Reviewer #3's Review (Sam Telford)

“This interesting manuscript by Naramsihan and colleagues reports on the identification of the early phase of deer tick feeding as that most likely to contain antigens that may induce protective immunity to tick bite. Previous work on anti-tick immunity has tended to focus on later stages (60-72 hours) of feeding, perhaps because it is easier to harvest tick saliva or dissect ticks for salivary gland extracts at that time. Although earlier reports suggested that there were significant differences in the repertoire of salivary antigens secreted over the duration of feeding by various ticks, this manuscript is the first to comprehensively demonstrate this using modern proteomic and genomic approaches. By using guinea pigs that had been repeatedly infested with ticks that were allowed to feed only for 24 hours, the authors demonstrate that ticks were rejected and spirochetal transmission was inhibited. Histopathology of attachment sites from the 24h immune animals that were reinfested confirmed the recruitment of basophils and mast cells, a hallmark of tick rejection. The work is, as usual from this group of researchers, comprehensive and technically superb, and justifies the conclusion that 24 hours of attachment is the most critical period for understanding antitick immunity.

Perhaps the most interesting observation was that passive transfer of serum from tick-immune rabbits into mice inhibited spirochetal transmission to those mice. Although passive transfer did not affect tick feeding, qPCR of the flaB gene of bladder and skin tissue indicated that spirochetal burden was diminished in those mice receiving the immune serum, suggesting that there are tick salivary components that are critical for transmission and perhaps independent of the feeding process. This suggestion would be much stronger if a table was presented comparing proportion of mice infected in the group receiving immune serum with that in those receiving control serum. Even though "clean ticks" were used to infest the rabbit that yielded the immune serum, was the reagent actually checked for antispirochetal antibodies anyway? It is not necessarily "puzzling" (end of page 13) that transmission was inhibited even when ticks fed for longer than 48 hours on the tick immune guinea pigs: spirochetal transmission might be affected by the volume of fluid being recycled via host saliva. That is, hemocoelic spirochetes are perfused by the influx of host plasma that is subsequently spit out and thus anything that inhibits this perfusion will reduce the number of spirochetes encountering the salivary glands or being carried out with the volume of saliva. It is premature to conclude that transmission and feeding might be found to be independent events (page 15, lines 11-14). Besides the probable mechanical function of perfusing spirochetes out of the tick by volumes of liquid, it may also be that the extent of salivation is important for inducing degrees of local immunomodulation of the site of the bite, e.g., by inhibiting the action of phagocytic cells or inactivating complement that might destroy spirochetes as they are being deposited during feeding.”

n.b. These are the general comments made by the reviewer when reviewing this paper. Specific points addressed during revision of the paper are not shown.