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Borrelia and Babesia in wild vertebrates, ticks, and humans in Florida

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

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Borrelia and Babesia in wild vertebrates, ticks, and humans in Florida

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  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. 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?

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

  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 ofLD 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

  17. 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

  18. Results: Borrelia burgdorferiflagellin 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)

  19. 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 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: Duval Big Talbot Island Raccoon 0/17

  20. 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).

  21. Results: lizard sampling and testing Broad-headed skink Florida Brown anole 8/18 (44) Fence lizard 2/4 (50) Glass lizard 3/9 (33) Scrub lizard 1/1 (100) Green anole 6/14 (43) Ground skink 7/17 (41) Race-runner 5/7 (71) Five-lined skink 2/11 (18) Gecko 3/8 (38) Total 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) 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 (Clark et al. 2005. Appl. Environ. Microbiol. 71: 2616-2625)

  22. 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)

  23. 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?

  24. 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 Locality ticks/hr. prevalence† ERI risk 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% 0.18 Referent 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)

  25. 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).

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

  27. 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

  28. Examples of Erythema Migrans

  29. Florida Lyme borreliosis patient

  30. 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% withFL 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

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

  32. 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

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

  34. 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

  35. 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

  36. 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

  37. 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 ticks UNF 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%

  38. 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

  39. 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?

  40. 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

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