Antigenic diversity in Theileria parva populations from sympatric cattle and African buffalo analysed using long read sequencing

East Coast fever (ECF) in cattle is caused by the Apicomplexan protozoan parasite , transmitted by the three-host tick . The African buffalo () is the natural host for but does not suffer disease, whereas ECF is often fatal in cattle. The genetic relationship between populations circulating in cattle and buffalo is poorly understood, and has not been studied in sympatric buffalo and cattle. This study aimed to determine the genetic diversity of populations in cattle and buffalo, in an area where livestock co-exist with buffalo adjacent to the Serengeti National Park, Tanzania. Three antigens (Tp1, Tp4, and Tp16), known to be recognized by CD8 and CD4 T cells in immunized cattle, were used to characterize genetic diversity of in cattle ( = 126) and buffalo samples ( = 22). Long read (PacBio) sequencing was used to generate full or near-full length allelic sequences. Patterns of diversity were similar across all three antigens, with allelic diversity being significantly greater in buffalo-derived parasites compared to cattle-derived (e.g., for Tp1 median cattle allele count was 9, and 81.5 for buffalo), with very few alleles shared between species (8 of 651 alleles were shared for Tp1). Most alleles were unique to buffalo with a smaller proportion unique to cattle (412 buffalo unique vs. 231 cattle-unique for Tp1). There were indications of population substructuring, with one allelic cluster of Tp1 representing alleles found in both cattle and buffalo (including the TpM reference genome allele), and another containing predominantly only alleles deriving from buffalo. These data illustrate the complex interplay between populations in buffalo and cattle, revealing the significant genetic diversity in the buffalo population, the limited sharing of parasite genotypes between the host species, and highlight that a subpopulation of is maintained by transmission within cattle. The data indicate that fuller understanding of buffalo population dynamics is needed, as only a comprehensive appreciation of the population genetics of populations will enable assessment of buffalo-derived infection risk in cattle, and how this may impact upon control measures such as vaccination

Genetic and antigenic variation of the bovine tick-borne pathogen Theileria parva in the Great Lakes region of Central Africa

