Skip to main content
SpringerLink
Log in

Constructing an ELISA for Detection of Anti-Borrelia in Wildlife and Agricultural Animals

  • Protocol
  • First Online:
Borrelia burgdorferi

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2742))

Abstract

Zoonotic diseases have major impacts on human and animal health, as well as being ecologically significant. Lyme Borreliosis or Lyme disease, caused by infection by pathogenic members of the Borrelia genus, is among these zoonotic diseases. Serology is one of the most accessible means for indirect surveillance of pathogen presence by monitoring the presence, abundance, and type of immune response to the pathogen or pathogen-associated epitopes. Serological surveillance of wild animals is important as wild animals are the primary reservoirs of many zoonotic diseases. Similarly, serological surveillance of agricultural animals is important due to their economic importance, in addition to animal welfare concerns. However, serology in any non-model animal such as wildlife or agricultural animals is difficult because serology necessarily relies on blood samples from the animals being tested. While companion or laboratory animals are generally sufficiently accustomed to humans that blood samples can be obtained, obtaining blood samples from wild or agricultural animals is more challenging. This initial challenge is compounded by the absence of validated serological tools to evaluate antibody titres in the sera. In this chapter, we provide methods for constructing an ELISA for the detection of anti-Borrelia antibodies in non-model animals, using studies on horses and cows as a proof of principle. The methods focus on the problems specific to non-model animals including obtaining sera, options for determining positive and negative controls without the ability to perform controlled infections, and methods for test optimization and validation.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Jones KE, Patel NG, Levy MA et al (2008) Global trends in emerging infectious diseases. Nature 451(7181):990–993. https://doi.org/10.1038/nature06536

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Dong Y, Zhou G, Cao W et al (2022) Global seroprevalence and sociodemographic characteristics of Borrelia burgdorferi sensu lato in human populations: a systematic review and meta-analysis. BMJ Glob Health 7(6):e007744. https://doi.org/10.1136/bmjgh-2021-007744

    Article  PubMed  PubMed Central  Google Scholar 

  3. Kučerová HL, Žákovská A, Marková J, Bártová E (2019) Detection of antibodies to Borrelia burgdorferi s.l. in wild small mammals and sensitivity of PCR and cultivation. Vet Microbiol 230:241–243. https://doi.org/10.1016/j.vetmic.2019.02.004

    Article  CAS  PubMed  Google Scholar 

  4. Zinck CB, Priest JM, Shutler D et al (2021) Detection of Borrelia spp., Ehrlichia canis, Anaplasma phagocytophilum, and Dirofilaria immitis in Eastern Coyotes (Canis latrans) in Nova Scotia, Canada. J Wildl Dis 57(3):678–682. https://doi.org/10.7589/JWD-D-20-00188

    Article  CAS  PubMed  Google Scholar 

  5. Burgess EC (1988) Borrelia burgdorferi infection in Wisconsin horses and cows. Ann N Y Acad Sci 539:235–243. https://doi.org/10.1111/j.1749-6632.1988.tb31857.x

    Article  CAS  PubMed  Google Scholar 

  6. Hahn CN, Mayhew IG, Whitwell KE (1996) A possible case of Lyme Borreliosis in a horse in the UK. Equine Vet J 28(1):84–88. https://doi.org/10.1111/j.2042-3306.1996.tb01595.x´

    Article  CAS  PubMed  Google Scholar 

  7. Burgess B (1988) British Columbia. Lyme disease in horses. Can Vet J 29(4):393-4.7

    Google Scholar 

  8. Tylewska-Wierzbanowska S, Chmielewski T (2022) Limitation of serological testing for Lyme Borreliosis: evaluation of ELISA and western blot in comparison with PCR and culture methods. Wien Klin Wochenschr 114(13–14):601–605

    Google Scholar 

  9. Chang YF, Novosol V, McDonough SP et al (2000) Experimental infection of ponies with Borrelia burgdorferi by exposure to Ixodid ticks. Vet Pathol 37(1):68–76. https://doi.org/10.1354/vp.37-1-68

    Article  CAS  PubMed  Google Scholar 

  10. Ben Said M, Belkahia H, Alberti A (2016) First molecular evidence of Borrelia burgdorferi sensu lato in goats, sheep, cattle and camels in Tunisia. Ann Agric Environ Med 23(3):442–447. https://doi.org/10.5604/12321966.1219184

    Article  CAS  PubMed  Google Scholar 

  11. George JE, Pound JM, Davey RB (2004) Chemical control of ticks on cattle and the resistance of these parasites to acaricides. Parasitology 129(Suppl):S353–S366. https://doi.org/10.1017/s0031182003004682

