Transcript Collection, Preservation, and Sporulation of Fecal Stages from Birds: Concepts, Methods and Challenges. J.R. Barta, J.D. Ogedengbe, and J. Cobean

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 Canada
[email protected]
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
Coccidia of Birds
Genera include:

Eimeria
(4ScX2Sz, Steida bodies)

Isospora
(2ScX4Sz, Steida bodies)

Tyzzeria
(0ScX8Sz, Steida bodies)

Caryospora
(1ScX8Sz, Steida bodies)
Isospora sp.
(Eimeriidae)
Speciose in virtually all avian
hosts and environments
Cystoisospora sp.
(Sarcocystidae)
Coccidia of Chickens - Galliformes
E.acervulina
Eimeria species
E. praecox

E. maxima
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
E. brunetti
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Morphological variation
Niche specialization
Reproductive isolation
Microallopatric speciation
E. mitis

E. mivati

E. necatrix

E. tenella
Other spp.
easy to grow, yes
tough to purify and keep
that way
tough to break for
parasite or nucleic acid
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’

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)

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

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
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

Coccidia of Birds - Challenges
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)
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0.78
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0.54
T gondi
Neos sp
N canNC 1
N caninum
C belli
C belli2
C orlovi
C suis
C ohioe
S mucosa
F glareoli
F microti
Guossia janae
E arnyi
E rioarri
E catron
Atoxoplasma
Isospora robini
E furonEF
E alabam
E faurei
E auburn
E ahsata
E crandal
E weybri
E ovinoi
E bovis
E scabra
E polita
E porci
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
E ten madur
E necat
E neca wTY
E praeAtt
E brun Att
E max prec
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
E antrozoi
E leucopi
E onycho
E reedi
E albigul
E arizon
E chaetod
E peromys
E chobot
E dipodo
C bigene
C bigene 2
E auritusi
E phalacro
E reichen 2
E reichen
E gruis
E tropi
Acknowledgements
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Central Isolation facility staff for technical support
M Aggie Fernando and Harry D. Danforth
Joanna and many other graduate and undergraduate
students