Use of Quantitative PCR To Measure Density of Borrelia burgdorferi in the Midgut and Salivary Glands of Feeding Tick Vectors

Abstract

Quantitative real-time PCR was used to assay spirochetes in feeding ticks. Spirochetes in tick midguts increased sixfold, from 998 per tick before attachment to 5,884 at 48 h of attachment. Spirochetes in tick salivary glands increased >17-fold, from 1.2 per salivary gland pair before feeding to 20.8 at 72 h postattachment. The period of the most rapid increase in the number of spirochetes in the salivary glands occurred from 48 to 60 h postattachment; this time period coincides with the maximal increase in transmission risk during nymphal tick feeding.Nymphal Ixodes scapularis ticks are the principal vectors of Lyme disease spirochetes (Borrelia burgdorferi sensu stricto) in North America. These spirochetes appear to be well adapted to their hosts (1) and tick vectors (12). Questing nymphal I. scapularis ticks contain spirochetes restricted mainly to the tick midgut. When an infected tick successfully finds a host and starts to take a blood meal, dramatic changes occur in the spirochete population: spirochetes multiply rapidly in the midgut of infected ticks, thus increasing the overall spirochete density (3). In parallel with increased density, a shift occurs in the dominant outer surface protein expression of the spirochetes, from OspA to OspC (4,17). Other proteins, such as the vlsE protein, become more heterogeneous upon tick attachment (12). Spirochetes then migrate through the hemolymph of the feeding nymphal tick to the salivary glands, where they are subsequently transmitted to the skin of the vertebrate host (2,12,21).A basic understanding of spirochete dynamics within feeding ticks is central to an appreciation of why nymphal I. scapularis ticks infected with B. burgdorferi sensu stricto removed during the first 2 days of attachment do not transmit infection to tick bite victims whereas those feeding for longer periods efficiently transmit infectious spirochetes (6,10,12). In addition, the efficacy of the newly licensed recombinant OspA human vaccine in use in the northeastern United States is apparently based on the ability of anti-OspA antibodies to enter the tick midgut and kill spirochetes before they can migrate to the salivary glands to be transmitted to the victims of tick bite (4, 5). This vaccine does not appear to act against the spirochetes within the vertebrate host (4,20). Thus, studies on the dynamics of spirochete populations in feeding ticks will improve our understanding of how ticks transmit spirochetes and how to prevent such transmission. A better appreciation of the interactions between ticks, hosts, and spirochetes may also lead to a better understanding of the epidemiology and ecology of these important human pathogens (11).Spirochete populations in ticks have been quantified principally through the use of microscopic tools. Rough estimates of the number of spirochetes viewed in tick smears through the use of standard epifluorescent, electron, or confocal microscopy have been reported (2, 12, 21). Recently, extremely sensitive and accurate quantitative fluorogenic-detection PCR ...

bptA(bbe16) is essential for the persistence of the Lyme disease spirochete,Borrelia burgdorferi, in its natural tick vector

“…The complemented strain (BbDTR653) also regained the ability to sustain spirochetal numbers through the molting process (P Ͻ 0.044), albeit not quite to wild-type levels ( Table 2). Of note, levels of wild-type PL133 dropped normally (40) in fed larvae during the 4-to 5-week time frame between larval tick engorgement and metamorphosis to the nymphal form (Fig. 2), whereas the bptA deficiency resulted in an accelerated loss of spirochetes within the first 2 weeks postrepletion ( Fig.…”

Quantitative analysis of Borrelia burgdorferi gene expression in naturally (tick) infected mouse strains

“…In light of the notion that needle exposure by no means reflects the situation of tick infection [23,25,54] and the highly improved real-time PCR technique to quantify B. burgdorferi in situ [24,27,43,46,48,62,63,64], we have now performed a detailed quantitative analysis of the spirochete burden and population dynamics in different tissues of mice following natural (tick) infection by B. burgdorferi s.s. (ZS7) for a period of 4 months postinfection (p.i.). For this purpose we have selected one cp26-encoded gene, ospC [40], and four previously described lp54-encoded genes, i.e.…”

Live imaging reveals a biphasic mode of dissemination of Borrelia burgdorferi within ticks

“…(25) and others (26) have demonstrated that borrelial shuttle vectors can be lost over time in the absence of selection during in vivo growth. We reasoned that insertion of a gfp expression cassette into cp26, a highly stable, endogenous borrelial plasmid (27,28), would prevent loss of the fluorescent reporter during the extensive replication of spirochetes that occurs following the nymphal blood meal (14,29), thereby enhancing our ability to track organisms in locales where bacterial burdens are known to be low (e.g., hemolymph and salivary glands) (15,18,30,31). To this end, Bb914 was generated by inserting gfp under the control of the constitutive flaB promoter (P flaB -gfp) into the bbb20-bbb21 intergenic region of cp26 in CE162, a virulent B. burgdorferi 297 clone (32) (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/ JCI39401DS1).…”