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Original article

Surveillance for Ixodes pacificus and the tick-borne pathogens Anaplasma phagocytophilum and Borrelia burgdorferi in birds from California’s Inner Coast Range Regina J. Dingler a , Stan A. Wright b , Ann M. Donohue c , Paula A. Macedo b , Janet E. Foley a,∗ a

Department of Medicine and Epidemiology, 1320 Tupper Hall, School of Veterinary Medicine, University of California, Davis, USA Sacramento-Yolo Mosquito & Vector Control District, 8631 Bond Road, Elk Grove, CA 95624, USA c Napa Mosquito Abatement District, 15 Melvin Road, American Canyon, CA 94503, USA b

a r t i c l e

i n f o

Article history: Received 19 August 2013 Received in revised form 2 February 2014 Accepted 6 February 2014 Available online xxx Keywords: Avian migration Disease ecology Ixodes pacificus Lyme disease Granulocytic anaplasmosis

a b s t r a c t We investigated the involvement of birds in the ecology of the western black-legged tick, Ixodes pacificus, and its associated zoonotic bacteria, Borrelia burgdorferi and Anaplasma phagocytophilum, at two interior coast-range study sites in northern California. Anaplasma phagocytophilum, the agent of granulocytic anaplasmosis (GA), and B. burgdorferi s.s., the agent of Lyme disease (LD), are tick-borne pathogens that are well established in California. We screened blood and ticks from 349 individual birds in 48 species collected in 2011 and 2012 using pathogen-specific PCR. A total of 617 immature I. pacificus was collected with almost three times as many larvae than nymphs. There were 7.5 times more I. pacificus at the Napa County site compared to the Yolo County site. Two of 74 (3%) nymphal pools from an Oregon junco (Junco hyemalis) and a hermit thrush (Catharus guttatus) and 4 individual larvae (all from Oregon juncos) were PCR-positive for B. burgdorferi. Blood samples from a golden-crowned sparrow (Zonotrichia atricapilla) and a European starling (Sturnus vulgaris) were positive for A. phagocytophilum DNA at very low levels. Birds that forage on ground or bark and nest on the ground, as well as some migratory species, are at an increased risk for acquiring I. pacificus. Our findings show that birds contribute to the ecologies of LD and GA in California by serving as a blood-meal source, feeding and transporting immature I. pacificus, and sometimes as a source of Borrelia infection. © 2014 Elsevier GmbH. All rights reserved.

Introduction Ticks may vector life-threatening or debilitating human and veterinary diseases. Borrelia burgdorferi, the agent of Lyme disease (LD), and Anaplasma phagocytophilum, the agent of granulocytic anaplasmosis (GA), are common in the United States. Despite being of medical importance, the ecologies of LD and GA are complex and incompletely understood. These two pathogens are present in various habitats across the northern hemisphere, they infect a wide range of hosts, and can be transmitted by multiple tick vectors. Much of the research to understand Ixodes spp.-borne disease ecology has focused on ticks, large mammalian hosts, rodents, and reptiles, while birds have received much less attention. It is important to understand whether birds may experience

∗ Corresponding author at: UC Davis School of Veterinary Medicine, 1320 Tupper Hall, Department of Medicine and Epidemiology, Davis, CA 95616, USA. Tel.: +1 530 754 9740; fax: +1 530 752 0414. E-mail address: [email protected] (J.E. Foley).

infection, what the risk factors are, and the implications such infection and tick infestation may have on people and domestic animals. Migratory birds are documented hosts of avian-specialist and non-specialist tick species. Long-distance dispersal of ticks and disease agents has been recorded for I. auritulus (Morshed et al., 2005), I. uriae (Olsen et al., 1995a), I. scapularis (formerly also referenced as I. dammini) (Smith et al., 1996; Scott et al., 2001; Ogden et al., 2008), and I. ricinus (Olsén et al., 1995b). In California, songbirds may be heavily parasitized by immature I. pacificus (Wright et al., 2000, 2006, 2011; Eisen et al., 2004b; Castro and Wright, 2007) which is a common human-feeding tick in California (Clover and Lane, 1995) and the primary bridge vector for A. phagocytophilum and B. burgdorferi in the state (Foley et al., 2004). A comprehensive list of all California host records for I. pacificus showed that immature I. pacificus commonly parasitize small mammals and reptiles, but that larvae were also recorded from 43 avian species and nymphs from 38 avian species (Castro and Wright, 2007). In the laboratory, immature I. pacificus showed a preference for lizards and rodents over avian hosts (Slowik and Lane, 2009).

http://dx.doi.org/10.1016/j.ttbdis.2014.02.002 1877-959X/© 2014 Elsevier GmbH. All rights reserved.

