Theileria parva genomics reveals an atypical apicomplexan genome

The discipline of genomics is setting new paradigms in research approaches to resolving problems in human and animal health. We propose to determine the genome sequence of Theileria parva, a pathogen of cattle, using the random shotgun approach pioneered at The Institute for Genomic Research (TIGR). A number of features of the T. parva genome make it particularly suitable for this approach. The G+C content of genomic DNA is about 31%, non-coding repetitive DNA constitutes less than 1% of total DNA and a framework for the 10-12 Mbp genome is available in the form of a physical map for all four chromosomes. Minisatellite sequences are the only dispersed repetitive sequences identified so far, but they are limited in distribution to 13 of 33 SfiI fragments. Telomere and sub-telomeric non-coding sequences occupy less than 10 kbp at each chromosomal end and there are only two units encoding cytoplasmic rRNAs. Three sets of distinct multicopy sequences encoding ORFs have been identified but it is not known if these are associated with expression of parasite antigenic diversity. Protein coding genes exhibit a bias in codon usage and introns when present are unusually short. Like other apicomplexan organisms, T. parva contains two extrachromosomal DNAs, a mitochondrial DNA and a plastid DNA molecule. By annotating the genome sequence, in combination with the use of microarray technology and comparative genomics, we expect to gain significant insights into unique aspects of the biology of T. parva. We believe that the data will underpin future research to aid in the identification of targets of protective CD8+ cell mediated immune responses, and parasite molecules involved in inducing reversible host leukocyte transformation and tumour-like behaviour of transformed parasitised cells.     

The genome of Theileria parva: some structural properties

The DNA of the protozoan Theileria parva, the causal agent of the bovine East Coast Fever, has been prepared at least 99% pure from the intra-erythrocytic form of the parasite. Its buoyant density was found to be 1.696 g/cm3 and its calculated G + C content was 36.7%. Fragmentation of T. parva by the restriction enzyme EcoRI provides some evidence of the presence of repetitive DNA sequences.     

Genome analysis of Theileria parva

Recent technological developments in the field of genome analyses have advanced our knowledge of the structures of prokaryotic and eukoryotic genomes. Examples of these range from small bacterial genomes, such as Escherichia coli, to the more complex genomes of Caenorhabditis elegans, Drosophila, humans and mouse. Here, Subhash Morzona and John Young review developments in mapping the genome of on economically important protozoan parasite o f cattle, Theileria parva. This map provides a framework for more detailed analysis of the genome structure o f this organism. The methodologies developed in constructing the map also have application to the mapping of other protozoan genomes.     

Whole genome sequencing and future breeding of rice

Contemporary crop improvement relies on the genetic analysis of progeny derived from a cross between different lines with contrasting phenotypes. Such analysis allowed positioning of genes for agronomically important traits, enabling development of DNA makers for marker-assisted selection (MAS). So far the identification of loci for desirable traits have been carried out by linkage analysis using DNA markers. This process required the development of DNA markers that are distributed over the genome as well as the genotyping of each progeny. Due to recent development in next generation sequencing (NGS) technology, whole genome sequencing (WGS) is becoming easier and cheaper. Using NGS, we developed a new method called MutMap that allows rapid isolation of useful alleles from rice mutant lines. An important feature of MutMap is that it does not require marker development. We foresee that the era of genetic markers will be eventually eclipsed by that of WGS applied to all the individuals in the breeding processes.     

Linkage of two distinct AT-rich minisatellites at multiple loci in the genome of Theileria parva

Minisatellite tandem repeat elements are well known components of vertebrate genomes, but have not yet been extensively characterized in lower eukaryotes. We describe two unusual, AT-rich minisatellites of the protozoan parasite Theileria parva whose sequences are unrelated to the G/C-rich `chi minisatellite superfamily' of vertebrate and plant genomes. The T. parva tandem repeats, one with a conserved sequence T_2-5ACACA (6–17 copies), and the other with a 6-bp core sequence of either ACTATA or TATACT associated with additional variable sequences in repeats of 10–17 bp (3–7 copies), were closely linked at more than 20 sites in the T. parva genome, separated by 390, 510 and 660 bp at three loci analysed in detail. Such linkage is without precedent in minisatellites so far analysed in other organisms. The minisatellite loci were widely dispersed on 13 out of 33 genomic SfiI fragments, on all four T. parva chromosomes and did not exhibit a telomeric bias in their distribution. Analysis of flanking sequences revealed no obvious conserved sequences between the five loci, or other multicopy repeat sequences outside the minisatellite regions. The T_2-5 ACACA minisatellite was highly effective as a multilocus fingerprinting probe for discrimination of T. parva isolates. Analysis of two individual minisatellite loci revealed variation between the genomic DNAs of two T. parva isolates in the copy number of the constituent repeats within the array, similar to that typical of vertebrate minisatellites.     

