A recent article1 demonstrated, underwell-controlled experimental conditions,marked inhibition of polymerase chainreaction (PCR) amplification of a Borreliaburgdorferi gene target by blood withinreplete Ixodes scapularis ticks. Thisphenomenon was not surprising, giventhe known inhibitory effects of bloodon PCR amplification, but the resultsclearly have implications for Lyme dis-ease physicians and diagnosticians seek-ing to verify B. burgdorferi infection ofticks collected from patients. Amplifi-cation was inhibited not only in ticks thatwere replete, but also in unengorgedticks, which contain small amounts ofhemoglobin retained through their pre-vious molt. These problems were com-pletely circumvented by DNA extrac-tion, using commercially available kits,prior to amplification.

These findings are important for ef-fective diagnosis, but have more globalsignificance for the application of nu-cleic acid amplification of pathogen andarthropod vector gene targets. PCRamplification of B. burgdorferi from ticksis important, but it is now possible toassess directly the genetic diversity of B. burgdorferi strains known to exist withinsingle ticks2. PCR can also be used to as-sess presence, prevalence and speciationof other pathogens within Ixodes ticks,including B. burgdorferi, Babesia microti,granulocytic ehrlichiae and encephalitisviruses3. Indeed, PCR has been used re-cently to demonstrate mixed infectionsof B. burgdorferi and Ehrlichia phago-cytophila within single I. ricinus ticks, andfurthermore, allowed speciation of theEhrlichia by sequencing the 16S rRNAamplicon4.

A recently published book providesa fascinating thesis on the complexityof the host–agent–arthropod interface5,and convincingly demonstrates that mostvectors are not simply ‘walking or flyingsyringes’. The relationship of B. burgdor-feri with I. scapularis and its mammalianhost is a particularly good example.Upon attachment of the tick to thehost, B. burgdorferi spirochetes withinthe tick midgut rapidly replicate and,within 48 h of tick attachment, pen-etrate the midgut wall and migrate tothe salivary glands, which in turn fa-cilitates entry into the host6. Host in-fection is enhanced by a number of ticksaliva-derived anti-inflammatory, anti-hemostatic, immunosuppressive and

other factors, whose secretion is stimu-lated by tick attachment and feeding7.There are concomitant and dynamicchanges in the spirochete as well, whichare triggered by a number of stimuli.One such stimulus is increased tem-perature, which influences upregulationof B. burgdorferi outer surface protein(Osp) C and members of the OspE/Fgene family8. By mechanisms that haveyet to be determined, spirochetes withinthe feeding tick downregulate OspA9.These events are no doubt reflectionsof the evolutionarily driven need forspirochetes to adapt rapidly to the vastlydifferent environments of the resting andfeeding arthropod and the mammalianhost. Indeed, spirochetes emerging fromthe feeding tick into the mammal arerefractory to the effects of passivelytransferred immune serum, suggestingthat they are prepared for a journey ofimmune evasion immediately upon entryto the host10. There is mounting evi-dence that a number of novel B. burg-dorferi gene products are expressedexclusively in the mammalian host11–14,and it is likely that others are up- ordownregulated in the flat (unfed) orfeeding tick.

How does the finding of PCR inhibi-tion within ticks relate to these events?It clears the way for a rich and poten-tially rewarding frontier for the in-vestigation of differential pathogen (orvector) gene expression, through PCR,reverse transcription-PCR (RT-PCR)and quantitative PCR, in the flat tick,the feeding tick, trans-stadial events,population dynamics, and co-infection.Now that the genome of B. burgdorferihas been sequenced and published15,

the real job of determining the functionof its many genes, including approxi-mately 150 lipoproteins, must begin.This pathogen has already providedintriguing insight to dramatic events ingene expression within the vector, andmore is surely to come.

References1 Schwartz, I. et al. (1997) Am. J. Trop. Med.

Hyg. 56, 339–3422 Guttman, D.S. et al. (1996) J. Clin. Microbiol.

34, 652–6563 Telford, S.R., III et al. (1997) Emerg. Infect. Dis.

3, 165–1704 Cinco, M. et al. (1997) J. Clin. Microbiol. 35,

3365–33665 Wikel, S.K. (1996) The Immunology of

Host–Ectoparasitic Arthropod Relationships,CAB International

6 Ribeiro, J.M.C. et al. (1987) J. Med. Entomol.24, 201–205

7 Ribeiro, J.M.C. et al. (1985) J. Exp. Med. 161,332–344

8 Stevenson, B., Schwan, T.G. and Rosa, P.A.(1995) Infect. Immun. 63, 4535–4539

9 deSilva, A. et al. (1996) J. Exp. Med. 183,271–275

10 deSilva, A. et al. (1998) J. Infect. Dis. 177,395–400

11 Wallich, R. et al. (1995) Infect. Immun. 63,3327–3335

12 Akins, D.R. et al. (1995) Mol. Microbiol. 18,507–520

13 Champion, C.I. et al. (1994) Infect. Immun. 62,2653–2661

14 Suk, K. et al. (1995) Proc. Natl. Acad. Sci. U. S. A.92, 4269–4273

15 Fraser, C.M. et al. (1997) Nature 390,580–586

Stephen W. Barthold is at the Center for Comparative Medicine, Schools of Medi-cine and Veterinary Medicine, University ofCalifornia Davis, One Shiels Avenue, Davis, CA 95616, USA. Tel: +1 530 752 7913,Fax: +1 530 752 7914, e-mail:swbarthold@ucdavis.edu

444 Parasitology Today, vol. 14, no. 11, 1998Copyright © 1998, Elsevier Science Ltd All rights reserved 0169–4758/98/$19.00 PII: S0169-4758(98)01332-5

Comment

Spirochetes, Ticks and DNAS.W. Barthold

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