Accepted Manuscript Title: Phylogeny of Theileria buffeli genotypes identified in the South African buffalo (Syncerus caffer) population Author: Mamohale E. Chaisi Nicola E. Collins Marinda C. Oosthuizen PII: DOI: Reference:
S0304-4017(14)00337-9 http://dx.doi.org/doi:10.1016/j.vetpar.2014.06.001 VETPAR 7278
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
21-1-2013 30-5-2014 1-6-2014
Please cite this article as: Chaisi, M.E., Collins, N.E., Oosthuizen, M.C.,Phylogeny of Theileria buffeli genotypes identified in the South African buffalo (Syncerus caffer) population, Veterinary Parasitology (2014), http://dx.doi.org/10.1016/j.vetpar.2014.06.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Phylogeny of Theileria buffeli genotypes identified in the South African buffalo
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(Syncerus caffer) population
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Mamohale E. Chaisia,b,*, Nicola E. Collinsa, Marinda C. Oosthuizena
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a
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Private Bag X04, Onderstepoort, 0110, South Africa
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b
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180, Lesotho;
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* Corresponding author. Tel. +27 735715876; fax: +27 125298312.
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E-mail address: [email protected] (M.E. Chaisi)
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Department of Biology, National University of Lesotho, Faculty of Science and Technology, Roma
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Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria,
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Abstract
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Theileria buffeli/orientalis is a group of benign and mildly pathogenic species of cattle and buffalo
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in various parts of the world. In a previous study, we identified T. buffeli in blood samples
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originating from the African buffalo (Syncerus caffer) in the Hluhluwe-iMfolozi Game Park (HIP)
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and the Addo Elephant Game Park (AEGP) in South Africa. The aim of this study was to
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characterize the 18S rRNA gene and complete internal transcribed spacer (ITS1-5.8S-ITS2) region
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of T. buffeli samples, and to establish the phylogenetic position of this species based on these loci.
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The 18S rRNA gene and the complete ITS region were amplified from DNA extracted from blood
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samples originating from buffalo in HIP and AEGP. The PCR products were cloned and the
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resulting recombinants sequenced. We identified novel T. buffeli-like 18S rRNA and ITS genotypes
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from buffalo in the AEGP, and novel Theileria sinensis-like 18S rRNA genotypes from buffalo in
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the HIP. Phylogenetic analyses indicated that the T. buffeli-like sequences were similar to T. buffeli
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sequences from cattle and buffalo in China and India, and the T. sinensis-like sequences were
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similar to T. sinensis 18S rRNA sequences of cattle and yak in China. There was extensive
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sequence variation between the novel T. buffeli genotypes of the African buffalo and previously
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described T. buffeli and T. sinensis genotypes. The presence of organisms with T. buffeli-like and T.
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sinensis-like genotypes in the African buffalo could be of significant importance, particularly to the
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cattle industry in South Africa as these animals might act as sources of infections to naïve cattle.
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This is the first report on the characterization of the full-length 18S rRNA gene and ITS region of T.
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buffeli and T. sinensis genotypes in South Africa. Our study provides invaluable information
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towards the classification of this complex group of benign and mildly pathogenic species.
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Keywords: Theileria buffeli/orientalis; Theileria sinensis; African buffalo; 18S rRNA; ITS
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1. Introduction
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Theileria buffeli/orientalis is a group of closely related parasites of cattle and buffalo. They have a
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cosmopolitan distribution and infect cattle and buffalo in Africa, Australia, New Zealand, Asia,
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Europe and the United States of America (USA) (Steward et al., 1996; Chae et al., 1998; Chansiri et
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al., 1999; M’ghirbi et al., 2008). The cosmopolitan distribution of these species has been attributed
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to the global movement of cattle (and buffalo) without any regard to infection, and their distribution
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mainly depends on the availability of a suitable tick vector (Chae et al., 1999a; Cossio-Bayugar et
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al., 2002). Haemaphysalis ticks act as vectors in Australia, Asia and Europe, but the vectors in
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Africa and the USA are still unknown (Yin et al., 2004; M’ghirbi et al., 2008). Benign isolates from
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Britain, Australia and the USA were initially designated as Theileria mutans as their pathology was
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similar to that of T. mutans (Chae et al., 1999a). However, further studies have indicated that T.
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mutans is an African species and is serologically and genetically distinct from other benign
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Theileria spp. (Chae et al., 1999b).
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Theileria sergenti and T. orientalis were first described from eastern Siberia , while T. buffeli was
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first described from the Asian water buffalo (Bubalus bubalis) (reviewed by Fujisaki et al., 1994).
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The classification of these benign parasites is still confusing and is complicated by their similar
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morphology, serology, vector transmission, geographical distribution, difficulties in obtaining pure
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isolates and incompletely understood life-cycles (Uilenberg et al., 1985; Uilenberg, 2011). It is still
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unclear if these organisms represent the same species or several different species. The term “T.
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sergenti” actually refers to a sheep parasite and was incorrectly used to name a parasite of cattle and
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buffalo (Fujisaki, 1992; Chae et al., 1999a; Uilenberg, 2011). T. buffeli refers to a cluster of benign
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species within the T. buffeli/orientalis group, based on molecular data and infectivity for buffalo.
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More data are awaited to have a better understanding of the diversity within the T. buffeli/orientalis
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group. In the meantime, we will use the T. buffeli-like and T. sinensis-like for the new Theileria
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genotypes characterized in this study.
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Another closely related species, T. sinensis, was recently described in China and is also regarded as
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a cause of bovine theileriosis in that country (Bai et al., 2002a; b, cited by Yin et al., 2004). T.
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sinensis is transmitted by Haemaphysalis qinghaiensis ticks and infects cattle, yak (Yin et al., 2004)
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and water buffalo (He et al., 2012) in China.
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Molecular biology studies based on the 18S ribosomal RNA (rRNA) gene, internal transcribed
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spacers (ITS), major piroplasm surface protein (MPSP) gene and other genetic markers have
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provided useful information on the epidemiology, diagnosis, taxonomy and phylogeny of these
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benign Theileria spp. in various parts of the world (Aktas et al., 2007; Altay et al., 2008; Allsopp et
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al., 1994; Chae et al., 1998; Chansiri et al., 1999; Cossio-Bayugar et al., 2002; Gubbels et al., 2000,
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2002; Sarataphan et al., 2003; M’ghirbi et al., 2008; Liu et al., 2010a, b; Wang et al., 2010; Kamau
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et al., 2011). We recently identified T .buffeli in some buffalo populations in South Africa (Chaisi et
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al., 2011). The aims of this study were to: (1) sequence the full-length 18S rRNA gene and
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complete ITS (ITS1-5.8S-ITS2) region of T. buffeli of buffalo from two geographically different
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areas in South Africa; (2) determine the level of genetic variation between novel T. buffeli-like and
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T. sinensis-like genotypes of the African buffalo and known T. buffeli and T. sinensis genotypes;
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and (3) to establish their phylogenetic positions based on their full-length 18S rRNA gene and
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complete ITS sequences.
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2. Materials and Methods
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2.1 DNA samples
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A molecular epidemiological survey based on the 18S rRNA gene was previously carried out to
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determine the occurrence of Theileria spp. from the African buffalo in different geographic areas in
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South Africa and Mozambique using the reverse line blot (RLB) hybridization assay (Chaisi et al.,
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2011). In that study, T. buffeli was identified from buffalo blood samples originating from the
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Hluhluwe-iMfolozi Game Park (HIP); these samples contained mixed Theileria spp. infections.
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Based on the RLB results, thirteen samples were selected for characterization of the full-length 18S
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rRNA gene and the complete ITS (ITS1-5.8S-ITS2) region (HIP/A2, HIP/A4, HIP/A36, HIP/B62,
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HIP/C5, HIP/C11, HIP/C13, HIP/C15, HIP/C18, HIP/C19, HIP/C23, HIP/C25, HIP/C27). T. buffeli
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is also known to occur in buffalo in the Addo Elephant Game Park (AEGP), Eastern Cape Province,
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South Africa (Milana Troskie, personal communication). The parasite 18S rRNA gene and ITS
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region were also characterized from seven samples originating from buffalo in the AEGP
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(AEGP/65, AEGP/66, AEGP/69, AEGP/70, AEGP/73, AEGP/74, AEGP/76).
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2.2 Amplification of the full-length 18S rRNA gene and complete ITS (ITS1-5.8S-ITS2) region
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The full length 18S rRNA genes from 11 samples (4 from HIP and 7 from AEGP) were
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successfully amplified by conventional PCR using forward primer Nbab-1F and reverse primer
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18SRev-TB (Oosthuizen et al., 2008). The reaction mixture and cycling conditions were as
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described by Chaisi et al. (2011). The resulting amplicons were purified using the QIAquick PCR
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Purification Kit (Qiagen, South Cross Biotechnology, South Africa).
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A nested PCR protocol was used to amplify the complete parasite ITS region from 17 samples (10
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from HIP and 7 from AEGP). The primary reaction contained 2.5 µl (~75 ng) genomic DNA, 0.1
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µM each of primer 1055F (5’- GGT GGT GCA TGG CCG-3’) and LSUR300 (5’-T(A/T)G CGC
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TTC AAT CCC-3’) (Holman et al., 2003), 1.5 mM MgCl2, 200 µM dNTPs, High Fidelity Enzyme
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blend (Roche Diagnostics, Mannheim, Germany) and nuclease-free water to a total volume of 25 µl.