BACKGROUND : Theileria parva causes East Coast fever (ECF), one of the most economically important tick-borne diseases of cattle in sub-Saharan Africa. A live immunisation approach using the infection and treatment method (ITM) provides a strong long-term strain-restricted immunity. However, it typically induces a tick-transmissible carrier state in cattle and may lead to spread of antigenically distinct parasites. Thus, understanding the genetic composition of T. parva is needed prior to the use of the ITM vaccine in new areas. This study examined the sequence diversity and the evolutionary and biogeographical dynamics of T. parva within the African Great Lakes region to better understand the epidemiology of ECF and to assure vaccine safety. Genetic analyses were performed using sequences of two antigencoding genes, Tp1 and Tp2, generated among 119 T. parva samples collected from cattle in four agro-ecological zones of DRC and Burundi. RESULTS : The results provided evidence of nucleotide and amino acid polymorphisms in both antigens, resulting in 11 and 10 distinct nucleotide alleles, that predicted 6 and 9 protein variants in Tp1 and Tp2, respectively. Theileria parva samples showed high variation within populations and a moderate biogeographical sub-structuring due to the widespread major genotypes. The diversity was greater in samples from lowlands and midlands areas compared to those from highlands and other African countries. The evolutionary dynamics modelling revealed a signal of selective evolution which was not preferentially detected within the epitope-coding regions, suggesting that the observed polymorphism could be more related to gene flow rather than recent host immune-based selection. Most alleles isolated in the Great Lakes region were closely related to the components of the trivalent Muguga vaccine. CONCLUSIONS : Our findings suggest that the extensive sequence diversity of T. parva and its biogeographical distribution mainly depend on host migration and agro-ecological conditions driving tick population dynamics. Such patterns are likely to contribute to the epidemic and unstable endemic situations of ECF in the region. However, the fact that ubiquitous alleles are genetically similar to the components of the Muguga vaccine together with the limited geographical clustering may justify testing the existing trivalent vaccine for cross-immunity in the region.Additional file 1: Table S1. Cattle blood sample distribution across agroecological zones.Additional file 2: Table S2. Nucleotide and amino acid sequences of Tp1 and Tp2 antigen epitopes from T. parva Muguga reference sequence.Additional file 3: Table S3. Characteristics of 119 T. parva samples obtained from cattle in different agro-ecological zones (AEZs) of The Democratic Republic of Congo and Burundi.Additional file 4: Figure S1. Multiple sequence alignment of the 11 Tp1 gene alleles obtained in this study.Additional file 5: Table S4. Estimates of evolutionary divergence between gene alleles for Tp1 and Tp2, using proportion nucleotide distance.Additional file 6: Table S5. Tp1 and Tp2 genes alleles with their corresponding antigen variants.Additional file 7: Table S6. Amino acid variants of Tp1 and Tp2 CD8+ T cell target epitopes of T. parva from DRC and Burundi.Additional file 8: Figure S2. Multiple sequence alignment of the 10 Tp2 gene alleles obtained in this study.Additional file 9: Table S7. Distribution of Tp1 gene alleles of T. parva from cattle and buffalo in the sub-Saharan region of Africa.Additional file 10: Table S8. Distribution of Tp2 gene alleles of T. parva from cattle and buffalo in the sub-Saharan region of Africa.Additional file 11: Figure S3. Neighbor-joining tree showing phylogenetic relationships among 48 Tp1 gene alleles described in Africa.Additional file 12: Figure S4. Phylogenetic tree showing the relationships among concatenated Tp1 and Tp2 nucleotide sequences of 93 T. parva samples from cattle in DRC and Burundi.This study is part of the PhD work supported by the University of Namur (UNamur, Belgium) through the UNamur-CERUNA institutional PhD grant awarded to GSA for bioinformatic analyses, interpretation of data and manuscript write up in Belgium. The laboratory aspects (molecular biology analysis) of the project were supported by the BecA-ILRI Hub through the Africa Biosciences Challenge Fund (ABCF) programme. The ABCF Programme is funded by the Australian Department for Foreign Affairs and Trade (DFAT) through the BecA-CSIRO partnership; the Syngenta Foundation for Sustainable Agriculture (SFSA); the Bill & Melinda Gates Foundation (BMGF); the UK Department for International Development (DFID); and the Swedish International Development Cooperation Agency (Sida). The ABCF Fellowship awarded to GAS was funded by BMGF grant (OPP1075938). Sample collection, field equipment and preliminary sample processing were supported through the “Theileria” project co-funded to the Université Evangélique en Afrique (UEA) by the Agence Universitaire de la Francophonie (AUF) and the Communauté Economique des Pays des Grands Lacs (CEPGL). The International Foundation for Science (IFS, Stockholm, Sweden) supported the individual scholarship awarded to GSA (grant no. IFS-92890CA3) for field work and part of field equipment to the “Theileria” project.http://www.parasitesandvectors.comam2020Veterinary Tropical Disease

A cattle graph genome incorporating global breed diversity

Despite only 8% of cattle being found in Europe, European breeds dominate current genetic resources. This adversely impacts cattle research in other important global cattle breeds, especially those from Africa for which genomic resources are particularly limited, despite their disproportionate importance to the continent's economies. To mitigate this issue, we have generated assemblies of African breeds, which have been integrated with genomic data for 294 diverse cattle into a graph genome that incorporates global cattle diversity. We illustrate how this more representative reference assembly contains an extra 116.1 Mb (4.2%) of sequence absent from the current Hereford sequence and consequently inaccessible to current studies. We further demonstrate how using this graph genome increases read mapping rates, reduces allelic biases and improves the agreement of structural variant calling with independent optical mapping data. Consequently, we present an improved, more representative, reference assembly that will improve global cattle research

Continent-wide genomic analysis of the African buffalo (Syncerus caffer)

The African buffalo (Syncerus caffer) is a wild bovid with a historical distribution across much of sub-Saharan Africa. Genomic analysis can provide insights into the evolutionary history of the species, and the key selective pressures shaping populations, including assessment of population level differentiation, population fragmentation, and population genetic structure. In this study we generated the highest quality de novo genome assembly (2.65 Gb, scaffold N50 69.17 Mb) of African buffalo to date, and sequenced a further 195 genomes from across the species distribution. Principal component and admixture analyses provided little support for the currently described four subspecies. Estimating Effective Migration Surfaces analysis suggested that geographical barriers have played a significant role in shaping gene flow and the population structure. Estimated effective population sizes indicated a substantial drop occurring in all populations 5-10,000 years ago, coinciding with the increase in human populations. Finally, signatures of selection were enriched for key genes associated with the immune response, suggesting infectious disease exert a substantial selective pressure upon the African buffalo. These findings have important implications for understanding bovid evolution, buffalo conservation and population management

Continent-wide genomic analysis of the African buffalo (<i>Syncerus caffer</i>)

