Reservoir competence of vertebrate hosts for anaplasma phagocytophilum.
Hersh, Michelle H.
McHenry, Diana J.
Schmidt, Kenneth A.
Ostfeld, Richard S.
|Publication:||Name: Emerging Infectious Diseases Publisher: U.S. National Center for Infectious Diseases Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2012 U.S. National Center for Infectious Diseases ISSN: 1080-6040|
|Issue:||Date: Dec, 2012 Source Volume: 18 Source Issue: 12|
Human granulocytic anaplasmosis (HGA), formerly known as human
granulocytic ehrlichiosis, is an emerging infectious disease in the
United States, Europe, and Asia (7,2). In the United States, most
reported cases are concentrated in north-central and northeastern
states. Patients with HGA typically have nonspecific febrile symptoms,
including fever, chills, headache, and myalgia (7). Most patients with
HGA respond well to antimicrobial drug treatment, but complications are
not uncommon and some cases are fatal (2). Because of difficulties in
diagnosis and lack of awareness of HGA by physicians and the public,
many cases are misdiagnosed, and national statistics likely dramatically
underreport this disease (7).
HGA is caused by a rickettsial bacterium, Anaplasma phagocytophilum (7), groups of which form dense aggregations in granulocytes (3). The bacterium is passed from host to host through the bite of an infected ixodid tick: Ixodes scapularis in the eastern and central United States and Ix. pacificus in the western United States (4-6). Serosurveys and molecular diagnostics within disease-endemic 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. Quantification of host species-specific 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.
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 floors 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 flash-frozen in liquid nitrogen and stored at -80[degrees]C.
DNA extraction was conducted as described (7). To amplify extracted DNA, we used protocols reported by Courtney et al. (70). Briefly, we used primers ApMSP2f and ApMSP2r and probe ApMSP2p, which are specific 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.
[FIGURE 1 OMITTED]
Ticks were considered positive for A. phagocytophilum if any 1 of 3 replicate samples showed amplified DNA for A. phagocytophilum relative to negative controls. Ticks with marginal results (i.e., moderate fluorescence) were tested a second time with the same primers and SYBR green dye. For these confirmatory 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[degrees]C through 85[degrees]C, raising the temperature by 0.5[degrees]C every 10 s. Positive controls consistently had melting point maxima of 80.5[degrees]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[degrees]C. Identity of sequences was confirmed by conducting BLAST searches (National Center for Biotechnology Information, Bethesda, MD, USA) of GenBank using the blastn algorithm (77). One hundred thirty-one of 140 fragments were identified 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 confirmatory test, ticks were considered positive for A. phagocytophilum. If all 3 replicates in the confirmatory 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 significantly among these 11 species (F = 2.294, df = 10,232, p = 0.014, by 2-way analysis of variance). Southern flying 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 [greater than or equal to] 2 years), reservoir competence of each species did not vary significantly from year to year (p>0.10 for all species tested, by analysis of variance or Kruskal-Wallis tests as appropriate) (Figure 2).
[FIGURE 2 OMITED]
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 (72). 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 (73), surveys of host exposure might not represent species-specific 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 field-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.
We thank Mitch Le Sage, Kelly Oggenfuss, Laura Cheney, Micah Strauss, and the 2008-2011 research assistants for laboratory and field assistance.
This study was supported by National Sciences Foundation Emerging Infectious Diseases grant 0813041.
Dr Keesing is an educator and scientist in the Program in Biology at Bard College in Annandale, NY, and an adjunct scientist at the Cary Institute of Ecosystem Studies in Millbrook, NY. Her major research interest is the ecology of infectious diseases.