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The thermal cycling programme was done at an initial denaturation at 96°C for 3 min, followed by
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30 cycles of denaturation at 94°C for 30 s; annealing at 50°C for 30 s; extension at 72°C for 3 min;
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a final extension at 72°C for 7 min and then hold at 4°C. Primers ITSF (5’-GAG AAG TCG TAA
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CAA GGT TTC CG-3’) and LSUR50 (5’-GCT TCA CTC GCC GTT ACT AGG-3’) (Holman et
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al., 2003) were used for the nested PCR. The reaction mixture was as above, except that 1 µl (~
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30ng) of the primary PCR product was used as template. The cycling conditions were also as above,
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except that annealing was done at 60°C for 30 s and extension was done at 72°C for 2 min. The
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nested PCR resulted in a number of non-specific bands. ITS amplicons of approximately 1200 bp
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were therefore excised from ethidium-bromide stained gels and purified using the QiAquick Gel
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Extraction Kit (Qiagen, Southern Cross Biotechnology, South Africa). Amplicons from four
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reactions per sample were pooled prior to cloning and/or sequencing to avoid Taq polymerase
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induced errors.
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2.3 Cloning and sequencing of the full-length 18S rRNA gene from HIP samples
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The HIP samples all contained mixed Theileria spp. infections and therefore the amplicons could
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not be directly sequenced. Purified 18S rRNA amplicons from the HIP samples were cloned into the
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pGEM-T Easy Vector and transformed into E. coli JM109 High Efficiency Competent cells
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(Promega, Madison, WI). At least 5 white colonies were selected per sample and recombinant
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plasmid DNA was extracted from overnight bacterial cultures using the High Pure Plasmid Isolation
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kit (Roche Diagnostics, Mannheim, Germany).
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The obtained plasmids were screened by sequencing using the ABI BigDyeTM Terminator Cycle
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Sequencing Ready Reaction kit (PE Applied Biosystems), 350 ng plasmid DNA and 3.2 pmol of
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primer RLB-F2 (Nijhof et al., 2003). The sequences obtained were subjected to a BLASTn
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(Altschul et al., 1990) similarity search. The full-length 18S rRNA genes from recombinant
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plasmids containing sequences that were closely similar to the published 18S rRNA gene sequences
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of T. buffeli or T. sinensis were then sequenced as described above using primers Nbab-1F, 18SRev-
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TB, RLB-R2, BT18S-2F, BT18S-3F, BT18S-4F, BT18S-4R, SP6, T7 (Oosthuizen et al., 2008;
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Chaisi et al., 2011). Sequencing was done on an ABI3100 genetic analyzer at the Agricultural
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Research Council-Onderstepoort Veterinary Institute (ARC-OVI), South Africa.
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2.3 Direct sequencing of the full-length 18S rRNA gene from AEGP samples
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Since T. buffeli is the only Theileria spp. known to infect buffalo in the AEGP, the 18S amplicons
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obtained from samples from this game park were directly sequenced. Full-length 18S rRNA gene
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amplicons were sequenced using ABI BigDyeTM Terminator Cycle Sequencing Ready Reaction kit
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(Life Technologies, South Africa), ~40 ng of PCR product and 3.2 pmol of each primer.
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Sequencing was done at the sequencing facility of the ARC-OVI, South Africa.
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2.4 Cloning and sequencing of the complete ITS (ITS1-5.8S-ITS2) region from HIP and AEGP
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samples
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Purified ITS amplicons were cloned as described above. Sequencing reactions were done using the
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ABI BigDyeTM Terminator Cycle Sequencing Ready Reaction kit (PE Applied Biosystems), ~300
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ng of plasmid DNA and 2 pmol each of primers ITSF, SP6 and T7. Sequencing was done on an
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ABI 3500XL genetic analyzer at Inqaba Biotechnologies, South Africa.
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2.5 Sequence and phylogenetic analyses
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The sequences were assembled and edited using the GAP4 program of the Staden package (version
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1.6.0 for Windows) (Bonfield et al., 1995). A BLASTn homology search of GenBank was done
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using the full length consensus sequences. These were aligned with 18S rRNA gene sequences
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(Table 1) or ITS sequences of related genera from GenBank using the MAFFT (multiple sequence
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alignment) v6 programme employing the FFT-NS-1 algorithm (Katoh et al., 2005). The alignments
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were manually examined and edited across their full-lengths, and then truncated to the size of the
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smallest sequence using BioEdit v7 (Hall, 1999). Heteroduplexes were eliminated from the
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alignments. A total of 58 (new and known) 18S rRNA gene sequences (1514 bp), and 30 ITS
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sequences (1510 bp) were analysed. Estimated evolutionary divergence was calculated by
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determining the number of nucleotide differences between similar sequences over a region of 1499
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and 1215 nucleotides for the 18S rRNA gene and ITS sequences, respectively. Nucleotide
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differences were also assessed in the V4 hypervariable region of the 18S rRNA sequences, and in
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the ITS1, 5.8S gene and ITS2 regions of the ITS sequences.
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Phylogenetic trees were inferred from the alignments by the neighbor-joining method (Saitou and
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Nei, 1987), maximum parsimony and maximum likelihood methods using PAUP* v4b10
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(Swofford, 2003). These were done in combination with bootstrapping (1000 replicates). Bayesian
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inference was done using MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2003), accessed via the
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Computational Biology Service Unit, Cornell University. For comparison of the phylogenetic trees,
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18S rRNA gene and ITS sequences of the same species or isolate were included, where possible.
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The 18S rRNA gene and ITS sequences of Babesia canis, B. caballi and B. orientalis were included
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as outgroups to root the phylogenetic trees. All consensus trees were edited using MEGA4 (Tamura
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et al., 2007).
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The near full-length 18S rRNA gene sequences have been deposited in GenBank under accession
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numbers JQ037779 – JQ037790. The complete ITS sequences have been deposited in GenBank
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under accession numbers JQ0377791 – JQ037795.
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3. Results
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3.1 Identification of T. buffeli-like and T. sinensis-like 18S rRNA gene sequences
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Amplification of the 18S rRNA gene from the HIP and AEGP samples resulted in single bands of
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approximately 1700 bp, as viewed on a 2% ethidium bromide stained agarose gel. These amplicons
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were purified, cloned and sequenced. A total of 12 (5 from HIP and 7 from AEGP) near full-length
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18S rRNA sequences were obtained (Table 1).
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The HIP sequences (GenBank accession nos. JQ037786 – JQ037790) ranged in length from 1582 to
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1592 bp.. A BLASTn homology search did not reveal any identical sequences in GenBank; the
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closest homology (98% and 99%) was found with 18S rRNA gene sequences of Theileria sp.
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(Thung Song) (AB000272), Theileria sp. type D (U97052), T. sinensis (EU277003), T. sinensis
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(EU27442) and Theileria sp. China (cattle) (AF036336).
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The seven 18S rRNA sequences from AEGP (GenBank accession nos. JQ037779 – JQ037785)
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were from 1587 to 1588 bp in length. BLASTn similarity searches of these sequences did not reveal
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any identical sequences, but they were most similar (99%) to unclassified T. buffeli 18S rRNA gene
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sequences from China (DQ104611 and HM538212) and India (EF126184).
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3.2 Sequence and phylogenetic analyses of the 18S rRNA genes
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Phylogenetic analyses using neighbor-joining, maximum parsimony, maximum likelihood and
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Bayesian inference, all yielded trees with almost identical topologies and high bootstrap or nodal
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support values, although there were differences within the branching of the clusters in some trees.
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The T. buffeli 18S rRNA sequences could be separated into two distinct clades comprising nine
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distinct clusters, and were clearly separated from other Theileria spp. (Figure 1).
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The first clade comprised clusters 1 to 5, and contained known T. buffeli 18S rRNA genotypes
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designated Types A, B/Ikeda, C/Medan, E/H/Ipoh and Warwick (Chae et al., 1998; 1999b; Chansiri
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et al., 1999; Gubbels et al., 2000; Yin et al., 2004).
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The second clade comprised clusters 6 to 9 and contained T. buffeli Type D and T. sinesis sequences
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of cattle and buffalo, as well as the novel T. buffeli-like 18S sequences from South African buffalo.
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These were designated as types SA1 and SA2 (Figure 1). Genotype SA1 comprised 18S rRNA gene
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sequences originating from the AEGP (cluster 6), and these sequences were closely related to those
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of T. buffeli from buffalo in China (DQ104611) (Liu et al., 2010a) and (HM538212) (unpublished),
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and India (EF126184) (unpublished) (cluster 7). Since the sequences from China and India do not
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fall under any of the designated T. buffeli genotypes, we indicated them as “unclassified” in Figure
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1. Genotype SA2 is composed of 18S rRNA gene sequences from the HIP (cluster 9), and is closely
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related to the T. buffeli type D/T. sinensis group (cluster 8).
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In order to estimate the genetic distance between the T. buffeli-like sequences, the novel genotypes
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were aligned with 17 known T. buffeli 18S rRNA gene sequences (representing the different
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genotypes), and compared along a region of 1499 bp. Sequence variation was observed both within
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and between the different T. buffeli genotypes. All seven novel sequences from AEGP were
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identical within this region and along their full lengths (results not shown). These sequences
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differed from those of the closely related unclassified genotypes by 9 – 12 bp, and from the novel
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sequences from HIP by 21 – 23 bp. The HIP sequences differed from each other by 2 – 7 bp, and
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from the T. buffeli type D/T. sinensis sequences by 11 - 16 bp. Sequences HIP/C23/a and HIP/C23/b
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were identical, while there was a 7 bp difference between sequences HIP/A4/c and HIP/A4/e. The
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greatest variation (~ 45 bp) was observed between sequence HIP/A4/c and Theileria sp. type E,
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which is from cattle isolates in the USA and South Korea (Chae et al., 1998).
