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Competence of American Robins as Reservoir Hosts for Lyme Disease

Spirochetes

Dania Richter,*† Spielman,* Komar,* Franz-Rainer Matuschka*†

* Harvard School of Public Health, Boston, Massachusetts, USA

† Charité, Medizinische Fakultät der Humboldt-Universität zu Berlin,

Germany

[Emerging Infectious Diseases 6(2), 2000. Centers for Disease Control]

Abstract

To explore the competence of American robins as a reservoir for Lyme disease

spirochetes, we determined the susceptibility of these birds to tickborne

spirochetes and their subsequent infectivity for larval vector ticks. Robins

acquired infection and became infectious to almost all xenodiagnostic ticks

soon after exposure to infected nymphal ticks. Although infectivity waned

after 2 months, the robins remained susceptible to reinfection, became

infectious again, and permitted repeated feeding by vector ticks. In

addition, spirochetes passaged through birds retained infectivity for

mammalian hosts. American robins become as infectious for vector ticks as do

reservoir mice, but infectivity in robins wanes more rapidly.

Introduction

Lyme disease spirochetes (Borrelia burgdorferi s.l.) perpetuate in cycles

involving rodent reservoir hosts, such as white-footed mice (Peromyscus

leucopus) in eastern North America[1] and Apodemus spp. mice in Eurasia[2].

Such hosts readily become infectious to vector ticks and appear to remain so

lifelong. They serve as natural reservoirs of infection because numerous

subadult vector ticks parasitize them, they are abundant, and they remain in

constant residence in tick-infested sites. Rats (Rattus norvegicus and R.

rattus)[3,4] and European dormice (Glis glis and Eliomys quercinus)[5,6] are

similarly competent and locally important hosts to these pathogens. Although

hares (Lepidus timidus) support infection with Lyme disease spirochetes in

Europe[7], the competence of rabbits for these spirochetes has not been

proven. A related spirochete, B. andersonii, appears to be specific to

American rabbits[8,9]. Ungulates, however, are not competent as hosts for

Lyme disease spirochetes. Deer in intensely enzootic sites fail to infect

vector ticks[10,11], and cattle and sheep exposed to infected vector ticks

never become infectious[12]. Lyme disease spirochetes infect diverse

mammals, but not all of them serve as competent hosts.

Certain birds may also contribute to the transmission of Lyme disease

spirochetes. Numerous vector ticks parasitize birds in nature, including

spirochete-infected larval ticks that presumably acquired infection from

these birds[13-19]. Seabirds appear to maintain such a cycle on Arctic

islands[20]. In an enzootic site, American robins (Turdus migratorius) are

considered to be an avian candidate as a reservoir of infection because they

are locally abundant, forage in ground and brush vegetation, and are

frequently infested by vector ticks[21]. Catbirds (Dumetella carolinensis)

in enzootic sites, however, appear not to infect vector ticks[22]. Although

spirochetes have been isolated from naturally infected European blackbirds

(Turdus merula)[15], a laboratory study failed to demonstrate reservoir

competence of these birds[23]; the reason for this discrepancy remains

unclear. Nymphal vector ticks occasionally become infected after feeding on

spirochete-exposed pheasants (Phasianus colchicus); these birds, however,

cannot contribute to transmission because larval ticks seem not to feed on

them, either in the laboratory or in nature[24,25]. Chickens (Gallus gallus)

become infectious, but only transiently and only when they are about a week

old[26]. Spirochetes inoculated by syringe can be detected in various

tissues of canaries (Serinus canaria), bobwhite quail (Colinus virginianus),

and Japanese quail (Coturnix coturnix)[27-29]. The competence of candidate

reservoir birds as hosts for tickborne spirochetes has not been thoroughly

evaluated.

Tickborne Lyme disease spirochetes may readily infect certain birds, and

these birds may subsequently infect numerous vector ticks. Therefore, we

determined the susceptibility of American robins to tickborne spirochetes

and their subsequent infectivity for larval deer ticks (Ixodes dammini,

which differ from I. scapularis[30]). American robins are suitable candidate

hosts because they are infested naturally by numerous vector ticks. We

evaluated the reservoir competence of captive robins and determined whether

they could subsequently be reinfested and reinfected by vector ticks and

whether spirochetes passaged through birds remain infective to rodents.

Materials and Methods

American robins were captured in mist nets in Brookline, MA (U.S. Fish and

Wildlife Scientific Collecting Permit# MB719506-0), and maintained at 20°

with a photoperiod of 16:8 hrs (L:D) (Animal Welfare Assurance# A-3431-01).

To establish that these birds had not already been infected by Lyme disease

spirochetes, nymphs derived from xenodiagnostic larval deer ticks that had

engorged on them were examined by dark-field microscopy. No

spirochete-infected ticks were found. The birds were held in captivity for 6

months before they were exposed to infected ticks.