Bard College, Annandale-on-Hudson, New York, USA (F. Keesing, M.H. Hersh, M. Tibbetts, D.J. McHenry); Cary Institute of Ecosystem Studies, Millbrook, NY, USA (F. Keesing, M.H. Hersh, S. Duerr, R.S. Ostfeld); Washington State University, Pullman, Washington, USA (J. Brunner); New York University, New York, New York, USA (M. Killilea); Union College, Schenectedy, NY, USA (K. LoGiudice); and Texas Tech University, Lubbock, TX, USA (K.A. Schmidt)
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Address for correspondence: Felicia Keesing, Bard College Program in Biology, Box 5000, Annandale-on-Hudson, NY, 12504, USA; email: firstname.lastname@example.org
Table 1. Host species tested for Anaplasma phagocytophiluum reservoir competence, southeastern New York, USA, 2008-2010 * Host species Common name Mammals Blarina brevicauda Northern short-tailed shrew Didelphis virginiana Virginia opossum Glaucomys volans Southern flying squirrel Mephitis mephitis Striped skunk Peromyscus leucopus White-footed mouse Procyon lotor Raccoon Sciurus carolinensis Eastern gray squirrel Sorex cinereus Masked shrew Tamias striatus Eastern chipmunk Tamiasciurus hudsonicus Eastern red squirrel Birds Catharus fuscescens Veery Dumetella carolinensis Gray catbird Hylocichla mustelina Wood thrush Turdus migratorius American robin No. Host species hosts hosts tested Mammals Blarina brevicauda 28 Didelphis virginiana 25 Glaucomys volans 4 Mephitis mephitis 1 Peromyscus leucopus 30 Procyon lotor 25 Sciurus carolinensis 20 Sorex cinereus 6 Tamias striatus 19 Tamiasciurus hudsonicus 15 Birds Catharus fuscescens 21 Dumetella carolinensis 14 Hylocichla mustelina 28 Turdus migratorius 18 No. Host species ticks tested Mammals Blarina brevicauda 529 Didelphis virginiana 501 Glaucomys volans 59 Mephitis mephitis 21 Peromyscus leucopus 571 Procyon lotor 484 Sciurus carolinensis 358 Sorex cinereus 41 Tamias striatus 300 Tamiasciurus hudsonicus 297 Birds Catharus fuscescens 427 Dumetella carolinensis 235 Hylocichla mustelina 496 Turdus migratorius 321 Mean no. Host species ticks sampled per host (range) Mammals Blarina brevicauda 18.9 (11-25) Didelphis virginiana 20.0 (11-25) Glaucomys volans 14.8 (6-25) Mephitis mephitis 21.0 (21-21) Peromyscus leucopus 19.0 (10-25) Procyon lotor 19.4 (10-25) Sciurus carolinensis 17.9 (10-25) Sorex cinereus 6.8 (4-10) Tamias striatus 15.8 (9-25) Tamiasciurus hudsonicus 19.8 (11-25) Birds Catharus fuscescens 20.3 (10-25) Dumetella carolinensis 16.8 (9-24) Hylocichla mustelina 17.7 (10-24) Turdus migratorius 17.8 (8-24) * Number of ticks tested per host can include samples from either natural body loads or experimental infestations, as described in the text, and is not representative of mean total body loads. Table 2. Host species infected with Anaplasma phagocytophilum southeastern New York, USA, 2008-2010 * No. hosts infected/ Host species no. tested (%) Mammals Blarina brevicauda 17/28 (61) Didelphis virginiana 9/25 (36) Glaucomys volanst 2/4 (50) Mephitis mephitist 1/1 (100) Peromyscus leucopus 15/30 (50) Procyon lotor 10/25 (40) Sciurus carolinensis 14/20 (70) Sorex cinereust 2/6 (33) Tamias striatus 10/19 (53) Tamiasciurus hudsonicus 7/15 (47) Birds Catharus fuscescens 9/21 (43) Dumetella carolinensis 7/14 (50) Hylocichla mustelina 14/28 (50) Turdus migratorius 6/18 (33) No. (%) ticks Host species infected Mammals Blarina brevicauda 67 (13) Didelphis virginiana 20 (4) Glaucomys volanst 5 (8) Mephitis mephitist 2 (10) Peromyscus leucopus 63 (11) Procyon lotor 17 (4) Sciurus carolinensis 19 (5) Sorex cinereust 4 (10) Tamias striatus 40 (13) Tamiasciurus hudsonicus 17 (6) Birds Catharus fuscescens 19 (4) Dumetella carolinensis 20 (9) Hylocichla mustelina 27 (5) Turdus migratorius 7 (2) Mean % infected ticks per Host species infected host (range) Mammals Blarina brevicauda 20 (4-56) Didelphis virginiana 13 (4-50) Glaucomys volanst 15 (14-16) Mephitis mephitist 10 Peromyscus leucopus 22 (4-50) Procyon lotor 9 (4-20) Sciurus carolinensis 8 (4-20) Sorex cinereust 23 (17-30) Tamias striatus 24 (6-46) Tamiasciurus hudsonicus 17 (4-73) Birds Catharus fuscescens 10 (4-25) Dumetella carolinensis 18 (4-33) Hylocichla mustelina 10 (4-25) Turdus migratorius 6 (4-11) * Infected hosts are those that transmitted A. phagocytophilum to [greater than or equal to] 1 Ixodes scapularis tick larva. ([dagger]) Host species with <10 individual hosts sampled.
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