American Black Bears as Hosts of Blacklegged Ticks ...

Journal of Medical Entomology Advance Access published July 12, 2015 VECTOR/PATHOGEN/HOST INTERACTIONS, TRANSMISSION

American Black Bears as Hosts of Blacklegged Ticks (Acari: Ixodidae) in the Northeastern United States CHRISTINE P. ZOLNIK,1,2 AMANDA M. MAKKAY,1 RICHARD C. FALCO,3 AND THOMAS J. DANIELS2,4

J. Med. Entomol. 1–8 (2015); DOI: 10.1093/jme/tjv092

ABSTRACT Ticks and whole blood were collected from American black bears (Ursus americanus Pallas) between October 2011 and October 2012 across four counties in northwestern New Jersey, an area where blacklegged ticks (Ixodes scapularis Say) and their associated tick-borne pathogens are prevalent. Adult American dog ticks (Dermacentor variabilis Say) were the most frequently collected tick species in late spring, whereas adult and nymphal blacklegged ticks were found in both the late spring and fall months. Additionally, for blacklegged ticks, we determined the quality of bloodmeals that females acquired from black bears compared with bloodmeals from white-tailed deer (Odocoileus virginianus Zimmerman), the most important host for the adult stage of this tick species. Measures of fecundity after feeding on each host species were not significantly different, suggesting that the bloodmeal a female blacklegged tick acquires from a black bear is of similar quality to that obtained from a white-tailed deer. These results establish the American black bear as both a host and quality bloodmeal source to I. scapularis. Thus, black bears may help support blacklegged tick populations in areas where they are both present. In addition, samples of black bear blood were tested for DNA presence of three tick-borne pathogens. Anaplasma phagocytophilum Foggie and Babesia microti Franca were found in 9.2 and 32.3% of blood samples, respectively. All blood samples were quantitative polymerase chain reaction-negative for Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt, & Brenner. Although circulating pathogens were found in blood, the status of black bears as reservoirs for these pathogens remains unknown. KEY WORDS Ixodes scapularis, Babesia microti, blacklegged tick, American black bear, Ursus americanus

The blacklegged tick (Ixodes scapularis Say) is an important disease vector in the United States, responsible for transmitting a variety of pathogens to humans, including Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt, & Brenner, Anaplasma phagocytophilum Foggie, and Babesia microti Franca, the causative agents of Lyme disease, human granulocytic anaplasmosis (HGA), and human babesiosis, respectively (Spielman 1976, Steere et al. 1978, Spielman et al. 1979, Steere and Malawista 1979, Pancholi et al. 1995). This tick species is most commonly found in the northeastern and upper Midwestern United States (DiukWasser et al. 2006, 2012), which include 14 states that account for 95% of Lyme disease cases nationally (CDC 2014b). Blacklegged ticks are generalist feeders with a large variety of potential host species, each of which differs in its status as a pathogen reservoir

1 Department of Biological Sciences, Fordham University, 441 East Fordham Rd., Bronx, NY, 10458. 2 Vector Ecology Laboratory, Louis Calder Center–Biological Field Station, Fordham University, PO Box 887, Armonk, NY 10504. 3 New York State Department of Health, Louis Calder Center, 53 Whippoorwill Rd., Armonk, NY, 10504. 4 Corresponding author, e-mail: [email protected].

(Keirans et al. 1996, Richter et al. 2000, Levin et al. 2002, Ginsberg et al. 2005, Brunner et al. 2008, Hersh et al. 2012). Consequently, some host species may be more important for maintaining disease cycles than others. A number of studies have focused on the importance of small mammals, birds, or white-tailed deer (Odocoileus virginianus Zimmermann) in supporting tick populations and maintaining pathogens (Watson and Anderson 1976; Main et al. 1981; Wilson et al. 1985, 1988; Daniels et al. 1993; Levin et al. 2002; Ginsberg et al. 2005; Brunner et al. 2008; Hersh et al. 2012), partly because of the abundance of those species in nature and partly because of their ease of capture for examination, or for white-tailed deer, their availability for examination during hunting season. However, little is known about the host status of the American black bear (Ursus americanus Pallas) for I. scapularis in the northeastern United States. Most studies of black bears as hosts for ticks in the United States (Henshaw and Birdseye 1911, King 1960, Jonkel 1971, Rogers 1975, Rogers and Rogers 1976, Manville 1978, Kazmierczak et al. 1988) were conducted before blacklegged ticks were discovered to be the primary vectors of the agents responsible for HGA, babesiosis, or Lyme disease (Spielman 1976, Steere et al. 1977), and thus before the importance of

