1. 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

2. 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

3. Rocky Mountain Spotted Fever Human Monocytic Ehrlichiosis (HME) Human Anaplasmosis (formerly HGE) Lyme Borreliosis Relapsing Fever Borreliosis? Babesiosis? Human Babesiosis? Major Tick-Borne Diseases in the Southeastern USA

4. Lyme disease   Most common vector-borne disease in U.S.   Over 23,700 cases reported in 2002 Lyme Borreliosis

5. 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

6. Geographic Distribution of LD in USA

7. Lyme Disease in Florida *2002 data are provisional; standard case definition began in 1991.

8. LD Spirochete, Borrelia burgdorferi

9. 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

10. LD Vector: Blacklegged (Deer) Tick, Ixodes scapularis

11. Geographic Distribution of LD Vectors

12. B. burgdorferi Life Cycle ??

13. LD Seasonal Distribution (USA overall)

14. LD Seasonal Risk Note: These estimates are based primarily on data from the northeastern USA

15. Materials and Methods Site and habitat selection

16. Materials and Methods Vertebrate and tick sampling

20. 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

21. Borrelia burgdorferi flagellin DNA among host-seeking adult ticks collected in northern Florida, 1999-2005 No. positive/ CountySiteTick species No. tested Prevalence Duval Univ. North Floridalone 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:

22. 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 Wood County Site opossum squirrel † mouse † rat mouse rat rat Total Duval 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: DuvalBig Talbot IslandRaccoon 0/17

23. 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).

25. 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 Florida8/18 (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 1/1 (100) NT22/33 (67) 1/1 (100) NT12/15 (80) NT49/68 (72) Total21/36 (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)

26. 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)

27. 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

28. 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 Borrelia ERI Mean no. RFG Borrelia ERI Locality ticks/hr.prevalence † ERIrisk ratio Alexander Springs 22.6 0% 0 ---- Guana River WMA 55.1 4.8%2.6 14.7 Juniper Springs 82.7 0% 0 ----- O’Leno State Park 92.9 2.8% 2.6 14.7 River Forest 19.4 0% 0 ----- Stephen Foster S.P. 15 0% 0 ----- Tomoka State Park 37 2.2%0.81 4.5 Univ. North FL 7.3 2.5%0.18Referent Total 27.5 2.0%0.55 ----- *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)

29. 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).

30. Geographic distribution of Borrelia spp. in ticks in Florida = B. burgdorferi positive site = B. lonestari positive site

31. 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

32. Examples of Erythema Migrans

33. Florida Lyme borreliosis patient

34. 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

35. Human Lyme borreliosis case in Florida   Treatment   Late signs/symptoms   Follow up   Discussion   Antibody test result   PCR/DNA sequence results Genetic heterogeneity? Multiple infection?

36. 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

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

38. 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

39. 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

40. 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

41. BAB1-4 PCR Prevalence in vertebrates and ticks in FL and SC Small mammals: Rodents(cotton rats 8/15 = 53%; other species 0/54) Raccoons15/17 = 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 UNF17/118 = 14.4% Guana River Site11/118 = 9.3% Lone star ticks8/198 = 4% American dog ticks3/81 = 4% Ixodes affinis4/54 = 7% Gulf Coast ticks1/24 = 4%

42. 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

43. 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?

44. 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 CONCLUSIONS