Understanding the ecology of outbreaks of diseases like anthrax as well as associations between landscape ecology and specific bacterial genotypes is essential for improving prevention and control of the disease in wildlife, livestock, and humans. The mechanisms through which the anthrax pathogen is transmitted at fine spatial and temporal scales are poorly characterized. This doctoral dissertation research project will combine molecular analysis of Bacillus anthracis, the causative bacterium of anthrax, with spatio-temporal epizootic data to explore anthrax dynamics in North America across different spatial and temporal scales, multiple genetic sub-lineages, and multiple mammalian host systems. The doctoral student will seek answers to the following questions: Do potential evolutionary changes in B. anthracis from infected animal hosts and necrophagous flies suggest mechanisms of pathogen transmission at a local scale? Are there associations among ecological conditions, epidemiology, and genetic diversity within outbreaks in different ecological settings and host systems? What are the broad scale spatio-temporal patterns of genetic diversity of B. anthracis across two important anthrax foci in North America? The student will use an existing collection of B. anthracis isolates and anthrax epizootic data from ecologically distinct geographic areas. New isolates and epizootic data will be collected from outbreaks occurring during the study period. High-resolution single-nucleotide repeat (SNR) analysis will characterize genetic diversity among and within multiple locus variable number of tandem repeat- (MLVA) defined genotypes across study sites. Trend surface analysis with vector velocity mapping will be used to analyze disease movement through host populations across the landscape and will be overlain with maps of genotypes to evaluate associations between genetic diversity and disease transmission.
This project will help fill important gaps in knowledge about anthrax transmission and will specifically evaluate whether genetic analysis can inform spatio-temporal models of anthrax transmission. Anthrax transmission is likely influenced by fine scale environmental characteristics, genetic lineage, host species and behaviors, and arthropods as well as other as-yet-unknown variables. This project will encompass multiple genetic lineages of the pathogen and multiple host systems, including a study area in Texas with annual, enzootic anthrax with wildlife and mixed wildlife/livestock host populations and a study area in Montana experiencing a reemergence of anthrax in wildlife. Project results will provide insight into the genetic diversity and evolution of an important spillover pathogen. The results also will inform public health strategies for prevention of wildlife, livestock, and human disease and will provide insight as to whether strategies should be tailored to the local ecology. As a Doctoral Dissertation Research Improvement award, this award also will provide support to enable a promising student to establish a strong independent research career.
Intellectual merit. Anthrax, a zoonosis caused by the bacterium Bacillus anthracis, occurs regularly in wildlife and livestock in the United States, resulting in substantial economic costs for veterinary care, diagnostics, and carcass disposal. We have yet to fully characterize the mechanisms for anthrax spread during outbreaks; such knowledge would better enable us to predict favorable conditions for an outbreak and implement early control measures. For this study, we set out to characterize the spatio-temporal patterns of an anthrax outbreak in white-tailed deer, Odocoileus virginianus, in West Texas and compare those results with a complementary genetic analysis of bacterial isolates of B. anthracis recovered from dead deer carcasses and the surrounding soils. We analyzed a dataset of carcass locations from a single large outbreak of anthrax from 2005 and analyzed 15 B. anthracis cultures using multi-locus variable number tandem repeat analysis (MLVA) and single nucleotide repeat (SNR) analysis. Existing additional, contemporary data about this deer population and the landscape made this outbreak ideal for investigating anthrax dynamics. Comparing spatial clustering of carcasses with deer movement data from a VHF telemetry study, we found that animals that died of anthrax likely had opportunity to come into contact with adjacent carcasses. Because carcass locations were also in areas where dense fly populations were likely, our results support the hypothesis that contamination of vegetation around carcasses from flies feeding on carcasses and depositing the materials in feces and emesis is important during outbreaks in this part of Texas. Genetic diversity of B. anthracis isolates was highest in areas of high transmission – those areas where deer carcasses were close together in space and time. Figure 1 illustrates the distribution of cases across the study ranch and the genetic diversity defined by SNR genotyping for this study. In addition to studying the genetic diversity within a single outbreak using MLVA/SNR typing, we also evaluated SNR diversity within genotypes across outbreaks in western Montana and several ranches in western Texas. In total, we analyzed 63 isolates from wildlife in the two states and mixed cattle/wildlife outbreaks in west Texas recovered from 2003 - 2012. We found that B. anthracis isolates recovered from the same geographic location over multiple years maintained a single and stable MLVA-25 genotype and that SNR diversity was greatest within the 2005 outbreak described above. We also confirmed that isolates from carcasses and surrounding soil may differ by SNR type, suggesting either animal carcass shed multiple SNR types or that some evolution occurred outside of the host. This is an area for further and significant research. We also confirmed that the SNR-type from a carcass may also be directly isolated from neighboring soil up to 1 year after an outbreak, suggesting a mechanism for long-term persistence of a genotype (MLVA type) or sub-genotype (SNR genotype) that may lead to subsequent cases or outbreaks. While the Texas isolates showed long-term stability in MVLA types, we showed greater diversity of SNR types within B. anthracis recovered from elk, Cervus elaphus, and bison, Bison bison bison associated with a single epizootic in 2008 in western Montana. In those strains, we confirmed that a single host, either elk or bison, carried multiple SNR types (2 sub-genotypes) and that bison from geographically separated regions of the outbreak area had differ MLVA-25 genotypes (unlike Texas, where we did not detect MLVA-25 diversity within a study ranch, though we did confirm it across the study area of multiple ranches). Broader impacts. Our studies provide new insight into anthrax outbreak dynamics and suggest that fine spatial and temporal scale sampling of future outbreaks will better characterize anthrax transmission. The study of a single outbreak confirmed through spatio-temporal analyses and strain genotyping that carcass distribution and bacterial diversity were likely due to animal movement patterns and carcass proximity. The study of multiple wildlife herds across west Texas and southwestern Montana confirmed B. anthracis strain diversity can differ across host species, with bison and elk having high SNR diversity and that individual deer herds in Texas see inter-annual stability within MLVA genotypes. These studies provide new information on the within-outbreak and inter-annual genetic diversity of B. anthracis and suggest that strain diversity may be associated with host behavior during the outbreak period. We confirmed that SNR types associated with an outbreak in one year can be recovered from the soil 1-year later, supporting the hypothesis that carcass sites from one outbreak can serve as a source of B. anthracis in future events. Benefits to society. Through the course of training a doctoral student, this work provides evidence that environmental contamination from carcasses may lead to additional cases within an outbreak. This suggests that outbreak cleanup efforts should include vegetation immediately around a carcass and that known previous carcass sites should be consider at-risk areas for future exposure to naïve animals.
