The incidence of Lyme disease (Borreliella burgdorferi) and anaplasmosis (Anaplasma phagocytophilum) are increasing in North America and Europe. The causative agents of these debilitating tick-transmitted infections are maintained in nature in an enzootic cycle involving Ixodes ticks and diverse mammals and birds. It has been postulated that predators directly or indirectly influence the dynamics of the enzootic cycle and disease incidence. Here, we demonstrate high seropositivity of eastern coyotes for B. burgdorferi and A. phagocytophilum. As coyotes become established in urban and suburban environments, interactions with humans, companion animals, and urban/suburban wildlife will increase. Knowledge of the pathogens that these highly adaptable predators are exposed to or carry, and their potential to influence or participate in enzootic cycles, is central to efforts to reduce the risk of tick-borne diseases in humans and companion animals.
KEYWORDS: Anaplasma, Borrelia, Borreliella, coyote, Ixodes, Lyme disease, Canis latrans, DbpA, Eastern coyotes, P44, VlsE, canines
ABSTRACT
Lyme disease and anaplasmosis are tick-borne bacterial diseases caused by Borreliella and Anaplasma species, respectively. A comprehensive analysis of the exposure of eastern coyotes (Canis latrans) in the northeastern United States to tick-borne pathogens has not been conducted. In this report, we assess the serological status of 128 eastern coyotes harvested in Pennsylvania in 2015 and 2017 for antibodies to Borreliella burgdorferi and Anaplasma phagocytophilum. Immunoblot and dot blot approaches were employed to test each plasma sample by using cell lysates and recombinant proteins as detection antigens. The results demonstrate high seropositivity incidences of 64.8% and 72.7% for B. burgdorferi and A. phagocytophilum, respectively. Antibodies to both pathogens were detected in 51.5% of the plasma samples, indicating high potential for coinfection. Antibodies to the B. burgdorferi proteins DbpB, VlsE, DbpA, BBA36, and OspF (BBO39) were detected in 67.2, 63.3, 56.2, 51.6, and 48.4% of the plasma samples, respectively. Antibodies to the A. phagocytophilum P44 and P130 proteins were detected in 72.7 and 60.9% of the plasma samples, respectively.
IMPORTANCE The incidence of Lyme disease (Borreliella burgdorferi) and anaplasmosis (Anaplasma phagocytophilum) are increasing in North America and Europe. The causative agents of these debilitating tick-transmitted infections are maintained in nature in an enzootic cycle involving Ixodes ticks and diverse mammals and birds. It has been postulated that predators directly or indirectly influence the dynamics of the enzootic cycle and disease incidence. Here, we demonstrate high seropositivity of eastern coyotes for B. burgdorferi and A. phagocytophilum. As coyotes become established in urban and suburban environments, interactions with humans, companion animals, and urban/suburban wildlife will increase. Knowledge of the pathogens that these highly adaptable predators are exposed to or carry, and their potential to influence or participate in enzootic cycles, is central to efforts to reduce the risk of tick-borne diseases in humans and companion animals.
OBSERVATION
The incidence of tick-borne diseases (TBDs) is increasing throughout North America and Europe (1, 2). In the eastern half of North America, the causative agents of Lyme disease (Borreliella burgdorferi) and anaplasmosis (Anaplasma phagocytophilum) are transmitted to mammals by Ixodes scapularis ticks. Established Ixodes populations have been reported in >50% of U.S. counties (1) and in pockets of western and eastern Canada (3). The spread of Ixodes tick populations has been attributed to climate change, land use patterns, landscape, food supply (acorn abundance), predator/prey relationships, and the population of mammalian and bird reservoirs (4). Tick-borne pathogens are maintained in nature by numerous mammalian species and birds. While Peromyscus leucopus (white-footed mouse) is often cited as the primary reservoir, evidence suggests that inconspicuous hosts, including shrews, play an even greater role in the maintenance of some tick-borne pathogens in nature (5). The identification of all potential reservoirs for TBDs is central to efforts that seek to interrupt their enzootic cycles in nature and thus decrease risk of disease in humans, companion animals, and wildlife.
Predators have been postulated to influence the dynamics of the tick-mammal enzootic cycles of TBDs (6). In the northeastern United States, the population of eastern coyotes (Canis latrans) has been steadily growing due in part to the extirpation of the eastern gray wolf (Canis lupus) (7) and the ability of these wild canids to rapidly adapt to suburban and urban environments. Coyotes are aggressive apex predators that displace, attack, and kill smaller predators, including the red fox (Vulpes vulpes) (8). In areas where coyotes are thriving and red foxes are declining, the infection prevalence of Ixodes nymphs for B. burgdorferi is increasing (4). This is due in part to the differing predation strategies of coyotes and red foxes. Red foxes are aggressive hunters that stockpile prey for future consumption. In contrast, coyotes hunt only when hungry and do not cache their kill. Hence, as coyote populations expand and red fox populations decline, an increase in low trophic zone mammalian hosts is expected, which will in turn lead to an increased risk for TBDs (6). The goal of this study was to conduct a comprehensive assessment of the serological status of eastern coyotes for B. burgdorferi and A. phagocytophilum.