    Article  CAS  PubMed  Google Scholar 

  12. Heyman P, Cochez C, Hofhuis A (2010) A clear and present danger: tick-borne diseases in Europe. Expert Rev Anti-Infect Ther 8(1):33–50. https://doi.org/10.1586/eri.09.118

    Article  PubMed  Google Scholar 

  13. Ji B, Collins MT (1994) Seroepidemiologic survey of Borrelia burgdorferi exposure of dairy cattle in Wisconsin. Am J Vet Res 55(9):1228–1231

    CAS  PubMed  Google Scholar 

  14. Post JE, Shaw EE, Wright SD (1988) Suspected Borreliosis in cattle. Ann N Y Acad Sci 539(1):488–488. https://doi.org/10.1111/j.1749-6632.1988.tb31916.x

    Article  Google Scholar 

  15. Sharma SP, Amanfu W, Losho TC (2000) Bovine Borreliosis in Botswana. Onderstepoort J Vet Res 67(3):221–223

    CAS  PubMed  Google Scholar 

  16. Stefanciková A, Adaszek Ł, Pet’ko B et al (2008) Serological evidence of Borrelia burgdorferi sensu lato in horses and cattle from Poland and diagnostic problems of Lyme Borreliosis. Ann Agric Environ Med 15(1):37–43

    Google Scholar 

  17. Zarkov I, Marinov M (2003) The Lyme disease: results of a serological study in sheep, cows and dogs in Bulgaria. Rev Médecine Vét 154(5):363–366

    Google Scholar 

  18. Zintl A, Moutailler S, Stuart P (2017) Ticks and Tick-borne diseases in Ireland. Ir Vet J 70:4. https://doi.org/10.1186/s13620-017-0084-y

    Article  PubMed  PubMed Central  Google Scholar 

  19. Lischer CJ, Leutenegger CM, Braun U, Lutz H (2000) Diagnosis of Lyme disease in two cows by the detection of Borrelia burgdorferi DNA. Vet Rec 146(17):497–499. https://doi.org/10.1136/vr.146.17.497

    Article  CAS  PubMed  Google Scholar 

  20. Parker JL, White KK (1992) Lyme Borreliosis in cattle and horses: a review of the literature. Cornell Vet 82(3):253–274

    CAS  PubMed  Google Scholar 

  21. Blowey RW, Carter SD, White AG, Barnes A (1994) Borrelia burgdorferi infections in UK cattle: a possible association with digital dermatitis. Vet Rec 135(24):577–578

    CAS  PubMed  Google Scholar 

  22. Wagner B, Freer H, Rollins A (2011) Development of a multiplex assay for the detection of antibodies to Borrelia burgdorferi in horses and its validation using Bayesian and conventional statistical methods. Vet Immunol Immunopathol 144(3–4):374–381. https://doi.org/10.1016/j.vetimm.2011.08.005

    Article  CAS  PubMed  Google Scholar 

  23. Burgess EC, Wachal MD, Cleven TD (1993) Borrelia burgdorferi infection in dairy cows, rodents, and birds from four Wisconsin dairy farms. Vet Microbiol 35(1–2):61–77. https://doi.org/10.1016/0378-1135(93)90116-o

    Article  CAS  PubMed  Google Scholar 

  24. Richter D, Matuschka FR (2006) Modulatory effect of cattle on risk for Lyme disease. Emerg Infect Dis 12(12):1919–1923. https://doi.org/10.3201/eid1212.051552

    Article  PubMed  PubMed Central  Google Scholar 

  25. Richter D, Matuschka FR (2010) Elimination of Lyme disease spirochetes from ticks feeding on domestic ruminants. Appl Environ Microbiol 76(22):7650–7652. https://doi.org/10.1128/AEM.01649-10

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Sperling JLH, Sperling FAH (2009) Lyme Borreliosis in Canada: biological diversity and diagnostic complexity from an entomological perspective. Can Entomol 141(6):521–549. https://doi.org/10.4039/n08-CPA04

    Article  Google Scholar 

  27. Stefancíková A, Stĕpánová G, Derdáková M et al (2002) Serological evidence for Borrelia burgdorferi infection associated with clinical signs in dairy cattle in Slovakia. Vet Res Commun 26(8):601–611. https://doi.org/10.1023/a:1020912618950

    Article  PubMed  Google Scholar 

  28. Divers TJ, Gardner RB, Madigan JE et al (2018) Borrelia burgdorferi infection and Lyme disease in North American horses: a consensus statement. J Vet Intern Med 32(2):617–632. https://doi.org/10.1111/jvim.15042. Arnold WK, Savage CR, Brissette CA et al (2016) RNA-Seq of Borrelia burgdorferi in Multiple Phases of Growth Reveals Insights into the Dynamics of Gene Expression, Transcriptome Architecture, and Noncoding RNAs. PLoS One 11(10):e0164165. https://doi.org/10.1371/journal.pone.0164165

