Drawing upon current understanding of the ecology of B. burgdorferi and B. miyamotoi, within the context of I. scapularis phenologies in the eastern U.S., we can extrapolate cautiously to interpret and better understand the results found here regarding B. andersonii and B. miyamotoi infections in I. dentatus ticks attached to passerine birds in the Upper Midwestern U.S. Transovarial transmission for B. burgdorferi is not known to occur in I. scapularis, and thus, larvae hatch uninfected. Larvae and nymphs become infected horizontally by feeding on infected hosts, resulting in infected nymphs and adults. The infection prevalence in questing adults is usually double that of questing nymphs in a given area and in part is attributed to each tick having two opportunities of becoming infected. If B. andersonii is similar to B. burgdorferi in that transovarial transmission does not occur, then through implementation of natural xenodiagnosis our study implicates six avian species as competent reservoirs for B. andersonii; the presumption is that infected larval ticks obtained their spirochete infections from the birds upon which they were feeding at the time the sample was taken. B. miyamotoi, on the other hand, is transovarially transmitted in I. scapularis, and little is known about the contribution of any reservoir species to its persistence via horizontal transmission. Thus, in addition to nymphs and adult ticks being infected, questing larvae can be infected as well. Little data exist on the infection prevalence of questing larvae, but in general, the infection prevalences of questing nymphs and adults are low and are similar[12, 18], suggesting that very little horizontal transmission occurs. While the reservoir competency of birds for B. miyamotoi using our study data cannot be established due to the potential for transovarial transmission, we suggest that I. dentatus tick and Northern Cardinals in particular may be important in the life history of B. miyamotoi due to the significant proportion of infected ticks contributed by this host species relative to its role in feeding ticks.
Tick phenology has implications for Borrelia species maintenance and infection dynamics of hosts. In eastern U.S. peak nymphal activity occurs in late spring/early summer. In the Northeast, there is a small peak of larval activity in late spring/early summer, but the main peak is in late summer[49–51]. This asynchronous sequence of nymphs host-seeking prior to larvae allows for hosts to become infected by nymphs, and then to serve as a source of infection to larvae later in the season and is believed to be a driving force in the high nymphal infection prevalence. Given the different modes of transmission between pathogens, in the Northeast peak B. burgdorferi transmission to hosts occurs in early summer, correlating with nymphal activity period, whereas peak B. miyamotoi transmission occurs in late summer, correlating with peak larval activity period. The tick phenology is different in the northern Midwest, however, where the late spring/early summer larval activity period is often the main peak, coincident with the nymphal activity peak[37, 48]. The more synchronous activity periods of nymphs and larvae is believed to results in some larvae feeding on hosts that have not yet been infected by nymphs, and therefore may contribute to lower nymphal infection prevalence in the Midwest compared to the Northeast. The synchronous phenology in the Midwest, however, may provide increased opportunities for co-feeding transmission, but presently its contribution is unknown.
In our study, we found that larval I. dentatus exhibited bimodal activity peaks in late spring/early summer and again in the fall, with nymphs active in the spring though mid-summer, similar to previous reports[52–54]. In the same way that I. scapularis nymphal activity prior to larval activity supports B. burgdorferi maintenance, the phenology we detected for I. dentatus is likely to support not only the low-level B. andersonii infection we report herein, but also our earlier findings of B. burgdorferi in the same ticks. However, because larval activity was bimodal with the first larval peak coincident with nymphal activity, pathogen prevalence may be reduced relative to what would be expected if the activity of the two life stages were completely asynchronous. As a potential to further augment pathogen prevalence in ticks, we suggest the concept of co-feeding transmission in this system requires further attention, since significant frequencies of simultaneous co-infestations of the same avian hosts with both nymphs and larvae occurred. For example, we detected a Song Sparrow that simultaneously harbored B. andersonii-infected nymphs and larvae. The infection prevalence of I. dentatus adults attached to rabbits interestingly was lower/not significantly different from the infection prevalence of nymphs attached to birds; this contradicts the increasing pattern of B. burgdorferi infection in nymphs and adults of I. scapularis. This may suggest that B. andersonii transmission dynamics to I. dentatus nymphs differs from that of B. burgdorferi transmission dynamics to I. scapularis nymphs. Alternatively, the actual infection prevalence of B. andersonii in adult I. dentatus may have been higher, as there were four other Borrelia-positive ticks for which we do not know the species identity.