Plasma samples from 128 eastern coyotes were screened for antibodies (Abs) to B. burgdorferi and A. phagocytophilum by using cell lysate immunoblot and recombinant protein dot blot approaches. The plasma samples were collected from coyotes harvested in U.S. Department of Agriculture (USDA)-sanctioned hunting and trapping events in the Commonwealth of Pennsylvania during 2015 and 2017 (Special Use: Scientific Study Permit no. 48548). All animal procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and in congruence with protocols approved by the Virginia Commonwealth University (VCU) Institutional Animal Care and Use Committee. Information on the collection sites, sex, and developmental stage of each animal is provided in Table 1. The initial screen for Abs to B. burgdorferi was done by screening individual immunoblot strips of cell lysates of B. burgdorferi strain B31 with all 128 plasma samples. Seventy-five of the 128 samples (58.6%) were seropositive for several B. burgdorferi proteins (Fig. 1A, representative data). To test for Abs to A. phagocytophilum, immunoblot strips of cell lysates of HL60 cells infected with A. phagocytophilum strain NCH-1 were screened. An initial screening of 19 plasma samples revealed that 73.7 and 57.9% were Ab positive for 44- and 130-kDa proteins, respectively. Screening of cell lysate immunoblot strips and recombinant P44 and P130 with antigen-specific antisera verified the identities of these immunoreactive proteins as the well characterized P44 (9) and P130 (10) antigens (Fig. 1C). It is important to note that while the actual molecular weight of P130 is 66.1 kDa, it migrates aberrantly upon SDS-PAGE due to its acidic pI of 3.8 (10).
TABLE 1.
Plasma sample ID |
Sexa | County (state sector)b |
Date (mo/day/yr) |
Longitude | Latitude | Age statusc |
A. phagocytophilum Ab result for P44/P130d |
B. burgdorferi Ab result by IB/DBe |
---|---|---|---|---|---|---|---|---|
MC2-121 | M | Clinton (NC) | 2/19/17 | 41.178125 | −77.433313 | A | +/+ | +/+ |
MC2-174 | NR | NR | 2/19/17 | NR | NR | NR | −/− | +/+ |
MC2-179 | F | Warren (NW) | 2/19/17 | 41.935572 | −79.537863 | A | −/− | +/+ |
SP0 | NR | NR | NR | NR | NR | NR | +/+ | +/+ |
SP2 | M | Wyoming (NE) | 1/23/15 | 41.514487 | −75.846361 | SA | +/+ | +/+ |
SP3 | M | Luzerne (NE) | 1/23/15 | 41.178429 | −76.237376 | A | +/+ | +/+ |
SP10 | F | Susquehanna (NE) | 1/24/15 | 41.669079 | −75.913765 | A | +/+ | +/+ |
SP11 | M | Wayne (NE) | 1/24/15 | 41.730075 | −75.388202 | A | +/+ | −/− |
SP12 | F | Wayne (NE) | 1/24/15 | 41.730075 | −75.388202 | A | +/+ | +/+ |
SP14 | F | Wayne (NE) | 1/24/15 | 41.730075 | −75.388202 | A | +/+ | +/+ |
SP15 | F | Wayne (NE) | 1/24/15 | 41.730075 | −75.388202 | A | +/+ | −/+ |
SP17 | M | Susquehanna (NE) | 1/24/15 | 41.678991 | −76.062398 | SA | +/+ | +/+ |
SP18 | M | Bradford (NE) | 1/24/15 | 41.667297 | −76.26158 | A | +/+ | +/+ |
SP20 | M | Wyoming (NE) | 1/25/15 | 41.614243 | −76.046592 | A | +/+ | +/+ |
SP21 | M | Luzerne (NE) | 1/25/15 | 41.113643 | −75.722647 | A | +/+ | +/+ |
SP24 | F | Susquehanna (NE) | 1/25/15 | 41.792753 | −75.689743 | A | −/− | −/− |
SP25 | M | Susquehanna (NE) | 1/25/15 | 41.669079 | −75.913765 | A | +/+ | +/+ |
SP26 | F | Wyoming (NE) | 1/25/15 | 41.485811 | −75.842651 | SA | +/+ | +/+ |
SP19 | F | Bradford (NE) | 1/24/15 | 41.667297 | −76.26158 | A | +/+ | −/+ |
MC12 | M | Clarion (NW) | 2/22/15 | 41.319663 | −79.391605 | J | +/+ | +/+ |
MC14 | M | Clarion (NW) | 2/22/15 | 41.319663 | −79.391605 | SA | +/+ | +/+ |
MC15 | M | Clearfield (NC) | 2/22/15 | 41.164035 | −78.384487 | A | +/+ | +/+ |
