COS 213-4 - Application of parallel PCR and DNA sequencing for detecting remnant host DNA from the prior blood meal of Ixodes scapularis

Background/Question/Methods

The blacklegged tick, Ixodes scapularis, is a generalist vector that feeds on many host species, which vary in their permissiveness to tick feeding and their reservoir competency for transmission of Borrelia burgdorferi, the agent of Lyme disease. However, our understanding of which hosts are responsible for maintaining populations of ticks and pathogens is biased by an historical focus on white-tailed deer (Odocoileus virginianus) and white-footed mice (Peromyscus leucopus), with considerably less attention paid to other species that may increase or decrease human exposure risk. Consequently, wildlife or habitat management strategies, which could help to reduce the spread of Lyme disease, cannot be widely implemented without first improving our knowledge of host species' contributions to local transmission cycles. The objective of this research is to develop a new amplicon sequencing protocol for determining the identity of the larval stage blood meal of the blacklegged tick.. We used parallel PCR with three general primer sets, combined with ultra-high throughput sequencing, to perform detection of host DNA. The approach was tested on DNA extracts from nymphal stage I. scapularis for which the larval blood meal host was known and on questing I. scapularis nymphs and adults for which the prior hosts were unknown.

Results/Conclusions

Among the eight mammal species from which engorged larvae were collected, 6/8 provided at least one tick which matched to our custom database. The best results were from the squirrel species, for which 6/10 ticks matched the genus Sciurus. Positive controls obtained from all bird species tested returned an accurate match to our custom database, however the highest taxonomic resolution attained was to the order Passeriformes. Our preliminary analysis of 38 adult I. scapularis field-collected from Rutland County, VT, indicated that nymphal and/or larval stage ticks were feeding from white-tailed deer. Each of our primer sets, targeting 12s mitochondrial, 16s mitochondrial and 18s rDNA returned matches with 100% identity to this species. This included 34/38 samples for the 12s primer set, 27/38 samples for the 16s primer set, and 38/38 samples for the 18s primer set. These ticks were collected from a single forest with evidence of heavy deer browsing, suggesting that this host many be important for maintaining juvenile I. scapularis populations in some locations. Our ongoing research indicates that PCR, followed by high throughput sequencing, may be a promising tool for high throughput blood meal host detection.