FIG. 5.

Multiple different msp2 variants are present in the polycistronic expression site in each population of A. marginale. The major variant type is conserved during passage of A. marginale between culture, acute erythrocyte stage infection, and tick salivary glands but is not conserved in persistent cattle infections. The expression site was amplified by PCR using primers which annealed 288 bp 3′ to the termination codon of msp2 (AB752) and to the intercistronic sequence between orf3 and orf4 (AB750) to generate a product of 2.9 kbp from A. marginale genomic DNA that contained msp2, orf2, and orf3. The PCR product was cloned in pCR-XL-TOPO vector (Invitrogen), and independent colonies containing a 2.9-kbp insert were selected for sequencing of cloned plasmid DNA. The hypervariable region of the msp2 gene was sequenced on both strands in seven independent clones derived by PCR amplification from genomic DNA of each of the A. marginale populations described in Fig. 1. DNA sequences were translated to amino acids, and the different variant sequences were aligned with PILEUP. The proportion of each sequence variant in that population is indicated in brackets; e.g., the major sequence variant detected in cultured A. marginale was variant A, which was found in three of seven independent clones of the expression site. Identical amino acids shared between all variants are indicated by a dash and are shown on the bottom row of the alignment. Variant types present in different A. marginale populations are indicated by identical symbols to the left of the sequence alignments.