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I. scapularis as a disease vector was known. More recently, studies have surveyed ticks on black bears in Georgia and Florida (Yabsley et al. 2009), and Louisiana (Leydet Jr and Liang 2013), but those studies are outside of the regions where the blacklegged tick is prevalent and its associated tick-borne diseases are endemic (Diuk-Wasser et al. 2006, Adams et al. 2014). Growth in black bear populations in the northeast (Pelton 1986, Williamson 2002), coupled with the generalist feeding nature of the blacklegged tick, increases the possibility that this large mammal may become a more important tick host in the future. In fact, black bears may already serve as important hosts for blacklegged ticks in this region, where current research on such relationships is absent from the literature. Additionally, although antibodies to both B. burgdorferi and A. phagocytophilum have been found in bear sera (Kazmierczak et al. 1988, Mortenson 1998, Schultz et al. 2002, Bronson et al. 2014), indicating exposure to infected tick vectors, identification of the pathogens themselves, as determined by the presence of pathogen DNA, in black bear blood is limited (Drazenovich et al. 2006, Stephenson et al. 2015). Likewise, published evidence of the presence of B. microti in black bear blood is lacking. The goal of this study was to determine the role black bears play as hosts to ticks and tick-borne pathogens in northwestern New Jersey. I. scapularis abundance and Lyme disease case numbers in this region are representative of the northeastern United States (Diuk-Wasser et al. 2006, CDC 2014c). This, in addition to an increase in both black bear population size and range expansion since the 1980s (Carr and Burguess 2011), provides an ideal location to explore the relationship between black bears, blacklegged ticks, and their associated pathogens. Host status of black bears was determined by sampling ticks found on captured animals, permitting engorged female blacklegged ticks from those animals to oviposit, and comparing fecundity of those ticks to that of engorged females that had fed on white-tailed deer (Odocoileus virginianus), a host known to be important for supporting and maintaining populations of this tick species and considered the most important host of adult I. scapularis (Watson and Anderson 1976; Main et al. 1981; Wilson et al. 1985, 1988, 1990; Daniels et al. 1993). Finally, this study explored the potential that these bears have in harboring and maintaining three human disease-causing pathogens that are transmitted by blacklegged ticks: B. burgdorferi, A. phagocytophilum, and B. microti. Results from this study will further our understanding of how black bears support I. scapularis populations and their associated human pathogens in a representative region of the northeastern United States. Materials and Methods Tick Collection: Host Status. In 2012, an ectoparasite survey of live-trapped black bears was conducted in collaboration with the New Jersey Department of Environmental Protection, Division of Fish and Wildlife (NJDFW). As part of that agency’s annual research