MC16 | M | Clearfield (NC) | 2/22/15 | 40.948667 | −78.478513 | A | +/+ | +/+ |
MC17 | F | Washington (SW) | 2/22/15 | 40.166354 | −80.259005 | J | +/+ | +/+ |
MC18 | M | Erie (NW) | 2/22/15 | 42.051004 | −79.942131 | SA | −/− | −/− |
MC19 | M | Erie (NW) | 2/22/15 | 41.935799 | −80.224812 | SA | −/− | −/− |
MC21 | M | Cumberland (SC) | 2/22/15 | 40.203461 | −77.309962 | A | +/+ | +/+ |
MC22 | M | Cumberland (SC) | 2/22/15 | 40.314718 | −76.98066 | SA | −/− | +/+ |
MC26 | M | Clearfield (NC) | 2/22/15 | 40.947123 | −78.214029 | A | +/+ | +/+ |
MC28 | F | Clearfield (NC) | 2/22/15 | 41.026246 | −78.31673 | J | +/+ | +/+ |
MC30 | M | Potter (NC) | 2/22/15 | 41.758617 | −78.132091 | A | +/+ | −/− |
MC32 | F | Fayette (SW) | 2/22/15 | 40.016783 | −79.588829 | J | −/− | −/− |
MC35 | M | Beaver (SW) | 2/22/15 | 40.589347 | −80.225357 | J | +/+ | −/− |
MC40-1 | M | Erie (NW) | 2/22/15 | 42.000261 | −80.318307 | A | −/− | +/+ |
MC40-2 | M | Centre (NC) | 2/22/15 | 40.847732 | −77.686139 | SA | −/− | +/+ |
MC48 | M | Tioga (NC) | 2/22/15 | 41.875854 | −77.401458 | SA | +/+ | +/+ |
MC49 | M | Mc Kean (NC) | 2/22/15 | 41.812173 | −78.480503 | A | −/− | −/+ |
MC53 | F | Clinton (NC) | 2/22/15 | 41.384684 | −77.545175 | J | +/+ | +/+ |
MC56 | M | Washington (SW) | 2/22/15 | 40.263001 | −80.187993 | A | −/− | +/+ |
MC57 | F | Allegheny (SW) | 2/22/15 | 40.382434 | −80.116141 | SA | −/− | +/+ |
MC86 | F | Northumberland (NE) | 2/22/15 | 40.75544 | −76.533517 | A | +/+ | +/+ |
MC87 | F | Pike (NE) | 2/22/15 | 41.463888 | −75.155292 | A | +/+ | +/+ |
MC90 | M | Wyoming (NE) | 2/22/15 | 41.525364 | −75.842013 | A | +/+ | +/+ |
MC97 | F | Mercer (NW) | 2/22/15 | 41.157502 | −80.089206 | A | −/− | −/− |
MC99 | F | Mercer (NW) | 2/22/15 | 41.186374 | −80.354815 | J | −/− | +/+ |
MC100 | M | Mercer (NW) | 2/22/15 | 41.186374 | −80.354815 | A | −/− | −/− |
MC113 | F | Butler (NW) | 2/22/15 | 41.157342 | −79.798464 | SA | +/− | −/− |
MC114 | M | Butler (NW) | 2/22/15 | 41.132894 | −79.852167 | A | +/+ | +/+ |
MC115 | F | Tioga (NC) | 2/22/15 | 41.748528 | −77.301304 | A | +/+ | +/− |
MC130 | NR | NR | 2/22/15 | NR | NR | NR | −/− | −/− |
MC131 | NR | Erie (NW) | 2/22/15 | 41.942181 | −79.985389 | A | −/− | +/+ |
MC148 | F | Clarion (NW) | 2/22/15 | 41.125935 | −79.558499 | J | +/+ | −/− |
MC2-1 | M | Clinton (NC) | 2/17/17 | 41.07909 | −77.412819 | SA | +/+ | −/− |
MC2-2 | M | Clearfield (NC) | 2/17/17 | 40.99839 | −78.341406 | A | +/+ | +/+ |
MC2-3 | F | Susquehanna (NE) | 2/17/17 | 41.267904 | −78.156443 | A | +/+ | +/+ |
MC2-4 | F | Indiana (SW) | 2/17/17 | 40.486455 | −79.451436 | SA | +/+ | −/− |
MC2-5 | M | Susquehanna (NE) | 2/17/17 | 41.724059 | −75.554157 | A | +/+ | −/− |
MC2-6 | M | Centre (NC) | 2/17/17 | 41.030891 | −77.949449 | J | +/+ | +/+ |
MC2-8 | F | Northumberland (NE) | 2/17/17 | 40.961519 | −76.664659 | A | +/+ | +/+ |
MC2-10 | F | Crawford (NW) | 2/17/17 | 41.63794 | −80.83697 | A | −/− | −/− |
MC2-11 | F | Crawford (NW) | 2/17/17 | 41.63794 | −80.83697 | A | −/− | −/+ |
MC2-13 | M | Crawford (NW) | 2/17/17 | 41.751677 | −80.368226 | J | +/+ | +/+ |
MC2-16 | M | Crawford (NW) | 2/17/17 | 41.63794 | −80.83697 | A | +/+ | −/− |
MC2-18 | F | Elk (NC) | 2/17/17 | 41.408709 | −78.434756 | A | +/+ | +/+ |
MC2-19 | M | Clearfield (NC) | 2/17/17 | 41.198563 | −78.770151 | A | +/+ | −/− |
MC2-20 | F | Clearfield (NC) | 2/17/17 | 41.161638 | −78.088013 | J | +/+ | +/+ |
MC2-21 | M | Crawford (NW) | 2/18/17 | 41.63794 | −80.83697 | SA | +/+ | +/+ |