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Most of the variation between the T. buffeli-like genotypes occurred in the V1 variable region
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(positions 70 – 140) and V4 hypervariable region (positions 490 – 560) of the gene. The probes
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used for the identification and differentiation of the different Theileria spp. and their variants using
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the RLB hybridization assay were designed to detect target sequences in the V4 region of the gene
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(Gubbels et al., 1999; 2000). Figure 2 shows the positions of the RLB probes that were designed for
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the detection of all T. buffeli 18S rRNA gene sequences and for specific detection of the T. buffeli
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types A, D and non-D (Gubbels et al., 1999; 2000). It is unlikely that the genotype-specific probes
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designed by Gubbels et al. (2000) will detect the novel South African genotypes identified in this
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study, due to nucleotide differences of 4 to 7 bp in the RLB probe target area (Figure 2).
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3.3 Sequence and phylogenetic analyses of the novel ITS sequences
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Amplicons of approximately 1200 bp, as viewed on a 2% agarose gel, were observed from the
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nested PCR products. Eleven ITS sequences were obtained from 10 samples from HIP. However,
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these were all identified as heteroduplex sequences, comprising chimaeric ITS sequences from the
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different Theileria species that were present in the HIP samples. In a PCR containing mixed
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templates, cross-hybridization of heterologous sequences leads to the formation of heteroduplexes
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during the plateau phase of the reaction when decreasing primer:template ratios no longer favor
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primer annealing (Suzuki et al., 1996). It is likely that the heteroduplexes formed during the primary
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PCR were further amplified during the secondary PCR, resulting in a bias towards heteroduplex
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sequences in the clones. Heteroduplex sequences were excluded from further analyses.
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Amplicons from five out of seven samples from AEGP were successfully sequenced. The complete
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ITS sequences (GenBank accession nos. JQ0377791 - JQ037795) differed in the lengths of their
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complete ITS region (952 – 1173 bp), as well as in the ITS1 (458 – 642 bp) and ITS2 (297 – 344
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bp) regions. The 5.8S gene was conserved amongst the sequences and shorter (187 bp) than the
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ITS1 and ITS2 regions. Unlike the novel 18S rRNA gene sequences from AEGP which were
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identical, there was polymorphism among the novel ITS sequences, with most of the variation
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occurring in the ITS1 region. This was mainly due to insertions or deletions of blocks of sequences.
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Three sequences (AEGP/65/ITS, AEGP/66/ITS, AEGP/69/ITS) were identical, while sequences
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AEGP/73/ITS and AEGP/73/ITS differed from each other at 4 positions, and differed from the
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other three sequences (AEGP/65, 66, 69) by 134 bp. There was extensive variation between these
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sequences and the homologous T. buffeli ITS sequences from China (107 – 164 bp), USA and Japan
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(159 – 200 bp), and T. sinensis ITS sequences (144 – 178 bp). These differences were in
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concordance with those obtained from analyses of similar genotypes/species of the 18S rRNA gene.
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Three distinct clusters of the T. buffeli ITS sequences were observed in all of the trees generated
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using neighbor-joining, maximum parsimony, maximum likelihood and Bayesian inference. A
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representative tree generated by Bayesian inference is shown in Figure 3. Cluster 1 comprised the T.
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buffeli sequences from the USA and Japan (Chitose). It has previously been shown that these two
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groups share identical 18S rRNA sequences (Figure 1, Chae et al., 1998, 1999b; Aktas et al., 2007)
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but different ITS (Figure 3; Aktas et al., 2007) and MPSP (Gubbels et al., 2000) sequences.
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Cluster 2 comprised the novel ITS sequences from AEGP and the T. buffeli ITS sequences from
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China. This clade is probably synonymous to that of the “unclassified” 18S rRNA sequences which
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also grouped together with AEGP 18S rRNA sequences (Figure 1). Cluster 3 contained the T.
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sinensis ITS sequences. The ITS sequences of Theileria cervi, Theileria uilenbergi and Theileria
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luwenshuni always grouped together, while the 18S rRNA gene sequences of these species were in
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separate clusters. Unlike the 18S rRNA sequences (Figure 1), the T. mutans ITS sequence grouped
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together with the Theileria parva and Theileria annulata sequences (Figure 3).
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4. Discussion
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In a previous study, RLB hybridization indicated that T. buffeli was the most commonly occurring
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species in buffalo in the HIP, mainly co-occurring with other Theileria spp. (Chaisi et al., 2011),
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and was the only Theileria sp. infecting buffalo in the AEGP (Milana Troskie, personal
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communication). In contrast, T. buffeli was not identified in buffalo samples from the Kruger
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National Park (KNP), Greater Limpopo Transfrontier Park (GLTP) and a private game farm
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bordering the KNP (Chaisi et al., 2011). The molecular characterization and phylogenetic analyses
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reported here indicate that there are at least two T. buffeli genotypes in the South African buffalo
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population and these are closely related to T. buffeli and T. sinensis from the water buffalo (Bubalus
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bubalis) and cattle in some parts of Asia.
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The distribution of different T. buffeli 18S rRNA genotypes (A, B, C, D, E, H) in buffalo and cattle
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has previously been reported from several parts of the world (Chae et al., 1998; 1999a, Chansiri et
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al., 1999, Gubbels et al., 2000; 2002). Types A, B, C, E and H are mainly cattle parasites and are
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considered to be of low to moderate pathogenicity. Type D (T. sinensis) infects water buffalo, yak
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and African buffalo and is probably not pathogenic in these animals, but it can cause severe disease
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when transmitted to cattle (Chae et al., 1999a). In the USA, Type A and D-like organisms have
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been associated with bovine theileriosis in Missouri (Stockham et al., 2000), Texas (Chae et al.,
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1999a) and Michigan (Cossio-Bayugar et al., 2002).
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Our phylogenetic analyses are in agreement with previous analyses of the 18S rRNA and MPSP
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genes of T. buffeli, which indicate that there is extensive variation between T. buffeli type D
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sequences and those of the other T. buffeli types (Chansiri et al., 1999; Gubbels et al., 2000; Yin et
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al. 2004; Liu et al., 2010b). These authors indicated that T. buffeli type D organisms may be
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genetically intermediate between the well-characterized pathogenic Theileria spp. (T. annulata, T.
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parva, T. lestoquardi, T. uilenbergi, T. luwenshuni) and the benign T. buffeli/orientalis spp.
295
Furthermore, maximum likelihood and parsimony analyses of 18S rRNA gene sequences of
296
Theileria spp. by Chansiri et al. (1999) grouped the T. buffeli type D sequences with the pathogenic
297
Theileria spp., whereas distance methods grouped them with those of the other T. buffeli sequences.
298
Randomly amplified polymorphic DNA (RAPD) profiles generated from Theileria sp. Thung Song
299
(a type D sequence) were different from those of the other benign T. buffeli-like species (Chansiri et
300
al., 1999). For these reasons, it was indicated that the classification of T. buffeli type D species is
301
questionable and should be investigated. Studies of type D sequences from cattle in China resulted
302
in the description of a new species, T. sinensis (Bai et al., 1995; 2002a; b).
303
Our findings indicate that suitable vectors for the transmission of T. buffeli are likely to be present
304
in South Africa. In Asia, T. buffeli/orientalis is transmitted by Haemaphysalis longicormis while T.
305
sinensis is transmitted by Haemaphysalis qinghaiensis (Yin et al., 2002; Liu et al., 2010b). There
306
are no reports of these ticks in South Africa. We previously speculated that Haemaphysalis silacea
307
is possibly the vector of the T. buffeli-like and T. sinensis-like organisms in South Africa due to its
308
geographical distribution in the KwaZulu-Natal and Eastern Cape provinces, but not in the north-
309
eastern areas of the country (Chaisi et al., 2011). However, the identification of T. buffeli type-D (T.
310
sinensis), type-C and type-B like organisms from the Limpopo province and Mozambique by Mans
311
et al. (2011) indicate the possibility of different and/or additional tick vectors. T. buffeli was also
312
identified as the most common Theileria spp. infecting cattle in Tunisia, and was more frequently
313
identified in the sub-humid zone of the country than in other climatic zones (M’ghirbi et al., 2008).
314
T. buffeli was identified from Boophilus annulatus, Hymaphysalis punctata, Ixodes ricinus,
315
Rhipicephalus bursa and Rhipicephalus turanicus in Tunisia (M’ghirbi et al., 2010), indicating that
316
a wide range of tick vectors may be responsible for transmission of the parasite in Africa. The
317
vectors of these organisms and their effect on the epidemiology of theileriosis in South Africa
318
should be established as buffalo might act as a source of infection to naïve cattle.
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The novel T. buffeli genotypes identified in this study are closely related to the T. buffeli Type D
320
group. Since some T. buffeli Type D organisms and the closely-related T. sinensis are known to
321
cause disease in cattle, it is important to be able to identify these organisms in South Africa,
322
especially in regions where theileriosis occurs, in order to determine if they play a role in the
323
epidemiology of the disease. T. buffeli infections in cattle and buffalo in South Africa have been
324
identified by the reverse line blot (RLB) hybridization assay using a species-specific probe that was
325
designed from the V4 hypervariable region of the 18S rRNA gene by Gubbels et al. (1999).
