Reservoir Competence of Vertebrate Hosts for Anaplasma phagocytophilum

Fourteen vertebrate species (10 mammals and 4 birds) were assessed for their ability to transmit Anaplasma phagocytophilum, the bacterium that causes human granulocytic anaplasmosis, to uninfected feeding ixodid ticks. Small mammals were most likely to infect ticks but all species assessed were capable of transmitting the bacterium, in contrast to previous findings.

zones show that many ground-dwelling vertebrate species are exposed to or infected with A. phagocytophilum (2). These data indicate that tick-to-host transmission rates are high and that infection is widespread in host communities.
However, few studies have examined rates of transmission from infected hosts to uninfected ticks, a trait known as the reservoir competence of these hosts. Quantifi cation of host species-specifi c reservoir competence can identify animals most responsible for producing infected ticks and therefore increasing risk for human exposure. Overall, robust quantitative information on reservoir competence is scarce and key hosts remain unstudied. We determined the reservoir competence for A. phagocytophilum of 14 species (10 mammals and 4 birds) in a disease-endemic region of the eastern United States.

The Study
All procedures were conducted after approval from the Cary Institute of Ecosystem Studies Institutional Animal Care and Use Committee. We conducted our research in Dutchess County, New York, a region where human cases of anaplasmosis are rapidly increasing. We trapped hosts on the property of the Cary Institute of Ecosystem Studies (Millbrook, NY, USA) during the peak abundance of larval blacklegged ticks (Ix. scapularis) during July-September in 2008, 2009, and 2010. Detailed methods have been reported (7).
We held members of 10 mammal and 4 bird species (Table 1) for 3 days in cages with wire mesh fl oors suspended over pans lined with wet paper towels. Ticks feeding on hosts were allowed to feed to repletion and drop from hosts into the pans, from which they were collected. In some cases, if hosts did not drop >10 ticks within 3 days, we infested them with unfed larval ticks following methods described (8). Because no evidence has been found for transovarial transmission of A. phagocytophilum (9) or of infection in larval ticks, these infestations likely did not affect host exposure to the pathogen. Hosts that had been infested were held for an additional 4 days, and engorged ticks were collected each day. All engorged larval ticks were held in moistened glass vials at constant temperature and humidity until they molted into the nymphal stage. Newly molted nymphs were fl ash-frozen in liquid nitrogen and stored at −80°C.
DNA extraction was conducted as described (7). To amplify extracted DNA, we used protocols reported by Courtney et al. (10). Briefl y, we used primers ApMSP2f and ApMSP2r and probe ApMSP2p, which are specifi c for the msp2 gene of A. phagocytophilum and generate a 77-bp fragment. Real-time PCR was performed by using a CFX96 Real-Time PCR System (Bio-Rad, Hercules, CA, USA) . We used extracted DNA from unfed larval ticks and ultrapure water as negative controls to account for potential contamination during the extraction and PCR processes, respectively. The cloned 77-bp fragment was used as a positive control. Barrier pipette tips were used throughout the process to prevent contamination. We conducted 3 replicate PCRs per tick.
Ticks were considered positive for A. phagocytophilum if any 1 of 3 replicate samples showed amplifi ed DNA for A. phagocytophilum relative to negative controls. Ticks with marginal results (i.e., moderate fl uorescence) were tested a second time with the same primers and SYBR green dye. For these confi rmatory tests, we included a melt curve analysis in which we determined the temperature at which half of the PCR products had denatured. PCR products were heated from 70°C through 85°C, raising the temperature by 0.5°C every 10 s. Positive controls consistently had melting point maxima of 80.5°C. Using a TOPO-TA Cloning Kit (Invitrogen, Carlsbad, CA, USA), we cloned and sequenced 140 fragments that had a melting point of 80.5°C. Identity of sequences was confi rmed by conducting BLAST searches (National Center for Biotechnology Information, Bethesda, MD, USA) of GenBank using the blastn algorithm (11). One hundred thirty-one of 140 fragments were identifi ed as A. phagocytophilum; the remaining 9 fragments either had poor-quality sequences or did not have the cloning vector inserted. If any replicate was positive in the confi rmatory test, ticks were considered positive for A. phagocytophilum. If all 3 replicates in the confi rmatory test showed marginal or negative results, the ticks were considered negative. Reservoir competence for each host species was calculated as the average percentage of ticks infected per individual host.
Using data for 4,640 ticks collected from 254 animals over 3 years, we assessed levels of reservoir competence for 14 host species (10 mammals and 4 birds) ( Table 1). Short-tailed shrews, white-footed mice, and eastern chipmunks had mean levels of reservoir competence >10% (Figure 1). All other hosts, including opossums, gray and red squirrels, and all 4 species of birds, had mean levels of reservoir competence ranging from 2% to 10%. Reservoir competence differed signifi cantly among these 11 species (F = 2.294, df = 10,232, p = 0.014, by 2-way analysis of variance). Southern fl ying squirrels, striped skunks, and masked shrews all transmitted A. phagocytophilum to ticks, but our sample sizes were too small to draw strong conclusions about reservoir competence. For species that we col-lected in abundant numbers in multiple years (≥4 animals in >2 years), reservoir competence of each species did not vary signifi cantly from year to year (p>0.10 for all species tested, by analysis of variance or Kruskal-Wallis tests as appropriate) (Figure 2).

Conclusions
Our data contradict several assumptions about the role of hosts in infecting ticks with A. phagocytophilum. First, the role of the white-footed mouse in infecting ticks has been controversial (2). Our data suggest that although the mouse is a major reservoir, short-tailed shrews and eastern chipmunks have comparable levels of reservoir competence. In addition, previous work has suggested that chipmunks, skunks, and opossums do not infect feeding ticks (12). At our sites, all of these species infected feeding ticks ( Table 2). Thus, the potential for these hosts to contribute to human risk for HGA should not be ignored.
Because hosts are capable of clearing A. phagocytophilum infections (13), surveys of host exposure might not represent species-specifi c probabilities of transmitting the pathogen to uninfected ticks. Instead, the role of particular species in contributing to the pool of infected ticks is best assessed by determining host reservoir competence using fi eld-captured animals that usually carry ticks. On the basis of the community of hosts we sampled, small mammals are most responsible for infecting uninfected larval ticks in nature, and this result is consistent across years.