MC2-23 | F | Centre (NC) | 2/18/17 | 41.086628 | −77.823244 | A | +/+ | +/+ |
MC2-24 | F | Tioga (NC) | 2/18/17 | 41.760218 | −77.293542 | SA | +/+ | +/+ |
MC2-25 | M | Warren (NW) | 2/18/17 | 41.653763 | −78.96286 | A | +/+ | +/+ |
MC2-26 | M | Warren (NW) | 2/18/17 | 41.653763 | −78.96286 | A | +/+ | +/+ |
MC2-28 | F | Clarion (NW) | 2/18/17 | 41.320065 | −79.391665 | J | +/+ | −/− |
MC2-29 | F | Venango (NW) | 2/18/17 | 41.284381 | −79.762418 | SA | +/− | −/− |
MC2-33 | M | Centre (NC) | 2/18/17 | 41.030891 | −77.949449 | J | +/+ | +/+ |
CR-7 | F | Columbia (NE) | 2/8/15 | 41.139421 | −76.477064 | SA | +/+ | −/− |
CR-4 | F | Clearfield (NC) | 2/8/15 | 40.883834 | −78.59515 | J | +/+ | −/− |
CR-6 | M | Tioga (NC) | 2/8/15 | 41.876717 | −76.97547 | A | −/− | −/− |
CR-9 | F | Cambria (SW) | 2/8/15 | 40.605372 | −78.802618 | SA | +/+ | +/+ |
CR-10 | M | Cambria (SW) | 2/8/15 | 40.605372 | −78.802618 | A | +/+ | −/− |
CR-11 | M | Jefferson (NW) | 2/8/15 | 41.097298 | −78.888108 | A | +/+ | +/+ |
CR-12 | M | Somerset (SW) | 2/8/15 | 40.007978 | −79.078024 | A | +/+ | −/− |
CR-13 | F | Washington (SW) | 2/8/15 | 40.743967 | −80.255714 | A | −/− | −/− |
CR-14 | F | Washington (SW) | 2/8/15 | 40.243967 | −80.255714 | SA | −/− | −/+ |
CR-15 | F | Schuylkill (SE) | 2/8/15 | 40.548175 | −76.384797 | A | −/− | +/+ |
CR-16 | M | Luzerne (NE) | 2/8/15 | 41.051704 | −76.221094 | SA | +/+ | −/− |
CR-17 | M | Luzerne (NE) | 2/8/15 | 41.051704 | −76.221094 | SA | +/+ | +/+ |
CR-18 | M | Cambria (SW) | 2/8/15 | 40.580221 | −78.606667 | SA | −/− | −/− |
MC2-34 | M | Centre (NC) | 2/18/17 | 41.030891 | −77.949449 | SA | +/+ | +/+ |
MC2-35 | M | Clearfield (NC) | 2/18/17 | 41.140848 | −78.270395 | SA | +/+ | +/+ |
MC2-36 | F | Bedford (SC) | 2/19/17 | 40.206744 | −78.522794 | SA | −/− | +/+ |
MC2-37 | M | Bedford (SC) | 2/19/17 | 40.206744 | −78.522794 | J | +/+ | +/+ |
MC2-39 | M | Huntingdon (SC) | 2/19/17 | 40.241508 | −78.088013 | SA | +/+ | −/+ |
MC2-41 | M | Warren (NW) | 2/19/17 | 41.945424 | −79.222558 | SA | −/− | +/+ |
MC2-44 | F | Monroe (NE) | 2/19/17 | 40.855663 | −75.456433 | SA | +/+ | +/+ |
MC2-47 | M | Elk (NC) | 2/19/17 | 41.427374 | −78.56094 | SA | +/+ | −/− |
MC2-48 | NR | NR | 2/19/17 | NR | NR | NR | −/− | +/+ |
MC2-51 | NR | NR | 2/19/17 | NR | NR | NR | +/− | +/+ |
MC2-52 | M | Clarion (NW) | 2/19/17 | 41.214251 | −79.375163 | A | +/+ | −/− |
MC2-62 | F | Somerset (SW) | 2/19/17 | 40.176389 | −78.959377 | A | −/− | −/− |
MC2-65 | M | Clearfield (NC) | 2/19/17 | 41.284381 | −79.762418 | A | +/+ | +/+ |
MC2-68 | M | Northumberland (NE) | 2/19/17 | 40.709529 | −76.842472 | A | +/− | +/+ |
MC2-70 | F | Northumberland (NE) | 2/19/17 | 40.709529 | −76.842472 | SA | +/+ | −/− |
MC2-73 | M | Pike (NE) | 2/19/17 | 41.186231 | −75.305459 | SA | +/+ | +/+ |
MC2-76 | F | Centre (NC) | 2/19/17 | 40.944512 | −77.444988 | A | +/+ | −/− |
MC2-79 | F | Crawford (NW) | 2/19/17 | 41.764223 | −80.367763 | J | −/− | −/− |
MC2-80 | M | Armstrong (SW) | 2/19/17 | 41.006628 | −79.323992 | J | +/+ | +/+ |
MC2-82 | F | Allegheny (SW) | 2/19/17 | 40.58605 | −80.029207 | A | +/− | +/+ |
MC2-83 | F | Clarion (NW) | 2/19/17 | 41.214251 | −79.375163 | J | +/+ | −/+ |
MC2-89 | M | Sullivan (NE) | 2/19/17 | 41.557347 | −76.502521 | A | +/+ | +/+ |
MC2-92 | F | Montour (NE) | 2/19/17 | 40.974178 | −76.627268 | SA | +/− | −/+ |
MC2-95 | M | Bradford (NE) | 2/19/17 | 41.656464 | −76.853293 | SA | +/+ | −/− |
MC2-100 | F | Monroe (NE) | 2/19/17 | 40.972418 | −75.219908 | A | +/+ | +/+ |
MC2-101 | NR | NR | 2/19/17 | NR | NR | NR | −/− | −/− |
