Determination of Yersinia pestis population structure as a model for low prevalence/epizootic disease dynamics

Abstract

Plague is a highly virulent zoonotic disease caused by the gram-negative bacterium Yersinia pestis. It is estimated that Y. pestis evolved only 1,500-20,000 years ago froth the mild enteric pathogen Yersinia pseudotuberculosis. Despite this recent emergence, Y. pestis has spread around the globe killing millions of people in three major pandemics and currently infects more than 200 mammal species world-wide. Plague was introduced in the U. S. from Asia in the early 1900s through shipping, and has established primarily in the west and southwest. Currently in the U.S., Y. pestis is most active in rodents in the Four-Comers region where frequent fluctuations in transmission rates result in the amplification of disease to epidemic (epizootic) proportions, or a decline to endemic (enzootic) states. During rodent epizootic events, Y. pestis occasionally spills over into humans causing severe and often fatal illness. It is hypothesized that these disease fluctuations are the result of changing rodent population densities and contact rates between hosts and/or disease vectors whose populations are affected by changes in temperature and moisture triggering cascades in terrestrial net primary productivity. While increasing evidence shows that plague can be influenced by factors like large-scale climatic patterns, the mechanisms of these disease cycles are unknown. Determining the spatial arrangement of disease foci using molecular genetic analyses will provide tools for increasing our understanding of disease dynamics. Genotyping Y. pestis isolates collected from geographically distinct areas that experience epizootic plague cycles may help us better understand the spatial dynamics of plague and the mechanisms behind epizootics. The objectives of this study focused on utilizing Y. pestis molecular diversity to identify epizootic sources of human plague infections, and to determine whether molecular typing of Y. pestis strains corresponds to purported geographic foci or hypotheses of epizootic spread in the Southwestern U. S. and in Kazakhstan, the hypothesized origin of Y. pestis. Isolates of Y. pestis were chosen from the collection at the Centers for Disease Control and Prevention in Fort Collins, CO and were collected during human plague case investigations, animal-based surveillance activities, and during an ecological study of Cynomys ludivicianus (black-tailed prairie dog) epizootics at the Pawnee National Grasslands during 2004, 2005, and 2006. Samples were chosen in clusters representing three geographic scales, and were analyzed to identify isolates that arose from the same epizootic source. The three geographic scales represented a close scale, an intermediate scale, and a distant scale. The molecular markers and the associated analyses used were variable number of tandem repeats (VNTRs) and multi-locus variable number of tandem repeat analysis (MLVA) respectively. The close geographic scale analysis consisted of Y. pestis isolates obtained from infected humans, and from environmental sources such as fleas and rodents collected during epidemiological investigations associated with these human cases. The objective of initial analyses was to determine whether MLVA could identify the epizootic event responsible for each human infection. Bacterial isolates collected from distinct clusters of human plague cases in New Mexico during a 1980s epidemic, and isolates collected from predefined foci in the central-Asian country of Kazakhstan were used to make inferences about Y. pestis population structure on intermediate scales. The distant geographic scale analyses were carried out using isolates from the three Y. pestis biovars, antiqua, medievalis, and orientalis, and the samples compared were from the U. S. and Kazakhstan. The last part of this study consisted of identifying single nucleotide polymorphisms (SNPs) in the Y. pestis genome. Molecular markers like SNPs are more stable than repeat elements and will provide a more adequate genotyping system for phylogenetic studies of Y. pestis than the current MLVA marker system. We determined that MLVA was useful for inferring Y. pestis isolate relationships on small and large geographic scales but was not useful for determining the population structure of epizootic foci. Molecular diversity detected using MLVA was extremely useful for the identification of epizootic sources of human plague infections, with isolates from the human and infective epizootic source inferring genetic relationships with greater than 70 % jackknife (JK) support in the phylogenetic trees. Biovars antiqua, medievalis, and orientalis also formed monophyletic clades when rooted with ancestral Yersinia pestoides isolates. Our intermediate scale analyses using MLVA were inconclusive. The stochastic variability generated by the mutation rates of the VNTR markers at intermediate scales made it impossible to determine the population structure and the arrangement of epizootic plague foci in the U. S. and in Kazakhstan. To address this problem, DNA microarrays were used to discover SNPs. These markers are commonly used in eukaryotic population studies because they have slow mutation rates, are distributed evenly throughout the genomes of most organisms, and are good genetic markers for testing hypotheses about hierarchy in populations. Our Y. pestis set chosen for SNP discovery represented isolates from two port cities in California and fifteen Colorado isolates from pre-defined plague foci. Colorado isolates were chosen from the foothills, the foothills/prairie interface, and the PNG to determine population structure among the three areas. Sixty-nine SNPs identified separate Y. pestis populations on the eastern and the western PNG. Isolates from the mountains and one isolate from the eastern plains were similar but weakly supported in phylogenetic analyses.