A Sporozoite-based vaccine for Theileria parva

East Coast fever, which is caused by Theileria parva infection in cattle, is of major economic importance in eastern and central Africa. Until recently, the only available method of immunization against East Coast fever was the infection with live sporozoites and simultaneous treatment with a long-acting oxytetracycline. This method has two major disadvantages: (I) it uses live organisms; and (2) the immunity engendered is parasite strain specific. In this article, Antony Musoke, Vishvonath Nene and Subhosh Morzoria review the progress made in developing an alternative method o f immunization based on a defined sporozoite antigen.     

Cryopreservation of infective particles of Theileria parva

Cryopreservation of infective particles of Theileria parva. International Journal for Parasitology3: 583–587. Infective particles of Theileria parva, the causative organism of East Coast fever of cattle, were obtained from infected Rhipicephalus appendiculatus ticks, either by using an in vitro feeding technique or by grinding the ticks in a suitable medium. If foetal calf serum containing 15% glycerol (v/v) was added to the infective material and it was then distributed either to glass capillary tubes (in vitro tick feed) or glass tubes (ground tick supernate) it could be slowly frozen to either ?80°C or ?196°C without loss of viability. Stabilates, tested by rapid thawing and inoculation into ECF-susceptible cattle, remained viable for up to a year at these temperatures.     

Whole genome sequencing and its applications in medical genetics

Fundamental improvement was made for genome sequencing since the next-generation sequencing (NGS) came out in the 2000s. The newer technologies make use of the power of massively-parallel short-read DNA sequencing, genome alignment and assembly methods to digitally and rapidly search the genomes on a revolutionary scale, which enable large-scale whole genome sequencing (WGS) accessible and practical for researchers. Nowadays, whole genome sequencing is more and more prevalent in detecting the genetics of diseases, studying causative relations with cancers, making genome-level comparative analysis, reconstruction of human population history, and giving clinical implications and instructions. In this review, we first give a typical pipeline of whole genome sequencing, including the lab template preparation, sequencing, genome assembling and quality control, variants calling and annotations. We compare the difference between whole genome and whole exome sequencing (WES), and explore a wide range of applications of whole genome sequencing for both mendelian diseases and complex diseases in medical genetics. We highlight the impact of whole genome sequencing in cancer studies, regulatory variant analysis, predictive medicine and precision medicine, as well as discuss the challenges of the whole genome sequencing.      

The antitheilerial effects of Theileria parva parva reaction and recovery sera in vitro

Peripheral blood leucocytes (PBL) of cattle were infected in vitro with the sporozoites of Theileria parva spp. The transformed cell lines were adapted to grow in sera from the PBL donors. The cattle were then infected with T. p. parva stabilate and either treated with parvaquone or the disease allowed to run its course. Sera harvested during severe disease reaction or early recovery were substituted for pre infection sera and caused the intracellular degeneration of the Theileria macroschizonts. Cell lines passaged in these sera died out as the parasites were eliminated. The antiparasitic effects of sera were short lived and were neither host nor parasite isolate restricted.     

Whole genome amplification and sequencing of a Daphnia resting egg

Resting eggs banks are unique windows that allow us to directly observe shifts in population genetics, and phenotypes over time as natural populations evolve. Though a variety of planktonic organisms also produce resting stages, the keystone freshwater consumer, Daphnia, is a well‐known model for paleogenetics and resurrection ecology. Nevertheless, paleogenomic investigations are limited largely because resting eggs do not contain enough DNA for genomic sequencing. In fact, genomic studies even on extant populations include a laborious preparatory phase of batch culturing dozens of individuals to generate sufficient genomic DNA. Here, we furnish a protocol to generate whole genomes of single ephippial (resting) eggs and single daphniids. Whole genomes of single ephippial eggs and single adults were amplified using Qiagen REPLI‐g Single Cell kit reaction, followed by NEBNext Ultra DNA Library Prep Kit for library construction and Illumina sequencing. We compared the quality of the single‐egg and single‐individual amplified genomes to the standard batch genomic DNA extraction in the absence of genome amplification. At mean 20× depth, coverage was essentially identical for the amplified single individual relative to the unamplified batch extracted genome (>90% of the genome was covered and callable). Finally, while amplification resulted in the slight loss of heterozygosity for the amplified genomes, estimates were largely comparable and illustrate the utility and limitations of this approach in estimating population genetic parameters over long periods of time in natural populations of Daphnia and also other small species known to produce resting stages.     

Whole genome sequencing of Thaumetopoea pityocampa revealed putative pesticide targets

Insect neuropeptides play a major role in the regulation of the physiological processes. Due to their versatile effects on the development of insects, their corresponding receptors, which are mostly G-protein coupled receptors, are considered as ideal targets for designing next-generation pesticides. In this study, we aimed to find neuropeptide receptors of pine processionary moth (Thaumetopoea pityocampa), a pest in the Mediterranean countries, that feeds on the needles of pine trees. To this aim, Whole Genome Shotgun sequencing technique was used. de novo assembly of the genome was performed using two different assemblers, SGA and MaSuRCA. The results of two assemblers were compared, and MaSuRCA assembler showed higher N50 length. To find some target GPCRs, sequences of Drosophila melanogaster and evolutionarily close species were used as blast queries in the assembled data. Five GPCRs were chosen from the genome and their expression was confirmed in the larval stage of the insect.     