MC2-104 | NR | NR | 2/19/17 | NR | NR | NR | −/− | −/− |
MC2-111 | F | Crawford (NW) | 2/19/17 | 41.860922 | −80.134784 | J | −/− | +/+ |
MC2-113 | M | Crawford (NW) | 2/19/17 | 41.860922 | −80.134784 | A | −/− | −/− |
MC2-119 | F | Erie (NW) | 2/19/17 | 42.000334 | −80.318119 | A | +/− | +/+ |
MC2-124 | M | Luzerne (NE) | 2/19/17 | 41.319399 | −76.012652 | SA | +/+ | +/+ |
MC2-125 | F | Luzerne (NE) | 2/19/17 | 41.314399 | −76.012652 | SA | +/+ | +/+ |
MC2-131 | F | Susquehanna (NE) | 2/19/17 | 41.685298 | −75.1562604 | SA | +/+ | −/− |
MC2-134 | F | Westmoreland (SW) | 2/19/17 | 40.164543 | −79.807261 | J | −/− | −/− |
MC2-141 | M | Potter (NC) | 2/19/17 | 41.872012 | −77.837218 | A | +/− | −/− |
MC2-146 | M | Crawford (NW) | 2/19/17 | 41.63794 | −80.83697 | A | −/− | −/− |
MC2-147 | F | Crawford (NW) | 2/19/17 | 41.718945 | −80.147558 | A | +/+ | −/− |
MC2-150 | F | Cambria (SW) | 2/19/17 | 40.618857 | −78.736284 | A | +/+ | +/+ |
MC2-155 | M | Centre (NC) | 2/19/17 | 40.847563 | −77.686109 | A | +/+ | −/− |
MC2-157 | NR | NR | 2/19/17 | NR | NR | NR | +/+ | −/− |
MC2-170 | F | Erie (NW) | 2/19/17 | 42.161082 | −79.989011 | A | +/+ | +/+ |
Abbreviations: M, male; F, female; NR, not reported.
The geographic sectors of the Commonwealth of Pennsylvania from which coyotes were harvested are indicated as follows: NW, Northwest; NC, North Central; NE, Northeast; SW, Southwest; SC, South Central; SE, Southeast.
Developmental stage (juvenile [J], subadult [SA], or adult [A]) was determined by assessment of tooth wear by wildlife biologists from the USDA and the PGC. This study was conducted under a Special Use: Scientific Study Permit (no. 48548) issued by the Pennsylvania Game Commission. NR, not reported.
For A. phagocytophilum, the Ab screening results for the P44 and P130 proteins are indicated as positive (+) or negative (−).
For the Ab screening for B. burgdorferi, a plus or minus indicates if an animal was positive or negative, respectively, by immunoblot (IB) and dot blot (DB) approaches.
With the initial finding that a high percentage of coyotes were seropositive for B. burgdorferi and A. phagocytophilum, the entire plasma panel was screened for Abs to individual proteins that are upregulated during spirochete residence in mammals or in ticks (reviewed in reference 11). The B. burgdorferi mammalian or infection-stage proteins VlsE, DbpA, DbpB, OspE (paralogs BBL39 and BBN38), and OspF (paralog BBR42) and the tick-stage OspA and OspB proteins were produced with hexahistidine tags, purified, and screened using a dot blot format. Sixty-four percent of the plasma samples harbored Ab to at least three of the six infection-stage antigens, and 50% had Abs to all six proteins (Fig. 2; Table 2). Abs to VlsE and DbpB were detected with the highest frequency. Abs to OspA, but not OspB, were detected in 6 of the 128 samples, but the reactivity was weak and considered equivocal (Fig. 2, sample MC2-134) . This is consistent with the downregulation of OspA and OspB at the tick-mammalian interface prior to transmission to mammals (12). The FhbB protein, a factor H-plasminogen binding protein produced by the human periopathogen Treponema denticola (13), served as a negative control, and as expected, the plasma samples were not reactive with this protein. The immunoscreening results obtained with each individual plasma sample are summarized in Table 1, and the results for each specific test antigen are summarized in Table 2. In Table 3, the data are presented in terms of age, gender, collection year, and state sector.