The colony of laboratory-raised deer ticks was originally isolated from

ticks captured in Ipswich, MA, and was maintained by feeding adults on

rabbits and subadults on outbred laboratory mice. To infect ticks, larvae

were fed on outbred laboratory mice infected with the N40 strain of B.

burgdorferi s.s. The resulting infected nymphal ticks were used to infect

the robins. Xenodiagnosis was used to determine whether host animals had

become infected by spirochetes; larval ticks were permitted to feed on them

and the resulting nymphs were examined for spirochetes by dark-field

microscopy. The larval ticks used for the xenodiagnosis were in their third

generation of continuous laboratory breeding and had not previously been

exposed to spirochete-infected hosts. Engorged ticks were held at 20±2° over

supersaturated MgSO4 in sealed desiccator jars with 16 hours of light per

day. Hatched or molted ticks remained in screened vials until they were

placed on hosts 2-4 months later.

To infest the robins with subadult ticks, each bird was restrained while

ticks were brushed onto its head and neck. Each bird was then placed in a

cotton bag suspended over water and kept in the dark for approximately 3

hours to limit their activity. The birds were then caged individually over

pans of water in cages with wire-mesh floors and fronts (Safeguard Products,

New Holland, PA). The contents of the pans were inspected twice a day, and

detached ticks were removed promptly. The birds were exposed three times to

nymphal ticks to test their susceptibility to repeated feeding (Figure 1).

The first and last of these infestations also served to infect them.

Xenodiagnostic larval ticks were permitted to feed on these robins nine

times during the study.

Figure 1. American robins with attached nymphal deer ticks (Ixodes dammini).

Results

To evaluate the reservoir competence of birds for Lyme disease spirochetes,

we exposed each of four American robins to the bites of 12

spirochete-infected nymphal deer ticks and determined the duration of their

subsequent infectivity for larval deer ticks. The presence of spirochetes in

these infecting nymphs was verified by dark-field microscopic examination of

the gut contents of the resulting adults; virtually all (97%) contained

spirochetes. Birds were exposed initially to xenodiagnostic larvae at the

same time they were exposed to the infecting nymphs. Xenodiagnosis was

repeated after two days and at intervals for six months. Although larval

ticks feeding simultaneously with the infected nymphs failed to acquire

spirochetes, two birds infected xenodiagnostic larvae within 6 days after

exposure to infected nymphs (Figure 2). All four birds were infectious to

xenodiagnostic ticks that detached 12 days after exposure and infected 88%

(±6.6) of ticks for at least another 3 weeks (Figure 2). Infectivity waned

by 2 months and disappeared by 6 months. Almost all the ticks became

infected that fed 2 and 4 weeks after the birds were infected (Table 1).

American robins are fully but transiently competent as hosts for Lyme

disease spirochetes.

Figure 2. Infectivity to larval vector ticks of four American robins exposed

to nymphal deer ticks infected with Lyme disease spirochetes on days 0 and

186. Each observation was recorded on the day on which each xenodiagnostic

test was complete.

We then determined whether American robins tolerate reinfection by tickborne

spirochetes. Six months after they were initially infected, when vector

ticks no longer acquired spirochetes from them, eight infected nymphs were

permitted to engorge on each bird. All four robins regained infectivity for

ticks within the next 2 weeks (Figure 2). Virtually all xenodiagnostic

larval ticks that fed on three of these reinfected birds acquired

spirochetes, and a third of them did so on the fourth bird. Although

spirochete-infected robins only transiently infect vector ticks, the birds

remain tolerant to reinfection.

The ability of nymphal deer ticks to reinfest American robins was evaluated

by recording their feeding success. On initial exposure, 96% of 48 nymphs

feeding on the four birds engorged successfully; 98% of 40 nymphs engorged 3

weeks later; and 82% of 32 nymphs engorged 6 months later, when the birds

were exposed for the third time. The robins appear to freely tolerate

repeated feeding by nymphal deer ticks.

To determine whether spirochetes passaged through birds retain infectivity

for mice, we permitted nymphs that had acquired infection as larvae from

birds to feed on mice. The ticks that were used had acquired spirochetes 3

weeks after their avian hosts had been infected. Each of four outbred

laboratory mice was exposed to six of the resulting nymphs. Two weeks later,

10 xenodiagnostic larval ticks were permitted to engorge on each of these

mice. All but one of the 40 resulting nymphal ticks acquired spirochetes

(Table 2). Spirochetes, therefore, remain infectious to mammals after a

tickborne passage through birds.

Discussion

A competent reservoir host for the agent of Lyme disease readily acquires

infection from vector ticks, permits spirochetes to proliferate, and readily

infects vector ticks. In white-footed mice, infection generally becomes

established after a single feeding by an infectious tick, and more than half

retain infection for approximately 6 months[31]. As many as three-quarters

of the larval ticks that feed on such mice acquire infection, which

subsequently wanes. Hamsters are similarly infectious[32]. The various

murids, glirids, voles, and sciurids that have been tested appear as

reservoir-competent for spirochetes as are cricetids[2,5,6,33]. Although

certain genospecies of the Lyme disease spirochetes are said to be more

mouse-adapted than others[34], no experimental evidence is available to

support this concept. Rodents, in general, are readily infected by Lyme

disease spirochetes, and most remain infectious to vector ticks for at least

6 months.

