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...
Background Studies have shown that drug-resistant tuberculosis (DR-TB) in South Africa (SA) is clonal and is caused mostly by transmission. Identifying transmission chains is important in controlling DR-TB. This study reports on the sentinel molecular surveillance data of Rifampicin-Resistant (RR) TB in SA, aiming to describe the RR-TB strain population and the estimated transmission of RR-TB cases. Method RR-TB isolates collected between 2014 and 2018 from eight provinces were genotyped using combination of spoligotyping and 24-loci mycobacterial interspersed repetitive-units-variable-number tandem repeats (MIRU-VNTR) typing. Results Of the 3007 isolates genotyped, 301 clusters were identified. Cluster size ranged between 2 and 270 cases. Most of the clusters (247/301; 82.0%) were small in size (< 5 cases), 12.0% (37/301) were medium sized (5–10 cases), 3.3% (10/301) were large (11–25 cases) and 2.3% (7/301) were very large with 26–270 cases. The Beijing genotype was responsible for majority of RR-TB cases in Western and Eastern Cape, while the East-African-Indian-Somalian (EAI1_SOM) genotype accounted for a third of RR-TB cases in Mpumalanga. The overall proportion of RR-TB cases estimated to be due to transmission was 42%, with the highest transmission-rate in Western Cape (64%) and the lowest in Northern Cape (9%). Conclusion Large clusters contribute to the burden of RR-TB in specific geographic areas such as Western Cape, Eastern Cape and Mpumalanga, highlighting the need for community-wide interventions. Most of the clusters identified in the study were small, suggesting close contact transmission events, emphasizing the importance of contact investigations and infection control as the primary interventions in SA.
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