Epidemic Spread of Lyme Borreliosis, Northeastern United States Kl�ra Hanincov�,*1 Klaus Kurtenbach,*2 Maria Diuk-Wasser,* Brandon Brei,* and Durland Fish* *Yale University, New Haven, Connecticut, USA
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 similar 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�34�N, 72�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) by 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) by using Tomahawk (Tomahawk, WI, USA) trap numbers 205 and 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-m2 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 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 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 (RDS) was computed for each capture time point as RDS = 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 (RSS) was computed as RSS equals Σ (RDS times C); C is the number of days between capture points. The number of nymphs infected with a genotype encountered by a host per season (RSIS) was calculated as RSIS equals IP/N times RSS; 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 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 prevalence 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 are that different coding systems can be applied to compare infection prevalence among various groupings of host species (e.g., mice vs. 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 χ2 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 (Table 2). Each of the 3 tested chipmunks produced 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 (CI) 1.63-110.41, p0.001).
Infection prevalences of B. burgdorferi in host-derived ticks also varied significantly among host species (Table 3). A considerably higher infection prevalence in ticks was observed for mice than for voles. 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, as was infection prevalence in ticks from voles compared to the pooled group.
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