More recently, ticks have been collected from multiple towns in Plymouth County including Rochester, Lakeville, Middleboro, Halifax and Bridgewater. To date, DNA extractions from 75 ticks collected in Rochester have been conducted. The GPS coordinates were recorded, and ticks were preserved in 100% ethanol. In the lab, the ticks were identified to genus and species, aged (adult/nymph/larvae) and sexed (if an adult). Once again, extracted total community DNA was utilized in PCR reactions using the bacterial specific primers 8F and 1492R to confirm successful bacterial DNA extraction. Once this was confirmed, these samples were utilized in PCR amplification assays with primers specific for both B. burgdorferi and Anaplasma spp. In order to assess specificity of the primer sets for the particular pathogen in question, DNA was extracted from a suite of both Gram-positive bacteria (Bacillus subtilis, Streptococcus epidermidis, Staphylococcus aureus and Micrococcus luteus) and Gram-negative bacteria (Escherichia coli and Proteus vulgaris). Two different, previously published primer sets were utilized to assess for the presence of B. burgdorferi. Neither of these primer sets had any cross-reactivity with the above mentioned bacteria, demonstrating that they were specific for the pathogen in question. Of the 75 ticks assayed, ~43% of them tested positive via PCR for B. burgdorferi.
Unfortunately, previously published primer sets for Anaplasma spp. proved to be less specific. One set of primers resulted in slight cross-reactivity with E. coli. This primer set did give stronger bands in the PCR for ~10% of samples. This data aligns well with infectivity rates of Anaplasma in ticks seen on the Cape. The second primer set gave a 100% infectivity rate. In order to confirm the prevalence of ticks infected with Anaplasma spp., additional, more specific, primer sets will be developed and tested.