Differential plague-transmission dynamics determine Yersinia pestis population genetic structure on local, regional, and global scales

Results

Regional Outbreak. Fleas from 19 of the 49 die-off colonies tested positive for plague; plague also was likely the causative agent of the other 30 die-offs (Fig. 1). Regional samples separated into seven distinct groups, which were strongly supported by branch-length and/or bootstrap data (Fig. 2B ). On average, branch lengths separating groups were three times longer than branch lengths within groups, and bootstrap values were >50% for six groups and ≥99% for four groups. Six groups were tightly clustered in geographic space; only the Sitgreaves Mountain group was not spatially grouped, and it had low bootstrap support as well as long within-group branch lengths.

Local Outbreak. Fleas were obtained from 352 of the 944 burrows sampled in the Ft. Valley outbreak; 149 burrows yielded plague-positive flea pools. MLVA analysis indicated two distinct genotypes each from 28 of the 149 burrows, for a total of 177 samples. Twenty-five unique MLVA genotypes were observed (Fig. 3A ). One genotype, FV-1, was dominant (71%) and widely distributed across the study area. It was particularly dominant in the eastern sectors, from which the outbreak is thought to have originated (Fig. 3B ). Fifteen genotypes were observed only once and were widely distributed across the area. Nine minor genotypes were observed multiple times and showed a strong tendency for spatial clustering. The topology, based on parsimony analysis, approaches that of a star phylogeny, with most genotypes only a single mutational step from the dominant FV-1 genotype (Fig. 3A ). Rooting such a phylogenetic topology is problematic, but the overall local dominance of FV-1, its presence in regionally adjacent locations (Fig. 2, Flagstaff group), and its dominance at the earliest local outbreak sites suggest it is the evolutionary root for this Y. pestis population. Only seven two-step branches are derived from the central FV-1 genotype, and these are spatially consistent with their proposed phylogenetic relationships. For example, the single FV-20 genotype is located immediately adjacent to several individual FV-4 genotypes, and the two FV-8 genotypes are located immediately adjacent to FV-2 genotypes (Fig. 3B ).

In Vitro Y. pestis Population. Twenty-six mutational events were observed across 10 different loci within this population (Table 1). Overall, single-repeat mutations were most common, and insertions were only slightly more common than deletions. The fastest mutating loci in vitro were also among those that varied the most in the Ft. Valley outbreak (Fig. 3A ) and regionally (data not shown). Slightly different mutation rates for loci with the same number of mutations are caused by missing data. Combined, the mutation rate for all 43 loci examined was 1.3 × 10^-3 mutations per generation.

Transmission Model. Given the most parsimonious topology for Ft. Valley genetic patterns (Fig. 3A ), 24 mutations occurred across the 177 observations in this outbreak. Our transmission model predicted 355 transmissions cycles for the 177 data points, or 2.0 transmission cycles per sample point. From empirical field data and burrow density at the Ft. Valley site (for the specific methodology, see Supporting Materials and Methods), we estimated that a total of 472 individual prairie dogs lived in this region, all of which died in the plague outbreak. If the entire population was included within our sampling, it would have represented 2.7 transmission cycles per sample point and 32 mutational events would have been expected. It is reasonable to believe the maximum-parsimony assumption is slightly underestimating mutational events and, also, not all prairie dog-flea transmission events are represented among our 177 sample points. If so, the already close predicted and expected values would be even closer.

Discussion

By using MLVA, we defined Y. pestis population structure on a landscape scale, which provides unique insights into plague ecology. First, plague is probably regionally widespread in mammalian reservoir populations. This pattern is difficult to examine, however, because the reservoirs are cryptic. Second, the lack of robust deeper phylogenetic topology (star phylogeny) and the clustering of similar genotypes in geographic space suggest the initial spread of plague across the landscape was probably quite rapid and possibly involved a single, common genotype, which later differentiated within local, dispersal-limited reservoir populations. Third, because plague-infected prairie dog colonies were interspersed with healthy colonies (Fig. 1), interspecific transmission events from the reservoir to prairie dogs probably occurred not once but multiple times. This pattern suggests these events are independent but coordinately triggered by environmental cues (9, 10). Fourth, in areas in which prairie dog colonies are located close to each other (<10 km) and separated by suitable grassland habitat (e.g., Fig. 2 A , within groups Hochderffer, Government Prairie, Mormon Lake, and Spring Valley), intercolony dispersal of plague is probably through infected prairie dogs. Indeed, samples from these four discrete grassland areas form distinct genetic groupings that are strongly supported by bootstrap data (Fig. 2B ). Fifth, the presence of unsuitable, forested habitat between colonies located close to each other (<10 km) likely prevents intercolony dispersal of plague through prairie dogs (e.g., Fig. 2 A , between groups Government Prairie and Spring Valley). Sixth, the presence of similar genotypes in prairie dog colonies that are separated by long distances (>10 km) and unsuitable habitat (e.g., Fig. 2 A , within groups Sitgreaves Mountain, Flagstaff, and San Francisco Peaks) is probably because of intercolony dispersal of plague-infected fleas through domestic dogs, coyotes, or other predator/scavengers (16, 17).

