Genotypic Diversity of Theileria orientalis Detected from Cattle Grazing in Kumamoto and Okinawa Prefectures of Japan

Abstract

ABSTRACT. Theileria orientalis is a benign protozoan species that is widely distributed in Japan, yet sometimes causes serious economic losses in the livestock industry. In this study, we conducted a molecular survey based on genes encoding the major piroplasm surface protein (MPSP) and p23 for T. orientalis detected in cattle grazing in southern areas of Japan, consisting of 2 farms in Kumamoto prefecture (Aso and Kuma districts) and 3 farms in Okinawa prefecture (Ishigaki, Iriomote, and Yonaguni Islands). High prevalence rates of T. orientalis infection were shown in all the cattle populations using the diagnostic MPSP-and p23-PCR assays. Phylogenetic analyses revealed 4 MPSP genotypes and 3 p23 genotypes. Furthermore, MPSP genotype-specific PCR methods were developed in this study and wide distributions of 5-district genotypes of T. orientalis were observed for the examined farms. Our results indicate that at least 5 types of T. orientalis exist in Kumamoto and Okinawa prefectures of Japan and that genotype-specific PCR assays are highly applicable for the quarantine of transported cattle and for epidemiological surveys of bovine theileriosis in Japan.

Assessment of sequence variability in a p23 gene region within and among three genotypes of the Theileria orientalis complex from south-eastern Australia

“…Oligonucleotide primers p23F1 (forward; 5 -ATATGGAAGGC-TACAAGGCC-3 ) and p23R1 (reverse; 5 -CAAGAGAGGCAACAA-CAACG-3 ) were designed by aligning all published p23 gene sequences (GenBank accession numbers AB021223, AB491348-AB491354, AB469176-AB469181, D84446 and D84447; Sako et al, 1999;Ota et al, 2009;Yokoyama et al, 2011) and used to specifically amplify a fragment (469 bp) of this gene by PCR from genotypes buffeli, chitose and ikeda of T. orientalis. The reagents and PCR conditions were optimised in a series of experiments; the final PCR was conducted in a 50 l volume containing 10 mM Tris-HCl (pH 8.4), 50 mM KCl (Promega, USA), 3.5 mM MgCl 2 , deoxynucleotide triphosphates (dNTPs; 200 M each), primers (35 pmol each; GeneWorks, Australia) and 1 U GoTaq polymerase (Promega) using the following protocol: 5 min at 95 • C, followed by 35 cycles of 15 s at 95 • C, 30 s at 64 • C and 30 s at 72 • C, followed by a final extension of 5 min at 72 • C. Following PCR, an aliquot (5 l) of each amplicon was examined on 1.5% w/v agarose gels and photographed using GelDoc system (BioRad, Hercules, CA, USA).…”

“…To date, the major piroplasm surface protein (mpsp), small subunit (SSU) of nuclear ribosomal RNA and 23-kDa piroplasm membrane protein (p23) genes as well as the first and second internal transcribed spacers of nuclear ribosomal DNA (ITS-1 and ITS-2) have been used to study the genetic composition of the T. orientalis complex (Sako et al, 1999;Gubbels et al, 2000;Aktas et al, 2006;Ota et al, 2009;Kamau et al, 2011b;Yokoyama et al, 2011). The most widely applied marker has been mpsp (Jeong et al, 2010;Altangerel et al, 2011;Islam et al, 2011;Kamau et al, 2011a;Khukhuu et al, 2011;Yokoyama et al, 2011Yokoyama et al, , 2012Cufos et al, 2012;Eamens et al, 2013;Perera et al, 2013Perera et al, , 2014Sivakumar et al, 2013). Employing this marker, at least 11 genotypes of T. orientalis (i.e., chitose or type 1; ikeda or type 2; buffeli or type 3; types 4-8; N-1; N-2 and N-3) have been defined (reviewed by Sivakumar et al, 2014).…”

Semiquantitative Multiplexed Tandem PCR for Detection and Differentiation of Four Theileria orientalis Genotypes in Cattle

“…(4)(5)(6). Presently, 11 genotypes of T. orientalis complex (designated Chitose or type 1, Ikeda or type 2, Buffeli or type 3, types 4 to 8, and N-1 to N-3) have been identified using a number of molecular markers, including major piroplasm surface protein (MPSP) (7,8), 23-kDa piroplasm membrane protein (p23) (9)(10)(11)60), small-subunit (SSU) rRNA gene (8,12,13), and/or the first and second internal transcribed spacers of nuclear ribosomal DNA (ITS-1 and ITS-2, respectively) (12,14). Of these genotypes, Ikeda and Chitose are recognized to be associated with clinical outbreaks of oriental theileriosis, mainly in the Asia-Pacific region (15)(16)(17)(18)(19)(20)(21).…”

Molecular Detection of <i>Theileria</i> Species in Sheep from Northern China

“…T. sergenti is an invalid name from a taxonomic viewpoint, since it has been used to previously describe a parasite of sheep [9,16]. Recently, some genotypes of T. orientalis that affected cattle have been identified in some countries, such as China [10], Thailand [13], Vietnam [7] and Japan [20]. However, little information was available for the occurrence of T. orientalis in sheep and goats.…”