326
Although the V4 hypervariable region is highly variable within T. buffeli, the RLB probe is
327
designed in a conserved area within this region and can therefore detect all known T. buffeli
328
genotypes; hence our initial detection of T. buffeli in buffalo from HIP and AEGP. Subsequently,
329
Gubbels et al. (2000) designed additional RLB probes for the specific detection of Type D, non-
330
type D, and Type A genotypes, and another probe was designed to detect all the other known T.
331
buffeli 18S rRNA genotypes (Ikeda, B, C, E, H, Warwick). The novel genotypes that we identified
332
in this study will not be detected by any of the genotype-specific probes designed by Gubbels et al.
333
(2000) due to nucleotide differences (4 – 7 bp) in the RLB probe area. There is therefore a need to
334
develop new RLB probes for the specific detection of the novel South African T. buffeli genotypes.
335
The ribosomal ITS region in eukaryotes is located between the genes coding for the small (18S) and
336
large (28S) ribosomal RNA subunits; the ITS spans the two ribosomal RNA transcribed spacers and
337
the 5.8S gene (ITS1-5.8S-ITS2) (Aktas et al., 2007). Unlike the rRNA genes, which are highly
338
conserved between closely related species, the spacer regions (ITS1 and ITS2) are subject to higher
339
evolutionary rates and are therefore more variable in length and nucleotide composition (Hills and
340
Dixon, 1991). The ITS region is thought to give a better resolution at the lower/subspecies level
341
(Gubbels et al., 2000) and is therefore more suitable for the discrimination of closely related species
342
and subspecies than the 18S rRNA gene. Furthermore, although T. buffeli (China) and T. buffeli
343
(USA), both Type A species, share identical 18S rRNA sequences they have different ITS
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sequences (Chae et al., 1998, 1999b; Aktas et al., 2007). As has previously been observed, the two
345
spacer regions (ITS1 and ITS2) from AEGP sequences were highly polymorphic in both length and
346
nucleotide composition, while the 5.8S region was highly conserved and was shorter than the spacer
347
regions. The ITS sequences from AEGP grouped together with Asian T. buffeli 18S rRNA and ITS
348
sequences to form a T. buffeli-like group, and correlated with the grouping revealed by phylogenetic
349
analysis of the novel T. buffeli 18S rRNA sequences from AEGP. Unfortunately sequencing of the
350
ITS region of the HIP clones was unsuccessful; had it been successful, we might have expected the
351
sequences to group together with the T. sinensis ITS sequences from China as was the case with the
352
18S rRNA sequences.
353
Based on the phylogenetic positions and nucleotide differences in the sequences of the 18S rRNA
354
gene and ITS region, it is possible that the two novel Theileria spp. genotypes from the South
355
African buffalo represent distinct species. However, additional molecular and biological data would
356
be required for such classification. Our results indicate the presence of two distinct clades of T.
357
buffeli organisms from different parts of the world. The first clade comprises T. buffeli types A, B,
358
C, E, H, Ikeda, Ipoh and Medan. Both the 18S rRNA gene and ITS sequences of these subtypes
359
form a monophyletic group of exclusively cattle pathogens. These sequences are clearly separated
360
from the second clade which contains the T. buffeli-like and T. sinensis-like sequences from buffalo
361
in South Africa, and the T. buffeli and T. sinensis (type D) sequences from buffalo and cattle in
362
Asia. The close relationship between T. buffeli identified in African buffalo and water buffalo, and
363
T. sinensis is likely due to the migration of cattle (with farmers) from the Far and Middle East to
364
Africa, and the importation of domestic and wild cattle from these regions as recently reported
365
(Decker et al., 2014).
366
In conclusion, we have established the phylogenetic position of T. buffeli-like organisms occurring
367
in buffalo in the AEGP based on 18S rRNA gene and ITS sequence, and that of T. sinensis-like
368
organisms of buffalo in the HIP. This study has confirmed that T. buffeli is a highly diverse and
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cosmopolitan species, and has indicated the presence of T. buffeli- and T. sinensis-like organisms in
370
the South African buffalo that are similar to those circulating in cattle, yak and water buffalo in
371
Asia, but are genetically distinct from T. buffeli from China and the USA. Whether this diversity
372
indicates the presence of different Theileria species of cattle and buffalo is still unclear and needs to
373
be clarified by more biological studies. The role of buffalo and other wildlife as reservoir hosts of
374
these species should be investigated as buffalo are known to be sources of many infectious diseases
375
of cattle in South Africa. Future studies should focus on animal transmission studies in order to
376
determine the tick vectors of the T. buffeli-like and T. sinensis-like genotypes, and on
377
epidemiological studies using new RLB probes that specifically detect and differentiate these novel
378
genotypes in hosts and tick vectors in South Africa. This study provides useful genetic information
379
towards the proper classification of this very complex group.
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380
Acknowledgements
382
This work was part of a PhD project that was funded by the South African National Research
383
Foundation (NRF ICD2006072000009) and UP Research Development Programme. It also falls
384
under the Belgian Directorate General for Development Co-operation Framework agreement
385
ITM/DGCD. We thank Lan He for her assistance in the lab and Milana Troskie for providing the
386
DNA of the AEGP samples. Buffalo blood samples were provided by Drs Roy Bengis, Fred
387
Potgieter and Dave Cooper.
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514
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Figure Legends
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Figure 1: Phylogenetic relationships of known T. buffeli sequences with novel T. buffeli-like and T.
521
sinensis-like 18S rRNA gene sequences from South Africa (underlined), as inferred by the
522
neighbor-joining method. GenBank accession numbers are indicated in parenthesis. Designated
523
cluster numbers are indicated in brackets. Bootstrap values are indicated at the nodes.
M
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519
524
Figure 2: Alignment of novel T. buffeli-like (blue) and T. sinensis-like (purple) 18S rRNA gene
526
sequences with known T. buffeli and T. sinensis sequences (black, green and pink), showing the V4
527
hypervariable region of the gene. Nucleotide positions identical to the sequence of Theileria sp.
528
type A (U9704) are indicated by dots. The regions where RLB probes were designed are
529
highlighted in grey. Nucleotide differences in the probe target sequences within the different
530
genotypes are underlined.
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531
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525
532
Figure 3: Phylogenetic relationships of novel T. buffeli-like ITS sequences from South Africa
533
(underlined) with known T. buffeli-like ITS sequences from GenBank (accession numbers in
534
parenthesis) as determined by Bayesian inference. Designated cluster numbers are indicated in
535
brackets. Posterior probabilities are indicated at the nodes of the tree.
536
25
Page 25 of 29
536
Country of origin
Reference
T. sp. type A T. sergenti T. sergenti T. buffeli T. buffeliMarula
U97047 AF081137 EU083802 AF236097 Z15106
USA, Korea,Japan China China China Kenya
Chae et al., 1998 Gubbels et al., 2000 Not published Gubbels et al., 2000 Allsopp et al., 1994
T. sp. type B T. sergenti (Ikeda) T. sp. type B1 T. sergenti (Ikeda)
U97048 AY661515 U97049 AB000271
USA, Korea, Japan Japan USA, Korea Japan
Chae et al., 1998 Atkas et al., 2007 Chae et al., 1998 Chansiri et a., 1999
Type C T. buffeli T. sp. Macheng T. sp. Xiaogan
U97051 DQ256380 DQ256381
Korea China China
Type D T. sinensis T. sinensis T. sp China (cattle) T. sp. Thung Song
U97052 EU277003 EU274472 AF036336 AB000270
USA, South Korea China China China Thailand
Type E Theileria sp. Ipoh T. sp. Hongan
U97053 AB000273 DQ286801
Korea Malaysia China
T. buffeli Warwick T. buffeli T. buffeli (cow) T. sp. Hubei T. buffeli
AB000272 FJ225391 DQ289795 DQ104610 AF236094
Australia Spain Spain China Australia
Chansiri et al., 1999 Gimerez et al., 2009 Criado et al., 2006 Liu et al., 2010a Gubbels et al., 2000
T. buffeli China T. buffeli T. buffeli Indian
DQ104611 HM538212 EF126184
China China India
Liu et al., 2010a Not published Not published
AEGP/65/18S AEGP/66/18S AEGP/69/18S AEGP/70/18S AEGP/73/18S AEGP/74/18S AEGP/76/18S HIP/A2/a HIP/A4/c HIP/A4/e HIP/C23/a HIP/C23/b
JQ037779 JQ037780 JQ037781 JQ037782 JQ037785 JQ037783 JQ037784 JQ037790 JQ037786 JQ037787 JQ037788 JQ037789
South Africa South Africa South Africa South Africa South Africa South Africa South Africa South Africa South Africa South Africa South Africa South Africa
This study This study This study This study This study This study This study This study This study This study This study This study
Type H
U97050
Korea
Chae et al., 1998
AB000274
Indonesia
Chansiri et al., 1999
537 538
cr
M
an
us
Chae et al., 1998 Liu et al., 2010a Liu et al., 2010a
d
te
Ac ce p
T. sp. Medan
ip t
Accession number
Chae et al., 1998 Not published Not published Gubbels et al., 2000 Chansiri et al., 1999 Chae et al., 1998 Chansiri et al., 1999 Liu et al., 2010a
Table 1: 18S rRNA gene sequences of T. buffeli-like isolates and field samples used for the construction of phylogenetic trees.
