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Ixodes ricinus tick saliva-activated transmission of Borrelia burgdorferi sensu stricto spirochetes was studied on the C3H/HeN mouse model. The influence of the feeding of uninfected nymphs on the proliferation and distribution of intradermally inoculated spirochetes was compared with the effect of co-inoculated saliva or salivary gland extract (SGE), respectively. Spirochete loads in murine tissues were evaluated using real-time q-PCR. SGE induced significantly increased spirochete numbers in the skin on the days 4 and 6 post-infection (p.i.). On the other hand, decreased bacterial load in the heart of SGE-treated mice was demonstrated in comparison with control animals. The inoculation of tick saliva increased spirochete load in the urinary bladder on day 6 p.i., while the number of spirochetes in the heart declined on day 6 p.i. The feeding of I. ricinus nymphs raised the spirochete load in the bladder on the days 4 and 6 p.i. On day 6, the number of spirochetes found in the heart was significantly lower than in controls. The prevalence of spirochetes in ticks infected by feeding on mice was more than 10 times higher when the mice were infected with the mixture of spirochetes and saliva or SGE, in comparison with spirochetes alone. The presence of SGE in the infectious inoculum increased the spirochete burden per tick from 0 to almost 28,000. Taken together, these results show a very early effect of tick saliva on the proliferation and distribution of Borrelia spirochetes in the host, probably due to the effect of saliva on the host innate immunity mechanisms.