Detection of Eimeria acervulina using the Polymerase Chain Reaction

Abstract

A polymerase chain reaction (PCR)-based assay was developed for the detection of Eimeria acervulina. Primers were designed to amplify a fragment of the EASZ240/160 sporozoite antigen gene. The PCR assay detected as few as 10 E. acervulina oocysts in a mixed population containing a total of 10(6) oocysts. No nonspecific reaction was observed with any other species of avian Eimeria known to occur in Australia. PCR products from genomic DNA were 237 bp larger than predicted from previously reported cDNA sequences. Sequencing of the product revealed the presence of a probable intron. This work demonstrates the potential of PCR-based assays for identification and detection of avian Eimeria. Potential uses include identification of minor species present in mixed infections and quality control in the production of live vaccines.

High-resolution electrophoretic procedures for the identification of fiveEimeria species from chickens, and detection of population variation

“…Individual amplicons were mixed with an equal volume of loading buffer (10 mM NaOH, 95% formamide, 0.05% bromophenol blue and 0.05% xylene cyanole) and their intensity verified on ethidium bromide-stained, 2.5% agarose-TBE gels using a 100 bp ladder (Promega) as a size marker. The lowest amount of Eimeria DNA required for effective amplification (for both primer sets) and visual detection on agarose gels was~5 pg (represents~5±50 oocysts), which is comparable with previous studies [5,6,12]. …”

Molecular diagnosis of Eimeria species affecting naturally infected Gallus gallus

“…Glass beads were used to rupture the oocyst wall; this procedure was essential for the success of the extraction. 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).…”

Sensitive and specific identification by polymerase chain reaction ofEimeria tenellaandEimeria maxima, important protozoan pathogens in laboratory avian facilities

“…Consequently, the development of molecular tools for identification and characterization of these parasites is important. Several polymerase chain reaction (PCR)-based assays targeting different regions of the Eimeria genome have been described, including 5S rRNA [6], small subunit rRNA [6,7], sporozoite antigen gene EASZ240/160 [8], internal transcribed spacer-1 (ITS-1) [9-12], and ITS-2 [13-15]. Nevertheless, the practical implementation of these methods in routine diagnostic and epidemiological studies of chicken coccidiosis [9,12] has been limited, and as such the assays are still regarded as experimental.…”

“…The oocyst wall of avian coccidia is particularly rigid and resistant to chemical and mechanical forces [2,17]. A number of methods to rupture the coccidial oocyst wall have been suggested [18-20], but the most widespread technique is to add glass beads to the oocyst suspension and then vortex until the glass-bead grinding ruptures the oocysts [8,16,21-23]. …”