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The Emerging Role of Molecular Markers in Coccidian Speciation

The Emerging Role of Molecular Markers in Coccidian Speciation . K. B. Miska, D. Motriuk-Smith , and R. S. Seville The 83 rd Annual Meeting of the American Society of Parasitologists 40 th Coccidosis Conference and Workshop June 29 th , 2008 Arlington, TX. Partial ORF470.

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The Emerging Role of Molecular Markers in Coccidian Speciation

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  1. The Emerging Role of Molecular Markers in Coccidian Speciation K. B. Miska, D. Motriuk-Smith, and R. S. Seville The 83rd Annual Meeting of the American Society of Parasitologists 40thCoccidosis Conference and Workshop June 29th, 2008 Arlington, TX

  2. Partial ORF470 • Reported to distinguish lineages that are oocyst residuum (OR) possessing or lacking (Zhao and Duszynski 2001) • Generated new partial ORF470 sequences (sciurid rodent hosts) • only E. callospermophilipossesses OR

  3. OR - sciurid host A ? OR + sciurid and murid host B OR - murid hosts NJ tree unpublished

  4. ITS-1 and ITS-2 • Reported to distinguish morphologically similar Eimeria species (Hnida and Duszynski 1999) in murid rodents • Generated new sequences to distinguish differences between morphologically similar species of Eimeria(sciurid rodent hosts) and/or diverse geographic locations • E. vilasiand E. pseudospermophili(3 hosts; WY) • E. lateralis(4 hosts; WY, AK, Siberia) • E. callospermophili (3 hosts; WY, CO)

  5. E.vilasi and E.pseudospermophili E.lateralis E.callospermophili NJ tree

  6. Materials and MethodsChallenge #1 • Collection of quality starting material • Identification of a pure sample (one host=one Eimeria species) • Repeated sucrose density centrifugation and floats • Collecting oocysts on the cover slide • Concentration of oocysts

  7. Materials and MethodsChallenge #2 • Disruption of the wall • Mechanical • Glass beads • Temperature • Repeated heating/freezing (liquid nitrogen) • Chemical • NaOCl, H2SO4 • Treatments unsuitable for wall digestion • sonication, ammonia, proteolitic enzymes

  8. Materials and Methods • Successful wall disruption allows for molecular work following standard protocols • DNA extraction with proteinase K treatment • Library construction • PCR

  9. The future? Requirement to submit sequence data with a description of a new species Creation of a standard list of phylogenetically informative sequences

  10. Acknowledgments • Funding • UW INBRE (NIH) • UW EPSCoR (NSF) • Lab members • Clint Oliver • Arik Smith • Dani Hofmann • Jason Casebolt • Jason Crouch • Stephanie Jensen

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