TABLE 2.
Species and/or protein (B31 ORF)a |
Description | % Ab-positive samples (no. of samples positive/total no.) |
Reference |
---|---|---|---|
B. burgdorferi | |||
OspA (BBA15) | Surface lipoprotein essential for survival in ticks; produced in culture and in ticks but not in mammals |
6.3 (8/128) (equivocal) | 16 |
OspB (BBA16) | Same as described above for OspA; forms an operon with ospA |
0 (0/128) | 16 |
VlsE (BBF0041) | Surface lipoprotein; functions in immune evasion; not produced during cultivation or in ticks; expressed in infected mammals |
63.3 (81/128) | 17 |
DbpA (BBA24) | Decorin binding protein; facilitates dissemination during early stage infection; produced in vitro and in vivo |
56.2 (72/128) | 18 |
DbpB (BBA25) | See information for DbpA above | 68.0 (87/128) | 18 |
OspE (BBL39) | Factor H binding protein; facilitates complement evasion; all LD spirochete strains encode multiple OspE paralogs |
15.6 (20/128) | 19 |
OspE (BBN38) | See information for BBL39 above | 16.4 (21/128) | 20 |
OspF (BBR42) | Expression upregulated in mammals; Ab to OspF has been suggested to be a marker for chronic infection; most strains produce multiple OspF paralogs |
43.0 (55/128) | 21 |
OspF (BBM38) | See information for BBR42 above | 40.6 (52/128) | 21 |
OspF (BBO39) | See information for BBR42 above | 48.4 (62/128) | 21 |
Mlp (BBA36) | Surface lipoprotein; expression upregulated in mammals; Mlp protein family member |
51.6 (66/128) | 22 |
Uncharacterized protein (BBK53) | Surface lipoprotein; uncharacterized | 18.8 (24/128) | |
P35 (BBA73) | Function unknown; surface protein; member of paralogous protein family 54; upregulated in mammals |
48.4 (62/128) | 23 |
Protein of unknown function (BB0238) |
Function unknown; required for infection in mammals | 27.3 (35/128) | 24 |
A. phagocytophilum | |||
P130 | Unique to A. phagocytophilum; localizes to the vacuolar membrane | 60.9 (78/128) | 10 |
P44 | Porin protein involved in immune evasion; homologs are found in A. platys and A. marginale |
72.7 (93/128) | 9 |
Aph_1235 | Specific to the infectious dense core cell | 3.3 (3/90) | 25 |
Asp14 | Adhesin; homologs are found in some Anaplasma and Ehrlichia species |
7.8 (7/90) | 26 |
OmpA | Adhesin; homologs are found in some Anaplasma and Ehrlichia species |
18.9 (17/90) | 26 |
FhbB | T. denticola FH binding protein; negative control | 0 (0/128) | 13 |
The ORF designations listed are those assigned to B. burgdorferi strain B31.
TABLE 3.
Parameter | % Ab positive (no. of positive samples/total no.) for: |
|
---|---|---|
B. burgdorferi | A. phagocytophilum | |
Gender | ||
Male | 65.6 (42/64) | 79.7 (51/64) |
Female | 65.4 (36/55) | 70.9 (39/55) |
Not reported | 55.6 (5/9) | 33.3 (3/9) |
Developmental stage | ||
Adult | 71.8 (46/64) | 75.0 (48/64) |
Subadult | 69.4 (25/36) | 75.0 (27/36) |
Juvenile | 80.0 (16/20) | 75.0 (15/20) |
Not reported | 62.5 (5/8) | 37.5 (3/8) |
State sector | ||
Northwest | 63.6 (21/33) | 57.6 (19/33) |
North Central | 75.9 (22/29) | 86.2 (25/29) |
Northeast | 82.9 (29/35) | 97.1 (34/35) |
Southwest | 58.8 (10/17) | 52.9 (9/17) |
South Central | 100 (5/5) | 60.0 (3/5) |
Southeast | 1/1a | 0/1a |
Not reported | 62.5 (5/8) | 37.5 (3/8) |
Collection yr | ||
2015 | 68.8 (42/61) | 68.9 (42/61) |
2017 | 62.1 (41/66) | 76.1 (51/66) |
Total for 2015 and 2017 | 64.8 (83/128) | 72.7 (93/128) |
Total for B. burgdorferi and A. phagocytophilum | 51.5 (72/128) |
aDue to sample size limitations, percentages would not be valid and thus were not calculated.
