Sequence heterogeneity in the equi merozoite antigen gene (ema-1) of Theileria equi and development of an ema-1-specific TaqMan MGBTM assay for the detection of T equi

Although a quantitative real-time PCR assay (qPCR) assay for the detection of Theileria equi has been developed and evaluated It is possible that additional as yet undetected 185 rRNA gene sequence variants may exist A qPCR assay targeting a different gene used in conjunction with the T equi 18S rRNA qPCR assay could assist in the detection of all T qui genotypes in field samples AT emu ema-1-specific qPCR (lieu et al 2003) was tested on 107 South African field samples 90 of which tested positive for T equi antibody using the immuno-fluorescent antibody test (IFAT) The qPCR assay performed poorly as T equi was detected in only 67 of the 90 IFAT-positive field samples at quantification cycle (C-q) values ranging from 27 to 39 95 Furthermore a high C-q value of 36 18 was obtained from DNA extracted from a South African in vitro-cultured T equi WL isolate [1 38% parasitized erythrocytes (PE)] when a low C-q value (indicative of a high T emu DNA concentration) was expected Approximately 600 bp of the ema-1 gene from 38 South African samples were sequenced and BLASTN analysis confirmed all sequences to be merozoite surface protein genes with an identity of 87 1-100% to previously published T equi ema-1 gene sequences Alignment of the sequences revealed extensive sequence variations in the target regions of the primers and probes (Ueti et al 2003) explaining the poor performance of the qPCR assay Based on these observations we developed a new TaqMan minor-groove binder (MGB(TM)) probe-based qPCR assay targeting a more conserved region of the ema-1 gene This assay was shown to be efficient and specific and the detection limit defined as the concentration at which 95% of T equi-positive samples are detected was determined to be 1 4 x 10(-4)% PE The two ema-1 assays were compared by testing 41 South African field samples in parallel The results suggested that the new assay was more sensitive than the original assay as T equi was detected in more samples and at lower C-q values when the new assay was used Phylogenetic analyses of the 18S rRNA gene sequences and ema-1 amino acid sequences from the same samples showed inconsistencies between the clades indicating that the T equi 18S rRNA genetic groups previously identified in South Africa may not represent distinct T emu lineages It is possible that the different T equi ema-1 genotypes could be related to antigenic variability and pathogenicity and may be associated with clinical differences in equine piroplasmosis cases but this remains to be elucidated (C) 2010 Elsevier B V All rights reserved