The standard of proof that has been applied to rodent reservoirs of

spirochetal infection has not previously been applied to candidate avian

reservoirs. Certain birds, including quail and canaries, can readily be

infected by syringe-inoculated spirochetes. Spirochetal DNA generally

becomes detectable in the viscera[27-29], and viable spirochetes can be

cultured from these tissues. Tickborne spirochetes infect pheasants[24] and

week-old chickens[26]. About a third of these chicks infect ticks, but they

remain infectious for only a week. Adult pheasants infect about a quarter of

vector ticks that engorge on them. European blackbirds appear not to be

susceptible to tickborne infection[23]. American robins, however, appear far

more reservoir-competent. Robins bitten by spirochete-infected nymphal ticks

subsequently infect larval ticks, and virtually all larvae become infected

after feeding on these birds throughout the following month. Infectivity

declines, however, and few ticks acquire infection from these birds after

approximately 3 months. Certain, but not all, bird species can readily be

infected by Lyme disease spirochetes and subsequently become highly but

transiently infectious.

A competent reservoir host tolerates feeding by both subadult stages of

vector ticks. Although nymphs feed readily on pheasants, larvae seldom

attach to these birds, either in nature or in the laboratory[24]. About as

many nymphal as larval deer ticks attach naturally to American robins[21].

Several times as many larvae as nymphs, however, feed on white-footed mice

in nature[35]. Nymphal vector ticks attach more readily to rats, dormice, or

the American reservoir host (P. leucopus) than to European mice (Apodemus

spp.) or voles[2,3,5,31]. Perhaps particular subadult stages of vector ticks

are more readily attracted to certain vertebrate animals than to others.

To be competent as reservoir hosts for Lyme disease spirochetes, an animal

must tolerate repeated feedings by vector ticks. White-footed or laboratory

mice serve as hosts to deer ticks almost as readily during their fifth

exposure as their first[36]. Voles are much less tolerant[37], and rabbits

(unpub. obs.) and guinea pigs[38] seldom tolerate more than one episode of

attachment. Vector ticks feed repeatedly on American robins as successfully

as on the natural rodent reservoirs of the Lyme disease spirochete.

Spirochete-infected larval ticks frequently are taken from birds in Lyme

disease-enzootic areas. Of 20 species of North American passerine birds that

were parasitized by larval vector ticks, 45% hosted spirochete-infected

larvae[13,18]. Spirochetes infected 10% to 40% of these batches of infected

ticks. In Europe, 81% of 16 bird species harbored infected ticks; of these

cohorts, 11% to 75% of the larval ticks were infected[17]. Because Lyme

disease spirochetes infect <1% of questing larval vector ticks captured in

nature and because inherited infection is exceedingly rare[39,40], these

larval ticks most likely acquired spirochetes from their avian hosts.

Nevertheless, the avian host-range of Lyme disease spirochetes remains to be

defined.

An effective reservoir of infection for a pathogen would generate for each

primary infection at least as many secondary infections. The contribution of

various alternative reservoir hosts for Lyme disease spirochetes, however,

can be compared by estimating the proportion of infections in nymphal vector

ticks that derive from a particular vertebrate population. This calculation

includes estimates of feeding density of vector ticks on the candidate

reservoir host, its local density, and its prevalence of infection[2,5]. At

least twice as many vector ticks appear to acquire infection from a

population of edible dormice (Glis glis) as from other rodents locally

abundant in Central Europe[5]. Similar evidence implicates white-footed mice

in eastern North America[1]. Information that would permit comparable

estimates of the reservoir significance of local populations of birds,

however, remains unavailable. Thus, it is still uncertain whether a bird,

although reservoir-competent for the spirochete, may serve as an important

reservoir in nature.

Because the prevalence of Lyme disease spirochetes in American robins has

not yet been determined for a representative enzootic site, the importance

of these birds as reservoir hosts remains speculative. Robins may contribute

to the force of transmission of the agent of Lyme disease, because these

locally abundant birds are reservoir-competent and may be infested by

numerous larval ticks. Of the larvae that feed on peridomestic birds in

North American enzootic sites, approximately three-quarters appear to

engorge on robins[21]. Although they may be about as abundant as

white-footed mice, robins appear to forage more frequently on lawns than do

mice[21]. The contribution of robins to the peridomestic risk for Lyme

disease may depend on the ability of engorged larval ticks detaching from

the birds to develop in such habitat. Questing nymphal ticks appear less

prevalent on lawns than on other vegetation in residential enzootic

sites[41]. Birds are more vagile than mice, which might dilute risk by

diffusing infected ticks into sites in which transmission is unlikely.

Alternatively, their vagility might seed new foci of transmission, if ticks

detach in sites that are suitable for their development. Certain migratory

passerines have been associated with long-distance dispersal of vector

ticks[17-19]. Robins may contribute to the emergence of Lyme disease in

previously unaffected sites to the extent that the season of their migration

overlaps with that of the activity of subadult vector ticks. Our finding

that American robins are reservoir-competent for Lyme disease spirochetes

warrants further epizootiologic studies, including estimates of the

prevalence of spirochetes in these birds in nature.