Biological and Transcriptomic Comparison of Two Immunologically Distinct Strains of Eimeria maxima (GS and M6) and Characterization of Their Glycophosphatidylinositol (GPI) Anchored Surface Antigen Expression

Al-Badri, Riadh Rahma Kazim
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University of Guelph

Two immunologically distinct strains of poultry coccidium Eimeria maxima, Guelph (GS) and M6 strains, were investigated. Paired in vivo experiments demonstrated that GS and M6 have prepatent periods of approximately 120 h followed by peak oocyst shedding at 144 150 h post inoculation. Fecundity of E. maxima M6 (12.8×103±1.95 oocysts shed/oocyst inoculated) was approximately twice that of GS (6.9×103±3.33) when inoculated with 1×103 infective oocysts per bird. Numerous sequential observations of synchronized populations of oocysts sporulating at 26°C showed no difference in the sporulation kinetics of the two strains; in both strains, sporogony was divided into five morphologically distinguishable stages whose abundance peaked at the following times during sporulation: unsporulated oocysts at 0 h; sporoblast anlagen at 18 h; sporoblasts without sporocyst walls at 22 h; and sporocysts without mature sporozoites at 38 h. Total RNA was isolated from four stages of sporogonic development (18 h, 22 h and 38 h of sporulation, and excysting sporozoites). These RNA samples were quantitatively pooled from each strain separately prior to selection of poly A mRNA that was then fragmented, end-labeled and pyrosequenced using an Illumina HiSeq 2000 sequencer. The resulting transcriptome sequences (~48.8×109bp total reads) were paired and de novo assembled. Ten thousands transcripts (5,000 from each strain) were searched against GenBank using blastx. A total of 2,067 transcripts of GS and 1,610 transcripts of M6 were assigned to putative biological function; ~60% of functionally annotated transcripts mapped to metabolic or cellular processes. GPI anchored surface antigens (SAgs) identified in GS (18 SAgs) and M6 (18 SAgs) belonged to four major multi copy gene families and 2 single copy loci. Relative expression of SAgs expressed by both strains was generally similar; however, 3 GPI-anchored SAgs were uniquely expressed by each of GS and M6. One multigene locus demonstrated strain-specific SAg expression that may explain the lack of cross immunity between these strains. This represents the first transcriptome data of sporulation of E. maxima and first comparison of immunologically distinct strains of any Eimeria sp. These data should aid in the search for antigenic targets that could be included in future subunit vaccines against these important agricultural parasites.

parasitology, coccidiosis, poultry diseases, molecular biology, transcriptomics