Evolution and diversity of secretome genes in the apicomplexan parasite Theileria annulata

<b>BACKGROUND</b>: Little is known about how apicomplexan parasites have evolved to infect different host species and cell types. Theileria annulata and Theileria parva invade and transform bovine leukocytes but each species favours a different host cell lineage. Parasite-encoded proteins secreted from the intracellular macroschizont stage within the leukocyte represent a critical interface between host and pathogen systems. Genome sequencing has revealed that several Theileria-specific gene families encoding secreted proteins are positively selected at the inter-species level, indicating diversification between the species. We extend this analysis to the intra-species level, focusing on allelic diversity of two major secretome families. These families represent a well-characterised group of genes implicated in control of the host cell phenotype and a gene family of unknown function. To gain further insight into their evolution and function, this study investigates whether representative genes of these two families are diversifying or constrained within the T. annulata population. <b>RESULTS</b>: Strong evidence is provided that the sub-telomerically encoded SVSP family and the host-nucleus targeted TashAT family have evolved under contrasting pressures within natural T. annulata populations. SVSP genes were found to possess atypical codon usage and be evolving neutrally, with high levels of nucleotide substitutions and multiple indels. No evidence of geographical sub-structuring of allelic sequences was found. In contrast, TashAT family genes, implicated in control of host cell gene expression, are strongly conserved at the protein level and geographically sub-structured allelic sequences were identified among Tunisian and Turkish isolates. Although different copy numbers of DNA binding motifs were identified in alleles of TashAT proteins, motif periodicity was strongly maintained, implying conserved functional activity of these sites. <b>CONCLUSIONS</b>: This analysis provides evidence that two distinct secretome genes families have evolved under contrasting selective pressures. The data supports current hypotheses regarding the biological role of TashAT family proteins in the management of host cell phenotype that may have evolved to allow adaptation of T. annulata to a specific host cell lineage. We provide new evidence of extensive allelic diversity in representative members of the enigmatic SVSP gene family, which supports a putative role for the encoded products in subversion of the host immune response

Evaluation of cytochrome b as a sensitive target for PCR based detection of T. annulata carrier animals

Bovine tropical theileriosis, caused by the tick-borne protozoan Theileria annulata, imposes a serious constraint upon breed improvement programmes and livestock production in tropical and sub-tropical regions of the world. Animals that recover from primary infection serve as carriers and play a critical role in the epidemiology of the disease, acting as reservoirs of infection. However, conclusive identification of carrier animals can be problematic. This study describes assessment of candidate target genes for PCR assay-based detection of T. annulata infected carrier animals. Following in silico screening and rejection of three major multi-copy gene families, an assay based on PCR amplification of a 312 bp segment of the T. annulata gene for cytochrome b (Cytob1 assay) was established. Sensitivity was evaluated using serial dilutions of blood obtained from experimentally infected calves, while specificity was confirmed by testing DNA representing twelve different T. annulata stocks and other Theileria and Babesia species. Direct comparison with other target genes and published data indicated that Cytob1 PCR-based assays provide the greatest level of sensitivity, combined with a high level of specificity and the ability to detect different T. annulata genotypes. It can be concluded that the cytochrome b gene is the optimal target for PCR amplification and its incorporation in a Reverse Line Blot Assay offers the most sensitive method yet devised to detect the parasite in carrier animals. The use of this assay will increase the accuracy of epidemiological studies aimed at improving disease control in endemically unstable regions. Keywords: Theileria annulata; PCR diagnosis; Tick-borne disease; Carrier state detectio