trapping survey, we visited 14 sites in the late spring (28 May–17 June) and 16 sites in the fall (20 September–26 October) across Sussex, Morris, Warren, and Passaic Counties, NJ (Fig. 1); one site was visited in both seasons. The research trapping months corresponded to nymphal (late spring or early summer) and adult (fall) blacklegged tick activity periods in the region (Falco et al. 2008). The bears in this study were trapped by NJDFW personnel under their standard operating procedure using Aldrich foot snares and culvert traps. A New Dart hand projector or Dan-Inject dart rifle (Wildlife Pharmaceuticals, Inc., Fort Collins, CO) was used for injecting anesthetic. The drug consisted of 200-mg/ml ketamine hydrochloride, and 450mg/ml xylazine hydrochloride and dosage was based on NJDFW estimation of body weight. This immobilization lasted on average 45–60 min, based on the animal’s metabolism and body location where the drug was administered. NJDFW personnel weighed, sexed, measured, ear tagged, and tattooed the bears, and collected biological samples from each bear including blood, tissue, and a tooth for aging, before the animal was released. As part of this project, a 10-min ectoparasite census was performed on one side of the head, ears, and neck (down to the shoulders) on each animal examined and all ectoparasites found were removed and placed in 70% ethanol for storage until life stage and species identification was conducted. Trapping and animal handling protocols were reviewed and sanctioned by the State of New Jersey, Department of Environmental Protection, and by Fordham University’s IACUC committee. Tick Collection: Fecundity. To compare the quality of bloodmeals that ticks acquire from bears with those from a known host, the white-tailed deer, fecundity of engorged female blacklegged ticks collected from both host species was evaluated. In October 2011, six engorged female blacklegged ticks were collected from four harvested white-tailed deer at a hunter check station in Westchester County, NY. In December 2011, five engorged female blacklegged ticks were collected from four harvested black bears at a hunter check station in Sussex County, NJ. Engorged ticks were returned to the laboratory, weighed, to the nearest 0.1 mg, and kept in an incubator at 95% humidity under a photoperiod of 12:12 [L:D] h until eggs were laid and hatched. All eggs produced, as well as subsequent larvae, were counted to determine the total number of eggs laid and percent hatching success (number of larvae per number of eggs, 100) per female. Previous tick studies have demonstrated that the size of a female tick’s egg mass (number of eggs laid) is a function of engorged female weight (Drummond et al. 1971, Drummond and Whetstone 1975, Koch and Dunn 1980, Gray 1981, Koch 1982); therefore, the number of eggs per milligram of female body weight was also calculated. A subset of 18–52 larvae (depending on egg hatching success) from each adult female tick was selected and measured as an additional index of female reproductive success; larger ticks generally are associated with increased survival (Daniels et al. 1989). By investing

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ZOLNIK ET AL.: BLACKLEGGED TICK HOST STATUS OF AMERICAN BLACK BEARS

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Fig. 1. Map of bear trapping and tick census locations in New Jersey. Counties with sampling sites are in gray and sampling sites are indicated as a triangle for summer or a circle for fall.

more of her resources per offspring, the maternal female may increase the likelihood that individual larvae will survive long enough to find a host. Scutal length and width were measured to the nearest 0.01 mm using a Leica MZ125 high-performance stereomicroscope (Leica Microsystems Ltd., Heerbrugg, Switzerland) with an Infinity 1 microscope camera (Lumenera Corp., Ottawa, ON, Canada) and NIS-Elements software, version D 4.13.04 (Nikon Instruments, Inc., Melville, NY). The scutum is a hard, sclerotized structure on the dorsal surface of Ixodid ticks and is

commonly used for tick measurements (Yeh et al. 1995) because its dimensions do not change during engorgement, or in response to moisture availability or during storage in ethanol, and thus it is an appropriate measure of comparison among individual ticks. Blood Collection and Pathogen Testing. A 4-ml sample of blood was collected from the femoral vein of each of 65 black bears sampled from 29 sites between 28 May and 12 November 2012. Collection tubes were coated with EDTA and DNA was extracted from whole blood using a Qiagen DNeasy Blood & Tissue Kit