539 540 541 26
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Figure 2
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S0304-4017(14)00337-9 http://dx.doi.org/doi:10.1016/j.vetpar.2014.06.001 VETPAR 7278
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
21-1-2013 30-5-2014 1-6-2014
Please cite this article as: Chaisi, M.E., Collins, N.E., Oosthuizen, M.C.,Phylogeny of Theileria buffeli genotypes identified in the South African buffalo (Syncerus caffer) population, Veterinary Parasitology (2014), http://dx.doi.org/10.1016/j.vetpar.2014.06.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Phylogeny of Theileria buffeli genotypes identified in the South African buffalo
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(Syncerus caffer) population
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Mamohale E. Chaisia,b,*, Nicola E. Collinsa, Marinda C. Oosthuizena
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a
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Private Bag X04, Onderstepoort, 0110, South Africa
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b
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180, Lesotho;
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* Corresponding author. Tel. +27 735715876; fax: +27 125298312.
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E-mail address: [email protected] (M.E. Chaisi)
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Department of Biology, National University of Lesotho, Faculty of Science and Technology, Roma
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Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria,
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Abstract
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Theileria buffeli/orientalis is a group of benign and mildly pathogenic species of cattle and buffalo
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in various parts of the world. In a previous study, we identified T. buffeli in blood samples
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originating from the African buffalo (Syncerus caffer) in the Hluhluwe-iMfolozi Game Park (HIP)
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and the Addo Elephant Game Park (AEGP) in South Africa. The aim of this study was to
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characterize the 18S rRNA gene and complete internal transcribed spacer (ITS1-5.8S-ITS2) region
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of T. buffeli samples, and to establish the phylogenetic position of this species based on these loci.
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The 18S rRNA gene and the complete ITS region were amplified from DNA extracted from blood
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samples originating from buffalo in HIP and AEGP. The PCR products were cloned and the
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resulting recombinants sequenced. We identified novel T. buffeli-like 18S rRNA and ITS genotypes
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from buffalo in the AEGP, and novel Theileria sinensis-like 18S rRNA genotypes from buffalo in
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the HIP. Phylogenetic analyses indicated that the T. buffeli-like sequences were similar to T. buffeli
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sequences from cattle and buffalo in China and India, and the T. sinensis-like sequences were
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similar to T. sinensis 18S rRNA sequences of cattle and yak in China. There was extensive
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sequence variation between the novel T. buffeli genotypes of the African buffalo and previously
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described T. buffeli and T. sinensis genotypes. The presence of organisms with T. buffeli-like and T.
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sinensis-like genotypes in the African buffalo could be of significant importance, particularly to the
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cattle industry in South Africa as these animals might act as sources of infections to naïve cattle.
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This is the first report on the characterization of the full-length 18S rRNA gene and ITS region of T.
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buffeli and T. sinensis genotypes in South Africa. Our study provides invaluable information
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towards the classification of this complex group of benign and mildly pathogenic species.
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Keywords: Theileria buffeli/orientalis; Theileria sinensis; African buffalo; 18S rRNA; ITS
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1. Introduction
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Theileria buffeli/orientalis is a group of closely related parasites of cattle and buffalo. They have a
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cosmopolitan distribution and infect cattle and buffalo in Africa, Australia, New Zealand, Asia,
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Europe and the United States of America (USA) (Steward et al., 1996; Chae et al., 1998; Chansiri et
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al., 1999; M’ghirbi et al., 2008). The cosmopolitan distribution of these species has been attributed
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to the global movement of cattle (and buffalo) without any regard to infection, and their distribution
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mainly depends on the availability of a suitable tick vector (Chae et al., 1999a; Cossio-Bayugar et
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al., 2002). Haemaphysalis ticks act as vectors in Australia, Asia and Europe, but the vectors in
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Africa and the USA are still unknown (Yin et al., 2004; M’ghirbi et al., 2008). Benign isolates from
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Britain, Australia and the USA were initially designated as Theileria mutans as their pathology was
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similar to that of T. mutans (Chae et al., 1999a). However, further studies have indicated that T.
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mutans is an African species and is serologically and genetically distinct from other benign
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Theileria spp. (Chae et al., 1999b).
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Theileria sergenti and T. orientalis were first described from eastern Siberia , while T. buffeli was
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first described from the Asian water buffalo (Bubalus bubalis) (reviewed by Fujisaki et al., 1994).
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The classification of these benign parasites is still confusing and is complicated by their similar
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morphology, serology, vector transmission, geographical distribution, difficulties in obtaining pure
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isolates and incompletely understood life-cycles (Uilenberg et al., 1985; Uilenberg, 2011). It is still
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unclear if these organisms represent the same species or several different species. The term “T.
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sergenti” actually refers to a sheep parasite and was incorrectly used to name a parasite of cattle and
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buffalo (Fujisaki, 1992; Chae et al., 1999a; Uilenberg, 2011). T. buffeli refers to a cluster of benign
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species within the T. buffeli/orientalis group, based on molecular data and infectivity for buffalo.
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More data are awaited to have a better understanding of the diversity within the T. buffeli/orientalis
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group. In the meantime, we will use the T. buffeli-like and T. sinensis-like for the new Theileria
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genotypes characterized in this study.
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Another closely related species, T. sinensis, was recently described in China and is also regarded as
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a cause of bovine theileriosis in that country (Bai et al., 2002a; b, cited by Yin et al., 2004). T.
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sinensis is transmitted by Haemaphysalis qinghaiensis ticks and infects cattle, yak (Yin et al., 2004)
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and water buffalo (He et al., 2012) in China.
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Molecular biology studies based on the 18S ribosomal RNA (rRNA) gene, internal transcribed
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spacers (ITS), major piroplasm surface protein (MPSP) gene and other genetic markers have
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provided useful information on the epidemiology, diagnosis, taxonomy and phylogeny of these
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benign Theileria spp. in various parts of the world (Aktas et al., 2007; Altay et al., 2008; Allsopp et
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al., 1994; Chae et al., 1998; Chansiri et al., 1999; Cossio-Bayugar et al., 2002; Gubbels et al., 2000,
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2002; Sarataphan et al., 2003; M’ghirbi et al., 2008; Liu et al., 2010a, b; Wang et al., 2010; Kamau
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et al., 2011). We recently identified T .buffeli in some buffalo populations in South Africa (Chaisi et
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al., 2011). The aims of this study were to: (1) sequence the full-length 18S rRNA gene and
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complete ITS (ITS1-5.8S-ITS2) region of T. buffeli of buffalo from two geographically different
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areas in South Africa; (2) determine the level of genetic variation between novel T. buffeli-like and
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T. sinensis-like genotypes of the African buffalo and known T. buffeli and T. sinensis genotypes;
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and (3) to establish their phylogenetic positions based on their full-length 18S rRNA gene and
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complete ITS sequences.
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2. Materials and Methods
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2.1 DNA samples
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A molecular epidemiological survey based on the 18S rRNA gene was previously carried out to
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determine the occurrence of Theileria spp. from the African buffalo in different geographic areas in
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South Africa and Mozambique using the reverse line blot (RLB) hybridization assay (Chaisi et al.,
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2011). In that study, T. buffeli was identified from buffalo blood samples originating from the
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Hluhluwe-iMfolozi Game Park (HIP); these samples contained mixed Theileria spp. infections.
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Based on the RLB results, thirteen samples were selected for characterization of the full-length 18S
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rRNA gene and the complete ITS (ITS1-5.8S-ITS2) region (HIP/A2, HIP/A4, HIP/A36, HIP/B62,
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HIP/C5, HIP/C11, HIP/C13, HIP/C15, HIP/C18, HIP/C19, HIP/C23, HIP/C25, HIP/C27). T. buffeli
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is also known to occur in buffalo in the Addo Elephant Game Park (AEGP), Eastern Cape Province,
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South Africa (Milana Troskie, personal communication). The parasite 18S rRNA gene and ITS
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region were also characterized from seven samples originating from buffalo in the AEGP
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(AEGP/65, AEGP/66, AEGP/69, AEGP/70, AEGP/73, AEGP/74, AEGP/76).
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2.2 Amplification of the full-length 18S rRNA gene and complete ITS (ITS1-5.8S-ITS2) region
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The full length 18S rRNA genes from 11 samples (4 from HIP and 7 from AEGP) were
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successfully amplified by conventional PCR using forward primer Nbab-1F and reverse primer
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18SRev-TB (Oosthuizen et al., 2008). The reaction mixture and cycling conditions were as
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described by Chaisi et al. (2011). The resulting amplicons were purified using the QIAquick PCR
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Purification Kit (Qiagen, South Cross Biotechnology, South Africa).
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A nested PCR protocol was used to amplify the complete parasite ITS region from 17 samples (10
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from HIP and 7 from AEGP). The primary reaction contained 2.5 µl (~75 ng) genomic DNA, 0.1
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µM each of primer 1055F (5’- GGT GGT GCA TGG CCG-3’) and LSUR300 (5’-T(A/T)G CGC
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TTC AAT CCC-3’) (Holman et al., 2003), 1.5 mM MgCl2, 200 µM dNTPs, High Fidelity Enzyme
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blend (Roche Diagnostics, Mannheim, Germany) and nuclease-free water to a total volume of 25 µl.