To screen for Abs to well characterized A. phagocytophilum proteins, recombinant P44, P130, Asp14, Aph_1235, and OmpA were generated and screened by dot blotting. Consistent with the results of the cell lysate immunoblot assays and with earlier reports that P44 is an immunodominant antigen (9), 72.7% of the plasma samples were P44 Ab positive. Ab to P130 and OmpA was detected in 60.9 and 18.9% of the plasma samples, respectively. Abs to the other proteins tested were detected in a low percentage of plasma samples (Table 2). It is important to note that Anaplasma platys, which also infects dogs and is transmitted by Rhipicephalus sanguineus ticks, produces homologs of P44. While there is significant sequence divergence between the A. phagocytophilum and A. platys P44 proteins, we cannot rule out the possibility that some animals had been infected or exposed to A. platys. However, in contrast to P44, P130 is unique to A. phagocytophilum, and thus Ab to P130 is a clear indicator of exposure to A. phagocytophilum. Note that clinical samples that may have allowed for a direct assessment of whether the individual animals were actively infected at the time of harvest were not available for analysis. However, the strong immunoreactivity of a majority of plasma samples with the cell lysates or recombinant proteins is consistent with either an active or recent infection in the animals at the time of harvest.
In summary, this study demonstrates that eastern coyotes have significant exposure to the causative agents of Lyme disease and anaplasmosis. The lifestyle habits of eastern coyotes would most certainly allow for frequent exposure to all developmental stages of Ixodes ticks, including larvae. This raises important questions as to the potential for coyotes to serve as reservoirs for tick-borne pathogens. While it remains to be determined if coyotes and other predators are competent reservoirs, if that proves to be the case, the results presented here have implications for the potential limitations of bait vaccine development efforts that are focused largely on targeting mice. As coyotes become increasingly urbanized and interact with humans, domestic canids, and suburban/urban wildlife, knowledge about the pathogens they may carry is important for understanding their potential contribution to the enzootic cycle of tick-borne pathogens.
ACKNOWLEDGMENTS
This study was supported in part through NIH grants R01 AI141801 (R.T.M. and J.A.C.) and R01 AI072683 (J.A.C. and R.T.M.), the Steven and Alexandra Cohen Foundation (R.T.M.), the Bay Area Lyme Foundation (R.T.M.), Zoetis (R.T.M.), and the VCU School of Medicine and VCU Health under the Value and Efficiency Teaching and Research (VETAR) program (R.T.M.).
REFERENCES
- 1.Eisen RJ, Eisen L, Beard CB. 2016. County-scale distribution of Ixodes scapularis and Ixodes pacificus (Acari: Ixodidae) in the continental United States. J Med Entomol 53:349–386. doi: 10.1093/jme/tjv237. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Sykes RA, Makiello P. 2017. An estimate of Lyme borreliosis incidence in Western Europe. J Public Health (Oxf) 39:74–81. doi: 10.1093/pubmed/fdw017. [DOI] [PubMed] [Google Scholar]
- 3.Gasmi S, Ogden NH, Lindsay LR, Burns S, Fleming S, Badcock J, Hanan S, Gaulin C, Leblanc MA, Russell C, Nelder M, Hobbs L, Graham-Derham S, Lachance L, Scott AN, Galanis E, Koffi JK. 2017. Surveillance for Lyme disease in Canada: 2009–2015. Can Commun Dis Rep 43:194–199. doi: 10.14745/ccdr.v43i10a01. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Ostfeld RS, Levi T, Keesing F, Oggenfuss K, Canham CD. 2018. Tick-borne disease risk in a forest food web. Ecology 99:1562–1573. doi: 10.1002/ecy.2386. [DOI] [PubMed] [Google Scholar]
- 5.Brisson D, Dykhuizen DE, Ostfeld RS. 2008. Conspicuous impacts of inconspicuous hosts on the Lyme disease epidemic. Proc Biol Sci 275:227–235. doi: 10.1098/rspb.2007.1208. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Levi T, Kilpatrick AM, Mangel M, Wilmers CC. 2012. Deer, predators, and the emergence of Lyme disease. Proc Natl Acad Sci U S A 109:10942–10947. doi: 10.1073/pnas.1204536109. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Hody JW, Kays R. 2018. Mapping the expansion of coyotes (Canis latrans) across North and Central America. Zookeys 759:81–97. doi: 10.3897/zookeys.759.15149. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Newsome TM, Ripple WJ. 2015. A continental scale trophic cascade from wolves through coyotes to foxes. J Anim Ecol 84:49–59. doi: 10.1111/1365-2656.12258. [DOI] [PubMed] [Google Scholar]
- 9.Caspersen K, Park JH, Patil S, Dumler JS. 2002. Genetic variability and stability of Anaplasma phagocytophila msp2 (p44). Infect Immun 70:1230–1234. doi: 10.1128/iai.70.3.1230-1234.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Huang B, Troese MJ, Howe D, Ye S, Sims JT, Heinzen RA, Borjesson DL, Carlyon JA. 2010. Anaplasma phagocytophilum APH_0032 is expressed late during infection and localizes to the pathogen-occupied vacuolar membrane. Microb Pathog 49:273–284. doi: 10.1016/j.micpath.2010.06.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Samuels DS. 2011. Gene regulation in Borrelia burgdorferi. Annu Rev Microbiol 65:479–499. doi: 10.1146/annurev.micro.112408.134040. [DOI] [PubMed] [Google Scholar]
