Genes encoding two <i>Theileria parva</i> antigens recognized by CD8⁺ T-cells exhibit sequence diversity in South Sudanese cattle populations but the majority of alleles are similar to the Muguga component of the live vaccine cocktail

<div><p>East Coast fever (ECF), caused by <i>Theileria parva</i> infection, is a frequently fatal disease of cattle in eastern, central and southern Africa, and an emerging disease in South Sudan. Immunization using the infection and treatment method (ITM) is increasingly being used for control in countries affected by ECF, but not yet in South Sudan. It has been reported that CD8⁺ T-cell lymphocytes specific for parasitized cells play a central role in the immunity induced by ITM and a number of <i>T</i>. <i>parva</i> antigens recognized by parasite-specific CD8⁺ T-cells have been identified. In this study we determined the sequence diversity among two of these antigens, Tp1 and Tp2, which are under evaluation as candidates for inclusion in a sub-unit vaccine. <i>T</i>. <i>parva</i> samples (<i>n</i> = 81) obtained from cattle in four geographical regions of South Sudan were studied for sequence polymorphism in partial sequences of the Tp1 and Tp2 genes. Eight positions (1.97%) in Tp1 and 78 positions (15.48%) in Tp2 were shown to be polymorphic, giving rise to four and 14 antigen variants in Tp1 and Tp2, respectively. The overall nucleotide diversity in the Tp1 and Tp2 genes was π = 1.65% and π = 4.76%, respectively. The parasites were sampled from regions approximately 300 km apart, but there was limited evidence for genetic differentiation between populations. Analyses of the sequences revealed limited numbers of amino acid polymorphisms both overall and in residues within the mapped CD8⁺ T-cell epitopes. Although novel epitopes were identified in the samples from South Sudan, a large number of the samples harboured several epitopes in both antigens that were similar to those in the <i>T</i>. <i>parva</i> Muguga reference stock, which is a key component in the widely used live vaccine cocktail.</p></div

Multiple amino acid sequence alignment of Tp1 and Tp2 antigen variants present in cattle from South Sudan.

<p>(A) Multiple sequence alignment of the four Tp1 antigen variants. Antigen variants Var-1, -3 and -9 were first described by Pelle et al. (2011) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171426#pone.0171426.ref017" target="_blank">17</a>]. (B) Multiple sequence alignment of 14 Tp2 antigen variants. The naming of the antigen variants follows the nomenclature by Pelle et al. (2011) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171426#pone.0171426.ref017" target="_blank">17</a>]. Antigen variants Var-1, -2 and -5 were first described by Pelle et al. (2011) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171426#pone.0171426.ref017" target="_blank">17</a>]. The CD8⁺ T-cell target epitopes are boxed and the polymorphic residues in the epitopes are shown in red. The frequency of each variant amongst the samples tested is indicated in square brackets. Residues conserved in all sequences are identified below the alignment (*). The shaded flanking regions are equivalent to the positions of the secondary (nested) PCR primers, and are not included in estimations of the percentage of the residues that are conserved.</p

Map of South Sudan showing the sampling sites.

<p>The four areas where cattle samples were collected are colour coded as follows: Bor = Blue; Juba = Orange; Yei = Green; Kajo keji = Red. Numbers in brackets indicate the number of cattle sampled at each site.</p

Principal component analysis (PCA) of Tp1 (A) and Tp2 (B).

<p>This diagram illustrates the relationship between the geographic origin of the samples and the Muguga strain. The proportion of variation in the dataset explained by the 1^st and 2^nd principal components is indicated in parentheses.</p

Median-Joining network of 9 and 15 haplotypes observed in <i>T</i>. <i>parva</i> population in South Sudan based on the polymorphic sites of (A) Tp1 and (B) Tp2 genes, respectively.

<p>The sizes of the circles are proportional to the haplotype frequencies. The origin of each haplotype is colour coded as follows: Bor = Yellow; Juba = Red; Yei = Blue; Kajo keji = Green; Median vector = Brown; <i>T</i>. <i>parva</i> Muguga = Black. The numbers in red in (A) represent the mutations that differentiate the haplotypes.</p

Neighbour-joining trees of Tp1 and Tp2 gene sequences indicating phylogeographic relationships among cattle derived <i>T</i>. <i>parva</i> isolates.

<p>The isolates are color-coded based on their geographic origin in South Sudan and alleles which are represented by these samples are shown in brackets. The colour codes are as follows: Bor = Blue; Juba = Orange; Yei = Green; Kajo keji = Red. Bootstrap values >50% are shown above the nodes. (A) Tree showing relationships between Tp1 gene sequences from 79 cattle isolates of <i>T</i>. <i>parva</i>. The TP03_0849 gene from the <i>T</i>. <i>parva</i> (Muguga) genome sequence was also included in the analysis (Tp1-F100-TpM). The sequence of <i>T</i>. <i>annulata</i> Tp1 homologue (TA17450) was used to root the tree. (B) Tree showing relationships among Tp2 gene sequences from 65 cattle-derived <i>T</i>. <i>parva</i> isolates. The TP01_0056 gene from <i>T</i>. <i>parva</i> (Muguga) genome sequence was also included in the analysis (Tp2-F100-TpM). The Tp2 homologous sequence from <i>T</i>. <i>annulata</i> (TA19865) was used to root the tree.</p

Genotyping 50kSNPs East African Shorthorn Zebu

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Results of significant predictors of haemonchosis deaths.

<p>Results of significant predictors of haemonchosis deaths.</p

Map of Western Kenya showing the 4 agro-ecological zones and the 20 study sub-locations (in red).

<p>The study area comprised sub-locations falling within a 45 km radius from Busia town where the IDEAL project laboratory was located.</p

Map showing number of deaths attributable to haemonchosis by sub-location.

<p>In total, 10 of 88 deaths were attributed to haemonchosis.</p