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The thermal cycling programme was done at an initial denaturation at 96°C for 3 min, followed by
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30 cycles of denaturation at 94°C for 30 s; annealing at 50°C for 30 s; extension at 72°C for 3 min;
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a final extension at 72°C for 7 min and then hold at 4°C. Primers ITSF (5’-GAG AAG TCG TAA
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CAA GGT TTC CG-3’) and LSUR50 (5’-GCT TCA CTC GCC GTT ACT AGG-3’) (Holman et
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al., 2003) were used for the nested PCR. The reaction mixture was as above, except that 1 µl (~
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30ng) of the primary PCR product was used as template. The cycling conditions were also as above,
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except that annealing was done at 60°C for 30 s and extension was done at 72°C for 2 min. The
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nested PCR resulted in a number of non-specific bands. ITS amplicons of approximately 1200 bp
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were therefore excised from ethidium-bromide stained gels and purified using the QiAquick Gel
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Extraction Kit (Qiagen, Southern Cross Biotechnology, South Africa). Amplicons from four
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reactions per sample were pooled prior to cloning and/or sequencing to avoid Taq polymerase
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induced errors.
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2.3 Cloning and sequencing of the full-length 18S rRNA gene from HIP samples
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The HIP samples all contained mixed Theileria spp. infections and therefore the amplicons could
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not be directly sequenced. Purified 18S rRNA amplicons from the HIP samples were cloned into the
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pGEM-T Easy Vector and transformed into E. coli JM109 High Efficiency Competent cells
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(Promega, Madison, WI). At least 5 white colonies were selected per sample and recombinant
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plasmid DNA was extracted from overnight bacterial cultures using the High Pure Plasmid Isolation
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kit (Roche Diagnostics, Mannheim, Germany).
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The obtained plasmids were screened by sequencing using the ABI BigDyeTM Terminator Cycle
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Sequencing Ready Reaction kit (PE Applied Biosystems), 350 ng plasmid DNA and 3.2 pmol of
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primer RLB-F2 (Nijhof et al., 2003). The sequences obtained were subjected to a BLASTn
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(Altschul et al., 1990) similarity search. The full-length 18S rRNA genes from recombinant
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plasmids containing sequences that were closely similar to the published 18S rRNA gene sequences
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of T. buffeli or T. sinensis were then sequenced as described above using primers Nbab-1F, 18SRev-
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TB, RLB-R2, BT18S-2F, BT18S-3F, BT18S-4F, BT18S-4R, SP6, T7 (Oosthuizen et al., 2008;
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Chaisi et al., 2011). Sequencing was done on an ABI3100 genetic analyzer at the Agricultural
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Research Council-Onderstepoort Veterinary Institute (ARC-OVI), South Africa.
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2.3 Direct sequencing of the full-length 18S rRNA gene from AEGP samples
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Since T. buffeli is the only Theileria spp. known to infect buffalo in the AEGP, the 18S amplicons
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obtained from samples from this game park were directly sequenced. Full-length 18S rRNA gene
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amplicons were sequenced using ABI BigDyeTM Terminator Cycle Sequencing Ready Reaction kit
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(Life Technologies, South Africa), ~40 ng of PCR product and 3.2 pmol of each primer.
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Sequencing was done at the sequencing facility of the ARC-OVI, South Africa.
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2.4 Cloning and sequencing of the complete ITS (ITS1-5.8S-ITS2) region from HIP and AEGP
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samples
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Purified ITS amplicons were cloned as described above. Sequencing reactions were done using the
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ABI BigDyeTM Terminator Cycle Sequencing Ready Reaction kit (PE Applied Biosystems), ~300
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ng of plasmid DNA and 2 pmol each of primers ITSF, SP6 and T7. Sequencing was done on an
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ABI 3500XL genetic analyzer at Inqaba Biotechnologies, South Africa.
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2.5 Sequence and phylogenetic analyses
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The sequences were assembled and edited using the GAP4 program of the Staden package (version
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1.6.0 for Windows) (Bonfield et al., 1995). A BLASTn homology search of GenBank was done
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using the full length consensus sequences. These were aligned with 18S rRNA gene sequences
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(Table 1) or ITS sequences of related genera from GenBank using the MAFFT (multiple sequence
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alignment) v6 programme employing the FFT-NS-1 algorithm (Katoh et al., 2005). The alignments
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were manually examined and edited across their full-lengths, and then truncated to the size of the
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smallest sequence using BioEdit v7 (Hall, 1999). Heteroduplexes were eliminated from the
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alignments. A total of 58 (new and known) 18S rRNA gene sequences (1514 bp), and 30 ITS
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sequences (1510 bp) were analysed. Estimated evolutionary divergence was calculated by
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determining the number of nucleotide differences between similar sequences over a region of 1499
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and 1215 nucleotides for the 18S rRNA gene and ITS sequences, respectively. Nucleotide
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differences were also assessed in the V4 hypervariable region of the 18S rRNA sequences, and in
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the ITS1, 5.8S gene and ITS2 regions of the ITS sequences.
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Phylogenetic trees were inferred from the alignments by the neighbor-joining method (Saitou and
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Nei, 1987), maximum parsimony and maximum likelihood methods using PAUP* v4b10
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(Swofford, 2003). These were done in combination with bootstrapping (1000 replicates). Bayesian
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inference was done using MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2003), accessed via the
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Computational Biology Service Unit, Cornell University. For comparison of the phylogenetic trees,
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18S rRNA gene and ITS sequences of the same species or isolate were included, where possible.
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The 18S rRNA gene and ITS sequences of Babesia canis, B. caballi and B. orientalis were included
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as outgroups to root the phylogenetic trees. All consensus trees were edited using MEGA4 (Tamura
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et al., 2007).
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The near full-length 18S rRNA gene sequences have been deposited in GenBank under accession
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numbers JQ037779 – JQ037790. The complete ITS sequences have been deposited in GenBank
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under accession numbers JQ0377791 – JQ037795.
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3. Results
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3.1 Identification of T. buffeli-like and T. sinensis-like 18S rRNA gene sequences
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Amplification of the 18S rRNA gene from the HIP and AEGP samples resulted in single bands of
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approximately 1700 bp, as viewed on a 2% ethidium bromide stained agarose gel. These amplicons
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were purified, cloned and sequenced. A total of 12 (5 from HIP and 7 from AEGP) near full-length
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18S rRNA sequences were obtained (Table 1).
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The HIP sequences (GenBank accession nos. JQ037786 – JQ037790) ranged in length from 1582 to
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1592 bp.. A BLASTn homology search did not reveal any identical sequences in GenBank; the
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closest homology (98% and 99%) was found with 18S rRNA gene sequences of Theileria sp.
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(Thung Song) (AB000272), Theileria sp. type D (U97052), T. sinensis (EU277003), T. sinensis
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(EU27442) and Theileria sp. China (cattle) (AF036336).
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The seven 18S rRNA sequences from AEGP (GenBank accession nos. JQ037779 – JQ037785)
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were from 1587 to 1588 bp in length. BLASTn similarity searches of these sequences did not reveal
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any identical sequences, but they were most similar (99%) to unclassified T. buffeli 18S rRNA gene
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sequences from China (DQ104611 and HM538212) and India (EF126184).
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3.2 Sequence and phylogenetic analyses of the 18S rRNA genes
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Phylogenetic analyses using neighbor-joining, maximum parsimony, maximum likelihood and
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Bayesian inference, all yielded trees with almost identical topologies and high bootstrap or nodal
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support values, although there were differences within the branching of the clusters in some trees.
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The T. buffeli 18S rRNA sequences could be separated into two distinct clades comprising nine
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distinct clusters, and were clearly separated from other Theileria spp. (Figure 1).
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The first clade comprised clusters 1 to 5, and contained known T. buffeli 18S rRNA genotypes
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designated Types A, B/Ikeda, C/Medan, E/H/Ipoh and Warwick (Chae et al., 1998; 1999b; Chansiri
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et al., 1999; Gubbels et al., 2000; Yin et al., 2004).
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The second clade comprised clusters 6 to 9 and contained T. buffeli Type D and T. sinesis sequences
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of cattle and buffalo, as well as the novel T. buffeli-like 18S sequences from South African buffalo.
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These were designated as types SA1 and SA2 (Figure 1). Genotype SA1 comprised 18S rRNA gene
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sequences originating from the AEGP (cluster 6), and these sequences were closely related to those
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of T. buffeli from buffalo in China (DQ104611) (Liu et al., 2010a) and (HM538212) (unpublished),
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and India (EF126184) (unpublished) (cluster 7). Since the sequences from China and India do not
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fall under any of the designated T. buffeli genotypes, we indicated them as “unclassified” in Figure
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1. Genotype SA2 is composed of 18S rRNA gene sequences from the HIP (cluster 9), and is closely
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related to the T. buffeli type D/T. sinensis group (cluster 8).
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In order to estimate the genetic distance between the T. buffeli-like sequences, the novel genotypes
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were aligned with 17 known T. buffeli 18S rRNA gene sequences (representing the different
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genotypes), and compared along a region of 1499 bp. Sequence variation was observed both within
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and between the different T. buffeli genotypes. All seven novel sequences from AEGP were
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identical within this region and along their full lengths (results not shown). These sequences
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differed from those of the closely related unclassified genotypes by 9 – 12 bp, and from the novel
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sequences from HIP by 21 – 23 bp. The HIP sequences differed from each other by 2 – 7 bp, and
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from the T. buffeli type D/T. sinensis sequences by 11 - 16 bp. Sequences HIP/C23/a and HIP/C23/b
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were identical, while there was a 7 bp difference between sequences HIP/A4/c and HIP/A4/e. The
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greatest variation (~ 45 bp) was observed between sequence HIP/A4/c and Theileria sp. type E,
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which is from cattle isolates in the USA and South Korea (Chae et al., 1998).
