Characterization of the A. marginale msp4 genotypes in cattle. The A. marginale msp4 coding region was amplified by PCR, sequenced and the frequency (%) for each genotype represented in (A) vaccinated and (B) control cattle. Dashed line represents time of last immunization shot. (C) The msp4 sequences of each genotype were aligned and compared to the reference sequence (GenBank accession number DQ000618). Sequence positions (the adenine in the translation initiation codon ATG corresponds to position 1) with polymorphisms were identified. Asterisks represent sequence positions identical to the reference sequence. (D) Distribution in the frequencies of the most abundant A. marginale msp4 genotypes in vaccinated and control cattle. Dashed line represents time of last immunization shot. A correlation analysis was conducted to analyze genotype frequencies in time using Excel and represented only when the correlation coefficient (R2) was ≥0.5.