The Lyme disease spirochete must undergo specific physiological changes in order to adjust and survive in the different environments encountered during its natural infectious cycle (reviewed in [5, 6]). Spirochetes must also migrate from the tick midgut to the salivary glands in order to be transmitted, a process that typically takes ~48 h [23, 26, 44–47]. We designed the present study to quantitatively assess the virulence of spirochetes derived from ticks before and after feeding on a vertebrate host. We needle-inoculated mice with infected tick material to bypass requirements specific for tick transmission and focus the comparison on the relative capabilities of these spirochetes to infect a mammalian host. Viable spirochetes in these inocula were quantified by colony formation in solid medium. A significant finding of this study was that 0/10 mice were infected with an inoculum of ~103 viable spirochetes from unfed ticks and only 1/15 mice became infected with inocula ranging from ~7×103 to 1×104 organisms from this source (Table 1). This outcome does not permit calculation of the ID 50 for spirochetes from unfed ticks, whereas we calculated an ID 50 of ~30 for spirochetes from fully engorged ticks. Increasing the inoculum 10-fold to 105 organisms in order to potentially determine an ID 50 for spirochetes in unfed ticks would require injecting the undiluted homogenate of ~ 30 unfed ticks per mouse (Table 1), which is technically limiting. When the outcomes of separate experiments are considered together (Tables 1 and 2), only 1/65 mice became infected following inoculation of ~ 10 to 104 spirochetes from unfed ticks, whereas 35/37 mice were infected by similar numbers of tick-derived spirochetes after the bloodmeal. Together these results demonstrate a large increase in the virulence of spirochetes when they have been “primed” by a blood meal. Previous studies have reported the lack of infectivity of B. burgdorferi from unfed ticks, but this outcome was attributed to potentially low numbers of bacteria in the inocula, which were difficult to accurately measure [20, 23]. However, our findings indicate that in addition to physical location and absolute number, the virulence of resident spirochetes is fundamentally different after tick feeding commences.

One well-characterized and major difference between B. burgdorferi in unfed versus fed ticks is the differential production of outer surface proteins, with OspA produced by spirochetes in unfed ticks and OspC induced during tick feeding [13, 41, 48]. The presence of OspA on spirochetes has been correlated with successful colonization of the tick midgut [49–51], while induction of OspC during tick feeding is an absolute requirement for B. burgdorferi to initiate infection of the vertebrate host [14, 15]. Therefore, since B. burgdorferi in unfed ticks do not produce OspC, it is perhaps not surprising that spirochetes persisting within an unfed tick environment would be unable to infect mice. However, we demonstrated that spirochetes derived from unfed ticks failed to infect mice even when they were engineered to constitutively produce OspC (Table 2). These data indicate that the presence of the virulence factor OspC is not sufficient to ‘prime’ spirochetes coming out of unfed ticks for productive mouse infection, and further demonstrate that induction of OspC is only one of several critical adaptive responses that spirochetes undergo during tick feeding to prepare for host infection [31, 32, 52].

There are plausible explanations for the observed lack or significant attenuation of virulence of spirochetes in the unfed tick vector other than OspC. The RpoN-RpoS regulatory cascade, which governs expression of many B. burgdorferi genes in the mammalian host, is shut off during spirochete acquisition by feeding ticks and remains off until it is activated during the next blood meal [11, 12, 32, 52]. A recent study by Iyer and colleagues utilized an amplification-microarray approach to compare the transcriptomes of mammalian host-adapted spirochetes with those in fed ticks or cultivated in vitro [31]. Significant differences were noted in the global patterns of gene expression among spirochetes from these distinct sources, particularly in various aspects of metabolism, nutrient uptake and chemotactic response [31]. These spirochetes were all in metabolically active states supported by nutrients present in the host, the fed tick or culture medium, whereas metabolically inactive spirochetes in unfed nymphal ticks (which we found to be non-infectious) were not part of this comparison because they do not provide enough material for microarray analysis. A direct comparison of global gene expression between spirochetes from fed and unfed ticks would be extremely insightful, however, and the substantially larger unfed adult tick, whose spirochete burden is similarly non-infectious (Table 1), but approximately 50-fold higher than that of an unfed nymph (data not shown), could represent a good source of material for such future analyses.

Genes with RpoS-dependent expression patterns like ospC (abundantly transcribed by spirochetes in the host and in fed nymphal ticks, but expressed at very low levels by spirochetes in fed larval ticks), should provide insight into virulence factors specifically induced for host infection rather than stimulated for cell growth by the bloodmeal [31, 32]. Surprisingly, of the 100 genes most abundantly expressed by spirochetes in fed nymphs, only ospC exhibited this anticipated pattern of putative virulence factor expression [31]. In addition, only a few members of the previously identified set of RpoS-dependent genes of B. burgdorferi [11] appear in the “top 100” list in fed nymphs, and of these, ospC is the only gene that is also highly expressed by host-adapted spirochetes [31]. It seems unlikely that metabolic state and OspC production, while both critically important, are the sole determinants of the infectious phenotype of spirochetes in fed versus unfed nymphs. Rather, less abundant gene products not highlighted by microarray analyses are also likely to play a key role in preparing B. burgdorferi for mammalian infection. Likewise, transcriptome comparisons do not identify post-transcriptional regulatory mechanisms that alter translation or protein turnover, which have been shown to play a role in modulation of RpoS function [53, 54]. Some spirochetal components, such as the integral outer membrane protein P66, are induced during tick feeding independently of the RpoN-RpoS regulon, yet are essential in the host [55, 56]. Finally, other B. burgdorferi factors that are specifically made during the tick starvation period could actively impede spirochete infectivity in the mammalian host [16, 24, 25, 27, 32, 53, 57, 58]. Thus, both the presence and absence of particular factors could contribute to the avirulent phenotype of spirochetes in unfed ticks.