Epidemic spread of Lyme

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Epidemic spread of Lyme borreliosis, Northeastern United States

by Klara Hanincova, Emerging Infectious Diseases > April, 2006

We examined the degree of host specialization of different strains

of Borrelia burgdorferi, the tickborne pathogen that causes Lyme

borreliosis in the northeastern United States. We first assessed the

genetic population structures of B. burgdorferi in ticks obtained

from different mammalian host species and in questing ticks sampled

in a woodland ecosystem in Connecticut. By comparing the patterns

found in our study with data from another cross-sectional study, we

demonstrate that B burgdorferi is a generalist microparasite and

conclude that efficient cross-species transmission of B. burgdorferi

is a key feature that has allowed the rapid spread of Lyme

borreliosis across the northeastern United States.

The evolution of specialization remains a major problem in ecology

and evolutionary biology; why some species are generalists and

others are specialists is not resolved (1,2). Like all organisms,

parasites have evolved to different levels of ecologic

specialization (3-5). The level of host specialization of parasites

is a key issue in infectious disease research because patterns of

cross-species transmission affect parasite dispersal and can

facilitate epidemics. West Nile virus is a recent example

illustrating that the utilization of many highly mobile host species

can enable a pathogen to disperse across an entire continent within

a few years (6). Multihost parasites are usually considered to be

generalists; however, this is not universally true, and several

examples exist in which generalist parasites are structured into

subpopulations that are host specialized (7). Theory predicts that

natural selection favors host specialization if hosts are abundant

and predictable, whereas generalist strategies evolve if hosts are

erratic (8).

Borrelia burgdorferi, the spirochetal agent of Lyme borreliosis (LB)

in the United States, is a tickborne zoonotic pathogen that infects

an expansive range of vertebrate species, involving diverse

mammalian and avian hosts (9-14). For this reason, it has been

suggested B. burgdorferi is likely less specialized than the other

genospecies that cause LB in Eurasia (15-18). Several loci of B.

burgdorferi are polymorphic (19), and balancing selection seems to

maintain the bacterium's diversity (20). Given the pronounced strain

structure of this bacterial species, natural selection possibly has

driven B. burgdorferi towards host specialization, and different

spirochete strains exploit different sets of vertebrate hosts (4,13).

The issue of vertebrate host specialization of B. burgdorferi is of

substantial public health importance. Since the reemergence of LB 3

decades ago, the disease has been spreading across the entire

northeastern United States and beyond (21,22). A condition necessary

for this dispersal has been the geographic expansion of its

principal and generalist tick vector, Ixodes scapularis. This

expansion is believed to be driven by large-scale reforestation and

an explosive growth of deer populations (21). Deer, however, do not

contribute directly to the dispersal of B. burgdorferi (23). Only

hosts that can infect ticks affect spirochete migration. If B.

burgdorferi were host specialized, the strains of this microparasite

would migrate differentially, resulting in geographic structuring of

this pathogen. Unrestricted cross-species transmission, in contrast,

would generate a spatially uniform population structure of B.

burgdorferi and substantially facilitate its dispersal. Information

on the level of host specialization of this multihost pathogen is

required to understand the patterns and mechanisms of the current

spread of LB.

We examined the level of host specialization of B. burgdorferi in

the northeastern United States by using a comparative approach. We

first assessed the genetic population structures of B. burgdorferi

in ticks obtained from different mammalian host species and in

questing ticks sampled in a woodland ecosystem at Lake Gaillard,

Branford, Connecticut. By comparing the patterns in our study with

data from another cross-sectional study carried out in a similiar

ecosystem in Millbrook, New York (13), we aimed to capture patterns

of cross-species transmission and to identify the niche breadth of

the various genotypes of B. burgdorferi.