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Most of the variation between the T. buffeli-like genotypes occurred in the V1 variable region
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(positions 70 – 140) and V4 hypervariable region (positions 490 – 560) of the gene. The probes
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used for the identification and differentiation of the different Theileria spp. and their variants using
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the RLB hybridization assay were designed to detect target sequences in the V4 region of the gene
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(Gubbels et al., 1999; 2000). Figure 2 shows the positions of the RLB probes that were designed for
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the detection of all T. buffeli 18S rRNA gene sequences and for specific detection of the T. buffeli
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types A, D and non-D (Gubbels et al., 1999; 2000). It is unlikely that the genotype-specific probes
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designed by Gubbels et al. (2000) will detect the novel South African genotypes identified in this
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study, due to nucleotide differences of 4 to 7 bp in the RLB probe target area (Figure 2).
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3.3 Sequence and phylogenetic analyses of the novel ITS sequences
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Amplicons of approximately 1200 bp, as viewed on a 2% agarose gel, were observed from the
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nested PCR products. Eleven ITS sequences were obtained from 10 samples from HIP. However,
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these were all identified as heteroduplex sequences, comprising chimaeric ITS sequences from the
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different Theileria species that were present in the HIP samples. In a PCR containing mixed
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templates, cross-hybridization of heterologous sequences leads to the formation of heteroduplexes
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during the plateau phase of the reaction when decreasing primer:template ratios no longer favor
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primer annealing (Suzuki et al., 1996). It is likely that the heteroduplexes formed during the primary
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PCR were further amplified during the secondary PCR, resulting in a bias towards heteroduplex
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sequences in the clones. Heteroduplex sequences were excluded from further analyses.
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Amplicons from five out of seven samples from AEGP were successfully sequenced. The complete
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ITS sequences (GenBank accession nos. JQ0377791 - JQ037795) differed in the lengths of their
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complete ITS region (952 – 1173 bp), as well as in the ITS1 (458 – 642 bp) and ITS2 (297 – 344
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bp) regions. The 5.8S gene was conserved amongst the sequences and shorter (187 bp) than the
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ITS1 and ITS2 regions. Unlike the novel 18S rRNA gene sequences from AEGP which were
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identical, there was polymorphism among the novel ITS sequences, with most of the variation
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occurring in the ITS1 region. This was mainly due to insertions or deletions of blocks of sequences.
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Three sequences (AEGP/65/ITS, AEGP/66/ITS, AEGP/69/ITS) were identical, while sequences
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AEGP/73/ITS and AEGP/73/ITS differed from each other at 4 positions, and differed from the
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other three sequences (AEGP/65, 66, 69) by 134 bp. There was extensive variation between these
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sequences and the homologous T. buffeli ITS sequences from China (107 – 164 bp), USA and Japan
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(159 – 200 bp), and T. sinensis ITS sequences (144 – 178 bp). These differences were in
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concordance with those obtained from analyses of similar genotypes/species of the 18S rRNA gene.
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Three distinct clusters of the T. buffeli ITS sequences were observed in all of the trees generated
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using neighbor-joining, maximum parsimony, maximum likelihood and Bayesian inference. A
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representative tree generated by Bayesian inference is shown in Figure 3. Cluster 1 comprised the T.
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buffeli sequences from the USA and Japan (Chitose). It has previously been shown that these two
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groups share identical 18S rRNA sequences (Figure 1, Chae et al., 1998, 1999b; Aktas et al., 2007)
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but different ITS (Figure 3; Aktas et al., 2007) and MPSP (Gubbels et al., 2000) sequences.
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Cluster 2 comprised the novel ITS sequences from AEGP and the T. buffeli ITS sequences from
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China. This clade is probably synonymous to that of the “unclassified” 18S rRNA sequences which
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also grouped together with AEGP 18S rRNA sequences (Figure 1). Cluster 3 contained the T.
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sinensis ITS sequences. The ITS sequences of Theileria cervi, Theileria uilenbergi and Theileria
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luwenshuni always grouped together, while the 18S rRNA gene sequences of these species were in
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separate clusters. Unlike the 18S rRNA sequences (Figure 1), the T. mutans ITS sequence grouped
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together with the Theileria parva and Theileria annulata sequences (Figure 3).
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4. Discussion
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In a previous study, RLB hybridization indicated that T. buffeli was the most commonly occurring
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species in buffalo in the HIP, mainly co-occurring with other Theileria spp. (Chaisi et al., 2011),
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and was the only Theileria sp. infecting buffalo in the AEGP (Milana Troskie, personal
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communication). In contrast, T. buffeli was not identified in buffalo samples from the Kruger
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National Park (KNP), Greater Limpopo Transfrontier Park (GLTP) and a private game farm
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bordering the KNP (Chaisi et al., 2011). The molecular characterization and phylogenetic analyses
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reported here indicate that there are at least two T. buffeli genotypes in the South African buffalo
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population and these are closely related to T. buffeli and T. sinensis from the water buffalo (Bubalus
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bubalis) and cattle in some parts of Asia.
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The distribution of different T. buffeli 18S rRNA genotypes (A, B, C, D, E, H) in buffalo and cattle
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has previously been reported from several parts of the world (Chae et al., 1998; 1999a, Chansiri et
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al., 1999, Gubbels et al., 2000; 2002). Types A, B, C, E and H are mainly cattle parasites and are
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considered to be of low to moderate pathogenicity. Type D (T. sinensis) infects water buffalo, yak
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and African buffalo and is probably not pathogenic in these animals, but it can cause severe disease
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when transmitted to cattle (Chae et al., 1999a). In the USA, Type A and D-like organisms have
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been associated with bovine theileriosis in Missouri (Stockham et al., 2000), Texas (Chae et al.,
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1999a) and Michigan (Cossio-Bayugar et al., 2002).
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Our phylogenetic analyses are in agreement with previous analyses of the 18S rRNA and MPSP
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genes of T. buffeli, which indicate that there is extensive variation between T. buffeli type D
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sequences and those of the other T. buffeli types (Chansiri et al., 1999; Gubbels et al., 2000; Yin et
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al. 2004; Liu et al., 2010b). These authors indicated that T. buffeli type D organisms may be
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genetically intermediate between the well-characterized pathogenic Theileria spp. (T. annulata, T.
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parva, T. lestoquardi, T. uilenbergi, T. luwenshuni) and the benign T. buffeli/orientalis spp.
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Furthermore, maximum likelihood and parsimony analyses of 18S rRNA gene sequences of
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Theileria spp. by Chansiri et al. (1999) grouped the T. buffeli type D sequences with the pathogenic
297
Theileria spp., whereas distance methods grouped them with those of the other T. buffeli sequences.
298
Randomly amplified polymorphic DNA (RAPD) profiles generated from Theileria sp. Thung Song
299
(a type D sequence) were different from those of the other benign T. buffeli-like species (Chansiri et
300
al., 1999). For these reasons, it was indicated that the classification of T. buffeli type D species is
301
questionable and should be investigated. Studies of type D sequences from cattle in China resulted
302
in the description of a new species, T. sinensis (Bai et al., 1995; 2002a; b).
303
Our findings indicate that suitable vectors for the transmission of T. buffeli are likely to be present
304
in South Africa. In Asia, T. buffeli/orientalis is transmitted by Haemaphysalis longicormis while T.
305
sinensis is transmitted by Haemaphysalis qinghaiensis (Yin et al., 2002; Liu et al., 2010b). There
306
are no reports of these ticks in South Africa. We previously speculated that Haemaphysalis silacea
307
is possibly the vector of the T. buffeli-like and T. sinensis-like organisms in South Africa due to its
308
geographical distribution in the KwaZulu-Natal and Eastern Cape provinces, but not in the north-
309
eastern areas of the country (Chaisi et al., 2011). However, the identification of T. buffeli type-D (T.
310
sinensis), type-C and type-B like organisms from the Limpopo province and Mozambique by Mans
311
et al. (2011) indicate the possibility of different and/or additional tick vectors. T. buffeli was also
312
identified as the most common Theileria spp. infecting cattle in Tunisia, and was more frequently
313
identified in the sub-humid zone of the country than in other climatic zones (M’ghirbi et al., 2008).
314
T. buffeli was identified from Boophilus annulatus, Hymaphysalis punctata, Ixodes ricinus,
315
Rhipicephalus bursa and Rhipicephalus turanicus in Tunisia (M’ghirbi et al., 2010), indicating that
316
a wide range of tick vectors may be responsible for transmission of the parasite in Africa. The
317
vectors of these organisms and their effect on the epidemiology of theileriosis in South Africa
318
should be established as buffalo might act as a source of infection to naïve cattle.
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The novel T. buffeli genotypes identified in this study are closely related to the T. buffeli Type D
320
group. Since some T. buffeli Type D organisms and the closely-related T. sinensis are known to
321
cause disease in cattle, it is important to be able to identify these organisms in South Africa,
322
especially in regions where theileriosis occurs, in order to determine if they play a role in the
323
epidemiology of the disease. T. buffeli infections in cattle and buffalo in South Africa have been
324
identified by the reverse line blot (RLB) hybridization assay using a species-specific probe that was
325
designed from the V4 hypervariable region of the 18S rRNA gene by Gubbels et al. (1999).
