Letter to the Editor

Inadequate Differentiation of Theileria orientalis Genotypes buffeli and ikeda in a Multiplexed Tandem PCR (MT-PCR) Assay Using the p23 Gene as a Marker

Hagos Gebrekidan, Robin B. Gasser, Abdul Jabbar
Brad Fenwick, Editor
DOI: 10.1128/JCM.02017-16

LETTER

Oriental theileriosis is a tick-borne disease of bovids caused by hemoprotozoan parasites within the Theileria orientalis complex. In the last 5 years, hundreds of outbreaks of oriental theileriosis were recorded in cattle from Australia and New Zealand (1–4), mainly ascribed to ikeda and chitose, pathogenic genotypes of all 11 currently known genotypes of T. orientalis (5). In 2015, we established and validated a multiplexed tandem PCR (MT-PCR) assay for the simultaneous detection, differentiation, and quantitation of four commonly prevalent pathogenic (chitose and ikeda) and nonpathogenic (buffeli and type 5) genotypes of T. orientalis in Australasia (6). This assay was designed by AusDiagnostics Pty. Ltd., Australia; in this assay, two primer pairs target the sequences of the first internal transcribed spacer and the p23 gene of T. orientalis for genotypes ikeda and buffeli, respectively, and two pairs target the major piroplasm surface protein (MPSP) gene for both chitose and type 5 to amplify regions of 70 to 115 bp (6).

Following its design, the MT-PCR was assessed to detect T. orientalis genotypes in blood samples from bovines in Australasia, and it performed well, achieving diagnostic specificities and sensitivities of 94.0 to 98.9% and 97.1 to 98.9%, respectively (6–9). However, when tested using samples from a number of locations in Ethiopia (10), Pakistan (11), and Vietnam (12), results revealed marked variations among peak melting temperatures and single-strand conformation polymorphism (SSCP) profiles for the genotype buffeli. Therefore, MT-PCR amplicons were sequenced and compared with previously published p23 sequences of T. orientalis (13–15).

Of 20 buffeli MT-PCR amplicons sequenced, 40% (8/20) represented genotype buffeli, whereas 60% (12/20) represented ikeda. Comparison of the p23 sequences characterized in this study with those published previously from Australia (13) and Japan (14, 15) revealed one buffeli sequence (42Crev) and seven ikeda sequences (67Brev, 22Brev, 7Drev, 37Brev, 47Brev, 57Crev, 62Brev) of T. orientalis (Fig. 1). Furthermore, the comparison showed that the sequence representing buffeli (42Crev) was identical to those reported previously from Australia (GenBank accession no. KM504986 and KM504987) and Japan (AB491349 and AB021223) (Fig. 1). However, it was different from those of accession no. KM504988 (one transversion, A↔ C) (Australia), AB469178 (one transition, A↔ C) (Japan), and AB491348 (three transitions, A↔ G or C↔ T) (Japan) (Fig. 1). Of the seven p23 sequences representing ikeda determined here (67Brev, 22Brev, 7Drev, 37Brev, 47Brev, 57Crev, 62Brev), nucleotide variability of 3.2 to 20% was linked mainly to the transitions C↔ T and A↔ G, and these sequences were more closely related to previously published p23 sequences for ikeda than for buffeli (Fig. 1).

FIG 1
FIG 1

Alignment of the p23 nucleotide sequences of the genotypes buffeli and ikeda of T. orientalis determined here (bold and underlined) with the reference sequences of buffeli, chitose, and ikeda. The unique number at the beginning of each reference sequence represents the GenBank accession number.

Furthermore, in order to verify the cross-reactivity exhibited by the p23 primer pair in MT-PCR, we tested all 20 samples using conventional PCR to amplify a longer region of the p23 gene (∼601 bp) using primers designed by Ota et al. (14). Results revealed that the samples that tested positive for genotype ikeda by MT-PCR also tested positive for ikeda using an amplification of a longer region of the p23 gene by conventional PCR, and the same was true for genotype buffeli. Hence, there was a concordance between the results of MT-PCR and conventional PCR in assigning genotypes buffeli and ikeda of T. orientalis using the p23 gene. To assess the relationship of p23 sequences obtained by sequencing of amplicons by conventional PCR, phylogenetic analyses were undertaken by compiling p23 sequences generated in this study and those available in GenBank representing genotypes buffeli, chitose, and ikeda of T. orientalis. Phylogenetic analyses revealed that p23 sequences of genotypes buffeli (GenBank accession no. KY235360 and KY235361) and ikeda (KY235356 to KY235359) grouped with previously published sequences of the respective genotypes (Fig. 2).

FIG 2
FIG 2

Genetic relationships of genotypes buffeli and ikeda of T. orientalis determined in this study. The relationships were inferred based on phylogenetic analyses of the p23 sequences determined herein (bold) and reference sequences using Bayesian inference (BI) and distance-based neighbor-joining (NJ) methods. Theileria annulata was used as the outgroup. There was a concordance in the topology between this NJ tree and that produced using BI (not shown). Nodal support (from left to right) is given as a posterior probability for BI and as a bootstrap value for NJ analyses. The scale bar indicates the number of inferred substitutions per nucleotide site.

The results indicate that the primer pair targeting the p23 gene region to amplify genotype buffeli of T. orientalis using MT-PCR is nonspecific, leading to a cross-amplification from genotype ikeda (Fig. 1). Currently, the assay specificity for genotype buffeli is linked to only a single nucleotide in the forward primer designed by AusDiagnostics Pty. Ltd., being insufficient to achieve selectivity from ikeda. Previously, when we tested DNA samples using the MPSP gene, we did not observe any significant variation in peak melting temperatures or SSCP profiles among MT-PCR amplicons from genotype buffeli (6). Additionally, we did not find any discrepancy in the assignments of three genotypes (buffeli, chitose, and ikeda) of T. orientalis using the MPSP gene or the p23 gene (6, 16). However, sequence variation in the p23 gene region identified in this study is likely due to population variation within T. orientalis (buffeli) in blood samples from cattle and buffaloes from locations outside Victoria, Australia (New South Wales and South Australia within Australia, and Ethiopia, New Zealand, and Pakistan) (7–12); at the time, the p23 gene had not been assessed for variation within T. orientalis outside Victoria, Australia (16).

The findings of the conventional PCR followed by DNA sequencing revealed that the p23 gene is a good molecular marker to differentiate genotypes of T. orientalis when larger fragments of the DNA are targeted. Therefore, based on the current evidence, it is concluded that the primers designed for the p23 gene by AusDiagnostics Pty. Ltd. do not allow the accurate differentiation of genotypes buffeli and ikeda within the T. orientalis complex.

Accession number(s).p23 nucleotide sequences of genotypes buffeli and ikeda of T. orientalis determined here are 42Crev_Buffeli, 67Brev_Ikeda, 22Brev_Ikeda, 7Drev_Ikeda, 37Brev_Ikeda, 47Brev_Ikeda, 57Brev_Ikeda, and 62Brev_Ikeda. The p23 sequences of genotype buffeli are GenBank accession no. KY235360 and KY235361, and those of genotype ikeda are KY235356 to KY235359.

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KEYWORDS

p23 gene
buffeli
cross amplification
diagnosis
ikeda
Theileria orientalis

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