Materials and Methods

Mammal Sampling

The fieldwork was carried out at Lake Gaillard (41[degrees]34'N, 72

[degrees]77'W), Connecticut, as described previously (24). Mammals

were captured alive at 2-week intervals from early June until late

August in 2002 and until mid-September in 2003. All trapping and

handling procedures were approved by the Yale University

Institutional Animal Care and Utilization Committee (Study Protocol

07596). Small mammals were trapped for 23 days/nights (432 trap

nights) using Sherman (Tallahassee, FL, USA) traps. In addition,

Pitfall traps were set up for 14 days/nights (98 trap nights) in

2003. Medium-sized mammals were captured for 27 days/nights (820

trap nights) and 25 days/nights (724 trap nights) using Tomahawk

(Tomahawk, WI, USA) traps no. 205 and no. 207, respectively. All

captured mammals were housed over pans of water for 72 hours to

recover engorged ticks. Ticks were allowed to molt to the next

developmental stage, determined to species, and stored in 70%

ethanol. Mammals were marked, sexed, and measured. Before handling,

mammals were anesthetized with ketamine hydrochloride or a

combination of ketamine hydrochloride and xylazine. After captivity,

mammals were released at their original location.

Host-seeking Ticks

Questing I. scapularis nymphs were collected over the same period

and in the same area where the mammals were captured by dragging the

vegetation with 1-[m.sup.2] drag cloths. Collected ticks were

preserved in 70% ethanol.

DNA Extraction and PCR

DNA was extracted from ticks according to a DNeasy Tissue Kit

protocol (Qiagen, Valencia, CA, USA) as described previously (25).

Ticks were screened for B. burgdorferi DNA by real-time Taqman

polymerase chain reaction (PCR) targeting the 16S rDNA of B.

burgdorferi (24). Positive samples were then subjected to a nested

PCR amplifying a fragment of the rrs (16S)-rrl (23S) intergenic

spacer of B. burgdorferi and sequenced (19).

Data Analysis

Infectivity of hosts to ticks was determined by identifying B.

burgdorferi in molted nymphs derived from mammals. Since

transovarial transmission of B. burgdorferi to larval I. scapularis

has not been demonstrated, infections found in molted nymphs were

assumed to be acquired from a host through feeding. A mammal,

therefore, was considered infectious to ticks if [greater than or

equal to] 1 nymphs that had fed, as larva, on that mammal tested

positive.

To evaluate the exposure of animals to infected nymphs for each host

species, the attachment rate of nymphs per animal per day (R

[D.sub.s]) was computed for each capture time point as R[D.sub.s]=

A/F; A is the mean number of feeding ticks per host, and F is the

average feeding time of I. scapularis nymphs which was

conservatively assumed to be 5 days (26). The minimum attachment

rate of nymphs per animal per season (R[s.sub.s]) was computed as R

[s.sub.s] = Z (R[D.sub.s] x C); C is the number of days between

capture points. The number of nymphs infected with a genotype

encountered by a host per season (RS[i.sub.s]) was calculated as RS

[i.sub.s] = IP/N x R[D.sub.s]; IP is the infection prevalence of a

genotype in field-collected questing nymphs, and N is the number of

nymphs tested. Since no data for May were obtained empirically, we

extrapolated the data on nymphal infestation obtained at the end of

the nymphal peak activity (i.e., end of June) and applied it to May.

This provided a conservative estimate of the total number of

infected nymphs a host encountered over the nymphal activity season.

Statistical Analysis

Differences in mean numbers of ticks per host were examined by using

the nonparametric Kruskal-Wallis test. Logistic regression was used

to estimate the infection prevalences in ticks or hosts and to

compare them among host species. Presence of B. burgdorferi

infection in a tick or host was the response variable in the model,

and a dummy variable for host species was used as the predictor. The

advantage of using logistic regression models for proportional data

is that different coding systems can be applied to compare infection

prevalence among various groupings of host species (e.g., mice

versus other hosts). Additionally, logistic models can control for

the fact that several ticks were collected from the same mammal and

were not independent samples. In this analysis, a cross-sectional

procedure (Stata xtlogit) was applied to control for the correlation

among ticks collected from the same mammal (27). To test for a

sample size effect on the number of genotypes found in a host

species, a Spearman rank correlation was performed between the

number of genotypes and the number of mammals sampled for each host

species. The differences in genotype frequency distributions were

estimated through exact nonparametric inference by the Fisher-

Freeman-Halton test (Monte Carlo testing). Pearson's [chi square]

test was used to compare the proportions of ticks infected with

different genotypes within and among host species. Data were

analyzed with Stata, version 8, (Stata Corporation, College Station,

TX, USA) and StataXact, version 6, (Cytel, San Diego, CA, USA).

