Initiation of a Sarcocystis neurona expressed sequence tag (EST) sequencing project: a preliminary report.

The researchers undertook a project to sequence expressed sequence tags (ESTs) for Sarcocystis neurona, a parasite that is the main cause of equine protozoal myeloencephalitis (EPM). This will provide around 7000-8000 ESTs that will aid in understanding the genetics and molecular characteristics of the parasite and will speed up the discovery of genes related to this protozoan pathogen, thereby aiding the development of improved diagnostic methods, treatments, and preventatives for EPM.

Expressed Sequence Tag (EST) Sequencing Project

• The process involved acquiring Poly(A)(+) RNA from culture-derived S. neurona merozoites and constructing a cDNA library in a unidirectional lambda phage cloning vector.
• The researchers selected 60 phage clones randomly from the library and identified cDNA inserts segregated in size from 0.4 to 4.0 kilobases (kb) with an average size of 1.23kb.

Analysis of the Clones

• All the 60 clones selected had recombinant cDNA inserts.
• The researchers performed single-pass sequencing, starting from the 5’ end, of the 60 amplified cDNAs, and yielded high-quality nucleotide sequence data from 53 of the clones.
• When comparing these ESTs with existing gene databases, there were significant matches for 10 of the ESTs, of which six resembled sequences from other Apicomplexa (specifically, Toxoplasma gondii).

Significance of the Research

• Notably, none of the ESTs seemed to have a mammalian origin, suggesting that the cDNAs in this library sourced predominately from parasite mRNA and not from mRNA of the bovine turbinate host cells.
• This gives a clear indication that the described cDNA library will greatly assist in generating a part of the planned ESTs from S. neurona.
• This sequencing project will substantially speed up the gene discovery for this protozoan pathogen, enhancing the development of improved diagnostic measures as well as treatment and prevention methods for EPM.
• The S. neurona ESTs will also contribute significantly to the large database of sequences from the Apicomplexa. Comparing these sequences could provide a wealth of knowledge about the biology and evolutionary history of this group of parasites.