Please cite this article in press as: Dingler, R.J., et al., Surveillance for Ixodes pacificus and the tick-borne pathogens Anaplasma phagocytophilum and Borrelia burgdorferi in birds from California’s Inner Coast Range. Ticks Tick-borne Dis. (2014), http://dx.doi.org/10.1016/j.ttbdis.2014.02.002

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Wild birds can become naturally and/or experimentally infected with B. burgdorferi, and some species are suspected of being reservoir-competent for this pathogen (Burgess, 1989; Kurtenbach et al., 1998a; Richter et al., 2000; Ginsberg et al., 2005). Spirochetes have been detected via polymerase chain reaction (PCR) of bird tissue (Lane et al., 2006; Wright et al., 2006) and cultured from blood, liver, and skin biopsies (Anderson et al., 1986; Humair et al., 1993; Mclean et al., 1993; Durden et al., 2001). Both B. burgdorferi s.l. and sensu stricto (s.s.) are frequently detected in ticks from wild-caught birds (Scott et al., 2010). Transmission of borreliae in immature avian ticks through a molt, with subsequent capacity to infect mammals, has been confirmed (Anderson et al., 1990). Fewer studies have examined the roles of birds in the ecology of A. phagocytophilum. Natural infections have been detected in the blood of European species by PCR (De La Fuente et al., 2005), and A. phagocytophilum and Ehrlichia-like agents have been detected in avian-fed ticks (Alekseev et al., 2001; Bjöersdorff et al., ˇ 2001; Daniels et al., 2002; De La Fuente et al., 2005; Spitalská et al., 2006, 2011; Ogden et al., 2008; Hildebrandt et al., 2010; Palomar et al., 2012). Anaplasma DNA in I. ricinus nymphs attached to migratory passerine birds in Russia was more common than from ticks removed from vegetation (Alekseev et al., 2001) and similarly, pooled avian-fed I. scapularis larvae were PCR-positive for A. phagocytophilum even though all 300 larval ticks collected by drag sampling at the New York collection site were negative (Daniels et al., 2002). Further, one of the I. scapularis larval pools collected from an American robin (Turdus migratorius) was PCR-positive for both A. phagocytophilum and B. burgdorferi (Daniels et al., 2002). Here, we investigate the role of terrestrial birds in harboring and transporting ticks and tick-borne infections in northern California. Early accounts at several sites in northern California found birds to have low tick burdens relative to sympatric mammal species, and undetectable to very low B. burgdorferi prevalence (Manweiler et al., 1990; Slowik and Lane, 2001). In contrast, Eisen et al. (2004b) reported that ground- or bark-foraging birds that also nest on the ground (guild I birds) from Hopland Research and Extension Center (HREC, Mendocino County), carried similar I. pacificus larval loads and at least 20-fold higher nymphal loads relative to dusky-footed woodrats (Neotoma fuscipes) and Peromyscus spp. mice. California is ideal for this study in that ticks, B. burgdorferi, and A. phagocytophilum are endemic in parts of the state and multiple studies have established many aspects of their ecologies. Moreover ecological heterogeneity and important north-south avian flyways serve to increase the interest in spatial patterns of ticks and tick-borne disease in the state. We know of no comprehensive state-wide accounting of the density of I. pacificus, although presence-absence literature documents these ticks along the Pacific coast and coast-range mountains from central to far northern California, in the Sierra Nevada foothills, and to a decreasing degree in transverse ranges and southern California coastal areas (Furman and Loomis, 1984). Preferred habitats are those with oak (Quercus spp.) woodland, although I. pacificus has been found among coast redwood (Sequoia sempervirens), in Douglas fir (Pseudotsuga menziesii) habitat, in chaparral, and many other ecosystems as well. Anaplasma phagocytophilum risk has been evaluated in redwood, montane hardwood, and blue oak/foothill pine habitats (Foley et al., 2005) while considerable comparative work shows highest risk of Lyme disease in oak-dominated sites (Eisen et al., 2004a). Using dogs as sentinels for both anaplasmosis and Lyme disease, highest prevalences occurred along the northern coast and Sierra Nevada foothill counties (Carrade et al., 2011), which coincides with the moderate risk status the CDC ranks for this area for Lyme disease (CDC, 2007). The prevalence of infection in ticks and hosts varies considerably across sites. In California, B. burgdorferi PCR-positive values have been reported from 3 to 25% in nymphal I. pacificus and 2–6% in adults, and for A. phagocytophilum, values range from