In contrast, the seasonal activity of nymphs relative to larvae may be less important for the maintenance of B. miyamotoi given the likely occurrence of transovarial transmission in I. dentatus. In addition to I. scapularis, B. miyamotoi has been found to be transovarially transmitted in I. pacificus, I. ricinus, and I. persulcatus. A majority of B. miyamotoi-infected ticks in our study were larvae removed from birds in the fall, a pattern that parallels the finding in which B. miyamotoi infection in mouse hosts rose toward the end of the summer coincident with the larval phenology. A majority of B. miyamotoi-infected ticks occurred on Northern Cardinals, which are permanent residents in Michigan that typically stay within 8 km of where they were hatched, thereby demonstrating local acquisition of the spirochete. The presence of B. miyamotoi-infected ticks on two Hermit Thrushes, which do not breed in the area, affords a mechanism for migratory importation of the spirochete. Interestingly, we did not find any adult I. dentatus infected with B. miyamotoi, but given the low infection prevalence found in nymphs (0.7%) and the number of I. dentatus found on rabbits (n=120), there might not have been enough power to detect an infected tick.
The phenologies of I. dentatus and H. leporispalustris were strikingly opposite; this pattern is not explained by associations of the tick species with spring and fall migrants versus local/breeding birds, respectively. We therefore used H. leporispalustris as a bioassay or sentinel tick species to increase our ability to detect Borrelia spp. among birds across a broader temporal period and in a greater number of samples than would have been afforded by assessment of only I. dentatus, a known Borrelia spp. vector. Indeed four of the 480 H. leporispalustris pools removed from birds were infected with B. andersonii, though the ability of H. leporispalustris to transmit these agents remains unknown.
A high level of genetic diversity of B. andersonii was present within birds, rabbits, and their ticks at this focal site, with twelve unique IGS strains present within 33 sequenced samples derived from ticks removed from 11 host species. This strain richness, when standardized by sample size, is similar to what we reported previously for B. burgdorferi in the same samples (25 IGS strains present within 72 sequenced samples). However, whereas the statistical analysis indicated that the sampling in this study captured most of the B. andersonii strains estimated to be present in the system, this was not true for B. burgdorferi, where strain richness was estimated to be an order of magnitude higher than what we detected. Furthermore, in comparing the amount of evolutionary distance that separates the B. andersonii strains to that which separates the B. burgdorferi found within the same samples, we found that the former is much greater (0.02 nucleotide substitutions per site) than the latter (0.005 nucleotide substitutions per site on aphylogram assuming the same model of evolution as above). These data suggest that the duration of establishment of B. andersonii may be greater than that of B. burgdorferi. In this study location, where I. scapularis is not endemic, differences in diversity between B. burgdorferi and B. andersonii is not likely explained by adaptation to another tick, but may relate to differences in host range and differences in contributions by migratory birds, transmission efficiency, or other unmeasured factors.
At the other end of the spectrum, all but one of the 12 bird-associated B. miyamotoi sequences were identical at the IGS locus to ‘Type 4’ that has been reported previously from I. scapularis nymphs in Connecticut and from wild turkey (Meleagris gallopavo) tissue and blood in Tennessee. The novel Michigan variant had a single nucleotide polymorphism from Type 4, and was different from two mammal-associated variants we previously reported from the ear tissue of a white-footed mouse (Peromyscus leucopus) and an eastern chipmunk (Tamias striatus) collected at different sites in Michigan (Figure3). This low diversity of B. miyamotoi agrees with what has been found previously in I. scapularis, I. ricinus, I. pacificus, and I persulcatus (for example[18, 44, 56]).
Comparison of our current results with our previous report of a minimum of 3.5, 3.1, and 20% B. burgdorferi infection prevalence in these bird ticks, rabbit ticks, and rabbit ears, respectively, confirms that individual mammals and birds are exposed to more than one Borrelia species. For instance, we amplified B. andersonii from the ear tissue of a rabbit that concurrently harbored B. burgdorferi- infected I. dentatus, and rabbits infected with B. burgdorferi harbored B. andersonii-infected ticks. We captured at least three birds that concurrently harbored ticks infected with B. andersonii and B. burgdorferi, and one bird that concurrently harbored ticks infected with B. miyamotoi and B. burgdorferi. Co-infections of I. scapularis with B. burgdorferi and B. miyamotoi has been reported previously: Barbour et al. found that the frequency of co-infections in 7,205 questing nymphs from across the northern U.S. was not different than expected; Ullmann et al. detected no coinfection in 250 I. scapularis nymphs from New Jersey, and Tokarz et al. found that all 7 B. miyamotoi-infected adult I. scapularis in a sample of 286 from New York were co-infected with B. burgdorferi. The ecological and epidemiologic significance of co-infections with two or more Borrelia species requires additional study.