326
Although the V4 hypervariable region is highly variable within T. buffeli, the RLB probe is
327
designed in a conserved area within this region and can therefore detect all known T. buffeli
328
genotypes; hence our initial detection of T. buffeli in buffalo from HIP and AEGP. Subsequently,
329
Gubbels et al. (2000) designed additional RLB probes for the specific detection of Type D, non-
330
type D, and Type A genotypes, and another probe was designed to detect all the other known T.
331
buffeli 18S rRNA genotypes (Ikeda, B, C, E, H, Warwick). The novel genotypes that we identified
332
in this study will not be detected by any of the genotype-specific probes designed by Gubbels et al.
333
(2000) due to nucleotide differences (4 – 7 bp) in the RLB probe area. There is therefore a need to
334
develop new RLB probes for the specific detection of the novel South African T. buffeli genotypes.
335
The ribosomal ITS region in eukaryotes is located between the genes coding for the small (18S) and
336
large (28S) ribosomal RNA subunits; the ITS spans the two ribosomal RNA transcribed spacers and
337
the 5.8S gene (ITS1-5.8S-ITS2) (Aktas et al., 2007). Unlike the rRNA genes, which are highly
338
conserved between closely related species, the spacer regions (ITS1 and ITS2) are subject to higher
339
evolutionary rates and are therefore more variable in length and nucleotide composition (Hills and
340
Dixon, 1991). The ITS region is thought to give a better resolution at the lower/subspecies level
341
(Gubbels et al., 2000) and is therefore more suitable for the discrimination of closely related species
342
and subspecies than the 18S rRNA gene. Furthermore, although T. buffeli (China) and T. buffeli
343
(USA), both Type A species, share identical 18S rRNA sequences they have different ITS
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sequences (Chae et al., 1998, 1999b; Aktas et al., 2007). As has previously been observed, the two
345
spacer regions (ITS1 and ITS2) from AEGP sequences were highly polymorphic in both length and
346
nucleotide composition, while the 5.8S region was highly conserved and was shorter than the spacer
347
regions. The ITS sequences from AEGP grouped together with Asian T. buffeli 18S rRNA and ITS
348
sequences to form a T. buffeli-like group, and correlated with the grouping revealed by phylogenetic
349
analysis of the novel T. buffeli 18S rRNA sequences from AEGP. Unfortunately sequencing of the
350
ITS region of the HIP clones was unsuccessful; had it been successful, we might have expected the
351
sequences to group together with the T. sinensis ITS sequences from China as was the case with the
352
18S rRNA sequences.
353
Based on the phylogenetic positions and nucleotide differences in the sequences of the 18S rRNA
354
gene and ITS region, it is possible that the two novel Theileria spp. genotypes from the South
355
African buffalo represent distinct species. However, additional molecular and biological data would
356
be required for such classification. Our results indicate the presence of two distinct clades of T.
357
buffeli organisms from different parts of the world. The first clade comprises T. buffeli types A, B,
358
C, E, H, Ikeda, Ipoh and Medan. Both the 18S rRNA gene and ITS sequences of these subtypes
359
form a monophyletic group of exclusively cattle pathogens. These sequences are clearly separated
360
from the second clade which contains the T. buffeli-like and T. sinensis-like sequences from buffalo
361
in South Africa, and the T. buffeli and T. sinensis (type D) sequences from buffalo and cattle in
362
Asia. The close relationship between T. buffeli identified in African buffalo and water buffalo, and
363
T. sinensis is likely due to the migration of cattle (with farmers) from the Far and Middle East to
364
Africa, and the importation of domestic and wild cattle from these regions as recently reported
365
(Decker et al., 2014).
366
In conclusion, we have established the phylogenetic position of T. buffeli-like organisms occurring
367
in buffalo in the AEGP based on 18S rRNA gene and ITS sequence, and that of T. sinensis-like
368
organisms of buffalo in the HIP. This study has confirmed that T. buffeli is a highly diverse and
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cosmopolitan species, and has indicated the presence of T. buffeli- and T. sinensis-like organisms in
370
the South African buffalo that are similar to those circulating in cattle, yak and water buffalo in
371
Asia, but are genetically distinct from T. buffeli from China and the USA. Whether this diversity
372
indicates the presence of different Theileria species of cattle and buffalo is still unclear and needs to
373
be clarified by more biological studies. The role of buffalo and other wildlife as reservoir hosts of
374
these species should be investigated as buffalo are known to be sources of many infectious diseases
375
of cattle in South Africa. Future studies should focus on animal transmission studies in order to
376
determine the tick vectors of the T. buffeli-like and T. sinensis-like genotypes, and on
377
epidemiological studies using new RLB probes that specifically detect and differentiate these novel
378
genotypes in hosts and tick vectors in South Africa. This study provides useful genetic information
379
towards the proper classification of this very complex group.
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Acknowledgements
382
This work was part of a PhD project that was funded by the South African National Research
383
Foundation (NRF ICD2006072000009) and UP Research Development Programme. It also falls
384
under the Belgian Directorate General for Development Co-operation Framework agreement
385
ITM/DGCD. We thank Lan He for her assistance in the lab and Milana Troskie for providing the
386
DNA of the AEGP samples. Buffalo blood samples were provided by Drs Roy Bengis, Fred
387
Potgieter and Dave Cooper.
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Figure Legends
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Figure 1: Phylogenetic relationships of known T. buffeli sequences with novel T. buffeli-like and T.
521
sinensis-like 18S rRNA gene sequences from South Africa (underlined), as inferred by the
522
neighbor-joining method. GenBank accession numbers are indicated in parenthesis. Designated
523
cluster numbers are indicated in brackets. Bootstrap values are indicated at the nodes.
M
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524
Figure 2: Alignment of novel T. buffeli-like (blue) and T. sinensis-like (purple) 18S rRNA gene
526
sequences with known T. buffeli and T. sinensis sequences (black, green and pink), showing the V4
527
hypervariable region of the gene. Nucleotide positions identical to the sequence of Theileria sp.
528
type A (U9704) are indicated by dots. The regions where RLB probes were designed are
529
highlighted in grey. Nucleotide differences in the probe target sequences within the different
530
genotypes are underlined.
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531
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525
532
Figure 3: Phylogenetic relationships of novel T. buffeli-like ITS sequences from South Africa
533
(underlined) with known T. buffeli-like ITS sequences from GenBank (accession numbers in
534
parenthesis) as determined by Bayesian inference. Designated cluster numbers are indicated in
535
brackets. Posterior probabilities are indicated at the nodes of the tree.
536
25
Page 25 of 29
536
Country of origin
Reference
T. sp. type A T. sergenti T. sergenti T. buffeli T. buffeliMarula
U97047 AF081137 EU083802 AF236097 Z15106
USA, Korea,Japan China China China Kenya
Chae et al., 1998 Gubbels et al., 2000 Not published Gubbels et al., 2000 Allsopp et al., 1994
T. sp. type B T. sergenti (Ikeda) T. sp. type B1 T. sergenti (Ikeda)
U97048 AY661515 U97049 AB000271
USA, Korea, Japan Japan USA, Korea Japan
Chae et al., 1998 Atkas et al., 2007 Chae et al., 1998 Chansiri et a., 1999
Type C T. buffeli T. sp. Macheng T. sp. Xiaogan
U97051 DQ256380 DQ256381
Korea China China
Type D T. sinensis T. sinensis T. sp China (cattle) T. sp. Thung Song
U97052 EU277003 EU274472 AF036336 AB000270
USA, South Korea China China China Thailand
Type E Theileria sp. Ipoh T. sp. Hongan
U97053 AB000273 DQ286801
Korea Malaysia China
T. buffeli Warwick T. buffeli T. buffeli (cow) T. sp. Hubei T. buffeli
AB000272 FJ225391 DQ289795 DQ104610 AF236094
Australia Spain Spain China Australia
Chansiri et al., 1999 Gimerez et al., 2009 Criado et al., 2006 Liu et al., 2010a Gubbels et al., 2000
T. buffeli China T. buffeli T. buffeli Indian
DQ104611 HM538212 EF126184
China China India
Liu et al., 2010a Not published Not published
AEGP/65/18S AEGP/66/18S AEGP/69/18S AEGP/70/18S AEGP/73/18S AEGP/74/18S AEGP/76/18S HIP/A2/a HIP/A4/c HIP/A4/e HIP/C23/a HIP/C23/b
JQ037779 JQ037780 JQ037781 JQ037782 JQ037785 JQ037783 JQ037784 JQ037790 JQ037786 JQ037787 JQ037788 JQ037789
South Africa South Africa South Africa South Africa South Africa South Africa South Africa South Africa South Africa South Africa South Africa South Africa
This study This study This study This study This study This study This study This study This study This study This study This study
Type H
U97050
Korea
Chae et al., 1998
AB000274
Indonesia
Chansiri et al., 1999
537 538
cr
M
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Chae et al., 1998 Liu et al., 2010a Liu et al., 2010a
d
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T. sp. Medan
ip t
Accession number
Chae et al., 1998 Not published Not published Gubbels et al., 2000 Chansiri et al., 1999 Chae et al., 1998 Chansiri et al., 1999 Liu et al., 2010a
Table 1: 18S rRNA gene sequences of T. buffeli-like isolates and field samples used for the construction of phylogenetic trees.
539 540 541 26
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