0 to 1% in nymphs and as high as 10% in adults (Foley et al., 2009; Kramer and Beesley, 1993; Holden et al., 2003; Tälleklint-Eisen and Lane, 1999; Lane et al., 2001). The highest prevalences are in far northern Humboldt County, where A. phagocytophilum prevalence by PCR has been reported as high as 80% in hosts such as western gray squirrels (Sciurus griseus) and gray foxes (Urocyon cinereoargenteus), although levels decline going south (Foley et al., 2004). At our study sites, annual surveillance by the local vector control districts regularly identifies questing adult and nymphal I. pacificus with a prevalence of B. burgdorferi in these ticks ranging from 0 to 8% (Paula A. Macedo, unpublished). The goals of the present study were to analyze the tick infestation rate and rates of infection with tick-borne pathogens in birds and ticks from 2 northern California counties where tick-borne pathogens have been documented previously in ticks, people, and reservoir hosts. We incorporated information on avian ecology, particularly feeding and nesting locations, migration, and habitat preference, into our statistical summaries to determine whether risk of tick infestation and tick-borne pathogen infection are associated with site of capture, migration, age, sex, bird species, or ecological guild.

Materials and methods Study sites Quail Ridge Reserve (QRR) (38◦ 29 N, 122◦ 08 W) in eastern Napa County is a 7839-km2 reserve administered by the John Muir Institute of the Environment at the University of California, Davis. Mist netting was done within Decker Canyon at ∼180 m, which is situated in a relatively moist part of the reserve near Lake Berryessa, with a small freshwater source at Dan’s Pond. Vegetation consists of riparian woodlands and dense oak woodland, with trees overgrown with lace lichen (Ramalina menziesii). Dominant plants include valley oak (Quercus lobata), interior live oak (Q. wislizeni), blue oak (Q. douglasi), gray pine (Pinus sabiniana), poison oak (Toxicodendron diversilobum), redbud (Cercis occidentalis), California wild grape (Vitis californica), toyon (Heteromeles arbutifolia), whiteleaf manzanita (Arctostaphylos Manzanita), hairy ceanothus (Ceanothus oliganthus), and sticky monkeyflower (Mimulus aurantiacus). The reserve is managed, however the landscape is allowed to exhibit some characteristics of oak woodlands, such that leaf litter clusters beneath dense woody vegetation and is sparse in open grassy areas. A prior study at QRR documented B. burgdorferi in 3 I. pacificus larvae, feeding on 2 Oregon juncos at this location in 2009 (Wright et al., 2011). Western gray squirrels (Sciurus griseus) and duskyfooted woodrats, 2 known reservoirs for GA and LD in California (Nieto and Foley, 2008; Salkeld et al., 2008; Brown and Lane, 1992; Lane et al., 2005), are common at QRR. Cache Creek Canyon Regional Park (CCCRP) (38◦ 54 N, 122◦  19 W) is approximately 2.8 km2 and located in northwestern Yolo County. All surveys were conducted at the middle campground site at ∼180 m elevation. Most of the park experiences intensive human recreational use, with manicured lawns for campground sites and access to Cache Creek, which flows year-round. A non-maintained strip of riparian vegetation within the campground consists of Fremont cottonwoods (Populus fremontii), willow (Salix spp.), and tamarisk (Tamarix chinensis). The nearby wild vegetation consists of oak woodlands, grasslands, and chaparral. There was less leaf litter surrounding the mist nets compared to the partially overgrown net locations at QRR. Like QRR, CCCRP has abundant I. pacificus (Wright et al., 1998; California Department of Public Health, 2012), western gray squirrels, and dusky-footed woodrats (Regina J. Dingler and Janet E. Foley, unpublished).

Please cite this article in press as: Dingler, R.J., et al., Surveillance for Ixodes pacificus and the tick-borne pathogens Anaplasma phagocytophilum and Borrelia burgdorferi in birds from California’s Inner Coast Range. Ticks Tick-borne Dis. (2014), http://dx.doi.org/10.1016/j.ttbdis.2014.02.002