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Altman DG, Bland JM (1994) Diagnostic tests. 1: sensitivity and specificity. BMJ 308(6943):1552. https://doi.org/10.1136/bmj.308.6943.1552

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Kurien BT, Scofield RH (eds) (2015) Western blotting: methods and protocols. Humana Press, New York

    Google Scholar 

  31. Carroll JA, Stewart PE, Rosa P et al (2003) An enhanced GFP reporter system to monitor gene expression in Borrelia burgdorferi. Microbiology (Reading) 149(Pt 7):1819–1828. https://doi.org/10.1099/mic.0.26165-0

    Article  CAS  PubMed  Google Scholar 

  32. Hyde JA, Trzeciakowski JP, Skare JT (2007) Borrelia burgdorferi alters its gene expression and antigenic profile in response to CO2 levels. J Bacteriol 189(2):437–445. https://doi.org/10.1128/JB.01109-06

    Article  CAS  PubMed  Google Scholar 

  33. Seshu J, Boylan JA, Gherardini FC, Skare JT (2004) Dissolved oxygen levels alter gene expression and antigen profiles in Borrelia burgdorferi. Infect Immun 72(3):1580–1586. https://doi.org/10.1128/IAI.72.3.1580-1586.2004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Tokarz R, Anderton JM, Katona LI, Benach JL (2004) Combined effects of blood and temperature shift on Borrelia burgdorferi gene expression as determined by whole genome DNA array. Infect Immun 72(9):5419–5432. https://doi.org/10.1128/IAI.72.9.5419-5432.2004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Brorson Ø, Brorson SH, Scythes J et al (2009) Destruction of spirochete Borrelia burgdorferi round-body propagules (RBs) by the antibiotic tigecycline. Proc Natl Acad Sci U S A 106(44):18656–18661. https://doi.org/10.1073/pnas.0908236106

    Article  PubMed  PubMed Central  Google Scholar 

  36. Feng J, Shi W, Zhang S et al (2016) A drug combination screen identifies drugs active against amoxicillin-induced round bodies of in vitro Borrelia burgdorferi persisters from an FDA drug library. Front Microbiol 7:743. https://doi.org/10.3389/fmicb.2016.00743

    Article  PubMed  PubMed Central  Google Scholar 

  37. Feng J, Li T, Yee R et al (2019) Stationary phase persister/biofilm microcolony of Borrelia burgdorferi causes more severe disease in a mouse model of Lyme arthritis: implications for understanding persistence, Post-treatment Lyme Disease Syndrome (PTLDS), and treatment failure. Discov Med 27(148):125–138

    PubMed  Google Scholar 

  38. Meriläinen L, Brander H, Herranen A et al (2016) Pleomorphic forms of Borrelia burgdorferi induce distinct immune responses. Microbes Infect 18(7–8):484–495. https://doi.org/10.1016/j.micinf.2016.04.002

    Article  CAS  PubMed  Google Scholar 

  39. Rudenko N, Golovchenko M, Kybicova K, Vancova M (2019) Metamorphoses of Lyme disease spirochetes: phenomenon of Borrelia persisters. Parasit Vectors 12(1):237. https://doi.org/10.1186/s13071-019-3495-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Frey A, Di Canzio J, Zurakowski D (1998) A statistically defined endpoint titer determination method for immunoassays. J Immunol Methods 221(1–2):35–41. https://doi.org/10.1016/s0022-1759(98)00170-7

    Article  CAS  PubMed  Google Scholar 

  41. Munro HJ, Ogden NH, Mechai S et al (2019) Genetic diversity of Borrelia garinii from Ixodes uriae collected in seabird colonies of the northwestern Atlantic Ocean. Ticks Tick Borne Dis 10(6):101255. https://doi.org/10.1016/j.ttbdis.2019.06.014

    Article  PubMed  Google Scholar 

  42. Ogden NH, Barker IK, Francis CM et al (2015) How far north are migrant birds transporting the tick Ixodes scapularis in Canada? Insights from stable hydrogen isotope analyses of feathers. Ticks Tick Borne Dis 6(6):715–720. https://doi.org/10.1016/j.ttbdis.2015.06.004

    Article  CAS  PubMed  Google Scholar 

  43. Scott JD (2016) Studies abound on how far north Ixodes scapularis ticks are transported by birds. Ticks Tick Borne Dis 7(2):327–328. https://doi.org/10.1016/j.ttbdis.2015.12.001