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(Qiagen, Redwood City, CA) according to the manufacturer’s protocol. Blood samples were tested for a 75-bp fragment of 23S rRNA gene specific for B. burgdorferi and a 77-bp fragment of the msp2 gene specific for A. phagocytophilum using a duplex quantitative polymerase chain reaction (PCR) protocol with absolute quantification developed by Courtney et al. (2004): specifically, we used 12.5 ml of 2 TaqMan Universal Master Mix II (Applied Biosystems, Foster City, CA), 1.25 ml of ddH2O, primers ApMSP2f and ApMSP2r and Bb23Sf and Bb23Sr at final concentrations of 0.9 and 0.7 mM each, respectively, probes ApMSP2p-VIC and Bb23Sp-FAM at final concentrations of 0.125 and 0.175 mM, respectively, and 2.5 ml of DNA template (Courtney et al. 2004). Thermal cycling conditions were 50 C for 2 min, 95 C for 10 min, and 40 cycles of 95 C for 15 s, followed by 60 C for 60 s with a plate read after each cycle. Blood samples were also tested for the presence of a 104-bp fragment of the 18S rRNA gene specific for B. microti using absolute quantification and the protocol of (Rollend et al. 2013): 12.5 ml of 2X TaqMan Universal Master Mix II (Applied Biosystems, Foster City, CA), 2.5 ml of ddH2O, primers Bm18Sf and Bm18Sr at final concentrations each of 0.9 mM, probe Bm18SpFAM at a final concentration of 0.2 mM and 5 ml of DNA template. Thermal cycling conditions were 50 C for 2 min, 95 C for 10 min, and 40 cycles of 95 C for 15 s followed by 59 C for 60 s with a plate read after each cycle. All pathogen testing was performed on an Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA). Statistical Analysis. A Shapiro–Wilk test was used to determine if the data met assumptions of normality and a Levene’s test was performed to verify homogeneity of variances. In cases where these assumptions were violated, nonparametric tests were used. For all analyses, P ¼ 0.05 was set as the significance level. Student’s t-tests (two-tailed) were used to determine if species and life stage of ticks differed by age and sex of bear hosts. Both fecundity and hatching success were assessed with a two-tailed Student’s t-test. Larval body size measurements (scutal length and width) did not meet assumptions of normality or homoscedasticity and were tested with a two-tailed Mann–Whitney U test to determine if maternal bloodmeal source had an effect on larval size. A Fisher’s exact test was used to compare number of infected blood samples and bear demographic data. All analyses were performed using R 3.1.2 (R Code Team 2014, Vienna, Austria). Results Host Status. In all, 45 black bears were examined for ectoparasites during the late spring sampling season and 29 bears were examined during the fall sampling season. All bears hosted at least one of two tick species: the blacklegged tick and the American dog tick (Dermacentor variabilis Say). The majority of D. variabilis were collected during the late spring and only adults of this species were found on bears (Table 1). In contrast, adult and nymphal blacklegged ticks were collected in

both the late spring and the fall seasons, with adult females outnumbering the males almost 2:1 in the summer, and 3:1 in the fall. Adult blacklegged ticks were also more abundant than nymphs in both seasons, and larval ticks were not found on any bear (Table 1). Additionally, the numbers and life stages of each tick species did not differ according to the sex or age of the bear hosts. Comparison of Host Bloodmeals. Mean numbers of eggs laid by engorged female blacklegged ticks collected from bears did not differ statistically from those laid by ticks collected from deer, when the engorged female tick’s weight was controlled for (t ¼ 1.72; df ¼ 9; P ¼ 0.119), nor did the mean rates of subsequent egg hatch differ significantly between females fed on bears versus those on deer (t ¼ 0.44; df ¼ 9; P ¼ 0.67). Comparisons of mean scutal lengths and widths between larvae (n ¼ 251) from five engorged females that fed on black bears and larvae (n ¼ 271) from six engorged females that fed on white-tailed deer showed no difference in scutal widths, but larval ticks with deer as the maternal bloodmeal source had significantly larger scutal lengths (Mann–Whitney U ¼ 278; P < 0.001; twotailed; Fig. 2). Tick-Borne Pathogen Presence. Of the 65 blood samples that were tested for tick-borne pathogens, all were negative for B. burgdorferi. Totals of 32.3 and 9.2% of the blood samples were positive for A. phagocytophilum and B. microti, respectively, but these pathogens were found to be independent of one another, as only two blood samples were coinfected with both pathogens. Prevalences of infection with these pathogens were similar across seasons and bear sexes; however, significantly more juvenile bears than adults were infected with A. phagocytophilum (Fisher’s exact test statistic value ¼ 0.001; P < 0.05). Discussion Although their status as a possible host for blacklegged ticks in the northeastern United States has remained largely unknown for the past 30 years, we found both nymphal and adult blacklegged ticks on black bears during both late spring and fall sampling seasons. The occurrence of adult blacklegged ticks on black bears in May and June was not unexpected because this stage is typically active in the northeastern United States during the fall and late spring (Fish 1993), although numbers are generally very low during the spring activity period. It is likely that the foraging behavior of bears, whose diet consists typically of forbs, tubers, bulbs, and newly emergent plants in the spring and soft mast items with high sugar content in the summer (Heidelberger 2003), may contribute to their propensity to pick up adult ticks that may still be present in the shrubs, woods, and forest edges in the late spring. Adult American dog ticks were found on black bears during the late spring sampling season in greater abundance than blacklegged ticks. Although D. variabilis is known to vector a number of pathogens, including the causative agents of Rocky Mountain spotted fever and

