Whole-genome sequencing of Theileria parva strains provides insight into parasite migration and diversification in the African continent.
ABSTRACT: The disease caused by the apicomplexan protozoan parasite Theileria parva, known as East Coast fever or Corridor disease, is one of the most serious cattle diseases in Eastern, Central, and Southern Africa. We performed whole-genome sequencing of nine T. parva strains, including one of the vaccine strains (Kiambu 5), field isolates from Zambia, Uganda, Tanzania, or Rwanda, and two buffalo-derived strains. Comparison with the reference Muguga genome sequence revealed 34 814-121 545 single nucleotide polymorphisms (SNPs) that were more abundant in buffalo-derived strains. High-resolution phylogenetic trees were constructed with selected informative SNPs that allowed the investigation of possible complex recombination events among ancestors of the extant strains. We further analysed the dN/dS ratio (non-synonymous substitutions per non-synonymous site divided by synonymous substitutions per synonymous site) for 4011 coding genes to estimate potential selective pressure. Genes under possible positive selection were identified that may, in turn, assist in the identification of immunogenic proteins or vaccine candidates. This study elucidated the phylogeny of T. parva strains based on genome-wide SNPs analysis with prediction of possible past recombination events, providing insight into the migration, diversification, and evolution of this parasite species in the African continent.
Project description:<i>Theileria parva</i> is a protozoan parasite transmitted by the brown-eared ticks, <i>Rhipicephalus appendiculatus</i> and <i>Rhipicephalus zambeziensis</i>. Buffaloes are the parasite's ancestral host, with cattle being the most recent host. The parasite has two transmission modes namely, cattle-cattle and buffalo-cattle transmission. Cattle-cattle <i>T. parva</i> transmission causes East Coast fever (ECF) and January disease syndromes. Buffalo to cattle transmission causes Corridor disease. Knowledge on the genetic diversity of South African <i>T. parva</i> populations will assist in determining its origin, evolution and identify any cattle-cattle transmitted strains. To achieve this, genomic DNA of blood and <i>in vitro</i> culture material infected with South African isolates (8160, 8301, 8200, 9620, 9656, 9679, Johnston, KNP2, HL3, KNP102, 9574, and 9581) were extracted and paired-end whole genome sequencing using Illumina HiSeq 2500 was performed. East and southern African sample data (Chitongo Z2, Katete B2, Kiambu Z464/C12, Mandali Z22H10, Entebbe, Nyakizu, Katumba, Buffalo LAWR, and Buffalo Z5E5) was also added for comparative purposes. Data was analyzed using BWA and SAMtools variant calling with the <i>T. parva</i> Muguga genome sequence used as a reference. Buffalo-derived strains had higher genetic diversity, with twice the number of variants compared to cattle-derived strains, confirming that buffaloes are ancestral reservoir hosts of <i>T. parva</i>. Host specific SNPs, however, could not be identified among the selected 74 gene sequences. Phylogenetically, strains tended to cluster by host with South African buffalo-derived strains clustering with buffalo-derived strains. Among the buffalo-derived strains, South African strains were genetically divergent from other buffalo-derived strains indicating possible geographic sub-structuring. Geographic sub- structuring was also observed within South Africa strains. The knowledge generated from this study indicates that to date, ECF is not circulating in buffalo from South Africa. It also shows that <i>T. parva</i> has historically been present in buffalo from South Africa before the introduction of ECF and was not introduced into buffalo during the ECF epidemic.
Project description:East Coast fever (ECF) is an acute fatal tick-borne disease of cattle caused by Theileria parva. It causes major losses in exotic and crossbreed cattle, but this could be prevented by a vaccine of T. parva if the vaccine is selected properly based on information from molecular epidemiology studies. The Muguga cocktail (MC) vaccine (Muguga, Kiambu 5 and Serengeti-transformed strains) has been used on exotic and crossbreed cattle. A total of 254 T. parva samples from vaccinated and unvaccinated cattle were used to understand the genetic diversity of T. parva in Malawi using partial sequences of the Tp1 and Tp2 genes encoding T. parva CD8+ antigens, known to be immunodominant and current candidate antigens for a subunit vaccine. Single nucleotide polymorphisms were observed at 14 positions (3.65%) in Tp1 and 156 positions (33.12%) in Tp2, plus short deletions in Tp1, resulting in 6 and 10 amino acid variants in the Tp1 and Tp2 genes, respectively. Most sequences were either identical or similar to T. parva Muguga and Kiambu 5 strains. This may suggest the possible expansion of vaccine components into unvaccinated cattle, or that a very similar genotype already existed in Malawi. This study provides information that support the use of MC to control ECF in Malawi.
