title: Investigating the diversity of the 18S SSU rRNA hyper-variable region of Theileria in cattle and Cape buffalo (Syncerus caffer) from southern Africa using a next generation sequencing approach.
abstract: Molecular classification and systematics of the Theileria is based on the analysis of the 18S rRNA gene. Reverse line blot or conventional sequencing approaches have disadvantages in the study of 18S rRNA diversity and a next-generation 454 sequencing approach was investigated. The 18S rRNA gene was amplified using RLB primers coupled to 96 unique sequence identifiers (MIDs). Theileria positive samples from African buffalo (672) and cattle (480) from southern Africa were combined in batches of 96 and sequenced using the GS Junior 454 sequencer to produce 825711 informative sequences. Sequences were extracted based on MIDs and analysed to identify Theileria genotypes. Genotypes observed in buffalo and cattle were confirmed in the current study, while no new genotypes were discovered. Genotypes showed specific geographic distributions, most probably linked with vector distributions. Host specificity of buffalo and cattle specific genotypes were confirmed and prevalence data as well as relative parasitemia trends indicate preference for different hosts. Mixed infections are common with African buffalo carrying more genotypes compared to cattle. Associative or exclusion co-infection profiles were observed between genotypes that may have implications for speciation and systematics: specifically that more Theileria species may exist in cattle and buffalo than currently recognized. Analysis of primers used for Theileria parva diagnostics indicate that no new genotypes will be amplified by the current primer sets confirming their specificity. T. parva SNP variants that occur in the 18S rRNA hypervariable region were confirmed. A next generation sequencing approach is useful in obtaining comprehensive knowledge regarding 18S rRNA diversity and prevalence for the Theileria, allowing for the assessment of systematics and diagnostic assays based on the 18S gene.
authors.affiliation: Parasites, Vectors and Vector-Borne Diseases, Agricultural Research Council-Onderstepoort Veterinary Institute, Onderstepoort 0110, South Africa; The Department of Veterinary Tropical Diseases, University of Pretoria, Pretoria, South Africa; Department of Life and Consumer Sciences, University of South Africa, South Africa. Electronic address: mansb@arc.agric.za.
authors.forename: Ben J
authors.initials: BJ
authors.lastname: Mans
authors.affiliation: Parasites, Vectors and Vector-Borne Diseases, Agricultural Research Council-Onderstepoort Veterinary Institute, Onderstepoort 0110, South Africa.
authors.forename: Ronel
authors.initials: R
authors.lastname: Pienaar
authors.affiliation: Parasites, Vectors and Vector-Borne Diseases, Agricultural Research Council-Onderstepoort Veterinary Institute, Onderstepoort 0110, South Africa.
authors.forename: John
authors.initials: J
authors.lastname: Ratabane
authors.affiliation: Parasites, Vectors and Vector-Borne Diseases, Agricultural Research Council-Onderstepoort Veterinary Institute, Onderstepoort 0110, South Africa.
authors.forename: Boitumelo
authors.initials: B
authors.lastname: Pule
authors.affiliation: Parasites, Vectors and Vector-Borne Diseases, Agricultural Research Council-Onderstepoort Veterinary Institute, Onderstepoort 0110, South Africa; School of Life Sciences, University of Kwa Zulu-Natal, South Africa.
authors.forename: Abdalla A
authors.initials: AA
authors.lastname: Latif
mesh_terms: D000353:Africa, Southern / Q000453:epidemiology; D000818:Animals; D002020:Buffaloes / Q000469:parasitology*; D002417:Cattle / Q000469:parasitology; D060085:Coinfection; D014644:Genetic Variation; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing; D058507:Host Specificity; D018512:Parasitemia / Q000662:veterinary; D012337:RNA, Ribosomal, 18S / Q000235:genetics*; D016415:Sequence Alignment; D016794:Theileria / Q000235:genetics* / Q000302:isolation & purification; D016797:Theileria parva / Q000235:genetics / Q000302:isolation & purification; D013801:Theileriasis / Q000453:epidemiology / Q000469:parasitology*
chemical_list: D012337:RNA, Ribosomal, 18S
keywords: 18S SSU; Diversity; Next-generation sequencing; Species; Theileria; Theileria parva