E. brunetti was never identified using the multiplex PCR in one- or two-tube formats. Accurate identification of Eimeria

spp. is important not only for the diagnosis of disease but also for management of subclinical infection, development and application of effective control strategies, and biological and epidemiological study ( Lee et al., 2010 and Sun et al., 2009). Traditionally, identification of Eimeria spp. has been based on the morphological characteristics of oocysts, parasite biology, Lapatinib datasheet clinical signs of the affected animals, and the typical macroscopic lesions assessed during necropsy ( Long and Joyner, 1984). However, in a natural setting mixed infections of different Eimeria spp. are commonly encountered and morphological characteristics and pathological changes may overlap, hindering accurate diagnosis and undermining detection of subclinical disease ( Long and Joyner, 1984 and Rice and Reid, 1973). Thus, it has been suggested that these methods should not be used in isolation selleck for differentiation of Eimeria

species ( Long and Joyner, 1984 and Lopez et al., 2007). Alternatives include molecular or computational approaches such as PCR, qPCR and the software COCCIMORPH. PCR assays capable of identifying and differentiating Eimeria spp. have been available for more than 20 years but, despite recognition as the ‘gold standard’ of detection for many pathogens, this technology is yet to replace traditional coccidial diagnostics ( Brook et al., 2008, Olano and Walker, 2011 and Stucki et al., 1993). Features of eimerian biology including the resistance of the oocyst wall to anything other than mechanical disruption, limiting access to template DNA (for most avian-infecting species), and PCR inhibition by the surrounding faecal material have discouraged use of PCR. While several PCR assays have been

described to identify specific Eimeria species very few studies have focused on the applicability of these techniques for identifying Eimeria spp. in commercially raised poultry throughout the world ( Carvalho et al., 2011a, Carvalho et al., 2011b, Frölich et al., 2013 and Haug et al., 2008). Development of a standardised protocol supporting medium throughput Endonuclease diagnostic sampling for Eimeria will enhance the value of such data while promoting the application of PCR and comparison between studies. Following collection of fresh environmental faecal samples we explored two DNA extraction procedures and the influence of residual faecal contamination. The inclusion of faecal material dramatically reduced PCR sensitivity with genomic DNA purified using the QIAamp DNA Stool kit, supporting the value of even a rudimentary pre-extraction parasite purification step. The cause of this inhibition remains unclear at present. The InhibitEx step of the Stool kit protocol is designed to adsorb substances that can degrade DNA and inhibit downstream enzymatic reactions and should minimise PCR inhibition.