Project description:Distinct pathogenic and epidemiological features underlie different <i>Theileria parva</i> strains resulting in different clinical manifestations of East Coast Fever and Corridor Disease in susceptible cattle. Unclear delineation of these strains limits the control of these diseases in endemic areas. Hence, an accurate characterization of strains can improve the treatment and prevention approaches as well as investigate their origin. Here, we describe a set of single nucleotide polymorphisms (SNPs) based on 13 near-complete mitogenomes of <i>T. parva</i> strains originating from East and Southern Africa, including the live vaccine stock strains. We identified 11 SNPs that are non-preferentially distributed within the coding and non-coding regions, all of which are synonymous except for two within the <i>cytochrome b</i> gene of buffalo-derived strains. Our analysis ascertains haplotype-specific mutations that segregate the different vaccine and the buffalo-derived strains except <i>T. parva-</i>Muguga and Serengeti-transformed strains suggesting a shared lineage between the latter two vaccine strains. Phylogenetic analyses including the mitogenomes of other <i>Theileria</i> species: <i>T. annulata</i>, <i>T. taurotragi</i>, and <i>T. lestoquardi</i>, with the latter two sequenced in this study for the first time, were congruent with nuclear-encoded genes. Importantly, we describe seven <i>T. parva</i> haplotypes characterized by synonymous SNPs and parsimony-informative characters with the other three transforming species mitogenomes. We anticipate that tracking <i>T. parva</i> mitochondrial haplotypes from this study will provide insight into the parasite's epidemiological dynamics and underpin current control efforts.
Project description: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 (?N) 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.
Project description:There are no commercially available vaccines against human protozoan parasitic diseases, despite the success of vaccination-induced long-term protection against infectious diseases. East Coast fever, caused by the protist Theileria parva, kills one million cattle each year in sub-Saharan Africa, and contributes significantly to hunger and poverty in the region. A highly effective, live, multi-isolate vaccine against T. parva exists, but its component isolates have not been characterized. Here we sequence and compare the three component T. parva stocks within this vaccine, the Muguga Cocktail, namely Muguga, Kiambu5 and Serengeti-transformed, aiming to identify genomic features that contribute to vaccine efficacy.We find that Serengeti-transformed, originally isolated from the wildlife carrier, the African Cape buffalo, is remarkably and unexpectedly similar to the Muguga isolate. The 420 detectable non-synonymous SNPs were distributed among only 53 genes, primarily subtelomeric antigens and antigenic families. The Kiambu5 isolate is considerably more divergent, with close to 40,000 SNPs relative to Muguga, including >8,500 non-synonymous mutations distributed among >1,700 (42.5 %) of the predicted genes. These genetic markers of the component stocks can be used to characterize the composition of new batches of the Muguga Cocktail.Differences among these three isolates, while extensive, represent only a small proportion of the genetic variation in the entire species. Given the efficacy of the Muguga Cocktail in inducing long-lasting protection against infections in the field, our results suggest that whole-organism vaccines against parasitic diseases can be highly efficacious despite considerable genome-wide differences relative to the isolates against which they protect.
Project description:<h4>Background</h4>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.<h4>Methods</h4>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.<h4>Results</h4>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<sub>ST</sub> = 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.<h4>Conclusions</h4>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.
Project description:Theileria parva (T. parva) is a protozoan parasite that causes East Coast fever (ECF). The disease is endemic in Burundi and is a major constraint to livestock development. In this study, the parasite prevalence in cattle in six regions namely; Northern, Southern, Eastern, Western, Central and North Eastern was estimated. Furthermore, the sequence diversity of p67, Tp1 and Tp2 genes was assessed coupled with the population genetic structure of T. parva using five satellite markers. The prevalence of ECF was 30% (332/1109) on microscopy, 60% (860/1431) on ELISA and 79% (158/200) on p104 gene PCR. Phylogenetic analysis of p67 gene revealed that only allele 1 was present in the field samples. Furthermore, phylogenetic analysis of Tp1 and Tp2 showed that the majority of samples clustered with Muguga, Kiambu and Serengeti and shared similar epitopes. On the other hand, genetic analysis revealed that field samples shared only two alleles with Muguga Cocktail. The populations from the different regions indicated low genetic differentiation (FST = 0.047) coupled with linkage disequilibrium and non-panmixia. A low to moderate genetic differentiation (FST = 0.065) was also observed between samples and Muguga cocktail. In conclusion, the data presented revealed the presence of a parasite population that shared similar epitopes with Muguga Cocktail and was moderately genetically differentiated from it. Thus, use of Muguga Cocktail vaccine in Burundi is likely to confer protection against T. parva in field challenge trials.
