The Emerging Role of The Emerging Role of Molecular Markers in Molecular Markers in Coccidian Speciation Coccidian Speciation

K. B. Miska, K. B. Miska, D. Motriuk-SmithD. Motriuk-Smith, , and R. S. Sevilleand R. S. Seville

The 83The 83rdrd Annual Meeting of the American Annual Meeting of the American Society of ParasitologistsSociety of Parasitologists

4040thth Coccidosis Conference and Workshop Coccidosis Conference and WorkshopJune 29June 29thth, 2008 , 2008

Arlington, TXArlington, TX

Partial ORF470Partial ORF470

Reported to distinguish lineages that Reported to distinguish lineages that are oocyst residuum (OR) possessing are oocyst residuum (OR) possessing or lacking (Zhao and Duszynski or lacking (Zhao and Duszynski 2001)2001)

Generated new partial ORF470 Generated new partial ORF470 sequences (sciurid rodent hosts)sequences (sciurid rodent hosts)• only only E. callospermophili E. callospermophili possesses ORpossesses OR

T.gondii

E.lancasterensis.FS

E.lancasterensis.EFS100

E.vilasi.WGS

E.pseudo.WGS100

53

E.ontarioensis.FS

E.lateralis.WGS

E.reedi

E.callo.MAR

E.callo.WGS

E.callo.BTPD99

100

74

E.arizonensis

E.onychomysis

E.albigulae86

100

100

67

95

37

E.langebarteli

E.separata

E.wenrichi.TV

E.sevilletensis

E.falciformis93

100

E.papillata

E.nieschulzi100

100

64

93

100

OR -murid hosts

OR +sciurid and murid host

OR -sciurid host

unpublished

?

A

B

NJ tree

ITS-1 and ITS-2ITS-1 and ITS-2

Reported to distinguish morphologically Reported to distinguish morphologically similar similar EimeriaEimeria species (Hnida and species (Hnida and Duszynski 1999) in murid rodentsDuszynski 1999) in murid rodents

Generated new sequences to distinguish Generated new sequences to distinguish differences between morphologically differences between morphologically similar species of similar species of Eimeria Eimeria (sciurid rodent (sciurid rodent hosts) and/or diverse geographic locationshosts) and/or diverse geographic locations• E. vilasi E. vilasi and and E. pseudospermophili E. pseudospermophili (3 hosts; WY)(3 hosts; WY)• E. lateralis E. lateralis (4 hosts; WY, AK, Siberia)(4 hosts; WY, AK, Siberia)• E. callospermophiliE. callospermophili (3 hosts; WY, CO) (3 hosts; WY, CO)

MaximaEFSLancaster1EFSLancaster2EFSLancaster4EFSLancaster5FSLancaster5

3351

FSLancaster3FSLancaster1FSLancaster4

3387

72

FSLancaster2EFSLancaster3

41

75

100

100

WGSVilasi1WGSPseudo4WGSPseudo1

58

MARPseudo1WGSPseudo3MARPseudo3

4799

90

100

UGVilasi5UGVilasi2UGVilasi1UGVilasi3UGVilasi4

3548

64

100

MARPseudo5MARPseudo2MARPseudo4

49100

100

100

61

WGSVilasi4WGSVilasi3WGSVilasi6WGSVilasi2

59100

97

WGSPseudo5WGSPseudo2

100

100

78

ACHMLateralis3BTPDLateralis3BTPDLateralis1BTPDLateralis6ACHMLateralis2WGSLateralis1WGSLateralis2WGSLateralis7WGSLateralis6

5761

78

57

94

WGSLateralis8ACHMLateralisSI7

96

64

91

ACHMLateralis1BTPDLateralis7BTPDLateralis2BTPDlateralis4

6758

76

77

88

100

AGSLateralis4AGSLateralis1AGSLateralis5AGSLateralis2AGSLateralis3

4239

49

100

100

MARCallo4WGSCallo6WGSCallo5WGSCallo2WGSCallo1WGSCallo63WGSCallo4

8589

87

61

MARCallo5MARCallo1MARCallo6MARCallo2

5843

100

44

99

BTPDCallo4BTPDCallo5BTPDCallo3BTPDCallo2BTPDCallo1

8153

34

100

100

100

99

TSOntario3TSOntario1TSOntario2

100100

100

100

E.vilasi and E.pseudospermophili

E.lateralis

E.callospermophili

NJ tree

Materials and MethodsMaterials and MethodsChallenge #1Challenge #1

Collection of quality starting Collection of quality starting materialmaterial

Identification of a pure sample (one Identification of a pure sample (one host=one host=one EimeriaEimeria species) species)

Repeated sucrose density Repeated sucrose density centrifugation and floatscentrifugation and floats

Collecting oocysts on the cover slideCollecting oocysts on the cover slide Concentration of oocystsConcentration of oocysts

Materials and MethodsMaterials and MethodsChallenge #2Challenge #2

Disruption of the wallDisruption of the wall MechanicalMechanical

• Glass beadsGlass beads TemperatureTemperature

• Repeated heating/freezing (liquid nitrogen)Repeated heating/freezing (liquid nitrogen) Chemical Chemical

• NaOCl, HNaOCl, H22SOSO44

Treatments unsuitable for wall Treatments unsuitable for wall digestiondigestion• sonication, ammonia, proteolitic enzymessonication, ammonia, proteolitic enzymes

Materials and MethodsMaterials and Methods

Successful wall disruption allows for Successful wall disruption allows for molecular work following standard molecular work following standard protocolsprotocols• DNA extraction with proteinase K DNA extraction with proteinase K

treatmenttreatment Library construction Library construction PCRPCR

The future?The future?

Requirement to submit sequence Requirement to submit sequence data with a description of a new data with a description of a new speciesspecies

Creation of a standard list of Creation of a standard list of phylogenetically informative phylogenetically informative sequences sequences

AcknowledgmentsAcknowledgments

Funding Funding • UW INBRE (NIH)UW INBRE (NIH)• UW EPSCoR (NSF)UW EPSCoR (NSF)

Lab membersLab members• Clint OliverClint Oliver• Arik SmithArik Smith• Dani HofmannDani Hofmann• Jason CaseboltJason Casebolt• Jason CrouchJason Crouch• Stephanie JensenStephanie Jensen