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Field sampling We visited each field site on 2 consecutive days approximately once each month from late February through late June in 2011 and 2012 in order to maximize sampling of avian year-round residents (YRR), summer residents (SR), migrants (M), and the last of the visiting winter residents (WR). YRR are found at the capture site year-round; WR migrate south in the fall to overwinter at the capture site; SR arrive at the capture site in spring for breeding; and M are transit through the site in spring or fall. Japanese-style mist nets (6 m × 12 m, 38-mm mesh) were erected within breaks in vegetation, near water, along wildlife corridors and trail edges, near hillsides and abrupt shifts in terrain, around man-made structures (e.g., a gazebo at CCCRP contained cliff swallow nests), and other spots where birds were active. Fifteen nets were opened at QRR and 10 at CCCRP each field day during morning hours (∼4–5 h per day). Nets were checked every 15–30 min depending on weather. Birds were identified to species, sex, and age if possible (Pyle, 1997). Morphological measurements were collected, and each bird was banded with an aluminum leg band provided by U.S. Geological Survey Bird Banding Laboratory (BBL) in Laurel, MD. A blood sample, never exceeding 1/10 ml per 10 g of body weight, was collected via jugular venipuncture using a 28-gauge needle. Blood samples were immediately transferred to heparin-coated tubes and held at 4 ◦ C prior to testing. Birds were inspected for ticks in the field using a 30–50× head-mounted lens. All ticks were removed using forceps and preserved in 70% ethyl alcohol. Birds were released near the point of capture. Very small birds [e.g., Anna’s hummingbird (Calypte anna)] or individuals showing signs of stress were either released from the nets without processing, or processed, but not bled. Upland game birds were not banded per California permit restrictions. Recaptured individuals previously banded during our study were recorded as an “old” recapture, while those banded prior to 2011, as part of other avian surveillance studies, were recorded as a “past” recapture. Any individual recaptured within the same 2-day sampling period was recorded as a “new” recapture on the second day and re-examined for ticks, but not rebled. All work was performed under the authority of the UC Davis Institutional Animal Care and Use Committee, and overseen by the Campus Attending Veterinarian. Collections were performed under BBL Permit # 22853 and California Department of Fish and Wildlife Scientific Collecting Permit # SC-002994. Tick identification Ticks were identified to species and stage using keys (Furman and Loomis, 1984; Webb et al., 1990; Durden and Keirans, 1996; Kleinjan and Lane, 2008). DNA extraction DNA was extracted from whole blood using the Qiagen DNeasy Blood & Tissue kit (Qiagen, Valencia, CA) with modifications. We diluted 30 ␮L whole blood into 300 ␮L of phosphate buffered saline (PBS) and centrifuged the sample. We added 5 ␮L of the pellet to 20 ␮L proteinase K and 175 ␮L PBS, mixed by vortexing, added 200 ␮L buffer AL, and vortexed prior to overnight incubation at 56 ◦ C. Then we added 200 ␮L of ethanol, mixed, and proceeded with extraction according to the manufacturer’s directions. We eluted DNA using 200 ␮L water, which was stored at −20 ◦ C. To reduce costs, up to 5 I. pacificus nymphs from an individual bird captured on the same date were pooled for testing. Also, we tested the 2 most engorged larval ticks from each bird, unless the host had only a single attached larval tick (in which case only the one tick was tested). However, if the corresponding bird blood

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tested PCR-positive for either pathogen or if larvae fed adjacent to infected nymphs, all larvae were also extracted and screened by PCR. Tick DNA was extracted with an ammonium hydroxide (NH4 OH) protocol. Ticks were air-dried, frozen in liquid nitrogen until brittle, and crushed using sterile wooden sticks. Then 100 ␮L of 0.7 M NH4 OH was added and the sample boiled for 15 min at 100 ◦ C, cooled for 30 s on ice, and boiled again for 15 min with the vial lids open to evaporate the ammonia. The liquid remaining in the tube was stored at −20 ◦ C. PCR testing Blood and I. pacificus samples were initially screened for B. burgdorferi and A. phagocytophilum DNA using real-time PCR (Drazenovich et al., 2006; Barbour et al., 2009). Each 12-␮L A. phagocytophilum PCR reaction contained 1 ␮L DNA, 1× Maxima® Probe/ROX (Fermentas International Inc., EU), 2 nmol of each primer (903f and 1024r), and 1 nmol probe (939 p). Each 12␮L B. burgdorferi PCR reaction contained 1 ␮L DNA, 1× Maxima® Probe/ROX, 2 nmol of each primer (forward and reverse), and 1 nmol probe (6FAM). Thermocycler conditions were set to 60 ◦ C for 30 s, 95 ◦ C for 10 min, 50 cycles at 95 ◦ C for 15 s, followed by 60 ◦ C for 1 min, and a post-read of 60 ◦ C for 30 s. For all PCR reactions, samples were considered positive if they had a cycle threshold (CT ) value

Surveillance for Ixodes pacificus and the tick-borne pathogens Anaplasma phagocytophilum and Borrelia burgdorferi in birds from California's Inner Coast Range.

We investigated the involvement of birds in the ecology of the western black-legged tick, Ixodes pacificus, and its associated zoonotic bacteria, Borr...
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