AbstractThe African buffalo (Syncerus caffer) is a wild bovid with a historical distribution across much of sub-Saharan Africa. Genomic analysis can provide insights into the evolutionary history of the species, and the key selective pressures shaping populations, including assessment of population level differentiation, population fragmentation, and population genetic structure. In this study we generated the highest qualityde novogenome assembly (2.65 Gb, scaffold N50 69.17 Mb) of African buffalo to date, and sequenced a further 195 genomes from across the species distribution. Principal component and admixture analyses provided surprisingly little support for the currently described four subspecies, but indicated three main lineages, in Western/Central, Eastern and Southern Africa, respectively. Estimating Effective Migration Surfaces analysis suggested that geographical barriers have played a significant role in shaping gene flow and the population structure. Estimated effective population sizes indicated a substantial drop occurring in all populations 5-10,000 years ago, coinciding with the increase in human populations. Finally, signatures of selection were enriched for key genes associated with the immune response, suggesting infectious disease exert a substantial selective pressure upon the African buffalo. These findings have important implications for understanding bovid evolution, buffalo conservation and population management

Impact of nationwide enhanced implementation of best practices in pancreatic cancer care (PACAP-1): A multicenter stepped-wedge cluster randomized controlled trial

Background: Pancreatic cancer has a very poor prognosis. Best practices for the use of chemotherapy, enzyme replacement therapy, and biliary drainage have been identified but their implementation in daily clinical practice is often suboptimal. We hypothesized that a nationwide program to enhance implementation of these best practices in pancreatic cancer care would improve survival and quality of life. Methods/design: PACAP-1 is a nationwide multicenter stepped-wedge cluster randomized controlled superiority trial. In a per-center stepwise and randomized manner, best practices in pancreatic cancer care regarding the use of (neo)adjuvant and palliative chemotherapy, pancreatic enzyme replacement therapy, and metal biliary stents are implemented in all 17 Dutch pancreatic centers and their regional referral networks during a 6-week initiation period. Per pancreatic center, one multidisciplinary team functions as reference for the other centers in the network. Key best practices were identified from the literature, 3 years of data from existing nationwide registries within the Dutch Pancreatic Cancer Project (PACAP), and national expert meetings. The best practices follow the Dutch guideline on pancreatic cancer and the current state of the literature, and can be executed within daily clinical practice. The implementation process includes monitoring, return visits, and provider feedback in combination with education and reminders. Patient outcomes and compliance are monitored within the PACAP registries. Primary outcome is 1-year overall survival (for all disease stages). Secondary outcomes include quality of life, 3- and 5-year overall survival, and guideline compliance. An improvement of 10% in 1-year overall survival is considered clinically relevant. A 25-month study duration was chosen, which provides 80% statistical power for a mortality reduction of 10.0% in the 17 pancreatic cancer centers, with a required sample size of 2142 patients, corresponding to a 6.6% mortality reduction and 4769 patients nationwide. Discussion: The PACAP-1 trial is designed to evaluate whether a nationwide program for enhanced implementation of best practices in pancreatic cancer care can improve 1-year overall survival and quality of life. Trial registration: ClinicalTrials.gov, NCT03513705. Trial opened for accrual on 22th May 2018

Profiling the immune epigenome across global cattle breeds

Background Understanding the variation between well and poorly adapted cattle breeds to local environments and pathogens is essential for breeding cattle with improved climate and disease-resistant phenotypes. Although considerable progress has been made towards identifying genetic differences between breeds, variation at the epigenetic and chromatin levels remains poorly characterized. Here, we generate, sequence and analyse over 150 libraries at base-pair resolution to explore the dynamics of DNA methylation and chromatin accessibility of the bovine immune system across three distinct cattle lineages. Results We find extensive epigenetic divergence between the taurine and indicine cattle breeds across immune cell types, which is linked to the levels of local DNA sequence divergence between the two cattle sub-species. The unique cell type profiles enable the deconvolution of complex cellular mixtures using digital cytometry approaches. Finally, we show distinct sub-categories of CpG islands based on their chromatin and methylation profiles that discriminate between classes of distal and gene proximal islands linked to discrete transcriptional states. Conclusions Our study provides a comprehensive resource of DNA methylation, chromatin accessibility and RNA expression profiles of three diverse cattle populations. The findings have important implications, from understanding how genetic editing across breeds, and consequently regulatory backgrounds, may have distinct impacts to designing effective cattle epigenome-wide association studies in non-European breeds