Spirochetes, Ticks and DNA

A recent article[1xSchwartz, I. et al. Am. J. Trop. Med. Hyg. 1997; 56: 339–342PubMedSee all References[1] demonstrated, under well-controlled experimental conditions, marked inhibition of polymerase chain reaction (PCR) amplification of a Borrelia burgdorferi gene target by blood within replete Ixodes scapularis ticks. This phenomenon was not surprising, given the known inhibitory effects of blood on PCR amplification, but the results clearly have implications for Lyme disease physicians and diagnosticians seeking to verify B. burgdorferi infection of ticks collected from patients. Amplification was inhibited not only in ticks that were replete, but also in unengorged ticks, which contain small amounts of hemoglobin retained through their previous molt. These problems were completely circumvented by DNA extraction, using commercially available kits, prior to amplification.These findings are important for effective diagnosis, but have more global significance for the application of nucleic acid amplification of pathogen and arthropod vector gene targets. PCR amplification of B. burgdorferi from ticks is important, but it is now possible to assess directly the genetic diversity of B. burgdorferi strains known to exist within single ticks[2xGuttman, D.S. et al. J. Clin. Microbiol. 1996; 34: 652–656PubMedSee all References[2]. PCR can also be used to assess presence, prevalence and speciation of other pathogens within Ixodes ticks, including B. burgdorferi, Babesia microti, granulocytic ehrlichiae and encephalitis viruses[3xTelford, S.R. III et al. Emerg. Infect. Dis. 1997; 3: 165–170Crossref | PubMedSee all References[3]. Indeed, PCR has been used recently to demonstrate mixed infections of B. burgdorferi and Ehrlichia phagocytophila within single I. ricinus ticks, and furthermore, allowed speciation of the Ehrlichia by sequencing the 16S rRNA amplicon[4xCinco, M. et al. J. Clin. Microbiol. 1997; 35: 3365–3366PubMedSee all References[4].A recently published book provides a fascinating thesis on the complexity of the host–agent-arthropod interface[5xSee all References[5], and convincingly demonstrates that most vectors are not simply "walking or flying syringes'. The relationship of B. burgdorferi with I. scapularis and its mammalian host is a particularly good example. Upon attachment of the tick to the host, B. burgdorferi spirochetes within the tick midgut rapidly replicate and, within 48 h of tick attachment, penetrate the midgut wall and migrate to the salivary glands, which in turn facilitates entry into the host[6xRibeiro, J.M.C. et al. J. Med. Entomol. 1987; 24: 201–205Crossref | PubMedSee all References[6]. Host infection is enhanced by a number of tick saliva-derived anti-inflammatory, anti-hemostatic, immunosuppressive and other factors, whose secretion is stimulated by tick attachment and feeding[7xRibeiro, J.M.C. et al. J. Exp. Med. 1985; 161: 332–344Crossref | PubMed | Scopus (178)See all References[7]. There are concomitant and dynamic changes in the spirochete as well, which are triggered by a number of stimuli. One such stimulus is increased temperature, which influences upregulation of B. burgdorferi outer surface protein (Osp) C and members of the OspE/F gene family[8xStevenson, B., Schwan, T.G., and Rosa, P.A. Infect. Immun. 1995; 63: 4535–4539PubMedSee all References[8]. By mechanisms that have yet to be determined, spirochetes within the feeding tick downregulate OspA[9xdeSilva, A. et al. J. Exp. Med. 1996; 183: 271–275Crossref | PubMed | Scopus (287)See all References[9]. These events are no doubt reflections of the evolutionarily driven need for spirochetes to adapt rapidly to the vastly different environments of the resting and feeding arthropod and the mammalian host. Indeed, spirochetes emerging from the feeding tick into the mammal are refractory to the effects of passively transferred immune serum, suggesting that they are prepared for a journey of immune evasion immediately upon entry to the host[10xdeSilva, A. et al. J. Infect. Dis. 1998; 177: 395–400Crossref | PubMedSee all References[10]. There is mounting evidence that a number of novel B. burgdorferi gene products are expressed exclusively in the mammalian host[11xWallich, R. et al. Infect. Immun. 1995; 63: 3327–3335PubMedSee all References, 12xAkins, D.R. et al. Mol. Microbiol. 1995; 18: 507–520Crossref | PubMedSee all References, 13xChampion, C.I. et al. Infect. Immun. 1994; 62: 2653–2661PubMedSee all References, 14xSuk, K. et al. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4269–4273Crossref | PubMedSee all References], and it is likely that others are up- or downregulated in the flat (unfed) or feeding tick.How does the finding of PCR inhibition within ticks relate to these events? It clears the way for a rich and potentially rewarding frontier for the investigation of differential pathogen (or vector) 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. burgdorferi has been sequenced and published[15xFraser, C.M. et al. Nature. 1997; 390: 580–586Crossref | PubMed | Scopus (1274)See all References[15], the real job of determining the function of its many genes, including approximately 150 lipoproteins, must begin. This pathogen has already provided intriguing insight to dramatic events in gene expression within the vector, and more is surely to come.