Cloning and characterization of the 5S rRNA genes from Eimeria spp. (Pengklonan dan pencirian gen 5S rRNA daripada Eimeria spp.)

Abstract Poultry coccidiosis is an economically important disease worldwide. It is caused by intracellular protozoan parasites of the genus Eimeria (phylum Apicomplexa). Traditional methods of relying on disease pathology or oocyst morphology have limitations particularly in detecting minor contaminating populations of Eimeria in chicken. Therefore, a DNA-based test using ribosomal RNA (rRNA) was chosen to identify a molecular marker to enable faster and more sensitive identification of a particular species. The 5S rRNA gene was chosen because of its high degree of conservation, ubiquity and the relative ease with which it can be cloned. The 5S rRNA genes from Eimeria spp. were amplified by the polymerase chain reaction (PCR) using purified DNAs of the sporozoites. TA Cloning method was used to clone the PCR products (600-900 bp) into plasmid vector pCR 2.1 (3.9 kb) and transformed into Escherichia coli strain TOP10F'. Recombinant plasmids with size of 4.6 -4.8 kb were found. Clones containing the inserts of the appropriate size were sequenced by automated sequencing whereby M13 forward and reverse primers were used. The 5S rRNA genes from the seven Eimeria species were successfully sequenced. The sequences obtained were then sent to the Basic Local Alignment Search Tool (BLAST) program and results showed that all sequences were identical to the 5S rRNA gene from other organism. Sequences of 726, 738, 697, 673, 732 and 931 bp were each shown by E. tenella, E. acervulina, E. praecox, E. maxima, E. brunetti and E. necatrix whilst E. mitis has two sequences of 710 and 592 bp. For each species, at least 2 clones of PCR-generated fragments were sequenced. The results indicated that the presence of unique amplified DNA segments could be exploited as molecular markers to identify Eimeria species of the chicken