Reservoir competence of vertebrate hosts for anaplasma phagocytophilum.
Subject: Birds
Disease transmission
Authors: Keesing, Felicia
Hersh, Michelle H.
Tibbetts, Michael
McHenry, Diana J.
Duerr, Shannon
Brunner, Jesse
Killilea, Mary
LoGiudice, Kathleen
Schmidt, Kenneth A.
Ostfeld, Richard S.
Pub Date: 12/01/2012
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
Accession Number: 313345651
Full Text: 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.

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 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]

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 (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.

Acknowledgments

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)

DOI: 10.3201/eid1812.120919

References

(1.) Demma LJ, Holman RC, McQuiston JH, Krebs JW, Swerdlow DL. Epidemiology of human ehrlichiosis and anaplasmosis in the United States, 2001-2002. Am J Trop Med Hyg. 2005;73:400-9.

(2.) Jin H, Feng W, Qian J. Epidemiology and control of human granulocytic anaplasmosis: a systematic review. Vector Borne Zoonotic Dis. 2012;12:269-74. http://dx.doi.org/10.1089/vbz.2011.0753

(3.) Dumler JS, Choi K, Garcia-Garcia J, Barat N, Scorpio D, Garyu J, et al. Human granulocytic anaplasmosis and Anaplasma phago cytophilum. Emerg Infect Dis. 2005;11: 1828-34. http://dx.doi. org/10.3201/eid1112.050898

(4.) Foley JE, Foley P, Brown RN, Lane RS, Dumler JS, Madigan JE. Ecology of granulocytic ehrlichiosis and Lyme disease in the western United States. J Vector Ecol. 2004;29:41-50.

(5.) Hodzic E, Fish D, Maretzki CM, De Silva AM, Feng S, Barthold SW. Acquisition and transmission of the agent of human granulocytic ehrlichiosis by Ixodes scapularis ticks. J Clin Microbiol. 1998;36:3574-8.

(6.) Kramer VL, Randolph MP, Hui LT, Irwin WE, Gutierrez AG, Vugia DJ. Detection of the agents of human ehrlichioses in ixodid ticks from California. Am J Trop Med Hyg. 1999;60:62-5.

(7.) Hersh MH, Tibbetts M, Strauss M, Ostfeld RS, Keesing F. Quantifying reservoir competence of wildlife host species for Babesia microti. Emerg Infect Dis. 2012;18:1951-7.

(8.) Keesing F, Brunner J, Duerr S, Killilea M, LoGiudice K, Schmidt K, et al. Hosts as ecological traps for the vector of Lyme disease. Proc Biol Sci. 2009;276:3911-9. http://dx.doi.org/10.1098/ rspb.2009.1159

(9.) Dunning Hotopp JC, Lin M, Madupu R, Crabtree J, Angiuoli SV, Eisen J, et al. Comparative genomics of emerging human ehrlichiosis agents. PLoS Genet. 2006;2:e21. http://dx.doi.org/10.1371/ journal.pgen.0020021

(10.) Courtney JW, Kostelnik LM, Zeidner NS, Massung RF. Multiplex real-time PCR for detection of Anaplasma phagocytophilum and Borrelia burgdorferi. J Clin Microbiol. 2004;42:3164-8. http:// dx.doi.org/10.1128/JCM.42.7.3164-3168.2004

(11.) Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403-10.

(12.) Levin ML, Nicholson WL, Massung RF, Sumner JW, Fish D. Comparision of the reservoir competence of medium-sized mammals and Peromyscus leucopus for Anaplasma phagocytophilum in Connecticut. Vector Borne Zoonotic Dis. 2002;2:125-36. http://dx.doi. org/10.1089/15303660260613693

(13.) Levin ML, Fish D. Immunity reduces reservoir host competence of Peromyscus leucopus for Ehrlichia phagocytophila. Infect Immun. 2000;68:1514-8. http://dx.doi.org/10.1128/IAI.68.3.1514 1518.2000

Address for correspondence: Felicia Keesing, Bard College Program in Biology, Box 5000, Annandale-on-Hudson, NY, 12504, USA; email: keesing@bard.edu
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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