Capture-based enrichment of Theileria parva DNA enables full genome assembly of first buffalo-derived strain and reveals exceptional intra-specific genetic diversity

Abstract

Theileria parva is an economically important, intracellular, tick-transmitted parasite of cattle. A live vaccine against the parasite is effective against challenge from cattle-transmissible T. parva but not against genotypes originating from the African Cape buffalo, a major wildlife reservoir, prompting the need to characterize genome-wide variation within and between cattle- and buffalo-associated T. parva populations. Here, we describe a capture-based target enrichment approach that enables, for the first time, de novo assembly of nearly complete T. parva genomes derived from infected host cell lines. This approach has exceptionally high specificity and sensitivity and is successful for both cattle- and buffalo-derived T. parva parasites. De novo genome assemblies generated for cattle genotypes differ from the reference by ~54K single nucleotide polymorphisms (SNPs) throughout the 8.31 Mb genome, an average of 6.5 SNPs/kb. We report the first buffalo-derived T. parva genome, which is ~20 kb larger than the genome from the reference, cattle-derived, Muguga strain, and contains 25 new potential genes. The average non-synonymous nucleotide diversity (πN) per gene, between buffalo-derived T. parva and the Muguga strain, was 1.3%. This remarkably high level of genetic divergence is supported by an average Wright’s fixation index (FST), genome-wide, of 0.44, reflecting a degree of genetic differentiation between cattle- and buffalo-derived T. parva parasites more commonly seen between, rather than within, species. These findings present clear implications for vaccine development, further demonstrated by the ability to assemble nearly all known antigens in the buffalo-derived strain, which will be critical in design of next generation vaccines. The DNA capture approach used provides a clear advantage in specificity over alternative T. parva DNA enrichment methods used previously, such as those that utilize schizont purification, is less labor intensive, and enables in-depth comparative genomics in this apicomplexan parasite.

South African Buffalo-Derived Theileria parva Is Distinct From Other Buffalo and Cattle-Derived T. parva

“…Available data so far supports the arguments that buffalo-derived T. parva is distinct from cattle-derived strains (Oura et al, 2011;Elisa et al, 2014) with cattle strains being less diverse, subset of the buffalo strains (Oura et al, 2003;Pelle et al, 2011). A similar study done by Palmateer et al (2020), also using bait capture found buffaloderived T. parva to be highly divergent from cattle-derived T. parva. While this latter genome would be of interest to use as an alternative reference genome to the Muguga genome, it still needs refinement with regard to assignment of contigs to the correct homologous chromosomes of the Muguga genome.…”

Antigenic Diversity in Theileria parva Populations From Sympatric Cattle and African Buffalo Analyzed Using Long Read Sequencing

“…The advent of genomic analysis has added further insight, with the initial sequencing of nine isolates indicating that the two genomes of buffalo-derived T. parva analyzed were divergent, with twice the number of single nucleotide polymorphisms (SNPs) compared to the T. parva reference genome (T. parva Muguga-originally isolated from a cow) compared to seven genomes of cattle-derived T. parva (Hayashida et al, 2013). This putative divergence has been recently supported by the first de novo genome assembly of a buffalo-derived T. parva isolate, which indicated a slightly larger genome, significantly high nonsynonymous nucleotide diversity and genome-wide F ST values compared to the T. parva Muguga genome, at levels compatible with those observed between species rather than within species (Palmateer et al, 2020).…”

“…However, the indications of separate clusters of genotypes that derive from either only buffalo or from both cattle and buffalo, alternatively suggest that this may be an example of incipient speciation, with divergence between cattle and buffalo-derived samples in process, i.e., comparable to but at an earlier stage of divergence than the very closely related T. sp. (buffalo) (Palmateer et al, 2020)-albeit that in T. sp. (buffalo) this is suggested to be due to adaptation to a different tick species rather than a different mammalian host (Bishop et al, 2015).…”

“…A further limitation of the current study is that by focusing on antigens recognized by the bovine immune system, we may be analyzing genes that are under particular selection pressures, which has the potential to confound other genetic signals, such as those associated with population substructuring by host. In order to more fully understand the population structure, ideally a larger sample set of both T. parva-positive cattle and buffalo would be required, and an approach such as sequence capture used to enable analysis of polymorphism at the whole genome level (Palmateer et al, 2020). Analysis of whole genome data would facilitate unbiased and thorough analysis of genetic diversity in cattle and buffalo-derived T. parva, and enable identification of signatures of selection that may underpin adaptation to differentiation to piroplasms in cattle, for example.…”

Unique Mitochondrial Single Nucleotide Polymorphisms Demonstrate Resolution Potential to Discriminate Theileria parva Vaccine and Buffalo-Derived Strains

“…However, the genetic underpinnings of these strain differences are yet to be fully unraveled, and a precise delineation of the various genotypes is lacking [ 23 ]. This is partly because of the parasite’s biology, which renders it technically unamenable for genomic studies, especially in obtaining pure parasite DNA free from host-DNA contamination [ 26 ]. An accurate determination of the origin (buffalo or cattle derived) and geographic spread of strains will help intervention and control efforts.…”