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Title: Genetic characterization of theileria equi infecting horses in North America: evidence for a limited source of U.S. introductions

Author
item HALL, CARINA - Northern Arizona University
item BUSCH, JOSEPH - Northern Arizona University
item PALMA-CAGLE, KRISTINA - Northern Arizona University
item Scoles, Glen
item Ueti, Massaro
item Kappmeyer, Lowell
item WAGNER, DAVID - Northern Arizona University

Submitted to: International Journal for Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/5/2013
Publication Date: 2/11/2013
Citation: Hall, C.M., Busch, J.D., Palma-Cagle, K.A., Scoles, G.A., Ueti, M.W., Kappmeyer, L.S., Wagner, D.M. 2013. Genetic characterization of theileria equi infecting horses in North America: evidence for a limited source of U.S. introductions. International Journal for Parasitology. doi:10.1186/PV.1756-3305-6-35.

Interpretive Summary: Theileria equi is a tick-transmitted blood-borne parasite that causes a disease known as equine piroplasmosis (EP) in horses and other equines (asses, donkeys, mules, zebra). This parasite is found throughout most of the world, but was eradicated from the United States by 1988 in a long and costly eradication campaign. Until outbreaks in Florida in 2008 and Texas in 2009 the U.S. has been considered to be free of EP. Analysis of ribosomal RNA gene sequences of samples of T. equi collected from the outbreaks and routine surveillance showed that most of the United States samples were most closely related to a South African strain. A single isolate from the United States-Mexico border was distinctly different from these but similar to a different set of South African strains. This is suggestive of a restricted source of introduction of this pathogen into the U.S. The genome sequence of T. equi was used to develop a DNA fingerprinting assay of 18 microsatellite markers for finer scale characterization of genetic variation between U.S. isolates of T. equi. We genotyped 37 U.S. samples including one sample from the 2008 Florida outbreak, 22 samples from the 2009 Texas outbreak as well as samples from routine screening in California, Colorado, Florida, Georgia, and Oklahoma. There was a moderate amount of genetic diversity among the samples, and we found strong population differentiation between the Texas and Georgia subpopulations. When applied to a broader cross section of worldwide isolates, these molecular tools will improve source tracking T. equi outbreaks and may be useful to help to prevent the spread of this tick-borne parasite.

Technical Abstract: Theileria equi is a tick-borne Apicomplexan hemoparasite that causes equine piroplasmosis (EP). This parasite has a worldwide distribution, but until recent outbreaks the United States has been considered to be free of EP. Maximum parsimony analysis of 18S rRNA gene sequences of North American T. equi samples revealed that they group into two distinct clades, one containing most of the United States samples (n=36) clustering with a South African sequence and the other containing a single isolate from the United States-Mexico border clustering with a distinctly different set of South African sequences. The placement of most U.S. T. equi by 18S into one monophyletic clade is suggestive of a restricted source of introduction of this pathogen into the U.S. Using the first complete genome sequence of T. equi we developed a DNA fingerprinting panel of 18 microsatellite markers for characterization of genetic variation in T. equi. We genotyped 37 U.S. samples and found a moderate amount of genetic diversity (3-9 alleles per locus). The 37 field samples were mostly from the 2009 Texas outbreak (n=22) though samples from five other states (California, Colorado, Florida, Georgia, and Oklahoma) were also included in this study. Using Weir and Cockerham’s FST estimator (Theta) we found strong population differentiation of the Texas and Georgia subpopulations (Theta = 0.414), which was supported by a neighbor-joining tree created with predominant haplotypes. Single-clone infections were found in 27 of the 37 samples (73%), allowing us to identify 15 unique genotypes. When applied to a broader cross section of worldwide isolates, these molecular tools will improve source tracking T. equi outbreaks and help to prevent the spread of this tick-borne parasite.