A simplified protocol for molecular identification of Eimeria species in field samples

Molecular diagnosis of Eimeria species affecting naturally infected Gallus gallus

“…The use of glass beads has been the most commonly used procedure for disruption of oocyst walls (Procunier et al, 1993;Molloy et al, 1998;Fernandez et al, 2003b). It is important to have the spheres of correct size, quantify the number of oocysts, take into account the contaminants in the sample and the time spent in disruption procedure (Haug et al, 2007). Other methods to rupture oocysts may also be employed, although the results are variable and large numbers of oocysts are often not affected.…”

“…Fernandez et al (2003b) found a minimum limit of 50 oocysts for detection of six species using the PCR multiplex, while the limit for E. tenella was found to be 100 oocysts both in individual and multiplex reactions. On the other hand, Haug et al (2007) managed to amplify individual samples containing between 2 and 10 E. tenella oocysts, using phenol/ chloroform for DNA extraction. In Japan, Kawahara et al (2008) used an average of 100 oocysts for the identification of five species of the genus Eimeria.…”

“…Failure to identify the field samples was possibly related to low-final DNA concentration of Eimeria species, associated with possible inhibitors that remained along in the sample after the extraction process. As indicated by Haug et al (2007), multiplex PCR is a technique that is more difficult to optimize, giving lower sensitivity and reproducibility. Furthermore, extraction of DNA from fecal samples may generate inhibitors of Taq DNA polymerase, negatively affecting the reactions.…”

“…For successful identification, it is necessary to have an efficient procedure for DNA extraction from oocysts; protocols with phenol/chloroform and commercial extraction kits are commonly used to break the rigid oocyst wall (Fernandez et al, 2003b;Meireles et al, 2004;Haug et al, 2007).…”

Prevalence of Eimeria species in scavenging native chickens of Shiraz, Iran

“…It is caused by one or several of seven Eimeria species infecting chickens (McDougald, 2003). These species differ in their localization in the gut and in their ability to induce morbidity and mortality (Haug et al, 2007;Morris et al, 2007). This parasitic infection occurs in the epithelial cells of the intestine, despite the advances in nutrition, chemotherapy, management and genetics.…”