Eimeria SPECIES
IDENTIFICATION IN FAECES AND LITTER SAMPLES FROM CHICKENS
A. Haug 1, 2, *, P. Thebo 1 and J.G.
Mattsson 1.1 Department of Parasitology (SWEPAR),
National Veterinary Institute and Swedish University
of Agricultural Sciences, Uppsala, Sweden 2 Department
of Pathology, National Veterinary Institute, Oslo,
Norway * anita.haug@vetinst.no
Coccidial infections
in chickens are caused by one or several Eimeria spp.
The oocyst wall of Eimeria spp. is particularly rigid
and resistant, which makes it difficult to achieve
an effective DNA extraction. A classic but time-consuming
method for Eimeria species identification is by first
breaking the oocysts by glass bead grinding, followed
by phenol DNA extraction and identification of species-specific
genomic regions by PCR. This study aimed to find a
fast, robust and efficient method for identifying
chicken Eimeria spp. in field samples. The methods
were evaluated according to inter-species variations
in detection level, repeatability, hands-on-time and
cost efficiency. Five methods for rupturing oocyst
walls were tested, including sonication, microwaves,
heating, pestle- and glass bead grinding. Two series
of suspensions containing faecal debris and with known
number of oocysts from E. mitis, E. praecox, E. maxima
and E. tenella were grinded with glass beads and pestle,
respectively. DNA was extracted from oocysts ruptured
with pestle using either commercial systems (GeneReleaser,
Qiagen Stoolkit and Prepman) or phenol extraction,
and then compared to the classical method. Detection
levels were evaluated by identifying species-specific
ITS-1 regions using optimized single species PCRs.
The StoolKit protocol showed poor reliability and
high variability in detection levels, and is expensive
and relatively time-consuming. Although the Prepman
protocol requires minimal hands-on-time and it is
cheap, detection levels were not consistent among
the species. The detection level for the GeneReleaser
protocol was very stable, both between species and
within the method, detecting less than 2 oocysts of
each species per PCR. The phenol DNA isolation method,
using either method for oocyst rupture, showed similar
results as the GeneReleaser protocol. Our results
suggest that isolation of DNA using the GeneReleaser
kit combined with a pestle grinder is a repeatable
and cost-efficient method with limited inter-species
variation in detection level. Importantly, it also
provides minimal hands-on-time in the pre-PCR process.