Sequence diversity of cytotoxic T cell antigens and satellite marker analysis of Theileria parva informs the immunization against East Coast fever in Rwanda

Abstract

Background: East Coast fever (ECF) caused by Theileria parva is endemic in Rwanda. In this study, the antigenic and genetic diversity of T. parva coupled with immunization and field challenge were undertaken to provide evidence for the introduction of ECF immunization in Rwanda.

Methods: Blood collected from cattle in the field was screened for T. parva using ELISA and PCR targeting the p104 gene. Tp1 and Tp2 gene sequences were generated from field samples and from Gikongoro and Nyakizu isolates. Furthermore, multilocus genotype data was generated using 5 satellite markers and an immunization challenge trial under field conditions using Muguga cocktail vaccine undertaken.

Results: Out of 120 samples, 44 and 20 were positive on ELISA and PCR, respectively. Antigenic diversity of the Tp1 and Tp2 gene sequences revealed an abundance of Muguga, Kiambu and Serengeti epitopes in the samples. A further three clusters were observed on both Tp1 and Tp2 phylogenetic trees; two clusters comprising of field samples and vaccine isolates and the third cluster comprising exclusively of Rwanda samples. Both antigens exhibited purifying selection with no positive selection sites. In addition, satellite marker analysis revealed that field samples possessed both shared alleles with Muguga cocktail on all loci and also a higher proportion of unique alleles. The Muguga cocktail (Muguga, Kiambu and Serengeti) genotype compared to other vaccine isolates, was the most represented in the field samples. Further low genetic sub-structuring (F_ST = 0.037) coupled with linkage disequilibrium between Muguga cocktail and the field samples was observed. Using the above data to guide a field immunization challenge trial comprising 41 immunized and 40 control animals resulted in 85% seroconversion in the immunized animals and an efficacy of vaccination of 81.7%, implying high protection against ECF.

Conclusions: Antigenic and genetic diversity analysis of T. parva facilitated the use of Muguga cocktail vaccine in field conditions. A protection level of 81.7% was achieved, demonstrating the importance of combining molecular tools with field trials to establish the suitability of implementation of immunization campaigns. Based on the information in this study, Muguga cocktail immunization in Rwanda has a potential to produce desirable results.

Keywords: ECF; Immunization; Muguga cocktail; Sub-structuring; Theileria parva; Tp1; Tp2.

Fig. 1

Map of Rwanda showing the location of study sites

Fig. 2

Theileria parva Tp1 neighbor-joining phylogenetic tree generated from 432 bp nucleotide sequences using MEGA 6 with 1000 bootstrap replicates. Rwanda Tp1 sequences are given in red and Muguga, Kiambu, Serengeti, Chitongo, Gikongoro and Nyakizu vaccine isolates are given in black and bold. Bootstrap values greater than 65% are shown on the phylogram. The outgroup used on the phylogenetic tree is Tp1 Theileria annulata (GenBank: TA17450)

Fig. 3

Theileria parva Tp2 neighbor-joining phylogenetic tree generated from 531 bp nucleotide sequences. MEGA 6 with 1000 bootstrap replicates was used to generate the phylogenetic tree. The sequences from Rwanda are given in red and the Muguga, Kiambu, Serengeti, Chitongo, Gikongoro and Nyakizu isolates in black and bold. Bootstrap values above 65% are given on the phylogram. Theileria annulata (GeneBank: TA19865) was utilized as the outgroup

Fig. 4

Median-joining network of Tp1 (a), Tp2 (b) and concatenated Tp1 and Tp2 sequences from Rwanda and Muguga cocktail, Chitongo, Gikongoro and Nyakizu isolates (c). The network compares the relatedness of the Rwanda field samples and the vaccine isolates. The frequency of the haplotype is represented by the size of the circle. Rwanda samples are given in yellow, Muguga cocktail in red, Chitongo in purple, Gikongoro in green and Nyakizu in brown. The size of the circles is proportional to the haplotype frequency

Fig. 5

Allele frequencies from field samples, Muguga, Kiambu and Serengeti (collectively referred to as Muguga cocktail), Chitongo, Gikongoro and Nyakizu vaccine isolates. Allele frequencies for loci MS39, MS25, MS7, ms9 and MS19 are presented in (a–e), respectively. On each locus, a high proportion of unique alleles is observed with only a few shared alleles. Muguga cocktail and Nyakizu isolates share alleles with Rwanda field samples on all loci while Gikongoro and Chitongo do not (a–e)

Fig. 6

Overall allele frequency from field samples, Muguga, Kiambu and Serengeti (together referred to Muguga cocktail), Chitongo, Gikongoro and Nyakizu vaccine isolates showing 41, 10, 8 and 6 unique alleles from Rwanda field samples, Chitongo, Gikongoro and Nyakizu vaccine isolates, respectively. A higher proportion of unique alleles is observed as compared to shared alleles. Predominant alleles were calculated as proportions of the total of each satellite marker and histograms were generated from the multi-locus genotype. The numbers on the x-axis represent the allele sizes in base pairs observed on each locus

Fig. 7

Principal components analysis (PCA) of Theileria parva field populations from Bugesera District, Eastern Rwanda and Muguga, Kiambu and Serengeti (collectively referred to as Muguga cocktail), Chitongo, Gikongoro and Nyakizu isolates showing a level of sub-structuring. Multilocus genotype data was used to construct the PCA and in each axis, the numbers show the variation in proportion in the population data set