Plague: Out of the Foothills Rebecca E. Colman1, Robert J. Brinkerhoff2, Adina Doyle1, Chris Ray3, Paul Keim1, Sharon K. Collinge3, and David M. Wagner1 1Northern Arizona University, Flagstaff, AZ; 2Yale University, New Haven, CT; 3University of Colorado at Boulder, Boulder, CO Study of CO plague over one season and it a collaboration with Sharon Collinge

Background Caused by the bacterium Yersinia pestis Obligate pathogen Cycles between rodents and their associated fleas Endemic in several foci around the world

Plague in North America Entered ~ 1900 at a California shipping port San Francisco area, maybe L.A. Characterized by large die-offs in rodent populations Increased human risk Mechanism for maintenance unknown Several theories but how it is sustained in North America is not clear

So what is the reservoir? NSF-NIH: Landscape Effects on Disease Dynamics in Prairie Dogs; 2002-2007; University of Colorado; $1,832,715 NSF-NIH: Ecological Drivers of Rodent-borne Disease Outbreaks: Trophic Cascades and Dispersal Waves; 2003-2009; University of New Mexico; $1,746,268 NSF-NIH: Plague As a Model for Low Prevalence/ epizootic Disease Dynamics; 2003-2009; Colorado State University; $1,281,000 TOTAL = $4.8 million

Plague in the US Was introduced into the US ~1900 and rapidly spread to the current distribution Has become endemic in the Southwest Cully and Williams 2001

Cycle: Enzootic / Epizootic

Study System: Boulder Colorado Sites with Y. pestis positive samples from June to September 2005

Study Questions Does plague come out of the Foothills “reservoir” hosts and infect the prairie dogs in the grasslands? Or is there another local reservoir that spills over into the prairie dog colonies?

Hypothetical Molecular Trees Out of the Foothills hypothesis Other local reservoir hypothesis F A E D B F A E D C B C

Collection Methods Rodents were trapped and combed for fleas DNA from individual fleas was extracted Extractions were screened with 2 PCR targets Plasmid target (pla) and chromosome target 78 samples were used for high resolution genetic analysis 150 samples were used for more ancestral genetic structure

Different Molecular Markers Fast Slow Mutation Rate 10-3 10-4 10-5 10-6 10-7 10-8 10-9 10-10 Complex VNTRs SNPs SNR / VNTRs

MLVA Multi-Locus Variable number tandem repeat Analysis Insertion Deletion

MLVA Examines 43 regions of Y. pestis genome with varying mutation rates Provide discrimination even among local outbreak samples

MLVA Results Shows population structure by site Interesting 5A dispersal event Reservoir sites are more genetically similar to each other than to the prairie dog sites except for 5A

MLVA Results

SNPs Single Nucleotide Polymorphisms Slow mutating markers compared to MLVA, therefore they are more stable markers Show more ancestral (historical) population structure

SNP Results Whole Genome Sequence comparisons Northern AZ Colorado Whole Genome Sequence comparisons SNPs are extremely rare Y. pestis Show historical divergence Samples fell out into two SNP groups

SNP and MLVA combined

Conclusion Geographic proximity does not equal genetic similarity

Implications Reservoir sites are distinct from each other based on MLVA Suggests plague is cycling between epizootics with no real gene flow Two SNP groups seen in one study season Suggests separate maintenance and dispersal of this disease on small spatial scale

Implications SNP group 2.2 (blue) – plague comes out of reservoirs (in the foothills) and infects prairie dog colonies (5A) However the other 2 prairie dog sites (MK and CR) are distinct and must have come from another introduction event Maybe out of the grassland habitat?

Acknowledgements A collaboration with Sharon Collinge’s group at UC Boulder Work supported by NIH grant #1R15-AI070183-01

Questions ?