su.Florida
FL121.Sw.Florida
SC17-3I.s.N.SouthCarolina
FL139.Sw.Florida
64
95
B.b.s.s.Tr293.Ir.Turkey
B.b.s.s.SCI2.Pg.GA.USA
FL53.Ov.Florida
B.b.s.s.MI2.Sh.FL.USA
43
65 B.b.s.s.B31.Is.NY.USA
B.sp.SCW-30h.Im.SC.USA
37
B.andersonii.21038.Id.USA
23
SC194.Sl.SouthCarolina
100
B.andersonii.19857.Sf.USA
B.andersonii.SI-10.Is.GA.USA
45
FL118.Su.Florida
85
FL187.Sl.Florida
85
SC89.Ei.SouthCarolina
20
SC170.El.SouthCarolina
19
SC152.Ac.SouthCarolina
63
B.bissettii.25015.Is.NY.USA
9
FL60.Sl.Florida
89
73
72
36
18
38
B.bissettii.SCGT8a.Im.SC.USA
B.bissettii.MI8.Sh.FL.USA
FL203.As.Florida
B.bissettii.DN127.Ip.CA.USA
B.japonica.HO14.Io.Japan
72
B.garinii.Ip90.Ip.Russia
39
34
B.sinica.CMN3.China
B.lusitaniae.PotiB2.Ir.Portugal
B.valaisiana.VS116.Ir.Switzerland
B.afzelii.ACA1.Hs.Sweden
B.lonestari.Aa.TX.USA
0.02
Relapsing Fever Borreliosis
Emergence of Lyme-like illness in eastern
USA (STARI: Master’s disease?)
Associated with bites of lone star ticks
RFG Borrelia spirochetes found in lone
star ticks via DNA tests
Named Borrelia barbouri /lonestari
Responsible for cryptic Lyme-like illnesses
in southern USA?
Environmental risk index (ERI*) data for relapsing fever group Borrelia and
adult lone star ticks at localities in Florida, March 1999-September 2000.
Locality
Alexander Springs
Guana River WMA
Juniper Springs
O’Leno State Park
River Forest
Stephen Foster S.P.
Tomoka State Park
Univ. North FL
Total
Mean no.
ticks/hr.
22.6
55.1
82.7
92.9
19.4
15
37
7.3
27.5
RFG Borrelia
prevalence†
0%
4.8%
0%
2.8%
0%
0%
2.2%
2.5%
2.0%
ERI
0
2.6
0
2.6
0
0
0.81
0.18
0.55
ERI
risk ratio
---14.7
----14.7
--------4.5
Referent
-----
*ERI = mean no. ticks encountered per hr. of collection effort x infection prevalence
† Tick infection status with Borrelia spirochetes determined by nested PCR DNA tests
(Clark 2004. J. Clin. Microbiol. 42: 5076-5086)
42
Neighbor-Joining tree based on
350-bp of the flagellin gene
amplified from Florida lone star
ticks. The tree was rooted with B.
burgdorferi B31 and Florida lone
star tick sample A.a. 4 pool.
Bootstrap values are percentages
of 1,000 replications. Florida B.
lonestari flagellin sequences were
more than 99% similar to B.
lonestari sequences in GenBank
(Clark 2004. J. Clin. Microbiol. 42:
5076-5086).
99
55
76
37
61
99
64
0.02
B.parkeri
B.turicatae
Borr.TXW1
B.hermsii
B.coriaceae
B.anserina
B.sp.Spain
57
B.hispanica
B.crocidurae
B.recurrentis
74
50
B.duttonii
B.miyamotoi
B.l.TX
9 B.l.TN
B.l.NJ
100
B.l.aa1
40
AA207FL
B.l.NC.MD
61
AA15POOLFL
78 AA18POOLFL
AA97FL
AA115FL
B.b.B31
100
AA4POOLFL
Geographic distribution of Borrelia spp. in ticks in Florida
= B. burgdorferi positive site
= B. lonestari positive site
Human Lyme borreliosis case in Florida
Adult female residing in JAX, FL
Tick bite in March 2003
Rash onset in April 2003
No travel outside JAX, FL/St. Mary’s GA region
Consultation in late May
Disseminated rash; no remarkable symptoms
Antibody testing
BSK blood culture
PCR testing
Examples of
Erythema
Migrans
Florida Lyme
borreliosis patient
PCR/DNA Sequence Analysis for human
Lyme borreliosis case in Florida
B.b.s.l. flaB (350-bp)
100% with B. andersonii strains (from lizards, I.
