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 incongruent 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. Theileria 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.Keywords: 18S SSU, Diversity, Next-generation sequencing, species, Theileria, Theileria parva 3 IntroductionThe Babesia and Theileria are part of the phylum Apicomplexa (Order Piroplasmorida) (Mans et al. 2015 A significant proportion of the systematic study of the Piroplasmorida comprise analysis of the 18S rRNA gene (Allsopp et al. 1994;Chae et al. 1999;Chansiri et al. 1999;Criado-Fornelio et al. 2004;Reichard et al. 2005;Criado et al. 2006;Altay et al. 2007;Bhoora et al. 2009). The reverse line blot (RLB) method (Gubbels et al. 1999), based on simultaneous detection of the 18S rRNA gene for various Babesia and Theileria species, has been extensively used for surveillance purposes (Georges et al. 2001; Almerìa et al. 2002;Oura et al. 2004;Nijhof et al. 2003Nijhof et al. , 2005Altay et al. 2007;M'ghirbi et al. 2008;Altay et al. 2008;Matjila et al. 2008;Oosthuizen et al. 2008; Almerìa et al. 2009;Salih et al. 2010;Yusufmia et al. 2010; Tomassone et al. 2012;Ceci et al. 2014;Githaka et al. 2014;Eygelaar et al. 2015;Njiiri et al. 2015). The method relies on the amplification of the V4 hypervariable region of the 18S rRNA gene, followed by hybridization t...

Elucidating cryptic dynamics of Theileria communities in African buffalo using a high‐throughput sequencing informatics approach

“…Importantly, the diversity of our communities is well supported by the existing literature. We detected almost all Theileria subtypes previously detected in southern KNP (Mans et al, ). We did not find T .…”

“…(sable) RLB probe crosshybridizes with T. velifera (Mans et al, ). Mans et al () used amplicon sequencing, using the Roche 454 platform, to describe the prevalence of Theileria spp. in South Africa, but did not detect T .…”

“…Disentangling the complex African buffalo— Theileria system poses two major challenges that previous studies using conventional approaches were unable to overcome: First, Theileria is taxonomically complex and classical taxonomists have difficulty distinguishing between species and haplotypes, requiring genetically distinct, yet closely related organisms, to remain distinguished as “subtypes” (reviewed in Mans et al, ). Importantly, subtypes are restricted by host specificity and geographic range, indicating important biological differences (Chaisi, Collins, Potgieter, & Oosthuizen, ; Mans, Pienaar, & Latif, ; Mans et al, ; Pienaar, Potgieter, Latif, Thekisoe, & Mans, ). Attempting to differentiate between subtypes using PCR‐ and antibody‐based approaches has been riddled with issues of crossreactivity (Mans et al, ).…”

“…Although rarely infected with Babesia spp. (Henrichs et al, 2016;Mans, Pienaar, Ratabane, Pule, & Latif, 2016), African buffalo have been reported to be simultaneously infected with multiple species of Theileria, encompassing a multitude of subtypes (Mans, Pienaar, & Latif, 2015). Infection with multiple Theileria spp., as opposed to single-species infections, results in dramatically different pathological disorders in cattle (Woolhouse et al, 2015), indicating that parasite interactions can adversely impact host health.…”

“…(sable) may have been previously reported in African buffalo as the T. sp. (sable) RLB probe crosshybridizes with T. velifera Mans et al (2016). used amplicon sequencing, using the Roche 454 platform, to describe the prevalence of Theileria spp.…”