Results

Mammal Trapping

Sampling over 2 years yielded 403 captures that included 222

individual mammals, representing 9 mammalian species of 6 families

(Muridae, Soricidae, Sciuridae, Mustelidae, Procyonidae, and

Didelphiidae) belonging to 4 orders (Rodentia, Insectivora,

Carnivora, and Marsupialia). Six species (white-footed mouse, pine

vole, eastern chipmunk, gray squirrel, Virginia opossum, and

raccoon) accounted for 98% of all mammals caught (Table 1).

Tick Infestation

Altogether, 9,032 immature ticks were collected from 399 captured

hosts. The most abundant tick species, I. scapularis, represented

99% (7,611 larvae and 1,373 nymphs) of all ticks examined. The

additional 3 species, I. texanus, Dermacentor variabilis, and

Amblyomma maculatum, comprised the remaining 1% and were omitted

from further analysis. The mean numbers of I. scapularis ticks per

host varied significantly among mammalian species for both larvae

and nymphs (Table 1).

B. burgdorferi Prevalence in Host-derived Ticks

Of the nymphs sampled from 62 mammals as engorged larvae, 1,117

specimens were screened for presence of B. burgdorferi. The number

of tested nymphs per host varied from 1 to 51, depending mainly on

the number of engorged larvae recovered. B. burgdorferi-positive

ticks were obtained from all 6 mammalian species.

Infection prevalence of B. burgdorferi in animals varied

significantly among host species (logistic regression, [chi square]

= 14.15, p<0.01) (Table 2). Each of the 3 tested chipmunks produced

[greater than or equal to] 1 infected nymphs and, therefore, this

species was excluded from the logistic regression model, since the

presence of a zero category (noninfectious chipmunks) produced

infinite odds ratios (OR), which precluded the estimation of the

model. No significant differences were found between voles,

squirrels, raccoons, and opossums. Hence, these species were pooled

and compared with mice. The proportion of infectious mice was

significantly higher than that of the pooled group of other host

species (logistic regression, OR 13.42, 95% confidence interval (el)

1.63-110.41, p<0.001).

Infection prevalences of B. burgdorferi in host-derived ticks also

varied significantly among host species (logistic regression, [chi

square] = 42.38, p<0.001) (Table 3). A considerably higher infection

prevalence in ticks was observed for mice than for voles (logistic

regression, OR 16.37, 95% CI 4.73-56.69, p<0.001). On the other

hand, no significant differences in tick infection prevalence were

found among raccoons, opossums, squirrels, and chipmunks. Therefore,

data for these host species were pooled into 1 group. Infection

prevalence in ticks from mice was significantly higher than in ticks

from the pooled group (logistic regression, OR 47.89, 95% CI 14.97-

153.23, p<0.001), as was infection prevalence in ticks from voles

compared to the pooled group (logistic regression, OR 2.92, 95% CI

1.16 7.34, p<0.001).

Population Structure of B. burgdorferi in Host-derived Ticks

A total of 205 B. burgdorferi infections in nymphs obtained from

mammals as engorged larvae could be sequenced successfully. The IGS

alleles were assigned to previously identified multilocus genotypes

(19), designated here as genotypes 1 to 9. A total of 8 genotypes

was shown (Tables 2-4). The white-footed mouse was the only host

species that transmitted all 8 genotypes to ticks. None of the

genotypes was transmitted by all host species. However, genotypes 1-

5 and 7 were found in ticks collected from as many as 5 host species

belonging to 3 different orders (Rodentia, Carnivora, Marsupialia).

Only mice were found to be infectious for genotype 6. No significant

relationship was found between the number of genotypes and the

number of sampled individuals of a host species (Spearman rank

correlation, [r.sub.s] = 0.5, p>0.05). The frequency distribution of

transmitted genotypes differed significantly among host species (6 x

8 Fisher-Freeman-Halton test, Fisher statistic = 41.93; Monte Carlo

p 0.05) (Table 2). The average number of genotypes per infectious

host was 2.4 (standard error [sE] = 0.3) for mice, 1.7 (SE = 0.3)

for opossums, 1.7 (SE = 0.7) for raccoons, 1.5 (SE = 0.5) for

squirrels, 1.3 (SE = 0.2) for voles, and 1.3 (SE = 0.3) for

chipmunks.