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Table 1. Total number of ticks, by species and life stage (mean per bear 6 SD), collected from American black bears in northern New Jersey during the late spring and fall sampling seasons in 2012 Sample season

No. of bears sampled

I. scapularis Adult

Late Spring Fall

45 29

161 (1.79 6 3.45) 178 (3.07 6 5.51)

D. variabilis

Nymph

Larva

Adult

Nymph

Larva

35 (0.78 6 2.34) 17 (0.58 6 1.35)

0 0

1,909 (21.21 6 14.68) 2 (0.3 6 0.13)

0 0

0 0

Fig. 2. Mean (6SE) scutal measurements (mm) for a subset of larvae derived from a black bear bloodmeal (n ¼ 251) or a white-tailed deer bloodmeal (n ¼ 271). Scutal length was significantly different based on maternal bloodmeal type (Mann– Whitney U ¼ 27844; P < 0.001).

tularemia (CDC 2014a), most human cases of these two diseases in the United States occur in the Midwestern and southeastern states (Taylor et al. 1991, Treadwell et al. 2000). The American dog tick’s role as a disease vector in northeastern forests is limited, but it is a nuisance pest of humans and animals in these areas. Its observed occurrence on black bears in high numbers suggests that bears may be an important host supporting dog tick populations in the region. Although a number of studies have indicated that molting success of immature blacklegged ticks varies based on host type (LoGiudice et al. 2003, Keesing et al. 2009, Brunner et al. 2011), little is known regarding the influence of hosts other than the white-tailed deer on adult fecundity. White-tailed deer are considered to be the most important host of adult I. scapularis in the northeastern and Midwestern states, based on both exclosure and removal studies, resulting in significantly decreased tick populations (Watson and Anderson 1976, Main et al. 1981; Wilson et al. 1985, 1988; Daniels et al. 1993). Consequently, we used this host species as the standard with which to compare the quality of black bear bloodmeals via two indices of subsequent tick fecundity. No differences were found in the fecundity of adult female blacklegged ticks that fed on black bears compared with those that fed on whitetailed deer, suggesting that black bear bloodmeals

provide a nutrient source that is comparable in quality to that provided by deer. Although black bear populations are considerably smaller than those of deer in the northeast, this large host may be contributing high numbers of replete female I. scapularis, which will then reproduce and contribute to maintaining the tick population. Furthermore, the movement of bears throughout the region may facilitate expansion of the blacklegged tick range into new areas. Recent studies have found Babesia spp. in the Japanese black bear (Ursus thibetanus japonicus) and the Hokkaido brown bear (Ursus arctos yesoensis; Jinnai et al. 2010, Ikawa et al. 2011), and our study identified B. microti in 9.2% of U. americanus blood samples tested. Phylogenetic analysis has shown B. microti to be a diverse species complex (Goethert and Telford 2003, Nakajima et al. 2009), and preliminary work based on short sequences has suggested that strains show a degree of host specialization, with one strain associated with human infections (Hersh et al. 2012). Future studies aimed at determining the strain of B. microti that black bears harbor may be warranted. Regarding the absence of B. burgdorferi in the blood samples we collected, both B. microti and A. phagocytophilum are intracellular parasites, infecting erythrocytes and leukocytes (neutrophils), respectively (Telford III et al. 1993, Dumler et al. 2001). This association