- 12.Caimano MJ, Groshong AM, Belperron A, Mao J, Hawley KL, Luthra A, Graham DE, Earnhart CG, Marconi RT, Bockenstedt LK, Blevins JS, Radolf JD. 2019. The RpoS gatekeeper in Borrelia burgdorferi: an invariant regulatory scheme that promotes spirochete persistence in reservoir hosts and niche diversity. Front Microbiol 10:1923. doi: 10.3389/fmicb.2019.01923. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.McDowell JV, Frederick J, Stamm L, Marconi RT. 2007. Identification of the gene encoding the FhbB protein of Treponema denticola, a highly unique factor H-like protein 1 binding protein. Infect Immun 75:1050–1054. doi: 10.1128/IAI.01458-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Huang B, Troese MJ, Ye S, Sims JT, Galloway NL, Borjesson DL, Carlyon JA. 2010. Anaplasma phagocytophilum APH_1387 is expressed throughout bacterial intracellular development and localizes to the pathogen-occupied vacuolar membrane. Infect Immun 78:1864–1873. doi: 10.1128/IAI.01418-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Izac JR, Camire AC, Earnhart CG, Embers ME, Funk RA, Breitschwerdt EB, Marconi RT. 2019. Analysis of the antigenic determinants of the OspC protein of the Lyme disease spirochetes: evidence that the C10 motif is not immunodominant or required to elicit bactericidal antibody responses. Vaccine 37:2401–2407. doi: 10.1016/j.vaccine.2019.02.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Bergström S, Bundoc VG, Barbour AG. 1989. Molecular analysis of the linear plasmid-encoded major surface proteins, OspA and OspB, of the Lyme disease spirochete Borrelia burgdorferi. Mol Microbiol 3:479–486. doi: 10.1111/j.1365-2958.1989.tb00194.x. [DOI] [PubMed] [Google Scholar]
- 17.Rogovskyy AS, Casselli T, Tourand Y, Jones CR, Owen JP, Mason KL, Scoles GA, Bankhead T. 2015. Evaluation of the importance of VlsE antigenic variation for the enzootic cycle of Borrelia burgdorferi. PLoS One 10:e0124268. doi: 10.1371/journal.pone.0124268. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Imai DM, Samuels DS, Feng S, Hodzic E, Olsen K, Barthold SW. 2013. The early dissemination defect attributed to disruption of decorin-binding proteins is abolished in chronic murine Lyme borreliosis. Infect Immun 81:1663–1673. doi: 10.1128/IAI.01359-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Metts MS, McDowell JV, Theisen M, Hansen PR, Marconi RT. 2003. Analysis of the OspE determinants involved in binding of factor H and OspE-targeting antibodies elicited during Borrelia burgdorferi infection in mice. Infect Immun 71:3587–3596. doi: 10.1128/iai.71.6.3587-3596.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Marconi RT, Sung SY, Hughes CA, Carlyon JA. 1996. Molecular and evolutionary analyses of a variable series of genes in Borrelia burgdorferi that are related to ospE and ospF, constitute a gene family, and share a common upstream homology box. J Bacteriol 178:5615–5626. doi: 10.1128/jb.178.19.5615-5626.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.McDowell JV, Sung SY, Price G, Marconi RT. 2001. Demonstration of the genetic stability and temporal expression of select members of the Lyme disease spirochete OspF protein family during infection in mice. Infect Immun 69:4831–4838. doi: 10.1128/IAI.69.8.4831-4838.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Yang XF, Hubner A, Popova TG, Hagman KE, Norgard MV. 2003. Regulation of expression of the paralogous Mlp family in Borrelia burgdorferi. Infect Immun 71:5012–5020. doi: 10.1128/iai.71.9.5012-5020.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Fikrig E, Barthold SW, Sun W, Feng W, Telford SR, III, Flavell RA. 1997. Borrelia burgdorferi P35 and P37 proteins expressed in vivo, elicit protective immunity. Immunity 6:531–539. doi: 10.1016/S1074-7613(00)80341-6. [DOI] [PubMed] [Google Scholar]
- 24.Kariu T, Sharma K, Singh P, Smith AA, Backstedt B, Buyuktanir O, Pal U. 2015. BB0323 and novel virulence determinant BB0238: Borrelia burgdorferi proteins that interact with and stabilize each other and are critical for infectivity. J Infect Dis 211:462–471. doi: 10.1093/infdis/jiu460. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Troese MJ, Kahlon A, Ragland SA, Ottens AK, Ojogun N, Nelson KT, Walker NJ, Borjesson DL, Carlyon JA. 2011. Proteomic analysis of Anaplasma phagocytophilum during infection of human myeloid cells identifies a protein that is pronouncedly upregulated on the infectious dense-cored cell. Infect Immun 79:4696–4707. doi: 10.1128/IAI.05658-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Kahlon A, Ojogun N, Ragland SA, Seidman D, Troese MJ, Ottens AK, Mastronunzio JE, Truchan HK, Walker NJ, Borjesson DL, Fikrig E, Carlyon JA. 2013. Anaplasma phagocytophilum Asp14 is an invasin that interacts with mammalian host cells via its C terminus to facilitate infection. Infect Immun 81:65–79. doi: 10.1128/IAI.00932-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.