The fragmented population structure of the outbreak host in this area, along with interspersed reservoir species habitat, allows a dynamic balance between the amplification of the pathogen and its long-term persistence, which seems ideal for a plague focus. Amplification of the pathogen can occur in a limited fashion that does not drive the susceptible host to extinction. At the same time, these amplification events also increase the likelihood of interspecific transmission back into the reservoir population, permitting survival of the pathogen between outbreaks in the susceptible host.

Despite the rapid progression of localized outbreaks, our findings demonstrate that Y. pestis population sizes during a single outbreak are large enough that mutational events are observed in multiple VNTR loci. These mutational events can provide insights into the dispersal of plague during an outbreak. The genotype diversity patterns in the outbreak described here (Fig. 3) are consistent with rapid wave-like dispersal by a single fit genotype (FV-1) followed by continued, localized Y. pestis population growth. The fitness of FV-1, which could be caused by either stochastic or adaptive processes (18), led to a dominant overall genotype but with diverse, locally derived genotype clusters. Overall, the phylogenetic relationships are spatially consistent. Either dispersal is slow enough that evolutionarily derived relationships are spatially conserved or dispersal is rapid but spares many of the prairie dogs during the initial plague progression. The differentiated population then could be associated with a second wave of prairie dog deaths; differentiated populations also may be associated with growth inside surviving, infected fleas.

Whereas the occurrence of VNTR mutations can provide insights into plague dispersal during an outbreak, understanding the rate at which these mutation events occur provides additional insights into transmission processes. The strong association between observed and expected VNTR mutation events indicates our model is approximating the natural transmission processes. Overall, the flea-prairie dog transmission model reasonably explains the Ft. Valley plague outbreak and illustrates that the generation of VNTR variation in vivo is not so different from that observed in vitro. However, if only the frequency and distribution of genotype FV-1 are considered, the model seems inadequate. The model would require FV-1 to be widely dispersed in only a few weeks and no mutations to occur during the dispersal. The probability of 124 successive transmissions without a mutation would be 1.7 × 10^-4 in our model. Thus, we believe the initial dispersal of the FV-1 contagion was not strictly a burrow-to-burrow progression but rather involved either a smaller number of mammalian hosts distributing plague-infected fleas widely or another completely different transmission mechanism. A small number of initially infective animals may have spread infected fleas across the entire outbreak area, causing rapid flea-borne transmission. Alternatively, plague can spread without vectors through respiratory droplets or feeding on infected carcasses. These more rapid transmission mechanisms would require smaller Y. pestis population sizes, reducing the likelihood of mutations. Regardless of the actual mechanism, our data would be consistent with an initial rapid dispersal by a few animals, followed by a slower, localized, vector-mediated process.

At both the landscape and local scales, the genetic data suggest very rapid plague transmission followed by slower localized differentiation, which is evidenced by long branch lengths accompanied by a lack of structure in the deeper branches of our phylogenetic hypotheses (Figs. 2 and 3). A “star phylogeny” is consistent with a common ancestor from which multiple lineages are derived simultaneously (19). In our data, these multiple lineages are spatially dispersed across our study areas at both the regional and local scales. At the local scale, the likely ancestral genotype is observable (FV-1), and its rapid dispersal occurred only weeks before complete extinction of the Ft. Valley prairie dog colony. In contrast, the regional common ancestor can only be postulated, but its wide dispersal must have been in the last century, because plague was first documented in Arizona in 1938 (8) and in North America in 1899 (4). It is interesting to note that this pattern of rapid and wide dispersal followed by localized differentiation is also observed at the global scale. During each of the three major plague pandemics that occurred over the last 1,500 years, there was rapid dispersal followed by regional establishment of plague foci that have existed, in some cases, for millennia (2).

We postulate that when plague encounters a susceptible rodent species that is both plague-naive and highly dense (perhaps social), there is rapid dispersal across the landscape. A natural consequence is a rapid decline in susceptible host density, which would extinguish the epidemic. In some cases, the rapid dispersal is followed by slower transmission cycles in mostly resistant or low-density hosts as the disease is established into a stable reservoir for future emergence. Whereas the pathogen would go extinct without the reservoir, it would also be very rare and limited in distribution without the explosive expansion in susceptible hosts. This dynamic balance explains the emergence and subsequent success of plague at the global and regional scales, and even in localized outbreaks.