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with blood cells likely facilitated our detection of DNA from these pathogens in blood samples. Conversely, B. burgdorferi is an extracellular parasite that circulates in blood for a relatively short time before invading a variety of other tissues in the host (Steere et al. 2004). Depending on the timing of spirochetemia in the host and when the blood sample is taken, numbers of spirochetes circulating in the blood of an infected host may be too low for detection because early infection usually manifests as localization in the skin before dissemination in other tissues occurs (Pal and Fikrig 2003, Steere et al. 2004). Thus, the apparent absence of B. burgdorferi in these bear blood samples was not unexpected. We found a significantly higher rate of A. phagocytophilum in juvenile bears compared with adults, which is consistent with a recent study conducted in northern California (Stephenson et al. 2015). This decline in infection with increasing age of the bear is possibly due to an increased acquired immunity resulting from previous exposure to infected ticks. Further work may be warranted to explore the immune response of black bears over time to A. phagocytophilum. Finally, host species may differ in their abilities to harbor and maintain tick-borne pathogens in their blood, or to transmit pathogens to feeding ticks (Richter et al. 2000, Levin et al. 2002, Ginsberg et al. 2005, Brunner et al. 2008). Although this study has shown that black bears may play a role in the ecology of blacklegged ticks in the northeast, questions regarding the reservoir competence of this host species remain to be answered. For instance, studies have found antibodies to B. burgdorferi and A. phagocytophilum in bear sera (Kazmierczak et al. 1988, Mortenson 1998, Schultz et al. 2002, Bronson et al. 2014, Stephenson et al. 2015), indicating that these bears have been exposed to ticks infected with these pathogens. Kazmierczak et al. (1988) found a Borrelia sp. morphologically and antigenically similar to B. burgdorferi in two of 14 blood samples from black bears in northern Wisconsin, and both Drazenovich et al. (2006) and Stephenson et al. (2015) identified A. phagocytophilum DNA in the blood of bears tested in California, indicating active infection, as well. Adelson et al. (2004) reported a 17% A. phagocytophilum infection rate in blacklegged ticks in a northwestern New Jersey county, which coupled with our reported infection rate of 32.3% of blood samples from NJ bears, suggests that, in areas where A. phagocytophilum-infected ticks and black bears coexist, these mammals can experience high rates of active infection. However, despite the fact that bears readily host blacklegged ticks and that the ticks themselves do well when feeding on bears, evidence of pathogen transmission from bears back to uninfected ticks is lacking. Previous studies have used infection of larval ticks collected from hosts as evidence that those hosts serve as competent reservoirs (Levine et al. 1985, Donahue et al. 1987, Daniels et al. 2002). We did not find larval blacklegged ticks on any of the bears in this study, but our sampling was conducted outside of the typical larval activity period (late summer; Fish 1993) in the northeast. Our findings of pathogens circulating in bear blood warrants attempts

to collect blacklegged tick larvae from bears in late summer to determine if the ticks acquired infection from these hosts. Given the difficulty of working with these large mammals, however, the likelihood of obtaining evidence of their reservoir status through experimental infestation is low. Until evidence regarding reservoir status is obtained, the ecological role of black bears in tick-borne disease cycles will remain uncertain.

Acknowledgment We would like to thank all personnel from the New Jersey Department of Environmental Protection, Division of Fish and Wildlife, who participated in tick collection, training, or both, in particular: Michael Madonia, Kim Tinnes, Joe Burke, Chuck Sliker, and Amy Schweitzer. Special thanks to Kelcey I. Burguess for study approval, advice, coordination, and overall support of the project. Also, thanks to Tony McBride for aid at check stations. Many thanks to Kam Truhn for larval measurements, to Grace Fredman for egg and larval counting, to Sam R. Telford III for providing B. microti controls, and to Denis Liveris for providing A. phagocytophilum and B. burgdorferi controls. This work was funded by Fordham University’s Calder Center Grant and the Graduate School of Arts and Sciences Fr. John McCloskey Summer Grant to CPZ. This article represents contribution number 259 of the Louis Calder Center—Biological Field Station, Fordham University.

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