Molecular epidemiology and evolution of Sarcocystis neurona, the etiologic agent of equine protozoal myeloencephalitis (EPM)

Equine Protozoal Myeloencephalitis (EPM) is a common, serious neurological disease of horses that can lead to disability and death. EPM is caused by the ubiquitous protozoan parasite Sarcocystis neurona. Horses acquire this parasite from the feces of opossums of the genus Didelphis when they ingest the sporocyst stage in contaminated food. Inadequate understanding of the parasite's species identity, genetic makeup, host range, and the mechanisms it uses to cause disease in horses has hampered effective diagnosis and management of the disease. The most basic information needed is how to determine which Sarcocystis strains are S. neurona and which are not and how genetic traits are transferred within the population. The main hypothesis investigated was that S. neurona undergoes genetic recombination during the sexual phase of parasite development leading to strains with variable genetic traits that may include increased virulence. The studies described here included the assembly of a large collection of S. neurona and other related cyst-forming coccidia, development of methods for purification of the sporocyst stage from intestinal cells of opossums and the merozoite stage from tissue culture and the use of DNA sequencing of the small subunit ribosomal RNA (SSUrRNA) gene and major surface antigen gene 1 (SAG1) as well as sequence of the 25/396 diagnostic marker. Amplified Fragment Length Polymorphism (AFLP) methodology was also used as a whole-genome fingerprinting method to develop a "species identity" or "genetic fingerprint" for S. neurona strains. This method was applied to characterize the available Sarcocystis strains that have been assessed by preliminary phenotypic and molecular marker methods such as PCR-RFLP as "neurona-like".; The contributions of the present work are: (1) evaluation of reproducible and sensitive methods for classification of strains of S. neurona from horses and opossums to the species and subspecies level based on SAG1 gene sequence data and AFLP markers; (2) elucidation of the nature and extent of genetic variation and phenotypic heterogeneity of Sarcocystis spp. isolated from horses, opossums, and other vertebrates; and (3) use of phylogenetic information to assess the distribution of S. neurona in equids and wild hosts and to predict the risk of introduction into horses. Methods developed in this work can be used to identify to the species and subspecies level any S. neurona isolate, to track sources of S. neurona for horses, and to test whether S. neurona undergoes genetic recombination to produce new strains with pathogenic potential.