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  Contributed Papers: Posters
Diagnosis and Epidemiology

 

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.


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