    Article  PubMed  Google Scholar 

  44. Scott JD, Fernando K, Banerjee SN et al (2001) Birds disperse ixodid (Acari: Ixodidae) and Borrelia burgdorferi-infected ticks in Canada. J Med Entomol 8(4):493–500. https://doi.org/10.1603/0022-2585-38.4.493

    Article  Google Scholar 

  45. Scott JD, Anderson JF, Durden LA (2012) Widespread dispersal of Borrelia burgdorferi-infected ticks collected from songbirds across Canada. J Parasitol 98(1):49–59. https://doi.org/10.1645/GE-2874.1

    Article  PubMed  Google Scholar 

  46. Smith RP Jr, Muzaffar SB, Lavers J et al (2006) Borrelia garinii in seabird ticks (Ixodes uriae), Atlantic Coast, North America. Emerg Infect Dis 12(12):1909–1912. https://doi.org/10.3201/eid1212.060448

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Branda JA, Lemieux JE, Blair L (2021) Detection of Borrelia burgdorferi cell-free DNA in human plasma samples for improved diagnosis of early Lyme Borreliosis. Clin Infect Dis 73(7):e2355–e2361. https://doi.org/10.1093/cid/ciaa858

    Article  CAS  PubMed  Google Scholar 

  48. Cerar T, Ruzić-Sabljić E, Glinsek U et al (2008) Comparison of PCR methods and culture for the detection of Borrelia spp. in patients with erythema migrans. Clin Microbiol Infect 14(7):653–658. https://doi.org/10.1111/j.1469-0691.2008.02013.x

    Article  CAS  PubMed  Google Scholar 

  49. Association of Public Health Laboratories (2021) Suggested reporting language, interpretation and guidance regarding Lyme disease serologic test results. Association of Public Health Laboratories. https://www.cdc.gov/lyme/diagnosistesting/index.html. Accessed 2 Jan 2023

  50. Pillai-Kastoori L, Schutz-Geschwender AR, Harford JA (2020) A systematic approach to quantitative Western blot analysis. Anal Biochem 593:113608. https://doi.org/10.1016/j.ab.2020.113608

    Article  CAS  PubMed  Google Scholar 

  51. Parikh R, Mathai A, Parikh S et al (2008) Understanding and using sensitivity, specificity and predictive values. Indian J Ophthalmol 56(1):45–50. https://doi.org/10.4103/0301-4738.37595

    Article  PubMed  PubMed Central  Google Scholar 

  52. Loss SR, Noden BH, Hamer GL, Hamer SA (2016) A quantitative synthesis of the role of birds in carrying ticks and tick-borne pathogens in North America. Oecologia 182(4):947–959. https://doi.org/10.1007/s00442-016-3731-1

    Article  PubMed  Google Scholar 

  53. Altman DG, Bland JM (1994) Statistics notes: diagnostic tests 2: predictive values. BMJ 309(6947):102. https://doi.org/10.1136/bmj.309.6947.102

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

The studies providing the data described here were funded by the Natural Sciences and Engineering Council and the Canadian Lyme Disease Foundation to VKL. Stipendiary support for some of the studies were provided by Mount Allison University and the Natural Sciences and Engineering Council to JB and EB. We would like to extend our gratitude to the horse owners and dairy farmers who enabled participation of their animals, as well as the New Brunswick and Nova Scotian veterinarians who aided in sample collection. We thank members of the Lloyd lab for comments and discussion.

Author information

Authors and Affiliations

  1. Department of Biology, Mount Allison University, Sackville, NB, Canada

    Julia Bland, Emma Bush & Vett Lloyd

  2. Atlantic Veterinary College, Charlottetown, PE, Canada

    Julia Bland, Caitlin McGowan & Emma Bush

  3. Nova Scotia, Society for Prevention of Cruelty to Animals (SPCA), Dartmouth, NS, Canada

    Caitlin McGowan

Authors
  1. Julia Bland
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Caitlin McGowan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Emma Bush
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Vett Lloyd
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Vett Lloyd .

Editor information

Editors and Affiliations

  1. Tezted Ltd, Jyväskylä, Finland

    Leona Gilbert

Rights and permissions

Reprints and permissions

Copyright information

© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Bland, J., McGowan, C., Bush, E., Lloyd, V. (2024). Constructing an ELISA for Detection of Anti-Borrelia in Wildlife and Agricultural Animals. In: Gilbert, L. (eds) Borrelia burgdorferi. Methods in Molecular Biology, vol 2742. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3561-2_4

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-3561-2_4

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3560-5

  • Online ISBN: 978-1-0716-3561-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation