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 HealthUniversity of North Florida

Topics for Discussion

Evidence of Borrelia and Babesia species in vertebrates and ticksPrimarily molecular dataGeographic distributionSpecies distribution and infection

prevalenceVertebrates: mammals and reptilesTicks

Borreliosis and babesiosis case reportsSummary of present data

Rocky Mountain Spotted FeverHuman Monocytic Ehrlichiosis (HME)Human Anaplasmosis (formerly HGE)Lyme BorreliosisRelapsing Fever Borreliosis?Human Babesiosis? Babesiosis?

Major Tick-Borne Diseases in the Southeastern USA

Lyme diseaseMost common vector-borne

disease in U.S.Over 23,700 cases reported in

2002

Lyme Borreliosis

Lyme Disease: Major ManifestationsSkin:

Erythema migrans rash; later disseminated rash

Musculoskeletal: Myalgias, arthralgias, recurrent arthritis in

large jointsNeurologic:

Headache, Bell’s palsy, concentrationCardiacConstitutional:

Flu-like symptoms, malaise, fatigue

Geographic Distribution of LD in USA

Lyme Disease in Florida

*2002 data are provisional; standard case definition began in 1991.

Reported Cases of Lyme Disease in Florida, 1985-2002*

(http://www.lyme.org/resources/stats/2000_04.html)

2 0 1 0

6 7

35

24

30 28

17

55 56

71

5954

43

79

0

10

20

30

40

50

60

70

80

90

1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002

Nu

mb

er

of

Ca

se

s

LD Spirochete, Borrelia burgdorferi

Lyme borreliosis group pathogensAt least 11 species (B. burgdorferi s.l.)

3 confirmed pathogensB. 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 MethodsSite and habitat selection

Materials and MethodsVertebrate and tick sampling

Methods: DNA TestingDNA extractions

Host-seeking adult ticksRodent ear tissueRaccoon, rodent, lizard blood (“Nobutos”)

Qiagen Dneasy Tissue kitEpicentre Masterpure kit

Screening PCR for B. burgdorferi s.l. flagellin (flaB)389-bp. nested PCR productDNA sequencing

Borrelia burgdorferi flagellin DNA among host-seeking adult ticks collected in northern Florida, 1999-2005

No. positive/County Site Tick species No. tested Prevalence

Duval Univ. North Florida lone star tick 4/118 3.4%

Lake Alexander Springs lone star tick 1/27 3.7%

River Forest lone star tick 0/35 0%

St. Johns Guana River lone star tick 0/63 0%

Species total lone star tick 19/622 3.1%

Duval Univ. North Florida blacklegged tick 5/108 4.6%

St. Johns Guana River blacklegged tick 5/108 4.6%

Species total blacklegged tick 10/216 4.6%(Clark 2004. J. Clin. Microbiol. 42: 5076-5086)

Results:

Prevalence of Borrelia burgdorferi flagellin DNA among small mammals collected in Florida, 1999.

Results: vertebrate sampling and testing

No. of PCR positive animals/no. tested (%) of each species

Virginia Flying Golden Rice Cotton Cotton WoodCounty Site opossum squirrel† mouse† rat mouse rat rat TotalDuval UNF 1/1 1/1 2/2 ** 22/25 2/2 ** 28/31

(100) (100) (100) (88) (100) (90)St. Johns Guana 0/1 ** 1/1 3/3 9/10 9/13 1/2 23/30

River (0) (100) (100) (90) (69) (50) (77)Total 1/2 1/1 3/3 3/3 31/34 11/15 1/2 51/61

(50) (100) (100) (100) (91) (73) (50) (84)

† = New host record(Clark 2004. J. Clin. Microbiol. 42: 5076-5086)

Additional data:Duval Big Talbot Island Raccoon

0/17

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

67

99

99

72

52

74

63

67

71

74

80

5667

91

40

18

52

25

54

56

0.02

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).

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

Broad-headed skink

 Brown anole

 Fence lizard

 Glass lizard

 Scrub lizard

 Green anole

 Ground skink

 Race-runner

Five-lined 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)

Results: lizard sampling and testing

(Clark et al. 2005. Appl. Environ. Microbiol. 71: 2616-2625)

FL204.As.Florida

FL71.Ac.Florida

FL66.Su.Florida

FL121.Sw.Florida

SC17-3I.s.N.SouthCarolina

FL139.Sw.Florida

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

B.b.s.s.B31.Is.NY.USA

B.sp.SCW-30h.Im.SC.USA

B.andersonii.21038.Id.USA

SC194.Sl.SouthCarolina

B.andersonii.19857.Sf.USA

B.andersonii.SI-10.Is.GA.USA

FL118.Su.Florida

FL187.Sl.Florida

SC89.Ei.SouthCarolina

SC170.El.SouthCarolina

SC152.Ac.SouthCarolina

B.bissettii.25015.Is.NY.USA

FL60.Sl.Florida

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

B.garinii.Ip90.Ip.Russia

B.sinica.CMN3.China

B.lusitaniae.PotiB2.Ir.Portugal

B.valaisiana.VS116.Ir.Switzerland

B.afzelii.ACA1.Hs.Sweden

B.lonestari.Aa.TX.USA

63

19

20

85

45

23

100

34

3972

18

9

73

38

36

72

89

85

37

43

65

95

17

25

47

23

39

64

0.02

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)

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?

Relapsing Fever Borreliosis

Environmental risk index (ERI*) data for relapsing fever group Borrelia and adult lone star ticks at localities in Florida, March 1999-September 2000.

Mean no. RFG Mean no. RFG BorreliaBorrelia ERI ERI

LocalityLocality ticks/hr. ticks/hr. prevalenceprevalence†† ERIERI risk ratiorisk ratio

Alexander SpringsAlexander Springs 22.6 22.6 0% 0% 0 0 ---- ----

Guana River WMAGuana River WMA 55.1 55.1 4.8% 4.8% 2.62.6 14.7 14.7

Juniper SpringsJuniper Springs 82.7 82.7 0% 0% 0 0 ----- -----

O’Leno State ParkO’Leno State Park 92.9 92.9 2.8% 2.8% 2.6 2.6 14.7 14.7

River ForestRiver Forest 19.4 19.4 0% 0% 0 0 ----- -----

Stephen Foster S.P.Stephen Foster S.P. 15 15 0% 0% 0 0 ----- -----

Tomoka State ParkTomoka State Park 37 37 2.2% 2.2% 0.810.81 4.5 4.5

Univ. North FLUniv. North FL 7.3 7.3 2.5% 2.5% 0.180.18 ReferentReferent

TotalTotal 27.5 27.5 2.0% 2.0% 0.550.55 ----- -----

*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)

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.parkeri

B.turicatae

Borr.TXW1

B.hermsii

B.coriaceae

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

AA4POOLFL100

42

99

50

74

57

99

37

55

76

61

64

100

9

40

61

78

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 ErythemaMigrans

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 resultsGenetic 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 MidwestBabesia microti most common agent in U.S.Babesia divergens in EuropeWA1 (B. divergens-like) in Pacific NorthwestMO1 (B. divergens-like) in Missouri

B. microti Life Cycle

B. microti image obtained from: http://medstat.med.utah.edu/parasitology/bmicrot.html

Babesiosis: Clinical FeaturesMany infections probably asymptomaticDisease manifestations

FeverChillsSweatingMyalgiasFatigueHepatosplenomegalyHemolytic anemia

Incubation period: 1 – 4 weeksDisease more severe in immunocompromised

Asplenic, elderly, HIV-infected

Babesiosis: Clinical FeaturesDiagnosis:

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 (cotton rats 8/15 = 53%; other species 0/54)Raccoons 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 ticksUNF 17/118 = 14.4%Guana River Site 11/118 = 9.3%

Lone star ticks 8/198 = 4%American dog ticks 3/81 = 4%Ixodes affinis 4/54 = 7%Gulf Coast ticks 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 FloridaBackground

Tampa area

Epidemiology

Confirmed tick species

DNA testing: tick and human blood

Results: B. microti strain? Other species?

Potentially pathogenic Borrelia and Babesia species

Human dataRare/unusual events?

Questions and answers

Clues to scope of risk in FL/Southeast

Research neededPresence, distribution, prevalence of tick-borne pathogens

Genetic data on strains from vertebrates, ticks

*More human case data

CONCLUSIONS