Whole genome sequencing and genome annotation of the wild edible mushroom,Russula griseocarnosa

Russula griseocarnosa is a species of edible ectomycorrhizal fungi with medicinal properties that grows in southern China. Total DNA was isolated from a fresh fruiting body of R. griseocarnosa and subjected to sequencing using Illumina Hiseq with the PacBio RS sequencing platform. Here, we present the 64.81 Mb draft genome map of R. griseocarnosa based on 471 scaffolds and 16,128 coding protein genes. The gene annotation of protein coding genes was used to obtain corresponding annotations by blastp. Phylogenetic analysis revealed a close evolutionary relationship of R. griseocarnosa to Heterobasidion irregulare and Stereum hirsutum in the core Russulales clade. The R. griseocarnosa genome encodes a repertoire of enzymes engaged in carbohydrate and polysaccharide metabolism, along with cytochrome P450s and secondary metabolite biosynthesis. The genome content of R. griseocarnosa provides insights into the genetic basis of its reported medicinal properties and serves as a reference for comparative genomics of fungi.     

A rapid and sensitive intracellular flow cytometric assay to identify Theileria parva infection within target cells

Theileria parva is an intracellular protozoan parasite transmitted by ticks that causes a fatal lymphoproliferative disease of cattle known as East Coast Fever. Vaccination against the disease currently relies on inoculation of the infective sporozoite stage of the parasite and simultaneous treatment with long-acting formulations of oxytetracycline. Sporozoites are maintained as frozen stabilates of triturated infected ticks and the method requires accurate titration of stabilates to determine appropriate dose rates. Titration has traditionally been undertaken in cattle and requires large numbers of animals because of individual variation in susceptibility to infection. An alternative tissue culture-based method is laborious and time consuming. We have developed a flow cytometric method for quantifying the infectivity of sporozoite stabilates in vitro based on the detection of intracellular parasite antigen. The method allows clear identification of parasitized cells with a high degree of sensitivity and specificity. Analysis of infected cells between 48 and 72 h post-infection clearly defines the potential transforming capability of different stabilates.     

Theileria parva: significance of leukocytes for infecting cattle

Using an artificial feeding technique, infective particles of Theileria parva were harvested in bovine blood in capillary tubes from prefed female Rhipicephalus appendiculatus over a 2-hr period. Inoculations of this blood feed pool invariably resulted in the establishment of patent East Coast fever in autogeneic or syngeneic cattle, i.e., the blood donors or their monozygotic twins, but not in unrelated animals. Mechanical passage of 4 × 10⁶ macroschizont-infected lymphoid cells, harvested from a heifer with East Coast fever, successfully induced patent theileriosis in the donor's monozygotic twin but not in a susceptible allogeneic bovid. The significance of parasite-cell association and histocompatibility of infected cells is discussed in relation to natural and mechanical transmission of Theileria parva.     

Whole genome amplification using single-primer PCR

Comprehensive genomic molecular analyses require relatively large DNA amounts that are often not available from forensic, clinical and other crucial biological samples. Numerous methods to amplify the whole genome have been proposed for cancer, forensic and taxonomic research. Unfortunately, when using truly random primers for the initial priming step, all of these procedures suffer from high background problems for sub-nanogram quantities of input DNA. Here we report an approach to eliminate this problem for PCR-based methods even at levels of DNA approaching that of a single cell.     

Whole genome sequencing of Ethiopian highlanders reveals conserved hypoxia tolerance genes

Background

Although it has long been proposed that genetic factors contribute to adaptation to high altitude, such factors remain largely unverified. Recent advances in high-throughput sequencing have made it feasible to analyze genome-wide patterns of genetic variation in human populations. Since traditionally such studies surveyed only a small fraction of the genome, interpretation of the results was limited.

Results

We report here the results of the first whole genome resequencing-based analysis identifying genes that likely modulate high altitude adaptation in native Ethiopians residing at 3,500 m above sea level on Bale Plateau or Chennek field in Ethiopia. Using cross-population tests of selection, we identify regions with a significant loss of diversity, indicative of a selective sweep. We focus on a 208 kbp gene-rich region on chromosome 19, which is significant in both of the Ethiopian subpopulations sampled. This region contains eight protein-coding genes and spans 135 SNPs. To elucidate its potential role in hypoxia tolerance, we experimentally tested whether individual genes from the region affect hypoxia tolerance in Drosophila. Three genes significantly impact survival rates in low oxygen: cic, an ortholog of human CIC, Hsl, an ortholog of human LIPE, and Paf-AHα, an ortholog of human PAFAH1B3.

Conclusions

Our study reveals evolutionarily conserved genes that modulate hypoxia tolerance. In addition, we show that many of our results would likely be unattainable using data from exome sequencing or microarray studies. This highlights the importance of whole genome sequencing for investigating adaptation by natural selection.