The frequency distributions of genotypes in host-derived ticks are

shown in Table 3. In most of the ticks obtained from mice, genotype

2 was identified, followed by genotype 3. Most of the ticks that had

fed on voles were found to carry genotype 1. On the other hand,

genotypes 8 and 4 were the most frequently detected variants in

ticks obtained from chipmunks and squirrels, respectively. Genotype

5 was the most common genotype found in ticks derived from raccoons,

and genotype 3 was the most frequent in ticks obtained from opossums.

Transmissibility of each genotype from infectious mammals to ticks

can be regarded as a fitness index of strains infecting hosts. The

values varied significantly within and among host species as shown

in Table 4.

B. burgdorferi in Field-collected Questing Nymphs and Exposure of

Animals

A total of 178 field-collected questing nymphs were screened for B.

burgdorferi. The overall infection prevalence was 39%. In this tick

population, the same 8 genotypes as in nymphs derived from the

animal pool were found. However, significant differences in the

genotype frequency distribution between these 2 tick populations

were observed (2 x 8 Fisher-Freeman-Halton, Fisher statistic =

19.93, Monte Carlo p<0.01) (Table 3). Questing nymphs were chosen to

estimate the exposure of hosts to B. burgdorferi genotypes. The

calculated values of exposure show that animals with higher nymphal

burdens were frequently exposed to >1 infected nymph. This value was

occasionally <1, reflecting the conservative estimation of exposure

(Table 5).

Comparative Analysis of B. burgdorferi Population Structures

Only 1 other study has analyzed the population structures of B.

burgdorferi in different vertebrate host species in the northeastern

United States (13). As in our study, transmissible infections in

hosts were determined through host-derived ticks. In contrast, the

population structures of B. burgdorferi were measured at the outer

surface protein C (ospC) locus. However, because the ospC locus and

the IGS used in our study are linked (19), the population structures

found in both studies may be compared. Genotypes 1-8 were present in

questing nymphs in each study, which indicates that the host

populations were exposed to a similar spectrum of spirochete

strains. Three rodent species, white-footed mice, chipmunks, and

squirrels, were captured in both studies and used for comparison.

The population structures of B. burgdorferi in each host species

were different in the 2 data sets. However, analysis of the combined

data set shows that, with the exception of genotypes 6 and 7 being

missing in both squirrel populations, all 9 major genotypes that

were prevalent in questing ticks were also found in the 3 rodent

species (Table 6).

Discussion

We explored the question of whether, and to what level, B.

burgdorferi is specialized to vertebrate host species. By analyzing

2 independent data sets obtained from cross-sectional field studies

in the northeastern United States (New York and Connecticut), we

show that most of the known genotypes of B. burgdorferi can, in

principle, infect a range of different rodent hosts. Furthermore,

our own data set indicates that several genotypes can infect as many

as 5 host species. This suggests that cross-species transmission of

B. burgdorferi among various mammalian species is common.

Several issues, however, need to be addressed before the level of

host specificity of B. burgdorferi can be confidently compared with

that of other microparasites. First, the level of host specificity

is generally dependent on the spatial and temporal scale of

observation (1,28). Our findings exemplify this notion, because the

combined data sets of the 2 studies analyzed in this study shows a

pattern of more relaxed host specificity than each of the data sets

would suggest on its own. Second, the " niche breadth " of a parasite

is influenced by the phylogenetic relationships of its hosts (29).

If, for example, a parasite infects a given number of hosts

belonging to different orders or classes, one would consider such a

parasite to be less specialized than a parasite that exploits the

same number of closely related species.

The analysis of the combined data sets obtained by the 2 field

surveys compared in this study shows that mice, chipmunks, and

squirrels (order Rodentia) are susceptible to most of the B.

burgdorferi genotypes described in the United States. Therefore, the

niche breadth of B. burgdorferi genotypes is not congruent with host

species. Furthermore, genotypes 1-5 and 7 can, at least transiently,

infect many additional, phylogenetically distant host species,

covering as many as 3 orders. This indicates that the niche breadth

of most B. burgdorferi genotypes in the United States is even wider

than the taxonomic unit of order.

