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).
FLGR11.Is(FL)
AA15pool(FL)
RET.KC9.On(FL)
FLTP1.Is(FL)
AA4Pool(FL)
FLCL3.Ia(FL)
RET.KC14.Gv(FL)
RET.KC1.Pg(FL)
FLNF26.Ia(FL)
B.b.ss.JD1.Is(MA)
RET.KC19.Dv(FL)
B.b.ss.SCI2.Pg(GA)
Bb.ss.SM1.Pg(GA)
B.b.ss.B31.Is(NY)
Bb.ss.HB19.HP(US)
B.b.ss.MI2.Pg(FL)
B.a.MOK3a.Id(MO)
B.a.21123.Id(US)
B.a.19857.Sf(US)
B.b.25015.Is(NY)
RET.FL42.Sh(FL)
RET.FL27.Sh(FL)
B.b.MI9.Pg(FL)
B.b.SCGT8a.I.m./N.f.(SC)
B.b.SCGT10.Nf(SC)
B.sp.MI8.Sh(FL)
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)
B.parkeri
B.turicatae
Borr.TXW1
B.hermsii
B.coriaceae
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).
B.anserina
B.sp.Spain
B.hispanica
B.crocidurae
B.recurrentis
B.duttonii
B.miyamotoi
B.l.TX
B.l.TN
B.l.NJ
B.l.aa1
AA207FL
B.l.NC.MD
AA15POOLFL
AA18POOLFL
AA97FL
AA115FL
B.b.B31
AA4POOLFL
0.02
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