Project description:Concerted evolution of multicopy gene families in vertebrates is recognized as an important force in the generation of biological novelty but has not been documented for the multicopy genes of protozoa. A multicopy locus, Tpr, which consists of tandemly arrayed open reading frames (ORFs) containing several repeated elements has been described for Theileria parva. Herein we show that probes derived from the 5'/N-terminal ends of ORFs in the genomic DNAs of T. parva Uganda (1,108 codons) and Boleni (699 codons) hybridized with multicopy sequences in homologous DNA but did not detect similar sequences in the DNA of 14 heterologous T. parva stocks and clones. The probe sequences were, however, protein coding according to predictive algorithms and codon usage. The 3'/C-terminal ends of the Uganda and Boleni ORFs exhibited 75% similarity and identity, respectively, to the previously identified Tpr1 and Tpr2 repetitive elements of T. parva Muguga. Tpr1-homologous sequences were detected in two additional species of Theileria. Eight different Tpr1-homologous transcripts were present in piroplasm mRNA from a single T. parva Muguga-infected animal. The Tpr1 and Tpr2 amino acid sequences contained six predicted membrane-associated segments. The ratio of synonymous to nonsynonymous substitutions indicates that Tpr1 evolves like protein-encoding DNA. The previously determined nucleotide sequence of the gene encoding the p67 antigen is completely identical in T. parva Muguga, Boleni, and Uganda, including the third base in codons. The data suggest that concerted evolution can lead to the radical divergence of coding sequences and that this can be a mechanism for the generation of novel genes.
Project description:Immunity to Theileria parva infection in cattle is often parasite stock specific. The antigenic diversity which is expressed at the schizont stage of the parasite together with a wild reservoir of the organism in buffalo has complicated the development of effective disease control by immunization. We have previously shown that about 70% of cattle inoculated with recombinant forms of p67, a sporozoite stage-specific surface antigen from the cattle-derived Muguga stock of the parasite, are immune to a homologous challenge. Thus, immune responses to p67 can play a role in immunity. The genes encoding this protein in five other parasite stocks have been sequenced. Here, we report that the p67 molecule encoded by four cattle-derived parasite stocks (Boleni, Uganda, Mariakani, and Marikebuni) that fall into different cross-immunity groups is identical in sequence to Muguga p67. The protein encoded by a buffalo-derived parasite exhibits 95% sequence identity with Muguga p67, the major difference being the presence of a 43-residue peptide insert. As predicted by these data, cattle inoculated with recombinant p67 can resist a heterologous cattle-derived parasite challenge. Seven of 12 cattle receiving a homologous Muguga challenge and 6 of 11 cattle receiving a heterologous Marikebuni challenge were immune to East Coast fever. These results extend earlier data suggesting that p67 is a conserved molecule and confirm its potential as a broad-spectrum vaccine antigen for the control of T. parva infection.
Project description:The extent of sequence diversity among the genes encoding 10 antigens (Tp1-10) known to be recognized by CD8+ T lymphocytes from cattle immune to Theileria parva was analysed. The sequences were derived from parasites in 23 buffalo-derived cell lines, three cattle-derived isolates and one cloned cell line obtained from a buffalo-derived stabilate. The results revealed substantial variation among the antigens through sequence diversity. The greatest nucleotide and amino acid diversity were observed in Tp1, Tp2 and Tp9. Tp5 and Tp7 showed the least amount of allelic diversity, and Tp5, Tp6 and Tp7 had the lowest levels of protein diversity. Tp6 was the most conserved protein; only a single non-synonymous substitution was found in all obtained sequences. The ratio of non-synonymous: synonymous substitutions varied from 0.84 (Tp1) to 0.04 (Tp6). Apart from Tp2 and Tp9, we observed no variation in the other defined CD8+ T cell epitopes (Tp4, 5, 7 and 8), indicating that epitope variation is not a universal feature of T. parva antigens. In addition to providing markers that can be used to examine the diversity in T. parva populations, the results highlight the potential for using conserved antigens to develop vaccines that provide broad protection against T. parva.