dentatus, others)
B.b.s.l. ospA (320-bp)
100% with FL 121 (scrub lizard from FL)
99.7% with SCW-30h (I.m. from bird in SC)
~96% with B.b. s.s. strains
B.b.s.l. p66 (275-bp)
100% with B. bissettii 25015
98% with B. bissettii in rodents from FL
Human Lyme borreliosis case in Florida
Treatment
Late signs/symptoms
Follow up
Discussion
Antibody test result
PCR/DNA sequence results
Genetic heterogeneity?
Multiple infection?
Human Babesiosis
Babesiosis
Malaria-like syndrome caused by Babesia spp.
(piroplasms) protozoans
First recognized in U.S. 1968
Hundreds of cases reported since, mostly in
Northeast, Upper Midwest
Babesia microti most common agent in U.S.
Babesia divergens in Europe
WA1 (B. divergens-like) in Pacific Northwest
MO1 (B. divergens-like) in Missouri
B. microti Life Cycle
B. microti image obtained from:
http://medstat.med.utah.edu/parasitology/bmicrot.html
Babesiosis: Clinical Features
Many infections probably asymptomatic
Disease manifestations
Fever
Chills
Sweating
Myalgias
Fatigue
Hepatosplenomegaly
Hemolytic anemia
Incubation period: 1 – 4 weeks
Disease more severe in immunocompromised
Asplenic, elderly, HIV-infected
Babesiosis: Clinical Features
Diagnosis:
Microscopic examination of thick/thin blood
smears
Antibody detection (indirect fluorescent
antibody (IFA) test
PCR for 18S rRNA gene (SSrDNA)
Treatment:
Clindamycin plus quinine
Atovaquone plus azithromycin
Babesia Research in Vertebrates and Ticks
Screening PCR:
18S SSU rRNA gene nested PCR
Primers BAB1/4 (~238-bp) + BAB2/3
(~154-bp) (Persing et al. 1992)
Confirmatory PCRs
Other 18S SSU rRNA gene nested PCRs
(500-1,000-bp)
Beta tubulin gene primers (modified from
Zamoto et al. 2004; 500-700-bp)
DNA Sequence Analysis
BAB1-4 PCR Prevalence in vertebrates and ticks in FL and SC
Small mammals:
Rodents
Raccoons
(cotton rats 8/15 = 53%; other species 0/54)
15/17 = 88%
Lizards:
All species 85/150 = 57% (7 genera and 8 of 10 species tested)
Anolis, Cnemidophorus, Eumeces, Hemidactylus, Ophisaurus,
Sceloporus, Scincella spp.
Ticks:
Blacklegged ticks
UNF
17/118 = 14.4%
Guana River Site 11/118 = 9.3%
Lone star ticks
American dog ticks
Ixodes affinis
Gulf Coast ticks
8/198 = 4%
3/81 = 4%
4/54 = 7%
1/24 = 4%
Babesia Sequence Data—Summary
18S SSU rRNA gene (154-238-bp)
Blacklegged, dog, Gulf Coast ticks, I. affinis; cotton
rats; lizards
~99% similar to B. microti s.s. (e.g. Gray strain)
Raccoons
~99% with MA USA raccoon strain of B. microti
Beta-tubulin gene (700-bp)
Cotton rats
~99% similar to B. microti s.s.
Raccoons
~99% with MA USA raccoon strain of B. microti
Human case of babesiosis in Florida
Background
Tampa area
Epidemiology
Confirmed tick species
DNA testing: tick and human blood
Results: B. microti strain? Other species?
CONCLUSIONS
Potentially pathogenic Borrelia and Babesia species
Human data
Rare/unusual events?
Questions and answers
Clues to scope of risk in FL/Southeast
Research needed
Presence, distribution, prevalence of tick-borne
pathogens
Genetic data on strains from vertebrates, ticks
*More human case data