Collection, preservation and sporulation of fecal stages from Birds: Concepts, Methods and Challenges

1. Collection, preservation and sporulation of fecal stages from Birds: Concepts, Methods and Challenges John R. Barta, Joseph D. Ogedengbe and Julie Cobean Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON Canadajbarta@uoguelph.ca

2. Coccidia of Birds - Concepts coccidia are speciose within avian hosts infections with multiple species simultaneously may be the norm not the exception type of host may be reflected in ‘hardiness’ of fecally obtained oocysts nature of feeding of host may affect method of in vitro excystation and nucleic acid isolation

3. Cystoisospora sp.(Sarcocystidae) Coccidia of Birds Genera include: Eimeria(4ScX2Sz, Steida bodies) Isospora(2ScX4Sz, Steida bodies) Tyzzeria(0ScX8Sz, Steida bodies) Caryospora(1ScX8Sz, Steida bodies) Speciose in virtually all avian hosts and environments Isospora sp. (Eimeriidae)

4. Coccidia of Chickens - Galliformes E.acervulina • Eimeria species • Morphological variation • Niche specialization • Reproductive isolation • Microallopatric speciation • easy to grow, yes • tough to purify and keep that way • tough to break for parasite or nucleic acid E. praecox E. maxima E. brunetti E. mitis E. mivati E. necatrix E. tenella Other spp.

5. Coccidia of Birds - Methods • Collection: • Oocysts usually obtained easily from fecal material (often mixed species present in sample) • Storage in potassium dichromate (2.5% w/v aqueous still best) • Keep at room temperature • Sporulation: • Soon as possible after collection • Avoid refrigeration before sporulation • Best at room temperature • Available oxygen important • ‘Shaken, not stirred’

6. Coccidia of Birds - Methods • Obtaining Viable Sporocysts for Sporozoites: • parasites should be surface sterilized and external lipids removed by bleach treatment (may not work/be necessary in some species in aquatic environments) • at least in Galliformes, require vigorous treatment to remove oocyst wall (glass beads or shearing; sonication is not useful here) • breaking needs to be done in 0.9% saline or PBS • sporocysts can be sorted from unbroken oocysts and debris using Nitex® print-screening cloth (monofilament best – pore sizes down to 5 µm depending on sporocyst size)

7. Coccidia of Birds - Methods • Excystation of Sporocysts: • excystation fluid is trypsin plus taurocholic acid (sodium taurocholate - artificial ‘bile salts’) • some coccidia (e.g. Eimeria maxima) prefer use of actual chicken bile (5% v/v) instead of taurocholate • Purification of Sporozoites: • filtration (Nitex or through glass wool) • density gradient centrifugation (e.g. isotonic Percoll – but not all species like this) • various published methods

8. Coccidia of Birds - Challenges • Species Identification/Verification: • overlap morphologically • overlap antigenically (ELISAs will not work to distinguish infections with different species; many monoclonal antibodies X-react) • easily contaminated with small numbers of another species(rigorous biocontainment plus decontamination required) • multiplex PCR identification based on SCAR markers is proving useful – of course, you need a well characterized species for that SCAR-based multiplex PCR identification of Eimeria spp. of chickens

9. Coccidia of Birds - Challenges • Isolation of Nucleic Material: • oocysts are tough • sonication is useless on oocysts • bleaching followed by glass beads most suitable for small samples (unless bleach sensitive) • 100 oocysts in 1 ml can be easily purified and broken for DNA using glass beads and a microfuge

10. Coccidia of Birds - Challenges T gondi Neos sp N canNC 1 N caninum C belli • Describing new species: • poor history of type deposition • few widely used molecular targets (discussion to follow) • Question of “What is a species??” needs to be addressed collectively • Suitability of genera and families is questionable • Genus Eimeria requires some revision(s) C belli2 C orlovi C suis C ohioe S mucosa 1.00 F glareoli F microti Guossia janae E arnyi E rioarri E catron 1.00 Atoxoplasma Isospora robini E furonEF E alabam 0.78 E faurei E auburn E ahsata E crandal E weybri E ovinoi 0.99 E bovis E scabra E polita 0.99 E porci 0.97 0.73 Cyc colob Cyc cerco Cyc Gom 40 Cyc papi Cyc sp Gom Cyc Gom 34 Cyc caye Cyc NP 233 E adene E ten drug 1.00 1.00 E ten madur E necat 0.50 E neca wTY 1.00 E praeAtt E brun Att E max prec 1.00 E max Att E acerv prec E acerv Sh E acerv Att E mitis E mivati E papil E falci E nieschul E sevill E langeb E scholty E separa E teleki 1.00 E antrozoi E leucopi E onycho E reedi E albigul E arizon E chaetod E peromys E chobot E dipodo 1.00 C bigene C bigene 2 E auritusi 1.00 E phalacro 0.54 E reichen 2 E reichen E gruis E tropi

11. Acknowledgements • Central Isolation facility staff for technical support • M Aggie Fernando and Harry D. Danforth • Joanna and many other graduate and undergraduate students Thank You