Genomic Characterization of Theileria luwenshuni Strain Cheeloo

Abstract

Theileria, a tick-borne intracellular protozoan, can cause infections of various livestock and wildlife around the world, posing a threat to veterinary health. Although more and more Theileria species have been identified, genomes have been available only from four Theileria species to date. Here, we assembled a whole genome of Theileria luwenshuni, an emerging Theileria, through next-generation sequencing of purified erythrocytes from the blood of a naturally infected goat. We designated it T. luwenshuni str. Cheeloo because its genome was assembled by the researchers at Cheeloo College of Medicine, Shandong University, China. The genome of T. lunwenshuni str. Cheeloo was the smallest in comparison with the other four Theileria species. T. luwenshuni str. Cheeloo possessed the fewest gene gains and gene family expansion. The protein count of each category was always comparable between T. luwenshuni str. Cheeloo and T. orientalis str. Shintoku in the Eukaryote Orthologs annotation, though there were remarkable differences in genome size. T. luwenshuni str. Cheeloo had lower counts than the other four Theileria species in most categories at level 3 of Gene Ontology annotation. Kyoto Encyclopedia of Genes and Genomes annotation revealed a loss of the c-Myb in T. luwenshuni str. Cheeloo. The infection rate of T. luwenshuni str. Cheeloo was up to 81.5% in a total of 54 goats from three flocks. The phylogenetic analyses based on both 18S rRNA and cox1 genes indicated that T. luwenshuni had relatively low diversity. The first characterization of the T. luwenshuni genome will promote better understanding of the emerging Theileria. IMPORTANCE Theileria has led to substantial economic losses in animal husbandry. Whole-genome sequencing data of the genus Theileria are currently limited, which has prohibited us from further understanding their molecular features. This work depicted whole-genome sequences of T. luwenshuni str. Cheeloo, an emerging Theileria species, and reported a high prevalence of T. luwenshuni str. Cheeloo infection in goats. The first assembly and characterization of T. luwenshuni genome will benefit exploring the infective and pathogenic mechanisms of the emerging Theileria to provide scientific basis for future control strategies of theileriosis.

Keywords: FISH; Theileria luwenshuni strain Cheeloo; genomic characteristics; phylogenetic analysis; whole-genome sequence.

FIG 1

Morphology and size of T. luwenshuni str. Cheeloo inside erythrocytes. (A) Wright-Giemsa staining of goat blood smears (1,000×). (B) Fluorescence in situ hybridization (FISH) results under fluorescence microscope of Theileria luwenshuni str. Cheeloo. From left to right represent the fluorescein isothiocyanate (FITC) channel (fluorescence channel), the DAPI channel, and the merge channel. The blue fluorescence is the nucleus of the Theileria and the green strong fluorescence is the target T. luwenshuni str. Cheeloo. (C) FISH results showing different shapes and sizes of T. luwenshuni str. Cheeloo in erythrocytes.

FIG 2

Genomic composition and evolution of T. luwenshuni str. Cheeloo. (A) Genome size and G+C content distribution of T. luwenshuni str. Cheeloo and the other four Theileria species with published complete genome sequences. Different Theileria species were indicated with different colors. (B) Bird’s eye view of the assembled genome of T. luwenshuni str. Cheeloo showing summary statistics. From the outer circle to the inner circle, six types of information: contig length, genes density, gene annotation (colors imply the KEGG of genes, A09100 Metabolism; A09120 Genetic Information Processing; A09130 Environmental Information Processing; A09140 Cellular Processes; A09150 Organismal Systems; A09160 Human Diseases; A09180 Brite Hierarchies; A09190 Not Included in Pathway or Brite), DNA sequencing data coverage, G+C skew value, and G+C content are labeled. (C) The maximum likelihood phylogenetic tree of Theileria species. The tree was inferred by Raxml based on 1,149 single-copy orthologs identified by orthofinder. A total of 1,000 alternative runs were used to calculate support values. Babesia microti and Toxoplasma gondii were two outgroup species to help root the tree. Different Theileria species were indicated with different colors. (D) An ultrametric tree of five Theileria species, with Babesia microti str. RI and Plasmodium ovale str. PocGH01 as the tree root. Numbers below the branches indicate gene family expansions/contractions, and the numbers above the branches show gene gains/losses.

FIG 3

Genome annotation and characterization of T. luwenshuni str. Cheeloo in comparison to other Theileria species. (A) KOG analysis of all predicted genes. The x axis indicates KOG categories and the y axis indicates the number of proteins. The x axis A to W represent: A, RNA processing and modification; B, chromatin structure and dynamics; C, energy production and conversion; D, cell cycle control, cell division, chromosome partitioning; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; J, translation, ribosomal structure and biogenesis; K, transcription; L, replication, recombination and repair; M, cell wall/membrane/envelope biogenesis; N, cell motility; O, posttranslational modification, protein turnover, chaperones; P, inorganic ion transport and metabolism; Q, secondary metabolites biosynthesis, transport and catabolism; R, general function prediction only; S, function unknown; T, signal transduction mechanisms; U, intracellular trafficking, secretion, and vesicular transport; V, defense mechanisms; W, extracellular structures. Y and Z represent nuclear structure and cytoskeleton, respectively. (B) Gene ontology (GO) terms assignment for proteins from five Theileria species at level 2. Results are summarized into three categories of cellular component, molecular function, and biological process. (C) GO terms with differential protein counts at level 3. (D) Venn diagram showing number of orthogroups found in different Theileria species.

FIG 4

Phylogenetic analysis of T. luwenshuni str. Cheeloo based on the full-length of the 18S rRNA gene and the partial cox1 gene. (A) Phylogenetic tree of Theileria based on 18S rRNA gene sequences. (B) Phylogenetic tree of Theileria based on cox1 gene sequences. The sequences obtained in this study are highlighted in red. The stars in panels A and B indicate the sequences from the assembled genome of T. luwenshuni str. Cheeloo. The asterisk (*) indicates the 18S rRNA gene sequences obtained in this study with GenBank accession numbers of OQ134880, OQ134882, OQ134884 to OQ134896, OQ134898 to OQ134906, and OQ134908 to OQ134919.