The issue of host specificity in B. burgdorferi, however, is more

complicated. Experimental work has shown that some isolates of B.

burgdorferi do not disseminate, or slowly disseminate, in mice (30).

Slowly disseminating strains are less efficiently transmitted to

ticks by mice (31). For this reason, it has previously been

suggested that such strains may occupy nonrodent or even

nonmammalian niches in nature, such as avian hosts (31). On the

other hand, certain strains of B. burgdorferi can infect both rodent

and avian hosts (10), which demonstrates that some strains of B.

burgdorferi are extreme generalists. In view of all ecologic and

experimental information available to date, we conclude that host

specificity of B. burgdorferi ranges from generalism to specialism,

depending on genetic background.

Several possible explanations exist for the discordance between the

data sets from New York and Connecticut. First, differences in the

local ecologic conditions could shape the local population

structures of B. burgdorferi in hosts (1). Furthermore, the 2 data

sets could represent snapshots of population structures that are

spatially and temporally variable due to stochastic effects or other

forces (32). In other words, the spirochete populations could be

dynamic. In fact, strong evidence exists for this scenario, since

pronounced temporal shifts in genotype frequency distribution of B.

burgdorferi within 2 years have been observed in questing adult

ticks (33). Considering that adult I. scapularis ticks have a

history of taking only 2 blood meals in 2 years (26), the scale of

this temporal variation is remarkable.

One of the most fundamental parameters in infectious disease biology

is the time scale of infectivity relative to host lifetime, which

affects the epidemic/endemic behavior of all microparasites (34). B.

burgdorferi infections in mice are believed to be lifelong (35). The

universality of this paradigm, however, has recently been challenged

by experimental studies in white-footed mice, which found that the

infectivity of some strains to ticks declines within a few weeks

(31,36). This feature is crucial in 2 ways. First, it indicates that

fitness of B. burgdorferi is a quantitative trait. This is

corroborated by our study that provides ample evidence for fitness

variation within and across diverse host species. Second, the

finding of declining infectivity shows that the transmission

kinetics of some B. burgdorferi strains is dynamic. Therefore, both

intrinsic transmission dynamics of B. burgdorferi strains in hosts

(37) and population fluctuations of the hosts (38) may result in

population fluctuations of B. burgdorferi. Time series analyses of

spirochete populations are required to clarify the scale of the

spatiotemporal dynamics of B. burgdorferi (32).

We are beginning to understand key molecular processes that enable

cross-species transmission of B. burgdorferi (11,16). Individual

strains of B. burgdorferi have been found to contain large arrays of

prophage-encoded outer surface proteins that differentially bind

complement control factors of a wide range of vertebrate species,

preventing the bacteria from being killed by innate immunity (11).

That the repertoire of these prophage genes determines the host

range of LB spirochetes has been hypothesized (11,16). B.

burgdorferi, thus, is 1 of the very few examples of zoonotic

pathogens for which a molecular mechanism of host-switch has been

proposed (39).

OspA serotypes 2 8, which comprise the Eurasian genospecies B.

afzelii and B. garinii, occupy distinct host niches, such as rodent

versus avian hosts (16). Here we demonstrate that B. burgdorferi

(OspA serotype 1) in the northeastern United States is much less

specialized than B. afzelii (serotype 2) and B. garinii (serotypes 3-

8), because the niche breadth of most of its genotypes covers a much

larger range of phylogenetically distant hosts than any of the other

OspA serotypes. The generalist strategy of B. burgdorferi is

consistent with its uniform population structure across much of the

northeastern United States (33). We may speculate that the

generalist strategy of B. burgdorferi echoes adaptation to

impoverished ecologic conditions in the past because of large-scale

habitat destruction in the northeastern United States in the course

of the post-Columbian settlement and during the industrial

revolution (8). We conclude that cross-species transmission of B.

burgdorferi is a key property that has allowed LB to spread rapidly

across the northeastern United States. Our study emphasizes that

accurate information on the degree of cross-species transmission is

necessary to understand and predict the spread of zoonotic pathogens.